Synthetic bioconjugates
阅读说明：本技术 合成的生物缀合物 (Synthetic bioconjugates ) 是由 约翰·埃里克·帕代里 茱莉娅·陈 萨曼莎·萨哈 于 2018-07-09 设计创作，主要内容包括：本发明提供了生物缀合物及其使用方法,该生物缀合物包含骨架和至少一个支链或非支链的肽,该肽具有至少一个通过间隔物共价结合至其的胶原结合单元。(The present invention provides bioconjugates comprising a scaffold and at least one branched or unbranched peptide having at least one collagen binding unit covalently bound thereto by a spacer, and methods of use thereof.)
1. A bioconjugate comprising a glycan and at least one binding unit of formula (I) covalently bound thereto:
((X1)mX2)nX3-L
(I)
wherein:
X1is an amino acid sequence comprising a collagen binding unit;
X2and X3Independently absent; an amino acid sequence having 1 to 15 amino acids; or group
l is a spacer of 5 to 20 amino acids selected from glycine (G), serine (S), arginine (R) and lysine (K), or a group
m is 1 or 2; and is
n is 1 or 2.
2. The bioconjugate of claim 1 wherein L is selected from the group consisting of GSGRR (SEQ ID NO:), GSGSGRR (SEQ ID NO:), GSGSGSGSRR (SEQ ID NO:), GSGSGSGRR (SEQ ID NO:), GSGSGSGSRR (SEQ ID NO:), GSGSGSGSGSGRR (SEQ ID NO:), GSGSGSGSGSGSRR (SEQ ID NO:), GSGSGSGSGSGSGRR (SEQ ID NO:), GSRRGS (SEQ ID NO:), GSGRRGSG (SEQ ID NO:), GSGRRRGSG (SEQ ID NO:), GSGRRR (SEQ ID NO:), GSGRRRR (SEQ ID NO:), GSGSGSRRR (SEQ ID NO:), GSGSGSRRRR (SEQ ID NO:), GSGSGSRRRRR (SEQ ID NO:), GSGSGSRRRRRR (SEQ ID NO:) and GSGSGSGSRRR (SEQ ID NO:).
3. The bioconjugate according to claim 1 wherein the bioconjugate comprises a glycan and from 1 to about 10 binding units of formula (I) covalently bound thereto.
4. The bioconjugate according to claim 1 wherein the bioconjugate comprises a glycan and from 1 to about 8 binding units of formula (I) covalently bound thereto.
5. The bioconjugate according to claim 1 wherein the bioconjugate comprises a glycan and about 5 binding units of formula (I) covalently bound thereto.
6. The bioconjugate according to any one of claims 1 to 5 wherein:
X2is an amino acid sequence comprising GSG; and is
X3Is an amino acid sequence comprising an XRR, wherein X is absent or a natural or unnatural amino acid having a side chain capable of forming an amide bond.
7. The bioconjugate according to any one of claims 1 to 6 wherein:
X2is an amino acid sequence comprising GSG; and is
X3Is an amino acid sequence comprising RR or KRR.
8. The bioconjugate according to any one of the preceding claims wherein the binding unit of formula (I) is X1-GSGSGSRR-。
9. The bioconjugate according to any preceding claim wherein X1Comprising 3 to about 20 amino acids.
10. The bioconjugate according to any preceding claim wherein X1Comprising 3 to about 10 amino acids.
11. The bioconjugate according to any preceding claim wherein X1Comprising amino acid sequence GQLYKSILY (SEQ ID NO:).
12. The bioconjugate according to any preceding claim wherein X1Comprising amino acid sequence RRANAALKAGELYKSILY (SEQ ID NO:).
13. The bioconjugate according to any preceding claim wherein X1Comprising amino acid sequence GELYKSILY (SEQ ID NO:).
14. The bioconjugate according to any preceding claim wherein X1Comprising amino acid sequence RRANAALKAGQLYKSILY (SEQ ID NO:).
15. The bioconjugate according to any preceding claim wherein X2Comprises 3 to 5 amino acids, and X3Comprising 2 to 4 amino acids.
16. The bioconjugate of any preceding claim, wherein the glycan comprises from about 1% to about 50% functionalization, or from about 5% to about 30% functionalization, or about 25% functionalization, wherein the percent (%) functionalization is determined by the percentage of disaccharide units on the glycan that are functionalized with the binding unit of formula (I).
17. The bioconjugate of claim 16, wherein the glycan comprises about 2.5% functionalization, or about 5% functionalization, or about 8% functionalization, or about 10% functionalization, or about 16% functionalization, or about 32% functionalization, wherein the percent (%) functionalization is determined by the percentage of disaccharide units on the glycan that are functionalized with the binding unit of formula (I).
18. The bioconjugate according to any preceding claim, wherein the glycan is heparin or a derivative thereof.
19. The bioconjugate according to any preceding claim wherein the bioconjugate comprises about 8 binding units of formula (I) covalently bound thereto.
20. The bioconjugate according to any preceding claim wherein the bioconjugate comprises about 5 binding units of formula (I) covalently bound thereto.
21. The bioconjugate according to claim 1 wherein the binding unit is RRANAALKAGELYKSILYGSGSGSRR-NHNH- (SEQ ID NO:).
22. The bioconjugate according to claim 1 wherein the binding unit is RRANAALKAGQLYKSILYGSGSGSRR-NHNH- (SEQ ID NO:).
23. The bioconjugate according to claim 1 wherein the binding unit is GQLYKSILYGSGSGSRR-NHNH- (SEQ ID NO:).
24. The bioconjugate of claim 1, wherein the binding unit is (GQLYKSILYGSG)2-KSGSRR-NHNH-。
25. A bioconjugate comprising heparin conjugated to GQLYKSILYGSGRRGSG (SEQ ID NO:) in a ratio of 1: 8.
26. A bioconjugate comprising heparin conjugated to GQLYKSILYGSGRRGSG (SEQ ID NO:) in a ratio of 1: 5.
27. The bioconjugate according to any preceding claim wherein heparin is conjugated to each of the peptides through a C-terminal glycine.
28. The bioconjugate of any preceding claim, wherein the bioconjugate has an EC of less than about 40 μ g/mL or less than about 20 μ g/mL50Binds to type I collagen.
29. The bioconjugate of any preceding claim, wherein the linking group comprises a hydrazide group (-NHNH-).
30. A binding unit of formula (II):
((X1)mX2)nX3-L
(II)
wherein:
X1is an amino acid sequence comprising a collagen binding unit;
X2and X3Independently absent; an amino acid sequence having 1 to 15 amino acids; or group
l is a spacer of 5 to 20 amino acids selected from glycine (G), serine (S), arginine (R) and lysine (K), or a group
m is 1 or 2; and is
n is 1 or 2;
with the proviso that the binding unit is not RRRKKIQGRSKR (SEQ ID NO:) or RRGGRKWGSFEG (SEQ ID NO:).
31. The binding unit according to claim 30, wherein:
X2is an amino acid sequence comprising glycine (G) and serine (S); and is
X3Is an amino acid sequence comprising glycine (G) and serine (S).
32. The binding unit according to claim 30, wherein:
X2is an amino acid sequence comprising GSG;
X3is an amino acid sequence comprising RR, KRR, GSG or KGSG (SEQ ID NO:).
33. The binding unit according to any one of claims 30 to 32, wherein X1Comprising 3 to about 20 amino acids.
34. The binding unit according to any one of claims 30 to 32, wherein X1Comprising 3 to about 10 amino acids.
35. The binding unit according to any one of claims 30 to 32, wherein X1Comprising amino acid sequence GQLYKSILY (SEQ ID NO:).
36. The binding unit according to any one of claims 30 to 32, wherein X1Comprising amino acid sequence RRANAALKAGELYKSILY (SEQ ID NO:).
37. The binding unit according to any one of claims 30 to 32, wherein X1Comprising 5 to 10 amino acids, X2Comprises 2 to 5 amino acids, and X3Comprising 3 to 5 amino acids.
38. A peptide having the amino acid sequence RRANAALKAGELYKSILYGSGSGSRR (SEQ ID NO:) or RRANAALKAGELYKSILYGSGSGSRR-NHNH2(SEQ ID NO: )。
39. A peptide having the amino acid sequence RRANAALKAGQLYKSILYGSGSGSRR (SEQ ID NO:) or RRANAALKAGQLYKSILYGSGSGSRR-NHNH2(SEQ ID NO: )。
40. A peptide having the amino acid sequence GQLYKSILYGSGSGSRR (SEQ ID NO:) or GQLYKSILYGSGSGSRR-NHNH2(SEQ ID NO: )。
41. A peptide having an amino acid sequence (GQLYKSILYGSG)2-KSGSRR or (GQLYKSILYGSG)2-KSGSRR-NHNH2。
42. A peptide having the amino acid sequence GQLYKSILYGSGRRGSG (SEQ ID NO:).
43. A method for treating peripheral vascular disease in a patient in need thereof, comprising administering an effective amount of the bioconjugate of any one of claims 1-29.
44. A method for treating fibrosis in a patient in need thereof comprising administering an effective bioconjugate of any one of claims 1-29.
Technical Field
The present invention provides bioconjugates comprising a scaffold and at least one branched or unbranched peptide having at least one collagen binding unit covalently bound thereto by a spacer.
Background
In tissue, cells are surrounded by an extracellular matrix (ECM) containing various macromolecules such as bioconjugates, collagen, hyaluronic acid, laminin, fibronectin, and the like. In mammals, bioconjugates are a major component of the extracellular matrix where they form large complexes with other bioconjugates, hyaluronic acid, and fibrous matrix proteins (e.g., collagen). As mammals age and in certain disease states, extracellular matrix in certain parts of the body (e.g., synovial joints, vitreous humor, spinal discs, skin, etc.) degrades, causing undesirable symptoms such as various forms of arthritis, loss of vision, and the like. In addition, some tissue injuries, such as vascular injury, corneal injury, and skin wounds, result in exposure of the extracellular matrix and/or its components (including collagen).
Summary of The Invention
It was surprisingly found that bioconjugates with certain bonds between glycans and peptides resulted in increased binding of certain peptides to their target. It was also found that having branched peptides resulted in improved binding. In particular, bioconjugates with certain spacers provide enhanced collagen binding activity.
The present invention provides bioconjugates comprising a glycan and at least one binding unit of formula (I) covalently bound thereto:
((X1)mX2)nX3-L
(I)
wherein:
X1is an amino acid sequence comprising a collagen binding unit;
X2and X3Independently absent; an amino acid sequence having 1 to 15 amino acids; or group
l is a spacer of 5 to 20 amino acids selected from glycine (G), serine (S), arginine (R) and lysine (K), or a group
m is 1 or 2; and is
n is 1 or 2.
In one embodiment, L is not GSGKRRGSG (SEQ ID NO:).
In one embodiment, X1Is the N-terminus of a peptide, and X3At the C-terminus of the peptide.
In one embodiment, formula (I) is not RRRKKIQGRSKR (SEQ ID NO:) or RRGGRKWGSFEG (SEQ ID NO:).
In one embodiment, the linking group is-NHNH-.
In one embodiment, the invention provides a functionalized peptide of the formula:
L-NHNH2
wherein L is a spacer of 5 to 20 amino acids selected from glycine (G), serine (S), arginine (R) and lysine (K), or a group
In one embodiment, L is-GSGSGSRR-NHNH- (SEQ ID NO:). The invention also provides peptides comprising GSGSGSGSRR (SEQ ID NO:).
The invention also provides a binding unit of formula (II):
((X1)mX2)nX3-L
(II)
wherein:
X1is an amino acid sequence comprising a collagen binding unit;
X2and X3Independently absent; an amino acid sequence having 1 to 15 amino acids; or group
l is a spacer of 5 to 20 amino acids selected from glycine (G), serine (S), arginine (R) and lysine (K), or a group
m is 1 or 2; and is
n is 1 or 2;
with the proviso that formula (II) is not RRRKKIQGRSKR (SEQ ID NO:) or RRGGRKWGSFEG (SEQ ID NO:).
In certain embodiments, m is 1 and n is 2. In certain embodiments, m is 2 and n is 1. In certain embodiments, m is 2 and n is 2.
In certain embodiments, X2And X3Independently is absent; an amino acid sequence having 1 to 15 amino acids selected from the group consisting of glycine (G), serine (S), arginine (R) and lysine (K); or group
In certain embodiments, X is when m and/or n is not 12And X3Comprises at least one arginine (R) or lysine (K). In certain embodiments, wherein when m and/or n is not 1, X2And X3Arginine (R) or lysine (K) is not included.
In certain embodiments, L comprises a hydrazide.
Drawings
Some aspects of the invention may be viewed through the figures. The following figures are included herein:
figure 1 shows a comparison of collagen binding of
Figure 2 shows a comparison of collagen binding of
Figure 3 shows a comparison of collagen binding of
Figure 4 shows a comparison of collagen binding of
Figure 5 shows a comparison of collagen binding of
FIG. 6 shows a comparison of collagen binding of some of the bioconjugates of FIGS. 1-5, and FIG. 7 shows that the corresponding EC were compared50Histogram of values for comparison.
Figure 8 shows a comparison of collagen binding of
Figure 9 shows a comparison of collagen binding of compound 16, compound 17 and compound 18.
Fig. 10 shows a comparison of collagen binding of bioconjugate compound 18 and compound 19.
