阅读说明：本技术 葡萄糖反应性胰岛素 (Glucose-responsive insulin ) 是由 D·H-C·周 于 2019-04-15 设计创作，主要内容包括：本公开涉及基于胰岛素的肽、制备所述肽的方法以及使用这些肽治疗糖尿病的方法。本摘要旨在作为用于在特定领域中搜索目的的浏览工具,而非意图限制本发明。(The present disclosure relates to insulin-based peptides, methods of making the peptides, and methods of using the peptides to treat diabetes. This abstract is intended as a browsing tool for search purposes in the particular field and is not intended to be limiting of the present invention.)

1. A peptide comprising an insulin a chain peptide and an insulin B chain peptide, wherein the insulin B chain peptide comprises at least 32 amino acid residues, and wherein at least three amino acid residues of the insulin B chain peptide are lysine residues.

2. The peptide of claim 1, wherein the insulin A chain peptide is at least 70% identical to a wild-type human insulin A chain peptide.

3. The peptide of claim 1, wherein the insulin A chain peptide comprises sequence GIVEQCCTSICSLYQLENYCN (SEQ ID NO: 1).

4. The peptide of claim 1, wherein the insulin A chain peptide comprises sequence GIVEQCCTSICSLYQLENYCG (SEQ ID NO: 3).

5. The peptide of claim 1, wherein the insulin B-chain peptide comprises at least 34 amino acid residues.

6. The peptide of claim 1 wherein the amino acid at position B29 is a lysine residue.

7. A peptide according to claim 6 wherein the lysine residue is unmodified.

8. The peptide of claim 1 wherein the amino acid at position B33 is a lysine residue.

9. A peptide according to claim 8 wherein the lysine residue is modified.

10. The peptide of claim 1 wherein the amino acid at position B34 is a lysine residue.

11. A peptide according to claim 10 wherein the lysine residue is modified.

12. The peptide of claim 1, wherein each of the amino acid at position B29, the amino acid at position B33, and the amino acid at position B34 is a lysine residue.

13. The peptide of claim 12, wherein the B29 lysine residue is unmodified, and wherein each of the B33 and B34 lysine residues is modified.

14. The peptide of claim 1, wherein the insulin B chain peptide comprises sequence FVNQHLCGSHLVEALYLVCGERGFFYTPKT (SEQ ID NO: 2).

15. The peptide of claim 1, wherein the insulin B chain peptide comprises sequence FVNQHLCGSHLVEALYLVCGERGFFYTPKTR (SEQ ID NO: 4), FVNQHLCGSHLVEALYLVCGERGFFYTPKTRR (SEQ ID NO: 5), or FVNQHLCGSHLVEALYLVCGERGFFYTPKTRRR (SEQ ID NO: 6).

16. The peptide of claim 1, wherein the insulin B chain peptide comprises sequence FVNQHLCGSHLVEALYLVCGERGFFYTPKTRKK (SEQ ID NO: 8).

17. The peptide of claim 1, wherein the insulin A chain peptide and the insulin B chain peptide are bonded by at least one disulfide bond.

18. The peptide of claim 1, wherein at least two of the lysine residues are located at the C-terminus of the insulin B-chain peptide.

19. The peptide of claim 1, wherein the peptide is a monomer.

20. The peptide of claim 1, wherein the insulin A chain peptide and the insulin B chain peptide are bonded by at least one disulfide bond, wherein the insulin A chain peptide comprises sequence GIVEQCCHRICSLYQLENYCN (SEQ ID NO: 1), and wherein the insulin B chain peptide comprises sequence FVNQHLCGSHLVEALYLVCGERGFFYTPKTRKK (SEQ ID NO: 8).

21. The peptide of claim 20, wherein one or both of the B33 lysine residue and the B34 lysine residue is modified.

22. A peptide comprising an insulin a chain peptide and an insulin B chain peptide, wherein said peptide is directly conjugated to at least one organoboronate group.

23. The peptide of claim 22, wherein the insulin A chain peptide comprises sequence GIVEQCCTSICSLYQLENYCN (SEQ ID NO: 1) or GIVEQCCTSICSLYQLENYCG (SEQ ID NO: 3), and wherein the insulin B chain peptide comprises sequence FVNQHLCGSHLVEALYLVCGERGFFYTPKT (SEQ ID NO: 2), FVNQHLCGSHLVEALYLVCGERGFFYTPKTRR (SEQ ID NO: 5) or FVNQHLCGSHLVEALYLVCGERGFFYTPKTRKK (SEQ ID NO: 8).

24. The peptide of claim 22, wherein each of the amino acid at position B29, the amino acid at position B33, and the amino acid at position B34 is a lysine residue.

25. The peptide of claim 24, wherein the B29 lysine residue is unmodified.

26. The peptide of claim 24, wherein each of the B33 and B34 lysine residues is modified.

27. The peptide of claim 22, wherein the peptide is directly conjugated to two organoboronate groups.

28. The peptide of claim 22, wherein the peptide is directly conjugated to at least one organoboronate group through a lysine residue.

29. The peptide of claim 22, wherein the organoboronate group has a structure represented by the formula:

wherein Z is selected from C (O) and SO₂；

Wherein Ar is¹Selected from 5-membered aryl, 5-membered heteroaryl, 6-membered aryl and 6-membered heteroaryl, and substituted with 0, 1, 2 or 3 groups independently selected from: halogen, -CN, -NO₂OH, -C1-C4 alkyl, -C1-C4 haloalkyl, -C1-C4 hydroxyalkyl, -C1-C4 alkoxy, -C1-C4 alkylamino and (C1-C4) (C1-C4) dialkylamino.

30. The peptide of claim 29, wherein Ar¹Selected from 6-membered aryl and 6-membered heteroaryl, and substituted with 0, 1, 2 or 3 groups independently selected from: halogen, -CN, -NO₂OH, -C1-C4 alkyl, -C1-C4 haloalkyl, -C1-C4 hydroxyalkyl, -C1-C4 alkoxy, -C1-C4 alkylamino and (C1-C4) (C1-C4) dialkylamino.

31. The peptide of claim 29, wherein the organoboronate group has a structure represented by the formula:

wherein R is^1a、R^1b、R^1c、R^1dAnd R^1eEach of which is independently selected from hydrogen, halogen, -CN, -NO₂OH, -C1-C4 alkyl, -C1-C4 haloalkyl, -C1-C4 hydroxyalkyl, -C1-C4 alkoxy, -C1-C4 alkylamino and (C1-C4) (C1-C4) dialkylamino,

with the proviso that R^1a、R^1b、R^1c、R^1dAnd R^1eOne of them is-B (OH)₂。

32. The peptide of claim 29, wherein R^1cis-B (OH)₂。

33. The peptide of claim 29, wherein R^1a、R^1b、R^1c、R^1dAnd R^1eIs halogen.

34. The peptide of claim 29, wherein R^1a、R^1b、R^1c、R^1dAnd R^1eOne of which is-F.

35. The peptide of claim 29, wherein R^1aIs halogen.

36. The peptide of claim 29, wherein R^1ais-F.

37. The peptide of claim 29, wherein the organoboronate group has a structure represented by the formula:

38. the peptide of claim 22, wherein the insulin A chain peptide comprises sequence GIVEQCCTSICSLYQLENYCG (SEQ ID NO: 3), wherein the insulin B chain peptide comprises sequence FVNQHLCGSHLVEALYLVCGERGFFYTPKTRKK (SEQ ID NO: 8), wherein the B29 lysine residue is unmodified, wherein each of the B33 lysine residue and the B34 lysine residue is directly conjugated to an organoboronate group, and wherein the organoboronate group has a structure represented by the formula:

39. a pharmaceutical composition comprising the peptide of claim 1 or claim 22 and a pharmaceutically acceptable carrier.

40. A method of treating diabetes in a subject, the method comprising administering to the subject a therapeutically effective amount of the peptide of claim 1 or claim 22, thereby treating diabetes in the subject.

41. The method of claim 40, wherein the subject is a mammal.

42. The method of claim 41, wherein the mammal is a human.

43. The method of claim 40, wherein diabetes is type I diabetes

44. The method of claim 40, wherein prior to the administering step, the subject has been diagnosed as in need of treatment for diabetes.

45. The method of claim 40, further comprising the step of identifying a subject in need of treatment for diabetes.

46. The method of claim 40, wherein diabetes is type I diabetes.

47. A method of modifying insulin receptor activation in at least one cell, the method comprising contacting the at least one cell with an effective amount of the peptide of claim 1 or claim 22, thereby increasing insulin receptor activation in the at least one cell.

48. The method of claim 47, wherein modification is increasing.

49. The method of claim 47, wherein the cell is human.

50. The method of claim 47, wherein contacting is by administration to a subject.

51. The method of claim 50, wherein prior to the administering step, the subject has been diagnosed as in need of treatment for diabetes.

52. A method of reducing blood glucose in a subject, the method comprising administering to the subject a therapeutically effective amount of the peptide of claim 1 or claim 22, thereby reducing blood glucose in the subject.

53. A method of preparing an insulin B-chain peptide, wherein the insulin B-chain peptide is directly conjugated to an organoboronate group, the method comprising: a step of reacting the peptide-bound insulin B chain resin with a phenylboronic acid having a structure represented by the following formula:

wherein Z is selected from C (O) and SO₂；

Wherein Ar is¹Selected from 5-membered aryl, 5-membered heteroaryl, 6-membered aryl and 6-membered heteroaryl, and substituted with 0, 1, 2 or 3 groups independently selected from: halogen, -CN, -NO₂OH, -OH, C1-C4 alkyl, C1-C4 haloalkyl, C1-C4 hydroxyalkyl, C1-C4 alkoxy, C1-C4 alkylamino and (C1-C4) (C1-C4) dialkylamino; and cleaving the resin, thereby producing the insulin B chain peptide.

