阅读说明：本技术 环糊精二聚体、其组合物及其用途 (Cyclodextrin dimers, compositions thereof, and uses thereof ) 是由 M·S·奧康诺尔 M·马兰加 M·科佩 C·A·T·M·B·汤姆 A·M·安德森 于 2020-01-03 设计创作，主要内容包括：描述了一类新的合成环糊精二聚体。示例性环糊精二聚体能够通过细胞内和细胞外靶向各种形式的胆固醇来治疗动脉粥样硬化斑块。还提供了通过用这种环糊精治疗来消耗胆固醇、胆固醇酯、7-酮基胆固醇和7-酮基胆固醇酯的动脉粥样硬化斑块的方法。还描述了对7-酮基胆固醇具有高特异性的二聚体子类。(A novel class of synthetic cyclodextrin dimers is described. Exemplary cyclodextrin dimers are capable of treating atherosclerotic plaques by targeting various forms of cholesterol both intracellularly and extracellularly. Also provided are methods of depleting atherosclerotic plaques of cholesterol, cholesterol esters, 7-ketocholesterol and 7-ketocholesterol esters by treatment with such cyclodextrins. Dimeric subclasses with high specificity for 7-ketocholesterol are also described.)

1. A cyclodextrin dimer having the structure:

CD-L-CD

wherein L passes through the C2 carbon of each CD subunit (instead of R)¹) And/or C3 carbon (instead of R)²) A large (sub-) surface attached to each CD molecule; wherein each CD has the structure of formula X:

Wherein L has a length of no more than 8 atoms, wherein said no more than 8 atoms are preferably each C, N, O or S;

wherein R1, R2 and R3 are each independently selected from H, methyl, hydroxypropyl, sulfobutyl, succinyl, quaternary ammonium such as-CH₂CH(OH)CH₂N(CH₃)₃ ⁺Alkyl, lower alkyl, alkylene, alkenyl, alkynyl, alkoxy, alkoxyalkyl, alkoxyalkoxyalkyl, alkylcarbonyloxyalkyl, alkylcarbonyl, alkylsulfonyl, alkylsulfonylalkyl, alkylamino, alkoxyamino, alkylsulfanyl, amino, alkylamino, dialkylamino, alkylaminoalkyl, dialkylaminoalkyl, aminoalkyl, aminoalkoxy, alkylsulfonylamino, aminocarbonyloxyalkyl, aminosulfonyl, ammonium, ammonia, alkylaminosulfonyl, dialkylaminosulfonyl, alkynylalkoxy, aryl, arylalkyl, arylsulfonyl, aryloxy, aralkyloxy, azido, bromo, chloro, cyanoalkyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, cycloalkylene, cycloalkylalkylene, deoxy, glucosyl, heteroalkyl, heteroaryl, heteroarylalkyl, alkoxy, alkylamino, dialkylamino, alkylaminoalkyl, aminoalkyl, amino, alkylamino, dialkylamino, alkylsulfonylamino, amino-alkoxy, dialkylamino, alkylsulfonyl, alkylamino, alkylsulfonyl, heteroaryl, alkylsulfonyl, heteroaryl, alkylsulfonyl, heteroaryl, alkylsulfonyl, or combinations thereof, wherein, Heteroarylsulfonyl, heteroaryloxy, heteroarylalkyloxy, heterocyclylalkoxy, halogen, haloalkyl, haloalkoxy, heterocyclylamino, heterocyclyl, heterocyclylalkyl, heterocyclyloxy, heterocyclylalkoxy, hydroxyalkoxy, hydroxyalkylamino, hydroxyalkylaminoalkyl, hydroxyalkyl, hydroxycarbonylalkyl, hydroxyalkyloxycarbonylalkyl, hydroxyalkyl, hydroxycycloalkyl, iodine, urea, carbamate, carboxyl, sulfate, sulfonyl, sulfonamide, nitro, nitrite, cyano, phosphate, phosphoryl, phenoxy, acetyl, a fatty acid such as palmitoyl, a monosaccharide or disaccharide, between 1 and 40 of which R1, R2 and R3 groups are not H, optionally between 1 and 28 of said R1, R2 and R3 groups are not H, optionally between 2 and 15 or between 4 and 20 of said R1, R2 and R3 groups are not H; and optionally the CD monomer has one or more additional substitutions.

2. A cyclodextrin dimer having the structure:

CD-L-CD

wherein L passes through the C2 carbon of each CD subunit (instead of R)¹) And/or C3 carbon (instead of R)²) A large (sub-) surface attached to each CD molecule; wherein each CD has the structure of formula X:

wherein L has a length of no more than 8 atoms, wherein said no more than 8 atoms are preferably each C, N, O or S;

wherein R1, R2 and R3 are each independently selected from H, methyl, hydroxypropyl, sulfobutyl, succinyl, maltosyl, carboxymethyl, quaternary ammonium (such as-CH)₂CH(OH)CH₂N(CH₃)₃ ⁺) Glucosyl, palmitoyl, phosphate, phosphoryl, amino, azido, sulfate, sulfonyl, alkyl, ethyl, propyl, isopropyl, butyl, isobutyl, bromo, chloro, wherein between 1 and 40 of said R1, R2, and R3 groups are not H, optionally between 1 and 28 of said R1, R2, and R3 groups are not H, optionally between 2 and 15 or between 4 and 20 of said R1, R2, and R3 groups are not H; and optionally the CD monomer has one or more additional substitutions.

3. A cyclodextrin dimer having the structure:

CD-L-CD

wherein L passes through the C2 carbon of each CD subunit (instead of R)¹) And/or C3 carbon (instead of R)²) A large (sub-) surface attached to each CD molecule; wherein each CD has a junction of formula XStructure:

wherein L has a length of no more than 8 atoms, wherein said no more than 8 atoms are preferably each C, N, O or S;

wherein R1, R2 and R3 are each independently selected from H, methyl, hydroxypropyl, sulfobutyl, succinyl, maltosyl, carboxymethyl, quaternary ammonium such as-CH₂CH(OH)CH₂N(CH₃)₃ ⁺Wherein between 1 and 40 of said R1, R2, and R3 groups are not H, optionally between 1 and 28 of said R1, R2, and R3 groups are not H, optionally between 2 and 15 or between 4 and 20 of said R1, R2, and R3 groups are not H; and is

Optionally the CD monomer has one or more additional substitutions.

4. A cyclodextrin dimer having the structure:

CD-L-CD

wherein L passes through the C2 carbon of each CD subunit (instead of R)¹) And/or C3 carbon (instead of R)²) A large (sub-) surface attached to each CD molecule;

wherein each CD has the structure of formula X:

wherein L has a length of no more than 8 atoms, wherein said no more than 8 atoms are preferably each C, N, O or S; the CD monomer is hydroxypropyl substituted with between 1 and 28 Hydroxypropyl (HP) groups, optionally between 2 and 15 HP groups or between 4 and 20 HP groups, preferably between 2 and 5 HP groups, and optionally the CD monomer has one or more additional substitutions.

5. A cyclodextrin dimer having the structure:

CD-L-CD

wherein L passes through the C2 carbon of each CD subunit (instead of R)¹) And/or C3 carbon (instead of R)²) A large (sub-) surface attached to each CD molecule;

wherein each CD has the structure of formula X:

wherein L has a length of no more than 8 atoms, wherein said no more than 8 atoms are preferably each C, N, O or S;

the CD monomer is methyl substituted with between 1 and 40 methyl (Me) groups, optionally between 2 and 15 Me groups or between 4 and 20 Me groups, preferably between 2 and 10 Me groups, and optionally the CD monomer has one or more additional substitutions.

6. A cyclodextrin dimer having the structure:

CD-L-CD

wherein L passes through the C2 carbon of each CD subunit (instead of R)¹) And/or C3 carbon (instead of R)²) A large (sub-) surface attached to each CD molecule;

wherein each CD has the structure of formula X:

wherein L has a length of no more than 8 atoms, wherein said no more than 8 atoms are preferably each C, N, O or S;

the CD monomer is sulfobutyl substituted with between 1 and 28 sulfobutyl groups, such as between 1 and 14 sulfobutyl groups, optionally between 2 and 10 sulfobutyl groups, preferably between 2 and 5 sulfobutyl groups, and optionally the CD monomer has one or more additional substitutions.

7. A cyclodextrin dimer having the structure:

CD-L-CD

wherein L passes through the C2 carbon of each CD subunit (instead of R)¹) And/or C3 carbon (instead of R)²) A large (sub-) surface attached to each CD molecule;

wherein each CD has the structure of formula X:

wherein L has a length of no more than 8 atoms, wherein said no more than 8 atoms are preferably each C, N, O or S;

the CD monomer is succinyl substituted with between 1 and 28 succinyl groups, optionally between 2 and 15 succinyl groups or between 4 and 20 succinyl groups, preferably between 2 and 5 succinyl groups, and optionally the CD monomer has one or more additional substitutions.

8. A cyclodextrin dimer having the structure:

CD-L-CD

wherein L passes through the C2 carbon of each CD subunit (instead of R)¹) And/or C3 carbon (instead of R)²) A large (sub-) surface attached to each CD molecule;

wherein each CD has the structure of formula X:

wherein L has a length of no more than 8 atoms, wherein said no more than 8 atoms are preferably each C, N, O or S;

the CD monomer is substituted with between 1 and 28 quaternary ammonium groups, optionally between 2 and 15 quaternary ammonium groups or between 4 and 20 quaternary ammonium groups, preferably 2 and 5 quaternary ammonium groups Wherein the quaternary ammonium group comprises-CH₂CH(OH)CH₂N(CH₃)₃ ⁺And optionally said CD monomer has one or more additional substitutions.

9. The cyclodextrin dimer of claim 1, wherein the R1, R2, and/or R3 subunit comprises one or more maltosyl groups.

10. The cyclodextrin dimer of claim 1, wherein the R1, R2, and/or R3 subunit comprises one or more carboxymethyl groups.

11. The cyclodextrin dimer of any one of claims 1-10, wherein:

l has the following structure:

each R is independently selected from H, X, SH, NH2 or OH, or is absent;

attachment of each CD to a linker is independently achieved by O, S or N attached to their C2 or C3 carbons, or by acetal attachment of two adjacent oxygens of the CD;

each X is a substituted or unsubstituted alkane, alkene, or alkyne;

each A is independently selected from a single, double or triple bond covalent bond, S, N, NH, O, or a substituted or unsubstituted alkane, alkene, or alkyne; and is

B is a substituted or unsubstituted 5 or 6 membered ring, S, N, NH, NR, O or absent.

12. The cyclodextrin dimer of any one of claims 1-10, wherein the linker is between 2 and 7 in length.

13. The cyclodextrin dimer of any one of claims 1-10, wherein the linker is between 3 and 6 in length.

14. The cyclodextrin dimer of any one of claims 1-10, wherein the linker is 2 or 3 in length.

15. The cyclodextrin dimer of any one of claims 1-10, wherein the linker is between 4 and 7 in length.

16. The cyclodextrin dimer of any one of claims 1-10, wherein the linker is between 4 and 6 in length.

17. The cyclodextrin dimer of any one of claims 1-10, wherein the linker is between 4 and 5 in length.

18. The cyclodextrin dimer of any one of claims 1-10, wherein the linker is 4 in length.

19. The cyclodextrin dimer of any one of claims 1-10, wherein the linker is an unsubstituted alkyl group.

20. The cyclodextrin dimer of any one of claims 1-10, wherein the linker is a substituted or unsubstituted butyl linker.

21. The cyclodextrin dimer of any one of claims 1-10, wherein the linker comprises a triazole.

22. The cyclodextrin dimer of any one of claims 1-10, wherein the linker comprises the structure: - (CH) ₂)_n1 (CH₂)_n2- (formula XI) in which n1 and n2 are eachBetween 1 and 8, such as between 1 and 4 each, preferably wherein n1 is 1 and n2 is 3.

23. The cyclodextrin dimer of any of claims 1-10, wherein the linker comprises any of the linkers shown in figure 8D, wherein the oxygen atom at each end of each linker forms part of the cyclodextrin monomer to which the linker is attached.

24. A cyclodextrin dimer having the structure:

CD-L-CD

wherein L passes through the C2 carbon of each CD subunit (instead of R)¹) And/or C3 carbon (instead of R)²) A large (sub-) surface attached to each CD molecule;

wherein each CD has the structure of formula X:

wherein L comprises a triazole and has a length of no more than 8 atoms, wherein said no more than 8 atoms are preferably each C, N, O or S;

each of the CD monomers is independently unsubstituted or optionally substituted.

25. The cyclodextrin dimer of claim 24, wherein the linker comprises the structure:

-(CH₂)_n1 (CH₂)_n2- (formula XI) wherein n1 and n2 are each between 1 and 8, such as between 1 and 4, preferably wherein n1 is 1 and n2 is 3.

26. The cyclodextrin dimer of claim 24 or claim 25, wherein the linker is between 4 and 7 in length.

27. The cyclodextrin dimer of claim 24 or claim 25, wherein the linker is between 4 and 6 in length.

28. The cyclodextrin dimer of claim 24 or claim 25, wherein the linker is between 4 and 5 in length.

29. The cyclodextrin dimer of any one of claims 1-29, further substituted with: (a) at least one methyl, hydroxypropyl, sulfobutyl, succinyl, or quaternary ammonium group such as-CH₂CH(OH)CH₂N(CH₃)₃ ⁺And/or (b) at least one alkyl, lower alkyl, alkylene, alkenyl, alkynyl, alkoxy, alkoxyalkyl, alkoxyalkoxyalkyl, alkylcarbonyloxyalkyl, alkylcarbonyl, alkylsulfonyl, alkylsulfonylalkyl, alkylamino, alkoxyamino, alkylsulfanyl, amino, alkylamino, dialkylamino, alkylaminoalkyl, dialkylaminoalkyl, aminoalkyl, aminoalkoxy, alkylsulfonylamino, aminocarbonyloxyalkyl, aminosulfonyl, alkylaminosulfonyl, dialkylaminosulfonyl, alkynylalkoxy, aryl, arylalkyl, arylsulfonyl, aryloxy, aralkoxy, cyanoalkyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, cycloalkylene, cycloalkylalkylene, heteroalkyl, heteroaryl, heteroarylalkyl, heteroarylsulfonyl, alkoxyalkylsulfonyl, alkoxyalkoxyalkoxyalkyl, alkoxyalkoxyalkoxyalkoxyalkoxyalkyl, alkoxyalkoxyalkoxyalkoxyalkoxyalkoxyalkyloxyalkyl, alkylamino, alkoxyalkyloxyalkyl, alkylsulfonylalkyl, alkoxyalkylsulfonylalkyl, alkoxyalkoxy, alkoxyalkylsulfonylalkyl, alkoxyalkoxy, alkoxyalkylsulfonylalkyl, alkoxyalkoxy, alkoxyalkylsulfonylalkyl, alkoxyalkoxy, and/or, Heteroaryloxy, heteroarylalkyloxy, heterocyclylalkoxy, halogen, haloalkyl, haloalkoxy, heterocyclylamino, heterocyclyl, heterocyclylalkyl, heterocyclyloxy, heterocyclylalkoxy, hydroxyalkoxy, hydroxyalkylamino, hydroxyalkylaminoalkyl, hydroxyalkyl, hydroxycarbonylalkyl, hydroxyalkyloxycarbonylalkyl, hydroxyalkyl, hydroxycycloalkyl, ureido, carbamate, carboxyl, sulfonamide, nitro, cyano, phenoxy, acetyl, ammonium, ammonia, azido, bromine, chlorine, deoxy, glucosyl, iodine, sulfate, sulfonyl, nitrite, phosphate, iodine, sulfate, sulfonyl, nitrite, ammonium, urea, carbamate, carboxyl, sulfonamide, nitro, cyano, phenoxy, acetyl, ammonium, amino, azido, bromine, chlorine, deoxy, glucosyl, iodine, sulfate, nitrite, phosphate, amino, nitro, amino, Phosphoryl, fatty acids such as palmitoyl groups, mono-or disaccharides, and/or (c) at least one methyl, hydroxypropyl, sulfobutyl, succinyl, maltosyl, carboxymethyl, quaternary ammonium (such as-CH)₂CH(OH)CH₂N(CH₃)₃ ⁺) Glucosyl, palmitoyl, phosphate, phosphoryl, amino, azido, sulfate, sulfonyl, alkyl, ethyl, propyl, isopropyl, butyl, isobutyl, bromo, chloro groups.

30. The cyclodextrin dimer of any of claims 1-29, having a structure according to any of formulas I-IX (shown in figures 3B-3J, respectively).

31. The cyclodextrin dimer of any of claims 1-30, wherein each R, if not otherwise indicated, is¹Each R²And each R³Independently selected from: (a) methyl, H, hydroxypropyl, sulfobutyl ether, succinyl-hydroxypropyl, quaternary ammonium such as-CH₂CH(OH)CH₂N(CH₃)₃ ⁺Carboxymethyl, carboxymethyl-hydroxypropyl, hydroxyethyl, maltosyl, acetyl, carboxyethyl, sulfate, sulfopropyl, sodium phosphate, or glucosyl; and/or (b) hydrogen, alkyl, lower alkyl, alkylene, alkenyl, alkynyl, alkoxy, alkoxyalkyl, alkoxyalkoxyalkyl, alkylcarbonyloxyalkyl, alkylcarbonyl, alkylsulfonyl, alkylsulfonylalkyl, alkylamino, alkoxyamino, alkylsulfanyl, amino, alkylamino, dialkylamino, alkylaminoalkyl, dialkylaminoalkyl, aminoalkyl, aminoalkoxy, alkylsulfonylamino, aminocarbonyloxyalkyl, aminosulfonyl, alkylaminosulfonyl, dialkylaminosulfonyl, alkynylalkoxy, aryl, arylalkyl, arylsulfonyl, aryloxy, aralkoxy, cyanoalkyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, cycloalkylene, cycloalkylalkylene, heteroalkyl, heteroaryl, heteroarylalkyl, heteroarylsulfonyl, heteroaryloxy, alkoxylalkyloxy, alkoxyalkyloxyalkyl, alkylcarbonyloxyalkyl, alkylcarbonylamino, alkylamino, alkylaminoalkyl, alkylaminosulfonyl, alkoxyalkoxy, cycloalkylalkylene, heteroaryloxy, cycloalkylalkylene, heteroalkyl, heteroaryl, heteroarylalkyl, heteroarylsulfonyl, heteroaryloxy, alkoxyalkyloxyalkyl, cycloalkylalkylene, heteroaryloxy, cycloalkylalkylene, heteroaryloxy, heteroarylalkylene, or, Heteroaralkyloxy, heterocyclylalkoxy, Halogen, haloalkyl, haloalkoxy, heterocyclylamino, heterocyclyl, heterocyclylalkyl, heterocyclyloxy, heterocyclylalkoxy, hydroxyalkoxy, hydroxyalkylamino, hydroxyalkylaminoalkyl, hydroxyalkyl, hydroxycarbonylalkyl, hydroxyalkyloxycarbonylalkyl, hydroxyalkyl, hydroxycycloalkyl, ureido, carbamate, carboxyl, sulfonamide, nitro, cyano, phenoxy or acetyl groups.

32. The cyclodextrin dimer of any one of claims 1-31, wherein L is attached to the C2 carbon of each CD monomer.

33. The cyclodextrin dimer of any one of claims 1-31, wherein L is attached to the C3 carbon of each CD monomer.

34. The cyclodextrin dimer of any of claims 1-31, wherein L is attached to C2 carbon of one CD monomer and C3 carbon of another CD monomer.

35. The cyclodextrin dimer of any of claims 1-34, wherein the cyclodextrin dimer exhibits greater affinity for 7KC than cholesterol, wherein optionally the greater affinity is determined by a turbidity test.

36. The cyclodextrin dimer of claim 35, wherein the cyclodextrin dimer exhibits at least 1.1-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, or 10-fold affinity for 7KC versus cholesterol.

37. A composition comprising a mixture of cyclodextrin dimers of any one of claims 1-36, and having an average degree of substitution between 2 and 10, such as between 4 and 8 or between 2 and 5; or having a degree of substitution of between 2 and 5, such as about 2, about 3, about 4, or about 5, of hydroxypropyl, sulfobutyl, succinyl, or quaternary ammonium groups; or have a degree of substitution of methyl groups of between 2 and 10, wherein the degree of substitution is measured by NMR or by mass spectrometry such as MALDI.

38. A composition comprising a mixture of cyclodextrin dimers according to claims 32, 33, and 34.

39. A pharmaceutical composition comprising a cyclodextrin dimer according to any one of claims 1-36 or a composition according to claim 37 or 38 and a pharmaceutically acceptable carrier.

40. The pharmaceutical composition of claim 39, wherein the cyclodextrin dimer is the only active ingredient in the composition.

41. The pharmaceutical composition of claim 39, consisting of or consisting essentially of the cyclodextrin dimer and the pharmaceutically acceptable carrier.

42. A method of treatment comprising administering to a subject in need thereof an effective amount of a cyclodextrin dimer according to any one of claims 1-36 or a composition according to any one of claims 37-41.

43. The method of claim 42, wherein the subject in need thereof suffers from a deleterious or toxic effect of 7 KC.

44. A method for reducing the amount of 7KC in a subject in need thereof, the method comprising administering to a subject in need thereof an effective amount of a cyclodextrin dimer according to any one of claims 1-36 or a composition according to any one of claims 37-41.

45. The method of any one of claims 42-44, wherein the cyclodextrin dimer is administered to the subject via parenteral (e.g., subcutaneous, intramuscular, or intravenous), topical, transdermal, oral, sublingual, or buccal administration.

46. The method of claim 45, wherein the cyclodextrin dimer is administered intravenously.

47. The method of any one of claims 42-46, comprising administering to the subject (a) between about 1mg and 20g, such as between 10mg and 1g, between 50mg and 200mg, or 100mg of the cyclodextrin dimer, or (b) between 1g and 10g of the cyclodextrin dimer, such as about 2g, about 3g, about 4g, or about 5g, or (c) between 50mg and 5g of the cyclodextrin dimer, such as between 100mg and 2.5g, between 100mg and 2g, between 250mg and 2.5 g.

48. The method of any one of claims 42-47, which prevents, treats, ameliorates symptoms of one or more of the following diseases: atherosclerosis/coronary artery disease, arteriosclerosis, coronary atherosclerosis due to calcified coronary lesions, heart failure (all stages), Alzheimer's disease, amyotrophic lateral sclerosis, Parkinson's disease, Huntington's disease, vascular dementia, multiple sclerosis, Smith-Lyme-Observation syndrome, infantile neuronal ceroid lipofuscinosis, lysosomal acid lipase deficiency, tendonoxanthomatosis, X-linked adrenoleukodystrophy, sickle cell disease, type A Niemann-pick disease, type B Niemann-pick disease, type C Niemann-pick disease, gaucher disease, Stargardt disease, age-related macular degeneration (dry), idiopathic pulmonary fibrosis, chronic obstructive pulmonary disease, cystic fibrosis, liver injury, liver failure, nonalcoholic steatohepatitis, Non-alcoholic fatty liver disease, irritable bowel syndrome, crohn's disease, ulcerative colitis, and/or hypercholesterolemia; wherein optionally the treatment is administered in combination with another therapy.

49. The method of any one of claims 42-47, which prevents, treats, ameliorates a symptom of atherosclerosis.

50. The method of claim 49, further comprising administering a second therapy to the subject, wherein the second therapy is administered simultaneously or sequentially in any order.

51. The method of claim 50, wherein the second therapy comprises one or more of an anti-cholesterol drug such as a fibrate or a statin, an anti-platelet drug, an anti-hypertensive drug, or a dietary supplement.

52. A method according to claim 51, wherein the statin is selected from the group consisting of ADVICOR (R) (niacin sustained release/lovastatin), ALTOPREV (R) (lovastatin sustained release), CADUET (R) (amlodipine and atorvastatin), CRESTOR (R) (rosuvastatin), JUVISYNC (R) (sitagliptin/simvastatin), LESCOL (R) (fluvastatin), LESCOL (fluvastatin sustained release), LIITIOR (R) (atorvastatin), LIVALO (R) (pitavastatin), MEVACOR (R) (lovastatin), PRAVACHOL (R) (pravastatin), SIMCOR (R) (niacin sustained release/simvastatin), VORYTIN (R) (ezetimibe/ZOR), and simvastatin (simvastatin).

53. The method of claim 51, wherein the second therapy comprises an anti-cholesterol drug and an anti-hypertensive drug.

54. A method of purifying an oxysterol, the method comprising: contacting a composition comprising an oxysterol with the cyclodextrin dimer of any one of claims 1-36, thereby solubilizing the oxysterol in the cyclodextrin dimer; and recovering the cyclodextrin dimer and solubilized oxysterol.

55. The method of claim 54, wherein the oxysterol comprises or consists of 7 KC.

56. The method of claim 54, further comprising measuring the amount or concentration of 7KC in the solubilized oxysterol, thereby determining the relative concentration of 7KC in the composition.

57. The method of claim 56, wherein the composition comprises a patient sample.

58. An in vitro method of removing oxysterol from a sample, said method comprising: contacting a sample comprising an oxysterol with a cyclodextrin dimer according to any one of claims 1-36, thereby solubilizing the oxysterol in the cyclodextrin dimer; and separating the sample from the cyclodextrin dimer and solubilized sterol, and optionally reintroducing the sample into the subject from which the sample was obtained.

59. A method of producing a reduced cholesterol product, the method comprising: contacting a product comprising cholesterol with a cyclodextrin dimer according to any of claims 1-36, thereby solubilizing the cholesterol in the cyclodextrin dimer; and removing the cyclodextrin dimer and solubilized cholesterol from the product.

60. The method of claim 59, wherein the product is a food product.

61. The method of claim 60, wherein the food product comprises meat and/or dairy products.

62. A method of preparing the cyclodextrin dimer according to any one of claims 1-23 or 29-36, comprising:

(a) reacting the beta-cyclodextrin protected on the primary side with a dialkylating agent to produce a primary side-protected beta CD dimer linked by a secondary surface, and optionally purifying the primary side-protected beta CD dimer;

(b) deprotecting the primary side-protected β CD dimer, thereby producing a deprotected β CD dimer, and optionally purifying the deprotected β CD dimer; and

(c) linking the deprotected CD to one or more hydroxypropyl, methyl, succinyl, sulfobutyl and/or quaternary ammonium (such as-CH)₂CH(OH)CH₂N(CH₃)₃ ⁺) A group, thereby producing the cyclodextrin dimer, and optionally purifying the cyclodextrin dimer.

63. The method of claim 62, wherein the beta-cyclodextrin protected on a major side comprises hepta (6-O-tert-butyldimethylsilyl) -beta-cyclodextrin.

64. The method of claim 62 or 63, wherein the dialkylating agent comprises a dibromoalkane, optionally 1, 4-dibromobutane.

65. The process of any one of claims 62-64, wherein step (a) is carried out under anhydrous conditions and/or with sodium hydride as a base.

66. The method of any one of claims 62-65, wherein the purifying in step (a) comprises direct phase chromatography and isocratic elution.

67. The process of any one of claims 62-66, wherein step (b) is carried out in Tetrahydrofuran (THF) with tetrabutylammonium fluoride.

68. The method of any one of claims 62-67, wherein the purifying in step (b) comprises direct phase chromatography and isocratic elution.

69. The method of any one of claims 62-68, wherein step (c) comprises reacting the deprotected CD dimers with a hydroxypropylation agent such as propylene oxide, a methylating agent such as methyl iodide, a succinylating agent such as succinic anhydride, a sulfobutylating agent such as 1, 4-butane sultone, and/or a quaternary ammonium linking agent such as glycidyl trimethylammonium chloride.

70. The method of any one of claims 62-69, wherein step (c) is performed under aqueous conditions, optionally comprising sodium hydroxide as a base.

71. The method of any one of claims 62-70, wherein the purifying in step (c) comprises one or more of ion exchange resin treatment, charcoal clarification, and dialysis.

72. A method of making the cyclodextrin dimer according to any of claims 24-36, comprising (a) reacting 2-O- (n-azidoalkyl) - β CD with 2-O- (n-alkyne) - β CD, thereby forming a β CD-triazole- β CD dimer having the structure β CD-alkyl 1-triazole-alkyl 2- β CD, and optionally (b) purifying the β CD-triazole- β CD dimer.

73. The method of claim 72, wherein step (a) is performed using a copper (I) catalyst, optionally about 15mM copper (I).

74. The method of claim 72 or 73, wherein step (a) is carried out in an aqueous solution.

75. The method of claim 74, wherein the aqueous solution comprises Dimethylformamide (DMF), optionally about 50% DMF (v/v).

76. The method of any one of claims 72-75, wherein step (b) comprises silica gel chromatography.

77. The process of any one of claims 72-76, further comprising producing the 2-O- (n-azidoalkyl) - β CD prior to step (a) by a process comprising: (1) reacting n-azido-1-bromo-alkane with β -cyclodextrin, optionally with a catalytic amount of lithium iodide, to form the 2-O- (n-azidoalkyl) - β CD; and (2) optionally purifying the 2-O- (n-azidoalkyl) - β CD.

78. The method of claim 77, wherein step (2) comprises silica gel chromatography.

79. The method of any one of claims 72-78, further comprising generating 2-O- (n-alkyne) - β CD prior to step (a) by a method comprising: (i) reacting n-bromo-1-alkyne with β -cyclodextrin, optionally with a catalytic amount of lithium iodide, to generate the 2-O- (n-alkyne) - β CD, and (ii) optionally purifying the 2-O- (n-alkyne) - β CD.

80. The method of claim 79, wherein step (2) comprises silica gel chromatography.

81. The method of claim 79 or 80, wherein step (1) is performed in dry DMSO.

82. The process of any one of claims 79 to 81, wherein the reaction in step (1) comprises lithium hydride.

83. The method of any one of claims 72-82, wherein the beta CD-triazole-beta CD dimer comprises the structure: CD- (CH)₂)n₁ (CH₂)_n2-CD (formula XII), wherein n1 is between 1 and 8, and/or n2 is between 1 and 8, such as n1 and n2 are each between 1 and 4, preferably wherein n1 is 1 and n2 is 3.

84. The method of claim 83, wherein n1 is 1, 2, 3, or 4, and/or n2 is 1, 2, 3, or 4.

85. The method of claim 84, wherein the triazole linker is between 5 and 8 in length.

86. The method of any one of claims 72-85, further comprising (c) hydroxypropylating the beta CD-triazole-beta CD dimer, thereby producing a cyclodextrin dimer, and optionally purifying the cyclodextrin dimer.

87. The method of claim 86, wherein step (c) comprises reacting the deprotected CD dimers with a hydroxypropylation agent such as propylene oxide, a methylating agent such as methyl iodide, a succinylating agent such as succinic anhydride, a sulfobutylating agent such as 1, 4-butane sultone, and/or a quaternary ammonium linking agent such as glycidyltrimethylammonium chloride.

88. The process of claim 86 or 87, wherein step (c) is carried out under aqueous conditions, optionally comprising sodium hydroxide as a base.

89. The method of any one of claims 86-88, wherein the purifying in step (c) comprises one or more of ion exchange resin treatment, charcoal clarification, membrane filtration, and dialysis.

90. Use of a cyclodextrin dimer according to any one of claims 24-28 in the synthesis of a cyclodextrin dimer according to any one of claims 1-23 or 29-36.

Background

7-ketocholesterol (7KC) is an oxysterol produced by the non-enzymatic reaction of oxygen radicals with cholesterol. 7KC can be formed in organisms or consumed in food, but it is potentially toxic and is considered to be useless to humans and other eukaryotes. Like cholesterol, 7KC is present in atherosclerotic plaques. 7KC is the most abundant non-enzymatically produced oxysterol in atherosclerotic plaques and can be a contributing factor to the pathogenesis of atherosclerosis and other aging diseases. It is believed that 7KC is also a contributing factor in the onset of lysosomal storage diseases, such as Niemann-Pick disease Type C (NPC).

Cyclodextrins (CD) are cyclic oligosaccharide polymers consisting of 6 (α CD), 7 (β CD) or 8 (γ CD) saccharide rings (fig. 1A). Alpha, beta and gamma cyclodextrins are the most common forms and have many medical, industrial, consumer and food related uses. Cyclodextrins have been used in a variety of applications, including as a food additive for dietary fiber. Cyclodextrins have also been commonly combined with active pharmaceutical ingredients, used in pharmaceutical compositions as nebulizers and as excipients for small hydrophobic drugs.

Hydroxypropyl- β -cyclodextrin (HP β CD) is one such β -cyclodextrin: wherein a number of Hydroxypropyl (HP) groups have been added to O2, O3, or O6 oxygen (or to atoms replacing the oxygen) on some or all of the seven glucose monomers that make up the β CD. Hydroxypropylation of cyclodextrins improves their water solubility and their safety, making them useful for a variety of purposes in the human body, especially as excipients for active drugs; this has been included in the FDA HP β CD GRAS (generally recognized safety) list. Most commercial HP β CDs have an average HP degree of substitution between 4 and 9, and all existing products contain a combination of number and position of substitutions, which is typically reflected in the average Degree of Substitution (DS) of the advertisement.

Other CD substitutions include methyl, succinyl, sulfobutyl, maltosyl, carboxymethyl, quaternary ammonium, and the like, which can yield CDs that are very soluble in water and have low cytotoxicity, whether they are charged or neutral groups. Commercially available CD's can have varying degrees of substitution, which can vary from only about 1 up to complete substitution (degree of substitution 21), depending on the particular substituent and supplier.

Disclosure of Invention

The present disclosure describes the design and testing of various dimers of Cyclodextrin (CD), including HP β CD dimer, methyl- β CD dimer, succinyl- β CD dimer, sulfobutyl- β CD dimer, and quaternary ammonium dimer, among others. Indicating that the affinity of certain dimers for 7KC and cholesterol was significantly increased compared to monomeric CD. Exemplary dimers represent a new class of linked and substituted cyclodextrin dimers with improved properties, including the ability to selectively interact with and solubilize sterols. Molecular modeling experiments described below show the predicted interaction mechanism. In addition, the working examples demonstrate the predictive ability of the novel substituted cyclodextrin dimers to solubilize sterols, including the selective solubilization of 7KC as compared to cholesterol.

In one aspect, the present disclosure provides CD dimers having the structure CD-L-CD, wherein each CD is a β -cyclodextrin, L is attached to the C2 or C3 carbon of each CD monomer, and one or both of the CD monomers is substituted with at least one functional group, such as methyl, Hydroxypropyl (HP), Sulfobutyl (SB), Succinyl (SUCC), Quaternary Ammonium (QA) (such as-CH₂CH(OH)CH₂N(CH₃)₃ ⁺) Or a combination thereof. Typically, each CD monomer consists of a glucose monomer in the D configuration. The CD dimer is substituted with a functional group, typically with a Degree of Substitution (DS) between 1 and 28, where the degree of substitution refers to the total number of functional group substitutions present on both CD subunits. The substitutions may be present on either or both of the CD subunits. On the shortest path through the linker connecting the two CD subunits of the cyclodextrin dimer, the linker length may be between 2-8 atoms long, such as between 4-8 atoms long. The linker may comprise an alkyl (e.g., butyl) linker and/or a triazole linker, the linkers being optionally substituted. Exemplary CD dimers are of formulae I-IX (shown in FIGS. 3B-3J, respectively). Optionally, the CD dimer is further substituted.

In another aspect, the present disclosure provides a β CD dimer having the structure CD-L-CD, wherein each CD is a β -cyclodextrin, L is attached to the C2 or C3 carbon of each CD monomer, and one or both of the CD monomers is substituted with at least one hydroxypropyl group. Typically, each CD monomer consists of a glucose monomer in the D configuration. The β CD dimer is substituted with Hydroxypropyl (HP), typically with a Degree of Substitution (DS) between 1 and 40, where the degree of substitution refers to the total number of substitutions present on both CD subunits. The substitutions may be present on either or both of the CD subunits. On the shortest path through the linker connecting the two CD subunits of the cyclodextrin dimer, the linker length may be between 4-8 atoms long. The linker may comprise an alkyl (e.g., butyl) linker and/or a triazole linker, the linkers being optionally substituted. Exemplary β CD dimers have formula I, II or III (shown in figures 3B-3D, respectively). Optionally, the β CD dimer is further substituted.

It is believed that 7KC is associated with complications of heart disease, cystic fibrosis, liver injury and failure, and hypercholesterolemia. When someone is affected by hypercholesterolemia, 7KC can diffuse through the cell membrane where it affects receptor and enzyme functions; an increased incidence of hypercholesterolemic dementia is associated with 7KC accumulation. In the liver, 7KC affects the perforation and porosity of the tissue, which increases with age. The 7KC also promotes transport of cytosolic NADPH oxidase components to the membrane in neutrophils (leukocytes) and enhances rapid production of reactive oxygen species. The pathogenesis of other aging diseases, such as age-related macular degeneration (AMD-dry), alzheimer's disease, and lysosomal storage diseases, such as niemann-pick disease type C (NPC), is also associated with increased 7KC levels. Oxysterols (including 7KC) are also associated with increased levels of free radicals, which in turn affect lipid circulation in cystic fibrosis. It is believed that the increase in free radicals caused by oxysterols (e.g., 7KC) is associated with apoptosis, cytotoxicity, impairment of endothelial function, and modulation of enzymes involved in inflammation and fatty acid metabolism.

7KC is formed by the non-enzymatic reaction of oxygen radicals with cholesterol, suggesting that its formation may not be favored. Indeed, it is believed that 7KC enhances free radical production everywhere in the body, but cardiac and vascular tissues are of particular interest. Free radicals affect cellular and enzymatic reactions that are important for cholesterol-mediated tissue damage, which is particularly important in these tissues; this is believed to enhance inflammation of the vasculature. It is believed that 7KC causes mitochondrial and lysosomal dysfunction by disrupting cell and organelle membrane function and is thought to be associated with increased frequency of foam cell formation from macrophages in atherosclerotic plaques. It is expected that the scavenging function of these macrophages may help to improve plaque, but when they are filled with cholesterol and oxysterol, they may become part of the plaque.