Figure 11 shows a comparison of collagen binding of
Figure 12, panels a and B, show the effect of compound 10 (panel B) on platelet binding to collagen compared to compound 1 (panel a).
Figure 13 shows the collagen content in the liver measured histologically by Sirius Red (Sirius Red) for
Figure 14 shows IVIS imaging showing the distribution of fluorescently labeled molecules in the kidney and bladder. Top images were acquired five minutes after IV injection and bottom images were acquired 1 hour after injection. The left image is the ventral side and the right image is the dorsal side of the mouse.
FIG. 15 shows the effect of the position of the first arginine residue from glycan (GAG) on collagen binding sequence GQLYKSILY (SEQ ID NO:).
FIG. 16 shows the effect of spacer length on collagen binding when attached to the collagen binding domain of GQLYKSILY (SEQ ID NO:).
FIG. 17 shows the effect of amino acid changes in the collagen binding domain of GQLYKSILY (SEQ ID NO:) on collagen binding. (A) The effect of replacing arginine with lysine or D-arginine is shown. (B) The collagen binding affinity of the spacer GSGSGSRR (SEQ ID NO:) compared to GQLYKSILYGSGSGSRR (SEQ ID NO:) is shown. (C) Showing the effect of inserting a specific sequence or chemical within the spacer. PAPARR (SEQ ID NO:) is Pro-Ala-Pro-Arg, AhxRR is 6-aminocaproic acid-Arg, PEG6RR is (polyethylene glycol)6-arginine, ahxRRAhx is 6-aminocaproic acid-arginine-6-aminocaproic acid.
FIG. 18 shows a second collagen binding assay test with GQLYKSILY (SEQ ID NO:) binding sequence with optimized spacer sequence compared to the binding sequence without spacer and the spacer sequence as a control.
FIG. 19 shows the effect of adding a GSGSGSRR (SEQ ID NO:) spacer sequence to the collagen binding domain WREPSFSALS (SEQ ID NO:) vWF-2 x.
Detailed Description
It is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.
1. Definition of
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. It should be noted that, as used herein and in the claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a peptide" includes a plurality of peptides.
As used herein, the following terms and abbreviations have the following meanings.
As used herein, the term "comprising" is intended to mean that the compositions and methods include the recited elements but not exclude other elements. "consisting essentially of … …" when used to define compositions and methods should be meant to exclude other elements that have any substantial effect on the combination for the intended use. Thus, a composition consisting essentially of the elements, as defined herein, would not exclude other materials or steps that do not materially affect the basic and novel characteristics of the invention. "consisting of … …" shall mean excluding other components and substantial method steps than trace elements. Aspects defined by each of these transitional terms are within the scope of the invention.
The term "about" when used in conjunction with a numerical designation (e.g., temperature, time, amount, and concentration, including ranges) means that the approximation can vary by (+) or (-) 10%, 5%, or 1%.
As used herein, the terms "bioconjugate," "peptidoglycan," and "proteoglycan," and "synthetic proteoglycan" are used interchangeably and refer to a synthetic conjugate comprising a glycan and one or more peptides covalently bonded to the glycan. The glycan moiety may be made synthetically or obtained from animal sources. In some embodiments, the peptide is covalently bound to the glycan through a hydrazide-carbonyl bond (i.e., -C (O) -NH-NH-C (O)). In some embodiments, the hydrazide-carbonyl bond is located between a terminal hydrazide group on the peptide and a carbonyl group on the glycan. In other embodiments, the hydrazide-carbonyl bond is between the terminal carbonyl group on the peptide and the hydrazide group on the glycan. In some embodiments, the term bioconjugate includes peptidoglycans.
As used herein, the term "glycan" refers to a compound having a plurality of glycosidically linked monosaccharides. In some embodiments, the glycan is a glycosaminoglycan (GAG) comprising a 2-amino sugar linked to uronic acid in an alternating manner and includes polymers such as heparin, heparan sulfate, chondroitin, keratin and dermatan. Accordingly, non-limiting examples of glycans that may be used in embodiments described herein include alginate, agarose, dextran sulfate, Chondroitin Sulfate (CS), Dermatan Sulfate (DS), heparan sulfate, heparin (Hep), keratin, keratan sulfate, and Hyaluronic Acid (HA), including derivatives thereof. In another embodiment, the molecular weight of the glycans is varied to tailor the effect of the bioconjugate (see, e.g., Radek, K.A., et al., Wound Repair Regen.,2009,17: 118-126; and Taylor, K.R., et al, J.biol. chem.,2005,280: 5300-5306). In one embodiment, the glycans are degraded by oxidation and alkaline elimination (see, e.g., Fransson, l.a., et al, eur.j.biochem.,1980,106:59-69) to provide degraded glycans having lower molecular weights (e.g., from about 10kDa to about 50 kDa). In some embodiments, the glycan is unmodified. In some embodiments, the glycan does not contain an oxidatively cleaved sugar ring, and therefore does not contain and does not contain an aldehyde functional group. In some embodiments, the glycans are derivatized.
As used herein, the term "derivatized glycan" is intended to include derivatives of glycans. For example, the derivatized glycan may include one or more chemical derivatives, such as, but not limited to, a partially N-desulfated derivative, a partially O-desulfated derivative, and/or a partially O-carboxymethylated derivative. For example, as used herein, the term "heparin" is intended to include heparin and derivatives thereof, such as, but not limited to, partial N-and/or partial O-desulfated heparin derivatives, partial O-carboxymethylated heparin derivatives, or combinations thereof. In some embodiments, the heparin is non-oxidized heparin (i.e., does not contain oxidatively cleaved sugar rings) and does not contain aldehyde functional groups.
As used herein, the terms "bonded" and "covalently bonded" may be used interchangeably to refer to one or more electron pairs shared by two atoms.
In one embodiment, the bioconjugates of the invention bind to collagen, either directly or indirectly. The term "binding" as used herein is intended to include interactions between molecules that can be detected using, for example, hybridization assays, surface plasmon resonance, ELISA, competitive binding assays, isothermal titration calorimetry, phage display, affinity chromatography, rheology, or immunohistochemistry. The term is also intended to include "binding" interactions between molecules. Binding may be "direct" or "indirect". "direct" binding includes direct physical contact between molecules. "indirect" binding between molecules includes direct physical contact of a molecule with one or more molecules at the same time. This binding may result in the formation of a "complex" containing the interacting molecules. "Complex" refers to the association of two or more molecules held together by covalent or non-covalent bonds, interactions, or forces.
As used herein, the term "composition" refers to a formulation containing at least one pharmaceutically active ingredient, including any solid form thereof, suitable for administration to an intended patient for therapeutic purposes. The composition may comprise at least one pharmaceutically acceptable component to provide improved formulation of the compound, for example a suitable carrier. In some embodiments, the composition is formulated as a film, gel, patch, or liquid solution. As used herein, the term "local" refers to non-systemic administration of a composition to the surface of the tissue and/or organ (internal or in some cases external) to be treated to obtain a local effect.
As used herein, the term "pharmaceutically acceptable" means that the indicated material is not of a nature that would cause reasonable caution in the physician avoiding administration to the patient, taking into account the amount used and/or the disease or condition to be treated and the respective route of administration. Typically pharmaceutically acceptable is substantially sterile. As used herein, the term "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, composition or carrier, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting any supplement or composition, or component thereof, from one organ or portion of the body to another organ or portion of the body, or delivering an agent to the interior surface of a vein.
As used herein, the term "solution" refers to solutions, suspensions, emulsions, drops, ointments, liquid lotions, sprays, and liposomes as are well known in the art. In some embodiments, the liquid solution contains an aqueous pH buffer that is resistant to changes in pH when small amounts of acid or base are added. In some embodiments, the liquid solution contains a lubricity enhancer.
As used herein, the term "polymer", "polymer matrix" or "polymeric agent" refers to a biocompatible polymeric material. The polymeric material described herein may comprise: such as sugars (e.g., mannitol), peptides, proteins, laminin, collagen, hyaluronic acid, ionic and non-ionic water soluble polymers; an acrylic polymer; hydrophilic polymers such as polyethylene oxide, polyoxyethylene-polyoxypropylene copolymer, and polyvinyl alcohol; cellulose polymers and cellulose polymer derivatives such as hydroxypropyl cellulose, hydroxyethyl cellulose, hydroxypropyl methylcellulose phthalate, methylcellulose, carboxymethyl cellulose, and etherified cellulose; poly (lactic acid), poly (glycolic acid), copolymers of lactic acid and glycolic acid, or other natural and synthetic polymers.
In some embodiments, the polymer matrix is absorbable, such as collagen, polyglycolic acid, polylactic acid, polydioxanone, and caprolactone. As used herein, the term "absorbable" refers to the ability of a material to be absorbed into the body. In other embodiments, the polymer is non-absorbable, such as polypropylene, polyester, or nylon.
As used herein, the term "pH buffer" refers to an aqueous buffer solution that resists changes in pH when small amounts of acid or base are added thereto. The pH buffered solution typically comprises a mixture of a weak acid and its conjugate base, and vice versa. For example, the pH buffered solution may comprise: phosphates such as sodium phosphate, sodium dihydrogen phosphate dihydrate, disodium hydrogen phosphate dodecahydrate, potassium phosphate, potassium dihydrogen phosphate, and dipotassium hydrogen phosphate; boric acid and borates such as sodium borate and potassium borate; citric acid and citrates, such as sodium citrate and disodium citrate; acetates such as sodium acetate and potassium acetate; carbonates such as sodium carbonate and sodium bicarbonate, etc. The pH adjusting agent may include, for example: acids such as hydrochloric acid, lactic acid, citric acid, phosphoric acid and acetic acid; and basic bases such as sodium hydroxide, potassium hydroxide, sodium carbonate, sodium bicarbonate, and the like. In some embodiments, the pH buffering agent is a Phosphate Buffered Saline (PBS) solution (i.e., containing sodium phosphate, sodium chloride, and in some formulations potassium chloride and potassium phosphate).
As used herein, the terms "peptide" and "peptide sequence" refer to straight or branched chain amino acids linked by peptide (or amide) bonds. In one embodiment, the peptide comprises from about 3 to about 120 amino acids, or from about 3 to about 110 amino acids, or from about 3 to about 100 amino acids, or from about 3 to about 90 amino acids, or from about 3 to about 80 amino acids, or from about 3 to about 70 amino acids, or from about 3 to about 60 amino acids, or from about 3 to about 50 amino acids, or from about 3 to about 40 amino acids, or from about 5 to about 120 amino acids, or from about 5 to about 100 amino acids, or from about 5 to about 90 amino acids, or from about 5 to about 80 amino acids, or from about 5 to about 70 amino acids, or from about 5 to about 60 amino acids, or from about 5 to about 50 amino acids, or from about 5 to about 40 amino acids, or from about 5 to about 30 amino acids, or from about 5 to about 20 amino acids, or from about 5 to about 10 amino acids.
In various embodiments described herein, these peptides may be modified by including one or more conservative amino acid substitutions in the binding unit. As is well known to those of ordinary skill in the art, altering any non-critical amino acid of a peptide by conservative substitutions should not significantly alter the activity of the peptide, as the side chain of the substituted amino acid should be capable of forming a bond and contact similar to the side chain of the amino acid that has been substituted. Non-conservative substitutions are also possible, as long as they do not significantly affect the binding activity of the peptide.
As used herein, the term "sequence identity" refers to the level of amino acid residue or nucleotide identity between two fetuses or between two nucleic acid molecules. When a position in the compared sequences is occupied by the same base or amino acid, then the molecules are identical at that position. A peptide (or polypeptide or peptide region) has a percentage (e.g., at least about 60%, or at least about 65%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 83%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 98% or at least about 99%) of "sequence identity" with another sequence means that when aligned, the percentage of bases (or amino acids) are the same in a comparison of the two sequences. It should be noted that for any sequence disclosed in this application ("reference sequence"), sequences having at least about 60%, or at least about 65%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 83%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 98%, or at least about 99% sequence identity to the reference sequence are also within the scope of the invention. Likewise, the invention also includes sequences having one, two, three, four, or five substitutions, deletions or additions of amino acid residues or nucleotides compared to the reference sequence. In some embodiments, in any one or more of the sequences specifically disclosed herein, the sequence may be modified by having one, two, or three amino group additions, deletions, and/or substitutions from each thereof.
As is well known in the art, a "conservative substitution" of an amino acid or a "conservatively substituted variant" of a peptide refers to an amino acid substitution that maintains: 1) the secondary structure of the peptide; 2) charge or hydrophobicity of amino acids; and 3) the bulkiness of the side chains or any one or more of these features. Illustratively, the well-known term "hydrophilic residue" relates to serine or threonine. "hydrophobic residue" refers to leucine, isoleucine, phenylalanine, valine, alanine, or the like. "positively charged residue" refers to lysine, arginine, ornithine, or histidine. "negatively charged residue" refers to aspartic acid or glutamic acid. Residues with "bulky side chains" refer to phenylalanine, tryptophan, or tyrosine, etc. A list of illustrative conservative amino acid substitutions is given in table 1.
TABLE 1
In some embodiments, the sequences described herein may be modified to replace one or more glutamic acid residues with glutamine and/or one or more aspartic acid residues with asparagine.