54. The method of claim 53, wherein the insulin B-chain peptide comprises sequence FVNQHLCGSHLVEALYLVCGERGFFYTPKTRKK (SEQ ID NO: 8).

55. The method of claim 53, wherein the insulin B-chain peptide comprises the amino acid at position B29 as an unmodified lysine residue.

56. The method of claim 53, wherein the insulin B-chain peptide comprises an amino acid at each of position B33 and position B34 as a modified lysine residue.

57. The method of claim 53, further comprising the step of coupling the insulin B chain peptide to an insulin A chain peptide.

Background

Since the discovery of insulin almost a century ago, many advances in insulin design have allowed the diabetic population to improve their glycemic control; however, The risk of hypoglycemia remains a major obstacle to tight glycemic control (Brown and Hirsch (2006) JAMA The Journal of The American Medical Association 295 (14): 1707-8; Frier, B.M, (2014) Nature Reviews Endocrinology 10 (12): 711). One problem is that commercially available insulin analogues are unable to modulate biological activity in response to circulating blood glucose and therefore have a narrow therapeutic index. To address this challenge, the concept of glucose-responsive insulin (GRI) or "smart" insulin was proposed to mimic glucose-stimulated insulin secretion in islet beta cells (Brown and Cerami (1979) Science 206 (4423): 1190-. To date, many studies to generate GRI have been developed using glucose triggers from: lectins (Kaarsholm et al (2018) Diabetes 67 (2): 299-308; Yang et al (2018) JCI Insight3(1)), glucose oxidases (Yu et al (2015) Proceedings of the National Academy of Sciences of the United States of America112 (27): 8260-5; Gu et al (2013) ACS Nano 7 (5): 4194-201), glucose transporters (Wang et al (2017) Advanced Materials 29(18)) and phenylboronic acid (PBA) (Guo et al (2015) Advanced Healthcare Materials 4 (12): 1796-1801; Chorisu et al (Proceedings of the Sciences of America112 (2408)). The glucose response characteristics using PBA are particularly useful because PBA is small in size compared to other sensors and is known to reversibly bind to cis-1, 2-or cis-1, 3-diols, such as glucose, thereby creating a negative charge on the boronic acid, a characteristic that can be used to alter insulin absorption characteristics. Therefore, chemically modified insulin derivatives are promising candidates for GRI design (Rege et al (2017) Current Opinion in Endocrinology, Diabetes and Obesity 24 (4): 267-278).

Insulin glargineIs a long-acting insulin commonly used by diabetic patients. The mechanism of sustained action of insulin glargine is due to the addition of two arginine residues to the B chain, which increases the isoelectric point (pI) of insulin to 6.7, thereby decreasing its solubility at physiological pH (Owens and Griffiths (2002) International Journal of Clinical Practice56 (6): 460-. Once injected, insulin glargine precipitates at the injection site and is very slowly converted to hexamers, dimers, and monomers for absorption, thereby allowing insulin to enter the bloodstream for a long period of time and stably in vivo. While insulin glargine has long-term benefits, the addition of glucose response characteristics to enhance glycemic control, while also preventing iatrogenic hypoglycemia, remains elusive. The present invention fulfills these needs and others.

Disclosure of Invention

In accordance with one or more objects of the present invention, as embodied and broadly described herein, in one aspect, the present invention relates to insulin-based peptides useful for treating diabetes.

Accordingly, the present invention discloses peptides comprising an insulin a chain peptide and an insulin B chain peptide, wherein the insulin B chain peptide comprises at least 32 amino acid residues and wherein at least three amino acid residues of the insulin B chain peptide are lysine residues.

The present invention discloses peptides comprising an insulin A chain peptide and an insulin B chain peptide, wherein the B chain peptide comprises substitutions at amino acids 10 and 20 and further comprises at least one substitution in the A chain peptide. In some cases, at least one substitution in the a chain peptide is T8H, T8Y, T8K, or S9R.

Also disclosed are peptides comprising an insulin A chain peptide and an insulin B chain peptide, wherein the peptides are directly conjugated to at least one organoboronate group.

Also disclosed are methods of making the disclosed peptides.

The present invention also discloses a method of preparing an insulin B-chain peptide, wherein the insulin B-chain peptide is directly conjugated to an organoboronate group, the method comprising: a step of reacting the peptide-bound insulin B chain resin with a phenylboronic acid having a structure represented by the following formula:

wherein Z is selected from C (O) and SO₂(ii) a Wherein Ar is¹Selected from 5-membered aryl, 5-membered heteroaryl, 6-membered aryl and 6-membered heteroaryl, and substituted with 0, 1, 2 or 3 groups independently selected from: halogen, -CN, -NO₂OH, -OH, C1-C4 alkyl, C1-C4 haloalkyl, C1-C4 hydroxyalkyl, C1-C4 alkoxy, C1-C4 alkylamino and (C1-C4) (C1-C4) dialkylamino; and cleaving the resin, thereby preparing the insulin B chain peptide.

Also disclosed are pharmaceutical compositions comprising a therapeutically effective amount of the disclosed peptides and a pharmaceutically acceptable carrier.

Also disclosed are methods of treating diabetes in a subject, comprising administering to the subject a therapeutically effective amount of the disclosed peptides, thereby treating diabetes in the subject.

Also disclosed are methods of modifying insulin receptor activation in at least one cell, the method comprising contacting the at least one cell with an effective amount of the disclosed peptide, thereby increasing insulin receptor activation in the at least one cell.

Also disclosed are methods of reducing blood glucose in a subject, comprising administering to the subject a therapeutically effective amount of the disclosed peptides, thereby reducing blood glucose in the subject.

Additional advantages of the disclosed methods and compositions will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the disclosed methods and compositions. The advantages of the disclosed methods and compositions will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention as claimed.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate several embodiments of the disclosed methods and compositions and, together with the description, serve to explain the principles of the disclosed methods and compositions.

Fig. 1A to 1C show representative schematic diagrams of Liquid Chromatography (LC) traces (top) and Mass Spectrometry (MS) spectra of compound No. 5 (insa (g), fig. 1A), compound No. 9 instb (fig. 1B) and compound No. 10 (smart glargine, fig. 1C). The compound numbers correspond to those shown in fig. 4B.

Figure 2 shows a representative image of an insulin analogue having a phenylboronic acid residue.

Fig. 3 shows a representative schematic illustrating the chemical synthesis of smart glargine insulin.

Fig. 4A and 4B show representative schematic diagrams illustrating the proposed glucose responsive smart insulin glargine design.

Fig. 5A to 5E show representative data relating to the characteristics of insulin derivatives.

Fig. 6A-6D show representative data relating to glucose clamp studies of insulin derivatives.

Fig. 7A and 7B show representative data illustrating the results of the insulin resistance test.

Fig. 8 shows representative images illustrating the design and synthesis of glucose-responsive insulin derivatives with improved glucose responsiveness.

Additional advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. The advantages of the invention will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention as claimed.

Detailed Description

The disclosed methods and compositions can be understood more readily by reference to the following detailed description of specific embodiments and the examples included therein, and to the figures and their previous and following description.

It is to be understood that, unless otherwise specified, the disclosed methods and compositions are not limited to specific synthetic methods, specific synthetic techniques, or specific reagents, and thus these may 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.

Disclosed are materials, compositions, and components useful for, in combination with, in preparation of, or products of the disclosed methods and compositions. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed that while specific reference of each various individual and various combinations and permutations of these compounds may not be explicitly disclosed, each is specifically contemplated and described herein. For example, if a peptide is disclosed and discussed, and a variety of modifications to a variety of molecules including the peptide are discussed, each and every combination and permutation of the peptide, and possible modifications, are specifically contemplated unless specifically indicated to the contrary. Thus, if a class of molecules A, B and C is disclosed as well as a class of molecules D, E and F and an example of a combination molecule a-D is disclosed, then each is individually and collectively contemplated even if not individually recited. Thus, in this example, combinations of each of A-E, A-F, B-D, B-E, B-F, C-D, C-E and C-F are specifically contemplated and should be considered disclosed from publications A, B and C; D. e and F; and exemplary combinations a-D. Likewise, any subset or combination of these is also specifically contemplated and disclosed. Thus, for example, subgroups A-E, B-F and C-E are specifically contemplated and should be considered disclosed in publications A, B and C; D. e and F; and exemplary combinations a-D. This concept applies to all aspects of this application including, but not limited to, steps in methods of making and using the disclosed compositions. Thus, if there are a plurality of additional steps that can be performed it is understood that each of these additional steps can be performed with any specific embodiment or combination of embodiments of the disclosed methods, and that each such combination is specifically contemplated and should be considered disclosed.

A. Definition of

It is to be understood that the disclosed methods and compositions are not limited to the particular methodology, protocols, and reagents described, as these may 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 limit the scope of the present invention which will be limited only by the appended claims.

It must be noted that, as used herein and in the appended 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 such peptides, reference to "the peptide" is a reference to one or more peptides and equivalents thereof known to those skilled in the art, and so forth.

As used herein, a plurality of items, structural elements, compositional elements, and/or materials may be presented in a common list for convenience. However, these lists should be construed as though each member of the list is individually identified as a separate and unique member. Thus, no individual member of such list should be construed as a de facto equivalent of any other member of the same list solely based on their presentation in a common group without indications to the contrary.

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

Ranges may be expressed herein as from "about" one particular value, and/or to "about" another particular value. Unless the context clearly dictates otherwise, when expressed, it is also specifically contemplated and considered to be a range from one particular value and/or to another particular value. Similarly, where values are expressed as approximations, by use of the antecedent "about," it will be understood that the particular value forms another specifically contemplated embodiment of the disclosure unless the context clearly dictates otherwise. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint, unless the context clearly dictates otherwise. Finally, it is to be understood that all individual values and subranges of values encompassed within the explicitly disclosed ranges are also specifically contemplated and should be considered disclosed unless the context clearly dictates otherwise. The foregoing applies regardless of whether some or all of these embodiments are explicitly disclosed in particular instances.