Exemplary embodiments provide treatment of diseases associated with and/or exacerbated by 7KC accumulation such as atherosclerosis, AMD, arteriosclerosis, coronary atherosclerosis due to calcified coronary lesions, heart failure (all stages), alzheimer's disease, amyotrophic lateral sclerosis, parkinson's disease, huntington's disease, vascular dementia, multiple sclerosis, Smith-lyme-oppez (Smith-Lemli-optiz) syndrome, infantile neuronal ceroid lipofuscinosis, lysosomal acid lipase deficiency, tendonoleucinosis, X-linked adrenoleukodystrophy, sickle cell disease, niemann-pick disease type a, niemann-pick disease type B, niemann-pick disease type C, Gaucher (Gaucher) disease, Stargardt disease, Gaucher's disease, and the like, Idiopathic pulmonary fibrosis, chronic obstructive pulmonary disease, cystic fibrosis, liver injury, liver failure, non-alcoholic steatohepatitis, non-alcoholic fatty liver disease, irritable bowel syndrome, Crohn's (Crohn's) disease, ulcerative colitis, and/or hypercholesterolemia or a hypercholesterolemia-associated dementia. Preferred cyclodextrin (e.g., HP β CD, Me β CD, SUCC β CD, QA β CD, or SB β CD) dimers are selective for 7KC (as compared to cholesterol). Preferably, the CD dimer preferentially solubilizes 7KC while minimizing or avoiding potentially harmful or toxic effects that may result from excessive removal of cholesterol.

Exemplary embodiments of the present invention provide the use of cyclodextrin (e.g., HP β CD, Me β CD, SUCC β CD, QA β CD, or SB β CD) dimers for the solubilization and/or removal of 7KC, which can be performed in vitro or in vivo.

In exemplary embodiments, the cyclodextrin (e.g., HP β CD, Me β CD, SUCC β CD, QA β CD, or SB β CD) dimer exhibits greater binding affinity and/or solubilization for 7KC than cholesterol. At sub-saturation concentrations, the specificity for 7KC was most pronounced relative to cholesterol, while at higher concentrations, the solubilization of both sterols could approach 100%. This specificity allows the use of such cyclodextrin dimers to preferentially solubilize and remove 7 KC.

In exemplary embodiments, the present disclosure provides a cyclodextrin dimer having the structure:

CD-L-CD

wherein L passes through the C2 carbon of each CD subunit (instead of R)¹) And/or C3 carbon (instead of R)²) A large (sub-) surface attached to each CD molecule;

wherein CD has the structure of formula X:

wherein L is no more than 8 atoms in length on the shortest path through the linker connecting the two CD subunits of the dimer, wherein said no more than 8 atoms are preferably each C, N, O or S;

And the CD is substituted with between 1 and 40 groups, such as between 1 and 28 groups, optionally between 2 and 15 or between 4 and 20 groups. The number of substitutions is R other than H¹、R²And/or R³The total number of groups. The CD may have one or more additional substitutions.

The R is¹、R²And R³Can each be independently selected from H, methyl, hydroxypropyl, sulfobutyl, succinyl, quaternary ammonium such as-CH₂CH(OH)CH₂N(CH₃)₃ ⁺Alkyl, lower alkyl, alkylene, alkenyl, alkynyl, alkoxy, alkoxyalkyl, alkoxyalkoxyalkyl, alkylcarbonyloxyalkyl, alkylcarbonyl, alkylsulfonyl, alkylsulfonylalkyl, alkylamino, alkoxyamino, alkylsulfanyl, amino, alkylamino, dialkylamino, alkylaminoalkyl, dialkylaminoalkyl, aminoalkyl, aminoalkoxy, alkylsulfonylamino, aminocarbonyloxyalkyl, aminosulfonyl, ammonium, ammonia, alkylaminosulfonyl, dialkylaminosulfonyl, alkynylalkoxy, aryl, arylalkyl, arylsulfonyl, aryloxy, aralkyloxy, azido, bromo, chloro, cyanoalkyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, cycloalkylene, cycloalkylalkylene, deoxy, glucosyl, heteroalkyl, heteroaryl, heteroarylalkyl, alkoxy, alkylamino, dialkylamino, alkylaminoalkyl, aminoalkyl, amino, alkylamino, dialkylamino, alkylsulfonylamino, amino-alkoxy, dialkylamino, alkylsulfonyl, alkylamino, alkylsulfonyl, heteroaryl, alkylsulfonyl, heteroaryl, alkylsulfonyl, heteroaryl, alkylsulfonyl, or combinations thereof, wherein, Heteroarylsulfonyl, heteroaryloxy, heteroarylalkyloxy, heterocyclylalkoxy, halogen, haloalkyl, haloalkoxy, heterocyclylamino, heterocyclyl, heterocyclylalkyl, heterocyclyloxy, heterocyclylalkoxy, hydroxyalkoxy, hydroxyalkylamino, hydroxyalkylaminoalkyl, hydroxyalkyl, hydroxycarbonylalkyl, hydroxyalkyloxycarbonylalkyl, hydroxyalkyl, hydroxycycloalkyl, iodine, urea, carbamate, carboxyl, sulfate, sulfonyl, sulfonamide, nitro, nitrite, cyano, phosphate, sulfonyl, sulfonamide, nitro, nitrite, cyano, hydroxyl, or a salt thereof, Phosphoryl, phenoxy, acetyl groups, fatty acids such as palmitoyl groups, mono-or disaccharides. In exemplary embodiments, the substitution is preferably a maltosyl group or a carboxymethyl group.

In exemplary embodiments, the R is¹、R²And/or R³The groups may each be independently selected from H, methyl, hydroxypropyl, sulfobutyl, succinyl, maltosyl, carboxymethyl, quaternary ammonium (such as-CH)₂CH(OH)CH₂N(CH₃)₃ ⁺) Glucosyl, palmitoyl, phosphate, phosphoryl, amino, azido, sulfate, sulfonyl, alkyl, ethyl, propyl, isopropyl, butyl, isobutyl, bromo, chloro, between 1 and 40, such as between 1 and 28, or optionally between 2 and 15 or between 4 and 20 of said R¹、R²And R³The group is not H.

In exemplary embodiments, the R is¹、R²And R³The groups may each be independently selected from H, methyl, hydroxypropyl, sulfobutyl, succinyl, maltosyl, carboxymethyl, quaternary ammonium (such as-CH)₂CH(OH)CH₂N(CH₃)₃ ⁺) Wherein between 1 and 40, such as between 1 and 28 of said R¹、R²And R³Radicals other than H, optionally between 2 and 15 or between 4 and 20 of said R¹、R²And R³The group is not H. The R is ¹、R²And R³The groups may include one or more maltosyl or carboxymethyl groups.

In further exemplary embodiments, the present disclosure provides a CD dimer having the structure:

CD-L-CD

wherein L passes through the C2 carbon of each CD subunit (instead of R)¹) And/or C3 carbon (instead of R)²) A large (sub-) surface attached to each CD molecule;

wherein CD has the structure of formula X:

wherein L is no more than 8 atoms in length on the shortest path through the linker connecting the two CD subunits of the dimer, wherein said no more than 8 atoms are preferably each C, N, O or S;

the CD is Hydroxypropyl (HP) substituted with between 1 and 28 HP groups, optionally between 2 and 15 or between 4 and 20 HP groups, preferably between 2 and 5 HP groups, and optionally the CD has one or more additional substitutions. The CD may comprise between 2 and 4 HP groups, or may comprise 2 HP groups, 3 HP groups, 4 HP groups, or 5 HP groups.

In further exemplary embodiments, the present disclosure provides a CD dimer having the structure:

CD-L-CD

wherein L passes through the C2 carbon of each CD subunit (instead of R)¹) And/or C3 carbon (instead of R)²) A large (sub-) surface attached to each CD molecule;

Wherein CD has the structure of formula X:

wherein L is no more than 8 atoms in length on the shortest path through the linker connecting the two CD subunits of the dimer, wherein said no more than 8 atoms are preferably each C, N, O or S;

the CD is methyl (Me) substituted with between 1 and 40 Me groups, optionally between 1 and 28 Me groups, optionally between 2 and 15 Me groups or between 4 and 20 Me groups, preferably between 2 and 10 Me groups, and optionally the CD has one or more additional substitutions. Without wishing to be bound by theory, it is believed that methyl groups are particularly suitable for substitution with a large number of substituents on such CD dimers, since the methyl groups are particularly small in size and therefore do not interfere with the entry of guests (such as 7KC or cholesterol) into the CD dimer binding cavity. Additionally, it is contemplated that one or more methyl substitutions may be added to any cyclodextrin dimer of the present disclosure, including in amounts greater than specified in the general formulae herein, e.g., up to a total of 40 non-hydrogen substituents when both non-methyl substituents and added methyl substituents are included.

In further exemplary embodiments, the present disclosure provides a CD dimer having the structure:

CD-L-CD

Wherein L passes through the C2 carbon of each CD subunit (instead of R)¹) And/or C3 carbon (instead of R)²) A large (sub-) surface attached to each CD molecule;

wherein CD has the structure of formula X:

wherein L is no more than 8 atoms in length on the shortest path through the linker connecting the two CD subunits of the dimer, wherein said no more than 8 atoms are preferably each C, N, O or S;

the CD is sulfobutyl substituted with between 1 and 28 sulfobutyl groups, such as between 1 and 14 sulfobutyl groups, optionally between 2 and 10 sulfobutyl groups, preferably between 2 and 5 sulfobutyl groups, and optionally the CD has one or more additional substitutions. The CD may have between 2 and 4 sulfobutyl groups, or may have 2 sulfobutyl groups, 3 sulfobutyl groups, 4 sulfobutyl groups, or 5 sulfobutyl groups.

In further exemplary embodiments, the present disclosure provides a CD dimer having the structure:

CD-L-CD

wherein L passes through the C2 carbon of each CD subunit (instead of R)¹) And/or C3 carbon (instead of R)²) A large (sub-) surface attached to each CD molecule;

wherein CD has the structure of formula X:

wherein L is no more than 8 atoms in length on the shortest path through the linker connecting the two CD subunits of the dimer, wherein said no more than 8 atoms are preferably each C, N, O or S;

The CD is succinyl substituted with between 1 and 28 succinyl groups, optionally between 2 and 15 succinyl groups or between 4 and 20 succinyl groups, preferably between 2 and 5 succinyl groups, and optionally the CD has one or more additional substitutions. The CD may comprise between 2 and 4 succinyl groups, or may comprise 2 succinyl groups, 3 succinyl groups, or 4 succinyl groups or 5 succinyl groups.

In further exemplary embodiments, the present disclosure provides a CD dimer having the structure:

CD-L-CD

wherein L passes through the C2 carbon of each CD subunit (instead of R)¹) And/or C3 carbon (instead of R)²) A large (sub-) surface attached to each CD molecule;

wherein CD has the structure of formula X:

wherein L is no more than 8 atoms in length on the shortest path through the linker connecting the two CD subunits of the dimer, wherein said no more than 8 atoms are preferably each C, N, O or S;

the CD is substituted with between 1 and 28 quaternary ammonium groups, optionally between 2 and 15 quaternary ammonium groups or between 4 and 20 quaternary ammonium groups, preferably between 2 and 5 quaternary ammonium groups, wherein the quaternary ammonium groups comprise-CH ₂CH(OH)CH₂N(CH₃)₃ ⁺Such as-CH₂CH(OH)CH₂N(CH₃)₃Cl, andoptionally the CD has one or more additional substitutions. The CD may comprise between 2 and 4 quaternary ammonium groups, or may comprise 2 quaternary ammonium groups, 3 quaternary ammonium groups, or 4 quaternary ammonium groups or 5 quaternary ammonium groups. It is to be understood that any pharmaceutically acceptable salt of the quaternary amine is included within the scope of the present disclosure.

L may have the following structure:

wherein each R is independently selected from H, X, SH, NH₂Or OH, or may be absent;

attachment of each CD to the linker is independently achieved through O, S or N attached to their C2 or C3 carbons, or through an acetal linkage of two adjacent oxygens of the CD.

Each X is a substituted or unsubstituted alkane, alkene, or alkyne;

each A is independently selected from a single, double or triple bond covalent bond, S, N, NH, O, or a substituted or unsubstituted alkane, alkene, or alkyne; and is

B is a substituted or unsubstituted 5 or 6 membered ring, S, N, NH, NR, O or absent.

The length of the linker may be between 2 and 7, between 3 and 6, between 4 and 7, between 4 and 6, between 4 and 5, or 4, or between 2 and 3.

The linker may be an unsubstituted alkyl group, such as an unsubstituted butyl group.

The linker may be a substituted or unsubstituted butyl linker.

The linker may comprise a triazole.

The joint may include the following structure:wherein n1 and n2 are each between 1 and 8 or between 1 and 4, preferably wherein n1 is 1 and n2 is 3.

In exemplary embodiments, when the linker L comprises a triazole, e.g., having the structure of formula XI, the linker L may be attached to the O2 position of each CD monomer, wherein n1 and n2 may each be between 0 and 8, such as each between 1 and 4; preferably, the total length of the joint may be 8 or less, such as 8, 7, 6, 5, 4, 3, or any range of values therein; and in a preferred embodiment, n1 is 1 and n2 is 3.

In exemplary embodiments, when the linker L comprises a substituted or unsubstituted alkyl group, preferably having a length of no more than 8 atoms, such as between 2 and 7, between 2 and 6, or between 4 and 7, or between 4 and 6, or between 4 and 5, or a length of 8, 7, 6, 5, 4, 3, or 2, or any numerical range therein, the linker L may be attached to the O2 position of each CD monomer, the O2 position of one CD monomer and the O3 position of another CD monomer, or the O3 positions of two CD monomers; wherein preferably the linker is a substituted or unsubstituted butyl group, more preferably an unsubstituted butyl group.

The linker may comprise a single point of attachment to each CD monomer. The linker may comprise a single point of attachment to one CD monomer and multiple (two or more) points of attachment to another CD monomer. The linker may comprise multiple points of attachment to each CD monomer (two or more points of attachment per CD monomer). The joints may include any of the joints shown in fig. 8D. It is to be understood that the linkers shown include an oxygen atom at each end which forms part of the cyclodextrin to which they are attached; to determine the length of the linker, these oxygen atoms are not considered part of the linker. In addition, for linkers attached to one or two cyclodextrin monomers at multiple positions, the linkage shown on the left is attached to one monomer and the linkage shown on the right is attached to another monomer.

In exemplary embodiments, the present disclosure provides a CD dimer having the structure:

CD-L-CD

wherein L is through each CDC2 carbon of radical (instead of R)¹) And/or C3 carbon (instead of R)²) A large (sub-) surface attached to each CD molecule;

wherein CD has the structure of formula X:

wherein L is a triazole and has a length of no more than 8 atoms, wherein said no more than 8 atoms are preferably each C, N, O or S;

The CD is substituted with between 0 and 28 groups, optionally 0 groups, or optionally the CD has one or more substitutions.

The joint may include the following structure:wherein n1 and n2 are each between 1 and 8 or between 1 and 4, preferably wherein n1 is 1 and n2 is 3.

The length of the linker may be between 3 and 7, between 3 and 6, between 4 and 7, between 4 and 6, or between 5 and 6.

The length of the linker may be between 4 and 5.

The cyclodextrin may be further substituted with: (a) at least one methyl, hydroxypropyl, sulfobutyl or succinyl group, and/or (b) at least one alkyl, lower alkyl, alkylene, alkenyl, alkynyl, alkoxy, alkoxyalkyl, alkoxyalkoxyalkyl, alkylcarbonyloxyalkyl, alkylcarbonyl, alkylsulfonyl, alkylsulfonylalkyl, alkylamino, alkoxyamino, alkylsulfanyl, amino, alkylamino, dialkylamino, alkylaminoalkyl, dialkylaminoalkyl, aminoalkyl, aminoalkoxy, alkylsulfonylamino, aminocarbonyloxyalkyl, aminosulfonyl, alkylaminosulfonyl, dialkylaminosulfonyl, alkynylalkoxy, aryl, arylalkyl, arylsulfonyl, aryloxy, aralkyloxy, cyanoalkyl, cycloalkyl, cycloalkenyl, cycloalkylalkylalkyl, substituted aryl, Cycloalkylene, cycloalkylalkylene, heteroalkyl, heteroaryl, heteroarylalkyl, heteroarylsulfonyl, heteroaryloxy, heteroarylalkyloxy, heterocyclylalkoxy, halogen, haloalkyl, haloalkoxy, heterocyclylamino, heterocyclyl, heterocyclylalkyl, heterocyclyloxy, heterocyclylalkoxy, hydroxyalkoxy, hydroxyalkylamino, hydroxyalkylaminoalkyl, hydroxyalkyl, hydroxycarbonylalkyl, hydroxyalkyloxycarbonylalkyl, hydroxyalkyl, hydroxycycloalkyl, ureido, carbamate, carboxyl, sulfonamido, nitro, cyano, phenoxy, acetyl, ammonium, ammonia, azido, bromine, chlorine, deoxy, glucosyl, iodine, sulfate, sulfonyl, nitrite, phosphate, phosphoryl, fatty acid such as a palmitoyl group, mono-or disaccharide, and/or (c) at least one methyl, di-or tri-saccharide, and/or (c) at least one alkyl, di-or tri-saccharide, and/or (d) at least one alkyl, di-or tri-saccharide, or (C) at least one alkyl, di-or tri-saccharide, and/or (C) at least one alkyl, di-saccharide, or a combination of an alkyl, a salt or a salt, or a salt, or a salt, or a salt, or a salt, or a salt, or a salt, or, Hydroxypropyl, sulfobutyl, succinyl, maltosyl, carboxymethyl, quaternary ammonium (such as-CH)₂CH(OH)CH₂N(CH₃)₃ ⁺) A glucosyl, palmitoyl, phosphate, phosphoryl, amino, azido, sulfate, sulfonyl, alkyl, ethyl, propyl, isopropyl, butyl, isobutyl, bromo, or chloro group.

The cyclodextrin dimer can have a structure according to any one of formulas I-IX (shown in figures 3B-3J, respectively).

Each R¹Each R²And each R³Can be independently selected from (a) methyl, H, hydroxypropyl, sulfobutyl ether, succinyl-hydroxypropyl, quaternary ammonium, carboxymethyl-hydroxypropyl, hydroxyethyl, maltosyl, acetyl, carboxyethyl, sulfate, sulfopropyl, sodium phosphate, or glucose; and/or (b) hydrogen, alkyl, lower alkyl, alkylene, alkenyl, alkynyl, alkoxy, alkoxyalkyl, alkoxyalkoxyalkyl, alkylcarbonyloxyalkyl, alkylcarbonyl, alkylsulfonyl, alkylsulfonylalkyl, alkylamino, alkoxyamino, alkylsulfanyl, amino, alkylamino, dialkylamino, alkylaminoalkyl, dialkylaminoalkyl, aminoalkyl, aminoalkoxy, alkylsulfonylamino, aminocarbonyloxyalkyl, aminosulfonyl, alkylalkylaminoAminosulfonyl, dialkylaminosulfonyl, alkynylalkoxy, aryl, arylalkyl, arylsulfonyl, aryloxy, aralkyloxy, cyanoalkyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, cycloalkylidene, cycloalkylalkylene, heteroalkyl, heteroaryl, heteroarylalkyl, heteroarylsulfonyl, heteroaryloxy, heteroaralkoxy, heterocyclylalkoxy, halogen, haloalkyl, haloalkoxy, heterocyclylamino, heterocyclyl, heterocyclylalkyl, heterocyclyloxy, heterocyclylalkoxy, hydroxyalkoxy, hydroxyalkylamino, hydroxyalkylaminoalkyl, hydroxyalkyl, hydroxycarbonylalkyl, hydroxyalkyloxycarbonylalkyl, hydroxyalkyl, hydroxycycloalkyl, urea, carbamate, carboxyl, sulfonamido, nitro, cyano, phenoxy, or acetyl groups.

L may be attached to the C2 carbon of each CD monomer, to the C3 carbon of each CD monomer, or to the C2 carbon of one CD monomer and the C3 carbon of another CD monomer. For linkers having multiple points of attachment to a single CD monomer, these points of attachment may be to the C2, C3, or a combination of C2 and C3 carbons of the monomer; specific arrangements may be advantageous depending on the reactions employed for their formation, purification steps and/or depending on the structure of the linker.

The cyclodextrin dimer may exhibit greater affinity for 7KC than cholesterol. The greater affinity may be determined using the turbidity test disclosed herein.

The cyclodextrin dimer may exhibit at least 1.1-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, or 10-fold affinity for 7KC over cholesterol. The cyclodextrin dimer may exhibit a relative turbidity of 7KC that is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50%, or more, less than the relative turbidity of cholesterol in a turbidity test.

In exemplary embodiments, the present disclosure provides a composition comprising a mixture of cyclodextrin dimers as disclosed herein, wherein optionally the average degree of substitution may be between 2 and 10, such as between 2 and 8, such as between 3 and 7, or between 2 and 5. The composition may comprise a mixture of CD dimers having a degree of substitution between 2 and 5 of hydroxypropyl, sulfobutyl, succinyl or quaternary ammonium groups, such as about 2, about 3, about 4, or about 5 of said substituents. The composition may comprise a mixture of CD dimers having a degree of substitution of methyl groups between 2 and 10. The degree of substitution can be measured by NMR. The degree of substitution can be measured by mass spectrometry (such as MALDI).

In exemplary embodiments, the present disclosure provides a composition comprising a mixture of cyclodextrin dimers (e.g., cyclodextrin dimers according to formulas I-III (shown in figures 3B-3D, respectively)) as disclosed herein.

In exemplary embodiments, the present disclosure provides a pharmaceutical composition comprising a cyclodextrin dimer or a composition thereof as disclosed herein and a pharmaceutically acceptable carrier. The cyclodextrin dimer may be the only active ingredient in the composition. The pharmaceutical composition can consist of, or consist essentially of, the cyclodextrin dimer and the pharmaceutically acceptable carrier.

In exemplary embodiments, the present disclosure provides a method of treatment comprising administering to a subject in need thereof an effective amount of a cyclodextrin dimer or a composition thereof as disclosed herein. A subject in need thereof may suffer from the deleterious or toxic effects of 7 KC.

In an exemplary embodiment, the present disclosure provides a method for reducing the amount of 7KC in a subject in need thereof, the method comprising administering to the subject in need thereof an effective amount of a cyclodextrin dimer as disclosed herein.

The cyclodextrin dimer may be administered to the patient via parenteral (e.g., subcutaneous, intramuscular, or intravenous), topical, transdermal, oral, sublingual, or buccal administration, preferably intravenous.

The method may comprise administering between about 1mg and 10g, such as between 10mg and 1g, between 50mg and 200mg, or 100mg of the cyclodextrin dimer to the patient. In exemplary embodiments, between 1g and 10g of cyclodextrin dimer, such as about 2g, about 3g, about 4g, or about 5g, may be administered. In exemplary embodiments, between 50mg and 5g of cyclodextrin dimer, such as between 100mg and 2.5g, between 100mg and 2g, between 250mg and 2.5g, for example about 1g, may be administered.

The methods can prevent, treat and/or ameliorate symptoms of one or more of the following diseases: atherosclerosis, arteriosclerosis, coronary atherosclerosis due to calcified coronary artery disease, heart failure (all stages), Alzheimer's disease, amyotrophic lateral sclerosis, Parkinson's disease, Huntington's disease, vascular dementia, multiple sclerosis, Smith-Lyme-Obtz syndrome, infantile neuronal ceroid lipofuscinosis, lysosomal acid lipase deficiency, tendonoxanthomatosis, X-linked adrenoleukodystrophy, sickle cell disease, type A Niemann-pick disease, type B Niemann-pick disease, type C Niemann-pick disease, gaucher disease, Stargardt disease, age-related macular degeneration (dryness), idiopathic pulmonary fibrosis, chronic obstructive pulmonary disease, cystic fibrosis, liver injury, liver failure, nonalcoholic steatohepatitis, nonalcoholic fatty liver disease, Irritable bowel syndrome, crohn's disease, ulcerative colitis and/or hypercholesterolemia, preferably atherosclerosis.

The method may further comprise administering a second therapy to the patient, wherein the second therapy may be administered simultaneously or sequentially in any order.

The second therapy may include one or more of an anti-cholesterol drug (such as a fibrate or statin), an anti-platelet drug, an antihypertensive drug, or a dietary supplement. The statin may include advicor (r) (niacin sustained release/lovastatin), altoprev (r) (lovastatin sustained release), caduet (amlodipine) and atorvastatin (atorvastatin)), crestor (r) (rosuvastatin), uviisync (r) (sitagliptin)/simvastatin (simvastatin), LESCOL (r) (fluvastatin), LESCOL XL (fluvastatin sustained release), lipitor (r) (simvastatin), lival (simvastatin), pitavastatin (mevastatin), mevacor (lovastatin), pravastatin (pravastatin), lovastatin, pravastatin (simutastatin) (simvastatin/simvastatin), or lovastatin (simvastatin/lovastatin).

The second therapy may include an anti-cholesterol drug and an anti-hypertensive drug.

In an exemplary embodiment, the present disclosure provides a method of purifying an oxysterol, the method comprising: contacting a composition comprising an oxysterol with a cyclodextrin dimer as disclosed herein, thereby solubilizing the oxysterol in the cyclodextrin dimer; and recovering the cyclodextrin dimer and solubilized oxysterol. The oxysterol comprises or consists of 7 KC. The method may additionally include measuring the concentration of 7KC in the solubilized oxysterol, thereby determining the relative concentration of 7KC in the composition. The composition may include a patient sample. The method can be used to determine a 7KC concentration in a patient sample, which 7KC concentration can be used for diagnosis and/or treatment planning.

In an exemplary embodiment, the present disclosure provides an in vitro method of removing oxysterol from a sample, the method comprising: contacting a sample comprising an oxysterol with a cyclodextrin dimer as disclosed herein, thereby solubilizing the oxysterol in the cyclodextrin dimer; and separating the sample from the cyclodextrin dimer and solubilized sterol.

In an exemplary embodiment, the present disclosure provides a method of producing a reduced cholesterol product, the method comprising: contacting a product comprising cholesterol with a cyclodextrin dimer as disclosed herein, thereby solubilizing the cholesterol in the cyclodextrin dimer; and removing the cyclodextrin dimer and solubilized cholesterol from the product. The product may be a food product, such as meat and/or milk.

In another aspect, the present disclosure provides a method of making a cyclodextrin dimer (such as a cyclodextrin dimer comprising an unsubstituted or substituted alkyl linker) as described herein, the method comprising: (a) reacting the beta-cyclodextrin protected on the primary side with a dialkylating agent to produce a primary side-protected beta CD dimer linked by a secondary surface, and optionally purifying the primary side-protected beta CD dimer; (b) deprotecting the primary side-protected β CD dimer, thereby producing a deprotected β CD dimer, and optionally purifying the deprotected β CD dimer; and (c) hydroxypropylating the deprotected β CD, thereby producing a cyclodextrin dimer, and optionally purifying the cyclodextrin dimer. The beta-cyclodextrin protected on the major side can include hepta (6-O-tert-butyldimethylsilyl) -beta-cyclodextrin. The dialkylating agent may comprise a dibromoalkane, optionally 1, 4-dibromobutane. Step (a) may be carried out under anhydrous conditions and/or with sodium hydride as the base. The purification in step (a) may comprise direct phase chromatography and isocratic elution. Step (b) may be carried out in Tetrahydrofuran (THF) together with tetrabutylammonium fluoride. The purification in step (b) may comprise direct phase chromatography and isocratic elution. Step (c) may comprise reacting the deprotected β CD dimers with a hydroxypropylation agent (such as propylene oxide), a methylating agent (such as methyl iodide), a succinylating agent (such as succinic anhydride), a sulfobutylating agent (such as 1, 4-butane sultone), and/or a quaternary ammonium linking agent (such as glycidyl trimethyl ammonium chloride).

Step (c) may be carried out under aqueous conditions, optionally including sodium hydroxide as a base. Step (c) may include one or more of ion exchange resin treatment, charcoal clarification and dialysis.

In another aspect, the present disclosure provides a method of preparing a cyclodextrin dimer (such as a cyclodextrin dimer comprising a triazole linker) as described herein, the method comprising: (a) reacting 2-O- (n-azidoalkyl) - β CD with 2-O- (n-alkyne) - β CD to form β CD-triazole- β CD dimer having the structure β CD-alkyl 1-triazole-alkyl 2- β CD, and optionally (b) purifying the β CD-triazole- β CD dimer. Step (a) may be performed using a copper (I) catalyst, optionally about 15mM copper (I). Step (a) may be carried out in an aqueous solution. The aqueous solution may comprise dimethyl ether(iv) Dimethylformamide (DMF), optionally about 50% DMF (v/v). Step (b) may comprise chromatography. The method may further comprise generating the 2-O- (n-azidoalkyl) - β CD prior to step (a) by a method comprising: (1) reacting n-azido-1-bromo-alkane with β -cyclodextrin, optionally with a catalytic amount of lithium iodide, to form the 2-O- (n-azidoalkyl) - β CD; and (2) optionally purifying the 2-O- (n-azidoalkyl) - β CD. Step (2) may comprise chromatography. The method may further comprise generating 2-O- (n-alkyne) - β CD prior to step (a) by a method comprising: (i) reacting n-bromo-1-alkyne with β -cyclodextrin, optionally with a catalytic amount of lithium iodide, to generate the 2-O- (n-alkyne) - β CD, and (ii) optionally purifying the 2-O- (n-alkyne) - β CD. Step (2) may comprise silica gel chromatography. Step (1) may be carried out in dry DMSO. The reaction in step (1) may comprise lithium hydride. The beta CD-triazole-beta CD dimer may comprise the structure: Wherein n1 may be between 1 and 8, and/or n2 may be between 1 and 8, optionally n1 may be 1, 2, 3 or 4, and/or n2 may be 1, 2, 3 or 4, preferably wherein n1 is 1 and n2 is 3. The triazole linker may be between 5 and 8 in length. The method may further comprise hydroxypropylating the beta CD-triazole-beta CD dimer, thereby producing a cyclodextrin dimer, and optionally purifying the cyclodextrin dimer. Step (c) may comprise reacting the beta CD-triazole-beta CD dimer with a hydroxypropylation agent (such as propylene oxide), a methylating agent (such as methyl iodide), a succinylating agent (such as succinic anhydride), a sulfobutylating agent (such as 1, 4-butanesultone), and/or a quaternary ammonium linking agent (such as glycidyltrimethylammonium chloride).

Step (c) may be carried out under aqueous conditions, optionally including sodium hydroxide as a base. The purification in step (c) may comprise one or more of ion exchange resin treatment, charcoal clarification, membrane filtration and dialysis.

Embodiments of the present invention provide compositions and methods for treating or preventing atherosclerosis. 7KC is the most abundant non-enzymatically produced oxysterol in atherosclerotic plaques and is believed to be a contributing factor to the pathogenesis of atherosclerosis. It is contemplated that treatment with the dimers of the CD of the invention (such as the HP β CD of the present disclosure or another CD) is beneficial for the prevention and/or reversal of atherosclerotic plaque formation.

Embodiments of the present invention provide compositions and methods for treating or preventing 7 KC-associated diseases and disorders. Such diseases and disorders include, but are not limited to, aging diseases such as atherosclerosis, AMD, arteriosclerosis, coronary atherosclerosis due to calcified coronary lesions, heart failure (all stages), alzheimer's disease, parkinson's disease, vascular dementia, chronic obstructive pulmonary disease, non-alcoholic fatty liver disease, and/or hypercholesterolemia-associated dementia. Other sporadic and/or congenital diseases associated with 7KC accumulation include huntington's disease, multiple sclerosis, smith-lyme-oppez syndrome, infantile neuronal ceroid lipofuscinosis, lysosomal acid lipase deficiency, amyotrophic lateral sclerosis, tendono-encephaloma disease, X-linked adrenoleukodystrophy, sickle cell anemia, niemann-pick disease type a, niemann-pick disease type B, niemann-pick disease type C, gaucher disease, stargardt disease, idiopathic pulmonary fibrosis, cystic fibrosis, liver injury, liver failure, non-alcoholic steatohepatitis, ulcerative colitis, crohn's disease, and other irritable bowel syndromes.

In another exemplary embodiment, the present disclosure provides a cyclodextrin dimer composition having a substituent degree of substitution between 1 and 40, such as between 1 and 28 or between 4 and 20, preferably between 2 and 15, wherein the substituent is selected from methyl, hydroxypropyl, sulfobutyl, succinyl, quaternary ammonium such as-CH, and the like₂CH(OH)CH₂N(CH₃)₃ ⁺Alkyl, lower alkyl, alkylene, alkenyl, alkynyl, alkoxy, alkoxyalkyl, alkoxyalkoxyalkyl, alkylcarbonyloxyalkyl, alkylCarbonyl, alkylsulfonyl, alkylsulfonylalkyl, alkylamino, alkoxyamino, alkylsulfanyl, amino, alkylamino, dialkylamino, alkylaminoalkyl, dialkylaminoalkyl, aminoalkyl, aminoalkoxy, alkylsulfonylamino, aminocarbonyloxyalkyl, aminosulfonyl, ammonium, ammonia, alkylaminosulfonyl, dialkylaminosulfonyl, alkynylalkoxy, aryl, arylalkyl, arylsulfonyl, aryloxy, aralkyloxy, azido, bromine, chlorine, cyanoalkyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, cycloalkylene, cycloalkylalkylene, deoxy, glucosyl, heteroalkyl, heteroaryl, heteroarylalkyl, heteroarylsulfonyl, heteroaryloxy, heteroarylalkyloxy, heterocyclylalkoxy, halogen, haloalkyl, haloalkoxy, heterocyclylamino, heterocyclyl, heterocyclylalkylthio, amino, heterocyclylalkoxy, amino, or amino, heterocyclylalkyl, heterocyclyloxy, heterocyclylalkoxy, hydroxyalkoxy, hydroxyalkylamino, hydroxyalkylaminoalkyl, hydroxyalkyl, hydroxycarbonylalkyl, hydroxyalkyloxycarbonylalkyl, hydroxyalkyl, hydroxycycloalkyl, iodine, urea, carbamate, carboxyl, sulfate, sulfonyl, sulfonamide, nitro, nitrite, cyano, phosphate, phosphoryl, phenoxy, acetyl, a fatty acid such as a palmitoyl group, a monosaccharide or a disaccharide, the composition comprising a cyclodextrin dimer having the structure CD-L-CD, wherein L is attached to the large (minor) surface of each CD molecule through the C2 carbon (instead of R1) and/or the C3 carbon (instead of R2) of each CD subunit; wherein each CD has the structure of formula X, wherein the substituents are located at one or more of R1, R2, and/or R3, wherein L has a length of no more than 8 atoms, wherein the no more than 8 atoms are preferably each C, N, O or S. The substituent may be carboxymethyl or maltosyl. The substituents are preferably methyl, hydroxypropyl, sulfobutyl, succinyl, quaternary ammonium (such as-CH) ₂CH(OH)CH₂N(CH₃)₃ ⁺). The degree of substitution can be determined by NMR. The degree of substitution can be determined by mass spectrometry, such as MALDI.

In another exemplary embodiment, the present disclosure provides a cyclodextrin diA dimer composition of cyclodextrins having a degree of substitution between 1 and 40, such as between 1 and 28 or between 4 and 20, preferably between 2 and 15, wherein the substituents are selected from methyl, hydroxypropyl, sulfobutyl, succinyl, maltosyl, carboxymethyl, quaternary ammonium (such as-CH)₂CH(OH)CH₂N(CH₃)₃ ⁺) Glucosyl, palmitoyl, phosphate, phosphoryl, amino, azido, sulfate, sulfonyl, alkyl, ethyl, propyl, isopropyl, butyl, isobutyl, bromo, or chloro, comprising a cyclodextrin dimer having the structure CD-L-CD, wherein L is attached to the large (sub-) surface of each CD molecule through the C2 carbon (instead of R1) and/or the C3 carbon (instead of R2) of each CD subunit; wherein each CD has the structure of formula X, wherein the substituents are located at one or more of R1, R2, and/or R3, wherein L has a length of no more than 8 atoms, wherein the no more than 8 atoms are preferably each C, N, O or S. The degree of substitution can be determined by NMR. The degree of substitution can be determined by mass spectrometry, such as MALDI.

In another exemplary embodiment, the present disclosure provides a cyclodextrin dimer composition having a substituent degree of substitution between 1 and 40, such as between 1 and 28 or between 4 and 20, preferably between 2 and 15, wherein the substituent is selected from methyl, hydroxypropyl, sulfobutyl, succinyl, maltosyl, carboxymethyl or quaternary ammonium (such as-CH)₂CH(OH)CH₂N(CH₃)₃ ⁺) The composition comprises a cyclodextrin dimer having the structure CD-L-CD, wherein L is attached to the large (minor) surface of each CD molecule through the C2 carbon (instead of R1) and/or the C3 carbon (instead of R2) of each CD subunit; wherein each CD has the structure of formula X, wherein the substituents are located at one or more of R1, R2, and/or R3, wherein L has a length of no more than 8 atoms, wherein the no more than 8 atoms are preferably each C, N, O or S. The degree of substitution can be determined by NMR. The degree of substitution can be determined by mass spectrometry, such as MALDI.