Collagen binding unit
As used herein, the term "collagen binding unit" is intended to refer to an amino acid sequence within a peptide that binds to collagen. "collagen binding" refers to interactions with collagen, which may include hydrophobic, ionic (charge), and/or van der waals interactions, such that a compound binds or interacts favorably with collagen. This binding (or interaction) is intended to be distinguished from covalent bonds and non-specific interactions with common functional groups, such that the peptide interacts with any substance comprising the functional group to which the peptide is bound on collagen. Any method known in the art can be used to test and assess the binding of peptides to collagen. See, for example, Li, Y., et al, Current Opinion in Chemical Biology,2013,17: 968-975, Helmes, B.A., et al, J.Am.chem.Soc.2009,131, 11683-11685, and Petsalaki, E., et al, PLoScout Biol,2009,5(3): e 1000335. In one embodiment, the peptide or the collagen binding unit of the peptide has a dissociation constant (K)d) Binding to collagen, the dissociation constant (K)d) Less than about 1mM, or less than about 900. mu.M, or less than about 800. mu.M, or less than about 700. mu.M, or less than about 600. mu.M, or less than about 500. mu.M, or less than about 400. mu.M, or less than about 300. mu.M, or less than about 200. mu.M, or less than about 100. mu.M. In certain embodiments, the collagen binding unit comprises up to about 120 amino acids.
The peptide may have amino acid homology to a portion of a protein that is normally or not normally involved in collagen fibril formation. In some embodiments, the peptides have homology or sequence identity to the amino acid sequence of a leucine-rich small bioconjugate, a platelet receptor sequence, or a protein that modulates collagen fibrillogenesis. In various embodiments, the peptide comprises an amino acid sequence selected from the group consisting of: RRANAALKAGELYKSILY (SEQ ID NO:), GELYKSILY (SEQ ID NO:), RRANAALKAGELYKCILY (SEQ ID NO:), GELYKCILY (SEQ ID NO:), RRANAALKAGQLYKSILY (SEQ ID NO:), GQLYKSILY (SEQ ID NO:), RRANAALKAGQLYKCILY (SEQ ID NO:), GQLYKCILY (SEQ ID NO:), RLDGNEIKR (SEQ ID NO:), AHEEISTTNEGVM (SEQ ID NO:), NGVFKYRPRYFLYKHAYFYPPLKRFPVQ (SEQ ID NO:), CQDSETRTFY (SEQ ID NO:), TKKTLRT (SEQ ID NO:), GLRSKSKKFRRPDIQYPDATDEDITSHM (SEQ ID NO:), SQVNPQP (SEQ ID NO:), SYIRIADTNIT ('). ID NO:), KELNLVYT (SEQ ID NO:), GSIT (SEQ ID NO:), GSITTIDVPWNV (SEQ ID NO:), GQLYKSILY (SEQ ID NO:, RRANAALKAGQLYKSILY (SEQ ID NO:, RVMHGLHLGDDE (SEQ ID NO), CRVMHGLHLGDDEC (SEQ ID NO:), RVMHGLHLGDDE (SEQ ID NO: or PVI:), or at least about 80% of the same sequence or about the same of the sequence as that of the sequence of SEQ ID NO, the sequence of SEQ ID NO, the sequence of the, Or at least about 85% sequence identity, or at least about 90% sequence identity, or at least about 95% sequence identity, or at least about 98% sequence identity, provided that the sequence is capable of binding to collagen.
In some embodiments, the peptide comprises an amino acid sequence having at least about 80%, or at least about 83%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 98%, or at least about 100% sequence identity to the collagen binding domain of von willebrand factor (vWF) or platelet collagen receptor described in Chiang, t.m., et al.j.biol.chem.,2002,277:34896-34901, Huizinga, e.g., et al, Structure,1997,5:1147-1156, Romijn, r.a., et al, j.biol.chem.,2003,278:15035-15039, and Chiang, et al, Cardio. A non-limiting example is WREPSFCALS (SEQ ID NO:), which is derived from vWF.
Various methods for screening peptide sequences for collagen binding affinity (or collagen binding domain/unit) are conventional in the art, other peptide sequences that show collagen binding affinity (or collagen binding unit) useful in the bioconjugates and methods disclosed herein include, but are not limited to, β AWHCTTKFPHHYCLYBip (SEQ ID NO: J), β AHHLTHYCFTBip (SEQ ID NO: J), β AHKCPWHLYTHYCFT (SEQ ID NO: J), etc., wherein Bip is biphenylalanine and β A is β -alanine (see Abd-Elgaliel, W.R. et al, Biomers, 2013,100(2), 167-Sci), GROGER (SEQ ID NO: 821, GMER (SEQ ID NO: J., WO 92), SEQ ID NO: 92, WO 11J., WLD, WO 92, WO 9J., WO 92, WO 11J., WO 92, WO 11J., WO 92, WO 11, WO 92, WO 11, WO 92, WO9, WO 11, WO 92, WO 11, WO 25J., WO 85, WO 11, WO 25J, WO 11, WO 85, WO9, WO 85, WO 11, WO9, WO 85, WO9, WO 85, WO9, WO 85, WO9, WO 85, WO9, WO 85, WO9, WO 85, WO9, WO 2, WO 85, WO 2, WO9, WO 85, WO9, WO 2, WO9, WO 2, WO9, WO 85, WO 2, WO9, WO 85, WO9, WO 85, WO 2, WO9, WO 2, WO9, WO 85, WO 2, WO9, WO 2, WO 9.
Other peptide sequences shown to have collagen binding affinity (or collagen binding units) useful in the bioconjugates and methods disclosed herein include, but are not limited to: LSELRLHEN (SEQ ID NO:)), LTELHLDNN (SEQ ID NO:), LSELRLHNN (SEQ ID NO:), LSELRLHAN (SEQ ID NO:), and LRELHLNNN (SEQ ID NO:) (see Fredrio, S., Angew. chem. int. Ed.2015,37, 10980-.
In some embodiments, the peptide comprises one or more sequences selected from the group consisting of: RVMHGLHLGDDE (SEQ ID NO:), D-amino acid EDDGLHLGHMVR (SEQ ID NO:), RVMHGLHLGNNQ (SEQ ID NO:), D-amino acid QNNGLHLGHMVR (SEQ ID NO:), RVMHGLHLGNNQ (SEQ ID NO:), GQLYKSILYGSG-4K2K (SEQ ID NO:) (a 4-branched peptide), GSGQLYKSILY (SEQ ID NO:), GSGGQLYKSILY (SEQ ID NO:), KQLNLVYT (SEQ ID NO:), CVWLWQQC (SEQ ID NO:), WREPSFSALS (SEQ ID NO:), GHRPLDKKREEAPSAPPPISGGGYR (SEQ ID NO:), and GHRPLNKKRQQAPSLRPAPPPISGGGYR (SEQ ID NO:).
Similarly, for collagen binding peptides, peptides derived from a phage display library selected for collagen can be generated. Peptides can be synthesized and evaluated for binding to collagen by any technique (e.g., SPR, ELISA, ITC, affinity chromatography, or other techniques known in the art). An example may be a biotin-modified peptide sequence (e.g. SILY)Biotin) Which is incubated on a microplate containing immobilized collagen. A dose-response binding curve can be generated using streptavidin chromophore to determine the ability of the peptide to bind to collagen.
In one embodiment, the peptide comprises one or more collagen binding units that bind to any one or more of collagen I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII, XIII, or XIV. In one embodiment, the peptide has a dissociation constant (K)d) Binding to type I collagen, the dissociation constant (K)d) Less than about 1mM, or less than about 900. mu.M, or less than about 800. mu.M, or less than about 700. mu.M, or less than about 600. mu.M, or less than about 500. mu.M, or less than about 400. mu.M, or less than about 300. mu.M, or less than about 200. mu.M, or less than about 100. mu.M. In one embodiment, the peptide has a dissociation constant (K)d) Binding to type II collagen, the dissociation constant (K)d) Less than about 1mM, or less than about 900. mu.M, or less than about 800. mu.M, or less than about 700. mu.M, or less than about 600. mu.M, or less than about 500. mu.M, or less than about 400. mu.M, or less than about 300. mu.M, or less than about 200. mu.M, or less than about 100. mu.M. In one embodiment, the peptide has a dissociation constant (K)d) Binding to type III collagen, the dissociation constant (K)d) Less than about 1mM, or less than about 900. mu.M, or less than about 800. mu.M, or less than about 700. mu.M, or less than about 600. mu.M, or less than about 500. mu.M, or less than about 400. mu.M, or less than about 300. mu.M, or less than about 200. mu.M, or less than about 100. mu.M. In one embodiment, the peptide has a dissociation constant (K)d) Binding to type IV collagen, the dissociation constant (K)d) Less than about 1mM, or less than about 900. mu.M, or less than about 800. mu.M, or less than about 700. mu.M, or less than about 600. mu.M, or less than about 500. mu.M, or less than about 400. mu.M, or less than about 300. mu.M, or less than about 200. mu.M, or less than about 100. mu.M.
In some embodiments, any of the sequences described herein can be modified such that any one or more amino acids (e.g., 1, 2,3, 4, or 5 amino acids) are added, deleted, or substituted therefrom. In some embodiments, the sequence is modified such that any one or more amino acids are substituted with alanine. In some embodiments, the sequence is modified such that any one or more 1-amino acids are replaced with the corresponding d-amino acid scan. In some embodiments, the sequence is modified such that any one or more valine is replaced by leucine, any one or more glutamic acid is replaced by glutamine, any one or more aspartic acid is replaced by asparagine, and/or any one or more arginine is replaced by glutamine.
In any of the embodiments described herein, the peptide having a collagen binding unit comprises any of the amino acid sequences described in the preceding paragraphs, or an amino acid sequence that is at least about 80%, or at least about 83%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 98%, or at least about 100% homologous to any of these amino acid sequences. In various embodiments, the peptide component of the synthetic bioconjugates can be modified by containing one or more conservative amino acid substitutions. As is well known to those of ordinary skill in the art, altering any non-critical amino acid of a peptide by conservative substitutions should not significantly alter the activity of the peptide, as the side chain of the substituted amino acid should be capable of forming a bond and contact similar to the side chain of the amino acid that has been substituted.
Peptides can also be selected by phage display, using positive selection for binding to hyaluronic acid. The collagen binding units may be determined by their interaction with collagen and measured by any technique for assessing molecular interactions (e.g., surface plasmon resonance, ELISA, competitive binding assays, isothermal titration calorimetry, affinity chromatography, rheology, and/or immunohistochemistry). Peptides that are considered "collagen-binding" can interact with collagen or collagen-containing tissues such that the interaction is not attributable to known chemically reactive groups. The interaction may be due to hydrophobic and charge interactions caused by amino acid residues in the peptide. The interaction can be measured by immobilizing collagen on a microplate and incubating with collagen binding units, followed by a detection technique (e.g., biotin-avidin with chromophores). The interaction can also be measured by mechanically influencing the collagen-containing fluid, gel or tissue incubated with the collagen binding unit or with the bioconjugate containing the collagen binding unit.
Bioconjugates
The present invention provides bioconjugates comprising a glycan and at least one binding unit of formula (I) covalently bound thereto:
((X1)mX2)nX3-L
(I)
wherein:
X1is an amino acid sequence comprising a collagen binding unit;
X2and X3Independently is absent; an amino acid sequence having 1 to 15 amino acids; or group Wherein p and q are each independently an integer from 1 to 10;
l is a spacer of 3 to 20 amino acids selected from glycine(G) Serine (S), arginine (R) and lysine (K), or a groupProvided that L comprises at least two arginines (R) within the first 5 amino acids from the glycan, and wherein L further comprises an optional linking group that covalently binds the peptide to the glycan;
m is 1 or 2; and is
n is 1 or 2.
In some embodiments, the present invention provides bioconjugates comprising a glycan and at least one binding unit of formula (I) covalently bound thereto:
((X1)mX2)nX3-L
(I)
wherein:
X1is an amino acid sequence comprising a collagen binding unit;
X2and X3Independently is absent; an amino acid sequence having 1 to 15 amino acids; or group
l is a spacer of 3 to 20 amino acids selected from glycine (G), serine (S), arginine (R) and lysine (K), or a group
m is 1 or 2; and is
n is 1 or 2.
In one embodiment, X1At the N-terminus of the peptideEnd, and X3Located at the C-terminus of the peptide.
In one embodiment, formula (I) is not RRRKKIQGRSKR (SEQ ID NO:) or RRGGRKWGSFEG (SEQ ID NO:).
In certain embodiments, m is 1 and n is 2. In certain embodiments, m is 2 and n is 1. In certain embodiments, m is 2 and n is 2.
In some embodiments, it is contemplated that L is a spacer of 3 to 20 amino acids selected from glycine (G), serine (S), arginine (R), and lysine (K), or a group
in some embodiments, X2And X3Independently is absent; an amino acid sequence having 1 to 15 amino acids selected from the group consisting of glycine (G), serine (S), arginine (R) and lysine (K); or group
In certain embodiments, X is when m and/or n is not 12And X3Comprises at least one arginine (R) or lysine (K). In certain embodiments, X is when m and/or n is not 12And X3Arginine (R) or lysine (K) is not included.
In some embodiments, X2、X3And L collectively comprises more than 2 arginines. In some embodimentsIn, X3Including two or more arginines. In some embodiments, L comprises more than two or more arginines.
In certain embodiments, X2Is 2 to 5 amino acids, wherein each amino acid is selected from glycine and/or serine. In some embodiments, X3Is 2 to 5 amino acids, wherein at least two amino acids are independently selected from arginine, lysine and/or natural or non-natural amino acids having a side chain capable of forming an amide bond.