Reference in the specification and the claims at the end to parts by weight of a particular element or component in a composition is expressed as a weight relationship between the element or component and any other element or component in the composition or article, with parts by weight being expressed in terms of the composition or article. Thus, in a compound containing 2 parts by weight of component X and 5 parts by weight of component Y, X and Y are present in a weight ratio of 2: 5 and always in such a ratio, regardless of whether additional components are contained in the compound.

Weight percent (wt.%) of a component, unless specifically stated to the contrary, is based on the total weight of the formulation or composition in which the component is included.

Throughout the description and claims of this specification, the word "comprise" and variations of the word, such as "comprises" and "comprising", means "including but not limited to" and is not intended to exclude, for example, other additives, components, integers or steps. In particular, in methods stated as including one or more steps or operations, it is specifically contemplated that each step includes what is listed (unless the step includes a limiting term such as "consisting of"), meaning that each step is not intended to exclude, for example, other additives, components, integers, or steps not listed in the step.

The terms "a chain peptide" and "B chain peptide" are interchangeable with "insulin a chain peptide" and "insulin B chain peptide".

The term "therapeutic" refers to a treatment, therapy, or drug that can treat a disease or condition or can ameliorate one or more symptoms associated with a disease or condition. As used herein, a therapeutic agent may refer to a therapeutic compound, including but not limited to a protein, peptide, nucleic acid (e.g., CpG oligonucleotide), small molecule, vaccine, allergic extract, antibody, gene therapy, other biological agent, or small molecule.

As used herein, the term "subject" or "patient" refers to any organism to which a peptide or composition of the invention can be administered, e.g., for experimental, diagnostic, and/or therapeutic purposes. Typical subjects include animals (e.g., mammals such as non-human primates and humans; avians, domestic or farm animals such as cats, dogs, sheep, goats, cattle, horses, and pigs, laboratory animals such as mice, rats, and guinea pigs, rabbits, fish, reptiles, zoos, and wildlife). Typically, a "subject" is an animal, including mammals, such as humans and primates; and so on.

As used herein, the term "treating" refers to partially or completely alleviating, ameliorating, palliating, delaying onset of, inhibiting or slowing progression of, reducing severity of, and/or reducing incidence of one or more symptoms or features of a particular disease, disorder, and/or condition. Treatment can be administered to a subject who does not exhibit signs of a disease, disorder, and/or condition and/or a subject who exhibits only early signs of a disease, disorder, and/or condition for the purpose of reducing the risk of developing a pathology associated with a disease, disorder, and/or condition. For example, the disease, disorder, and/or condition may be type 1 diabetes or any other insulin-related condition.

As used herein, the terms "prevent" or "preventing" mean to suppress, alleviate, avoid, prevent, deter or hinder the occurrence of something, especially premature effects. It is to be understood that where reduction, inhibition, or prevention is used herein, the other two terms used herein are also specifically disclosed unless specifically indicated otherwise.

As used herein, the term "diagnosis" means having been physically examined and found by a skilled person, e.g., a physician, to have a condition that can be diagnosed or treated by a compound, composition, or method disclosed herein. In some aspects of the disclosed methods, prior to the administering step, the subject has been diagnosed as in need of treatment for a disease or disorder, such as, for example, diabetes. As used herein, the phrase "identified as in need of treatment for a disorder" and the like refers to selecting a subject based on the need to treat the disorder. It is contemplated that, in one aspect, the identification can be performed by a person other than the person making the diagnosis. It is also contemplated that, in another aspect, administration may be by a human who subsequently performs the administration.

As used herein, the terms "administering" and "administration" refer to any method of providing a pharmaceutical product to a subject. Such methods are well known to those skilled in the art and include, but are not limited to, oral administration, transdermal administration, administration by inhalation, nasal administration, topical administration, intravaginal administration, ocular administration, otic administration, intracranial administration, rectal administration, and parenteral administration, including injectable, such as intravenous administration, intraarterial administration, intramuscular administration, and subcutaneous administration. The administration may be continuous or intermittent. In various aspects, the article of manufacture can be administered therapeutically; i.e., administered to treat an existing disease or condition. In other various aspects, the article can be applied prophylactically; i.e., administration to prevent a disease or condition.

The term "contacting" as used herein means bringing the disclosed compounds together with a cell, target receptor, or other biological entity in such a way that the compounds can be directed; i.e., through the target's own interaction, or indirectly; that is, the activity of a target (e.g., receptor, cell, etc.) is affected by interaction with another molecule, cofactor, factor, or protein upon which the target activity depends.

As used herein, the terms "effective amount" and "effective amount" refer to an amount sufficient to achieve a desired result or to be effective against an undesirable condition. For example, "therapeutically effective amount" refers to an amount sufficient to achieve a desired therapeutic result or an effect on an undesirable symptom, but generally insufficient to cause an adverse side effect. The specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the condition being treated and the severity of the condition; the specific composition used; the age, weight, health, sex, and diet of the patient; the time of administration; the route of administration; the rate of excretion of the particular compound used; the duration of the treatment; drugs used in combination or by chance with the particular compound used and similar factors well known in the medical arts. For example, it is well within the skill of the art to start doses of the compound at levels below those required to achieve the desired therapeutic effect and to gradually increase the dosage until such time as the desired effect is achieved. If desired, the effective daily dose may be divided into multiple doses for administration purposes. Thus, a single dose composition may contain such an amount or submultiples thereof to make up a daily dose. In the case of any contraindication, the dosage may be adjusted by the individual physician. The dosage may vary and may be administered in one or more doses per day for one or more days. Appropriate dosages for a given class of pharmaceutical products can be found in the literature guidelines. In other various aspects, the article of manufacture can be administered in a "prophylactically effective amount" (i.e., an amount effective for preventing a disease or condition).

The term amino acid "modified" or "modified" amino acid refers to a substitution of an amino acid, or a derivative of an amino acid by the addition and/or removal of chemical groups to and from an amino acid, and includes substitutions by any of the 20 amino acids commonly found in human proteins, as well as atypical or non-naturally occurring amino acids. Commercial sources of atypical amino acids include Sigma-Aldrich (Milwaukee, Wis.), ChemPep Inc. (Miami, Fla.), and Genzyme Pharmaceuticals (Cambridge, Mass.). Atypical amino acids can be purchased from commercial suppliers, synthesized de novo, or chemically modified or derived from naturally occurring amino acids.

As used herein, an amino acid "substitution" refers to the replacement of one amino acid residue by a different amino acid residue. The substituted amino acid can be any of the 20 amino acids commonly found in human proteins, as well as atypical or non-naturally occurring amino acids.

Compounds according to the present disclosure may use alkoxy, amino acid, etc. groups as prodrug forming moieties to form prodrugs of hydroxy or amino functional groups. For example, the hydroxymethyl position may form a monophosphate, diphosphate, or triphosphate, and these phosphates may again form prodrugs. The preparation of such prodrug derivatives is discussed in various literature sources (examples are: Alexander et al, J.Med.chem.1988, 31, 318; Aligas-Martin et al, PCT WO 2000/041531, page 30). The nitrogen function converted in the preparation of these derivatives is one (or more) of the nitrogen atoms of the compounds of the present disclosure.

"derivatives" of the compounds disclosed herein are pharmaceutically acceptable salts, prodrugs, deuterated forms, radiolabeled forms, isomers, solvates and combinations thereof. Reference herein to "a combination" refers to a derivative that falls into at least two of the following groups: pharmaceutically acceptable salts, prodrugs, deuterated forms, radiolabeled forms, isomers, and solvates. Examples of radiolabeled forms include compounds labeled with tritium, phosphorus-32, iodine-129, carbon-11, fluorine-18, and the like.

"pharmaceutically acceptable salts" refers to derivatives of the disclosed compounds wherein the parent compound is modified by making acid or base salts thereof. The compounds of the present disclosure form acid addition salts with a variety of organic and inorganic acids, and include physiologically acceptable salts that are often used in pharmaceutical chemistry. Such salts are also part of the present disclosure. Typical inorganic acids used to form such salts include hydrochloric, hydrobromic, hydroiodic, nitric, sulfuric, phosphoric, hypophosphorous, and the like. Salts derived from organic acids such as: aliphatic mono-and dicarboxylic acids, phenyl substituted alkanoic, hydroxyalkanoic and hydroxyalkanedioic acids, aromatic acids, aliphatic sulfonic acids and aromatic sulfonic acids. Thus, such pharmaceutically acceptable salts include acetate, phenyl acetate, trifluoroacetate, acrylate, ascorbate, benzoate, chlorobenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, methylbenzoate, orthoacetoxybenzoate, naphthalene-2-benzoate, bromide, isobutyrate, phenylbutyrate, beta-hydroxybutyrate, butyne-1, 4-dioate, hexyne-1, 4-dioate, decanoate, octanoate, chloride, cinnamate, citrate, formate, fumarate, glycolate, heptanoate, hippurate, lactate, malate, maleate, hydroxymaleate, malonate, mandelate, methanesulfonate, nicotinate, isonicotinate, oxalate, nitrate, oxalate, dihydrogenate, dihydrogena, Phthalates, terephthalates, phosphates, monohydrogen phosphates, dihydrogen phosphates, metaphosphates, pyrophosphates, propiolates, propionates, phenylpropionates, salicylates, sebacates, succinates, suberates, sulfates, hydrogensulfates, pyrosulfates, sulfites, hydrogensulfites, sulfonates, benzene-sulfonates, p-bromobenzenesulfonates, chlorobenzenesulfonates, ethanesulfonates, 2-hydroxyethanesulfonates, methanesulfonates, naphthalene-1-sulfonates, naphthalene-2-sulfonates, p-toluenesulfonates, xylenesulfonates, tartrates, and the like.