In another exemplary embodiment, the present disclosure provides a cyclodextrin dimer composition having a degree of substitution of hydroxypropyl substituents between 1 and 40, such as between 1 and 28 or between 4 and 20, preferably between 2 and 15, more preferably between 2 and 5, and even more preferably between 2 and 4, comprising a cyclodextrin dimer having the structure CD-L-CD, wherein L is attached to the large (minor) surface of each CD molecule through the C2 carbon (instead of R1) and/or the C3 carbon (instead of R2) of each CD subunit; wherein each CD has the structure of formula X, wherein the substituents are located at one or more of R1, R2, and/or R3, wherein L has a length of no more than 8 atoms, wherein the no more than 8 atoms are preferably each C, N, O or S. The degree of substitution can be determined by NMR. The degree of substitution can be determined by mass spectrometry, such as MALDI.

In another exemplary embodiment, the present disclosure provides a cyclodextrin dimer composition having a methyl substituent degree of substitution between 1 and 40, such as between 1 and 28 or between 4 and 20, preferably between 2 and 15, more preferably between 2 and 10, comprising a cyclodextrin dimer having the structure CD-L-CD, wherein L is attached to the large (minor) surface of each CD molecule through the C2 carbon (instead of R1) and/or the C3 carbon (instead of R2) of each CD subunit; wherein each CD has the structure of formula X, wherein the substituents are located at one or more of R1, R2, and/or R3, wherein L has a length of no more than 8 atoms, wherein the no more than 8 atoms are preferably each C, N, O or S. The degree of substitution can be determined by NMR. The degree of substitution can be determined by mass spectrometry, such as MALDI.

In another exemplary embodiment, the present disclosure provides a cyclodextrin dimer composition having a degree of substitution of sulfobutyl substituents between 1 and 40, such as between 1 and 28 or between 4 and 20, preferably between 2 and 15, more preferably between 2 and 5, and even more preferably between 2 and 4, comprising a cyclodextrin dimer having the structure CD-L-CD, wherein L is attached to the large (minor) surface of each CD molecule through the C2 carbon (instead of R1) and/or the C3 carbon (instead of R2) of each CD subunit; wherein each CD has the structure of formula X, wherein the substituents are located at one or more of R1, R2, and/or R3, wherein L has a length of no more than 8 atoms, wherein the no more than 8 atoms are preferably each C, N, O or S. The degree of substitution can be determined by NMR. The degree of substitution can be determined by mass spectrometry, such as MALDI.

In another exemplary embodiment, the present disclosure provides a cyclodextrin dimer composition having a succinyl substituent degree of substitution between 1 and 40, such as between 1 and 28 or between 4 and 20, preferably between 2 and 15, more preferably between 2 and 5, and even more preferably between 2 and 4, comprising a cyclodextrin dimer having the structure CD-L-CD, wherein L is attached to the large (minor) surface of each CD molecule through the C2 carbon (instead of R1) and/or the C3 carbon (instead of R2) of each CD subunit; wherein each CD has the structure of formula X, wherein the substituents are located at one or more of R1, R2, and/or R3, wherein L has a length of no more than 8 atoms, wherein the no more than 8 atoms are preferably each C, N, O or S. The degree of substitution can be determined by NMR. The degree of substitution can be determined by mass spectrometry, such as MALDI.

In another exemplary embodiment, the present disclosure provides a cyclodextrin dimer composition having a quaternary ammonium substituent (preferably-CH)₂CH(OH)CH₂N(CH₃)₃ ⁺) A degree of substitution between 1 and 40, such as between 1 and 28 or between 4 and 20, preferably between 2 and 15, more preferably between 2 and 5, and even more preferably between 2 and 4, the composition comprising a cyclodextrin dimer having the structure CD-L-CD, wherein L is attached to the large (minor) surface of each CD molecule through the C2 carbon (instead of R1) and/or the C3 carbon (instead of R2) of each CD subunit; wherein each CD has the structure of formula X, wherein the taking Substituents are located at one or more of R1, R2, and/or R3, wherein L has a length of no more than 8 atoms, wherein the no more than 8 atoms are preferably each C, N, O or S. The degree of substitution can be determined by NMR. The degree of substitution can be determined by mass spectrometry, such as MALDI.

In another exemplary embodiment, the present disclosure provides a cyclodextrin dimer composition having a degree of substitution between 0 and 40, comprising a cyclodextrin dimer having the structure CD-L-CD, wherein L is attached to the large (minor) surface of each CD molecule through the C2 carbon (instead of R1) and/or the C3 carbon (instead of R2) of each CD subunit; wherein each CD has the structure of formula X, optionally substituted with one or more substituents, wherein L has a length of no more than 8 atoms, wherein the no more than 8 atoms are preferably each C, N, O or S. The cyclodextrin dimer compositions can be used to synthesize cyclodextrin dimer compositions substituted with one or more substituents. The degree of substitution can be determined by NMR. The degree of substitution can be determined by mass spectrometry, such as MALDI.

The linker L may have the following structure:

wherein each R is independently selected from H, X, SH, NH2 or OH, or is absent;

attachment of each CD to the linker is independently achieved through O, S or N attached to their C2 or C3 carbons, or through an acetal linkage of two adjacent oxygens of the CD.

Each X is a substituted or unsubstituted alkane, alkene, or alkyne;

each A is independently selected from a single, double or triple bond covalent bond, S, N, NH, O, or a substituted or unsubstituted alkane, alkene, or alkyne; and is

B is a substituted or unsubstituted 5 or 6 membered ring, S, N, NH, NR, O or absent.

The length of the linker may be between 2 and 7. The length of the linker may be between 3 and 6. The length of the linker may be 2 or 3. The length of the linker may be between 4 and 7. The length of the linker may be between 4 and 6. The length of the linker may be between 4 and 5. The length of the joint may be 4.

The linker may be a substituted or unsubstituted alkyl group, such as an unsubstituted alkyl group, e.g., an unsubstituted butyl group. The linker may comprise a triazole.

The linker may comprise the following structure: n1 and n2 may each be between 0 and 8, such as each between 1 and 4. Preferably, the total length of the joint may be 8 or less, such as 8, 7, 6, 5, 4, or any range of values therein. In a preferred embodiment, n1 is 1 and n2 is 3.

In exemplary embodiments, when the linker L comprises a triazole, e.g., having the structure of formula XI, the linker may be attached to the O2 position of each CD monomer, wherein n1 and n2 may each be between 0 and 8, such as each between 1 and 4; preferably, the total length of the joint may be 8 or less, such as 8, 7, 6, 5, 4, or any numerical range therein; and in a preferred embodiment, n1 is 1 and n2 is 3.

In exemplary embodiments, when the linker L comprises a substituted or unsubstituted alkyl group, preferably having a length of no more than 8 atoms, such as between 2 and 7, between 2 and 6, or between 4 and 7, or between 4 and 6, or between 4 and 5, or a length of 8, 7, 6, 5, 4, 3, or 2, or any numerical range therein, the linker L may be attached to the O2 position of each CD monomer, the O2 position of one CD monomer and the O3 position of another CD monomer, or the O3 positions of two CD monomers; wherein preferably the linker is a substituted or unsubstituted butyl group, more preferably an unsubstituted butyl group.

The linker may comprise any of the linkers shown in fig. 8D, wherein the oxygen atoms shown at each end of each linker form part of the cyclodextrin monomer to which the linker is attached.

The cyclodextrin dimer composition may comprise further substitution of the cyclodextrin dimer with: (a) at least one methyl, hydroxypropyl, sulfobutyl, succinyl, or quaternary ammonium group (such as-CH)₂CH(OH)CH₂N(CH₃)₃ ⁺) And/or (b) at least one alkyl, lower alkyl, alkylene, alkenyl, alkynyl, alkoxy, alkoxyalkyl, alkoxyalkoxyalkyl, alkylcarbonyloxyalkyl, alkylcarbonyl, alkylsulfonyl, alkylsulfonylalkyl, alkylamino, alkoxyamino, alkylsulfanyl, amino, alkylamino, dialkylamino, alkylaminoalkyl, dialkylaminoalkyl, aminoalkyl, aminoalkoxy, alkylsulfonylamino, aminocarbonyloxyalkyl, aminosulfonyl, alkylaminosulfonyl, dialkylaminosulfonyl, alkynylalkoxy, aryl, arylalkyl, arylsulfonyl, aryloxy, aralkoxy, cyanoalkyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, cycloalkylene, cycloalkylalkylene, heteroalkyl, heteroaryl, heteroarylalkyl, heteroarylsulfonyl, alkoxyalkylsulfonyl, alkoxyalkoxyalkoxyalkyl, alkoxyalkoxyalkoxyalkoxyalkoxyalkyl, alkoxyalkoxyalkoxyalkoxyalkoxyalkoxyalkyloxyalkyl, alkylamino, alkoxyalkyloxyalkyl, alkylsulfonylalkyl, alkoxyalkylsulfonylalkyl, alkoxyalkoxy, alkoxyalkylsulfonylalkyl, alkoxyalkoxy, alkoxyalkylsulfonylalkyl, alkoxyalkoxy, alkoxyalkylsulfonylalkyl, alkoxyalkoxy, and/or, Heteroaryloxy, heteroarylalkyloxy, heterocyclylalkoxy, halogen, haloalkyl, haloalkoxy, heterocyclylamino, heterocyclyl, heterocyclylalkyl, heterocyclyloxy, heterocyclylalkoxy, hydroxyalkoxy, hydroxyalkylamino, hydroxyalkylaminoalkyl, hydroxyalkyl, hydroxycarbonylalkyl, hydroxyalkoxycarbonylalkyl, hydroxyalkyl, hydroxycycloalkyl, ureido, carbamate, carboxyl, sulfonamide, nitro, cyano, phenoxy, acetyl, ammonium, ammonia, azido, bromine, chlorine, deoxy, glucosyl, iodine, sulfate, sulfonyl, nitrite, phosphate, phosphoryl, fatty acid such as palmitoyl, mono-or disaccharide, and/or (c) at least one methyl, hydroxypropyl, sulfobutyl, succinyl, maltosyl, carboxymethyl, quaternary ammonium (such as-CH). ₂CH(OH)CH₂N(CH₃)₃ ⁺) Glucosyl, palmitoyl, phosphate, phosphoryl, amino, azido, sulfate, sulfonyl, alkyl, ethyl, propyl, isopropyl, butyl, isobutyl, bromo, chloro groups.

The cyclodextrin dimer composition can comprise a cyclodextrin dimer having a structure according to any one of formulas I-IX (shown in figures 3B-3J, respectively).

Where not otherwise indicated, each R1, each R2, and each R3 can be independently selected from (a) methyl, H, hydroxypropyl, sulfobutyl ether, succinyl-hydroxypropyl, quaternary ammonium (such as-CH)₂CH(OH)CH₂N(CH₃)₃ ⁺) Carboxymethyl, carboxymethyl-hydroxypropyl, hydroxyethyl, maltosyl, acetyl, carboxyethyl, sulfate, sulfopropyl, sodium phosphate, or glucosyl; and/or (b) hydrogen, alkyl, lower alkyl, alkylene, alkenyl, alkynyl, alkoxy, alkoxyalkyl, alkoxyalkoxyalkyl, alkylcarbonyloxyalkyl, alkylcarbonyl, alkylsulfonyl, alkylsulfonylalkyl, alkylamino, alkoxyamino, alkylsulfanyl, amino, alkylamino, dialkylamino, alkylaminoalkyl, dialkylaminoalkyl, aminoalkyl, aminoalkoxy, alkylsulfonylamino, aminocarbonyloxyalkyl, aminosulfonyl, alkylaminosulfonyl, dialkylaminosulfonyl, alkynylalkoxy, aryl, arylalkyl, arylsulfonyl, aryloxy, aralkoxy, cyanoalkyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, cycloalkylene, cycloalkylalkylene, heteroalkyl, heteroaryl, heteroarylalkyl, heteroarylsulfonyl, heteroaryloxy, alkoxylalkyloxy, alkoxyalkyloxyalkyl, alkylcarbonyloxyalkyl, alkylcarbonylamino, alkylamino, alkylaminoalkyl, alkylaminosulfonyl, alkoxyalkoxy, cycloalkylalkylene, heteroaryloxy, cycloalkylalkylene, heteroalkyl, heteroaryl, heteroarylalkyl, heteroarylsulfonyl, heteroaryloxy, alkoxyalkyloxyalkyl, cycloalkylalkylene, heteroaryloxy, cycloalkylalkylene, heteroaryloxy, heteroarylalkylene, or, Heteroaralkoxy, heterocyclylalkoxy, halogen, haloalkyl, haloalkoxy, heterocyclylamino, heterocyclyl, heterocyclylalkyl, heterocyclyloxy, heterocyclylalkoxy, hydroxyalkoxy, hydroxyalkylamino, hydroxyalkylaminoalkyl, hydroxyalkyl, hydroxycarbonylalkyl, hydroxyalkyloxycarbonylalkyl, hydroxyalkyl, hydroxycycloalkyl, ureido, carbamate, carboxyl, sulfonamide, nitro, cyano, phenoxy, or acetyl groups.

The linker L may be attached to the C2 carbon of each CD monomer. The linker L may be attached to the C3 carbon of each CD monomer. The linker L may be attached to C2 carbon of one CD monomer and C3 of another CD monomer.

The cyclodextrin dimer composition can exhibit a greater affinity for 7KC than cholesterol, wherein optionally the greater affinity is determined by a turbidity test.

The cyclodextrin dimer composition can exhibit at least 1.1-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, or 10-fold affinity for 7KC over cholesterol. The cyclodextrin dimer may exhibit a relative turbidity of 7KC that is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50%, or more, less than the relative turbidity of cholesterol in a turbidity test.

The degree of substitution may be 2. The degree of substitution may be 3. The degree of substitution may be 4. The degree of substitution may be 5. The degree of substitution may be 6. The degree of substitution may be 7. The degree of substitution may be 8. The degree of substitution may be 9. The degree of substitution may be 10.

The cyclodextrin dimer composition can comprise a mixture of cyclodextrin dimer molecules each having a different number of substituents and/or different linker attachment points, wherein the average degree of substitution of the composition is specified.

In another aspect, the present disclosure provides a pharmaceutical composition comprising a cyclodextrin dimer composition as disclosed herein and a pharmaceutically acceptable carrier. The pharmaceutical composition may be suitable for administration to a subject, for example parenteral (e.g., subcutaneous, intramuscular or intravenous), topical, transdermal, oral, sublingual or buccal administration, preferably intravenous or subcutaneous administration, more preferably intravenous administration. The cyclodextrin dimer composition may be the only active ingredient in the composition. The pharmaceutical composition can consist of, or consist essentially of, the cyclodextrin dimer and the pharmaceutically acceptable carrier.

In another aspect, the present disclosure provides a method of treatment comprising administering to a subject in need thereof an effective amount of a cyclodextrin dimer composition as disclosed herein. The subject may suffer from a deleterious or toxic effect of 7KC or a condition associated with a deleterious or toxic effect of 7 KC.

In another aspect, the present disclosure provides a method for reducing the amount of 7KC in a subject in need thereof, the method comprising administering to the subject an effective amount of a cyclodextrin dimer composition as disclosed herein or a pharmaceutical composition comprising a cyclodextrin dimer composition as disclosed herein.

The cyclodextrin dimer composition can be administered to the subject via parenteral (e.g., subcutaneous, intramuscular, or intravenous), topical, transdermal, oral, sublingual, or buccal administration, preferably intravenous administration.

The method may comprise administering to the subject (a) between about 1mg and 20g, such as between 10mg and 1g, between 50mg and 200mg, or 100mg of the cyclodextrin dimer composition, or (b) between 1g and 10g of the cyclodextrin dimer composition, such as about 2g, about 3g, about 4g, or about 5g, or (c) between 50mg and 5g of the cyclodextrin dimer composition, such as between 100mg and 2.5g, between 100mg and 2g, between 250mg and 2.5 g.

The methods may be used to prevent, treat or ameliorate symptoms of one or more of the following diseases: atherosclerosis/coronary artery disease, arteriosclerosis, coronary atherosclerosis due to calcified coronary lesions, heart failure (all stages), Alzheimer's disease, amyotrophic lateral sclerosis, Parkinson's disease, Huntington's disease, vascular dementia, multiple sclerosis, Smith-Lyme-Observation syndrome, infantile neuronal ceroid lipofuscinosis, lysosomal acid lipase deficiency, tendonoxanthomatosis, X-linked adrenoleukodystrophy, sickle cell disease, type A Niemann-pick disease, type B Niemann-pick disease, type C Niemann-pick disease, gaucher disease, Stargardt disease, age-related macular degeneration (dry), idiopathic pulmonary fibrosis, chronic obstructive pulmonary disease, cystic fibrosis, liver injury, liver failure, nonalcoholic steatohepatitis, Non-alcoholic fatty liver disease, irritable bowel syndrome, crohn's disease, ulcerative colitis, and/or hypercholesterolemia; wherein optionally the treatment is administered in combination with another therapy. The method may comprise administering a second therapy to the subject, wherein the second therapy is administered simultaneously or sequentially in any order.

The methods can be used to prevent, treat or ameliorate the symptoms of atherosclerosis. The cyclodextrin dimer composition can be administered in combination with another therapy for treating or preventing atherosclerosis, such as an anti-cholesterol drug, an anti-hypertensive drug, an anti-platelet drug, a dietary supplement, or surgical or behavioral intervention, including but not limited to those described herein. The anti-cholesterol drug may include a fibrate or a statin, an anti-platelet drug, an anti-hypertensive drug, or a dietary supplement. The statin may include advicor (r) (niacin sustained release/lovastatin), altoprev (r) (lovastatin sustained release), caduet (r) (amlodipine and atorvastatin), crestor (r) (rosuvastatin), juvsync (r) (sitagliptin/simvastatin), LESCOL (r) (fluvastatin), LESCOL XL (fluvastatin sustained release), lipitor (r) (atorvastatin), livalo (r) (pitavastatin), mevacor (r) (lovastatin), pravachol (r) (pravastatin), simcor (r) (niacin sustained release/simvastatin), vorin (r) (ezetimibe/simvastatin), or zocor (r) (simvastatin).

The methods may be used to prevent, treat or ameliorate the symptoms of dry age-related macular degeneration. The methods can be used to prevent, treat or ameliorate symptoms of Stargardt disease. The cyclodextrin dimer composition may be administered in combination with another therapy for treating or preventing dry AMD or stargardt disease, such as LBS-008(Belite Bio) (a non-retinoic acid antagonist of retinol binding protein 4), AREDS supplement formulations (comprising vitamins C and E, beta-carotene ト, zinc, and copper), AREDS2 supplement formulations (including supplement formulations with vitamins C and E, zinc, copper, lutein, zeaxanthin, and omega-3 fatty acids, or combinations thereof).

The methods can be used to prevent, treat or ameliorate the symptoms of niemann-pick disease. The cyclodextrin dimer composition may be administered in combination with another therapy for treating or preventing niemann-pick disease, such as one or more of miglustat (zavesca (r)), HP β CD (trap CD, VTS-270), and physical therapy.

The methods can be used to prevent, treat or ameliorate the symptoms of alzheimer's disease. The cyclodextrin dimer composition can be administered in combination with another therapy for treating or preventing alzheimer's disease, such as cholinesterase inhibitors (aricept (r), exelon (r), razadyne (r)), and memantine (namenda (r)), or a combination thereof.

The methods may be used to prevent, treat or ameliorate the symptoms of heart failure. The cyclodextrin dimer composition may be administered in combination with another therapy for treating or preventing heart failure, such as one or more aldosterone antagonists, ACE inhibitors, ARBs (angiotensin II receptor blockers), ARNI (angiotensin receptor-enkephalinase inhibitors), beta-blockers, vasodilators, calcium channel blockers, digoxin, diuretics, cardiac pumping drugs, potassium, magnesium, selective sinus node inhibitors, or combinations thereof.

In another aspect, the present disclosure provides a method of making a cyclodextrin dimer composition (such as a cyclodextrin dimer composition comprising an unsubstituted or substituted alkyl linker) as described herein, the method comprising: (a) reacting the beta-cyclodextrin protected on the primary side with a dialkylating agent to produce a primary side-protected beta CD dimer linked by a secondary surface, and optionally purifying the primary side-protected beta CD dimer; (b) deprotecting the primary side-protected β CD dimer, thereby producing a deprotected β CD dimer, and optionally purifying the deprotected β CD dimer; and (c) hydroxypropylating the deprotected β CD, thereby producing a cyclodextrin dimer composition, and optionally purifying the cyclodextrin dimer composition. The beta-cyclodextrin protected on the major side can include hepta (6-O-tert-butyldimethylsilyl) -beta-cyclodextrin. The dialkylating agent may comprise a dibromoalkane, optionally 1, 4-dibromobutane. Step (a) may be carried out under anhydrous conditions and/or with sodium hydride as the base. The purification in step (a) may comprise direct phase chromatography and isocratic elution. Step (b) may be carried out in Tetrahydrofuran (THF) together with tetrabutylammonium fluoride. The purification in step (b) may comprise direct phase chromatography and isocratic elution. Step (c) may comprise reacting the deprotected β CD dimers with a hydroxypropylation agent (such as propylene oxide), a methylating agent (such as methyl iodide), a succinylating agent (such as succinic anhydride), a sulfobutylating agent (such as 1, 4-butane sultone), and/or a quaternary ammonium linking agent (such as glycidyl trimethyl ammonium chloride). The cyclodextrin dimer composition can be a cyclodextrin dimer composition as disclosed herein. The cyclodextrin dimer composition may have a degree of substitution of substituents between 1 and 40, such as between 1 and 28 or between 4 and 20, preferably between 2 and 15, more preferably between 2 and 5 or between 2 and 10.

Step (c) may be carried out under aqueous conditions, optionally including sodium hydroxide as a base. Step (c) may include one or more of ion exchange resin treatment, charcoal clarification and dialysis.

In another aspect, the present disclosure provides a method of making a cyclodextrin dimer composition (such as a cyclodextrin dimer composition comprising a triazole linker) as described herein, the method comprising: (a) reacting 2-O- (n-azidoalkyl) - β CD with 2-O- (n-alkyne) - β CD to form β CD-triazole- β CD dimer having the structure β CD-alkyl 1-triazole-alkyl 2- β CD, and optionally (b) purifying the β CD-triazole- β CD dimer. Step (a) may be performed using a copper (I) catalyst, optionally about 15mM copper (I). Step (a) may be carried out in an aqueous solution. The aqueous solution may comprise Dimethylformamide (DMF), optionally about 50% DMF (v/v). Step (b) may comprise chromatography. The method may further comprise generating the 2-O- (n-azidoalkyl) - β CD prior to step (a) by a method comprising: (1) reacting n-azido-1-bromo-alkanes with beta-cyclodextrinsReacting, optionally with a catalytic amount of lithium iodide, to form said 2-O- (n-azidoalkyl) - β CD; and (2) optionally purifying the 2-O- (n-azidoalkyl) - β CD. Step (2) may comprise chromatography. The method may further comprise generating 2-O- (n-alkyne) - β CD prior to step (a) by a method comprising: (i) reacting n-bromo-1-alkyne with β -cyclodextrin, optionally with a catalytic amount of lithium iodide, to generate the 2-O- (n-alkyne) - β CD, and (ii) optionally purifying the 2-O- (n-alkyne) - β CD. Step (2) may comprise silica gel chromatography. Step (1) may be carried out in dry DMSO. The reaction in step (1) may comprise lithium hydride. The beta CD-triazole-beta CD dimer composition may comprise the following structure: Wherein n1 may be between 1 and 8, and/or n2 may be between 1 and 8, optionally n1 may be 1, 2, 3 or 4, and/or n2 may be 1, 2, 3 or 4, preferably wherein n1 is 1 and n2 is 3. The triazole linker may be between 5 and 8 in length. The method can further include hydroxypropylating the beta CD-triazole-beta CD dimer composition, thereby producing a cyclodextrin dimer composition, and optionally purifying the cyclodextrin dimer composition. Step (c) may comprise reacting the beta CD-triazole-beta CD dimer with a hydroxypropylation agent (such as propylene oxide), a methylating agent (such as methyl iodide), a succinylating agent (such as succinic anhydride), a sulfobutylating agent (such as 1, 4-butanesultone), and/or a quaternary ammonium linking agent (such as glycidyltrimethylammonium chloride). The cyclodextrin dimer composition can be a cyclodextrin dimer composition as disclosed herein. The cyclodextrin dimer composition may have a degree of substitution of substituents between 1 and 40, such as between 1 and 28 or between 4 and 20, preferably between 2 and 15, more preferably between 2 and 5 or between 2 and 10.

Step (c) may be carried out under aqueous conditions, optionally including sodium hydroxide as a base. The purification in step (c) may comprise one or more of ion exchange resin treatment, charcoal clarification, membrane filtration and dialysis.

In another aspect, the disclosure provides a pharmaceutical composition comprising the CD (such as HP β CD or another CD of the disclosure) dimer.

In another aspect, the present disclosure provides a pharmaceutical composition comprising a cyclodextrin dimer as disclosed herein and a hydrophobic drug. The hydrophobic drug may include hormones or sterols, such as estrogens, estrogen analogs, and the like. The cyclodextrin dimer may be present in an amount effective to solubilize the hydrophobic drug.

The phrase "pharmaceutically acceptable" is used herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in entering a living organism or living biological tissue, preferably without significant toxicity, irritation, or allergic response. The invention includes methods comprising administering a cyclodextrin dimer to a patient, wherein the cyclodextrin dimer is contained within a pharmaceutical composition. The pharmaceutical compositions of the invention are formulated with pharmaceutically acceptable carriers, excipients, and other agents that provide suitable transfer, delivery, tolerance, and the like. Many suitable formulations may be present in formulations known to Pharmaceutical chemists, such as Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa. These preparations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid-containing (cationic or anionic) vesicles (such as LIPOFECTIN) ^TM) DNA conjugates, anhydrous absorbent pastes, oil-in-water and water-in-oil emulsions, emulsions of carbowax (polyethylene glycol of various molecular weights), semi-solid gels, and semi-solid mixtures of carbowax. See also (Powell et al]，J.Pharm.Sci. Technol.，52：238-311，(1998))。

As used herein, the phrase "pharmaceutically acceptable carrier" generally refers to a pharmaceutically acceptable composition, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, talc magnesium, calcium or zinc stearate, or stearic acid), or solvent encapsulating material for introducing an active agent into the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Examples of suitable aqueous and nonaqueous carriers that can be used in the pharmaceutical compositions of the invention include, for example, water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), vegetable oils (such as olive oil), and injectable organic esters (such as ethyl oleate), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating material, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.

Other examples of materials that can serve as pharmaceutically acceptable carriers include: (1) sugars such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered gum tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients such as cocoa butter and suppository waxes; (9) oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols such as glycerol, sorbitol, mannitol, and polyethylene glycol; (12) esters such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) ringer's solution; (19) ethanol; (20) a pH buffer solution; (21) polyesters, polycarbonates and/or polyanhydrides; and (22) other non-toxic compatible materials employed in pharmaceutical formulations.

Various adjuvants such as wetting agents, emulsifying agents, lubricating agents (e.g., sodium lauryl sulfate and magnesium stearate), coloring agents, sequestering agents, coating agents, sweetening agents, flavoring agents, preserving agents, and antioxidants may also be included in the pharmaceutical compositions. Some examples of pharmaceutically acceptable antioxidants include: (1) water-soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, and the like; (2) oil-soluble antioxidants such as ascorbyl palmitate, Butylated Hydroxyanisole (BHA), Butylated Hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal chelating agents such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like. In some embodiments, the pharmaceutical formulation comprises an excipient selected from, for example, cellulose, liposomes, micelle-forming agents (e.g., bile acids), and polymeric carriers (e.g., polyesters and polyanhydrides). Suspensions, in addition to the active compounds, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof. Prevention of the action of microorganisms on active compounds can be ensured by the incorporation of various antibacterial and antifungal agents (e.g., parabens, chlorobutanol, phenol sorbic acid, and the like). It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like, in the compositions. In addition, prolonged absorption of the injectable pharmaceutical form can be brought about by the inclusion of agents which delay absorption, such as aluminum monostearate and gelatin.

The pharmaceutical formulations of the present invention may be prepared by any of the methods known in the pharmaceutical art. The amount of active ingredient (i.e., a CD dimer, such as HP β CD dimer or another CD dimer of the present disclosure) that can be combined with a carrier material to produce a single dosage form will vary depending on the host treated and the particular mode of administration. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect. The amount of active compound may be in the range of about 0.1% to 99.9%, more typically, in the range of about 80% to 99.9%, and more typically, about 99%. The amount of active compound may be in the range of about 0.1% to 99%, more typically in the range of about 5% to 70%, and more typically in the range of about 10% to 30%. In an exemplary embodiment, a dosage form is provided for intravenous administration in an aqueous solution having a concentration of between 0.5% and 0.001%, such as between 0.12% and 0.0105%, for example about 0.01% (W/V). In an exemplary embodiment, a dosage form is provided for intravenous administration in an aqueous solution having a concentration of between 2.5% and 0.25%, such as between 2% and 0.5%, for example about 1% (W/V). In an exemplary embodiment, the dosage form provides intravenous administration of up to 500mL of a 1% solution (W/V), resulting in a dose of up to 5 grams.

In exemplary embodiments, the cyclodextrin dimer may be administered to a patient in an amount of between 1mg and 10g, such as between 10mg and 1g, between 100mg and 500 mg. In an exemplary embodiment, about 400mg of cyclodextrin dimer may be administered. In exemplary embodiments, between 1g and 10g of cyclodextrin dimer, such as about 2g, about 3g, about 4g, or about 5g, may be administered. In exemplary embodiments, between 50mg and 5g of cyclodextrin dimer, such as between 100mg and 2.5g, between 100mg and 2g, between 250mg and 2.5g, for example about 1g, may be administered.

Exemplary embodiments provide a single dosage form that may comprise the aforementioned amounts of cyclodextrin dimers, which may be packaged for separate administration, optionally in addition to a pharmaceutically acceptable carrier or excipient. The total amount of the cyclodextrin dimer in the single dosage form may be provided as described above, for example between 1mg and 10g, such as between 10mg and 1g, between 100mg and 500mg, between 1g and 10g of cyclodextrin dimer, between about 50mg and 5g, between 100mg and 2.5g, between 100mg and 2g, between 250mg and 2.5g, such as about 1g, 2g, about 3g, about 4g, or about 5 g.

Formulations of the invention suitable for oral administration may be in the form of capsules, cachets, pills, tablets, lozenges (using flavoring agents, typically sucrose and acacia or tragacanth), powders, granules, or as solutions or suspensions in aqueous or non-aqueous liquids, or as oil-in-water or water-in-oil liquid emulsions, or as elixirs or syrups, or as pastilles (using inert bases such as gelatin and glycerin, or sucrose and acacia) and/or as mouthwashes and the like, each form containing a predetermined amount of a compound of the invention as the active ingredient. The active compounds can also be administered in the form of a bolus, electuary or paste.

Methods of making these formulations or compositions generally include the step of admixing a compound of the present invention with a carrier and, optionally, one or more adjuvants. For solid dosage forms (e.g., capsules, tablets, pills, powders, granules, buccal tablets, etc.), the active compound may be admixed with a finely divided solid carrier and usually formed, such as by pelleting, tabletting, granulating, pulverizing or coating. Typically, the solid carrier may comprise, for example, sodium citrate or dicalcium phosphate, and/or any of the following: (1) fillers or extenders such as starch, lactose, sucrose, glucose, mannitol, and/or silicic acid; f2) binding agents, for example carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and/or acacia; (3) humectants, such as glycerol; (4) disintegrating agents such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) slow solvents, such as paraffin; (6) absorption enhancers such as quaternary ammonium compounds and surfactants such as poloxamers and sodium lauryl sulfate; (7) wetting agents such as, for example, cetyl alcohol, glycerol monostearate and nonionic surfactants; (8) absorbents such as kaolin and bentonite clay; (9) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, zinc stearate, sodium stearate, stearic acid and mixtures thereof; (10) a colorant; and (11) controlled release agents such as crospovidone or ethylcellulose. In the case of capsules, tablets and pills, the pharmaceutical compositions may also comprise buffering agents. Solid compositions of a similar type may also be employed as fillers in soft and hard shell gelatin capsules using excipients such as lactose or milk sugar, as well as high molecular weight polyethylene glycols and the like.

Tablets may be prepared by compression or moulding, optionally with one or more accessory ingredients. Compressed tablets may be prepared using binders (e.g., gelatin or hydroxypropylmethyl cellulose), lubricants, inert diluents, preservatives, disintegrating agents (e.g., sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), surface active agents or dispersing agents.

Tablets and other solid dosage forms of the active agents, such as capsules, pills, and granules, may optionally be scored or prepared using coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical formulating art. The dosage form may also be formulated to provide sustained or controlled release of the active ingredient therein using, for example, hydroxypropylmethylcellulose in varying proportions to provide the desired release profile, other polymer matrices, liposomes and/or microspheres. Alternatively, the dosage form may be formulated for rapid release, e.g., lyophilized.

Generally, the dosage form must be sterile. To this end, the dosage form may be sterilized, for example, by filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which may be dissolved in sterile water or some other sterile injection medium prior to use. The pharmaceutical composition may also comprise a sunscreen and may be a composition that releases one or more active ingredients, optionally in a delayed manner, only or preferably in certain parts of the gastrointestinal tract. Examples of embedding compositions that may be used include polymeric substances and waxes. The active compound may also, if appropriate, be in microencapsulated form with one or more of the above-mentioned excipients.

Liquid dosage forms are typically pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups or elixirs of the active agent. In addition to the active ingredient, the liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1, 3-butylene glycol, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.

Dosage forms particularly for topical or transdermal administration may be in the form of, for example, powders, sprays, ointments, pastes, creams, lotions, gels, solutions or patches. Ophthalmic formulations, such as ophthalmic ointments, powders, solutions, and the like, are also contemplated herein. The active compound may be mixed under sterile conditions with a pharmaceutically acceptable carrier, and with any preservatives, buffers, or propellants which may be necessary. In addition to the active compounds of the present invention, the topical or transdermal dosage forms may also contain one or more excipients, such as those selected from the group consisting of: animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide and mixtures thereof. Sprays can also contain conventional propellants such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons (such as butane or propane).

For the purposes of the present invention, transdermal patches may provide the advantage of allowing controlled delivery of the compounds of the present invention into the body. Such dosage forms may be prepared by dissolving or dispersing the compound in a suitable medium. Absorption enhancers may also be included to increase the flux of the compound across the skin. The rate of such flux can be controlled by providing a rate controlling membrane or dispersing the compound in a polymer matrix or gel.

Pharmaceutical compositions of the invention suitable for parenteral administration typically comprise one or more compounds of the invention in combination with one or more of the following: pharmaceutically acceptable sterile isotonic aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions prior to use, may contain sugars, alcohols, antioxidants, buffers, bacteriostats or solutes which render the formulation isotonic with the blood of the intended recipient.

In some cases, to prolong the effect of a drug, it may be desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This can be achieved by using a liquid suspension of crystalline or amorphous material which is poorly water soluble. The rate of absorption of the drug then depends on its dissolution rate, which in turn may depend on crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered drug form is achieved by dissolving or suspending the drug in an oil vehicle.

Injectable depot forms can be prepared by forming a microencapsulated matrix of the active compound in a biodegradable polymer, such as polylactide-polyglycolide. Depending on the ratio of drug to polymer and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly (orthoesters) and poly (anhydrides). Depot injectable formulations can also be prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissues.

The pharmaceutical composition may also be in the form of a microemulsion. In the form of a microemulsion, the bioavailability of the active agent may be improved. See (Dorunoo et al, Drug Development and Industrial Pharmacy, 17 (12): 1685-.

The pharmaceutical compositions may also contain micelles formed by the compounds of the invention, wherein the micelles have an average diameter of less than about 100nm, and at least one amphiphilic carrier. In some embodiments, the micelles have an average diameter of less than about 50nm, or an average diameter of less than about 30nm, or an average diameter of less than about 20 nm.

Although any suitable amphiphilic vehicle is contemplated herein, an amphiphilic vehicle is generally a vehicle that has been granted Generally Recognized As Safe (GRAS) status and that can both solubilize the compounds of the present invention and microemulsify the solution at a later time when it contacts a complex aqueous phase, such as that present in living biological tissues. Generally, amphiphilic components meeting these requirements have an HLB (hydrophilic to lipophilic balance) value of 2-20 and their structure contains straight chain aliphatic groups in the range of C-6 to C-20. Some examples of amphiphiles include pegylated fatty glycerides and polyethylene glycols.

Particularly preferred amphiphilic carriers are saturated and monounsaturated pegylated fatty acid glycerides, such as those obtained from various fully or partially hydrogenated vegetable oils. Such oils may advantageously consist of fatty acid triglycerides, fatty acid diglycerides and fatty acid monoglycerides of the corresponding fatty acids, with particular preference being given toThe fatty acid composition of (1) comprises 4-10% of capric acid, 3-9% of capric acid, 40-50% of lauric acid, 14-24% of myristic acid, 4-14% of palmitic acid and 5-15% of stearic acid. Another useful class of amphiphilic carriers includes partially esterified sorbitan and/or sorbitol, as well as saturated or monounsaturated fatty acids (SPAN series) or corresponding ethoxylated analogs (TWEEN series). Commercially available amphiphilic carriers are specifically contemplated, including A series of,OrPEG-monooleate, PEG-dioleate, PEG-monolaurate and dilaurate, lecithin, polysorbate 80.