In some embodiments, X2Is 2 to 5 amino acids, wherein each amino acid is selected from glycine and/or serine. In some embodiments, X3Is 2 to 5 amino acids, wherein each amino acid is selected from glycine and/or serine.
In some embodiments, X2、X3And/or the total number of amino acid residues in L is 6 or more. In some embodiments, X2、X3And/or the total number of amino acid residues in L is 8 or more. In some embodiments, X2、X3And/or the total number of amino acid residues in L is 6 to 15.
In some embodiments, L comprises the amino acid sequence GSGSGSRR (SEQ ID NO:). Accordingly, in certain embodiments, the invention provides amino acids comprising the amino acid sequence GSGSGSGSRR (SEQ ID NO:).
In some embodiments, L comprises the amino acid sequence GSGSGSRRGS (SEQ ID NO:). Accordingly, in certain embodiments, the invention provides amino acids comprising the amino acid sequence GSGSGSRRGS (SEQ ID NO:).
In some embodiments, L is an amino acid sequence selected from the group consisting of seq id nos: GSGRR (SEQ ID NO:), GSGSGRR (SEQ ID NO:), GSGSGSGSRR (SEQ ID NO:), GSGSGSGRR (SEQ ID NO:), GSGSGSGSRR (SEQ ID NO:), GSGSGSGSGSGRR (SEQ ID NO:), GSGSGSGSGSGSRR (SEQ ID NO:), GSGSGSGSGSGSGRR (SEQ ID NO:), GSRRGS (SEQ ID NO:), GSGRRGSG (SEQ ID NO:), GSGRRRGSG (SEQ ID NO:), GSGRRR (SEQ ID NO:), GSGRRRR (SEQ ID NO:), GSGSGSRRR (SEQ ID NO:), GSGSGSRRRR (SEQ ID NO:), GSGSGSRRRRR (SEQ ID NO:), GSGSGSRRRRRR (SEQ ID NO:) and GSGSGSGSRRR (SEQ ID NO:).
The invention also provides compositions comprising a glycan and at least one compound of formula X1Bioconjugates of binding units of GSGSGSGSRR, wherein X1Is an amino acid sequence comprising a collagen binding unit covalently linked thereto.
The invention also provides compositions comprising a glycan and at least one compound of formula X1Bioconjugates of binding units of-GSGSGSGSRR-NHNH-, wherein X1Is an amino acid sequence comprising a collagen binding unit covalently linked thereto.
The invention also provides compositions comprising a glycan and at least one compound of formula X1Bioconjugates of binding units of GSGSGSGSRR, wherein X1Is an amino acid sequence comprising a collagen binding unit covalently linked thereto.
The invention also provides compositions comprising a glycan and at least one compound of formula X1Bioconjugates of binding units of-GSGSGSGSRR-NHNH-, wherein X1Is an amino acid sequence comprising a collagen binding unit covalently linked thereto.
The invention also provides a bioconjugate comprising a glycan as described herein and a collagen binding unit, wherein L is a spacer of 5 to 20 amino acids selected from glycine (G), serine (S), arginine (R) and lysine (K); radical (I)
In certain embodiments, the peptide comprises amino acid sequence X comprising a collagen binding unit1And X2、X3And L to control the number and arrangement (e.g., linear or branched) of binding units within the peptide and the distance between each binding unit and the amino acid used to conjugate to the glycan.
In certain embodiments, formula (I) comprises a-c (o) -NH-c (o) - (i.e., hydrazide-carbonyl) bond to the glycan. In certain embodiments, L comprises a hydrazide. -C (O) -NH-NH-C (O) -may comprise carbonyl-C (O) -groups from amino acids of L and/or carbonyl-C (O) -groups from glycans.
In certain embodiments, peptides are bound to glycans, e.g., dermatan sulfate, by using oxidative chemistry to cleave one or more sugar rings within the glycan backbone, thereby providing aldehyde binding sites on the glycan. The aldehyde binding site is then used to conjugate the peptide (e.g., via a-C (o) -NH-N ═ C bond).
In some embodiments, the peptide is bound to the glycan through a hydrazide-carbonyl bond, wherein the carbonyl group of the hydrazide-carbonyl is an exocyclic carbonyl group present on the glycan. Exocyclic carbonyl groups may be present on the native glycan or the glycan modification may be modified to include such functional groups. Such methods are described in detail below. It is expected that the beneficial effects (e.g., increased binding affinity) exhibited by bioconjugates bound in this manner are due, at least in part, to the glycans not containing oxidatively cleaved sugar rings.
In some embodiments, the bioconjugate can comprise a polymer backbone (e.g., a biocompatible polymer other than a glycan) that is composed of any monomer or combination of monomer units so long as at least one (in some
In some embodiments of the bioconjugates described herein, the glycan can be alginate, chondroitin, dermatan sulfate, heparan sulfate, heparin, dextran sulfate, or hyaluronic acid. In one embodiment, the glycan is dermatan sulfate. In one embodiment, the glycan is not dermatan sulfate. In another embodiment, the glycan is chondroitin sulfate. In another embodiment, the glycan is heparin. Various molecular weights of heparin, for example, from a single disaccharide unit of about 650-700Da to a glycan of about 50kDa, may be used in the bioconjugates described herein. In some embodiments, the heparin is about 10 to about 20 kDa. In some embodiments, the heparin is up to about 15, or about 16, or about 17, or about 18, or about 19, or about 20 kDa.
In one embodiment, the bioconjugate includes a peptide having a collagen binding unit that binds to one or more of collagen I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII, XIII, or XIV. In one embodiment, the collagen binding unit promotes or inhibits fibrillogenesis when bound to collagen. In one embodiment, the collagen binding unit does not promote or inhibit fibrillogenesis when bound to collagen. In some embodiments, the peptide binds to type I collagen. In other embodiments, the peptide binds to type IV collagen. In some embodiments, one or more peptides having a particular binding affinity for collagen may be used in the bioconjugates described herein. For example, a synthetic bioconjugate can comprise at least one peptide having binding affinity for type I collagen and at least one peptide having binding affinity for type IV collagen. In another embodiment, the peptide has binding affinity for type I collagen. In another embodiment, the peptide has binding affinity for type IV collagen. In some embodiments, the peptide has binding affinity for type II collagen. In some embodiments, the peptide has binding affinity for type III collagen. In some embodiments, the peptide binds to more than one type of collagenWherein the relative affinity for each collagen type may vary. In one embodiment, the collagen binding unit has a dissociation constant (K)d) Binding to collagen, the dissociation constant (K)d) Less than about 1mM, or less than about 900. mu.M, or less than about 800. mu.M, or less than about 700. mu.M, or less than about 600. mu.M, or less than about 500. mu.M, or less than about 400. mu.M, or less than about 300. mu.M, or less than about 200. mu.M, or less than about 100. mu.M.
The total number of binding units bonded to the glycan may vary depending on the desired properties of the bioconjugate. In some embodiments, the total number of binding units present in the bioconjugate is about 1 to about 50, or about 1 to about 40, or about 1 to about 30, or about 1 to about 25, or about 2 to about 30, or about 2 to about 25, or about 3 to about 25, or about 4 to about 25, or about 5 to about 30, or about 1 to about 25, or about 2 to about 25, or about 11 to about 14, or about 1 to about 8, or about 1 to about 5, or about 1, or about 2, or about 3, or about 4, or about 5, or about 6, or about 7, or about 8 binding units. In some embodiments, the bioconjugate comprises about 10 to about 40 binding units. In other embodiments, the bioconjugate comprises about 5 to about 30 binding units. In some embodiments, the bioconjugate includes less than about 20 binding units. In some embodiments, the bioconjugate includes less than about 18 binding units. In various embodiments, the bioconjugate comprises about 4 to about 18 binding units. In some embodiments, the bioconjugate includes less than about 15 binding units. In some embodiments, the bioconjugate includes less than about 10 binding units. In some embodiments, the bioconjugate includes less than about 30 binding units. In some embodiments, the bioconjugate includes less than about 25 binding units. In some embodiments, the bioconjugate comprises about 5 to about 40, or about 10 to about 40, or about 5 to about 20, or about 4 to about 18, or about 10, or about 11, or about 18, or about 20 binding units, or about 25 binding units, or about 30 binding units, or about 40 binding units, or about 50 binding units. In some embodiments, the bioconjugate includes about 2 binding units, about 4 binding units, or about 8 binding units.
The binding unit as described herein further comprises a hydrazide moiety for conjugation to the glycan. The hydrazide group may be attached to the binding unit at any suitable point of attachment, e.g., the C-terminus, the N-terminus, or through a side chain on the amino acid. For example, when the binding unit is bound to the glycan through a side chain of an amino acid of the binding unit, the side chain is glutamic acid or aspartic acid. Hydrazines (-NHNH) that can be bound to a carbonyl group (e.g., C-terminal carbonyl) present on an amino acid in a peptide sequence2) Form a hydrazide therebetween.
In certain embodiments, the bioconjugate comprises a glycan covalently bound to a binding unit of formula (I):
((X1)mX2)nX3-L
(I)
wherein, X1And L is as defined herein, includes an amino acid sequence X comprising 2 to about 5 amino acids2And/or X3The amino acid is selected from glycine (G), serine (S), arginine (R), lysine (K) and natural or unnatural amino acids having a side chain capable of forming an amide bond. For example, X2And/or X3Can comprise amino acid sequences such as GS (SEQ ID NO:), GSG (SEQ ID NO:), GSGS (SEQ ID NO:), RG (SEQ ID NO:), RGS (SEQ ID NO:), RGSG, RGSGS (SEQ ID NO:), RR (SEQ ID NO:), RRG (SEQ ID NO:), RRS (SEQ ID NO:), RRGS (SEQ ID NO:), RRGSG (SEQ ID NO:), RRGSGS (SEQ ID NO:), RGR (SEQ ID NO:), RSR (SEQ ID NO:), RRR (SEQ ID NO:), etc.
In some embodiments, X2And/or X3Is an amino acid sequence that comprises more than one binding site (which may be linear or branched) such that more than one peptide sequence can bind thereto, thereby creating a branched construct. Furthermore, since the peptide may be bound to the glycan through a terminal or non-terminal amino acid moiety, the peptide will be branched when bound to the glycan through a non-terminal amino acid moiety. The binding sites may be the same or different, and may be any suitable binding siteE.g., amine or carboxylic acid moieties, to which the desired peptide sequence can be bound (e.g., via an amide bond). Thus, in some embodiments, X2And/or X3Contains one or more lysine, glutamic acid or aspartic acid residues. For example, X2And/or X3Amino acid sequences such as KGSG (SEQ ID NO:), KKGSG (SEQ ID NO:), or KKKGSG (SEQ ID NO:), etc., provide 2,3, or 4 binding sites, respectively.
In some embodiments, X2And/or X3Comprising one or more amino acids containing side chains capable of linking to further peptide or collagen binding units. Exemplary amino acids for inclusion in such spacers include, but are not limited to, lysine, glutamic acid, aspartic acid, and the like. In some embodiments, X2And/or X3Comprising one or more amino acid sequences of formula KXX, wherein each X is independently a natural or unnatural amino acid. Specific examples of amino acid sequences that may be used alone or in combination to prepare branched constructs include, but are not limited to, KRR, KKK, (K)nGSG and sum (KRR)n-KGSG, wherein n is 0 to 5, or 1, 2,3, 4, or 5.
X is shown in the following table2And/or X3Schematic representation of (a).
In some embodiments, the hydrazide group is bonded to the N-terminus of the peptide. In some embodiments, the hydrazide group is bonded to the C-terminus of the peptide. In some embodiments, the hydrazide group is bonded to the side chain of an amino acid in the peptide, such as glutamic acid and/or aspartic acid.
In any of the embodiments described herein, the number of peptides per glycan is an average, where certain bioconjugates in the composition may have more peptides per glycan and certain bioconjugates have less peptides per glycan. Accordingly, in some embodiments, the number of peptides as described herein is the average number in the composition of the bioconjugate. For example, in some embodiments, the bioconjugate is a composition wherein the average number of peptides per glycan is about 5. In other embodiments, the average number of peptides per glycan is about 6, or about 7, or about 8, or about 9, or about 10, or about 11, or about 12, or about 13, or about 14, or about 15, or about 16, or about 17, or about 18, or about 19, or about 20, or about 25, or about 30.
In some embodiments, the number of peptides per glycan can be described as "percent (%) functionalized", based on the percentage of disaccharide units functionalized with peptides on the glycan backbone. For example, the total number of available disaccharide units present on a glycan can be calculated by dividing the molecular weight (or average molecular weight) of the glycan (e.g., about 25kDa up to about 70kDa, or even about 100kDa) by the molecular weight of the individual disaccharide unit (e.g., about 550-800Da, or about 650-750 Da). In embodiments where the glycans do not contain a conventional "disaccharide unit" (e.g., alginic acid), the total number of available disaccharide units present on the glycans used in the calculations shown herein can be calculated by dividing the molecular weight (or average molecular weight) of the glycan by the molecular weight of the individual saccharide unit, and multiplying by 2.