It is to be understood that, unless otherwise indicated, the compounds of the present disclosure relate to all optical isomers and stereoisomers at the various possible atoms of the molecule. The compounds may be isolated or prepared as their pure enantiomers or diastereomers by crystallization, chromatography, or synthesis.

The term "leaving group" refers to an atom (or group of atoms) having electron withdrawing capability that can be replaced with a stable species while serving as a bonding electron. Examples of suitable leaving groups include sulfonates, including triflate, mesylate, tosylate, brosylate and halides.

As used herein, the term "substituted" is intended to include all permissible substituents of organic compounds. In a broad aspect, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic and aromatic and nonaromatic substituents of organic compounds. Exemplary substituents include, for example, those described below. For suitable organic compounds, the permissible substituents can be one or more and the same or different. For purposes of this disclosure, a heteroatom (such as nitrogen) may have a hydrogen substituent and/or any permissible substituents of organic compounds described herein that satisfy the valencies of the heteroatom. The present disclosure is not intended to be limited in any way by the permissible substituents of organic compounds. Furthermore, the terms "substituted" or "substituted" include the implicit proviso that such substitution is in accordance with the allowed valency of the substituted atom or substituent, and that the substitution results in a stable compound, e.g., a compound that does not spontaneously undergo transformation (such as by rearrangement, cyclization, elimination, etc.). It is also contemplated that, in certain aspects, individual substituents may be further optionally substituted (i.e., further substituted or unsubstituted), unless expressly stated to the contrary.

In the various terms defined, "A¹”、“A²”、“A³"and" A⁴"is used herein as a generic symbol to denote various specific substituents. These symbols may be any substituent, but are not limited to those disclosed herein, and when they are defined as certain substituents in one instance, they may be defined as some other substituents in another instance.

The term "alkyl" as used herein is a branched or unbranched saturated hydrocarbon group of 1 to 24 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, sec-pentyl, neopentyl, hexyl, heptyl, octyl, nonyl, decyl, dodecyl, tetradecyl, hexadecyl, eicosyl, tetracosyl and the like. Alkyl groups may also be substituted or unsubstituted. The alkyl group may be substituted with one or more groups including, but not limited to, optionally substituted alkyl, cycloalkyl, alkoxy, amino, ether, halide, hydroxy, nitro, silyl, sulfo-oxo, or thiol as described herein. "lower alkyl" is an alkyl group containing one to six (e.g., one to four) carbon atoms.

Throughout the specification, "alkyl" is generally used to refer to both unsubstituted alkyl and substituted alkyl; however, substituted alkyl is also specifically referred to herein as recognizing one or more specific substituents on the alkyl. For example, the term "haloalkyl" specifically refers to an alkyl group substituted with one or more halides (e.g., fluorine, chlorine, bromine, or iodine). The term "alkoxyalkyl" specifically refers to an alkyl group substituted with one or more alkoxy groups as described below. The term "alkylamino" specifically refers to an alkyl group substituted with one or more amino groups, etc., as described below. When "alkyl" is used in one instance and a specific term such as "alkyl alcohol" is used in another instance, it is also not intended to imply that the term "alkyl" nor refers to a specific term such as "alkyl alcohol" or the like.

This practice is also applicable to the other groups described herein. That is, in addition, when terms such as "cycloalkyl" refer to unsubstituted and substituted cycloalkyl moieties, the substituted moieties may be specifically identified herein; for example, a particular substituted cycloalkyl group can be referred to as, e.g., "alkylcycloalkyl". Similarly, a substituted alkoxy group can be specifically referred to as, for example, "haloalkoxy," and a particular substituted alkenyl group can be, for example, "alkenyl alcohol," and the like. Again, practice using generic terms such as "cycloalkyl" and specific terms such as "alkylcycloalkyl" is not intended to imply that the generic term nor the specific term is included.

The term "cycloalkyl" as used herein is a non-aromatic carbon-based ring consisting of at least three carbon atoms. Examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, norbornyl, and the like. The term "heterocycloalkyl" is a type of cycloalkyl group as defined above and is included within the meaning of the term "cycloalkyl" wherein at least one carbon atom of the ring is replaced by a heteroatom such as, but not limited to, nitrogen, oxygen, sulfur, or phosphorus. Cycloalkyl and heterocycloalkyl groups may be substituted or unsubstituted. Cycloalkyl and heterocycloalkyl groups may be substituted with one or more groups including, but not limited to, optionally substituted alkyl, cycloalkyl, alkoxy, amino, ether, halide, hydroxy, nitro, silyl, sulfo-oxo, or mercapto as described herein.

The term "polyalkylene" as used herein is a compound having two or more CH groups attached to each other₂A group. The polyalkylene may be represented by the formula- (CH)₂)_a-represents, wherein "a" is an integer from 2 to 500.

The terms "alkoxy" and "alkoxy" as used herein refer to an alkyl or cycloalkyl group bonded through an ether linkage; that is, "alkoxy" may be defined as-OA¹Wherein A is¹Is alkyl or cycloalkyl as defined above. "alkoxy" also includes polymers of the above alkoxy groups, i.e., the alkoxy group can be, for example, -OA¹-OA²or-OA¹-(OA²)_a-OA³Wherein "a" is an integer of 1 to 200 and A¹、A²And A³Is alkyl and/or cycloalkyl.

The term "alkenyl" as used herein is a hydrocarbon group of 2 to 24 carbon atoms having a structural formula containing at least one carbon-carbon double bond. Asymmetric structures such as (A)¹A²)C＝C(A³A⁴) It is intended to include both the E and Z isomers. This may be assumed in the structural formulae herein where asymmetric olefins are present, or may be explicitly indicated by the bond symbol C ═ C. The alkenyl group may be substituted with one or more groups including, but not limited to, optionally substituted alkyl, cycloalkyl, alkoxy, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone, azido, nitro, silyl, sulfo-oxo, or thiol as described herein.

The term "cycloalkenyl" as used herein is a non-aromatic carbon-based ring consisting of at least three carbon atoms and containing at least one carbon-carbon double bond (i.e., C ═ C). Examples of cycloalkenyl groups include, but are not limited to, cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclopentadienyl, cyclohexenyl, cyclohexadienyl, norbornenyl, and the like. The term "heterocycloalkenyl" is a type of cycloalkenyl group as defined above, and is included within the meaning of the term "cycloalkenyl" where at least one carbon atom of the ring is replaced with a heteroatom such as, but not limited to, nitrogen, oxygen, sulfur, or phosphorus. Cycloalkenyl and heterocycloalkenyl groups can be substituted or unsubstituted. The cycloalkenyl group and heterocycloalkenyl group can be substituted with one or more groups including, but not limited to, optionally substituted alkyl, cycloalkyl, alkoxy, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone, azido, nitro, silyl, sulfo-oxo, or thiol as described herein.

The term "alkynyl" as used herein is a hydrocarbon group of 2 to 24 carbon atoms having a structural formula containing at least one carbon-carbon triple bond. The alkenyl group can be unsubstituted or substituted with one or more groups including, but not limited to, optionally substituted alkyl, cycloalkyl, alkoxy, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone, azido, nitro, silyl, sulfo-oxo, or thiol as described herein.

The term "cycloalkynyl" as used herein is a non-aromatic carbon-based ring consisting of at least seven carbon atoms and containing at least one carbon-carbon triple bond. Examples of cycloalkynyl include, but are not limited to, cycloheptynyl, cyclooctynyl, cyclononynyl, and the like. The term "heterocycloalkynyl" is a type of cycloalkenyl group as defined above and is included within the meaning of the term "cycloalkynyl" wherein at least one carbon atom of the ring is replaced by a heteroatom such as, but not limited to, nitrogen, oxygen, sulfur, or phosphorus. Cycloalkynyl and heterocycloalkynyl can be substituted or unsubstituted. Cycloalkynyl and heterocycloalkynyl may be substituted with one or more groups including, but not limited to, optionally substituted alkyl, cycloalkyl, alkoxy, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone, azido, nitro, silyl, sulfo-oxo, or thiol as described herein.

The term "aryl" as used herein is a group containing any carbon-based aromatic group, including but not limited to benzene, naphthalene, phenyl, biphenyl, phenoxybenzene, and the like. The term "aryl" also includes "heteroaryl," which is defined as a group containing an aromatic group having at least one heteroatom incorporated into the ring of the aromatic group. Examples of heteroatoms include, but are not limited to, nitrogen, oxygen, sulfur, and phosphorus. Likewise, the term "non-heteroaryl", also included in the term "aryl", defines a group containing an aromatic group that does not contain heteroatoms. The aryl group may be substituted or unsubstituted. The aryl group can be substituted with one or more groups including, but not limited to, optionally substituted alkyl, cycloalkyl, alkoxy, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone, azido, nitro, silyl, sulfo-oxo, or thiol as described herein. The term "biaryl" is a specific type of aryl group and is included in the definition of "aryl". Biaryl refers to two aryl groups joined together via a fused ring structure (e.g., naphthalene) or two aryl groups connected via one or more carbon-carbon bonds (e.g., biphenyl).

The term "aldehyde" as used herein is represented by the formula-c (o) H. Throughout the specification, "C (O)" is a shorthand notation for carbonyl (i.e., C ═ O).

The term "amine" or "amino" as used herein is represented by the formula-NA¹A²Is shown in the specification, wherein A¹And A²May independently be hydrogen or an alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl or heteroaryl group as described herein.