CD (such as HP β CD or another CD of the present disclosure) dimer may be administered by any suitable means. Preferred routes of administration include parenteral (e.g., subcutaneous, intramuscular or intravenous), topical, transdermal, oral, sublingual or buccal administration. The administration may be ocular (e.g., in the form of eye drops), intravitreal, retroorbital, subretinal, subscleral administration, which may be preferred in the case of ocular disorders such as AMD.

The CD (such as HP β CD or another CD of the present disclosure) dimer may be administered to a subject, or may be used in vitro, for example, to cells or tissues that have been removed from an animal. The cells or tissue may then be introduced into the subject, whether the subject from which the cells or tissue were removed or another individual (preferably of the same species).

The subject (i.e., patient) to be treated is typically an animal, typically a mammal, preferably a human. The subject can be a non-human animal including all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dogs, cats, cows, horses, chickens, amphibians, and reptiles. In some embodiments, the subject is a livestock animal, such as cattle, swine, sheep, poultry, and horses, or a companion animal, such as dogs and cats. The subject may be genetically male or female. The subject can be of any age, such as elderly (typically, at least or greater than 60, 70, or 80 years of age), transition age subjects from elderly to adult, transition age subjects from adult to pre-adult, and pre-adult subjects, including adolescents (e.g., 13 years of age to 16, 17, 18, or 19 years of age maximum), children (typically, under 13 years of age or before the onset of puberty), and infants. The subject may also be a human of any race or genotype. Some examples of human ethnicities include caucasians, asians, hispanic, african americans, american native residents, emmetrettes, and pacific islands. The methods of the invention may be more suitable for certain ethnic groups, such as caucasian, especially the northern european population and asian population.

The present disclosure includes further substitutions of the dimeric CDs described herein (such as HP β CD or another CD of the present disclosure). The chemical modification may be performed before or after dimerization. Chemical modification of cyclodextrins can be performed directly on native β -cyclodextrin rings by reacting chemical reagents (nucleophiles or electrophiles) with appropriately functionalized cyclodextrins (Adair-Kirk [ et al ], nat. med., 14 (10): 1024-5, (2008)); (Khan, [ et al ], chem.Rev., 98 (5): 1977-. To date, more than 1,500 cyclodextrin derivatives have been prepared by chemical modification of natural cyclodextrins. Cyclodextrins can also be prepared by de novo synthesis starting from glucopyranose-linked oligopyranosides. This synthesis can be accomplished by using various chemical reagents or biological enzymes such as cyclodextrin transglycosylase. A summary of chemically modified cyclodextrins as drug carriers in drug delivery systems is described, for example, (Stella, [ et al ], toxicol. Electrically neutral cyclodextrins are described in U.S. patent nos. 3,453,259 and 3,459,731, the disclosures of which are incorporated herein by reference in their entirety. Other derivatives include cyclodextrins with cationic properties, as disclosed in U.S. patent No. 3,453,257; insoluble cross-linked cyclodextrins, as disclosed in U.S. patent No. 3,420,788; and cyclodextrins with anionic properties, as disclosed in U.S. patent No. 3,426,011, the disclosures of which are hereby incorporated by reference in their entirety. In cyclodextrin derivatives having anionic properties, carboxylic acids, phosphorous acids, phosphinic acids, phosphonic acids, phosphoric acids, thiophosphonic acids, thiosulfinic acids and sulfonic acids have been attached to the parent cyclodextrin as disclosed, for example, in U.S. patent No. 3,426,011. Sulfoalkyl ether cyclodextrin derivatives are also described, for example, in U.S. patent No. 5,134,127, the disclosure of which is hereby incorporated by reference in its entirety. In some embodiments, the cyclic oligosaccharide may have two or more monosaccharide units replaced with a triazole ring that may be synthesized by an azide-alkyne wheaten (Huisgen) cycloaddition reaction ((Bodine, [ et al ], j.am. chem. soc., 126 (6): 1638-9, (2004)).

The dimeric cyclodextrins of the present disclosure are linked by a linker. Methods that can be used to attach the CD subunit to the linker are described in the working examples. Additional methods of attaching CD subunits to linkers are known in the art. (Georgeta [ et al ], J.Bioact.Combat. pol., 16: 39-48. (2001)), (Liu [ et al ], Acc.chem.Res., 39: 681- & 691.(2006)), (Ozmen [ et al ], J.mol.Catal.B-enzym., 57: 109- & 114. (2009)), (Trotta [ et al ], composite.interface, 16: 39-48.(2009)), each of these documents being hereby incorporated by reference in its entirety. For example, a linker group containing a moiety that reacts with a hydroxyl group (e.g., a carboxyl group, which may be activated by a carbodiimide) may react with a cyclodextrin to form a covalent bond thereon. In another example, one or more hydroxyl groups of the cyclodextrin can be activated by known methods (e.g., tosylation) to react with a reactive group (e.g., an amino group) on the linker.

Typically, the linker initially contains two reactive moieties that react and bond with each CD monomer. In one embodiment, the linker is first attached to the cyclodextrin to produce an isolated linker-cyclodextrin compound, and then the remaining reactive portion of the linker in the linker-cyclodextrin compound is subsequently reacted with a second cyclodextrin. The second reactive moiety of the linker may be protected during the reaction of the first reactive group, although protection may not be employed where the first and second reactive moieties of the linker react differently with the two molecules. The linker can react with both molecules simultaneously to link them together. In other embodiments, the linker may have additional reactive groups to attach to other molecules.

Many linkers are known in the art. When the group has or has been functionalized with a group that can react with and attach to a reactive linker, such linkers can be used to attach any of a number of groups together. Some groups capable of reacting with a di-reactive linker include amino, thiol, hydroxyl, carboxyl, ester, and haloalkyl groups. For example, when each group to be attached has at least one amino group, an amino-amino coupling reagent may be employed to attach the cyclic oligosaccharide to the polysaccharide (or, for example, any of these groups are attached to a fluorophore or to each other). Some examples of amino-amino coupling agents include diisocyanates, alkyl dihalides, dialdehydes, disuccinimidyl suberate (DSS), disuccinimidyl tartrate (DST), and disuccinimidyl tartrate (sulfo-DST), all of which are commercially available. In other embodiments, an amino-thiol coupling agent may be used to link a thiol group of one molecule to an amino group of another molecule. Some examples of amino-thiol coupling agents include 4- (N-maleimidomethyl) -cyclohexane-1-carboxylic acid succinimidyl ester (SMCC) and 4- (N-maleimidomethyl) -cyclohexane-1-carboxylic acid sulfosuccinimidyl ester (sulfo-SMCC). In still other embodiments, a thiol-thiol coupling agent may be employed to attach groups having at least one thiol group.

In some embodiments, the linker is as small as a single atom in length (e.g., - -O- -, - -CH2- -, or- -NH- -linkage) or two or three atoms in length (e.g., amine, urea, urethane, ester, carbonate, sulfone, ethylene, or trimethylene linkage). In other embodiments, the linker provides greater freedom of movement through a length of at least four, five, six, seven, or eight atoms, and up to, for example, a length of 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, or 30 atoms. Preferred linker lengths are between 2 and 12 atoms, or between 4 and 8 atoms. In an exemplary embodiment, the linker is a C4 alkyl group, which may be unsubstituted. In exemplary embodiments, the linker comprises a triazole.

Atherosclerosis of arteries

The exemplary cyclodextrin dimers described herein are useful in the prevention or treatment of disease (such as atherosclerosis). The combination of cyclodextrin dimer and one or more active agents, such as those described herein (e.g., hypolipidemic agents such as statins), may be used to treat any atherosclerosis and signs, symptoms, or complications of atherosclerosis. Atherosclerosis (also known as arteriosclerotic vascular disease or ASVD, also known as coronary artery disease or CAD) is a condition in which the walls of the arteries thicken due to the accumulation of fatty substances, such as cholesterol. Atherosclerosis is a chronic disease that can remain asymptomatic for decades. It is a syndrome affecting arterial blood vessels, a chronic inflammatory reaction of the arterial wall, believed to be caused mainly by the accumulation of macrophage leukocytes, and promoted by low density lipoproteins (plasma proteins carrying cholesterol and triglycerides) in the case where functional High Density Lipoproteins (HDL) are unable to properly remove fat and cholesterol from macrophages. It is commonly referred to as arteriosclerosis or obstruction. It is caused by the formation of multiple plaques within the artery.

The pathobiology of atherosclerotic lesions is complex, but in general, stable atherosclerotic plaques tend to be asymptomatic, rich in extracellular matrix and smooth muscle cells, while unstable plaques are rich in macrophages and foam cells, and the extracellular matrix separating the lesion from the arterial lumen (also known as the fibrous cap) is generally weak and easily ruptured. Rupture of the fibrous cap exposes thrombogenic materials (such as collagen) to the circulation and ultimately causes thrombosis in the lumen. After formation, intraluminal thrombi can completely occlude arteries (e.g., coronary occlusion), but more of the time they break away, enter the circulation and can eventually occlude smaller downstream vessel branches, resulting in thromboembolism (e.g., stroke is often caused by thrombosis in the carotid artery). In addition to thromboembolism, chronic dilated atherosclerotic lesions may result in complete closure of the lumen. Chronic dilated lesions are usually asymptomatic until the luminal stenosis is so severe that one or more downstream tissues are under supplied with blood that ischemia results.

These complications of advanced atherosclerosis are chronic, slowly progressive and cumulative. In some cases, soft plaque suddenly ruptures, resulting in the formation of a thrombus that rapidly slows or stops blood flow, resulting in the death (infarction) of the tissue supplied by the artery. Coronary thrombosis of the coronary arteries is also a common complication that can lead to myocardial infarction. Occlusion of the brain by an artery may lead to a stroke. In advanced atherosclerotic disease, claudication may occur due to insufficient blood supply to the legs, often caused by a combination of narrowing and narrowing of the aneurysmal segment by clots.

Atherosclerosis can affect the entire arterial tree, but the risk of larger high pressure vessels (such as coronary arteries, renal arteries, femoral arteries, cerebral arteries, and carotid arteries) is generally higher.

Signs, symptoms, and complications of atherosclerosis include, but are not limited to, an increase in plasma total cholesterol, VLDL-C, LDL-C, free cholesterol, cholesterol esters, triglycerides, phospholipids, and the presence of lesions (e.g., plaques) in the arteries, as discussed above. In some cases, an increase in cholesterol (e.g., total cholesterol, free cholesterol, and cholesterol esters) may be seen in one or more of plasma, aortic tissue, and aortic plaque.

Some individuals may be predisposed to atherosclerosis. Accordingly, the present disclosure relates to methods of administering cyclodextrin dimers alone or in combination with one or more additional therapeutic agents (e.g., hypolipidemic agents, such as statins) to a subject to prevent atherosclerosis or their signs, symptoms, or complications. In some embodiments, a subject predisposed to atherosclerosis may exhibit one or more of the following characteristics: age, family history of heart disease, biological disorders, high blood cholesterol. In some embodiments, the biological condition comprises a high level of low density lipoprotein cholesterol in the blood (LDL-C), a low level of high density lipoprotein cholesterol in the blood (HDL-C), hypertension, insulin resistance, diabetes, overweight, obesity, sleep apnea, one or more contributing lifestyle choices, and/or one or more contributing habitual behaviors. In some embodiments, the behavioral habit includes smoking and/or drinking. In some embodiments, the lifestyle selection includes an inactive lifestyle and/or a high stress level.

Exemplary embodiments provide for the administration of cyclodextrin dimers of the present disclosure to a patient having atherosclerosis, optionally in combination with one or more additional agents. The patient may exhibit one or more signs or symptoms of atherosclerosis. Atherosclerosis may be diagnosed from one or more of doppler ultrasound, ankle brachial index, electrocardiogram, stress test, angiogram (optionally with cardiac catheterization), Computed Tomography (CT), Magnetic Resonance Angiography (MRA), or other methods of imaging arteries or measuring blood flow.

Exemplary embodiments provide for administering a therapy comprising a cyclodextrin dimer of the present disclosure in combination with one or more additional therapies. These combination therapies for treating atherosclerosis may include a cyclodextrin dimer of the present disclosure administered in combination with another therapy for treating or preventing atherosclerosis, such as an anti-cholesterol drug, an anti-hypertensive drug, an anti-platelet drug, a dietary supplement, or a surgical or behavioral intervention, including but not limited to those described below. Additional combination therapies include the CD dimers of the present disclosure and another therapy for treating heart failure, such as one or more aldosterone antagonists, ACE inhibitors, ARBs (angiotensin II receptor blockers), ARNI (angiotensin receptor-enkephalinase inhibitors), beta-blockers, vasodilators, calcium channel blockers, digoxin, diuretics, cardiac pump drugs, potassium, magnesium, selective sinus node inhibitors, or combinations thereof. A combination therapy for treating dry age-related macular degeneration (AMD) or stargardt disease includes the CD dimers of the present disclosure and another therapy for treating AMD, such as LBS-008(Belite Bio) (a non-retinoic acid antagonist of retinol binding protein 4), AREDS supplement formulations (including vitamins C and E, beta-carotene ト, zinc and copper), AREDS2 supplement formulations (including supplement formulations with vitamins C and E, zinc, copper, lutein, zeaxanthin, and omega-3 fatty acids, or combinations thereof). Combination therapies for the treatment of alzheimer's disease include the CD dimers of the present disclosure and one or more cholinesterase inhibitors (aricept (r), exelon (r), razadyne (r)) and memantine (namenda (r)) or combinations thereof. Combination therapy for niemann-pick disease includes CD dimer and one or more of meglumine (zavesca (r)), HP β CD (trap sol cyclic, VTS-270), and physical therapy of the present disclosure. The combination therapy may be administered simultaneously, substantially simultaneously, or sequentially in any order. The combination therapy may be co-administered in a single formulation, or separately, optionally in combination, in a dosage kit or package containing each drug, e.g., in a convenient pre-measured form in which one or more single doses of each drug are provided in the combination. The combination therapy may exhibit a synergistic effect, wherein the effect of the combination therapy exceeds the effect of the individual treatments alone. Although combination therapy typically includes administering an effective amount of CD dimer and combination therapy, combination therapy may allow for effective treatment at lower doses of CD and/or combination therapy, which may advantageously reduce side effects associated with conventional (non-combination) doses.

Combination therapy may include therapies for treating or preventing diseases or disorders associated with atherosclerosis (such as coronary artery disease, angina pectoris, heart attack, cerebrovascular disease, transient ischemic attack, and/or peripheral artery disease). Combination therapy may include therapies for treating or preventing conditions that may lead to atherogenesis and/or a poor prognosis, such as hypertension, hypercholesterolemia, hyperglycemia, and diabetes.

In an exemplary embodiment, the cyclodextrin dimers of the invention are co-administered with an anti-cholesterol drug, such as a fibrate or a statin, for example, advicor (r) (niacin sustained release/lovastatin), altoprev (r) (lovastatin sustained release), caduet (r) (amlodipine and atorvastatin), crestor (r) (rosuvastatin), juusinc (r) (sitagliptin/simvastatin), LESCOL (r) (fluvastatin), LESCOL XL (fluvastatin sustained release), lipitor (r) (atorvastatin), livalo (r) (pitavastatin), mevacor (r) (lovastatin), pravachol (r) (pravastatin), simcor (r) (niacin sustained release/simvastatin), vytorin (r) (ezetimibe/simvastatin), and/or simvastatin zor (r). The anti-cholesterol agent may be administered in an amount effective to prevent or treat hypercholesterolemia.

In exemplary embodiments, the cyclodextrin dimers of the invention are co-administered with an antiplatelet agent (e.g., aspirin).

In exemplary embodiments, the cyclodextrin dimers of the invention are co-administered with an antihypertensive drug. Exemplary antihypertensive drugs include beta blockers, Angiotensin Converting Enzyme (ACE) inhibitors, calcium channel blockers, and/or diuretics.

In exemplary embodiments, the cyclodextrin dimers of the invention are co-administered with dietary supplements such as one or more of the following: alpha-linolenic acid (ALA), barley, beta-sitosterol, black tea, psyllium, calcium, cocoa, cod liver oil, coenzyme Q10, fish oil, folic acid, garlic, green tea, niacin, oat bran, omega-3 fatty acids such as eicosapentaenoic acid (EPA) and/or docosahexaenoic acid (DHA), sitostanol and/or vitamin C.

Exemplary combination therapies also include patient behavioral and/or lifestyle interventions including counseling and/or supporting smoking cessation, exercise, and healthy diets such as low-density lipoprotein (LDL) low and optionally high-density lipoprotein (HDL) high diets.

Exemplary combination therapies also include surgical interventions such as angioplasty, stenting, or both.

The methods of the invention are useful for treating or preventing atherosclerosis in a human subject. In some cases, in addition to exhibiting atherosclerosis, the patient is healthy. For example, the patient may not exhibit any other risk factors for cardiovascular, thrombosis, or other diseases or conditions at the time of treatment. In other cases, however, the patient is selected based on being diagnosed with or at risk of developing a disease or condition caused by or associated with atherosclerosis. For example, at or prior to administration of the pharmaceutical composition of the invention, the patient may be diagnosed or identified as at risk of developing a cardiovascular disease or condition, such as coronary artery disease, acute myocardial infarction, asymptomatic carotid atherosclerosis, stroke, peripheral arterial occlusive disease, and the like. In some cases, the cardiovascular disease or condition is hypercholesterolemia.

In other cases, a patient may be diagnosed or identified as at risk of developing atherosclerosis at the time of or prior to administration of the pharmaceutical composition of the invention.

In still other instances, the patient to be treated using the methods of the invention is selected based on one or more factors selected from age (e.g., age greater than 40, 45, 50, 55, 60, 65, 70, 75, or 80 years), race, gender (male or female), exercise habits (e.g., regular, non-exercise), other past medical conditions (e.g., type II diabetes, hypertension, etc.), and current medication status (e.g., currently taking statins, such as cerivastatin, atorvastatin, simvastatin, pitavastatin, rosuvastatin, fluvastatin, lovastatin, pravastatin, etc., a beta blocker, niacin, etc.).

Drawings

In the following figures, the following abbreviations are used: me or ME or Me or met: a methyl group; SB: a sulfobutyl group; QA ═ quaternary ammonium, e.g., -CH₂CH(OH)CH₂N(CH₃)₃ ⁺Such as-CH₂CH(OH)CH₂N(CH₃)₃Cl; SUCC: a succinyl group; DMSO, DMSO: dimethyl sulfoxide (DMSO).

Figure 1a. structure of Cyclodextrin (CD), a cyclic oligosaccharide polymer consisting of 6 (aCD), 7 (β CD) or 8 (γ CD) saccharide rings (from left to right). All sugar rings in all CDs are D-glucose molecules.

FIGS. 1B-1J structures of substituted CDs.

FIG. 1B, wherein R¹、R²And R³Is a substituent group, and is a substituent group,

FIG. 1℃ beta. CD (DS0), i.e., each R¹、R²And R³Is a hydrogen atom, and is,

FIG. 1D. hydroxypropyl CD (DS4),

FIG. 1E methyl β CD (DS6),

FIG. 1F sulfobutyl BCD (DS4),

FIG. 1G. quaternary ammonium (DS 3),

FIG. 1H succinyl (DS 1),

FIG. 1I carboxymethyl (DS4), and

FIG. 1J. the maltosyl (DS 1) group is substituted at the C2, C3, or C6 position of BCD.

Figure 2a. solubilization of various cholesterol derivatives by HP β CD (DS 4.5) monomer assessed by relative turbidity, where 100 is defined as the absorbance of an aqueous suspension containing 300uM sterol tested in PBS. FIG. 2A shows the results for cholesterol (diamonds), 7KC (squares), vitamin D2 (triangles), vitamin D3(X) and desmosterol (+). In this and subsequent figures, the data points are connected by a smooth curve to aid in viewing the results.

Figure 2b. solubilization of various sterols by hydroxypropyl- β -cyclodextrin (DS 4.5) monomer evaluated by relative turbidity, where 100 is defined as the absorbance of an aqueous suspension containing 300uM sterol tested in PBS. FIG. 2B depicts the results for 7-ketocholesterol (7KC (with straight X)), 4- β -hydroxycholesterol (4-BOH (squares)), 25-hydroxycholesterol (25OH (triangles)), cholesterol epoxide (diamonds), and 27-hydroxycholesterol (27OH (circles)).

Figure 2c solubilization of 7KC by various forms of hydroxypropyl- β -cyclodextrin monomer assessed by relative turbidity. DS is the average number of hydroxypropyl substitutions per molecule.

Figure 2d solubilization of cholesterol by various forms of hydroxypropyl- β -cyclodextrin monomer assessed by relative turbidity. DS is the average number of hydroxypropyl substitutions per molecule.

Figure 2e predicted relative affinities of HP β CD molecules calculated by molecular docking. DS represents the number of hydroxypropyl substitutions per molecule.

Figure 2f solubilization of cholesterol in vitro by Me β CD of various degrees of substitution evaluated by relative turbidity.

Fig. 2g. solubilization of 7KC in vitro by Me β CD of various degrees of substitution evaluated by relative turbidity.

Figure 2h solubilization of cholesterol in vitro by various monomeric β CDs assessed by relative turbidity.

Figure 2i. solubilization of 7KC in vitro by various monomeric β CDs assessed by relative turbidity.

Figure 3a. structure of HP β CD dimers of the disclosure. The β -cyclodextrin monomers are linked via a large (sub-) surface, i.e., a linker is attached to the C2 or C3 carbon of each CD subunit. The HP substitution is attached to the C2, C3, and/or C6 carbons (typically in combination).

Figure 3b, formula i, C2-C2 cyclodextrin dimer with triazole linker.

Figure 3C, formula ii, C2-C3 cyclodextrin dimer with triazole linker.

Figure 3d, formula iii, C3-C3 cyclodextrin dimer with triazole linker.

Figure 3e formula iv sub-surface attached methyl substituted BCD with linker L.

Figure 3f formula v subsurface-linked sulfobutyl-substituted BCD with linker L. Sodium salts are depicted in the figure, but other salts are also included within the compounds of the present disclosure.

Figure 3g, formula vi, subsurface-attached succinyl-substituted BCD with linker L.

Figure 3h formula vii a subsurface-linked maltosyl-substituted BCD with a linker L.

Figure 3i formula viii subsurface-linked quaternary ammonium-substituted BCD with linker L.

Figure 3j. formula IX. has a subsurface-linked carboxymethyl substituted BCD of linker L. Sodium salts are depicted in the figure, but other salts are also included within the compounds of the present disclosure.

Figure 4a structural model of HP β CD monomer association with sterol (top panel) or HP β CD butyl linked dimer association with sterol (bottom panel). This is shown as a graphical representation of the monomer-sterol and dimer-sterol guest interactions.

Figure 4b predicted relative affinities of butyl and triazole linked dimers for cholesterol and 7 KC. Docking calculations were performed for various degrees of hydroxypropylation of the linked HP β CD dimers.

Fig. 4c. description of the measured values used for the molecular dynamics simulation. The nomenclature of cyclodextrins and sterols is included in the figure to define the O4 atom (marked with an arrow) of CD, the minor and major surfaces of CD, and the head and tail groups of sterols. The angle between the O4 plane and the ligand indicates the degree of nesting of the ligand within the CD cavity. 30 degrees corresponds to the solubilizing "up" configuration (head of sterol associated with minor surface of CD and tail associated with major surface) and 150 degrees corresponds to the solubilizing "down" configuration (tail of sterol associated with minor surface of CD and head associated with major surface).

Fig. 4 d.md simulation of ds0 β CD: distance between the centroid of all O4 oxygens and the centroid of the ligands in the GROMOS force field in the up and down ligand orientations for native (i.e. unsubstituted) monomeric β CD (upper panel); the angle between the vector perpendicular to the plane formed by the O4 atoms of the CD and the major axis of the ligand (middle panel); Lonana-Jones (Lennard-Jones) and coulomb energy of the interaction between cyclodextrin and ligand (lower panel). In the figures included between 4D and 4LL, the light lines indicate cholesterol results and the dark lines indicate 7KC results.

Figure 4e solubilization of ligand by native DS0 monomer β CD in GROMOS force field.

Fig. 4f visual traces of 7KC and cholesterol complexed with native DS0 β CD (GROMOS force field) in two orientations.

Figure 4g. distance between the centroids of all O4 oxygens and ligands in AMBER force field, in up and down ligand orientations, for the natural monomer DS0 β cyclodextrin; the angle between the vector perpendicular to the plane formed by the O4 atoms of the CD and the major axis of the ligand; the Lanna-Jones and the Coulomb energy of the interaction between the cyclodextrin and the ligand.

Figure 4h solubilization of ligand by native DS0 monomer β CD in AMBER force field.

Figure 4i visual traces of 7KC and cholesterol complexed with native DS0 β CD (AMBER force field) in two orientations. Abbreviations used: "ms": in microseconds.

Figure 4j. distance between the centroid of all O4 oxygens and the centroid of the ligands in the GROMOS force field in up and down ligand orientations for the translated native monomeric beta cyclodextrin (DS 0); the angle between the vector perpendicular to the plane formed by the O4 atoms of the CD and the major axis of the ligand; the Lanna-Jones and the Coulomb energy of the interaction between the cyclodextrin and the ligand.

Figure 4k solubilization of ligands by translated monomer β CD in GROMOS force field.

Fig. 4l visual traces of 7KC and cholesterol complexed with translated native (DS0) β CD in GROMOS force field.

Figure 4m. distance between the centroids of all O4 oxygens and ligands in AMBER force field, in upward and downward ligand orientation, for the translocated native monomeric beta cyclodextrin (DS 0); the angle between the vector perpendicular to the plane formed by the O4 atoms of the CD and the major axis of the ligand; the Lanna-Jones and the Coulomb energy of the interaction between the cyclodextrin and the ligand.

Figure 4n. solubilization of ligands by translated monomer β CD in AMBER force field.

Figure 4o. visual traces of 7KC and cholesterol complexed with native DS0 β CD (AMBER force field) in two orientations.

Figure 4p. distance between the centroid of all O4 oxygens and the centroid of the ligands in the GROMOS force field in up and down ligand orientations for native DS0 monomeric beta cyclodextrin; the angle between the vector perpendicular to the plane formed by the O4 atoms of the CD and the major axis of the ligand; the Lanna-Jones and the Coulomb energy of the interaction between the cyclodextrin and the ligand.

Figure 4q solubilization of ligands by native monomer β CD in GROMOS force field.

Figure 4r. visual traces of 7KC and cholesterol complexed with native monomeric β CD (GROMOS force field) in two orientations.

Figure 4s. distance between the centroid of all O4 oxygen and the centroid of the ligand in both the up and down ligand orientations in the AMBER force field; the angle between the vector perpendicular to the plane formed by the O4 atoms of the CD and the major axis of the ligand; the Lanner-Jones and Coulomb energies of the interaction between HP β CD DS5 and cholesterol or 7 KC.

Figure 4t. solubilization of ligand by HP β CD DS5 in AMBER force field.

Figure 4u. visual traces of 7KC and cholesterol complexed with HP β CD DS5(AMBER force field) in two orientations.

Figure 4v. distance between the centroid of all O4 oxygen and the centroid of the ligand in the case of translation, in the up and down ligand orientations, in the GROMOS force field; the angle between the vector perpendicular to the plane formed by the O4 atoms of the CD and the major axis of the ligand; the Lanner-Jones and Coulomb energies of the interaction between HP β CD DS5 and cholesterol or 7 KC.

Figure 4w. solubilization of ligands by translated monomeric HP β CD in GROMOS force field.

Figure 4x. visual traces of 7KC and cholesterol complexed with a translated monomeric HP β CD DS5(GROMOS force field) in two orientations.

Figure 4y. the distance between the centroid of all O4 oxygen and the centroid of the ligand in the up and down ligand orientations, in the case of translation, in the AMBER force field; the angle between the vector perpendicular to the plane formed by the O4 atoms of the CD and the major axis of the ligand; the Lanner-Jones and Coulomb energies of the interaction between HP β CD DS5 and cholesterol or 7 KC.

Figure 4z. solubilization of ligand by translated monomer HP β CD DS5 in AMBER force field.

Figure 4aa visual traces of 7KC and cholesterol complexed with the translated monomer DS5 HP β CD (AMBER force field) in two orientations.

FIG. 4BB. distances between the centroid of all O4 oxygens and the centroid of ligands in the GROMOS force field, in up and down ligand orientations; the angle between the vector perpendicular to the plane formed by the O4 atoms of the CD and the major axis of the ligand; Lanner-Jones and Coulomb energies of the interaction between butyl dimerized HP β CD DS5 and cholesterol or 7 KC.

Fig. 4cc solubilization of 7KC and cholesterol by butyl dimerized HP β CD DS5 in GROMOS force field.

Fig. 4DD. visual traces of 7KC and cholesterol complexed with butyl dimerized DS5 HP β CD (GROMOS force field) in two orientations.

Figure 4ee distance between the centroid of all O4 oxygens and the centroid of the ligand in the AMBER force field, in both up and down ligand orientations, for butyl dimerized HP β CD DS 5; the angle between the vector perpendicular to the plane formed by the O4 atoms of the CD and the major axis of the ligand; the Lanna-Jones and the Coulomb energy of the interaction between the cyclodextrin and the ligand.

FIG. 4FF. solubilization of ligand by butyl dimerized HP β CD DS5 in AMBER force field.

Figure 4gg visual traces of 7KC and cholesterol complexed with butyl dimerized HP β CD DS5(AMBER force field) in two orientations.

Figure 4HH. distances between the centroid of all O4 oxygens and the centroid of the ligands for dimerized DS5 hydroxypropyl β cyclodextrin in the GROMOS force field, in case of translation, in up and down ligand orientations; the angle between the vector perpendicular to the plane formed by the O4 atoms of the CD and the major axis of the ligand; the Lanna-Jones and the Coulomb energy of the interaction between the cyclodextrin and the ligand.

FIG. 4II solubilization of 7KC and cholesterol by shifted butyl dimerized DS5 HP β CD in a GROMOS force field.

Figure 4jj visual traces of 7KC and cholesterol complexed with translated butyl dimerized DS5 HP β CD (GROMOS force field) in two orientations.

Figure 4KK. distances between the centroid of all O4 oxygens and the centroid of the ligands for butyl dimerized DS5 hydroxypropyl β cyclodextrin in AMBER force field, in case of translation, in up and down ligand orientations; the angle between the vector perpendicular to the plane formed by the O4 atoms of the CD and the major axis of the ligand; the Lanna-Jones and the Coulomb energy of the interaction between the cyclodextrin and the ligand.

FIG. 4LL. solubilization of 7KC and cholesterol by shifted butyl dimerized DS5 HP β CD in AMBER force field.

Fig. 4MM. visual traces of 7KC and cholesterol complexed with shifted butyl dimerized DS5 HP β CD (AMBER force field) in two orientations.

Figure 4NN. distances between the centroids of all O4 oxygens and the centroids of ligands for unsubstituted (DS0) butyl dimerized beta cyclodextrin in GROMOS force field, in up and down ligand orientations; the angle between the vector perpendicular to the plane formed by the O4 atoms of the CD and the major axis of the ligand; the Lanna-Jones and the Coulomb energy of the interaction between the cyclodextrin and the ligand.

Fig. 4OO. visual traces of 7KC and cholesterol complexed with unsubstituted (DS0) butyl dimerized beta CD (AMBER force field) in two orientations.

FIG. 4 MD analysis of PP. triazole-linked DS0 cyclodextrins. The angle between the vector perpendicular to the plane formed by the O4 atoms of the CD and the major axis of the ligand in the GROMOS force field in the up and down ligand orientations for the unsubstituted (DS0) dimerized beta cyclodextrin; and the lanna-jones and coulomb energy of the interaction between the cyclodextrin and the ligand.

FIG. 4QQ. visual traces of 7KC and cholesterol complexed with triazole dimerized DS0 β CD in two orientations.

FIG. 4 MD analysis of RR. triazole-linked DS4 HP β CD. The angle between the vector perpendicular to the plane formed by the O4 atoms of the CD and the major axis of the ligand in the GROMOS force field in the upward and downward ligand orientations for the translated dimerized DS4 hydroxypropyl β cyclodextrin; and the lanna-jones and coulomb energy of the interaction between the cyclodextrin and the ligand.

FIG. 4SS. visual traces of a 100ns interaction between triazole-linked DS4 hydroxypropyl β CD dimer and 7 KC/cholesterol in two orientations.

Figure 5a. predicted relative affinities of a number of possible dimerized Me β CD molecules calculated by molecular docking. Butyl (left) and triazole (right) linked dimers affinity for sterols. Docking calculations were performed for linked Me β CD dimers of various degrees of methylation. Cholesterol (dotted line) and 7KC (solid line).

Md simulation describes 100ns interaction between butyl-linked DS4 methyl β CD dimer and 7 KC/cholesterol in both up and down orientations. Legend: 7KC (dark line) and cholesterol (light gray line), the dotted line indicates downward orientation, and the solid line indicates upward orientation.

Figure 5c visual traces of butyl-linked DS4 methyl β CD dimer and 7 KC/cholesterol in both up and down orientations.

Md simulation describes 100ns interaction between triazole-linked DS4 methyl β CD dimer and 7 KC/cholesterol in both up and down orientations. As shown, for example, in fig. 5B.

Figure 5e visual traces of triazole-linked DS4 methyl β CD dimer and 7 KC/cholesterol in both up and down orientations.

Figure 6a. predicted relative affinities of a number of possible dimerized sulfobutylated β CD molecules calculated by molecular docking. Affinity of butyl and triazole linked dimers for sterols. Docking calculations were performed for the linked SB β CD dimers for various degrees of sulfobutylation. Cholesterol (dotted line) and 7KC (solid line).

Figure 6b. md simulation describes 100ns interaction between butyl-linked DS4 sulfobutyl β CD dimer and 7 KC/cholesterol in both up and down orientations. As shown, for example, in fig. 5B.

Figure 6c visual traces of butyl-linked DS4 sulfobutyl β CD dimer and 7 KC/cholesterol in both up and down orientations.

Md simulation describes 100ns interaction between triazole-linked DS4 sulfobutyl β CD dimer and 7 KC/cholesterol in both up and down orientations. As shown, for example, in fig. 5B.

FIG. 6E visual traces of triazole-linked DS4 sulfobutyl β CD dimer and 7 KC/cholesterol in both up and down orientations.

Figure 7a. md simulation describes a 100ns interaction between the butyl-linked DS4 quaternary ammonium β CD dimer and 7 KC/cholesterol in both the up and down orientations. As shown, for example, in fig. 5B.

Figure 7b visual traces of butyl-linked DS4 quaternary ammonium β CD dimer and 7 KC/cholesterol in both up and down orientations.

Md simulation describes 100ns interaction between the triazole-linked DS4 quaternary ammonium β CD dimer and 7 KC/cholesterol in both up and down orientations. As shown, for example, in fig. 5B.

Figure 7d visual traces of triazole-linked DS4 quaternary ammonium β CD dimer and 7 KC/cholesterol in both up and down orientations. As shown, for example, in fig. 5B.

Figure 8a. different hydroxypropylation sites of ds8 and DS4 triazole and butyl linked dimers, including hydroxypropylation of only small or large surfaces. Docking calculations were performed on various hydroxypropylation sites in the HP β CD dimer to determine the effect of changing the position of the hydroxypropyl group on sterol binding. The site of hydroxypropylation is variable in practice due to the randomness of the substitution onto a roughly symmetrical molecule. The designations "C", "D" and "E" refer to different (one to the other) variant structures that have an equal distribution of HP groups between the small and large surfaces of the CD monomer. Legend: the upper (light grey) bars represent the cholesterol values and the lower (dark) bars represent the 7KC values.

Figure 8b different lengths of alkyl-linked HP β CD DS5 dimers. Docking calculations were performed for various degrees of hydroxypropylation and for various lengths of carbon-only linkers. The bars in each group are sequentially DS20, DS16, DS12, DS8, DS4 and DS0 from top to bottom.

Figure 8c different lengths of triazole-linked HP β CD DS5 dimer. The docking calculations were performed for triazole linkers of different lengths by varying the number of carbon atoms on either side of the triazole ring. The length of each side of the linker is distinguished by n1 or n2, cholesterol is represented by striped bars, and 7KC is a solid bar. The bars in each group are sequentially N1-2 and 7KC from top to bottom; n1 ═ 2 and cholesterol; n1 ═ 3 and 7 KC; n1 ═ 3 and cholesterol; n1 ═ 4 and 7 KC; n1 ═ 4 and cholesterol.

Figure 8d. linker was tested by docking calculation (figure 8E) to determine linker-dependent changes in sterol binding. In contrast to the four-carbon linker (linker W, where n ═ 3 carbons) and triazole linked dimers (linker U, where n ═ 1 carbons, and linker V, where n ═ 1 carbons), the linked HP β CD dimer combinations of hydroxypropyl DS4 and DS8 dimers were based on the addition of various side chains, rings, double bonds and/or substitution of sulfur, nitrogen and/or oxygen atoms of the linker combination.

Figure 8e docking results for various HP β CD dimers with different linkers. Preference of the linked HP β CD dimer 7KC for hydroxypropyl DS4 and DS8 dimers over the four carbon linker (linker W, where n ═ 3 carbons) and triazole linked dimer (linker U, where n ═ 1 carbons, and linker V, where n ═ 1 carbons) based on linkers a-W (fig. 8D). Legend: the upper (light grey) bars represent the cholesterol values and the lower (dark) bars represent the 7KC values.

Figure 8f effect of CD attachment sites on molecular docking projections of triazole-linked and butyl-linked dimers on cholesterol and 7KC projection affinities. Docking calculations were performed on dimers linked by symmetric butyl and triazole linkers to give three possible linkages. C2-C2, C3-C3 and C2-C3, C2-C3 is the same as the dimer linked with C3-C2 due to the symmetry of the linker. Legend: the upper (light grey) bars represent the cholesterol values and the lower (dark) bars represent the 7KC values.