In some embodiments, the number of disaccharide units available present on the glycan is about 10 to about 80, or about 10 to about 70, or about 15 to about 70, or about 20 to about 70, or about 30 to about 70, or about 35 to about 70, or about 40 to about 70, or about 10 to about 75, or about 15 to about 75, or about 20 to about 75, or about 30 to about 75, or about 35 to about 75, or about 40 to about 75, or about 10 to about 50, or about 20 to about 50, or about 25 to about 50, or about 10 to about 35, or about 15 to about 35, or about 20 to about 35, or about 10 to about 30, or about 15 to about 30, or about 20 to about 30, or about 15, or about 20, or about 25, or about 30, or about 35, or about 40, or about 45, or about 50, or about 55, or about 60, or about 65, or about 70.
Thus, in some embodiments, the glycan contains from about 1 to about 50, or from about 5 to about 40% functionalization, or from about 5 to about 30% functionalization, or from about 1 to about 15% functionalization, or from about 1 to about 5% functionalization, or from about 5 to about 15% functionalization, or from about 10 to about 25% functionalization, or from about 25 to about 40% functionalization, or about 32% functionalization, or about 25% functionalization, or about 16% functionalization, or about 10% functionalization, or about 8% functionalization, or about 5% functionalization, or about 2.5% functionalization, wherein the percentage (%) of functionalization is determined by the percentage of disaccharide units on the glycan functionalized with the peptide. In some embodiments, the percent (%) of functionalization of the glycan is about 1% to about 50%, or about 3% to about 40%, or about 5% to about 30%, or about 10% to about 20%, or about 1%, or about 2%, or about 5%, or about 10%, or about 15%, or about 20%, or about 25%, or about 30%, or about 35%, or about 40%, or about 45%, or about 50%, or about 55%, or about 60%, or about 65%, or about 70%, or about 75%, or about 80%, or about 85%, or about 90%, or about 95%, or about 100%.
In certain embodiments, the glycan is functionalized with a branched peptide. In some embodiments, the peptide has 2-4 branches. Thus, in certain embodiments, the glycan comprises from about 1 to about 50% functionalization, or from about 5 to about 40% functionalization, or from about 5 to about 30% functionalization, or from about 1 to about 15% functionalization, or from about 1 to about 5% functionalization, or from about 5 to about 15% functionalization, or from about 10 to about 25% functionalization, or from about 25 to about 40% functionalization, or about 2.5% functionalization, or about 5% functionalization, or about 8% functionalization, or about 10% functionalization or about 16% functionalization, or about 32% functionalization, wherein percent (%) functionalization is determined by the percentage of disaccharide units on the glycan functionalized with the peptide.
In some embodiments, provided herein is a composition comprising a bioconjugate as described herein and a peptide, wherein the peptide is closely associated (e.g., by ionic bonding) with the bioconjugate. In some embodiments, bioconjugate aggregates can be formed thereby. It is contemplated that the bioconjugate aggregate (comprising the bioconjugate and the non-covalently bound peptide) can comprise 1% to 200% functionalization (determined by the percentage of disaccharide units on the glycan functionalized with the peptide). In some embodiments, the percent (%) of functionalization of the bioconjugate is about 1% to about 50%, or about 3% to about 40%, or about 5% to about 30%, or about 10% to about 20%, or about 1%, or about 2%, or about 5%, or about 10%, or about 15%, or about 20%, or about 25%, or about 30%, or about 35%, or about 40%, or about 45%, or about 50%, or about 55%, or about 60%, or about 65%, or about 70%, or about 75%, or about 80%, or about 85%, or about 90%, or about 95%, or about 100%.
It is contemplated that any glycan may be used in the various embodiments described herein, including but not limited to alginate, agarose, dextran sulfate, Chondroitin Sulfate (CS), Dermatan Sulfate (DS), heparan sulfate, heparin (Hep), keratin, keratan sulfate, and Hyaluronic Acid (HA). The glycans can be naturally occurring or chemically derivatized, such as, but not limited to, partially N-desulfated derivatives, partially O-desulfated derivatives, and/or partially O-carboxymethylated derivatives.
In some embodiments, the glycan is unmodified. In some embodiments, the glycan does not contain an oxidatively cleaved sugar ring, and therefore does not contain an aldehyde functional group. It is expected that the beneficial effects exhibited by the bioconjugates disclosed herein may be attributed, at least in part, to the glycans not comprising oxidatively cleaved saccharide rings.
Such attachment may result from binding a hydrazide group on the peptide to a carbonyl group (e.g., a carboxylic acid group or an activated derivative thereof) on the glycan, or alternatively binding a hydrazide group on the glycan to a carbonyl group (e.g., a carboxylic acid group or an activated derivative thereof) on the peptide. In some embodiments, the hydrazide-carbonyl bond is located between the terminal hydrazide group on the peptide and the carbonyl group on the glycan.
In one embodiment, the glycan is heparin, wherein heparin may include heparin derivatives such as, but not limited to, partially N-and/or partially O-desulphated heparin derivatives, partially O-carboxymethylated heparin derivatives, or combinations thereof. In some embodiments, the heparin is non-oxidized heparin (i.e., does not contain oxidatively cleaved sugar rings) and does not contain aldehyde functional groups. Heparin derivatives and/or methods of providing heparin derivatives, such as partially N-desulfated heparin and/or partially O-desulfated heparin (i.e., 2-O and/or 6-O-desulfated heparin), are known in the art (see, e.g., Kariya et al, J.biol. chem.,2000,275: 25949-. It is also contemplated that partial O-carboxymethylated heparin (or any glycan) derivatives, such as may be prepared according to Prestwich et al (US 2012/0142907; US 2010/0330143), may be used to provide the bioconjugates disclosed herein.
In some embodiments, the molecular weight of the glycans is altered to modulate the effect of the bioconjugate (see, e.g., Radek, K.A., et al., Wound Repair Regen.,2009,17: 118-. In another embodiment, the glycans are degraded by oxidation and alkaline elimination (see, e.g., Fransson, l.a., et al, eur.j.biochem.,1980,106:59-69) to provide degraded glycans having lower molecular weights (e.g., from about 10kDa to about 50 kDa).
In one embodiment, the glycan is Dermatan Sulfate (DS). The biological functions of DS are extensive and include the binding and activation of the growth factors FGF-2, FGF-7 and FGF-10, which promote endothelial cell and keratinocyte proliferation and migration. In some embodiments, the dermatan sulfate ranges in weight from about 10kDa to about 70 kDa. In one embodiment, the dermatan sulfate has a molecular weight of about 46 kDa. In another embodiment, dermatan sulfate is degraded by oxidation and alkaline elimination (see, e.g., Fransson, L.A., et al, Eur.J.biochem.,1980,106:59-69) to provide degraded dermatan sulfate having a lower molecular weight (e.g., about 10 kDa).
In another embodiment, the glycan is heparin. Various molecular weights of heparin may be used in the bioconjugates described herein, for example from a single disaccharide unit of about 650-700Da to about 50 kDa. In some embodiments, the heparin is from about 10 to about 20 kDa. In some embodiments, the heparin is up to about 15, or about 16, or about 17, or about 18, or about 19, or about 20 kDa. In some embodiments, heparin may be oxidized without cleaving one or more sugar rings (see, e.g., Wang, et al, biomacromolecules 2013,14(7): 2427-.
In one embodiment, the bioconjugate comprises a glycan and about 5 to about 10, or about 5 binding units, wherein the binding units comprise at least one of RRANAALKAGELYKSILY (SEQ ID NO:) or RRANAALKAGELYKSILYGSG (SEQ ID NO:) and are bound to the glycan through a hydrazide-carbonyl bond. The binding units may be within the same or different peptides. In some embodiments, the hydrazide-carbonyl bond is located between the terminal hydrazide group on the peptide and the carbonyl group on the glycan. In one embodiment, the glycan is heparin. In some embodiments, the heparin does not contain oxidatively cleaved sugar rings and therefore does not contain aldehyde functionality.
In one embodiment, the bioconjugate comprises a glycan and about 5 to about 10, or about 8, or about 5 binding units, wherein the binding units comprise at least one sequence of GQLYKSILY (SEQ ID NO:) and are bound to the glycan through a hydrazide-carbonyl bond. In one embodiment, the bioconjugate comprises a glycan and about 5 to about 10, or about 8, or about 5 binding units, wherein the binding units comprise at least one sequence of GQLYKSILYGSGSGSRR (SEQ ID NO:) and are bound to the glycan through a hydrazide-carbonyl bond. In one embodiment, the bioconjugate comprises a glycan and about 5 to about 10, or about 8, or about 5 binding units, wherein the binding units comprise at least one sequence of GQLYKSILYGSGSGSRR-NHNH- (SEQ ID NO:) and are bound to the glycan through a hydrazide-carbonyl bond. In some embodiments, the hydrazide-carbonyl bond is located between the terminal hydrazide group on the binding unit and the carbonyl group on the glycan. In one embodiment, the glycan is heparin. In some embodiments, the heparin does not contain oxidatively cleaved sugar rings and therefore does not contain aldehyde functionality.
It has been found that the addition of an amino acid to the C-terminal end of the collagen binding unit GQLYKSILY is positively correlated with an increase in binding affinity (fig. 17). It is contemplated that increasing the distance between the glycan and the binding site may reduce steric hindrance between regions of the bound complex. Once the spacer reaches a length sufficient to reduce steric repulsion between the regions of bound complex, there may be a diminishing effect on further spacer addition. Increasing the spacer length beyond this threshold may even reduce the affinity.
As shown in fig. 15, the position of the arginine residue in the spacer sequence also affects binding affinity. Positioning the first arginine closer to the glycan backbone increases binding affinity. This may be due to the positive charge shielding the binding site from the negatively charged backbone.
In some embodiments, the collagen binding unit comprises sequence GQLYKSILY (SEQ ID NO:), wherein one amino acid is substituted with a synthetic or naturally occurring D-or L-amino acid residue. In some embodiments, the collagen binding unit comprises the sequence XQLYKSILY (SEQ ID NO:), GXLYKSILY (SEQ ID NO:), GQXYKSILY (SEQ ID NO:), GQLXXKSILY, GQLYXSILY, GQLYKXILY (SEQ ID NO:), GQLYKSXLY (SEQ ID NO:), GQLYKSIXY (SEQ ID NO:), or GQLYKSILX (SEQ ID NO:), wherein X can be any amino acid residue (D or L). Each of these sequences may be immediately followed by a spacer of any of the embodiments listed herein. In some embodiments, X is L-alanine. In some embodiments, the spacer is GSGSGSRR (SEQ ID NO:).
IN some embodiments, the binding unit and spacer comprise the sequences LKAGQLYKSILYHHLHSGSGSGSRR (SEQ IN NO:), KAGQLYKSILYHHLHSYGSGSGSRR (SEQ IN NO:), GQLYKSILYHHLHSYQNSKPGSGSGSRR (SEQ IN NO:).
IN some embodiments, the binding units and spacers comprise the sequences GQLYKSILYGQLYKSILYGSGSGSRR (SEQ IN NO:), GQLYKSILYGSGQLYKSILYGSGSGSRR (SEQ IN NO:), GQLYKSILY-Ahx-GQLYKSILYGSGSGSRR (SEQ IN NO:), or (GQLYKSILY)2KGSGSGSRR(SEQ IN NO:)。
2. Synthesis of bioconjugates
The peptides used herein may be purchased from commercial sources or partially or fully synthesized using methods well known in the art (e.g., chemical and/or biotechnological methods). In some embodiments, the peptide is synthesized according to solid phase peptide synthesis protocols well known in the art. In another embodiment, the peptide is synthesized on a solid support according to the well-known Fmoc protocol, cleaved from the support with trifluoroacetic acid and purified by chromatography, according to methods known to those skilled in the art. In other embodiments, the peptides are synthesized using biotechnological methods well known to those skilled in the art. In one embodiment, the DNA sequence encoding the amino acid sequence information of the desired peptide is ligated into an expression plasmid (e.g., a plasmid that binds an affinity tag for affinity purification of the peptide) by recombinant DNA techniques known to those skilled in the art, the plasmid is transfected into a host organism for expression, and the peptide is then isolated from the host organism or growth medium, e.g., by affinity purification. Recombinant DNA techniques are described in Sambrook et al, "Molecular Cloning: A Laboratory Manual", 3rd Edition, Cold Spring harbor Laboratory Press, (2001) (which is incorporated herein by reference) and are well known to those skilled in the art.
As shown in
In one embodiment, the peptide coupling reaction is performed by an activated glycan intermediate by reacting the carboxylic acid moiety of the glycan with a coupling agent (e.g., a carbodiimide reagent such as, but not limited to, N '-Dicyclohexylcarbodiimide (DCC), N' -Diisopropylcarbodiimide (DIC), 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC), etc.) to form an O-acylisourea intermediate. Such carbodiimide chemistry is well known in the art, and suitable coupling agents are commercially available. The O-acylisourea intermediate is contacted with the desired peptide to produce a bioconjugate. The glycan may be contacted with an activating agent prior to or in the presence of the peptide. In some embodiments, the reaction is carried out in the presence of N-hydroxysuccinimide (NHS) or derivatives thereof. In some embodiments, the peptide sequence may be modified to include a reactive moiety (e.g., a hydrazide functional group) to facilitate a coupling reaction with the glycan or O-acylisourea intermediate thereof. In addition, in some cases where one or more amino acids in the peptide contain reactive functional groups (e.g., carboxylic acid side chains), standard protecting group chemistry can be used to protect one or more side chains to facilitate the coupling reaction. In addition, non-amino acid spacers may be used alone or in combination with amino acid spacers (e.g., aminocaproic acid).