The term "alkylamino" as used herein is represented by the formula-NH (-alkyl), wherein alkyl is as described herein. Representative examples include, but are not limited to, methylamino, ethylamino, propylamino, isopropylamino, butylamino, isobutylamino, (sec-butyl) amino, (tert-butyl) amino, pentylamino, isopentylamino, (tert-pentyl) amino, hexylamino, and the like.

The term "dialkylamino" as used herein is represented by the formula-N (-alkyl)₂Wherein alkyl is as described herein. Representative examples include, but are not limited to, dimethylamino, diethylamino, dipropylamino, diisopropylamino, dibutylamino, diisobutylamino, di (sec-butyl) amino, di (tert-butyl) amino, dipentylamino, diisopentylamino, di (tert-pentyl) amino, dihexylamino, N-ethyl-N-methylamino, N-methyl-N-propylamino, N-ethyl-N-propylamino, and the like.

The term "carboxylic acid" as used herein is represented by the formula-c (o) OH.

The term "ester" as used herein is represented by the formula-OC (O) A¹or-C (O) OA¹Is shown in the specification, wherein A¹May be an optionally substituted alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl or heteroaryl group as described herein. The term "polyester" as used herein is represented by the formula- (A)¹O(O)C-A²-C(O)O)_a-or- (A)¹O(O)C-A²-OC(O))_a-is represented by, wherein A¹And A²May independently be an optionally substituted alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl or heteroaryl group as described herein and "a" is an integer from 1 to 500. The term "polyester" is used to describe a group produced by the reaction between a compound having at least two carboxylic acid groups and a compound having at least two hydroxyl groups.

The term "ether" as used herein is represented by formula A¹OA²Is shown in the specification, wherein A¹And A²May independently be an optionally substituted alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl or heteroaryl group as described herein. The term "polyether" as used herein is represented by the formula- (A)¹O-A²O)_a-is represented by, wherein A¹And A²May independently be an optionally substituted alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl or heteroaryl group as described herein and "a" is an integer from 1 to 500. Examples of polyether groups include polyethylene oxide, polypropylene oxideAlkanes and polybutylene oxides.

The term "halide" as used herein refers to the halogens fluorine, chlorine, bromine and iodine.

The term "heterocycle" as used herein refers to monocyclic and polycyclic aromatic or non-aromatic ring systems in which at least one ring member is not carbon. Heterocycles include pyridine, pyrimidine, furan, thiophene, pyrrole, isoxazole, isothiazole, pyrazole, oxazole, thiazole, imidazole, oxazole (including 1, 2, 3-oxadiazole, 1, 2, 5-oxadiazole and 1, 3, 4-oxadiazole), thiadiazole (including 1, 2, 3-thiadiazole, 1, 2, 5-thiadiazole and 1, 3, 4-thiadiazole), triazole (including 1, 2, 3-triazole, 1, 3, 4-triazole), tetrazole (including 1, 2, 3, 4-tetrazole and 1, 2, 4, 5-tetrazole), pyridine, pyridazine, pyrimidine, pyrazine, triazine (including 1, 2, 4-triazine and 1, 3, 5-triazine), tetrazine (including 1, 2, 4, 5-tetrazine), pyrrolidine, piperidine, piperazine, morpholine, azetidine, Tetrahydropyran, tetrahydrofuran, dioxane, and the like.

The term "hydroxy" as used herein is represented by the formula-OH.

The term "ketone" as used herein is represented by formula A¹C(O)A²Is shown in the specification, wherein A¹And A²May independently be an optionally substituted alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl or heteroaryl group as described herein.

The term "azide" as used herein is represented by the formula-N₃And (4) showing.

The term "nitro" as used herein is represented by the formula-NO₂And (4) showing.

The term "nitrile" as used herein is represented by the formula-CN.

The term "silyl" as used herein is represented by the formula-SiA¹A²A³Is shown in the specification, wherein A¹、A²And A³May independently be hydrogen or an optionally substituted alkyl, cycloalkyl, alkoxy, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl or heteroaryl group as described herein.

The term "sulfo-oxo" as used herein is represented by the formula-S (O) A¹、-S(O)₂A¹、-OS(O)₂A¹or-OS (O)₂OA¹Is shown in the specification, wherein A¹May be hydrogen or an optionally substituted alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl or heteroaryl group as described herein. Throughout the specification, "S (O)" is a shorthand notation of S ═ O. The term "sulfonyl" as used herein refers to a compound of the formula-S (O)₂A¹A sulfo-oxo group represented by¹May be hydrogen or an optionally substituted alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl or heteroaryl group as described herein. The term "sulfone" as used herein is represented by formula A¹S(O)₂A²Is shown in the specification, wherein A¹And A²May independently be an optionally substituted alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl or heteroaryl group as described herein. The term "sulfoxide" as used herein is represented by formula A¹S(O)A²Is shown in the specification, wherein A¹And A²May independently be an optionally substituted alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl or heteroaryl group as described herein.

The term "thiol" as used herein is represented by the formula-SH.

"R" as used herein¹”、“R²”、“R³”、“Rⁿ"(wherein n is an integer) may independently have one or more of the groups listed above. For example, if R¹Is a straight chain alkyl group, one of the hydrogen atoms of the alkyl group may be optionally substituted with a hydroxyl group, an alkoxy group, an alkyl group, a halide, or the like. Depending on the group selected, the first group can be introduced into the second group, or alternatively, the first group can be pendant (i.e., attached) to the second group. For example, for the phrase "alkyl group comprising an amino group," the amino group can be introduced into the backbone of the alkyl group. Alternatively, the amino group may be attached to the backbone of the alkyl group. If a first group is intercalated or attached to a second group, the nature of the selected group or groups will be determined.

As described herein, the compounds of the present invention may contain "optionally substituted" moieties. Generally, the term "substituted" whether preceded by the term "optionally" or not, means that one or more hydrogens of the designated moiety are replaced with a suitable substituent. Unless otherwise indicated, an "optionally substituted" group may have suitable substituents at each substitutable position of the group, and when more than one position in any given structure may be substituted with more than one substituent selected from a particular group, the substituents at each position may be the same or different. The combinations of substituents envisaged by the present invention are preferably those which allow the formation of stable or chemically feasible compounds. It is also contemplated that, in certain aspects, individual substituents may be further optionally substituted (i.e., further substituted or unsubstituted), unless expressly stated to the contrary.

The term "stable" as used herein refers to a compound that is substantially unchanged when subjected to conditions that allow the compound to be produced, detected, and in some aspects recovered, purified, and used for one or more of the purposes disclosed herein.

Suitable monovalent substituents on the substitutable carbon atom of the "optionally substituted" group are independently halogen; - (CH)₂)_0-4R^o；-(CH₂)_0-4OR^o；-O(CH₂)_0-4R^o，-O-(CH₂)_0-4C(O)OR^o；-(CH₂)_0-4CH(OR^o)₂；-(CH₂)_{0- 4}SR^o；-(CH₂)_0-4Ph, which may be represented by R^oSubstitution; - (CH)₂)_0-4O(CH₂)_0-1Ph, which may be represented by R^oSubstitution; -CH ═ CHPh, which may be substituted by R^oSubstitution; - (CH)₂)_0-4O(CH₂)_0-1-a pyridyl group, which may be substituted by R^oSubstitution; -NO₂；-CN；-N₃；-(CH₂)_0-4N(R^o)₂；-(CH₂)_0-4N(R^o)C(O)R^o；-N(R^o)C(S)R^o；-(CH₂)_0-4N(R^o)C(O)NR^o ₂；-N(R^o)C(S)NR^o ₂；-(CH₂)_0-4N(R^o)C(O)OR^o；-N(R^o)N(R^o)C(O)R^o；-N(R^o)N(R^o)C(O)NR^o ₂；-N(R^o)N(R^o)C(O)OR^o；-(CH₂)_0-4C(O)R^o；-C(S)R^o；-(CH₂)_0-4C(O)OR^o；-(CH₂)_0-4C(O)SR^o；-(CH₂)_0-4C(O)OSiR^o ₃；-(CH₂)_0-4OC(O)R^o；-OC(O)(CH₂)_0-4SR-，SC(S)SR^o；-(CH₂)_0-4SC(O)R^o；-(CH₂)_0-4C(O)NR^o ₂；-C(S)NR^o ₂；-C(S)SR^o；-SC(S)SR^o，-(CH₂)_0-4OC(O)NR^o ₂；-C(O)N(OR^o)R^o；-C(O)C(O)R^o；-C(O)CH₂C(O)R^o；-C(NOR^o)R^o；-(CH₂)_0-4SSR^o；-(CH₂)_0-4S(O)₂R^o；-(CH₂)_0-4S(O)₂OR^o；-(CH₂)_0-4OS(O)₂R^o；-S(O)₂NR^o ₂；-(CH₂)_0-4S(O)R^o；-N(R^o)S(O)₂NR^o ₂；-N(R^o)S(O)₂R^o；-N(OR^o)R^o；-C(NH)NR^o ₂；-P(O)₂R^o；-P(O)R^o ₂；-OP(O)R^o ₂；-OP(O)(OR^o)₂；SiR^o ₃；-(C_1-4Straight or branched alkylene) O-N (R)^o)₂(ii) a Or- (C)_1-4Straight or branched alkylene) C (O) O-N (R)^o)₂Wherein each R is^oMay be substituted as defined below and independently is hydrogen, C_1-6Aliphatic, -CH₂Ph，-O(CH₂)_0-1Ph，-CH₂- (5-to 6-membered heteroaryl ring), or having 0 to 4 heteroatoms independently selected from nitrogen, oxygen, or sulfurA 5-to 6-membered saturated, partially unsaturated, or aryl ring, or, although defined above, two independently occurring R^oTogether with one or more of their intervening atoms, form a 3-to 12-membered saturated, partially unsaturated, or aryl monocyclic or bicyclic ring having 0 to 4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, which may be substituted as defined below.