Fig. 8g. asymmetric joint variation of the connection points. Docking calculations were performed on dimers linked by asymmetric four atom linkers C, D, K, N and R (see fig. 8D). For these asymmetric linkers, there are four possible connections: C2-C2, C3-C3, C2-C3 and C3-C2. In these cases, C3-C2 differs from C2-C3 due to the asymmetry of the linker. Legend: each set of bars represents, from top to bottom, cholesterol with a C3/C2 linkage; cholesterol with a C2/C3 linkage; 7KC with C3/C2 linkage and 7KC with C2/C3 linkage.

Md simulation describes 100ns interaction (linker O) between nitrogen-linked DS4 hydroxypropyl β CD dimer and 7 KC/cholesterol in two orientations. As shown, for example, in fig. 5B.

Figure 8i. visual traces of nitrogen-linked DS4 hydroxypropyl BCD dimer and 7 KC/cholesterol in two orientations (linker O).

Figure 9a predicted 7KC specificity for a number of linked dimers determined by molecular docking. The 7KC specificity was maintained over multiple linker and substitution types of the β CD dimer. The order of the bars within each group is from left to right: sulfobutyl (DS 4); hydroxypropyl (DS 4); methyl (DS 4); quaternary ammonium (DS 4); succinyl (DS 4); carboxymethyl (DS 4); maltosyl (DS 4).

FIG. 9B sterol affinities (DS4) for alkyl linkers of various lengths with hydroxypropyl, methyl, and sulfobutyl substitution; as modeled by molecular docking. The order of the bars within each group is from top to bottom: methyl, sulfobutyl and hydroxypropyl.

FIG. 9C sterol affinities (DS4) for triazole linkers of various lengths with hydroxypropyl, methyl, and sulfobutyl substitution; as modeled by molecular docking. The sequence of the bars is shown in fig. 9B.

FIG. 9D. predicted 7KC specificity of butyl and triazole linked beta CD dimers for multiple substitution positions; as modeled by molecular docking. The X-axis is the fold affinity of 7KC relative to cholesterol. In each group, the upper bar represents triazole and the lower bar represents butyl.

Figure 9e docking screening of other β CD variants. Specificity was observed for butyl and triazole linked beta CD dimer, even for the substituted combination of 7 KC; as modeled by molecular docking. The X-axis is the fold affinity of 7KC relative to cholesterol. The sequence of bars is shown in figure 9D.

FIG. 10A. Synthesis strategy for the linkage of hydroxypropylated dimer to a 1, 4-dibromobutane-based linker unit (resulting in butyl-linked HP. beta. CD dimer).

Figure 10b. synthetic strategy for the linkage of hydroxypropylated dimer to a 3-azido-1-bromo-propane based linker unit (resulting in triazole-linked HP β CD dimer).

Tlc analysis was used to assess reaction progress and conversion.

MALDI spectrum of TBDMS- β CD-BUT- β CD-TBDMS.

Figure 10e.tlc analysis was used to assess reaction progress and conversion.

Fig. 10f MALDI spectrum of synthetic large surface butyl-linked β -cyclodextrin (β CD-BUT- β CD) DS ═ 0.

FIG. 10G MALDI spectra of synthesized large surface butyl-linked hydroxypropyl β -cyclodextrin HP (. beta.CD-BUT-. beta.CD) DS-3. Some peaks were unlabeled due to crowding, but exhibited the expected molecular weight.

FIG. 10H MALDI spectra of synthesized large surface butyl-linked hydroxy-propyl β -cyclodextrin HP (. beta.CD-BUT-. beta.CD) DS-6. Some peaks were unlabeled due to crowding, but exhibited the expected molecular weight.

FIG. 10I MALDI spectra of synthetic large surface butyl-linked hydroxy-propyl β -cyclodextrin HP (. beta.CD-BUT-. beta.CD) DS-8.

FIG. 10J. of HP (β CD-BUT- β CD)¹H-NMR spectrum (D2O, 298K) and signals are marked.

Structure of one of the expected isomers of hp (β CD-BUT- β CD) DS8, and nomenclature of the linker.

FIG. 10 DEPT edited HSQC spectra (D2O, 298K) of HP (β CD-BUT- β CD).

Hp (β CD-BUT- β CD) DEPT edited HSQC spectrum, and assignment of linker frequencies determined by heatmap (D2O, 298K).

FIG. 10 DEPT edited HSQC spectra of HP (. beta.CD-BUT-. beta.CD), and complete assignment (D2O, 298K).

Fig. 10o. MALDI spectrum of synthesized large surface triazole-linked β -cyclodextrin (β CD- (triazole) 1-BCD, DS ═ 0).

FIG. 10P MALDI spectra of synthesized large surface triazole-linked beta-cyclodextrin HP (. beta.CD-triazole-. beta.CD) DS-3. Some peaks were unlabeled due to crowding, but exhibited the expected molecular weight.

FIG. 10Q is a MALDI spectrum of synthesized large surface triazole-linked beta-cyclodextrin HP (. beta.CD-triazole-. beta.CD) DS-7. Some peaks were unlabeled due to crowding, but exhibited the expected molecular weight.

Hp (β CD-triazole- β CD) DEPT edited HSQC spectrum, and linker assignment (D2O, 298K). DS-7 (left) and TLC, and linker fraction (right).

Tlc plate shows reaction monitoring and spot assignment.

FIG. 10 T.MALDI spectrum of 2-O-propargyl- β -CD.

FIG. 10 U.2-O-propargylOf the group-beta-CD¹H-NMR spectrum, and partial peak sorting (DMSO-d6, 298K).

FIG. 10V.BCD- (triazole)₁Of the-BCD dimer¹H-NMR spectrum (D)₂O，298K)。

FIG. 10W.HP (β CD-triazole- β CD)¹H-NMR spectrum (D2O, 298K) and signals are marked. Molecules corresponding to figure 16B and labeled CD-triazole-CD DS3 elsewhere.

FIG. 10X.HP (β CD-triazole- β CD)¹H-NMR spectrum (D2O, 298K) and signals are marked. Corresponding to the molecule labeled CD-triazole-CD DS6 in fig. 16B.

FIG. 10Y.HP (β CD-triazole- β CD)¹H-NMR spectrum (D2O, 298K) and signals are marked. Corresponding to the molecule labeled CD-triazole-CD DS7 in fig. 16B.

Figure 11a. synthesis scheme of methylated β CD dimer.

Figure 11b. tlc analysis was used to assess reaction progress and conversion.

Fig. 11c MALDI spectrum of final compound obtained by the reaction in (a).

Fig. 11d. MALDI spectrum of final compound obtained by the reaction in (B).

FIG. 11E MALDI spectrum of final compound obtained by reaction in (C).

Fig. 11f MALDI spectrum of final compound obtained by the reaction in (D).

Figure 11g superimposed MALDI spectrum of reaction traces. Reaction a (DS0), reaction B (DS1), reaction C (DS2) and reaction D (DS4, 5, 6).

MALDI spectrum of me- (β CD-triazole- β CD) dimer.

FIG. 11I amplification of MALDI spectra of the Me- (. beta.CD-triazole-. beta.CD) dimer.

Structure and atom numbering of one possible isomer of me- (β CD-triazole- β CD) dimer.

Fig. 11k. HNMR spectra of me- (β CD-triazole- β CD) dimer, and complete assignment of frequency.

Figure 11l. HNMR spectra and integrals of me- (β CD-triazole- β CD) dimer.

Fig. 11 DEPT edited HSQC spectrum of me- (β CD-triazole- β CD) dimer, and full partition.

FIG. 11 COSY-NMR spectrum and distribution of Me- (. beta.CD-triazole-. beta.CD) dimer.

Figure 12a. synthesis scheme for sulfobutylated β CD dimers.

Tlc analysis was used to assess the reaction progress and conversion of the SB- β CD assay.

Figure 12c. overlay fingerprint chromatography analysis for evaluation of DS of SB- β CD test reaction a.

Figure 12d. overlay fingerprint chromatography analysis for evaluation of DS of SB- β CD test reaction B.

MALDI of SB- β CD dimer (low DS).

One possible isomer of the sb- β CD dimer, and atom numbering.

FIG. 12G HNMR spectra and complete assignment of sulfobutylated dimers (low DS) (D20; 298K).

FIG. 12H HNMR spectra and integrals (D20; 298K) of sulfobutylated dimers (low DS). The NMR based DS value calculations are shown.

Fig. 12 DEPT edited HSQC spectrum of sb dimer (low DS), and complete assignment (D2O, 298K).

FIG. 12J COSY spectrum of SB dimer (low DS), and complete partition (D2O, 298K).

MALDI spectrum of sb dimer (high DS).

Structure and atom numbering of one possible isomer of sb dimer (DS 3).

Fig. 12 m.hnmr spectra of sb dimer (high DS) and full assignment (D20, 298K).

HNMR spectra and integrals (D20, 298K) of sb dimers (high DS). The NMR based DS value calculations are shown.

Fig. 12 Dept edited HSQC spectrum of sb dimer (high DS), and full assignment (D20, 298K).

FIG. 12P COSY spectrum of SB dimer (high DS) and complete partition (D20, 298K).

Figure 13a. synthesis scheme for quaternary ammonium β -cyclodextrin dimers.

FIG. 13B MALDI spectra of quaternary ammonium β -cyclodextrin dimer reaction A.

Figure 13c MALDI spectrum of quaternary ammonium β -cyclodextrin dimer reaction B.

FIG. 13D MALDI spectra of quaternary ammonium β -cyclodextrin dimer reaction C.

Figure 13e MALDI spectrum of quaternary ammonium β -cyclodextrin dimer reaction D.

Figure 13f MALDI spectrum of quaternary ammonium β -cyclodextrin dimers.

Fig. 13g structure and atom numbering of one possible QA dimer isomer (DS 3).

HNMR spectra and complete assignment of the h.qa dimer (D20, 298K).

Fig. 13 HNMR spectra and integrals of i.qa dimers (D20, 298K). The NMR based DS value calculations are shown.

Fig. 13j. DEPT edited HSQC spectrum and complete assignment of qa dimer (D20, 298K).

COSY spectrum and partial assignment of qa dimer (D20, 298K).

Figure 14a. synthesis scheme for succinylated dimers.

MALDI of succinylated dimer reaction a.

MALDI of succinylated dimer reaction B.

FIG. 14D MALDI of succinylated dimer reaction C.

FIG. 14E MALDI of succinylated dimer reaction D.

MALDI of succinylated dimers.

Fig. 14g structure and atom numbering of one possible SUCC dimer isomer (DS 3).

Fig. 14h HNMR spectra and full assignment of succinylated dimers (D20, 298K).

Fig. 14i HNMR spectra and integrals (D20, 298K) of succinylated dimers. The NMR based DS value calculations are shown.

Figure 14j DEPT edited HSQC spectrum and complete assignment of succinylated dimer (D20, 298K).

FIG. 14K COSY spectrum and partial assignment of succinylated dimer (D20, 298K).

Figure 15a. 7KC extracellular fluid concentration after incubation with DS8 HP β CD dimer.

Figure 15b. 7KC blood cell efflux concentration after incubation with HP β CD monomer.

Figure 15c. plasma cholesterol was not interfered with by incubation with HP β CD dimer. Plasma cholesterol was measured by mass spectrometry to determine the cholesterol efflux from blood cells caused by incubation with HP β CD dimer.

Fig. 15d. hemolytic assay as a measure of potential cytotoxicity of various butyl and triazole linked HP β CD and methyl dimers.

Figure 15e hemolytic assay as various triazole linked β CD dimers: a measure of the potential cytotoxicity of unsubstituted β CD, SB β CD (low and high DS), QA β CD and succinylated β CD dimers.

Figure 16a. butyl linked HP β CD dimer was far superior to monomeric HP β CD in solubilizing 7KC and cholesterol. Dimers of about 3, about 6 and about 8 degrees of substitution were tested.

Figure 16b. triazole linked HP β CD dimer was far superior to monomeric HP β CD in solubilizing 7KC and cholesterol. Dimers of 0, about 3, about 5 and about 6 degrees of substitution were tested. HPBCD represents the monomer HP β CD, while CD-triazole-CD represents the triazole-linked dimer with the indicated degree of substitution.

FIG. 16C solubilization of various cholesterol derivatives and oxysterol by butyl-linked HP β CD dimer (DS-8). The results for cholesterol, 7-ketocholesterol (7KC), vitamin D2, vitamin D3, desmosterol, 27-hydroxycholesterol (27OH), 4- β -hydroxycholesterol (4BOH), 25-hydroxycholesterol (25OH) and cholesterol epoxide are depicted.

FIG. 16D solubilization of compounds by butyl-linked HPBCD dimer (DS-8). The sterol hormones tested were estradiol, estriol, estrone, pregnenolone and progesterone.

FIG. 16E. butyl-linked HP β CD dimer (DS-3) ("DS 3 butyl dimer") has affinity and specificity for 7 KC. HPBCD represents monomeric HP β CD.

FIG. 16F. triazole-linked HP β CD dimer (DS-3) has affinity and specificity for 7 KC.

FIG. 16G. the effect of triazole-linked Me β C dimer (DS-3) ("methyl dimer DS 3") in solubilizing 7KC and cholesterol was similar to HP β CD dimer (DS-3) ("HPBCD dimer DS 3").

FIG. 16H. triazole-linked unsubstituted β CD ("CD-triazole-CD DS 0"), triazole-linked SB β CD dimer (DS-3.4) ("SB CD-triazole-CD DS 3.4"), triazole-linked Qa β CD dimer (DS-2) (QA CD-triazole-CD DS 2 "), and triazole-linked succinylated β CD dimer (DS-2) (" SUCC CD-triazole-CD DS 2 ") are specific for 7KC in vitro versus cholesterol. Triazole-linked SB β CD dimer (DS-14.6) ("SB CD-triazole-CD DS 14.6) has a lower affinity for both cholesterol and 7 KC.

Definition of

Unless otherwise indicated, the following terms used in this application, including the specification and claims, have the definitions set forth herein.

As used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise.

The length of the joint. As used herein, the length of the linker or "linker length" used interchangeably refers to the number of atoms of the linker on the shortest path through the linker connecting the two CD subunits of the cyclodextrin dimer. For clarity, the length of the linker does not include the oxygen atom (or other atoms that may replace the oxygen) of each CD subunit to which the linker is attached. For example, in fig. 3B, the linker length is 3+ n1+ n2, which reflects the shortest path through the triazole ring. For linkers attached to one or both of the cyclodextrin monomers at multiple points, the linker length is the shortest path connecting the two cyclodextrins in all possible pathways that can begin and end at different positions in each cyclodextrin.

Head-to-head cyclodextrin dimers. As used herein, the term "head-to-head cyclodextrin dimer" refers to a CD dimer in which two CD monomers are linked by the large (minor) surface of the cyclodextrin, typically via the C2 and/or C3 carbons of each CD monomer.

Tail-to-tail cyclodextrin dimers. As used herein, the term "tail-to-tail cyclodextrin dimer" refers to a CD dimer in which two CD monomers are linked on the small (major) surface of the cyclodextrin molecule, typically via the C6 carbon of each CD monomer.

Head-to-tail cyclodextrin dimers. As used herein, the term "head-to-tail cyclodextrin dimer" refers to a CD dimer in which two CD monomers are linked at opposite ends, i.e., one monomer is linked from a small (major) surface, typically through a C6 carbon, and the other monomer is linked from a large (minor) surface, typically via a C2 and/or C3 carbon.

Degree of Substitution (DS). As used herein, "degree of substitution" or "DS" refers to the number of given sub-groups bonded to a monomer or dimer. For example, Me β CD DS3 refers to β CD having O2, O3, or O6 with an average of 3 methyl R groups attached to CD, while HP β CD DS3 represents a monomer or dimer of O2, O3, or O6 with an average of 3 hydroxypropyl groups attached to CD. When referring to CD dimers, unless otherwise indicated, DS is used to refer to the overall average substitution of the two constituent monomers including all substituents (e.g., all inclusive in the case of mixed substituents such as mixed hydroxypropyl and methyl substituents). Terms such as "degree of substitution of substituent X" and the like refer to the average number of substituents X per CD dimer, i.e., excluding other substituents that may be present. DS can be measured by mass spectrometry (e.g., matrix-assisted laser desorption/ionization, "MALDI") or by NMR. MALDI is preferred for cyclodextrin derivatives containing substituents that give a more typical Gaussian distribution of ions in the mass spectrum, as shown, for example, by the methyl, hydroxypropyl, and sulfobutyl substituents in FIGS. 10G-10I, 10P-10Q, 11C-11G, 11I, 12E, and 12K. The average DS determined by MALDI is calculated by averaging the peak heights of the peaks corresponding to each DS species of the CD in question. In other cases, a less regular ion peak pattern may occur, for example, due to the formation of various adductions, fragmentation, elimination products, and the like. Other mass spectrometry techniques can be employed to potentially circumvent these problems. Alternatively, NMR can be used to determine DS values, which is preferred for succinyl and quaternary ammonium groups given the more complex MS spectra observed by MALDI. The calculation of the average Degree of Substitution (DS) is then done by identifying the peak to which the proton from the core dimer corresponds and scaling the measurements for the first time to correspond the peak area to the known number of such protons in the structure. The signals corresponding to the protons in the substituent groups are then examined and appropriately scaled to obtain an average degree of substitution. In the simpler case, a well-resolved peak corresponding to the proton of the substituent is determined and has been scaled as described above, then divided by the number of protons represented in that peak to give the average number of substituents. For example, for the hydroxypropyl substituent, the peak corresponding to 14 protons in the core structure (anomeric region of glucopyranose) was identified and its signal normalized to 14, then the peak corresponding to 3 protons of the methyl substituent was identified and finally the area of the peak was divided by 3 to obtain the average number of hydroxypropyl groups present per molecule. In other cases, the substituent peak and the cyclodextrin core peak can be very close or overlapping. In this case, the number of contributing protons in the cyclodextrin core structure is determined, then subtracted from the peak area (the peak area has been scaled to the integrated area of 1 per proton), and then the remaining area is divided by the number of contributing protons to obtain the average degree of substitution. For example, for the methyl substituent (as shown in FIGS. 11K-11L), three methyl hydrogens of the substituent and a cluster of peaks corresponding to a set of 86 protons of the core cyclodextrin dimer structure were identified. As in the case of the hydroxypropyl substituent, the peaks corresponding to 14 protons in the core structure (anomeric region of glucopyranose) were identified and their signals normalized to 14; the peak area containing methyl hydrogen and core cyclodextrin hydrogen was determined to be 92.77, and 6.77 was obtained by subtracting the signal from 86 protons in the core cyclodextrin structure; and divided by 3 protons per methyl group, the average degree of substitution was estimated to be 2.26. For HP and ME substituted CD, the integral is divided by 3, for QA, the integral is divided by 9, for SB, the integral is divided by 2, and for SUCC, the integral is divided by 4. The above calculations are directly applicable to other substituent types based on the identification of the peak corresponding to the proton in the substituent structure. DS calculations using NMR are shown in FIGS. 10X-Y, 11L, 12H, 12N, 13I and 14I. A CD composition, such as a CD dimer composition (as defined below), may comprise a mixture of individual molecules substituted with different numbers of substituents, in which case the DS value is expressed as the average (median) number of substitutions. The fractional DS value reflects the case where the median value can be between integer substitutions. Unless otherwise indicated, the integer DS values represent CD compositions having that DS number when rounded to the nearest integer. For example, DS4 refers to a DS value of at least 3.5 and less than 4.5.

Average degree of substitution of hydroxypropyl groups. As used herein, the term "average degree of substitution of a hydroxypropyl group" refers to the degree of substitution as defined above, ignoring any substituents other than the hydroxypropyl group. Likewise, reference to an average degree of substitution for a given substituent means that the average degree of substitution is as defined above, ignoring other substituent types.

Hydroxypropyl (HP or HP) substituted Cyclodextrins (CD). As used herein, the term "hydroxypropyl-substituted cyclodextrin" or "HP-substituted CD" refers to a cyclodextrin linked to a hydroxypropyl group (i.e., -CH2-CH (oh) -CH 3). Typically, the HP group is attached to an oxygen atom (most commonly with a combination of these attachment sites) that is attached to a C2, C3, and/or C6 carbon atom of the CD.

Hydroxypropyl- β -cyclodextrin, abbreviated HP β CD, HPBCD, HP-BCD, HP- β CD, 2-HP β CD and similar terms, refers to β -cyclodextrin substituted with one or more hydroxypropyl groups (i.e., -CH2-CH (oh) -CH3), typically attached to an oxygen atom attached to the C2, C3 and/or C6 carbons of CD (most commonly with a combination of these attachment sites).

Hydroxypropyl- β -cyclodextrin dimer, abbreviated to HP (CD-L-CD) or HP (β CD-L- β CD) HP and similar terms, refers to hydroxypropyl- β -cyclodextrin dimer covalently linked by linker L. There may be a particular average number of substitutions, for example, DS4 indicates that there are an average of 4 HP groups. Additional substitutions may be present as further described herein.

Similar conventions are also used for other substituted cyclodextrins and cyclodextrin dimers, such as methyl (Me), Quaternary Ammonium (QA), Succinyl (SUCC), Sulfobutyl (SB), and the like. Thus, for example, Me β CD refers to methyl- β -cyclodextrin. Similarly, methyl- β -cyclodextrin dimer is sometimes abbreviated as Me (CD-L-CD) or Me (β CD-L- β CD) Me and similar terms, which refer to methyl- β -cyclodextrin dimer covalently linked through linker L. Specific average substitution numbers may be present, for example, DS4 indicates that an average of 4 Me groups are present. Additional substitutions may be present as further described herein.

A cyclodextrin dimer composition. As used herein, the term "cyclodextrin dimer composition" or "CD dimer composition" refers to a mixture of cyclodextrin dimers, e.g., CD dimers substituted with a different number of the same substituents. In general, the CD dimer compositions are characterized by a specified degree of substitution with a specified substituent. The CD dimer composition may result from a synthetic process in which substituents are added to the CD dimer in a random fashion due to the substantially symmetrical nature of the CD molecule, such that the number and position of substituents for a single CD molecule will vary. In addition, the CD dimer composition may include a mixture of single molecules with different linker attachment sites (e.g., O2 to O2, O2 to O3, O3 to O2, or O3 to O3), or the linker attachment sites may be uniform (e.g., only O2 to O2, only O2 to 03, only O3 to O2, or only O3 to O3). The degree of substitution of the CD dimer composition may be determined by NMR and/or mass spectrometry, e.g., as described above.

The term "specifically binds" or the like means that the molecule (e.g., cyclodextrin dimer of the present disclosure) forms a complex with a binding partner (e.g., cholesterol (such as oxysterol, e.g., 7KC)) that is relatively stable under physiological conditions. Methods for determining whether a molecule specifically binds to a binding partner are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, and the like. In exemplary embodiments, the cyclodextrin dimers of the present disclosureBetween about 5 μ M and about 100 μ M, between about 10 μ M and about 90 μ M, between about 20 μ M and about 80 μ M, between about 30 μ M and about 70 μ M, between about 40 μ M and about 60 μ M, between about 0.5 μ M and about 50 μ M, between about 1 μ M and about 40 μ M, between about 2 μ M and about 30 μ M, between about 3 μ M and about 20 μ M, between about 4 μ M and about 10 μ M, less than about 1000 μ M, less than about 500 μ M, less than about 300 μ M, less than about 200 μ M, less than about 100 μ M, less than about 90 μ M, less than about 80 μ M, less than about 70 μ M, less than about 60 μ M, less than about 50 μ M, less than about 40 μ M, less than about 30 μ M, less than about 20 μ M, less than about 10 μ M, less than about 5 μ M, less than about 4 μ M, less than about 3 μ M, K of less than about 2 μ M, less than about 1 μ M, or less than about 0.5 μ M _DBinding to cholesterol, oxysterol or 7 KC.

The affinity for 7KC was greater than for cholesterol. As used herein, the term "greater affinity for 7KC than cholesterol" refers to a compound (e.g., cyclodextrin) that has a greater ability to solubilize 7KC than cholesterol. It can also be predicted by molecular docking, predicted by molecular dynamics simulations or measured by calorimetry. In exemplary embodiments, the cyclodextrin dimer has a binding affinity for 7KC that is at least 1.5 fold, at least 2 fold, at least 3 fold, at least 4 fold, at least 5 fold, at least 8 fold, at least 10 fold, at least 15 fold, at least 20 fold, at least 30 fold, or at least 50 fold greater than its binding affinity for cholesterol, as may optionally be determined by comparing the concentration at which 50% of 7KC is solubilized in suspension, e.g., using the procedure described in the working examples herein. In exemplary embodiments, the cyclodextrin dimer has a binding affinity for 7KC that is at least 1.1-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, or 10-fold greater than its binding affinity for cholesterol, optionally by using a calculated or measured binding affinity (K) for cholesterol _D) Divided by the calculated binding affinity for 7 KC.

Greater affinity for one compound than for another, e.g., greater affinity for 7KC than for cholesterol, can be determined using a "turbidity test" performed on an aqueous suspension containing 3% ethanol, 300uM sterol in PBS and 1mM cyclodextrin to be tested. This single concentration of cyclodextrin was used to normalize the test results. For testing, the samples were incubated at 37C for 30 minutes and then the absorbance at 350nm was measured, for example, using a spectrophotometer plate reader. Relative turbidity was determined by dividing the turbidity measured in the presence of cyclodextrin by the baseline turbidity without cyclodextrin. If the relative turbidity of the 7KC suspension is less than that of the cholesterol solution, then the affinity of a given cyclodextrin for 7KC is greater than that for cholesterol.

A hydrophobic drug. As used herein, the term "hydrophobic drug" refers to a drug that is insoluble in water in the absence of some detergent or other solvent. Hydrophobic drugs include, but are not limited to, hormones such as estrogen, progesterone, and testosterone. The cyclodextrin dimers of the present disclosure can be used as excipients for hydrophobic drugs. Additional exemplary hydrophobic drugs include dextromethorphan hydrobromide (DXM), diphenhydramine hydrochloride (DPH), lidocaine hydrochloride (LDC), heparin, benfluthiazine, acyclovir, revaprazan, curcumin, and Testosterone Propionate (TP), among others. The cyclodextrin dimer may be present in an amount sufficient to increase the solubility of the molecule and/or to facilitate better drug delivery. The molecular ratio of drug to cyclodextrin can be 1: 1 or greater than 1: 1.

An amount effective to solubilize the hydrophobic drug. As used herein, the phrase "an amount effective to solubilize the hydrophobic drug" refers to the concentration of a substance (e.g., cyclodextrin dimer) that is capable of solubilizing the hydrophobic drug, typically in an aqueous composition such as Phosphate Buffered Saline (PBS) or water. Solubilization can be determined spectrophotometrically or by other means known in the art. Solubilization can be determined at room temperature, physiological temperature (37 ℃) or another suitable temperature (e.g., between 0 and 4 ℃).

"alkyl" means a monovalent straight or branched chain saturated hydrocarbon moiety consisting only of carbon and hydrogen atoms having from one to twelve carbon atoms.

"lower alkyl" refers to an alkyl group having one to six carbon atoms, i.e., C3 alkyl. Examples of alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, isobutyl, sec-butyl, tert-butyl, pentyl, n-hexyl, octyl, dodecyl and the like.

"alkylene" means a straight or branched chain saturated divalent hydrocarbon radical having from one to twelve carbon atoms or a branched chain saturated divalent hydrocarbon radical having from three to six carbon atoms, such as methylene, ethylene, 2-dimethylethylene, propylene, 2-methylpropylene, butylene, pentylene, and the like.

"alkenyl" means a straight-chain monovalent hydrocarbon group having two to twelve carbon atoms or a branched-chain monovalent hydrocarbon group having three to twelve carbon atoms that contains at least one double bond. Examples of alkenyl groups include, but are not limited to, vinyl (vinyl), -CH ═ CH2), 1-propenyl (-CH ═ CH-CH3), 2-propenyl (allyl, -CH ═ CH2) moieties, including, but not limited to, methoxy, ethoxy, isopropoxy, and the like.

"alkoxyalkyl" means a moiety of the formula Ra-O-Rb-, wherein Ra is alkyl and Rb is alkylene as defined herein. Exemplary alkoxyalkyl groups include, for example, 2-methoxyethyl, 3-methoxypropyl, 1-methyl-2-methoxyethyl, 1- (2-methoxyethyl) -3-methoxy-propyl, and 1- (2-methoxyethyl) -3-methoxypropyl.

"Alkoxyalkoxyalkyl" means a group of the formula-R-O-R '-O-R ", wherein R and R' are each alkylene, and R" is alkyl as defined herein.

"Alkylcarbonyloxyalkyl" means a group of the formula-R-O-C (O) -R ', wherein R is alkylene and R' is alkyl as defined herein.

"alkylcarbonyl" means a moiety of the formula-R '-R ", wherein R' is-C (═ O) -, and R" is alkyl as defined herein.

"alkylsulfonyl" means a moiety of the formula-R '-R ", wherein R' is-SO 2-, and R" is alkyl as defined herein.

"Alkylsulfonylalkyl" means a moiety of the formula-R ' -R ', where R ' is alkyl, R ' is-SO 2-, and R ' is alkyl as defined herein.

"alkylamino" means a moiety of the formula-NR-R ', wherein R is hydrogen or alkyl, and R' is alkyl as defined herein.

"Alkoxyamino" means a moiety of the formula-NR-OR 'where R is hydrogen OR alkyl and R' is alkyl as defined herein.

"Alkylsulfanyl" means a moiety of the formula-SR, where R is alkyl as defined herein.

By "alkali metal ion" is meant a monovalent ion of a group I metal, such as lithium, sodium, potassium, rubidium, or cesium, preferably sodium or potassium.

By "alkaline earth metal ion" is meant a divalent ion of a group II metal, such as beryllium, magnesium, calcium, strontium, or barium, preferably magnesium or calcium.

"amino" means a group-NR 'R "where R' and R" are each independently hydrogen or alkyl. Thus, "amino" as used herein encompasses "alkylamino" and "dialkylamino".

"alkylaminoalkyl" means a group-R-NHR ', where R is alkylene and R' is alkyl. Alkylaminoalkyl groups include methylaminomethyl, methylaminoethyl, methylaminopropyl, ethylaminoethyl, and the like.

"Dialkylaminoalkyl" means a group-R-NR 'R "wherein R is an alkylene group and R' and R" are alkyl groups as defined herein. Dialkylaminoalkyl groups include dimethylaminomethyl, dimethylaminoethyl, dimethylaminopropyl, N-methyl-N-ethylaminoethyl, and the like.

"aminoalkyl" means a group-R-R ', wherein R' is amino and R is alkylene as defined herein. "aminoalkyl" includes aminomethyl, aminoethyl, 1-aminopropyl, 2-aminopropyl and the like.

"aminoalkoxy" means a group-OR-R1, wherein R' is amino and R is alkylene as defined herein.

"Alkylsulfonylamino" means a moiety of the formula-NR 'SO 2-R, wherein R is alkyl and R' is hydrogen or alkyl.

"aminocarbonyloxyalkyl" or "carbamoylalkyl" means the group R-O-C (═ O) -R ', where R' is amino and R is alkylene as defined herein.

"aminosulfonyl" means a group-SO 2-NR 'R "where R' and R" are each independently hydrogen or alkyl. Thus, "aminosulfonyl" as used herein encompasses "alkylaminosulfonyl" and "dialkylaminosulfonyl".

"Alkynylalkoxy" means a group of the formula-O-R-R ', wherein R is alkylene and R' is alkynyl as defined herein.

"aryl" means a monovalent cyclic aromatic hydrocarbon moiety consisting of a monocyclic, bicyclic, or tricyclic aromatic ring. The aryl group may be optionally substituted, as defined herein. Examples of aryl moieties include, but are not limited to, optionally substituted phenyl, naphthyl, phenanthryl, fluorenyl, indenyl, pentenyl, azulenyl, oxydiphenyl, biphenyl, methylenediphenyl, aminodiphenyl, diphenylthio, diphenylsulfonyl, diphenylisopropylidene, benzodioxanyl, benzofuranyl, benzodioxy, benzopyranyl, benzoxazinyl, benzoxazinonyl, benzopiperidinyl, benzopiperazinyl, benzopyrrolidinyl, benzomorpholinyl, methylenedioxyphenyl, ethylenedioxyphenyl, and the like, including partially hydrogenated derivatives thereof.

"arylalkyl" and "aralkyl" are used interchangeably to mean the group-RaRb, where Ra is an alkylene group and Rb is an aryl group as defined herein; examples of arylalkyl groups are, for example, phenylalkyl groups such as benzyl, phenethyl, 3- (3-chlorophenyl) -2-methylpentyl and the like.

"arylsulfonyl" means a group of the formula-SO 2-R, where R is aryl as defined herein.

"aryloxy" means a group of the formula-O-R, wherein R is aryl as defined herein.

"aralkyloxy" or "arylalkyloxy" means a group of the formula-O-R-R ", wherein R is alkylene and R' is aryl as defined herein.

"cyanoalkyl" means a moiety of the formula-R '-R', where R 'is alkylene as defined herein, and R' is cyano or nitrile.

"cycloalkyl" means a monovalent saturated carbocyclic moiety consisting of a monocyclic or bicyclic ring. Cycloalkyl groups may be optionally substituted with one or more substituents, wherein each substituent is independently hydroxy, alkyl, alkoxy, halo, haloalkyl, amino, monoalkylamino, or dialkylamino, unless otherwise specifically stated. Examples of cycloalkyl moieties include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and the like, including partially unsaturated derivatives thereof.

"cycloalkenyl" means a monovalent unsaturated carbocyclic moiety consisting of a monocyclic or bicyclic ring containing at least one double bond. The cycloalkenyl can be optionally substituted with one or more substituents, wherein each substituent is independently hydroxy, alkyl, alkoxy, halo, haloalkyl, amino, monoalkylamino, or dialkylamino, unless otherwise specifically stated. Examples of cycloalkenyl moieties include, but are not limited to, cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclohexenyl, cycloheptenyl.

"cycloalkylalkyl" means a moiety of the formula-R '-R ", wherein R' is alkylene, and R" is cycloalkyl as defined herein.

"cycloalkylene" means a divalent saturated carbocyclic group consisting of a single ring or a bicyclic ring. Cycloalkylene groups may be optionally substituted with one or more substituents, wherein each substituent is independently hydroxy, alkyl, alkoxy, halo, haloalkyl, amino, monoalkylamino, or dialkylamino, unless otherwise specifically stated.

"cycloalkylalkylene" means a moiety of the formula-R '-R "-where R' is alkylene and R" is cycloalkylene as defined herein.

"heteroalkyl" means an alkyl group as defined herein in which one, two or three hydrogen atoms have been replaced by a substituent independently selected from-ORa, -NRbRc and-s (o) nRd (where n is an integer from 0 to 2), wherein the point of attachment of the heteroalkyl group is connected through a carbon atom, wherein Ra is hydrogen, acyl, alkyl, cycloalkyl or cycloalkylalkyl; rb and Rc are independently from each other hydrogen, acyl, alkyl, cycloalkyl or cycloalkylalkyl; and when n is 0, Rd is hydrogen, alkyl, cycloalkyl or cycloalkylalkyl; and when n is 1 or 2, Rd is alkyl, cycloalkyl, cycloalkylalkyl, amino, acylamino, monoalkylamino or dialkylamino. Representative examples include, but are not limited to, 2-hydroxyethyl, 3-hydroxypropyl, 2-hydroxy-1-hydroxymethylethyl, 2, 3-dihydroxypropyl, 1-hydroxymethylethyl, 3-hydroxybutyl, 2, 3-dihydroxybutyl, 2-hydroxy-1-methylpropyl, 2-aminoethyl, 3-aminopropyl, 2-methylsulfonylethyl, aminosulfonylmethyl, aminosulfonylethyl, aminosulfonylpropyl, methylaminosulfonylmethyl, methylaminosulfonylethyl, methylaminosulfonylpropyl, and the like.

"heteroaryl" means a monocyclic or bicyclic group having 5 to 12 ring atoms, wherein at least one aromatic ring contains one, two or three ring heteroatoms selected from N, O or S, the remaining ring atoms being C, wherein the attachment point of the heteroaryl group will be on the aromatic ring. The heteroaryl ring may be optionally substituted, as defined herein. Examples of heteroaryl moieties include, but are not limited to, optionally substituted imidazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, oxadiazolyl, thiadiazolyl, pyrazinyl, thienyl, benzothienyl, thiophenyl, furanyl, pyranyl, pyridyl, pyrrolyl, pyrazolyl, pyrimidinyl, quinolinyl, isoquinolinyl, benzofuranyl, benzothienyl, benzothiopyranyl, benzimidazolyl, benzoxazolyl, benzooxadiazolyl, benzothiazolyl, benzothiadiazolyl, benzopyranyl, indolyl, isoindolyl, triazolyl, triazinyl, quinoxalinyl, purinyl, quinazolinyl, quinolizinyl, naphthyridinyl, pteridinyl, carbazolyl, azaazazolylRadical diazaRadicals, acridinyl radicals and the like, including partially hydrogenated derivatives thereofAnd (4) living things.

"heteroarylalkyl" or "heteroaralkyl" means a group of the formula-R ', wherein R is alkylene and R' is heteroaryl as defined herein.

"Heteroarylsulphonyl" means a group of formula-SO 2-R, wherein R is heteroaryl as defined herein.

"heteroaryloxy" means a group of the formula-O-R, wherein R is heteroaryl as defined herein.

"Heteroaralkyloxy" means a group of the formula-O-R-R ", wherein R is alkylene and R' is heteroaryl as defined herein.

"Heterocyclylalkoxy" means a group of the formula O-R-R ', where R is alkylene and R' is heterocyclyl as defined herein.