In some embodiments, the bioconjugates are derived from modified glycan derivatives (e.g., heparin) (scheme 2). Various glycan derivatives suitable for use in the bioconjugates described herein are known in the art, such as partially N-and partially O-desulfated heparin (i.e., 2-O and/or 6-O-desulfated heparin, see, e.g., Kariya et al, J.biol.chem.,2000,275: 25949-one 5958; Lapierre, et al. glycobiology,1996,6(3): 355-one 366). An exemplary method is shown in
In certain embodiments, bioconjugates can be prepared according to
3. Application method
3.1 failure of transplantation
One example of the present disclosure provides methods and related compositions for improving the success rate and/or reducing failure of surgical bypass surgery. Bypass grafts are used as a form of treatment for arterial occlusions in Coronary Artery Disease (CAD) and Peripheral Artery Disease (PAD). As used herein, the term "treating" refers to preventing, curing, reversing, attenuating, alleviating, minimizing, inhibiting, suppressing, and/or arresting one or more clinical symptoms of a disease or disorder. Approximately 500,000 Coronary Artery Bypass Grafts (CABG) and over 70,000 peripheral bypass grafts are performed annually in the united states. Most commonly, autologous vascular grafts are typically harvested from saphenous veins.
Despite the prevalence of surgical bypass using autologous vein grafts to restore blood flow, there are a number of Vein Graft Failures (VGFs) in both CAD and PAD. The failure rate of vein transplantation reaches 50% only in the periphery within 5 years. Due to technical factors and acute thrombosis, 5% to 10% of venous grafts fail shortly after implantation, with an additional 20% to 30% of cases with a potentially intermediate failure (3 to 24 months) and potentially leading to expensive monitoring, re-interventional procedures and amputations. In a twenty year experience at the woman's brigade hospital, CLI patients (n ═ 1219) have a 29% incidence of vein graft failure at 12 months. The consequences of vein graft failure are often severe for the patient, including recurrent ischemic symptoms, debilitating surgery, and loss of limbs. To date, drug therapy and technological innovation have had little impact on reducing vein graft failure.
It is expected that damage to the fragile endothelial layer of a vein graft catheter, whether caused by vein graft harvesting, preservation of media, over-manipulation to prepare a bypass, or by ischemia and reperfusion injury, results in a platelet-mediated inflammatory response within the vessel wall after implantation. This endothelial injury and ECM-platelet activation cascade can be delayed by VGF in the early stages of acute inflammation and thrombosis, or by the formation of neointimal hyperplasia. Thus, limiting the exposure of the subcutaneous matrix within the vein graft to circulating platelets after implantation can help reduce acute vessel wall inflammation, improve re-epithelialization and limit excessive neointimal hyperplasia that may lead to vessel occlusion and VGF. Bioconjugates as described herein may be used as vein graft preservation solutions for patients with cardiovascular disease with surgical bypass of autologous vein grafts. Bioconjugates as described herein and compositions comprising the same can be used to treat and/or prevent coronary artery disease and/or peripheral artery disease in a patient in need thereof.
Thus, according to one example of the present disclosure, there is provided a method of preparing a vascular graft (e.g., a venous graft) by contacting the inner wall of a segment of a blood vessel with a solution containing a synthetic bioconjugate of the invention. One way to achieve said contact is to soak the section in said solution. The conditions of such contacting can vary, but can be readily determined based on the concentration of the synthetic bioconjugate and the characteristics of the blood vessel, such that an appropriate amount of the synthetic bioconjugate binds to the inner wall. Vascular grafts prepared in this manner are also within the scope of the present disclosure.
Once the graft is prepared, it can be implanted into a patient in need thereof. Surgical bypass surgery can be readily performed by medical professionals. Once implanted, synthetic bioconjugates that bind to the inner wall of the graft can help reduce acute vessel wall inflammation, improve graft re-epithelialization and limit excessive neointimal hyperplasia of the graft, thereby reducing graft failure.
In one embodiment, when the graft has been treated with a synthetic bioconjugate as described above, a solution of the synthetic bioconjugate may be injected into the interior lumen of the graft during or after the bypass surgery procedure so that the synthetic bioconjugate will bind to the interior wall of the graft. In one aspect, the injection is completed before blood flow resumes or begins to pass through the graft. In another aspect, the injection is completed shortly after blood flow is restored or initiated (e.g., within 10 minutes, within 5 minutes, or within 1 minute).
In some embodiments, the method is effective to inhibit negative remodeling of blood vessels. Coronary artery disease, also known as ischemic or coronary heart disease, effectively restricts blood flow to the heart when atherosclerosis occurs in a portion of the smooth, flexible lining within the coronary arteries (the arteries that supply blood to the heart muscle). Peripheral arterial disease, also known as atherosclerosis or arteriosclerosis, is a disease that occurs in arteries of the circulatory system. Negative remodeling involves the physiological or pathological response of the vessel to a stimulus, resulting in a reduction in the diameter of the vessel and the diameter of the lumen. Such stimulation may be provided by, for example, blood flow modification or angioplasty procedures. In some embodiments, injection of the bioconjugates described herein, and compositions comprising the same, results in an increase in vessel diameter of about 10%, 20%, 30%, 40%, 60%, 70%, 80%, 95%, or any of the above, as compared to the diameter of a vessel that has not been injected. Negative remodeling can be quantified, for example, by angiography as the percentage of diametric stenosis at a lesion (or disease site). Another method of determining the extent of remodeling involves measuring the area of the outer elastic lamina within the lesion using intravascular ultrasound (IVUS). IVUS is a technique that can image the outer elastic lamina as well as the lumen of the blood vessel. In some embodiments, the negative remodeling is associated with a vascular interventional procedure, such as angioplasty, stenting, or atherectomy. Accordingly, bioconjugates as described herein and compositions comprising the same can be injected before, during and/or after vascular interventional procedures. In some embodiments, there is provided a method of treating a stenosis, or occlusion within the popliteal femoral artery in a patient in need thereof, comprising applying a solution to an interior wall of a lumen before, during, and/or after balloon angioplasty, wherein the solution comprises an effective amount of a bioconjugate as described herein or a composition comprising the same.
Accordingly, the present invention provides a method of inhibiting negative remodeling in a blood vessel (e.g., an artery) in an individual in need thereof, the method comprising injecting an effective amount of a bioconjugate described herein, or a composition comprising the same, into the blood vessel wall or tissue surrounding the blood vessel wall. In some embodiments, the bioconjugate or composition is injected at or near a site of potential or actual negative remodeling (e.g., no more than about 2, 1, or 0.5cm from the site). In some embodiments, the nanoparticle composition is injected away from a potential or actual negative remodeling site (e.g., at least about any one of 1, 2,3, 4, 5, 6, 7, 8,9, or 10cm away from the site). In some embodiments, the injection is through a catheter with a needle. In some embodiments, the site is a coronary artery or a peripheral artery. In some embodiments, the artery is selected from the group consisting of a renal artery, a cerebral artery, a pulmonary artery, and a leg artery. In some embodiments, the artery is a balloon-injured artery. Further examples include, but are not limited to, abdominal aorta, anterior tibial artery, aortic arch, arcuate artery, axillary artery, brachial artery, carotid artery, celiac artery, cyclofibular artery, common hepatic artery, common iliac artery, deep femoral artery (deep femoral artery), deep palmar artery arch, dorsal digital artery, plantar dorsal artery, external carotid artery, external iliac artery, facial artery, femoral artery, inferior mesenteric artery, internal iliac artery, intestinal artery, lateral below knee artery, lateral above knee artery, metacarpal artery, peroneal artery, popliteal artery, posterior tibial artery, deep femoral artery (profunda femoris artery), pulmonary artery, radial artery, renal artery, splenic artery, subclavian artery, superficial metacarpal artery arch, superior mesenteric artery, superior auxiliary ulnar artery, and/or ulnar artery. In some embodiments, the artery is part of a coronary vessel.
In one embodiment, the bioconjugate used in the above method comprises heparin and about 5 to about 10, or about 5 peptides, wherein the peptides comprise at least one sequence of GQLYKSILYGSGSGSRR (SEQ ID NO:). In one embodiment, the bioconjugate used in the above method comprises heparin and about 5 to about 10, or about 5 peptides, wherein the peptides comprise at least one sequence of GQLYKSILYGSGSGSRR (SEQ ID NO:) and bind to the heparin through a hydrazide-carbonyl bond.
3.2 fiberization
In one embodiment, provided herein are bioconjugates and methods for preventing and/or treating fibrosis. Fibrosis is an inflammatory disease in which inflammatory cells migrate into tissues and organs, resulting in a cellular response that leads to scarring. Fibrosis can often occur in many tissues in the body due to inflammation or injury. By preventing inflammatory cell extravasation, fibrosis can be reduced or prevented.
In one embodiment, the bioconjugates and methods provided herein can be used to prevent and/or treat pulmonary fibrosis. In the lung, types of fibrosis include pulmonary fibrosis, such as cystic fibrosis and idiopathic pulmonary fibrosis. Pulmonary fibrosis is a respiratory disease in which scars are formed in lung tissue, causing serious respiratory problems. Scar formation results in thickening of the wall and a reduction in oxygen supply in the blood. As a result, the patient suffers from permanent shortness of breath.
In one embodiment, the bioconjugates and methods provided herein can be used to treat liver fibrosis, liver fibrosis can be caused by a variety of conditions, including chronic alcohol exposure, Hepatitis B Virus (HBV) infection, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), Hepatitis C Virus (HCV) infection, Wilson's disease, α -1-antitrypsin deficiency, hemochromatosis, primary biliary cirrhosis, primary sclerosing cholangitis, and autoimmune hepatitis.
Cirrhosis is fibrosis in the liver, where the liver fails to function properly due to long-term damage. Typically, the disease will develop slowly over months or years. Early, there are usually no symptoms. As the disease worsens, the person may become tired, weak, itchy, swollen lower legs, yellow skin, prone to bruising, abdominal dropsy, or spider vessels form on the skin. Fluids accumulated in the abdomen may spontaneously infect. Other complications include hepatic encephalopathy, esophageal or gastric vein dilation bleeding, and liver cancer. Hepatic encephalopathy can lead to confusion and possibly even loss of consciousness. Cirrhosis can lead to liver dysfunction. The following symptoms or characteristics are a direct consequence of liver dysfunction and thus may also be treated or ameliorated by the compositions and methods of the present disclosure.
Direct interactions between Hepatic Stellate Cells (HSCs) and tumor cells have been shown to promote tumor growth through a variety of mechanisms. Thus, targeting HSCs to reduce or eliminate their tumor supporting role presents a potential therapeutic strategy for the prevention, inhibition, or treatment of hepatocellular carcinoma (HCC). In certain embodiments, there is provided a method of preventing or inhibiting the development of hepatocellular carcinoma (HCC) in a patient in need thereof, comprising administering to the patient an effective amount of a bioconjugate described herein. In certain embodiments, the development of hepatocellular carcinoma (HCC) is the result of cirrhosis. In certain embodiments, the method comprises inhibiting hepatic stellate cell proliferation and/or fibrotic phenotypic shift. In certain embodiments, the bioconjugate is administered locally to the liver, for example during Transcatheter Arterial Chemoembolization (TACE).
Spider angiomas or spider nevi are vasculopathies consisting of a central arteriole surrounded by many smaller vessels, occurring as a result of an increase in estradiol. Palmar erythema is the redness of the palm at the prominences of the greater and lesser thenar regions, and is also due to increased estrogen. Gynecomastia or breast gland enlargement without cancer in men is caused by an increase in estradiol, which can occur in up to two-thirds of patients. Hypogonadism (decreased sex hormones manifested as impotence), infertility, decreased libido and testicular atrophy may be caused by primary gland damage or by hypothalamic/pituitary function compression. Hypogonadism is associated with cirrhosis of the liver caused by alcoholism and hemochromatosis. The liver size of a cirrhosis patient may be increased, normal or decreased.
In one embodiment, the bioconjugates and methods provided herein can be used to prevent and/or treat renal fibrosis. Renal fibrosis can be caused by acute or sustained damage to the kidney. Damage may lead to excessive deposition of extracellular matrix. Over time, this may lead to kidney failure, requiring the patient to undergo dialysis or kidney transplantation.
Ascites is a fluid accumulation in the peritoneal cavity that can cause abdominal side blunting. This is seen as the circumference of the abdomen increases. Hepatic halitosis is a musty odor caused by an increase in dimethyl sulfide content. Jaundice is the yellowing of skin and mucous membranes due to increased bilirubin. In addition, cirrhosis increases resistance to blood flow and high pressure of the portal system, resulting in portal hypertension.
In one embodiment, the bioconjugates and methods provided herein can be used to prevent and/or treat fibrosis in the heart. Fibrosis in the heart occurs in the form of atrial fibrosis, endocardial fibrosis, or myocardial infarction. Glial scars are fibrosis in the brain. Other types of fibrosis include, but are not limited to, joint fibrosis (knee, shoulder, other joints), Crohn's disease (bowel), Dupuytren's contractures (hands, fingers), keloids (skin), mediastinal fibrosis (soft tissue of mediastinum), myelofibrosis (bone marrow), peloney's disease (penis), nephrogenic systemic fibrosis (skin), progressive massive fibrosis (lung), retroperitoneal fibrosis (soft tissue behind the peritoneum), scleroderma/systemic sclerosis (skin, lung), and certain forms of adhesive capsulitis (shoulder).