At R^oSuitable monovalent substituents on (or two independently occurring R)^oThe ring formed together with the intervening atoms thereof) is independently halogen, - (CH)₂)_0-2R^·- (halogenated R)^·)、-(CH₂)_0-2OH、-(CH₂)_0-2OR^·、-(CH₂)_0-2CH(OR^·)₂(ii) a -O (halo R)^·)、-CN、-N₃、-(CH₂)_0-2C(O)R^·、-(CH₂)_0-2C(O)OH、-(CH₂)_0-2C(O)OR^·、-(CH₂)_0-2SR^·、-(CH₂)_{0- 2}SH、-(CH₂)_0-2NH₂、-(CH₂)_0-2NHR^·、-(CH₂)_0-2NR^· ₂、-NO₂、-SiR^· ₃、-OSiR^· ₃、-C(O)SR^·、-(C_1-4Straight OR branched alkylene) C (O) OR^·or-SSR^·Wherein each R is^·Is unsubstituted or is preceded by "halo" and is independently selected from C_1-4Aliphatic, -CH₂Ph，-O(CH₂)_0-1Ph or a 5 to 6 membered saturated, partially unsaturated or aryl ring having 0 to 4 heteroatoms independently selected from nitrogen, oxygen or sulfur. At R^oSuitable divalent substituents on the saturated carbon atom of (a) include ═ O and ═ S.

Suitable divalent substituents on the saturated carbon atom of the "optionally substituted" group include the following: is one of O, S and NNR^* ₂、＝NNHC(O)R^*、＝NNHC(O)OR^*、＝NNHS(O)₂R^*、＝NR^*、＝NOR^*、-O(C(R^* ₂))_2-3O-or-S (C (R)^* ₂))_2-3S-, wherein each independently occurs R^*Selected from hydrogen, C_1-6Aliphatic (which may be substituted as defined below), or an unsubstituted 5-to 6-membered saturated, partially unsaturated, or aryl ring having 0 to 4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. Suitable divalent substituents bound to the carbon substitutable at the ortho position of the "optionally substituted" group include: -O (CR)^* ₂)_2-3O-, wherein each independently occurs R^*Selected from hydrogen, C_1-6Aliphatic (which may be substituted as defined below), or an unsubstituted 5-to 6-membered saturated, partially unsaturated, or aryl ring having 0 to 4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

R^*Suitable substituents on the aliphatic radical of (A) include halogen, -R^·- (halogenated R)^·)、-OH、-OR^·-O (halo R)^·)、-CN、-C(O)OH、-C(O)OR^·、-NH₂、-NHR^·、-NR^· ₂or-NO₂Wherein each R is^·Is unsubstituted or substituted, where preceded by "halo", with only one or more halogen, and is independently C_1-4Aliphatic, -CH₂Ph，-O(CH₂)_0-1Ph, or a 5 to 6 membered saturated, partially unsaturated or aryl ring having 0 to 4 heteroatoms independently selected from nitrogen, oxygen or sulfur.

Suitable substituents on the substitutable nitrogen of the "optionally substituted" group include OrEach of whichIndependently hydrogen, C substituted as defined below_1-6unsubstituted-OPh, or an unsubstituted 5-to 6-membered saturated, partially unsaturated, or aryl ring having 0 to 4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or two independently occurring, although the definition is given aboveTogether with one or more of their intervening atoms form a 3-to 12-membered saturated, partially unsaturated or aryl monocyclic or bicyclic ring having 0 to 4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

Suitable substituents on the aliphatic radical of (A) are independently halogen, -R^·- (halogenated R)^·)、-OH、-OR^·-O (halo R)^·)、-CN、-C(O)OH、-C(O)OR^·、-NH₂、-NHR^·、-NR^· ₂or-NO₂Wherein each R is^·Is unsubstituted or substituted, where preceded by "halo", with only one or more halogen, and is independently C₁-₄Aliphatic, -CH₂Ph，-O(CH₂)_0-1Ph, or a 5 to 6 membered saturated, partially unsaturated or aryl ring having 0 to 4 heteroatoms independently selected from nitrogen, oxygen or sulfur.

The term "organic residue" defines a carbon-containing residue (i.e., a residue comprising at least one carbon atom) and includes, but is not limited to, a carbon-containing group, residue, or group as defined above. The organic residue may contain various heteroatoms or be bonded to another molecule through heteroatoms (including oxygen, nitrogen, sulfur, phosphorus, and the like). Examples of organic residues include, but are not limited to, alkyl or substituted alkyl, alkoxy or substituted alkoxy, mono-or di-substituted amino, amido, and the like. The organic residue may preferably contain 1 to 18 carbon atoms, 1 to 15 carbon atoms, 1 to 12 carbon atoms, 1 to 8 carbon atoms, 1 to 6 carbon atoms, or 1 to 4 carbon atoms. In another aspect, the organic residue can comprise 2 to 18 carbon atoms, 2 to 15 carbon atoms, 2 to 12 carbon atoms, 2 to 8 carbon atoms, 2 to 4 carbon atoms, or 2 to 4 carbon atoms

Synonyms for the term "residue" are the term "group (radial)" as used in the specification and the final claims, which refers to a fragment, group or substructure of a molecule described herein, regardless of how the molecule is prepared. For example, the 2, 4-thiazolidinedione group in a particular compound has the following structure:

regardless of whether the thiazolidinedione is used to prepare the compound. In some embodiments, a group (e.g., an alkyl group) may be further modified by bonding thereto one or more "substituent groups" (i.e., a substituted alkyl group). The number of atoms in a given group is not critical to the invention unless indicated to the contrary elsewhere herein.

The term "organic group" as defined and used herein contains one or more carbon atoms. The organic group can have, for example, 1 to 26 carbon atoms, 1 to 18 carbon atoms, 1 to 12 carbon atoms, 1 to 8 carbon atoms, 1 to 6 carbon atoms, or 1 to 4 carbon atoms. In another aspect, the organic group can have 2 to 26 carbon atoms, 2 to 18 carbon atoms, 2 to 12 carbon atoms, 2 to 8 carbon atoms, 2 to 6 carbon atoms, or 2 to 4 carbon atoms. The organic group often has hydrogen bonded to at least some of the carbon atoms of the organic group. An example of an organic group that does not contain inorganic atoms is 5, 6, 7, 8-tetrahydro-2-naphthyl. In some embodiments, the organic group may contain 1 to 10 inorganic heteroatoms bonded thereto or therein, including halogens, oxygen, sulfur, nitrogen, phosphorus, and the like. Examples of organic groups include, but are not limited to, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, mono-substituted amino, di-substituted amino, acyloxy, cyano, carboxy, alkoxycarbonyl, alkylcarboxamide, substituted alkylcarboxamide, dialkylcarboxamide, substituted dialkylcarboxamide, alkylsulfonyl, alkylsulfinyl, thioalkyl, thiohaloalkyl, alkoxy, substituted alkoxy, haloalkyl, haloalkoxy, aryl, substituted aryl, heteroaryl, heterocycle, or substituted heterocycle groups, wherein the terms are defined elsewhere herein. Some non-limiting examples of organic groups that include heteroatoms include alkoxy groups, trifluoromethoxy groups, acetoxy groups, dimethylamino groups, and the like.

The term "inorganic group" as defined and used herein does not contain carbon atoms and therefore only contains atoms other than carbon. Inorganic groups include bonded combinations of atoms selected from the group consisting of hydrogen, nitrogen, oxygen, silicon, phosphorus, sulfur, selenium, and halogens (such as fluorine, chlorine, bromine, and iodine), which atoms may be present alone or bonded together in chemically stable combinations thereof. The inorganic group has 10 or fewer, or preferably one to six or one to four, inorganic atoms as listed above bonded together. Examples of inorganic groups include, but are not limited to, amino, hydroxyl, halogen, nitro, mercapto, sulfate, phosphate, and the like, which are generally known inorganic groups. The inorganic group does not have a metal element of the periodic table of elements (such as an alkali metal, an alkaline earth metal, a transition metal, a lanthanide metal or an actinide metal) bonded therein, although for anionic inorganic groups (anionic inorganic groups such as sulfate, phosphate, etc.), these metal ions can sometimes act as pharmaceutically acceptable cations. The inorganic group does not contain a metalloid element such as boron, aluminum, gallium, germanium, arsenic, tin, lead, or tellurium or an inert gas element unless explicitly stated otherwise elsewhere herein.

The compounds described herein may contain one or more double bonds, and thus may give rise to cis/trans (E/Z) isomers as well as other conformational isomers. Unless stated to the contrary, the present invention includes all such possible isomers as well as mixtures of such isomers.

Unless stated to the contrary, a formula having a chemical bond shown only as a solid line rather than a wedge or dashed line encompasses each possible isomer, e.g., each enantiomer and diastereomer, as well as mixtures of isomers, such as racemic or non-racemic mixtures. The compounds described herein may contain one or more asymmetric centers and, thus, may give rise to diastereomers and optical isomers. Unless stated to the contrary, the present invention includes all such possible diastereomers and racemic mixtures thereof, substantially pure resolved enantiomers thereof, all possible geometric isomers thereof, and pharmaceutically acceptable salts thereof. Mixtures of stereoisomers as well as isolated specific stereoisomers are also included. During the course of the synthetic procedures used to prepare such compounds, or in the use of racemization or epimerization procedures known to those skilled in the art, the products of such procedures can be mixtures of stereoisomers.