The terms "halo", "halogen" and "halide" are used interchangeably to refer to the substituents fluorine, chlorine, bromine, or iodine. In some embodiments, halo refers to a fluoro substituent.

"haloalkyl" means an alkyl group, as defined herein, wherein one or more hydrogens have been replaced with the same or different halogen. In some embodiments, the haloalkyl is a fluoroalkyl; in some embodiments, the haloalkyl is a perfluoroalkyl. Exemplary haloalkyl groups include-CH 2Cl, -CH2CF3, -CH2CCl3, perfluoroalkyl groups (e.g., -CF3), and the like.

"haloalkoxy" means a moiety of the formula-OR, wherein R is a haloalkyl moiety as defined herein. In some embodiments, the haloalkoxy group is fluoroalkoxy; in some embodiments, the haloalkoxy group is a perfluoroalkoxy group. An exemplary haloalkoxy group is difluoromethoxy.

"Heterocyclylamino" means a saturated ring in which at least one ring atom is N, NH or N-alkyl, and the remaining ring atoms form an alkylene group.

"heterocyclyl" means a monovalent saturated moiety consisting of one to three rings incorporating one, two, or three or four heteroatoms (selected from nitrogen, oxygen or sulfur). The heterocyclyl ring may be optionally substituted, as defined herein. Heterocyclyl moietiesExamples of (A) include, but are not limited to, optionally substituted piperidinyl, piperazinyl, homopiperazinyl, azaExamples of the substituent include a substituent such as a phenyl group, a naphthyl group, a phenanthryl group, a thienyl group, a naphthyl group, a thienyl group, and the like.

"Heterocyclylalkyl" means a moiety of the formula-R-R ', where R is alkylene and R' is heterocyclyl as defined herein.

"Heterocyclyloxy" means a moiety of formula-OR, wherein R is heterocyclyl as defined herein.

"Heterocyclylalkoxy" means a moiety of the formula-OR-R 'where R is alkylene and R' is heterocyclyl as defined herein.

"Hydroxyalkoxy" means a moiety of the formula-OR, wherein R is hydroxyalkyl as defined herein.

"Hydroxyalkylamino" means a moiety of the formula-NR-R ', wherein R is hydrogen or alkyl, and R' is hydroxyalkyl as defined herein.

"Hydroxyalkylaminoalkyl" means a moiety of the formula-R-NR '-R ", wherein R is alkylene, R' is hydrogen or alkyl, and R" is hydroxyalkyl as defined herein.

"hydroxyalkyl" means an alkyl moiety as defined herein substituted by one or more, preferably one, two or three hydroxyl groups, provided that the same carbon atom carries no more than one hydroxyl group. Representative examples include, but are not limited to, hydroxymethyl, 2-hydroxyethyl, 2-hydroxypropyl, 3-hydroxypropyl, 1- (hydroxymethyl) -2-methylpropyl, 2-hydroxybutyl, 3-hydroxybutyl, 4-hydroxybutyl, 2, 3-dihydroxypropyl, 2-hydroxy-1-hydroxymethylethyl, 2, 3-dihydroxybutyl, 3, 4-dihydroxybutyl and 2- (hydroxymethyl) -3-hydroxypropyl.

"Hydroxycarbonylalkyl" or "carboxyalkyl" means a group of the formula-R- (CO) -OH, wherein R is alkylene as defined herein.

"Hydroxyalkyloxycarbonylalkyl" or "hydroxyalkoxycarbonylalkyl" means a group of the formula-R-C (O) -O-R-OH wherein each R is alkylene and may be the same or different.

"hydroxyalkyl" means an alkyl moiety as defined herein substituted by one or more, preferably one, two or three hydroxyl groups, provided that the same carbon atom carries no more than one hydroxyl group. Representative examples include, but are not limited to, hydroxymethyl, 2-hydroxyethyl, 2-hydroxypropyl, 3-hydroxypropyl, 1- (hydroxy-5-methyl) -2-methylpropyl, 2-hydroxybutyl, 3-hydroxybutyl, 4-hydroxybutyl, 2, 3-dihydroxypropyl, 2-hydroxy-1-hydroxymethylethyl, 2, 3-dihydroxybutyl, 3, 4-dihydroxybutyl and 2- (hydroxymethyl) -3-hydroxypropyl.

"Hydroxycycloalkyl" means a cycloalkyl moiety as defined herein in which one, two or three hydrogen atoms in the cycloalkyl group have been substituted by a hydroxy substituent. Representative examples include, but are not limited to, 2-hydroxy-cyclohexyl, 3-hydroxy-cyclohexyl, or 4-hydroxy-cyclohexyl, and the like.

"Urea" or "ureido" means a group of the formula-NR ' -C (O) -NR "R '", wherein R, R "and R '" are each independently hydrogen or alkyl.

"carbamate" means a group of the formula-O-C (O) -NR 'R', wherein R 'and R' are each independently hydrogen or alkyl.

"carboxy" means a group of the formula-C (O) OH.

"sulfonamide" means a group of the formula-SO 2-NR 'R "wherein R', R", and R "are each independently hydrogen or alkyl.

"nitro" means-NO 2.

"cyano" means-CN.

"phenoxy" means a benzene ring substituted with at least one-OH group.

"acetyl" means-C (═ O) -CH 3.

"Cn-m-" is used as a prefix before a functional group, where "n" and "m" represent integer values (i.e., 0, 1, 2, 12), such as C1-12-alkyl or C5-12-heteroaryl. The prefix indicates the number or range of numbers of carbon atoms present in the functional group. For ring systems, the prefix indicates the number of ring atoms or a range of numbers of ring atoms, whether the ring atoms are carbon or heteroatoms. Where the functional group constitutes cyclic and acyclic portions (i.e., "arylalkyl" is made up of aryl and alkyl portions), the prefix is used to indicate how many carbon and ring atoms are present in total. For example, for arylalkyl, "C7-arylalkyl" can be used to represent "phenyl-CH 2-". For certain functional groups, zero carbon atoms may be present, such as C0-aminosulfonyl (i.e., -SO2-NH2, with both potential R groups being hydrogen), "0" meaning that no carbon atoms are present.

"peptide" means an amide derived from two or more amino acids by the combination of an amino group and a carboxyl group of one acid. "Monopeptide" means a single amino acid, "dipeptide" means an amide compound comprising two amino acids, "tripeptide" means an amide compound comprising three amino acids, and so on. The C-terminus of a "peptide" may be linked to another moiety via an ester functionality.

"optionally substituted" when used in combination with "aryl", "phenyl", "heteroaryl", "cyclohexyl" or "heterocyclyl" means aryl, phenyl, heteroaryl, cyclohexyl or heterocyclyl optionally independently substituted with one to four substituents, preferably one or two substituents, selected from alkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, hydroxyalkyl, halo, nitro, cyano, hydroxy, alkoxy, amino, acylamino, monoalkylamino, dialkylamino, haloalkyl, haloalkoxy, heteroalkyl, -COR (where R is hydrogen, alkyl, phenyl or phenylalkyl), -CR ' R ") n-COOR (where n is an integer from 0 to 5, R ' and R" are independently hydrogen or alkyl, and R is hydrogen, alkyl, cycloalkyl, cycloalkylalkyl, phenyl or phenylalkyl) or- (CR ' R ") n-CONRaRb (where n is an integer from 0 to 5, r' and R "are independently hydrogen or alkyl, and Ra and Rb are independently from each other hydrogen, alkyl, cycloalkyl, cycloalkylalkyl, phenyl or phenylalkyl).

"leaving group" means a group having the meaning normally associated therewith in synthetic organic chemistry, i.e., an atom or group that is replaceable under substitution reaction conditions. Examples of leaving groups include, but are not limited to, halogen, alkane-or arylenesulfonyloxy, such as methanesulfonyloxy, ethanesulfonyloxy, thiomethyl, benzenesulfonyloxy, toluenesulfonyloxy, and thiophenyloxy, dihalophosphonoxy, optionally substituted benzyloxy, isopropoxy, acyloxy, and the like.

By "modulator" is meant a molecule that interacts with a target. Interactions include, but are not limited to, agonists, antagonists, and the like, as defined herein.

"optional" or "optionally" means that the subsequently described event or circumstance may, but need not, occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

"disease" and "disease state" mean any disease, disorder, symptom, condition, or indication.

By "inert organic solvent" or "inert solvent" is meant a solvent that is inert under the reaction conditions described in connection therewith, including, for example, benzene, toluene, acetonitrile, tetrahydrofuran, N-dimethylformamide, chloroform, dichloromethane (methylene chloride or dichloromethane), dichloroethane, diethyl ether, ethyl acetate, acetone, methyl ethyl ketone, methanol, ethanol, propanol, isopropanol, tert-butanol, dioxane, pyridine, and the like. Unless stated to the contrary, the solvent used in the reaction of the present disclosure is an inert solvent.

By "pharmaceutically acceptable" is meant that the compositions used to prepare the pharmaceutical compositions are generally safe, non-toxic and neither biologically nor otherwise desirable, and include those that are pharmaceutically acceptable for veterinary use as well as for human use.

By "pharmaceutically acceptable salt" of a compound is meant a salt that is pharmaceutically acceptable as defined herein and has the desired pharmacological activity of the parent compound. Such salts include: acid addition salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like; or an acid addition salt formed with an organic acid such as acetic acid, benzenesulfonic acid, benzoic acid, camphorsulfonic acid, citric acid, ethanesulfonic acid, fumaric acid, glucoheptonic acid, gluconic acid, glutamic acid, glycolic acid, hydroxynaphthoic acid, 2-hydroxyethanesulfonic acid, lactic acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, muconic acid, 2-naphthalenesulfonic acid, propionic acid, salicylic acid, succinic acid, tartaric acid, p-toluenesulfonic acid, trimethylacetic acid, and the like; or salts formed when an acidic proton present in the parent compound is replaced by a metal ion, such as an alkali metal ion, an alkaline earth metal ion, or an aluminum ion; or a complex formed with an organic or inorganic base. Acceptable organic bases include diethanolamine, ethanolamine, N-methylglucamine, triethanolamine, trimethylamine, tris (hydroxymethyl) aminomethane and the like. Acceptable inorganic bases include aluminum hydroxide, calcium hydroxide, potassium hydroxide, sodium carbonate, and sodium hydroxide. Preferred pharmaceutically acceptable salts are those formed from acetic acid, hydrochloric acid, sulfuric acid, methanesulfonic acid, maleic acid, phosphoric acid, tartaric acid, citric acid, sodium, potassium, calcium, zinc and magnesium. All references to pharmaceutically acceptable salts include the solvent addition forms (solvates) or crystal forms (polymorphs) of the same acid addition salt as defined herein. In general, when a particular salt is included in a structure or formula herein, it is understood that other pharmaceutically acceptable salts may be substituted within the scope of the present disclosure, for example, in the case of the quaternary ammonium salt of formula VIII, a chloride or another negative ion or combination of ions may be included, and similarly, in the carboxymethyl sodium salt of formula IX, the sodium shown may be substituted with another positive ion.

"protecting group" or "protecting group" means a group that selectively blocks one reactive site in a multifunctional compound such that a chemical reaction can be selectively carried out at another unprotected reactive site in the sense normally associated therewith in synthetic chemistry. Certain processes of the present disclosure rely on protecting groups to block reactive nitrogen and/or oxygen atoms present in the reactants. For example, the terms "amino protecting group" and "nitrogen protecting group" are used interchangeably herein and refer to those organic groups intended to protect a nitrogen atom from undesirable reactions during synthesis. Exemplary nitrogen protecting groups include, but are not limited to, trifluoroacetyl, acetamido, benzyl (Bn), benzyloxycarbonyl (benzyloxycarbonyl, CBZ), p-methoxybenzyloxycarbonyl, p-nitrobenzyloxycarbonyl, tert-Butoxycarbonyl (BOC), and the like. One skilled in the art will know how to select groups so as to be easily removed and have the ability to withstand subsequent reactions.

"subject" refers to mammals and non-mammals. Mammal means any member of the mammalia class, including but not limited to humans; non-human primates, such as chimpanzees and other apes and monkey species; farm animals such as cows, horses, sheep, goats, and pigs; domestic animals such as rabbits, dogs, and cats; laboratory animals, including rodents, such as rats, mice and guinea pigs; and the like. Examples of non-mammals include, but are not limited to, birds and the like. The term "subject" does not denote a particular age or gender.

By "therapeutically effective amount" is meant an amount of a compound that, when administered to a subject to treat a disease state, is sufficient to effect such treatment of the disease state. The "therapeutically effective amount" will vary depending on the compound, the disease state being treated, the severity of the disease being treated, the age and relative health of the subject, the route and form of administration, the judgment of the attending physician or veterinarian, and other factors.

When referring to a variable, the terms "those defined above" and "those defined herein" are incorporated by reference into the broad definition of the variable as well as the preferred, more preferred and most preferred definitions (if any).

"treatment (treatment) of a disease state" includes: (i) preventing a disease state, i.e., such that clinical symptoms of the disease state do not develop in a subject who may be exposed to or predisposed to the disease state, but do not yet experience or display symptoms of the disease state; (ii) inhibiting the disease state, i.e., arresting the development of the disease state or its clinical symptoms; or (iii) relieving the disease state, i.e., causing temporary or permanent regression of the disease state or its clinical symptoms.

In the structures herein, any open valency appearing on a carbon, oxygen, sulfur or nitrogen atom indicates the presence of a hydrogen atom.

Examples

Example 1 solubilization of Compounds by HP β CD

Example 1The ability of HP β CD (DS4.5) monomer to solubilize various sterols, vitamins, oxyputrols and steroid hormones was demonstrated (fig. 2A-B). Lower turbidity indicates greater ability to solubilize a given sterol. FIGS. 2A-B show the solubilization of various sterols and sterol derivatives by HP β CD (DS4.5) monomer by relative turbidity assessment.

We also tested variants of HP β CD by testing the number of a series of hydroxypropyl groups on HP β CD. The range we tested is 3.7 to 21 (maximum number of possible substitutions). Although the data is noisy, the ability to solubilize 7KC and cholesterol was reduced with greater degree of substitution (fig. 2C-2D). This is supported by the widely substituted molecular docking on the monomeric HP β CD (fig. 2E). Monomers and sterols were designed in PyMOL based on known chemical characteristics. The most likely position of each hydroxypropyl was used and the top 20 conformations were considered in determining the affinity score for each pair. If any atom of the sterol passes through the plane formed by the O4 oxygen of the cyclodextrin, the conformation is included in the calculation. HP β CDs with lower DS showed a preference for solubilization of 7KC over cholesterol, indicating that they are specific for 7 KC. Without being bound by theory, a possible explanation is the availability of the maximum number of hydroxyl groups at position 7 on the 7KC to hydrogen bond with a ketone group, which theory is not required to practice the present invention.

Example 2 computational modeling of the interaction of Cyclodextrin monomers and dimers with Cholesterol and 7KC

SUMMARY

This example describes molecular modeling and computational simulations aimed at exploring the mechanism of CD binding to sterols, predicting the relative binding capacity of cyclodextrin dimers to cholesterol and 7KC, and identifying cyclodextrin dimers predicted to have a higher affinity for 7KC than cholesterol. It is speculated that the configuration in which the sterol is completely surrounded by CD or CD dimers isolates the hydrophobic sterol from the hydrophilic solvent, thereby bringing the sterol into solution.

For initial docking analysis (FIG. 2E [ monomer)]4B [ dimer ]]) Using a computer modeling program PyMOL (PyMOL molecular graphics System, version 2.0)LLC.) construction of HP β CD monomers and dimers at various levels of substitution, and then use the expansion package AutoDock Vina (Trott [ et al.)]C omput. chem., 31 (2): 455-61.(2010)) (developed by the Scripps Research Institute (La Jolla, Calif., USA)) to mimic the interaction between these putative CD molecules and 7KC or cholesterol. Autodock Vina is a molecular docking software with significant accuracy and speed improvements over previous Autodock 4. The software can predict non-covalent binding between molecules, approximating the standard chemical potential of the system using scoring functions to predict energetically favorable conformations and binding affinities. It was generally found that hydroxypropyl dimer and monomers of DS 2-6 showed the best specificity for 7 KC.

Molecular dynamics simulations were performed using GROMACS 2018(University of grongen, Netherlands; Bekker [ et al ], World Scientific (1993); and Berendsen [ et al ], comp.phs.comm., 91: 43-56.(1995), et al), and docking simulations of binding of 7KC or cholesterol by three derivatives of β -cyclodextrin were also performed using AutoDock Vina: natural monomer (DS0) beta-cyclodextrin (β CD), monomeric hydroxypropyl- β -cyclodextrin (DS5, HP β CD) and dimeric DS5 hydroxypropyl- β -cyclodextrin, where two HP β CD monomers are linked via a butyl chain through the O2 oxygen of DS2 monomer and the O3 oxygen of DS3 monomer, thus the total DS is 5. Both ligands are asymmetric, so both up and down orientations of the ligands are simulated. These simulations were then repeated in the AMBER force field and at the translation position to determine which position/force field produced the most informative data for these novel molecules (initial MD analysis, fig. 4D-MM). It has been determined that the GROMOS force field at the initial position most effectively captures CD dimer interactions with sterols, and therefore this force field and position are used for subsequent abbreviated MD modeling of other CD dimers (subsequent MD analysis, FIG. 4 NN-SS; 5B-C; 6B-7B).

In general, it was found that the addition of hydroxypropyl resulted in complex instability, but it was more specific for 7KC than cholesterol than was observed for native unsubstituted β CD. This is because 7KC can form and reform some stable complex in both the up and down orientations, while cholesterol is less likely to form a stable complex, probably because β CD does not appear to be fully encapsulated by β CD as 7KC does, especially in the down orientation. Dimerization of β CDs significantly increases the affinity for sterol targets (such as 7KC and cholesterol). This is clearly seen by the formation of a stable dimer complex (all ligands and orientations have strong interaction energies) in which the ligand nests inside the hydrophobic core of the CD dimer, thus allowing the ligand to be solubilized in aqueous solution.

To further analyze the effect of small modifications on β CD dimers, additional docking and molecular dynamics simulations were performed for various linkers and degrees of substitution for HP β CD (fig. 8). We extended the analysis to include other selected substitution types and other selected linkers (fig. 9) and found that among the tested, in general, DS showed the best specificity for 7KC at-2-6, applicable to a wide range of substitution and linker types.

Based on this extended computational analysis, we believe that dimerization of β CDs is critical for forming a robust soluble complex with sterols, regardless of the type or position of substituents or linkers used. Extensive dimerized β CD molecules have been tested and show that for many forms of substitution and linker, even though they are chemically very different from each other compared to the β CD monomeric form, a higher affinity for sterols is maintained.

Calculation method

Initial docking simulation

We have developed a method using AutoDock Vina that allows predictions of cyclodextrin binding to various sterols to be made on a computer more quickly and easily without analyzing the entire trajectory, which is time consuming and computationally intensive. Applying this technique to cyclodextrin systems enables us to perform hundreds of docking simulations with many different cyclodextrins we designed. This type of computational modeling presents us with possible interactions between different cyclodextrins and different sterols, while generating steric information and binding affinity data.

These conformational predictions can be modeled for a number of different sterols and/or derivatives of CD so that potential mechanical features can be revealed. We have several preliminary theories combined and we wish to use computational techniques for testing. We developed different models for HP β CD to test our binding theory:

Monomer-sterol association: we tested the affinity of the monomers to sterols for comparison to association with dimers to help determine whether sterols are more likely to bind to monomers or dimers of HP β CD and whether these monomers exhibit specificity for 7KC or cholesterol (fig. 4A).

Linked dimer-sterols: to eliminate the need for multiple steps and to test new potential molecules, two monomers were covalently linked to various types of linkers and associated with sterols to investigate the affinity and specificity for these pre-linked dimers (fig. 4A).

In order to make the output of these documents comparable to each other, a scoring system was developed for the complexation with sterols, in which the most favorable affinity was adjusted depending on whether the dimers were head-to-head (where applicable) and whether the sterols were actually in the barrel of the HP β CD cavity. This number of "complex conformations" (up to twenty conformations) is then added to the absolute value of the most favorable affinity; that is, a configuration of 15/20 that produces complexation with sterols (head-to-head and/or sterols within the CD cavity) and an association with an optimal affinity of-10 kJ/mol would result in 25 points (| -10| +15 ═ 25). For this calculation, a ligand is considered to be in a complex if any atom on the ligand crosses the plane formed by the O4 atom of the CD, regardless of the angle or degree of insertion into the cavity. The resulting value is referred to as the "affinity score".

We then extended the docking analysis to include a variety of different types of substitutions (including substitutions with charged groups) and linkers to determine whether 7KC specificity is affected by these factors. Sulfobutyl and methyl substitutions with triazole and butyl linkers were tested across the entire DS range of 0-20 and showed a pattern similar to hydroxypropyl, with DS with the highest 7KC specificity being approximately 4 (fig. 5A and 6A). Thus, other cyclodextrins (such as quaternary ammonium and carboxymethylated cyclodextrins) were tested only at low DS (. about.4).

Initial molecular dynamics simulation (FIG. 4D-MM)

An initial set of simulations were performed in GROMOS 54a7 and AMBER 99SB force fields using gromac 2018(University of grongen, Netherlands) so that two repetitions of these simulations helped determine the consistency of the observed interactions. The two repeats of each of the three CD molecules and each orientation of the ligand are then repeated with a different initial structure in which the ligand is displaced to determine the dependence of these calculations on the initial structure and the force field. The resulting 48 hydroxypropyl dimer traces were then analyzed using the GROMACS tool.

Unlike docking, molecular dynamics allows the simulated molecules to interact in a time-dependent manner, rather than making a simple snapshot of the energetically favorable conformation as docking provides. For the first three CD-sterol complexes, the time for each simulation was extended to one microsecond (which is a very long time for MD simulation), providing sufficient time for the complexes to stabilize. The output was then analyzed to determine the distance between the centroid of all O4 atoms (the center of the CD cavity for dimers and monomers) and the centroid of the ligand, the angle between the vector perpendicular to the plane formed by the O4 atoms of the CD and the major axis of the ligand (see fig. 4C), and the lanner-jones and coulomb energy of the interaction between the cyclodextrin and the ligand.

Thus, distance represents the proximity of the ligand to the cyclodextrin, angle represents the degree of nesting of the ligand within the CD cavity, and the energy of interaction represents the strength of the interaction between the two molecules (the stronger the negative interaction energy, the stronger the interaction). Figure 4C shows how the "angle" measurement is used to determine the degree of shielding of the ligand from the surrounding water molecules: zero or 180 degrees indicates that the ligand is perfectly perpendicular to the cyclodextrin plane, while 90 degrees indicates that the ligand is parallel to the CD plane and therefore does not complex in the cavity. For these simulations, we chose 30 degrees to correspond to the initial, complexed "up" configuration (head of sterol associated with the minor surface of the CD, tail associated with the major surface, insertion of the entire ligand into the cavity of the CD) and 150 degrees to the initial, complexed "down" configuration (tail of sterol associated with the minor surface of the CD, head associated with the major surface, insertion of the entire ligand into the cavity of the CD). Note that for dimers, only the plane of one CD monomer is considered to be at the angle between CD and ligand, but if the dimer is fully formed, this plane will reflect the plane of the sister monomer.

The number of water molecules within ligand 3A over time was determined to determine the degree of screening of the ligand from the surrounding solvent by CD. It is speculated that more water molecules around the ligand will indicate that it is not sufficiently shielded by the surrounding water and therefore not in solution. All these simulations extend to 1 microsecond (1000ns), which should be long enough to accurately describe the interaction between CD and sterol.

Long-term preliminary analysis provides evidence that the simulation correctly captures the interaction of CD monomers and dimers with sterol ligands and thus can be extended to other CD monomers and dimers without resorting to such laborious methods.

Additional molecular dynamics simulation (FIGS. 4NN-SS, 5B-C, 6B-C, 7A-B, 8H-I)

From the initial HP β CD simulations, it was concluded that the GROMOS force field in the non-translated position produced the best, most dynamic results for these complexes. This lengthy initial analysis is important to establish precedent for modeling these new molecules, and therefore allows for shorter, more targeted simulations of other types of dimers. Therefore, extension of molecular dynamics analysis with various types of linkers and substitutions has shown promise. First, a docking calculation was performed for the DS range of methyl (fig. 5A) and sulfobutyl (fig. 6A) β CD dimers. This indicates that low DS (-4) shows the most promising results in terms of dimers with the best 7KC specificity. Thus, additional MD simulations were performed for the DS4 β CD dimer with triazole and butyl linker (FIGS. 4RR-SS, 5B-C, 6B-C, 7A-B). We also simulated the DS0 β CD dimer (FIG. 4 NN-QQ). These simulations were run for 100ns and only the angle and energy of interaction were analyzed to assess the major difference or similarity between these molecular interactions and the interaction with the butyl-linked hydroxypropyl dimer.

Additional docking simulation

After preliminary simulations demonstrated similar prospects for substitution and the range of possible linkers, the same docking techniques described above were used to screen for more linkers, substitutions, and even substitution positions. This analysis indicates that the actual dimerization of β CDs, regardless of linker or substitution type, largely, if not completely, conveys the effectiveness of these molecules.

Calculation results and conclusions

Butt joint:

we first examined whether HP β CD can bind cholesterol and 7KC as a monomer (fig. 2E), and then we examined whether HP β CD can bind cholesterol and 7KC as a dimer (fig. 4B).

We found that HP β CD monomer (fig. 2E) has high affinity for both cholesterol and 7KC at low Degree of Substitution (DS), but appears to have reduced affinity for both sterols with increasing DS. This is probably due to the crowding of the hydroxypropyl group, which does not allow the sterol to enter the core of the monomer. In addition, fewer hydroxyl groups on the inner surface of the CD are available for hydrogen bonding to carbonyl groups on the 7 KC. The best specificity (rather than the best affinity) was considered as a superscript of DS4, with preference for 7KC extending from DS2 to DS6 and conversion to cholesterol for DS7 and greater. After DS10, little affinity was observed in these models.

The butyl-linked dimer showed higher affinity for sterols compared to monomeric CD, with optimal affinity/specificity for 7KC at dimerized DS10 and DS4 (fig. 4B). However, for these calculations, this specificity appears to exist only in the dimer of a particular DS, and the variation between different DSs shown in these calculations is very important. Triazole-linked dimers generally showed better specificity, with similar affinity to butyl-linked dimers, except at DS 6. It is speculated that this specificity is due to additional hydrogen bonding with the 7KC between the nitrogen contributing hydrogen bond and the hydrogen bond accepting ketone of the 7 KC.

Initial molecular dynamics analysis:

FIG. 4D-O supports the following assumptions: the native (unsubstituted, DS0) monomer CD is capable of complexing with 7KC and cholesterol in both the up and down orientations, although 7KC remains more stable complexed in the down orientation than cholesterol, and vice versa in the up orientation. Cholesterol showed a small change throughout the upward trajectory, showing how cholesterol exited the CD multiple times in the upward orientation and re-associated with it (note the large angle change at approximately 150ns around which cholesterol rotated to associate in the opposite orientation) (fig. 4D). This angular change indicates that the downward orientation is significantly more stable, so that cholesterol leaves the lumen and rotates 180 degrees before reassociating, and the overall affinity for cholesterol is very high, as it is able to accomplish this large movement in the simulation.

On the other hand, once the complex breaks in either orientation, the 7KC does not re-associate, but the downward orientation is significantly more stable at more than half the trajectory, supporting the assumption that the 7KC favors the downward orientation. This indicates that both 7KC and cholesterol favor downward orientation, with the head group associated with the major surface and the tail associated with the minor surface, but in this favorable conformation, only cholesterol is able to actually leave and re-associate with the CD. This may explain why native CD solubilizes cholesterol and its derivatives well, but does not show specificity for 7 KC. This slight preference of the natural monomer β CD for cholesterol was expected and was consistent with published experimental results (Zidovetzki et al, biochim. biophysis. acta., 1768 (6): 1311-1324.(2007)), and was further enhanced by the number of water molecules around the ligand (fig. 4E); cholesterol is much less hydrated than 7KC, especially in the "up" orientation.

AMBER force fields (fig. 4G-4I) showed a significant enhancement of the interaction between native β CD and sterols. Over the entire track, both ligands in both orientations remain inside the cyclodextrin ring, and little preference for 7KC or cholesterol was observed. The AMBER force field shows a stronger, longer interaction between the two molecules compared to the GROMOS force field, and the solubilization of sterols by native β CD in AMBER force field appears to be the same in both up and down orientation between the two ligands. Despite this strong and stable interaction, the AMBER force field may not be able to fully capture the interaction between β CD and sterol because the complex is not broken at all. To fully elucidate the interactions that occur, some movement must be made, but this is good evidence that a strong complex is indeed formed between the two molecules.

Even when the ligand translates deeper within the CD cavity (fig. 4J-O), the native complex is still effectively formed in both force fields, albeit less consistent for GROMOS than for AMBER. The GROMOS force field showed significant preference for the "up" orientation of 7KC and for the "down" orientation of cholesterol, whereas only AMBER showed strong interactions between the two ligands and CD. This suggests that 7KC and cholesterol have similar and strong interactions with native β CD, which is consistent with experimental data, but the orientation of the ligands does appear to differ in the observed complexation. These slight differences in trajectory are detailed below.

Monomer DS5 HP β CD (fig. 4P-AA) showed a CD and sterol interaction in the GROMOS force field that is not consistent with native CD, but, as shown in fig. 4P, also appeared to favor the downward orientation of 7 KC. The AMBER force fields (fig. 4S, Y) again show stronger, more consistent interactions, but the stable complexes formed in both force fields remain the same. In summary, we can see that the addition of hydroxypropyl groups to cyclodextrin monomers makes the two ligands less likely to form complexes in both force fields, but 7KC is able to form and reform stable complexes more stably than cholesterol. In general, cholesterol appears to form complexes with HP β CD less readily than 7KC for both force fields, and more water molecules are able to contact cholesterol, indicating the preference of HP β CD for 7 KC. This is clear in fig. 4R, since the 7KC complex is formed and reformed in a "down" orientation, while cholesterol is not complexed. The visual trace also shows how 7KC strongly favors the "up" orientation, but complexes are still formed in the "down" orientation of about 500 ns.

As the ligand translates, HP β CD can complex with both sterols more efficiently as the original position of the ligand is embedded deeper into the cavity of the CD. For this translational trajectory in GROMOS (fig. 4V), the preference for 7KC over cholesterol was more pronounced compared to previous simulations, since 7KC was able to form a stable complex in both orientations, whereas cholesterol could only form a stable complex in the "down" orientation. Furthermore, the upwardly oriented 7KC starts outside the cavity and is able to associate with the cavity and form a very stable complex within 300 ns. The AMBER force field again showed that the interaction between HP β CD and sterol was significantly stronger, but still formed the same stable complex and favoured the upward orientation of both ligands, and overall slightly less water surrounding 7KC over the entire track (see figure 4T). Presumably, this is because the "down" orientation shows more of the head group of the sterol protruding from the lumen than the "up" orientation. This is consistent with our experimental data (fig. 2) as it has been shown that HP β CD monomer has some specificity for 7KC while still forming a stable, apparently soluble complex with 7KC and cholesterol. All of these simulations will be described in detail below.

Our novel butyl-linked DS5 hydroxypropyl- β -cyclodextrin dimer was then modeled with 7KC and cholesterol in GROMOS and AMBER force fields as shown in fig. 4 BB-MM. Comparison of these traces with the monomeric HP β CD and native β CD traces provides clear evidence that the dimeric form binds sterols significantly more reliably than its corresponding hydroxypropylated or non-hydroxypropylated monomer. This is consistent with our experimental data (fig. 16). The angles, distances and energies around the ligand, as well as the water molecules, are all more stable and in a significantly more solubilizing configuration than the monomer simulation. The GROMOS force field showed less than five angstroms between the ligand centroid and the CD when the complex was fully formed in the downward orientation (fig. 4BB), while the monomers in the GROMOS force field consistently showed an upward orientation of 5-10 angstroms between the molecules when the complex was formed. The AMBER force field also shows a very strong interaction between the sterol and dimerized CD, with the energy of the interaction approaching-300 kJ/mol in the downward orientation compared to the monomer at about-150 kJ/mol (fig. 4 BB). This suggests that the dimer forms a very strong, stable complex with both ligands, especially in the down orientation, and especially compared to monomeric β CD.

AMBER force field results (fig. 4EE, KK) support the finding of the GROMOS force field simulation that dimerization of HP β CD produces stronger, more stable interactions between CD and sterols, the distance between the two molecules is small, and the interaction energy is large. Dimerized CDs also consistently showed less than five water molecules around the ligand, particularly in the downward orientation, while monomeric CDs showed ten water molecules around the ligand in the upward orientation (fig. 4 CC). While this can sometimes be achieved for monomers with 7KC and cholesterol, the overall presence of water around the sterol has been greatly reduced by dimerization. Dimerization of HP β CDs also delivers some specificity for 7KC, which is evident because 7KC remains associated with at least one of the two linked CDs throughout the entire track, regardless of force field or translation, while cholesterol typically dissociates from the two monomers in at least a portion of the track and even forms a distorted head-to-tail dimer configuration in which cholesterol cannot be fully encapsulated by dimers. These trajectories will be described in detail in the next section.

These simulations provide strong evidence that dimerization of HP β CD promotes complexation with sterols by forming an encapsulation complex that isolates hydrophobic sterols from surrounding water molecules. The data indicate that dimeric HP β CD has overall higher sterol affinity than the monomer, and it has a preference for 7KC, since the association time of 7KC with at least one CD is significantly longer than cholesterol. We can conclude from this approach that while strong complex formation in the AMBER force field is good evidence of the rationality of our complex formation and stability, more valuable information can be gathered from the GROMOS force field. This is because, unlike AMBER, the GROMOS force field shows dynamic interactions between molecules, not just an incredibly (possibly impractical) stable complex.

Details of the 48 traces of hydroxypropyl- β -cyclodextrin dimer are described below, with each trace being one microsecond in length.

Detailed description of initial molecular kinetic trajectories (fig. 4):

native monomers β CD and 7KC, upward orientation, GROMOS force field:

in fig. 4F, the 7KC begins with the cephalad inserted into the CD cavity and the caudal of the extended subsurface. At 134ns, the complex breaks and the 7KC moves towards the sub-surface, spinning out of the cavity. It then remains associated with the secondary surface, moving the head group into and out of the cavity until the complex is fully dissociated at 150ns and the 7KC moves around the frame, re-associating with the primary surface. The 7KC continues to associate and disassociate with the major surface but does not re-enter the cavity in the remainder of the track.

Natural monomers β CD and cholesterol, oriented upwards, GROMOS force field:

4F shows that cholesterol (up) starts in the tail, is inserted into the CD cavity, and the cephalad extends out of the subsurface. The complex breaks at about 150ns and the "angle" of the cholesterol changes greatly because the cholesterol leaves the lumen and spins outward, parallel to the cyclodextrin, then reassociates in the opposite direction, with the tail extending out of the subsurface. Cholesterol then reinserts into the head group and cycles around 200ns between insertion into the head group and becoming parallel to the CD, which is seen in fig. 4D as a change in angle, energy and distance (upward) of cholesterol. At about 300ns, the complex completely fragmented (corresponding to the labeling of cholesterol in fig. 4D), and cholesterol randomly moved around the CD molecule. The two molecules are briefly re-associated for about one nanosecond at 310ns, where the cholesterol is parallel to the major surface of the CD. The cholesterol then resumes random movement until it is re-associated with the subsurface for about two nanoseconds at 330ns, while the cholesterol tail is loosely inserted into the CD cavity. The cholesterol is then flipped at about 400ns to associate the head group with the CD cavity, which configuration remains relatively stable with periodic association and dissociation of the head group until the complex is again broken at about 560 ns. At this point, cholesterol transiently moves randomly around the CD, and then the tail is associated with the subsurface of the CD. At 580ns, the tail of cholesterol is tightly inserted into the CD molecule, with its head group extending from the CD subsurface. The complex then breaks again at 582ns, up to 610ns, at which point the complex is re-formed again by the head group inserted from the subsurface. The complex breaks again at approximately 680ns, reforms at 750ns, then breaks again at 880ns, reforms at 920ns, and then continues to break and reform approximately every 10ns (but always associates as shown by 920 ns) until the trajectory ends. The fact that cholesterol completely leaves the CD cavity and then reassociates within the simulated time indicates that the procedure is able to self-associate the two molecules without being affected by any external environment. This provides strong evidence that this interaction is reasonable, recurring, and can be captured effectively by simulation.

Native monomers β CD and 7KC, down-oriented, GROMOS force field:

the 7KC begins with the tail inserted into the CD cavity and the head base extending beyond the major surface in fig. 4F. The complex remains in this conformation as the 7KC moves and tilts back and forth in the lumen. Complex fracture did not occur until 600ns, at which point the 7KC quickly exited the cavity and rotated to the subsurface. The 7KC continued to float around the mock frame, briefly binding to CD in the conformation periodically, similar to 720 ns. In general, the complex will remain in the dissociated state until the end of the simulation. Despite this dissociation, the complex was stable for 600ns, indicating that once 7KC entered the CD cavity, it was held in the CD cavity by the interaction force. This trace can be quantified in fig. 4D, since the 7KC (up) curve remains relatively flat until about 600ns, which is where the complex breaks and assumes random movement.