The compositions and methods of the invention are expected to be useful in the prevention and/or treatment of any of these diseases or the symptoms or features associated with these diseases. The development of fibrosis involves the stimulated cells laying connective tissue, including collagen and glycosaminoglycans. The bioconjugates of the present disclosure can interact with collagen or glycosaminoglycans and thus disrupt the formation of this excess connective tissue. Bioconjugates can also protect the endothelial barrier. This may be an interaction with the extracellular matrix exposed as a result of microvascular damage. Protecting the endothelial barrier prevents inflammatory cells from infiltrating the tissue causing excessive ECM deposition, which leads to fibrotic tissue. Thus, the bioconjugate can prevent, inhibit, delay and/or reverse fibrosis.
In certain embodiments, the fibrosis is post-ischemic, post-infectious, or idiopathic (e.g., kidney, liver, heart, lung). See, e.g., Guerrot, D., et al, Fibrogenesis & tissue repair 5.Suppl 1(2012): S15 and Yamaguchi, I., et al, Nephron Experimental neuropathology 120.1(2012): e20-e 31. In certain embodiments, the fibrosis is retroperitoneal. In certain embodiments, the fibrosis is cutaneous (e.g., scleroderma). See, for example, Maurer, B., et al, annals of the rhematic diseases (2013), anrheumdis-2013.
In one embodiment, the disease is not acute tubular necrosis, diabetic chronic renal failure, lupus nephritis, renal fibrosis, or acute glomerulonephritis. In one embodiment, the disease is not Idiopathic Pulmonary Fibrosis (IPF), chronic obstructive pulmonary disease, asthma, or emphysema.
In certain embodiments, the fibrosis is caused by, or associated with, a lysosomal storage disease, including, but not limited to, Fabry (Fabry) disease, Gaucher (Gaucher) disease, Niemann-Pick (Niemann-Pick) disease, and Hunter (Hunter) syndrome (mucopolysaccharidosis). Thus, in certain embodiments, provided herein are methods of preventing fibrosis caused by or associated with a lysosomal storage disease in a patient in need thereof.
In one embodiment, provided herein is the use of a bioconjugate disclosed herein for preventing or treating fibrosis. In one embodiment, provided herein is the use of a bioconjugate disclosed herein for the preparation of a medicament for the prevention or treatment of fibrosis. In one embodiment, provided herein is the use of a bioconjugate disclosed herein for preventing or treating liver fibrosis. In one embodiment, provided herein is the use of a bioconjugate disclosed herein for the prevention or treatment of pulmonary fibrosis. In one embodiment, the bioconjugate used in the above method comprises heparin and about 5 to about 10, or about 5 peptides, wherein the peptides comprise at least one sequence of GQLYKSILYGSGSGSRR (SEQ ID NO:), or amino acid sequences having one, two or three amino acid additions, deletions and/or substitutions from each thereof. In one embodiment, the bioconjugate used in the above method comprises heparin and about 5 to about 10, or about 5 peptides, wherein the peptides comprise at least one sequence of GQLYKSILYGSGSGSRR (SEQ ID NO:). In one embodiment, the peptide is bound to heparin or other glycans through a hydrazide-carbonyl bond.
In one embodiment, provided herein is a method for preventing or treating liver or pulmonary fibrosis in a patient in need thereof, comprising administering to the patient an effective amount of a bioconjugate comprising heparin and about 5 to about 10, or about 5 peptides, wherein the peptides comprise at least one sequence of GQLYKSILYGSGSGSRR (SEQ ID NO:). In one embodiment, provided herein is the use of a bioconjugate disclosed herein to prevent or treat liver fibrosis or pulmonary fibrosis in a patient in need thereof. In one embodiment, an effective amount of the bioconjugate is administered.
Also provided herein are methods of preventing and/or treating vasculitis. Vasculitis is defined by inflammation of the vessel wall and forms the basis of pathology for different populations of various disease entities. Vasculitis is one of the intractable pathological conditions commonly observed in autoimmune diseases, and many of its causes are intractable to conventionally used therapeutic methods such as steroids and immunosuppressants. Healds for vasculitisIn combination, inflammation occurs in arteries of various sizes, and fever, muscle and joint pain, vascular obstruction, skin ulcers, and multiple mononeuritis may develop. The method can be used to treat vasculitis, such as macrovasculitis (LVV), Mesovasculitis (MVV), small vasculitis (SVV), variable vasculitis (VVVV), Single Organ Vasculitis (SOV), vasculitis associated with systemic disease, and/or vasculitis associated with a possible etiology. Non-limiting examples of macrovasculitis (LVV) include Takayasu Arteritis (TAK) and Giant Cell Arteritis (GCA). Non-limiting examples of Medium Vessel Vasculitis (MVV) include polyarteritis nodosa (PAN) and Kawasaki (KD). Non-limiting examples of Small Vessel Vasculitis (SVV) include anti-neutrophil cytoplasmic antibody (ANCA) associated vasculitis (AAV), Microscopic Polyangiitis (MPA), granulomatous polyangiitis (Wegener's) (GPA), eosinophilic granulomatous polyangiitis (Churg-Strauss) (EGPA), immune complex SVV, anti-glomerular basement membrane (anti-GBM) disease, glomerulovasculitis (CV), IgA vasculitis (Henoch-
In one embodiment, provided herein is a method of preventing and/or treating vascular vasculitis. In one embodiment, provided herein are methods of preventing and/or treating small vessel vasculitis, comprising anti-neutrophil cytoplasmic antibody (ANCA) associated vasculitis (AAV), Microscopic Polyangiitis (MPA), granulomatous polyangiitis (Wegener's) (GPA), eosinophilic granulomatous polyangiitis (Churg-Strauss) (EGPA), immune complex SVV, anti-glomerular basement membrane (anti-GBM) disease, glomerulovasculitis (CV), IgA vasculitis (Henoch-
Combination therapy
In some embodiments, the compositions of the present disclosure may be used in combination with a second agent useful for preventing or treating fibrosis. Thus, in one embodiment, a combination, composition, kit or kit is provided comprising any composition of the present disclosure and one or more such second agents. In one embodiment, any of the methods of treatment of the present disclosure further comprises administering one or more such second agents.
The second agent can be any drug or biological agent useful for preventing, treating, or otherwise ameliorating the symptoms of fibrosis. Non-limiting examples include: steroids, such as predonine; reducing agents, such as N-acetylcysteine; anti-fibrotic drugs such as pirfenidone and nintedanib; immunosuppressive drugs such as corticosteroids, cyclophosphamide, azathioprine, methotrexate, penicillin, cyclosporin a and FK 506; and other agents, such as colchicine, IFN-gamma, and mycophenolate mofetil.
In some embodiments, the compositions of the present disclosure may be used in combination with a second agent useful for preventing or treating vasculitis. Thus, in one embodiment, a combination, composition, kit or kit is provided comprising any composition of the present disclosure and one or more such second agents. In one embodiment, any of the methods of treatment of the present disclosure further comprises administering one or more such second agents.
The second agent can be any drug or biological agent useful for preventing, treating, or otherwise ameliorating the symptoms of vasculitis. Non-limiting examples include Prednisone, cyclophosphamide (Cytoxan), methylprednisolone, methotrexate sodium, Medrol (pak), Medrol, dexamethasone, prednisolone, DexPak, Deltasone, cortisone, Prednisone enhancer (Prednisone Intenol), dexamethasone sodium phosphate, Orapred ODT, Trexall, Rheumatrex, methotrexate sodium (PF),
4. Composition comprising a metal oxide and a metal oxide
In one embodiment, the bioconjugate is administered in a composition. The present disclosure provides compositions comprising a bioconjugate and a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are known to those of ordinary skill in the art and include water or saline. As is known in the art, the components and their relative amounts are determined by the intended use and method of delivery. The diluent or carrier used in the composition may be selected such that they do not reduce the desired effect of the bioconjugate. Examples of suitable compositions include aqueous solutions, such as in isotonic saline, 5% glucose. Other well known pharmaceutically acceptable liquid carriers may also be used, such as alcohols, glycols, esters and amides. In certain embodiments, the compositions further comprise one or more excipients such as, but not limited to, ionic strength modifiers, solubility enhancers, sugars (such as mannitol or sorbitol), pH buffers, surfactants, stabilizing polymers, preservatives, and/or co-solvents.
In certain embodiments, the composition is an aqueous solution. Aqueous solutions are suitable for use in composition formulations because of the ease of formulation, and the ease with which these compositions can be administered by instillation of the solution. In certain embodiments, the composition is a suspension, a viscous or semi-viscous gel, or other type of solid or semi-solid composition. In some embodiments, the composition is in the form of foams, ointments, liquid detergents, gels, sprays, and liposomes as are well known in the art. Alternatively, topical administration is infusion of the provided bioconjugate to a treatment site by a device selected from a pump-catheter system, a continuous or selective release device, or an adhesive barrier (adhesive barrier). In certain embodiments, the composition is a solution that is applied directly to or in contact with the inner wall of a vein or artery. In some embodiments, the composition comprises a polymer matrix. In other embodiments, the composition is absorbable. In certain embodiments, the composition comprises a pH buffering agent. In some embodiments, the composition contains a lubricity enhancer.
In certain embodiments, a polymer matrix or polymeric material is used as a pharmaceutically acceptable carrier for the composition. The polymeric materials described herein can comprise natural or non-natural polymers, such as sugars, peptides, proteins, laminin, collagen, hyaluronic acid, ionic and non-ionic water soluble polymers; an acrylic polymer; hydrophilic polymers such as polyethylene oxide, polyoxyethylene-oxypropylene copolymers, and polyvinyl alcohol; cellulose polymers and cellulose polymer derivatives such as hydroxypropyl cellulose, hydroxyethyl cellulose, hydroxypropyl methylcellulose phthalate, methylcellulose, carboxymethyl cellulose and etherified cellulose; poly (lactic acid), poly (glycolic acid), copolymers of lactic acid and glycolic acid, or other polymeric agents, both natural and synthetic. In certain embodiments, the compositions provided herein are formulated into a film, gel, foam, or other dosage form.
Suitable ionic strength modifiers include, for example, glycerol, propylene glycol, mannitol, glucose, dextrose, sorbitol, sodium chloride, potassium chloride, and other electrolytes.
In certain embodiments, the solubility of the bioconjugate may need to be enhanced. In such cases, the solubility can be enhanced by using suitable formulation techniques, for example incorporating solubilizing agent ingredients such as mannitol, ethanol, glycerol, polyethylene glycol, propylene glycol, poloxamers and others known in the art.
In certain embodiments, the composition contains a lubricity enhancer. As used herein, a lubricity enhancer refers to one or more pharmaceutically acceptable polymeric materials capable of altering the viscosity of a pharmaceutically acceptable carrier. Suitable polymeric materials include, but are not limited to: ionic and non-ionic water-soluble polymers; hyaluronic acid and its salts, chondroitin sulfate and its salts, dextran, gelatin, chitosan, gellan (gellan), other bioconjugates or polysaccharides, or any combination thereof; cellulose polymers and cellulose polymer derivatives such as hydroxypropyl cellulose, hydroxyethyl cellulose, hydroxypropyl methylcellulose phthalate, methylcellulose, carboxymethyl cellulose and etherified cellulose; collagen and modified collagen; galactomannans such as guar gum, locust bean gum and tara gum, as well as polysaccharides derived from the above natural gums and similar natural or synthetic gums containing mannose and/or galactose moieties as the main structural components (e.g. hydroxypropyl guar); gums, such as tragacanth and xanthan gum; gellan gum; alginic acid and sodium alginate; chitosan; an ethylene polymer; hydrophilic polymers such as polyethylene oxide, polyoxyethylene-polyoxypropylene copolymer and polyvinyl alcohol; carboxyvinyl polymers or crosslinked acrylic polymers, e.g. "carbomer" family polymers (e.g. as can be obtained in Carbopol)TMCarboxy polyalkylene commercially available under the trademark); and various other viscous or viscoelastic substances. In one embodiment, the lubricity enhancer is selected from the group consisting of hyaluronic acid, dermatan, chondroitin, heparin, heparan, keratin, dextran, chitosan, alginate, agarose, gelatin, hydroxypropyl cellulose, hydroxyethyl cellulose, hydroxypropyl methyl celluloseCellulose, hydroxypropyl methylcellulose phthalate, methylcellulose, carboxymethylcellulose and etherified cellulose, polyvinyl alcohol, polyvinylpyrrolidone, povidone, carbomer 941, carbomer 940, carbomer 971P, carbomer 974P, or a pharmaceutically acceptable salt thereof. In one embodiment, the lubricity enhancer is administered simultaneously with the bioconjugate. Alternatively, in one embodiment, the lubricity enhancer and bioconjugate are administered sequentially. In one embodiment, the lubricity enhancer is chondroitin sulfate. In one embodiment, the lubricity enhancer is hyaluronic acid. The lubricity enhancer may alter the viscosity of the composition.
For further details on the structure, chemical and physical properties of the above lubricity enhancers see, for example, US5,409,904, US 4,861,760 (gellan gum), US 4,255,415, US 4,271,143 (carboxyvinyl polymer), WO94/10976 (polyvinyl alcohol), WO 99/51273 (xanthan gum) and WO 99/06023 (galactomannan). The use of non-acidic lubricity enhancers, such as neutral or basic agents, is proposed to help achieve the desired pH of the formulation.