Many organic compounds exist in optically active forms that are capable of rotating the plane of plane polarized light. In describing optically active compounds, the prefixes D and L or R and S are used to denote the absolute configuration of a molecule with respect to its chiral center. The prefixes d and 1 or (+) and (-) are used to denote the rotational label of the plane polarized light of the compound, where (-) or denotes that the compound is left-handed. Compounds with (+) or d prefixes are dextrorotatory. For a given chemical structure, these compounds, referred to as stereoisomers, are identical except that they are mirror images that cannot overlap each other. Specific stereoisomers may also be referred to as enantiomers, and mixtures of such isomers are often referred to as enantiomeric mixtures. A50: 50 mixture of enantiomers is referred to as a racemic mixture. Many of the compounds described herein may have one or more chiral centers and thus may exist in different enantiomeric forms. If desired, chiral carbon (C: (C))^*) May be designated by an asterisk. When the bond to the chiral carbon is depicted as a straight line in the formula, it is understood that both the (R) and (S) configurations of the chiral carbon, and thus both enantiomers and mixtures thereof, are included in the formula. As used in the art, when it is desired to specify an absolute configuration with respect to a chiral carbon, one of the bonds to the chiral carbon may be described as a wedge (a bond to an atom above a plane), while the others may be described as a series or wedge of short parallel lines (a bond to an atom below a plane). The Cahn-Inglod-Prelog system can be used to map (R) or (S)The configuration is designated as a chiral carbon.

When the disclosed compounds contain one chiral center, the compounds exist in two enantiomeric forms. Unless expressly stated to the contrary, the disclosed compounds include both enantiomers and mixtures of enantiomers, such as the specific 50: 50 mixture referred to as a racemic mixture. Enantiomers can be resolved by methods known to those skilled in the art, such as the Formation of Diastereomeric salts that can be separated, for example, by crystallization (see, CRC Handbook of Optical resolution via diastereometric Salt Formation by David Kozma (CRC Press, 2001)); forming a diastereoisomeric derivative or complex which may be separated, for example, by crystallization, gas-liquid or liquid chromatography; selective reaction of one enantiomer with an enantiomer-specific reagent, e.g. enzymatic esterification; or for example gas-liquid or liquid chromatography on a chiral support (e.g. silica with bound chiral ligand) or in the presence of a chiral solvent in a chiral environment. It will be appreciated that where the desired enantiomer is converted to another chemical entity by one of the separation methods described above, further steps may liberate the desired enantiomeric form. Alternatively, specific enantiomers can be synthesized by asymmetric synthesis using optically active reagents, substrates, catalysts or solvents, or by converting one enantiomer into another by asymmetric transformation.

Designation of a specific absolute configuration at a chiral carbon in a disclosed compound is understood to mean that the designated enantiomeric form of the compound can be provided in enantiomeric excess (e.e.). As used herein, enantiomeric excess is the presence of a particular enantiomer by greater than 50%, e.g., greater than 60%, greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95%, greater than 98%, or greater than 99%. In one aspect, a designated enantiomer is substantially free of the other enantiomer. For example, the "R" form of a compound can be substantially free of the "S" form of the compound, and thus in enantiomeric excess of the "S" form. Conversely, the "S" form of a compound may be substantially free of the "R" form of the compound, and thus in enantiomeric excess of the "R" form.

When the disclosed compounds have two or more chiral carbons, they can have more than two optical isomers and can exist in diastereomeric forms. For example, when two chiral carbons are present, the compound may have up to four optical isomers and two pairs of enantiomers ((S, S)/(R, R) and (R, S)/(S, R)). The enantiomeric pairs (e.g., (S, S)/(R, R)) are mirror image stereoisomers of each other. Stereoisomers that are not mirror images (e.g., (S, S) and (R, S)) are diastereomers. Diastereoisomeric pairs may be separated by methods known to those skilled in the art (e.g. chromatography or crystallization), and the individual enantiomers in each pair may be separated as described above. Unless expressly excluded otherwise, the disclosed compounds include each diastereomer of such compounds and mixtures thereof.

The compounds described herein contain atoms in both their natural isotopic abundance and unnatural abundance. The disclosed compounds may be isotopically labeled or isotopically substituted compounds, which are identical to those recited, but for the following facts: one or more atoms of which are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Examples of isotopes that can be incorporated into compounds of the invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine and chlorine, such as²H、³H、¹³C、¹⁴C、¹⁵N、¹⁸O、¹⁷O、³⁵S、¹⁸F and³⁶and (4) Cl. Compounds also include prodrugs thereof, and pharmaceutically acceptable salts of the compounds or of the prodrugs containing the aforementioned isotopes and/or other isotopes of other atoms are within the scope of this invention. Certain isotopically-labeled compounds of the present invention (e.g., incorporation of a radioactive isotope such as³H and¹⁴those of C) can be used in drug and/or stromal tissue distribution assays. The tritiation (i.e.,³H) and carbon-14 (i.e.,¹⁴C) isotopes are particularly preferred because of their ease of useIn preparation and detection. In addition, the heavy chain is formed via heavier isotopes such as deuterium (i.e.,²H) substitution may provide certain therapeutic advantages resulting from greater metabolic stability (e.g., prolonged in vivo half-life or reduced dosage requirements), and thus may be preferred in some circumstances. Isotopically labeled compounds of the present invention and prodrugs thereof can generally be prepared by carrying out the following procedure by substituting a readily available isotopically labeled reagent for a non-isotopically labeled reagent.

The compounds described in the present invention may exist in the form of solvates. "solvate" refers to a compound that is formed by the interaction of a solvent and a solute, and includes hydrates. Solvates are generally crystalline solid adducts containing stoichiometric or non-stoichiometric proportions of solvent molecules in the crystal structure. In some cases, the solvent used to prepare the solvate is an aqueous solution, and the solvate is then often referred to as a hydrate. The compounds may exist as hydrates, which may be obtained, for example, by crystallization from a solvent or from an aqueous solution. In this regard, one, two, three or any number of solvate or water molecules may be combined with the compounds according to the present invention to form solvates and hydrates. Unless stated to the contrary, the present invention includes all such possible solvates.

The term "co-crystal" means a physical association of two or more molecules that confers their stability through non-covalent interactions. One or more components of such molecular complexes provide a stable framework in the crystal lattice. In some cases, guest molecules are incorporated into the Crystal lattice as anhydrates or solvates, see, e.g., "Crystal Engineering of the Composition of Pharmaceutical drugs. Do Pharmaceutical Co-crystals retrieval a New Path to Improved therapeutics? "Almarasson, O.et al, The Royal Society of Chemistry, 1889-. Examples of co-crystals include p-toluenesulfonic acid and benzenesulfonic acid.

Chemical species are known to form solids that exist in different ordered states, which are referred to as polymorphs or modifications. The differently modified polymorphic substances may vary greatly in their physical properties. The compounds according to the invention may exist in different polymorphs and may thus be metastable for specific modifications. Unless stated to the contrary, the present invention includes all such possible polymorphs.

In some aspects, the structure of a compound may be represented by the formula:

it is understood to be equivalent to the following formula:

where n is typically an integer. Namely, RⁿIs understood to mean five independent substituents, R^n(a)、R^n(b)、R^n(c)、R^{n (d)}、R^n(e). In each such case, five RⁿEach of which may be hydrogen or the listed substituents. By "independent substituent" is meant that each R substituent may be independently defined. For example, if in one instance R^n(a)Is halogen, then R^n(b)In this case not necessarily halogen.

In some other aspects, the structure of the compound can be represented by the formula:

wherein R is^yRepresents for example selected from A¹、A²And A³Is understood to be equivalent to a group of formula:

again, by "independent substituent" it is meant that each R substituent may be independently defined. For example, if in one instance R^y1Is A¹Then R is^y2In this case not necessarily A¹。

In some other aspects, the structure of the compound can be represented by the formula:

wherein, for example, Q comprises three substituents independently selected from hydrogen and a, which is understood to be equivalent to the formula:

again, by "independent substituent" is meant that each Q substituent is independently defined as hydrogen or a, which is understood to be equivalent to a group of the formula:

certain materials, compounds, compositions, and components disclosed herein are commercially available or can be readily synthesized using techniques well known to those skilled in the art. For example, starting materials and Reagents for preparing the disclosed compounds and compositions can be obtained from commercial suppliers such as Aldrich Chemical co., (Milwaukee, Wis.), Acros Organics (Morris Plains, n.j.), Fisher Scientific (Pittsburgh, Pa.), or Sigma (st.louis, Mo.) or prepared by methods known to those of skill in the art according to methods set forth in documents such as Fieser and Fieser's Reagents for Organic Synthesis, volumes 1-17 (John Wiley and Sons, 1991); rodd's Chemistry of Carbon Compounds, Vol.1-5 and appendix (Elsevier Science Publishers, 1989); organic Reactions, Vol.1-40 (John Wiley and Sons, 1991); march's Advanced Organic Chemistry, (John Wiley and Sons, 4 th edition); and Larock's Comprehensive Organic Transformations (VCH Publishers Inc., 1989).

Unless explicitly stated otherwise, it is in no way intended that any method set forth herein be construed as requiring that its steps be performed in a specific order. Thus, in the claims or specification, where a method claim does not actually recite an order to be followed by its steps or where a method claim does not specifically recite an order to be limited to a specific order, it is not intended that an order be inferred, in any respect. This applies to any non-express basis for interpretation, including logical matters with respect to step arrangements or operational flows, obvious meanings derived from grammatical organization or punctuation, or numbers or types of embodiments described in the specification.