Natural monomers β CD and cholesterol, down-oriented, GROMOS force field:

in fig. 4F, cholesterol begins in the downward position at the head base inside the CD cavity and the tail extending out of the subsurface. This remained stable until about 125ns of cholesterol was rotated out of the cavity, but cholesterol continued to insert the head base periodically from the subsurface into the CD cavity for the next 200 ns. At about 340ns, the complex completely breaks down and the cholesterol flies around the mock frame until re-associating with the subsurface at about 560ns in the same manner as before. Then, cholesterol dissociates and re-associates parallel to the major surface after about 30 ns. The cholesterol then oscillates between associating with the major surface in this manner and randomly floating throughout the remainder of the trajectory.

Natural monomers β CD and 7KC, upward orientation, AMBER force field:

the interactions seen in the AMBER force field of fig. 4I support a strong solubilization of sterols by the native monomer β CD. Both ligands in both orientations remain within the cyclodextrin ring throughout the entire track, with little preference for 7KC or cholesterol. The 7KC (up) starts at the center of the molecule within the CD cavity, with the head group extending slightly beyond the minor surface and the tail group extending slightly beyond the major surface. The 7KC remained tightly fitting within the CD cavity throughout the track, with a slight rocking back and forth visible in fig. 4G, with a slight change in the overall flat line, indicating that a stable conformation had formed and did not break. This is also consistent with experimental data, although the AMBER force field shows a stronger, longer interaction between the two molecules than the GROMOS force field.

Natural monomers β CD and cholesterol, upward orientation, AMBER force field:

cholesterol starts from the center of the molecule within the CD cavity (up) and the head group extends slightly from the minor surface, while the tail group extends slightly from the major surface in fig. 4I. The composite remains stable throughout the track; cholesterol never leaves the lumen or changes orientation, it simply sways back and forth within the lumen. These small position changes correspond to the small projections in fig. 4G, in particular the angular cross-section.

Natural monomers β CD and 7KC, down orientation, AMBER force field:

starting at the center of the molecule within the CD cavity, the 7KC (down) in fig. 4I, extends slightly beyond the major surface at the head and slightly beyond the minor surface at the tail. The composite remains stable throughout the track; the 7KC never leaves the cavity or changes orientation, it simply sways back and forth within the cavity. These small position changes correspond to the small projections in fig. 4G, in particular the angular cross-section.

Natural monomers β CD and cholesterol, down-oriented, AMBER force field:

figure 4I shows cholesterol (down) starting from the center of the molecule within the CD cavity, with the head group extending slightly beyond the major surface and the tail group extending slightly beyond the minor surface. The composite remains stable throughout the track; cholesterol never leaves the lumen or changes orientation, it simply sways back and forth within the lumen. These small position changes correspond to the small projections in fig. 4G, in particular the angular cross-section.

Translated native monomers β CD and 7KC, upward orientation, GROMOS force field:

in fig. 4L, the 7KC starts at the molecular center within the CD cavity, with the head group extending slightly beyond the minor surface and the tail group extending slightly beyond the major surface. When the 7KC is removed from the secondary surface and rotated to associate parallel to the secondary surface, the composite will remain stable until about 710 ns. The 7KC is then fully rotated to insert the head base such that the head base extends toward the major surface at 715ns and the tail extends from the minor surface. The 7KC then associated and dissociated the head group several times with the CD cavity until the complex completely broke at about 850 ns. In the remainder of the trajectory, the complex remains in the dissociated state.

Translated native monomers β CD and cholesterol, oriented upwards, GROMOS force field:

fig. 4L shows that cholesterol begins to associate with the CD, with the head group extending out of the minor surface and the tail extending from the major surface. When cholesterol moves to the CD major surface, the complex dissociates at about 120ns, inserts into the head group, and rotates in and out of the cavity on the major side until it dissociates completely again at about 160 ns. At about 163ns, cholesterol re-associates with the minor surface portion until after 5ns it rotates back to the major surface. The cholesterol is then switched between sub-or major surface association and random movement until the complex reforms at the very end of the trajectory for the last three nanoseconds. This continuous formation and deformation of the complex on the computer indicates that it has a strong tendency to form in reality.

Translated native monomers β CD and 7KC, down-oriented, GROMOS force field:

fig. 4L shows 7KC starting from the downward position with the head base extending out of the main surface. At 40ns, 7KC exited the cavity and was associated parallel to the minor surface, reinserted the headgroup after 2ns, and then exited again. The complex completely broke at 45ns, when 7KC floated around the mock frame and re-associated with the major surface at 47ns, transiently inserted the headgroup, and then rotated back parallel to the surface until the complex again broke at 51 ns. The complex reforms at 210ns with the head group inserted from the subsurface and the tail extending outward, as in the original conformation, and when 7KC is again withdrawn from the CD and associated parallel to the subsurface, the complex remains stable up to 268 ns. The complex again breaks completely, but reforms briefly at 360 ns. Thereafter, the 7KC occasionally associates parallel to one of the two surfaces in the conformation as at 710ns, but does not re-enter the CD cavity. The trace is somewhat blurred because the 7KC only associates with the cavity within 100ns, but the complex is still free to form in the simulation, suggesting that it is likely to form in reality, even though the interaction forces seem to be less consistent.

Translated native monomers β CD and cholesterol, down-oriented, GROMOS force field:

in fig. 4L, cholesterol begins with a head group associated with the major flank, and the tail extends out of the minor surface. Cholesterol swings left and right in the lumen until the complex breaks at about 15 ns. Cholesterol reinserts into the head group at 17ns and continues to rotate between parallel to the minor surface of the CD and inserted into the cavity from the minor side (always the head group) until the complex is truly dissociated at about 675 ns. This suggests a strong interaction and tendency of cholesterol to form a superficially stable complex with native β CD, but the complex does not re-associate once cholesterol is completely dissociated from the CD subsurface at 675 ns.

Translational natural monomers β CD and 7KC, upward orientation, AMBER force field:

in fig. 4O, 7KC begins with a head base extending from the minor surface and a tail extending from the major surface. The composite remained stable throughout the track, but the 7KC did show greater curvature than the downward orientation-in most of the track, the 7KC remained curved around the CD ring.

Translated native monomers β CD and cholesterol, upward orientation, AMBER force field:

fig. 4O shows how cholesterol starts extending from the head base out of the minor surface and the tail out of the major surface. The composite remains stable throughout the track. Cholesterol does not substantially move back and forth in the cavity, but the angle within the cavity remains relatively constant.

Translated native monomers β CD and 7KC, downward orientation, AMBER force field:

the 7KC begins with a head base extending from the major surface and a tail extending from the minor surface, as shown in fig. 4O. The composite remained stable throughout the track and the 7KC did not bend significantly within the CD cavity as shown by the horizontal and stable curves in fig. 4M.

Translated natural monomers β CD and cholesterol, down-oriented, AMBER force field:

fig. 4O shows how cholesterol starts at the head base extending out of the major surface and the tail extending out of the minor surface. The complex remained stable throughout the track and cholesterol did not bend significantly within the CD cavity as shown by the level and stability curves in figure 4M.

Monomeric hydroxypropyl β CD and 7KC, oriented upwards, GROMOS force field:

the 7KC begins in an upward position (fig. 4R) with a tail inside the HP β CD cavity and a head extending from the minor surface. At about 13ns, 7KC rotated out of the sub-surface and association proceeded parallel to the surface. At 28ns, the head group of 7KC reassociates in the cavity, but rotates back multiple times, keeping the 7KC associated parallel to the subsurface, until the complex is completely broken about 47 ns. The 7KC then rotates between association parallel to one surface or random movement around the frame until the rest of the trajectory. No stable complex was formed.

Monomeric hydroxypropyl β CD and cholesterol, oriented upward, GROMOS force field:

in fig. 4R, cholesterol begins with a tail inserted into the CD cavity and a head group extending from the subsurface. The complex will remain stable for up to about 3ns as the cholesterol is spun off the secondary surface, parallel to the CD, and then around the primary surface for another 7 ns. Cholesterol then moves randomly around the mock frame, occasionally associated parallel to the major or minor surfaces, but it has not been stable within the cavity except for a brief time of about 300 ns. This lack of strong association is clearly seen by the strong change in fig. 4P and supported by experimental evidence.

Monomeric hydroxypropyl β CD and 7KC, oriented downward, GROMOS force field:

fig. 4R shows that the 7KC starts slightly from the outside of the cell cavity and initially flies randomly around the simulation box. At 29ns, 7KC had associated the head group within the HP β CD cavity, with the tail extending from the subsurface. This state remains stable until 35ns when the complex is completely dissociated. The complex remains dissociated for up to 320ns, and re-forms with the head group in the cavity and the tail extending out of the subsurface. Complexes remain associated until about 470ns, dissociate again until the end of the trajectory.

Monomeric hydroxypropyl β CD and cholesterol, oriented downward, GROMOS force field:

figure 4R shows cholesterol in a downward position starting with a tail inserted into the lumen and a head extending beyond the major surface. The complex remains stable until about 300ns, when cholesterol is spun out of the subsurface and associates parallel to the CD, and then the complex breaks completely and dissociates from the CD. Cholesterol then migrates around the CD, sometimes associating parallel to the minor surface, and finally associating with the major surface at about 100 ns. Cholesterol then continues to move randomly around the CD, sometimes associating or rotating with the surface, just as it enters the cavity, similar to the conformation at 275ns, but cholesterol does not completely re-enter the cavity at any appreciable time. These traces indicate a preference for upward orientation, where the only stable complexes formed are 7 KC-up (formed independently in the simulation after complete dissociation), and cholesterol-down (stable from the original conformation). This indicates a strong preference for 7KC in the upward orientation, and interaction with cholesterol in the downward orientation.

Monomeric hydroxypropyl β CD and 7KC, upward orientation, AMBER force field:

the 7KC (up) starts at the center of the molecule within the CD cavity and extends slightly beyond the head group of the minor surface, while the tail group extends slightly beyond the major surface. Fig. 4U shows how 7KC remains in the cavity of the HP β CD throughout the track, swinging slightly up and down, but never extending either end far out of the cavity. The complex is not destroyed.

Monomeric hydroxypropyl β CD and cholesterol, upward orientation, AMBER force field:

the AMBER force field showed more consistent interactions and a more stable complex compared to the GROMOS force fields of native and HP β CD. In fig. 4U, cholesterol (up) starts at the center of the molecule within the CD cavity and extends slightly beyond the head group of the minor surface, while the tail group extends slightly beyond the major surface. The composite remains stable throughout the track; cholesterol never leaves the lumen or changes orientation, it simply sways back and forth within the lumen. As shown in FIG. 4S, the most favorable conformation occurs for 500- "700 ns, but cholesterol and CD are still complexed throughout the entire track. These small position changes correspond to the small projections in fig. 4S, especially in the angular cross-section.

Monomeric hydroxypropyl β CD and 7KC, downward orientation, AMBER force field:

the 7KC (down) starts at the center of the molecule within the CD cavity and extends slightly beyond the head group of the major surface, while the tail group extends slightly beyond the minor surface. Fig. 4U shows how the 7KC head protrudes from the cavity rather than being in an upward orientation, but the composite remains intact throughout the track. This preference for upward orientation can be seen in fig. 4S, as the "up" plot changes less than the "down" plot, but in GROMOS, both still vary significantly less than the HP β CD.

Monomeric hydroxypropyl β CD and cholesterol, oriented downward, AMBER force field:

figure 4U shows that cholesterol (down) starts inside the CD cavity and extends out of the head group of the major surface, while the tail group extends slightly out of the minor surface. Notably, the head group of cholesterol sometimes extends far beyond the abluminal in the upward orientation, but the complex remains stable throughout the trajectory. Cholesterol never leaves the lumen completely or changes orientation. These small position changes correspond to the small projections in fig. 4S, in particular the angular cross-section. The lateral movement through the CD cavity is significantly more and the radial shaking is also less compared to other composites.

Translated monomers hydroxypropyl β CD and 7KC, oriented upwards, GROMOS force field:

fig. 4X shows that 7KC translated in an upward orientation begins with insertion into the tail of the CD cavity and the cephalad extending out of the subsurface. The 7KC was spun out of the cavity at about 105ns, then the 7KC oscillated about every 5-10ns between insertion of the head base into the CD and parallel to the CD, seemed to take more time in the conformation of the head base in the cavity. At about 415ns, the structure will sit with the inserted head group until it breaks again and dissociates completely at 700 ns. Then, when the head group of 7KC inserts itself into the large surface of the CD, the complex remains dissociated in the rest of the track, except for a short reassociation at 726 ns. The interaction energy here is temporally comparable to the interaction energy at 400ns for complex formation. Since the complex appears to be easily formed and broken, this interaction is likely to be real, strong, and can be captured by simulation.

Translated monomers hydroxypropyl β CD and cholesterol, oriented upwards, GROMOS force field:

cholesterol begins at the head base inserted into the lumen and at the tail extending out of the major surface. The complex stabilizes for 60ns until cholesterol is spun off the subsurface and associates parallel to the CD. Cholesterol then completely exited the CD and randomly moved around the mock frame until the tail re-associated with the CD cavity at about 215ns, with the head group extending from the subsurface again. This remained stable for about 30ns until cholesterol again exited the CD, then rapidly re-associating the head group in the CD cavity at 280ns, this time with the head group in the cavity and the tail extending from the subsurface. The complex remains stable in the rest of the trajectory. This indicates that the complex formed at the end of the trace is very stable and is likely to form as shown in figure 4X.

Translated monomers hydroxypropyl β CD and 7KC, down-oriented, GROMOS force field:

7KC translated in a downward orientation, starting with the cephalad inserted into the CD cavity and the caudal extending out of the subsurface. The 7KC was spun out of the cavity at about 105ns, then the 7KC oscillated about every 5-10ns between insertion of the head base into the CD and parallel to the CD, seemed to take more time in the conformation of the head base in the cavity. At about 415ns, the structure will sit with the inserted head group until it breaks again and dissociates completely at 700 ns. Then, in the rest of the trajectory, the complex remains dissociated, as shown in fig. 4X.

Translated monomer hydroxypropyl β CD and cholesterol, down-oriented, GROMOS force field:

in the downward orientation of the translated cholesterol of the HP β CD, cholesterol begins with a tail inserted into the CD cavity and a head base extending out of the major surface. The complex breaks at 50ns, but cholesterol remains associated with the major surface, and the tails periodically enter and leave the cavity before completely dissociating at 88 ns. The cholesterol molecules then associate with the minor side of the CD, and then resume random movement around the mock box. The trajectory cycles between association with one of the two surfaces and random movement until 215ns, at which time the cholesterol tail re-enters the cavity from the major side for the next 25 ns. Cholesterol then resumes random movement around the CD. At 275 nanoseconds, the head group of cholesterol enters the cavity from the major surface and remains there until the complex is completely dissociated at about 410 ns. At this point, the cholesterol moves randomly around the mock frame until approximately 490ns when the cholesterol rotates to the subsurface and inserts the head base into the cavity. When cholesterol is removed from the cavity, spun and reinserted with its tail group from the subsurface into the cavity, the complex will remain in this conformation until about 530 ns. At 540ns, cholesterol had recovered random movement. Cholesterol is never reinserted into the cavity, but will typically be tightly associated with either surface of the CD. Since cholesterol does not form a stable complex with HP β CD for any significant time, the interaction between HP β CD and cholesterol appears to be transient, not as strong as the interaction between HP β CD and 7KC, even in the translated position as shown in fig. 4X.

Translational monomer hydroxypropyl β CD and 7KC, upward orientation, AMBER force field:

fig. 4AA shows that the 7KC begins with the head base extending out of the major surface and the tail of the surface facing away from the minor surface, with the center of the 7KC being located in the cavity of the CD. The composite remained stable throughout the track and the 7KC did not bend significantly within the CD cavity as shown by the horizontal and stable curves in figure 4Y.

Translated monomers hydroxypropyl β CD and cholesterol, upward orientation, AMBER force field:

fig. 4AA shows that cholesterol starts at the cephalic base extending out of the major surface and at the caudal portion of the surface facing away from the minor surface, with the center of the cholesterol being located in the cavity of the CD. The complex remains stable throughout the track and, as shown by the level and stability curves in fig. 4Y, cholesterol does not significantly bend or move around within the CD cavity.

Translational monomer hydroxypropyl β CD and 7KC, downward orientation, AMBER force field:

fig. 4AA shows that 7KC begins with the head base extending significantly beyond the major surface and the tail of the surface facing away from the minor surface, but the tail is entirely within the cavity. The composite remained stable throughout the track and the 7KC did not bend significantly within the CD cavity as shown by the horizontal and stable curves in figure 4Y. 7KC does not exhibit much more lateral motion in this orientation than in the upward orientation.

Translational monomeric hydroxypropyl β CD and cholesterol, down-oriented, AMBER force field:

fig. 4AA shows that cholesterol starts at the cephalic base extending out of the major surface and at the caudal portion of the surface facing away from the minor surface, with the center of the cholesterol being located in the cavity of the CD. The complex remains stable throughout the entire trajectory, but cholesterol does migrate significantly within the lumen, usually with only tail association and head groups extending out of the CD. This can be observed in fig. 4Y, since the downward orientation varies more than the upward orientation, especially over distance.

Dimerized hydroxypropyl β CD and 7KC, oriented upward, GROMOS force field:

in fig. 4DD, 7KC starts inside the dimer and remains a good cage. Dimer begins to stretch at about 100ns, and despite this stretching, 7KC remains in the bucket within both CDs. At 111ns the head group dissociates from its monomer (in this discussion the term "monomer" refers to the CD subunit, although it is part of a covalently linked dimer), while the tail remains associated with the cavity of the other monomer, after 5ns the head group of 7KC continues to interact with the large surface of one monomer (rather than the cavity), while the tail remains anchored in the other monomer. At 120ns, the tail releases its monomer, while the head group inserts itself into the cavity of another monomer. This configuration remains stable, with the sterol-related monomers oscillating around the empty monomers until the end of the trajectory.

Dimerized hydroxypropyl β CD and cholesterol, oriented upward, GROMOS force field:

the cholesterol (up) trajectory begins with cholesterol encapsulated in dimers. The dimer begins to bend at about 22ns, but cholesterol moves and remains in the dimer cavity. At about 200ns, the monomer associated with the cholesterol head group is cleaved and dissociates from the dimer, but cholesterol remains associated with one of the monomers (head group aligned with the minor surface and tail aligned with the major surface). This configuration remained unchanged until cholesterol was completely dissociated from the cavity and rotated towards the sub-surface at 355 ns. Then, cholesterol remains between the two monomers, occasionally with the head group loosely associated with one monomer, until it completely leaves and floats around the mock box. Cholesterol continues to interact intermittently with one of the CD monomers, but as shown in figure 4DD, the dimer-cholesterol complex is never completely reformed.

Dimerized hydroxypropyl β CD and 7KC, down-oriented, GROMOS force field:

7KC is in the down position, as shown in FIG. 4DD, and begins to remain caged inside the dimer. When one monomer is stretched away from the other, the dimer does not begin to deform until about 600ns, while 7KC remains between the two. At about 820ns, 7KC dissociates the tail from one monomer while the head group remains in the cavity of the other monomer. This configuration remains stable, with the sterol-related monomers oscillating around the empty monomers until the end of the trajectory.

Dimerized hydroxypropyl β CD and cholesterol, down-oriented, GROMOS force field:

cholesterol in the downward position (fig. 4DD) began inside the dimer cage. At about 50ns, the complex begins to stretch and twist, but cholesterol is anchored inside the dimer throughout the trajectory. This is clear in fig. 4BB, since the curve of cholesterol angle is very horizontal and stable over the entire trajectory. This is the only complex in the GROMOS force field analysis that remains intact over the entire trajectory. Figure 4BB shows the molecular dynamics analysis of our novel butyl-linked hydroxypropyl DS5 β -cyclodextrin dimer with 7KC and cholesterol to form a very stable complex. Comparison of these figures with those of the monomeric HP β CD provides clear evidence that the dimeric form consistently binds sterols more reliably and more strongly than its monomeric counterpart. The downward orientation, energy, angle and distance were all kept very consistent and varied very little, indicating that a stable, clearly soluble complex of 7KC and cholesterol was present and did not change significantly over time. The same change can also be seen in the upward direction, but the change is slightly greater, especially for cholesterol. This indicates that 7KC binds most efficiently in the downward orientation, strongly preferring this orientation as shown by the angular reversal of the upward orientation at about 350 ns. This is where 7KC leaves the dimer and reassociates in the downward orientation. Cholesterol also does this, but it is less stable than 7KC formation, indicating that cholesterol does not appear to have the ability to form a more stable down-complex from a less stable up-complex, whereas 7KC does.

Dimerized hydroxypropyl β CD and 7KC, upward orientation, AMBER force field:

fig. 4GG details how the 7KC remains nested within the cavity formed by the two monomers throughout the track. The complex is somewhat curved and the 7KC moves slightly within the cavity, but the 7KC remains complexed with the CD dimer over the entire track.

Dimerized hydroxypropyl β CD and cholesterol, upward orientation, AMBER force field:

in fig. 4GG, cholesterol remains nested between two monomers throughout the trajectory. The monomers remain associated with each other and the cholesterol-complex bends but never breaks.

Dimerized hydroxypropyl β CD and 7KC, downward orientation, AMBER force field:

fig. 4GG shows that 7KC remains nested within the cavity formed by the two monomers throughout the track. The complex is somewhat curved and the 7KC moves slightly within the cavity, but the 7KC remains complexed with the CD dimer over the entire track.

Dimerized hydroxypropyl β CD and cholesterol, down orientation, AMBER force field:

figure 4GG shows that cholesterol remains nested within the cavity formed by the two monomers throughout the trajectory. The complex is somewhat tortuous and cholesterol moves slightly within the cavity, but cholesterol remains complexed with CD dimers throughout the track.

Translational dimeric hydroxypropyl β CD and 7KC, upward orientation, GROMOS force field:

fig. 4JJ shows that the dimerization complex starts with 7KC translated in an upward orientation, closely nested in the cavities of two CD monomers. The composite stretches at about 140ns, producing a first change at this point in fig. 4GG, but reforms very quickly. As shown in the variation of fig. 4GG, the composite continues to stretch and deform periodically, but the 7KC remains in both cavities until the tail releases its monomer at about 700ns, and the 7KC does not re-enter both cavities simultaneously in the rest of the track.

Translational dimeric hydroxypropyl β CD and cholesterol, upward orientation, GROMOS force field:

figure 4JJ shows that cholesterol translated in the upward orientation begins to complex with dimers that begin to twist at about 100ns (much larger than the 7KC complex). The large angle change in fig. 4GG for cholesterol at approximately 180ns results when one monomer associated with head cholesterol through the minor surface is completely inverted to the other side of the second monomer, such that the slightly deformed minor surface of the second monomer (associated with cholesterol) is associated to the slightly deformed major surface, resulting in a slightly deformed head-to-tail dimer. However, such head-to-tail dimers never fully form complexes with cholesterol, while the head group of cholesterol remains associated with the monomer with which it was originally associated. This is the only trajectory that produces head-to-tail dimers, and this configuration does not appear to be effective in forming complexes with cholesterol.

Translational dimeric hydroxypropyl β CD and 7KC, down-oriented, GROMOS force field:

the shifted 7KC at the down position began to associate with the two monomers in the center of the CD dimer. FIG. 4JJ shows that one monomer stretches away from the tail of 7KC at 230ns, then the 7KC leaves the dimer completely at 355ns (note that this is also where the consensus breaks in FIG. 4 GG). At 400ns, 7KC re-associates the head group with one monomer. In the remainder of the trace, the head of the 7KC remained associated with this monomer, but the tail did not reinsert into the second monomer.

Translational dimeric hydroxypropyl β CD and cholesterol, down-oriented, GROMOS force field:

cholesterol translates in a downward orientation, forming a complex with CD dimers for approximately 162 ns. At this point, the dimerized complex begins to stretch and deform, and then the head group of cholesterol releases its monomer at 190 ns. At 210ns, cholesterol is not associated with the lumen of either monomer, but remains between the two separate monomers. Cholesterol remains in close association with the dimer until complete dissociation at 320 ns. As shown in fig. 4JJ, cholesterol does not re-enter the two lumens, nor does the dimerized complex completely reform on the rest of the trajectory, but cholesterol sometimes associates the head group with the subsurface of one monomer as in the 640ns configuration.

Translational dimeric hydroxypropyl β CD and 7KC, upward orientation, AMBER force field:

fig. 4MM details how 7KC is nested within a cavity formed by two monomers throughout the track. The composite is slightly curved around 7KC, but 7KC remains in almost exactly the same position throughout the track.

Translational dimeric hydroxypropyl β CD and cholesterol, upward orientation, AMBER force field:

figure 4MM details how cholesterol is nested within a cavity formed by two monomers throughout the track. The complex and cholesterol will move slightly in the trajectory, particularly with the monomer associated with the cholesterol head, but cholesterol is never completely dissociated from either monomer. Cholesterol was complexed with CD dimers throughout the track.

Translational dimeric hydroxypropyl β CD and 7KC, downward orientation, AMBER force field:

fig. 4MM details how 7KC is nested within a cavity formed by two monomers throughout the track. The composite is slightly curved around 7KC, but 7KC remains in almost exactly the same position throughout the track. Over the entire track, 7KC complexed with CD dimer.

Translational dimerized hydroxypropyl β CD and cholesterol, down-oriented, AMBER force field:

figure 4MM details how cholesterol is nested within a cavity formed by two monomers throughout the track. The complex bends slightly around the cholesterol, but the cholesterol remains in almost exactly the same position throughout the trajectory. Cholesterol was complexed with CD dimers throughout the track.

In addition, short analyses were performed on DS0 β CD dimer with butyl and triazole linkers (FIG. 4NN-QQ) and hydroxypropyl dimer with triazole linker (FIG. 4 RR-SS). The DS0 simulations show that the triazole linker to some extent disrupts the stability of the complex, however, this allows some additional specificity of 7KC to be delivered. Slightly different but still strong and favourable interactions are predicted to be good for both linker types.

Triazole-linked HP β CD dimer (fig. 4RR) showed slightly weaker interaction than butyl-linked hydroxypropylated dimer and strong preference for 7KC in the down-orientation. Cholesterol interactions are weaker than with 7KC, indicating some specificity for 7KC, while 7KC is the most stable complex formed to date in the downward orientation. The addition of the triazole group stabilized the 7KC in the down orientation, while all other complexes broke at some point in time.

Additional MD analysis

In addition, simplified MD analyses were also performed at DS4 for triazole and butyl linked methyl β CD, sulfobutyl β CD, and quaternary ammonium β CD (FIGS. 5B-C, 6B-C, 7A-B). The methyl dimer showed the most stable complex with the butyl linker and appeared to favor upward orientation in both linker cases, however the interaction was very similar for the two methyl dimers tested. It is difficult to distinguish which is more effective in practice, but both types of linker are susceptible to forming complexes with the two ligands used for methyl substitution. The traces indicate that the head group of 7KC is not entirely within the lumen of the dimer, but remains stable between the two sister monomers. The complex with 7KC in the down orientation remained associated for about 50ns before the 7KC moved out of the cavity and only the head group was associated with one monomer in the remaining traces.

The negatively charged sulfobutyl dimer showed a pattern similar to methyl and hydroxypropyl dimers, with the triazole linker producing a slightly unstable complex, then allowing 7KC specificity. The charged bulky sulfobutyl group appears to interact very favorably with both 7KC and cholesterol, but in the case of the two linkers, the only complex capable of destruction is that of cholesterol. This suggests that sulfobutyl dimer may have very good specificity for 7KC compared to methyl and hydroxypropyl.

To further evaluate the use of charged substituent groups, MD analysis was performed on DS4 positively charged quaternary ammonium β CD. These traces demonstrate strong binding between QA β CD and sterols, since neither linker releases sterols at any point. The powerful energy of interaction and association with at least one sister monomer over the entire trajectory of the ligand and linker suggests that DS4 QA β CDs are well suited to bind sterols and solubilize them, just like other types of substitutions.

In the final MD analysis, HP β CD with a single O-linker was tested (FIG. 8H). The O-linked dimer (fig. 8H) showed good 7KC specificity, since only the upwardly oriented 7KC remained complexed throughout 100 ns. The strength of the interaction energy of the O linkage is slightly lower compared to the butyl linkage, but since both cholesterol complexes are 100ns cleaved, the overall specificity for linker O seems to be better. The interactions were similar to butyl-linked dimers, but they appeared to have better 7KC specificity, apparently due to the interaction of the nitrogen in the linker with the carbonyl group of 7 KC.

Additional docking screens

Docking simulations enabled us to rapidly model many different possible molecules without synthesizing them. For this reason, many different substitution types, linker types, substitution numbers and substitution positions were "screened" using these docking techniques (FIGS. 8-9). This screen enabled us to determine whether certain modifications gave better or worse specificity to 7 KC.

Figure 8E depicts the evaluation of the dependence of our HP β CD dimer on linker composition and attachment points, changes in hydroxypropylation sites, changes in linker length, and different chemical compositions of linkers. Linker attachment sites have been tested on a third machine because the linker attachment sites are not easily controlled during cyclodextrin chemical synthesis. Docking calculations were performed for various hydroxypropylation sites (fig. 8A), carbon-only linkers of various lengths (chain lengths of two to eight carbons, fig. 8B) and triazole linkers (different values of n1 and n2 around the triazole ring, fig. 8C), and different points of attachment to O2 and/or O3 oxygen of dimerized HP β CD (fig. 8F-G), as well as different linker types (fig. 9A). The results show that when the position of the hydroxypropyl group is varied, there is little effect on 7KC preference and little effect on total sterol binding. Linker lengths between 3 and 5 carbons showed the greatest affinity and specificity for 7KC (fig. 8B).

The various triazole linkers modeled in AutoDock are shown in figure 8C. For these linked dimers, n1 refers to the number of carbon atoms to the right of the ring, and n2 refers to the number of carbon atoms to the left of the azido ring. From these results, the change in length of the triazole linker on each side of the loop was predicted to be less than 4 with maximum affinity for 7 KC.

In fig. 8E, we performed docking calculations for the HP β CD dimer with 7KC for 23 possible alternative linkers (shown in fig. 8D). From these results, it was predicted that most of the tested linked dimers maintained good affinity for 7 KC.

We also considered the fact that the linker may be attached to the minor surface of the cyclodextrin at the C2 or C3 carbon atom. We tested whether this would affect the predicted affinity by molecular docking (fig. 8F). We also investigated whether there could be more significant differences in the affinity of sterols attached to the variable attachment site via asymmetric linkers. These calculations indicate that for all three possible ligation sites, there is a tendency to bind 7KC and cholesterol, which are present in roughly equal numbers in a typical synthesis. These calculations show the propensity to bind 7KC and cholesterol for all four possible linkages present in the synthesis of dimers linked by five different asymmetric linkers. Overall, we observed no significant difference between the C2 and C3 attachment sites.

Our molecular modeling revealed that there was a difference in the level of specificity for 7KC for different substitution numbers. Of particular interest are linked HP β CDs containing 3, 4 or 5 hydroxypropyl groups, which show the greatest specificity for 7KC in any butyl dimer we model (fig. 4B). We synthesized a variety of butyl and triazole linked HP β CD dimers, including DS-3. Consistent with our predictions, the specificity of HP β CD-butyl-DS 3 and HP β CD-triazole-DS 3 for 7KC was greater than cholesterol (fig. 16A-C).

After completing the hydroxypropyl CD dimer docking analysis, various CD dimers with different degrees of substitution and various linkers for 7KC and cholesterol were docked to see how these factors affect the binding of 7KC and cholesterol (fig. 5A, 6A and 9). Methyl and sulfobutyl substitutions from DS1 to DS20 were tested using butyl and triazole linkers (fig. 5A, 6A) and the results were encouraging enough to stimulate other molecular dynamics analyses, and ultimately synthesis.

We observed in fig. 5A and 6A that 7KC specificity was best at low DS (2-6) for sulfobutyl and methyl substitutions. DS4 Me β CD and SB β CD behaved most similarly to HP β CD DS5, with 7KC solubilizing well but not cholesterol. It appears that with the increase of both linkers and all substituted DSs, the 7KC specificity becomes less and less pronounced. When it appeared that-DS 4 achieved the maximum 7KC specificity for all substitution types tested, only DS4 was tested along with the other linker types.

Substitutions other than hydroxypropyl, methyl, or sulfobutyl were tested only at low DS using only butyl, triazole, linker O, and linker R (fig. 9A). While some linkers or substitution types do show more or less specificity than others, the vast majority still show at least some specificity for 7 KC. This indicates that the 7KC specificity in the compounds tested does not depend on the type of linker or substitution, but on the number of substitutions on the β CD ring. While some substitution patterns do show negative specificity for a few linker patterns, the average 7KC specificity is still much greater than 0 for these 23 linkers and the seven low DS (4) substitution patterns.

Using molecular docking, we were able to test how the length of the triazole or alkyl linker affects the 7KC specificity of cyclodextrin dimers containing hydroxypropyl, methyl and sulfobutyl substitutions (fig. 9B-C). We show that as the linker length increases, the specificity decreases. Without intending to be bound by theory, it is believed that for longer linkers, the CD subunits may be separated by greater distances and thus take less time in a conformation that is capable of effectively encapsulating molecules of 7KC or cholesterol size. From these results, we conclude the following: dimers with linker lengths that allow the guest (7KC or cholesterol) to fit both CD subunits will show more of such molecular solubilization, e.g., linker lengths of 7 atoms or less.

We also tested whether the specificity of CD dimers for 7KC was dependent on substitution position by creating many different substitution patterns with sulfobutyl, hydroxypropyl and methyl substitutions and combinations of the three (fig. 9D-E). We found that when a single substitution type or even multiple substitution types are present on a CD dimer, 7KC specificity is largely retained when the DS is about 4. The type and position of these substitutions have little effect on the specificity of 7 KC. The results of docking simulations indicate that, although both the linker and the composition of the substitutions affect the ability of a given CD to solubilize guests, the degree of specificity for 7KC depends largely on the number of substitutions on the CD loop. As can be seen in FIGS. 4B and 5A-B, the butyl-linked dimer showed the highest specificity for 7KC at methyl, sulfobutyl and hydroxypropyl degrees of substitution of approximately DS 2-5. This also applies to triazole linkers, supporting the following idea: i.e., a variety of linkers and substitution types exhibit similar specificity for degrees of substitution between 2 and 5, and for 7 KC. In addition, 23 different linkers and 14 different substitution patterns/combinations were also docked to determine if the linker or substitution pattern had an effect on 7KC specificity (fig. 9A). Both assays showed differences in the degree of specificity of 7KC, but the average specificity was still much greater than zero.

Docking and molecular dynamics screening was performed to determine whether certain linker types or number, type and position of substitutions affect 7KC specificity. The only modification that has a large impact on binding (affinity) is the actual dimerization of the cyclodextrin (figure 2E, dimer shows much better sterol binding compared to the docked monomer). In contrast, the number of substitutions present on the dimer had the greatest effect on 7KC binding specificity. Docking simulations indicate that once β CDs are dimerized and substituted with approximately 4 compatible functional groups, specificity for 7KC will be maintained in most cases for many different substitution types, patterns and linkers.

Since the methyl, sulfobutyl and hydroxypropyl groups are quite different from each other, and the range of linkers tested contains significant variability, we believe that it is reasonable that other substitution types that have linker lengths similar to the sterol guest behave similarly to the butyl-linked CD dimer with the hydroxypropyl group. While substitutions and linker types may have some impact on other properties such as solubility and toxicity, specificity for 7KC is also expected to exist for such other molecules.

Example 3 Synthesis of HP β CD substituted Cyclodextrin dimer

FIGS. 3A-D show the molecules to be synthesized in FIG. 10 below.

This example describes the synthesis of a substituted cyclodextrin dimer, first linked through a butyl linker, then linked through a triazole-containing linker.

For the DS measurements, 1H and 2DNMR spectra were recorded on a 600MHz VarianVXR-600 using the residual solvent signal as an internal reference. Samples were incubated in DMSO-d₆/D₂O dissolved to clarify the structure. FID signals were recorded with at least 16 scans to obtain a spectral window of at least between 0ppm to +10 ppm. The average Degree of Substitution (DS) can be calculated by integrating the anomeric regionFourteen (fourteen is the anomeric proton of the β -cyclodextrin dimer) and the integral of the alkyl region is divided by three (see fig. 10J).

Overview of Synthesis and characterization

HP(βCD-BUT-βCD)

Preparation of hydroxypropylated β -cyclodextrin dimer was accomplished by a three-step synthesis (see fig. 10A). The starting material was a monomeric beta-cyclodextrin (TBDMS-. beta.CD, CycloLab, Budapest, Hungary) protected on the main side by a tert-butyldimethylsilyl group.

Subsurface dimerization was achieved by using TBDMS- β CD, anhydrous conditions and sodium hydride as base. The dialkylating agent was added dropwise to the heterogeneous reaction mixture and reacted thoroughly at room temperature.

The main side-protected β CD dimer (TBDMS- β CD-BUT- β CD-TBDMS) was purified by chromatography using an isocratic elution (chloroform: methanol: water ═ 50: 8: 0.8(v/v/v) as eluent). MALDI analysis of the compound confirmed the identity of the product (fig. 10D).

Desilylation was performed with tetrabutylammonium fluoride in THF at room temperature. Using isocratic elution (1, 4-dioxane: NH)₃As eluent 10: 7(v/v) was purified by chromatography of β CD dimer (β CD-BUT- β CD). MALDI and TLC analysis of the compounds confirmed the identity of the product (fig. 10E-10F).

Hydroxypropylation of β CD dimer was achieved under aqueous conditions by using sodium hydroxide as a base at room temperature. Purification of hydroxypropylated β CD dimer (HP (β CD-BUT- β CD)) is based on ion exchange resin treatment, charcoal clarification and extensive dialysis. MALDI and NMR analysis of the compounds confirmed the identity and structure of the product (FIGS. 10G-10N).