In some embodiments, the bioconjugate can be combined with minerals, amino acids, sugars, peptides, proteins, vitamins (such as ascorbic acid) or laminin, collagen, fibronectin, hyaluronic acid, fibrin, elastin, or aggrecan or growth factors (such as epidermal growth factor, platelet-derived growth factor, transforming growth factor β, or fibroblast growth factor), and glucocorticoids (such as dexamethasone) or viscoelasticity modifiers (such as ionic and non-ionic water soluble polymers; acrylic acid polymers; hydrophilic polymers (such as polyethylene oxide, polyoxyethylene-polyoxypropylene copolymer, and polyvinyl alcohol), cellulosic polymers and cellulosic polymer derivatives (such as hydroxypropyl cellulose, hydroxyethyl cellulose, hydroxypropyl methyl cellulose phthalate, methyl cellulose, carboxymethyl cellulose, and etherified cellulose); poly (lactic acid), poly (glycolic acid), copolymers of lactic acid and glycolic acid, or other polymers, both natural and synthetic).
pH buffers suitable for use in the compositions of the present invention include, for example, acetate, borate, carbonate, citrate, and phosphate buffers, as well as hydrochloric acid, sodium hydroxide, magnesium oxide, potassium dihydrogen phosphate, bicarbonate, ammonia, carbonic acid, hydrochloric acid, sodium citrate, citric acid, acetic acid, disodium hydrogen phosphate, borax, boric acid, sodium hydroxide, diethylbarbituric acid, and proteins, as well as various biological buffers, such as TAPS, Bicine, Tris, Tricine, HEPES, TES, MOPS, PIPES, diarsonate, or MES. In certain embodiments, a suitable buffering system (e.g., sodium phosphate, sodium acetate, sodium citrate, sodium borate, or boric acid) is added to the composition to prevent pH drift under storage conditions. In some embodiments, the buffer is a Phosphate Buffered Saline (PBS) solution (i.e., containing sodium phosphate, sodium chloride, and in some formulations potassium chloride and potassium phosphate). The specific concentration will vary depending on the reagent used. In certain embodiments, a pH buffering system (e.g., sodium phosphate, sodium acetate, sodium citrate, sodium borate, or boric acid) is added to maintain the pH at about
Surfactants are used in the compositions to deliver higher concentrations of bioconjugates. Surfactants act as solubilization inhibitors and stabilize colloidal dispersions, such as micellar solutions, microemulsions, emulsions and suspensions. Suitable surfactants include polysorbates, poloxamers,
In certain embodiments, a stabilizing polymer (i.e., a demulcent) is added to the composition. The stabilising polymer should be an ionic/charged polymer, more particularly a polymer carrying a negative charge on its surface, which may exhibit a zeta potential of (-)10-50mV to achieve physical stability and be able to form a dispersion in water (i.e. water solubility). In one embodiment, the stabilizing polymer comprises a polyelectrolyte or polyelectrolytes from the family of crosslinked polyacrylates (if more than one), such as carbomers and
In one embodiment, the composition comprises an agent that increases the permeability of the bioconjugate to the extracellular matrix of the blood vessel. Preferably, the agent that increases permeability is selected from benzalkonium chloride, saponins, fatty acids, polyoxyethylene fatty ethers, fatty acid alkyl esters, pyrrolidones, polyvinylpyrrolidone, pyruvic acid, pyroglutamic acid or mixtures thereof.
The bioconjugates can be sterilized to remove unwanted contaminants including, but not limited to, endotoxins and infectious agents. Sterilization techniques that do not adversely affect the structure and the biopsychoppy properties (biotical property) of the bioconjugate can be used. In certain embodiments, bioconjugates can be sterilized and/or disinfected using conventional sterilization techniques, including propylene oxide or ethylene oxide treatment, sterile filtration, gas plasma sterilization, gamma radiation, electron beam, and/or peracid (e.g., peracetic acid) sterilization. In one embodiment, the bioconjugate can be subjected to one or more sterilization processes. Alternatively, the bioconjugate can be packaged in any type of container including a plastic or foil package and can be further sterilized.
In some embodiments, preservatives are added to the compositions to prevent microbial contamination during use. Suitable preservatives for incorporation into the compositions include benzalkonium chloride, benzoic acid, alkyl parabens, alkyl benzoates, chlorobutanol, chlorocresol, cetyl alcohol, fatty alcohols (e.g., cetyl alcohol), organometallic compounds of mercury (e.g., acetate, phenylmercuric nitrate, or phenylmercuric borate), diazolidinyl urea, diisopropyl adipate, dimethylpolysiloxane, salts of EDTA, vitamin E, and mixtures thereof. In certain embodiments, the preservative is selected from benzalkonium chloride, chlorobutanol, dimethyldodecylbenzylammonium bromide, methylparaben, propylparaben, phenylethyl alcohol, disodium edetate, sorbic acid, or polyquaternium-1. In certain embodiments, the composition comprises a preservative. In some embodiments, the preservative is present in an amount of about 0.001% to about 1.0% w/v. In certain embodiments, the composition is preservative-free and is referred to as "preservative-free. In some embodiments, the unit dosage composition is sterile, but preservative-free.
In some embodiments, separate or sequential administration of the bioconjugate and other agent is advantageous for delivery of the composition. In certain embodiments, the bioconjugate and other agent can be administered at different dosing frequencies or intervals. For example, the bioconjugate may be administered daily, while other agents may be administered less frequently. In addition, bioconjugates and other agents can be administered using the same route of administration or different routes of administration, as will be apparent to those skilled in the art.
Any effective protocol for administration of the bioconjugate can be used. For example, the bioconjugate can be administered as a single dose or as a multiple dose daily regimen. In addition, weekly alternating therapy, for example, for one to five days, may be used as an alternative to daily treatment.
In various embodiments, the bioconjugate can be administered topically, such as through a membrane, gel, patch, or liquid solution. In some embodiments, the composition is provided in the form of a buffered sterile aqueous solution. In certain embodiments, the solution has a viscosity of from about 1 to about 100 centipoise (cps), or from about 1 to about 200cps, or from about 1 to about 300cps, or from about 1 to about 400 cps. In some embodiments, the solution has a viscosity of about 1 to about 100 cps. In certain embodiments, the solution has a viscosity of about 1 to about 200 cps. In certain embodiments, the solution has a viscosity of about 1 to about 300 cps. In certain embodiments, the solution has a viscosity of about 1 to about 400 cps. In certain embodiments, the solution is in the form of an injectable liquid solution. In other embodiments, the composition is formulated as a viscous liquid (i.e., a viscosity of several hundred to several thousand cps), a gel, or an ointment. In these embodiments, the bioconjugate is dispersed or dissolved in a suitable pharmaceutically acceptable carrier.
Exemplary compositions for use with bioconjugates for catheter-based delivery may comprise: a) a synthetic bioconjugate as described herein; b) a pharmaceutically acceptable carrier; c) a polymer matrix; d) a pH buffering agent to provide a pH in the range of about
An exemplary formulation may comprise: a) a bioconjugate as described herein; b) a pharmaceutically acceptable carrier; c) a polymer matrix; (ii) a And d) a pH buffer to provide a pH in the range of about
Exemplary compositions contemplated by the present disclosure may also be for administration by injection, including aqueous or oily suspensions or emulsions in sesame, corn, cottonseed or peanut oil, as well as elixirs with mannitol, dextrose or sterile aqueous solutions and similar pharmaceutical vehicles. Aqueous solutions in saline are also routinely used for injection, but are less preferred herein. Ethanol, glycerol, propylene glycol, liquid polyethylene glycols and the like (and suitable mixtures thereof), cyclodextrin derivatives and vegetable oils may also be used. For example, proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants. The action of microorganisms can be prevented by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.
Sterile injectable solutions are prepared by the following method: the required amounts of the components are added to the appropriate solvent with the various other ingredients described above (as required), followed by filter sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and the freeze-drying technique which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
In preparing a pharmaceutical composition comprising a bioconjugate described herein, the active ingredient is typically diluted by an excipient or carrier and/or enclosed within a carrier which may be in the form of a capsule, sachet, paper or other container. When the excipient serves as a diluent, it may be a solid, semi-solid, or liquid material (as above) that acts as a vehicle, carrier, or medium for the active ingredient. Thus, the compositions may be in the form of films, gels, patches, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols (in solid form or in a liquid medium), ointments containing, for example, up to 10% by weight of the active compound, soft and hard gelatin films, gels, patches, sterile injectable solutions, and sterile packaged powders.
Some examples of suitable excipients include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, sterile water, syrup, and methyl cellulose. The formulation may additionally comprise: lubricants, such as talc, magnesium stearate and mineral oil; a wetting agent; emulsifying and suspending agents; preservatives, such as methyl and propyl hydroxybenzoate; a sweetener; and a flavoring agent.
Films for drug delivery are well known in the art and include non-toxic, non-irritating polymers, free of leachable impurities such as polysaccharides (e.g., cellulose, maltodextrin, etc.). In some embodiments, the polymer is hydrophilic. In other embodiments, the polymer is hydrophobic. The film adheres to the tissue to which it is applied and is slowly absorbed into the body over a period of about a week. The polymers used in the thin film dosage forms described herein are absorbable and exhibit sufficient peel, shear and tensile strength as is well known in the art. In some embodiments, the membrane is injectable. In certain embodiments, the membrane is administered to the patient before, during, or after the surgical intervention.
As used herein, a gel refers to a solid, gelatinous substance that may have properties ranging from soft and weak to hard and tough. As is well known in the art, a gel is a non-fluid colloidal or polymeric network that is expanded throughout its volume by a fluid. A hydrogel is a gel that comprises a network of hydrophilic polymer chains, sometimes in the form of a colloidal gel, in which water is the dispersion medium. Hydrogels are highly absorbent and may contain large amounts of water, e.g., greater than 90% water. In some embodiments, the gels described herein comprise a natural or synthetic polymer network. In some embodiments, the gel comprises a hydrophilic polymer matrix. In other embodiments, the gel comprises a hydrophobic polymer matrix. In some embodiments, the gel has a degree of flexibility that is very similar to natural tissue. In certain embodiments, the gel is biocompatible and absorbable. In certain embodiments, the gel is administered to the patient before, during, or after the surgical procedure.
Liquid solutions as used herein refers to solutions, suspensions, emulsions, drops, ointments, liquid lotions, sprays, liposomes as are well known in the art. In some embodiments, the liquid solution contains an aqueous pH buffer that resists changes in pH when small amounts of acid or base are added. In certain embodiments, the liquid solution is administered to the patient before, during, or after the surgical intervention.
An exemplary formulation may comprise: a) one or more bioconjugates as described herein; b) a pharmaceutically acceptable carrier; and c) a hydrophilic polymer as a matrix network, wherein the composition is formulated as a viscous liquid (i.e., a viscosity of several hundred to several thousand centipoise), gel, or ointment. In these embodiments, the bioconjugate is dispersed or dissolved in a suitable pharmaceutically acceptable carrier.
In certain embodiments, the bioconjugate or a composition comprising the same is lyophilized prior to, during, or after formulation. Thus, also provided herein are lyophilized compositions comprising a bioconjugate or a lyophilized composition comprising a bioconjugate as described herein.
5. Dosage form
Suitable dosages of bioconjugates can be determined by standard methods, for example by establishing dose-response curves in laboratory animal models or clinical trials, and can vary significantly depending on the patient condition, the disease state being treated, the route of administration and tissue distribution, and the possibility of co-using other therapeutic approaches. The effective amount administered to the patient is based on the body surface area, the weight or mass of the patient, and the physician's assessment of the patient's condition. In various exemplary embodiments, the dose is in the range of about 0.01 μ g to about 10 g. For example, for systemic delivery, the dose may be about 10g, or about 5g, or about 1 g. In other exemplary embodiments, an effective dose ranges from about 100 μ g to about 10g per dose, or from about 100 μ g to about 1g per dose, or from about 100 μ g to about 500mg per dose, from about 0.01 μ g to about 100mg per dose, or from about 100 μ g to about 50mg per dose, or from about 500 μ g to about 10mg per dose, or from about 1mg to about 100mg per dose, or from about 1mg to 500mg per dose, or from about 1mg to 200mg per dose, or from about 10mg to 100mg per dose, or from about 10mg to 75mg per dose, or from about 10mg to 50mg per dose, or about 10mg per dose, or about 20mg per dose, or about 30mg per dose, or about 40mg per dose, or about 50mg per dose, or about 60mg per dose, or about 70mg per dose, or about 80mg per dose, or about 90mg per dose, or about 100mg per dose. In any of the various embodiments described herein, an effective dose ranges from about 0.01 μ g to about 1000mg per dose, from 1 μ g to about 100mg per dose, from about 100 μ g to about 1.0mg, from about 50 μ g to about 600 μ g, from about 50 μ g to about 700 μ g, from about 100 μ g to about 200 μ g, from about 100 μ g to about 600 μ g, from about 100 μ g to about 500 μ g, from about 200 μ g to about 600 μ g, or from about 100 μ g to about 50mg, or from about 500 μ g to about 10mg per dose, or from about 1mg to about 10mg per dose.
In some embodiments, the composition is packaged in a multi-dose form. Preservatives are therefore required to prevent microbial contamination during use. In certain embodiments, a suitable preservative as described above may be added to the composition. In some embodiments, the composition contains a preservative. In certain embodiments, the preservative is present in an amount of about 0.001% to about 1.0% w/v. In some embodiments, the unit dosage composition is sterile, but preservative-free.
Examples
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