HP (beta CD-triazole-beta CD)

The preparation of hydroxypropylated β -cyclodextrin dimers linked to one triazole moiety through the subsurface can be performed in a four-part procedure (fig. 10B). The first part is the preparation of the azide linker (3-azido-1-bromopropane) since this reagent is not commercially available. The second part is to prepare two beta CD monomers, namely 2-O-propargyl-beta-CD and 2-O- (3-azidopropyl) -beta-CD, respectively. The third synthesis part is the stacking of dimer-cores formed by copper-assisted azide-alkyne cycloaddition, and the last part is the preparation of a series of 2-hydroxypropylated triazole-linked dimers according to classical alkylation methods.

In particular, the preparation of the azide linker can be achieved by severely limiting the amount of sodium azide and by extending the time of addition of the limiting reagent. The azide-linker was then characterized by NMR spectroscopy and TLC (fig. 10R).

The synthesis of both monomers was accomplished by using lithium hydride as the selective base for the secondary side deprotonation. In particular, according to this method, only the hydroxyl group located at C2 is activated. As a result, the monomers prepared by this method were substituted only at O2 (they were single isomers). Both monomers were characterized by NMR spectra MALDI and TLC (FIG. 10S-U).

The dimer-core is then prepared by reacting the two monomers. The resulting compound, the single isomer (BCD- (triazole), was characterized by NMR spectroscopy (FIG. 10V) and MALDI (FIG. 10O)₁-BCD DS＝0)。

Hydroxypropylation of BCD-triazole-BCD was accomplished using propylene oxide and basic aqueous solution. The series of hydroxypropylated compounds was characterized by MALDI (FIGS. 10P-Q).

Detailed description of the Synthesis (HP (. beta.CD-BUT-. beta.CD))

Step 1: subsurface dimerization of TBDMS-beta CD

Dried TBDMS-. beta.CD (10g, 5.17mmol) was solubilized in THF (400mL) under an inert atmosphere and sodium hydride (2.5g, 50mmol) was added carefully in portions (over 30 min). The addition of sodium hydride results in the formation of hydrogen and a strong foaming of the suspension. After stirring for 15 minutes, the reaction mixture gelled and stirring became difficult. To break the gel, the reaction mixture was heated until a gentle reflux occurred and kept at reflux for 30 minutes. The yellowish heterogeneous suspension becomes more stirrable and the gel-like structure disappears. The reaction mixture was cooled to room temperature with a water bath. The alkylating agent 1, 4-dibromobutane (1.25mL, 2.25g, 10.5mmol) was added dropwise (15 min) and the reaction mixture turned dark orange in color.

The brown suspension was stirred under an inert atmosphere overnight. Conversion was estimated by TLC to be between 10% and 15% (eluent: chloroform: methanol: water 50: 10: 1, v/v/v, see figure 10C) and was considered acceptable for work-up.

The reaction mixture was quenched with methanol (30mL), concentrated under reduced pressure (about 20mL), and precipitated with water (200 mL). The crude reaction product was filtered on a sintered glass filter and washed thoroughly with water (3X 300 mL). Subjecting the crude product to reaction in the presence of KOH and P₂O₅(12.1g) was dried in a drying oven to a constant weight.

The crude reaction product was purified by chromatography, the product containing fractions were collected based on TLC analysis and evaporated under reduced pressure until dryness (fig. 10C) to give a white substance, which was purified in the presence of KOH and P₂O₅Dried in a drying cabinet to constant weight (TBDMS-beta CD-BUT-beta CD-TBDMS, 3.5 g).

Step 2: deprotection of TBDMS-beta CD butyl linked dimers

Dried TBDMS- β CD-BUT- β CD-TBDMS (3.5g, 0.89mmol) was solubilized in THF (250mL) under inert atmosphere and tetrabutylammonium fluoride (8.75g, 33.47mmol) was added in one portion to the light yellow solution. After stirring at room temperature for 30 minutes, the color of the reaction mixture turned dark green. The reaction mixture was stirred at room temperature overnight. TLC analysis (1, 4-dioxane: NH) ₃10: 7(v/v)) indicated that the reaction was not complete and a second portion of tetrabutylammonium fluoride (4g, 13.3mmol) was added to the vessel. The reaction mixture was heated to gentle reflux and refluxed for two hours. The reaction conversion at this stage was complete, as TLC did not detect any starting material. The reaction mixture was cooled to room temperature, concentrated under reduced pressure (to about 10mL), and methanol (200mL) was added to give a white precipitate. The solid was filtered off, analyzed by TLC and in the presence of KOH and P₂O₅(1.2g) was dried in a dry box until constant weight was obtained. According to TLC analysis, this material contained a negligible (. ltoreq.3%) amount of tetrabutylammonium fluoride. The mother liquor was concentrated under reduced pressure (to about 10mL) and purified by chromatography (eluent: 1, 4-dioxane: NH₃10: 7v/v), the fractions containing the product were collected and evaporated under reduced pressure until dryness to give a white substance,white substance is put in the presence of KOH and P₂O₅Dried in a drying cabinet to constant weight (. beta.CD-BUT-. beta.CD, 0.55 g).

And step 3: hydroxypropylation of beta CD-BUT-beta CD

β CD-BUT- β CD (0.5g, 0.21mmol) was suspended in water (10mL), sodium hydroxide (0.1g, 2.5mmol) was added to the reaction vessel, and the color of the mixture became a pale yellow solution. The reaction mixture was cooled with a water bath (10 ℃ C.) and propylene oxide (0.5mL, 0.415g, 7.14mmol) was added in one portion. The reaction vessel was flushed with argon, sealed and stirred at room temperature for two days. The reaction mixture was concentrated under reduced pressure until a viscous slurry was obtained, which was precipitated with acetone (50 mL). The white solid was filtered on a sintered glass filter and washed thoroughly with acetone (3X 15 mL). The material was solubilized with water (50mL), treated with ion exchange resin (to remove salts), clarified with charcoal, filtered through a membrane and dialyzed against purified water for one day. The retentate was evaporated under reduced pressure until dryness to give a white solid (0.8 g).

Detailed description of the Synthesis (HP (. beta.CD-triazole-. beta.CD))

Step 1: preparation of azido-linkers

1, 3-dibromopropane (10mL, 20.18g, 0.1mol) was solubilized in 40mL of DMSO with vigorous stirring. A solution of sodium azide (6.7g, 0.1mol) was prepared in DMSO (240mL) and added dropwise (addition for 2 hours) to a solution of dihalopropane. The solution was stirred at room temperature overnight. The crude reaction product is then extracted with n-hexane (3X 100mL), the collected n-hexane phase is back-extracted with water (3X 50mL) and the organic phase is carefully evaporated under reduced pressure (strictly at 40 ℃ C. and 400mbar, otherwise the target compound may distill off). The residue oil was purified by chromatography (n-hexane-EtAc 98: 2 as eluent, isocratic elution). The appropriate fractions were collected and concentrated under reduced pressure to give the target compound as a viscous oil (which can be stored under an inert atmosphere in a dark refrigerated container). Compounds were observed by the following steps: the TLC plate was immersed in triphenylphosphine solution (10%) in dichloromethane for about 15s, dried below 60 ℃, immersed in ninhydrin ethanol solution (2%) for about 15s, and finally dried below 60 ℃. The target compound appears as a purple spot on the TLC plate.

Step 2.1: preparation of 2-O-propargyl-beta CD

Lithium hydride (212mg, 26.432mmol) was added to a solution of β -cyclodextrin (20g, 17.62mmol) in anhydrous DMSO (300 mL). The resulting suspension was brought to room temperature N₂Stirring was continued until clear (12-24 hours). Propargyl bromide (1.964mL, 17.62mmol) and a catalytic amount of lithium iodide (about 20mg) were then added and the mixture was stirred at 55 ℃ for 5 hours in the absence of light. TLC (10: 5: 2 CH)₃CN-H₂O-25％v/v NH₃Aqueous solution) was used to characterize the product and showed spots corresponding to mono-propargylated and non-propargylated β -cyclodextrin, respectively. The solution was poured into acetone (3.2L), and the precipitate was filtered and washed thoroughly with acetone. The resulting solid was transferred to a round bottom flask and dissolved in a minimum volume of water. Silica gel (40g) was added and the solvent was removed under vacuum until a powdery residue was obtained. The crude mixture was applied on top of a column of silica gel (25X 6cm) and chromatographed (10: 5: 2 CH)₃CN-H₂O-25％v/v NH₃Aqueous solution) to give 2-O-propargyl- β -CD as a solid after freeze-drying. 2-O-propargyl- β -CD was analyzed by MALDI and NMR (FIG. 10T and FIG. 10U).

Step 2.2: synthesis of 2-O- (3-azidopropyl) -beta CD

Lithium hydride (212mg, 26.432mmol) was added to a solution of β -cyclodextrin (20g, 17.62mmol) in anhydrous DMSO (300 mL). The resulting suspension was brought to room temperature N ₂Stirring was continued until clear (12-24 hours). Then 3-azido-1-bromopropane (3mL) and a catalytic amount of lithium iodide (about 20mg) were added, and the mixture was stirred at 55 ℃ for 5 hours in the absence of light. TLC (10: 5: 2 CH)₃CN-H₂O-25％v/vNH₃Aqueous solution) was used to characterize the product and showed spots corresponding to 2-O- (3-azidopropyl) - β CD and β CD. The solution was poured into acetone (3.2L), and the precipitate was filtered and washed thoroughly with acetone. The resulting solid was transferred to a round bottom flask and dissolved in a minimum volume of water. Silica gel (40g) was added and the solvent was removed under vacuum until obtainedA powdery residue. The crude mixture was applied on top of a silica gel column and chromatographed (10: 5: 2 CH)₃CN-H₂O-25％v/v NH₃Aqueous solution) to give 2-O- (3-azidopropyl) - β -CD as a solid after drying.

And step 3: synthesis of beta-CD-triazole-beta-CD dimer

2-O-propargyl-beta-CD and 2-0- (3-azidopropyl) -beta-CD were suspended in water (300mL) with vigorous stirring (each at a concentration between about 8-12 mM). Dimethylformamide (DMF) (about 300mL) was added to the suspension to completely dissolve the heterogeneous mixture (addition of DMF is a slightly exothermic process). Copper bromide (2g, 13.49mmol) was added to the solution. The suspension was stirred at room temperature for 1 hour. The reaction was monitored by TLC and was expected to be complete after about 1 hour (eluent: CH) ₃CN∶H₂O∶NH₃10: 5: 2). The crude reaction product was filtered and the mother liquor was concentrated under reduced pressure (60 ℃). The gel-like material was diluted with water and silica (15g) was added. The heterogeneous mixture was concentrated to dryness under reduced pressure. The crude mixture was applied on top of a silica gel column and chromatographed (10: 5: 2 CH)₃CN-H₂O-25％v/v NH₃Aqueous solution) and dried to give BCD- (triazole)₁-BCD dimer. Pair of BCD- (triazole) by NMR₁Preparation of BCD dimer (fig. 10V).

And 4, step 4: HP (beta CD-triazole-beta CD)

Beta CD- (triazole) obtainable according to steps 1-3 above or by other methods₁- β CD dimer (1g, 0.418mmol) was suspended in water (50mL), sodium hydroxide (DS3 ═ 0.32g, 8 mmol; DS6 ═ 0.74g, 18.5 mmol; DS7 ═ 0.87g, 21.75mmol) was added to the reaction vessel, and the mixture turned to a light yellow solution. The reaction mixture was cooled through a water bath (10 ℃) and propylene oxide (DS3 ═ 0.49mL, 0.42g, 7.25 mmol; DS6 ═ 1.21mL, 1.04g, 17.9 mmol; DS7 ═ i.46ml, 1.7g, 29.3mmol) was added in one portion. The reaction vessel was flushed with argon, sealed and stirred at room temperature for two days. The solution was concentrated under reduced pressure until a viscous slurry was obtained, which was precipitated with acetone (50 mL). Will whiten The colored solid was filtered on a sintered glass filter and washed thoroughly with acetone (3X 15 mL). The material was solubilized with water (50mL), treated with ion exchange resin (to remove salts), clarified with charcoal, filtered through a membrane and dialyzed against purified water for one day. The retentate was evaporated under reduced pressure until dryness to give a white solid (0.8 g). The HP (β CD-triazole- β CD) products were analyzed by NMR (fig. 10W, 10X and 10Y) and their respective degrees of substitution were calculated as shown.

Example 4 Synthesis of methyl-substituted Cyclodextrin dimer

Figure 3E shows the molecule to be synthesized.

This example describes the synthesis of methyl substituted cyclodextrin dimers with triazole-containing linkers.

Methyl (beta CD- (triazole)₁- β CD) dimer (exemplary Synthesis)

The preparation of methylated β -cyclodextrin dimer was done in a one-step reaction (see fig. 11A). Beta CD- (triazole)₁The β CD dimer core was prepared according to the synthetic strategy described in example 3 above.

Synthesis of

Beta CD- (triazole) is stirred vigorously₁The-. beta.CD dimer core (1.1g, 0.46mmol) was suspended in deionized H₂O (100mL), and sodium hydroxide (0.35g, 8.8mmol) was added. The resulting pale yellow suspension was stirred for 30 minutes until complete solubilization. Methyl iodide (0.5mL, 1.14g, 8.03mmol) was added in one portion with vigorous stirring when the temperature of the pale yellow clear solution stabilized at about 20 deg.C (note: methyl iodide was not miscible with the reaction mixture, therefore vigorous stirring may improve efficiency). The reaction mixture was stirred at room temperature for 24 hours and then treated with ion exchange resin: h + resin (6g) and OH- (6g) resin were added to the solution, stirred for 15 minutes and filtered off (resin was washed with 3X 15mL of deionized water). The resulting filtrate (final pH 7) was clarified with activated charcoal: activated carbon (0.2g) was added to the solution with vigorous stirring, stirred for 30 minutes and filtered off (the activated carbon pad was washed with 3X 15mL of deionized water). The colorless solution was evaporated under reduced pressure (40 ℃ C.) to give the title compound (about 1g) as a white powder.

Characterization of

The progress of the reaction was monitored by TLC (FIG. 11B), and the resulting material was characterized by MALDI-TOF and NMR analysis, as shown in FIGS. 11C-N.

Example 5 Synthesis of sulfobutyl substituted Cyclodextrin dimer

Figure 12F shows the molecule to be synthesized.

This example describes the synthesis of sulfobutyl substituted cyclodextrin dimers with triazole-containing linkers.

The preparation of SB-dimer was completed in a one-step reaction (fig. 12A).

Synthesis (SB Low DS)

Beta CD- (triazole) 1-beta CD dimer core (1.2g, 0.5mmol) was suspended in deionized H with vigorous stirring₂O (60 mL). To the mixture was added sodium hydroxide (0.39g, 9.75mmol) and the resulting solution was heated at 60 ℃. Butane sultone (0.88mL, 1.17g, 8.6mmol) was added dropwise at 60 deg.C, and the solution was heated at the same temperature for 3 hours. The reaction was then heated to 90 ℃ for an additional 1 hour to destroy residual butane sultone. The reaction mixture was cooled and treated with an ion exchange resin. Cation exchange resin (H + resin, 2g) and anion exchange resin (OH-resin, 2g) were added to the solution, stirred for 15 minutes and filtered off (resin washed with 3X 15mL of deionized water). The resulting filtrate (final pH 7) was clarified with activated charcoal: activated carbon (0.3g) was added to the solution with vigorous stirring, stirred for 30 minutes and filtered off (the activated carbon pad was washed with 3X 15mL of deionized water).

The colorless solution was evaporated under reduced pressure (40 ℃ C.) to give a white powder (1.47 g).

Characterization of

The reaction was monitored by TLC analysis (FIG. 12B), and the resulting material was characterized by MALDI-TOF and NMR analysis, as shown in FIGS. 12C-K.

Synthesis (high DS)

(beta CD- (triazole) 1-beta CD) dimer core (1.2g, 0.5mmol) was suspended in deionized H with vigorous stirring₂O (60 mL). Sodium hydroxide (1.22g, 30.5 mmo) was added to the mixturel) and the resulting solution is heated at 60 ℃. Butane sultone (2.8mL, 3.72g, 27.35mmol) was added dropwise at 60 deg.C, and the solution was heated at the same temperature for 3 hours. The reaction was then heated at 90 ℃ for an additional 1 hour to destroy residual butane sultone. The reaction mixture was cooled and treated with an ion exchange resin. Cation exchange resin (H + resin, 4g) and anion exchange resin (OH-resin, 4g) were added to the solution, stirred for 15 minutes and filtered off (resin washed with 3X 15mL of deionized water). The resulting filtrate (final pH 7) was clarified with activated charcoal: activated carbon (0.5g) was added to the solution with vigorous stirring, stirred for 30 minutes and filtered off (the activated carbon pad was washed with 3X 15mL of deionized water). The colorless solution was evaporated under reduced pressure (40 ℃ C.) to give a white powder (1.51 g).

Characterization of

The resulting material was characterized by MALDI-TOF and NMR analysis as shown in FIG. 12M-P.

Example 6 Synthesis of Quaternary ammonium substituted Cyclodextrin dimers

FIGS. 3I and 13G show the molecules to be synthesized.

This example describes the synthesis of a quaternary ammonium substituted cyclodextrin dimer with a triazole-containing linker.

Quaternary ammonium (beta CD- (triazole)₁- β CD) dimer (exemplary Synthesis)

The preparation of QA-dimer was accomplished in a one-step reaction (see FIG. 13A). Beta CD- (triazole)₁The β CD dimer core was prepared according to the synthetic strategy described in example 2 above.

Synthesis of

(BCD- (triazole) 1-BCD) dimer core (1.2g, 0.5mmol) was suspended in deionized H2O (100mL) with vigorous stirring and sodium hydroxide (0.39g, 9.8mmol) was added. The resulting pale yellow suspension was stirred for 30 minutes until complete solubilization. The temperature of the pale yellow clear solution was stabilized at 5-10 ℃ and glycidyltrimethylammonium chloride (1.17mL, 1.32g, 8.7mmol) was added in one portion with vigorous stirring. The reaction mixture was stirred at room temperature for 24 hours, then the temperature of the solution was stabilized at 5-10 ℃ and a second portion of glycidyltrimethylammonium chloride (0.4mL, 0.45g, 3mmol) was added. The reaction mixture was heated at 50 ℃ for 3 hours, then cooled and treated with ion exchange resin: h + resin (6g) and OH- (6g) resin were added to the solution, stirred for 15 minutes and filtered (resin was washed with 3X 15mL of deionized water). The resulting filtrate (final pH 7) was clarified with activated charcoal: activated carbon (0.2g) was added to the solution with vigorous stirring, stirred for 30 minutes and filtered off (the activated carbon pad was washed with 3X 15mL of deionized water). The colorless solution was evaporated under reduced pressure (40 ℃ C.) to give the title compound (about 800mg) as a white powder.

Characterization of

The resulting material was characterized by MALDI-TOF and NMR analysis as shown in FIGS. 13B-K.

In the case of QA-BCD derivatives, the typical gaussian distribution with the regular pattern observed for randomly substituted derivatives is absent, while irregular fragmentation patterns can be detected. The identification/assignment of these irregular peaks is very complex because simple fragmentation patterns cannot be predicted. The irregular pattern observed in the MALDI spectrum is likely due to the instability of the trimethylammonium moiety under the experimental conditions. In particular, the elimination product (see fig. 2) is the result of partial cleavage of trimethylammonium, while the demethylation product (see fig. 2) is the result of stepwise cleavage of the methyl group from the cationic side chain. The following conclusions can be reasonably drawn: MALDI conditions are not suitable for determination of DS for QA-BCD derivatives because non-informative peaks are generated during laser desorption. However, the DS of the QA-BCD derivative can be determined by NMR (fig. 13I), and is estimated to be about 2.1.

Example 7 Synthesis of succinyl substituted Cyclodextrin dimer

FIGS. 3G and 14G show the molecules to be synthesized. The preparation of succinyl-substituted dimer (Succ-dimer) was completed in a one-step reaction (fig. 14A).

Synthesis of

Under vigorous stirring and an inert atmosphere, the (β CD- (triazole) 1- β CD) dimer core (1.2g, 0.5mmol) was suspended in pyridine (23 mL). However, to increase the solubility of the (. beta.CD- (triazole) 1-. beta.CD) dimer, the suspension was heated at 40 ℃ for 1 hour, and complete solubilization was not achieved. A second portion of pyridine (23mL) was added to the suspension, but dilution did not further increase the solubility of the (β CD- (triazole) 1- β CD) dimer. Succinic anhydride (0.1g, 1mmol) was added at room temperature and the reaction mixture was stirred for 24 hours. The crude reaction product was concentrated under reduced pressure, solubilized in water (no clear solution obtained) (50mL) and treated with ion exchange resin: h + resin (2g) and OH- (2g) resin were added to the solution, stirred for 15 minutes and filtered (resin was washed with 3X 15mL of deionized water). The resulting filtrate (final pH 7) was clarified with activated charcoal: activated carbon (0.5g) was added to the solution with vigorous stirring, stirred for 30 minutes and filtered off (the activated carbon pad was washed with 3X 15mL of deionized water). The colorless solution was evaporated under reduced pressure (40 ℃ C.) to give the title compound (about 900mg) as a white powder.

Characterization of

The resulting material was characterized by MALDI-TOF and NMR analysis as shown in FIGS. 14B-K.

Just as with the QA-dimer, MALDI analysis proved to be unfavorable for DS determination, and DS was determined by NMR (fig. 14I), and was estimated to be about 2.1.

Example 8 extraction of 7KC and Cholesterol from blood cells Using beta CD dimer and monomer

Method

Blood was collected from healthy volunteers by licensed phlebotomists. Test substance alone or PBS (negative control) was added to whole blood at various concentrations and incubated at 37C for 3 hours. The blood was then centrifuged and serum collected. The sera were frozen and then processed for mass spectrometry.

Plasma free 7-ketocholesterol was determined by LC-MS/MS after protein precipitation, extraction with acetonitrile and derivatization with a novel Quaternary Aminooxy (QAO) mass labeling Reagent (ampliflex Keto Reagent (AB Sciex, Framingham, MA, USA)) that has been used for testosterone analysis (Star-Weinstock [ et al ], Analytical Chemistry, 84 (21): 9310-.

A50 μ L sample of plasma was spiked with 0.5ng of 7-ketocholesterol (Toronto Research Chemicals, North York, Ontario, Calif.) prepared in ethanol at 0.1 ng/. mu.L. Samples were treated with 250 μ L acetonitrile, vortex mixed, and centrifuged at 12,000 × g for 10 minutes to remove protein. The supernatant was dried in vacuo and then treated with 75 μ L of QAO reagent. Working reagents were prepared by mixing 0.7mL of amplifiex ketone reagent with 0.7mL of amplifiex ketone diluent to prepare a 10mg/mL stock solution. The stock solution was then diluted 1: 4 with 5% acetic acid in methanol to a final working concentration of 2.5 mg/mL. The mixture was allowed to react at room temperature for two days, then subjected to LC-MS/MS analysis.

1 to 100ng/ml of 7-ketocholesterol standard (Toronto Research Chemicals, North York, Ontario, Calif.) was prepared in charcoal-eluted plasma SP1070(Golden West Biological, Temecula, Calif., USA) and phosphate buffered saline. Residual 7-ketocholesterol was detected in the eluted plasma, and therefore a standard from PBS was used.

QAO-7-ketocholesterol derivatives were analyzed by positive mode electrospray ionization (ESI) using a 4000Q-TRAP tandem/triple quadrupole linear ion TRAP mass spectrometer (SCIEX, Framingham, MA, USA). The mass spectrometer was connected to a Shimadzu (Columbia, MD) SIL-20AC XR autosampler, followed by 2 LC-20AD XR LC pumps.

The instrument operates in the following settings: supply voltages 4500kV, GS 150, GS250, CUR 20, TEM 550 and CAD gas medium. Compounds were quantified by Multiple Reaction Monitoring (MRM) and switching was optimized by infusion of pure derivatized compounds as shown in table 1 below. Bold face conversion was used for quantification.

The use of Gemini 3. mu.C 6-phenylThe separation was carried out on a 100X 2mm column (Phenomenex, Torrance, CA, USA) maintained at 35 ℃ using a Shimadzu (Columbia, MD) CTO-20AC column oven. Flow rate of gradient mobile phase is 0 5ml/min and consists of two solvents, a: 0.1% formic acid in water, B: 0.1% formic acid in acetonitrile. The initial concentration of solvent B was 20%, then increased linearly to 60% B in 10 minutes, then increased linearly to 95% B in 0.1 minutes, held for 3 minutes, then dropped back to the initial 20% B in 0.1 minutes, and held for 4 minutes. The retention time of 7-ketocholesterol was 8.46 minutes.

Data were acquired using Analyst 1.6.2(SCIEX, Framingham, MA, USA) and analyzed using multi quant 3.0.1(SCIEX, Framingham, MA, USA) software. Sample values were calculated from a standard curve derived from the ratio of the peak area ratio of analyte to internal standard to analyte concentration, fitted to a 1/x weighted linear equation. The lower limit of quantitation was 1ng/mL, the accuracy was 102%, and the precision (relative standard deviation) was 8.5%. The signal-to-noise ratio (S/N) was 19: 1. At a concentration of 100ng/mL, the accuracy was 98%, the precision was 0.5%, and the S/N was 24: 1.

Results

FIGS. 15A and 15B show that HP β CD dimer (DS-8 determined by MALDI and NMR, see FIGS. 10I and 10J) can remove 7KC from blood cells (whole blood) more efficiently than HP β CD monomer. This is an ex vivo assay on human subjects which allows us to obtain results which can predict the effect on human patients more accurately than in non-human animal experiments. Figure 15C shows that this did not significantly affect plasma cholesterol levels. This means that HP β CD dimer cannot remove large amounts of cholesterol from blood cells. Removal of excess cholesterol from cells may result in disruption of cell and organelle membranes and cell death. We hope to directly investigate this and therefore do the haemolysis assay.

Example 9 hemolysis caused by high concentrations of Cyclodextrin dimer only

Method

For the test solutions, the amount of PBS was varied according to the concentration of cyclodextrin tested. Samples were tested in triplicate. 50 μ L of blood was added to each sample along with PBS and cyclodextrin solution (stock solution also made of PBS) to the appropriate concentration in a final volume of 200 ul. 5% Triton X-100 was used as a positive control and PBS was a negative control. Once all samples were mixed, the samples were placed in a 37C incubator with agitation for three hours. The positive control was 100% hemolyzed with Triton X-100 detergent. Once the samples were removed from incubation, they were diluted by the same fold in a 96-level plate and normalized to the positive control absorbance (approximately 1.1). The absorbance was read at 540 nm. The average of the samples was then corrected by subtracting the negative control. The experiment was performed three times, with error bars being the standard error of the mean (Melanga [ et al ], Journal of Pharmaceutical Sciences, 105 (9): 2921-31.(2016)), (Kiss [ et al ], European Journal of Pharmaceutical Sciences, 40 (4): 376-80. (2010)).

FIGS. 15D-15E show that the butyl and triazole linked dimers are still very low toxic to blood cells and have no apparent toxicity in the pharmacological range of less than 1 mM. FIG. 15D shows hemolysis of butyl-linked HP-dimers of three different DSs (DS is determined by MALDI in FIGS. 10G-10I and DS is confirmed by NMR in FIG. 10J), DS-3 triazole-linked HP-dimers (characterized in FIGS. 10P and 10W; based on MALDI labeling) and DS-3 triazole-linked Me-dimers (characterized in FIGS. 11I and 11L). At higher concentrations, only three butyl-linked dimers showed measurable hemolysis. In fig. 15E, we tested hemolysis of other various substitutions of triazole-linked β CD dimers. We tested unsubstituted quaternary ammonium (DS-2, characterized in FIG. 13I), succinyl (DS-2, characterized in FIG. 14I), and sulfobutyl (DSes characterized by NMR and MALDI in FIGS. 12E, 12H, 12K, and 12N; MALDI DSes were used in the labeling). Only up to 7.5mM of unsubstituted dimer was tested, at which concentration we could detect approximately 5% hemolysis. Other dimers were only tested up to 5mM and no significant hemolysis was detected at any of the tested concentrations.

It appears that at high concentrations, the triazole dimerized form of β CD is less hemolytic than the tested HP β CD butyl dimer, but the linker and all substitution types show very low cleavage, indicating low toxicity.

EXAMPLE 10 solubilization of sterols and sterol-like Compounds by Cyclodextrin dimers

Solubilization of lipophilic compounds was tested by the dimers described in examples 2-6. Test compounds include cholesterol precursors (desmosterols), other oxysterols, steroid hormones, and sterol vitamins.

Method for in vitro solubility determination (turbidity determination)

Stock solutions of sterols (including oxysterol, hormones and vitamins) were suspended in 100% ethanol. Final concentration of suspension: 3% ethanol, 300uM sterol, in PBS and various concentrations of cyclodextrin. The samples were incubated at 37C for 30 minutes and then the absorbance was measured at 350nm in a spectrophotometer plate reader. Samples were prepared in quadruplicate using a Beckman Biomek 2000 liquid handler and plates with hydrophilic coatings were used to minimize binding of sterols to the well surfaces. All experiments were performed 3 or more times and the error bars are the standard error of the mean.

The haze values were normalized to the percent haze measured in the absence of cyclodextrin.

Results

We tested our novel dimers against 7-ketocholesterol in an in vitro spectroscopic assay. In fig. 16A, DS3 is a butyl-linked dimer with an average of about 3 hydroxypropyl groups (quantified by MALDI in fig. 10G), DS6 is a butyl-linked dimer with an average of about 6 substitutions (MALDI, fig. 10H), and DS8 is a butyl-linked dimer with an average of about 8 hydroxypropyl substitutions (MALDI fig. 10I). Various concentrations of HP β CD dimer were tested and sterol concentration was kept constant at 300 μ M throughout. HP (CD-triazole-CD) is the average number of substitutions determined by MALDI for triazole-linked cyclodextrin dimer (FIG. 10P), while HP (CD-But-CD) represents the butyl-linked dimer of the DS.

FIGS. 16A-B show that all of the HP β CD dimers we synthesized solubilized 7KC and cholesterol more efficiently than the HP β CD monomers. This is consistent with our computational model and predictions that show how two linked monomers completely surround a sterol, how it is protected from water, maintain binding for a long time, and how it is recovered if lost. For some low concentrations of dimer, it is possible to compare the solubilization achieved by the high concentration of monomer with an approximation of the same solubilization achieved at a molarity of about 1/10. This means that the affinity for cholesterol/7 KC may be about 10 times higher than that of the monomer, although we must wait for the results of other experiments to determine the affinity constant rigorously. We then further sought to determine whether these dimerized HP β CDs were able to bind 7KC with favorable affinity.

We found that several different HP β CD dimers did bind 7KC favorably (fig. 16A-B). Figure 16B shows that triazole dimer labeled DS 3 binds 7KC with greater specificity than either DS 6 or DS 7 dimer. These DS values were determined by MALDI. We have further found that these HP β CDs bind 7KC more favorably than cholesterol. We note that some dimers appear to solubilize 7KC more favorably than others and have investigated this in fig. 16E-H.

As described above in fig. 15C, we found that in human blood, the DS8 HP β CD dimer removed a large amount of 7KC from donor cells, while serum cholesterol levels did not appear to be disturbed. This means that, although the affinity for cholesterol may result in cholesterol being removed from the cells at the tested concentrations, it is not sufficient to interfere with plasma cholesterol levels within the normal range.

Figures 16C-D show how dimers interact with various other sterols and steroid hormones with different affinities, as defined by relative turbidity.

FIG. 16C shows HP (β CD- (butyl)₁- β CD) dimer efficiently encapsulates vitamin D3 (cholecalciferol) but not vitamin D2. It has been previously observed that beta CD monomer can encapsulate vitamin D3(Szejtli et al ]Drugs of the Future, 9: 675-676.(1984)), but our dimer appeared to solubilize vitamin D3 more efficiently than the HP β CD monomer (FIG. 2A and FIG. 16℃ note that in the dimer experiments, the concentration range was 10-fold smaller).

We also wanted to test the ability of our dimers to solubilize oxysterols other than 7 KC.

Figure 16C shows that HP β CD-butyl linked dimer (DS8) solubilized various oxysterols to various degrees. It appears that cholesterol epoxide is well solubilized.

Figure 16D demonstrates the ability of butyl dimer to bind various hormones. Just like the monomer HP β CD, our dimer binds 3 estrogens well. It should be noted that although the solubilization of progesterone seems to be dramatic here, the solubility of progesterone is naturally much greater than the other hormones tested, and therefore, in this case, this method of normalizing the data is somewhat fraudulent.

We observed that the dimer with the lowest DS was more specific for 7KC than cholesterol, so we performed a more detailed analysis of the least substituted molecules per attached dimer. FIGS. 16E and 16F provide a more detailed presentation of the two HP dimers that show the best specificity for 7 KC. We demonstrate in more detail that two head-to-head linked cyclodextrin dimers with 3HP substitution solubilize 7KC more preferentially than cholesterol. These dimers exhibit significant affinity and specificity for 7KC at concentrations below 0.5 mM.

We further noted that CD dimers substituted with another group conferring solubility and low toxicity greatly increased the affinity of CD for 7KC (fig. 16G-H). The methylated triazole linked dimer contains a similar number of substitutions (about 3) as the HP β CD dimer of figure 16F. We retested the HP β CD DS3 dimer and the methyl DS3 dimer, and found that they had significantly similar capacity to solubilize 7KC and cholesterol, while maintaining similar specificity for 7 KC.

Based on the following predictions: i.e., dimeric CD with other substituents of similar degree of substitution, will also bind 7KC and cholesterol with similar affinity and specificity, synthesizing a novel substituted triazole-linked dimer (examples 5-7 above). We utilized a set of charged functional groups (quaternary ammonium (QA), Sulfobutyl (SB), and Succinyl (SUCC)) commonly used as substitutions on cyclodextrins. These low-substituted compounds gave comparable or improved affinity and specificity for 7KC (fig. 16H) compared to unsubstituted hydroxypropyl or methyl substituted triazole linked dimers (fig. 16B, fig. 16E-G). In contrast, highly substituted SB dimers do not bind cholesterol or 7KC well. This is probably due to the many bulky SB groups that restrict access to the binding cavity of the CD dimer.

Combining the turbidity data of the monomer and dimer with the calculated data, we can draw two general conclusions: the low degree of substitution (probably most important for the subsurface) promotes the specificity of certain interactions, especially 7 KC. Modeling data suggests that hydrogen bonding between subsurface hydroxyl groups and 7-keto groups may contribute to this specificity. Furthermore, in general, modeling data indicates that large steric substitutions can indiscriminately block access to the cavity of any potential guest molecule if present at sufficiently high DS levels. Thus, non-bulky steric groups (such as methyl groups) added to CD dimers at high substitution levels are predicted to bind sterol molecules (such as cholesterol and 7KC) with high affinity but selectivity to 7KC is not particularly high compared to cholesterol, while low-substituted methyl- β -cyclodextrin dimers are predicted to bind 7KC with high specificity compared to cholesterol. In contrast, cyclodextrin dimers containing large steric substitutions (such as SB) are predicted to bind 7KC with greater specificity than cholesterol at low substitution levels, but at high substitution levels, either cholesterol or 7KC cannot be bound, nor other sterols, due to blocking access to the binding cavity. Less hindered groups (such as HP) are predicted to behave similarly to SB, but more HP groups are generally required than SB groups to prevent entry into the cavity.

From the above results, we predicted that randomly methyl substituted bCD dimers preferentially bind 7KC rather than cholesterol up to at least the substitution level of DS 10. Beyond this DS level, specificity towards 7KC is progressively reduced compared to cholesterol due to the reduced number of hydroxyl groups on the subsurface available for hydrogen bonding to 7KC as the degree of methyl substitution increases; however, binding to 7KC and cholesterol may still occur.

In contrast, it is predicted that the randomly SB substituted β CD dimer binds 7KC preferentially to cholesterol until the substitution level is at least DS 4 to DS 5, and the hydroxyl groups in the secondary surface again form hydrogen bonds with 7KC and promote stronger binding relative to cholesterol. However, beyond this level of DS, specificity for 7KC can gradually decrease, and in addition binding to 7KC and cholesterol and other similar guest molecules is expected to decrease due to steric interference with guest entry into the β CD cavity. In our data, DS exceeding 14 appears to almost eliminate binding to cholesterol or 7 KC.

For similar reasons, HP substituted dimers are expected to preferentially bind 7KC over cholesterol until the substitution level is at least DS 4 or DS 5, whereas above this level binding specificity for 7KC of up to about DS 20 is predicted to decrease progressively upon binding of the two compared to cholesterol, and binding to 7KC and cholesterol greater than DS 20 is expected to decrease due to steric interference with guest entry into the β CD cavity.

It is predicted that SUCC-substituted and QA-substituted β CD dimers also preferentially bind 7KC over cholesterol until the substitution level is at least DS 4 or DS 5, and the hydroxyl groups in the secondary surface again hydrogen bond with 7KC and promote stronger binding relative to cholesterol. Beyond this DS level, however, specificity for 7KC may decrease, and in addition binding to 7KC and cholesterol is expected to decrease gradually due to steric interference on guest entry into the β CD compartment at certain DS levels (which may exceed DS 15).

Our wet laboratory data validated these models as follows: we used all commonly used substitutions (. about.DS 3-4) on various synthetic β CD dimers in small amounts to demonstrate the specificity of 7KC versus cholesterol. Increasing the DS of the HP group to greater than 4 up to 8 reduces the affinity for 7KC but does not reduce the affinity for cholesterol. Increasing DS of SB dimer to about 15 severely reduces binding to both cholesterol and 7 KC.