The present invention discloses the components to be used in preparing the compositions of the present invention as well as the compositions themselves to be used in the methods disclosed herein. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed that while specific reference of each various individual and collective combinations and permutation of these compounds may not be explicitly disclosed, each is specifically contemplated and described herein. For example, if a particular compound is disclosed and discussed, the discussion is intended to be illustrative of the many modifications that can be made to the molecules comprising the compound, and specifically contemplates each and every combination and permutation of the compounds and possible modifications unless specifically indicated to the contrary. Thus, if a class of molecules A, B and C is disclosed as well as a class of molecules D, E and F and examples of combination molecules A-D are disclosed, then even if each is not individually recited as being combined in individually and collectively contemplated meanings, publications A-E, A-F, B-D, B-E, B-F, C-D, C-E and C-F are considered. Also, any subset or combination of these is disclosed. Thus, for example, subgroups A-E, B-F and C-E will also be considered disclosed. This concept applies to all aspects of this application including, but not limited to, steps in methods of making and using the compositions of the present invention. Thus, if a variety of additional steps can be performed, it is understood that each of these additional steps can be performed with any specific embodiment or combination of embodiments of the methods of the present invention.

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 the disclosed methods and compositions belong. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the methods and compositions of the present invention, the methods, devices, and materials particularly useful are as described. The publications cited herein and the materials cited therein are hereby specifically incorporated by reference. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention. No admission is made that any reference constitutes prior art. The discussion of the references states what the authors assert, and the applicants reserve the right to challenge the accuracy and pertinency of the cited documents. It should be clearly understood that, although a number of publications are mentioned herein, such references do not constitute a commitment to: any of these documents form part of the common general knowledge in the art.

B. Peptides

In one aspect, the invention discloses peptides comprising an insulin a chain peptide and an insulin B chain peptide, wherein the insulin B chain peptide comprises at least 32 amino acid residues, and wherein at least three amino acid residues of the insulin B chain peptide are lysine residues.

In one aspect, the invention also discloses peptides comprising an insulin a chain peptide and an insulin B chain peptide, wherein the peptides are directly conjugated to at least one organoboronate group.

Wild-type insulin contains an a chain peptide and a B chain peptide. The wild type human insulin A chain is represented by sequence GIVEQCCTSICSLYQLENYCN (SEQ ID NO: 1). The wild type human insulin B chain is represented by sequence FVNQHLCGSHLVEALYLVCGERGFFYTPKT (SEQ ID NO: 2).

In various aspects, the insulin a chain peptide and the insulin B chain peptide are bonded by at least one disulfide bond. In another aspect, the insulin a chain peptide and the insulin B chain peptide are bonded by at least two disulfide bonds.

In various aspects, the disclosed peptides are monomeric. In other words, in various aspects, the disclosed peptides are less likely to form dimers, tetramers, hexamers, etc. than wild-type insulin.

In various aspects, the insulin a chain peptide is at least 70% identical to a wild-type human insulin a chain peptide. In some cases, the insulin a chain peptide is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to the wild-type human insulin a chain peptide. In some cases, percent identity can be achieved by deleting one or more amino acids from the N-terminus or C-terminus of the disclosed peptides.

In various aspects, the insulin A chain peptide comprises sequence GIVEQCCTSICSLYQLENYCN (SEQ ID NO: 1). In another aspect, the insulin A chain peptide comprises sequence GIVEQCCTSICSLYQLENYCG (SEQ ID NO: 3).

In various aspects, the insulin B chain peptide is at least 70% identical to a wild-type human insulin B chain peptide. In another aspect, the insulin B chain peptide is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% identical to a wild-type human insulin B chain peptide. In another aspect, percent identity can be achieved by deleting one or more amino acids from the N-terminus or C-terminus of the disclosed peptides. In another aspect, percent identity can be achieved by adding one or more amino acids from the N-terminus or C-terminus of the disclosed peptides.

In various aspects, the insulin B-chain peptide comprises at least 33 amino acid residues. In another aspect, the insulin B-chain peptide comprises at least 34 amino acid residues.

In various aspects, the amino acid at position B29 is a lysine residue. In another aspect, the B29 lysine residue is modified. In another aspect, the B29 lysine residue is unmodified.

In various aspects, the amino acid at position B33 is a lysine residue. In another aspect, the B33 lysine residue is modified. In another aspect, the B33 lysine residue is unmodified.

In various aspects, the amino acid at position B34 is a lysine residue. In another aspect, the B34 lysine residue is modified. In another aspect, the B34 lysine residue is unmodified.

In various aspects, the amino acid at position B29 and the amino acid at position B33 are lysine residues. In another aspect, the amino acid at position B29 and the amino acid at position B34 are lysine residues. In another aspect, the amino acid at position B33 and the amino acid at position B34 are lysine residues. In another aspect, the amino acid at position B29, the amino acid at position B33, and the amino acid at position B34 are lysine residues.

In various aspects, the B29 lysine residue is unmodified, and each of the B33 and B34 lysine residues are modified. In another aspect, the B33 lysine residue is unmodified, and each of the B29 and B34 lysine residues are modified. In another aspect, the B34 lysine residue is unmodified, and each of the B29 and B33 lysine residues are modified. In another aspect, each of the B29, B33, and B34 lysine residues is modified. In another aspect, each of the B29, B33, and B34 lysine residues is unmodified.

In various aspects, the insulin B chain peptide comprises sequence FVNQHLCGSHLVEALYLVCGERGFFYTPKT (SEQ ID NO: 2). In another aspect, the insulin B chain peptide comprises the sequence FVNQHLCGSHLVEALYLVCGERGFFYTPKTR (SEQ ID NO: 4), FVNQHLCGSHLVEALYLVCGERGFFYTPKTRR (SEQ ID NO: 5), or FVNQHLCGSHLVEALYLVCGERGFFYTPKTRRR (SEQ ID NO: 6). In another aspect, the insulin B chain peptide comprises sequence FVNQHLCGSHLVEALYLVCGERGFFYTPKTR (SEQ ID NO: 4) or FVNQHLCGSHLVEALYLVCGERGFFYTPKTRRR (SEQ ID NO: 6). In another aspect, the insulin B chain peptide comprises sequence FVNQHLCGSHLVEALYLVCGERGFFYTPKTRR (SEQ ID NO: 5) or FVNQHLCGSHLVEALYLVCGERGFFYTPKTRRR (SEQ ID NO: 6). In another aspect, the insulin B chain peptide comprises sequence FVNQHLCGSHLVEALYLVCGERGFFYTPKTR (SEQ ID NO: 4). In another aspect, the insulin B chain peptide comprises sequence FVNQHLCGSHLVEALYLVCGERGFFYTPKTRR (SEQ ID NO: 5). In another aspect, the insulin B chain peptide comprises sequence FVNQHLCGSHLVEALYLVCGERGFFYTPKTRRR (SEQ ID NO: 6).

In various aspects, the insulin B chain peptide comprises sequence FVNQHLCGSHLVEALYLVCGERGFFYTPKTRK (SEQ ID NO: 7). In another aspect, the insulin B chain peptide comprises sequence FVNQHLCGSHLVEALYLVCGERGFFYTPKTRKK (SEQ ID NO: 8).

In various aspects, at least one of the lysine residues is located C-terminal to the insulin B chain peptide. In another aspect, at least two of the lysine residues are located C-terminal to the insulin B chain peptide. In another aspect, at least three of the lysine residues are located C-terminal to the insulin B chain peptide.

In various aspects, the insulin A chain peptide and the insulin B chain peptide are bonded by at least one disulfide bond, the insulin A chain peptide comprises sequence GIVEQCCHRICSLYQLENYCN (SEQ ID NO: 1), and the insulin B chain peptide comprises sequence FVNQHLCGSHLVEALYLVCGERGFFYTPKTRKK (SEQ ID NO: 8). In another aspect, one or both of B33 lysine residues and B34 lysine residues are modified. In another aspect, one of the B33 lysine residue and B34 lysine residue is modified. In another aspect, the B33 lysine residue is modified. In another aspect, the B34 lysine residue is modified. In another aspect, both of the B33 lysine residue and the B34 lysine residue are modified.

In various aspects, the insulin A chain peptide comprises the sequence GIVEQCCTSICSLYQLENYCN (SEQ ID NO: 1) or GIVEQCCTSICSLYQLENYCG (SEQ ID NO: 3) and the insulin B chain peptide comprises the sequence FVNQHLCGSHLVEALYLVCGERGFFYTPKT (SEQ ID NO: 2), FVNQHLCGSHLVEALYLVCGERGFFYTPKTRR (SEQ ID NO: 5) or FVNQHLCGSHLVEALYLVCGERGFFYTPKTRKK (SEQ ID NO: 8). In another aspect, the insulin A chain peptide comprises sequence GIVEQCCTSICSLYQLENYCN (SEQ ID NO: 1) or GIVEQCCTSICSLYQLENYCG (SEQ ID NO: 3) and the insulin B chain peptide comprises sequence FVNQHLCGSHLVEALYLVCGERGFFYTPKTRR (SEQ ID NO: 5) or FVNQHLCGSHLVEALYLVCGERGFFYTPKTRKK (SEQ ID NO: 8). In another aspect, the insulin A chain peptide comprises sequence GIVEQCCTSICSLYQLENYCN (SEQ ID NO: 1) or GIVEQCCTSICSLYQLENYCG (SEQ ID NO: 3) and the insulin B chain peptide comprises sequence VNQHLCGSHLVEALYLVCGERGFFYTPKTRKK (SEQ ID NO: 8).

In various aspects, the insulin A chain peptide comprises sequence GIVEQCCTSICSLYQLENYCN (SEQ ID NO: 1) and the insulin B chain peptide comprises sequence FVNQHLCGSHLVEALYLVCGERGFFYTPKT (SEQ ID NO: 2), FVNQHLCGSHLVEALYLVCGERGFFYTPKTRR (SEQ ID NO: 5) or FVNQHLCGSHLVEALYLVCGERGFFYTPKTRKK (SEQ ID NO: 8). In another aspect, the insulin A chain peptide comprises sequence GIVEQCCTSICSLYQLENYCG (SEQ ID NO: 3) and the insulin B chain peptide comprises sequence FVNQHLCGSHLVEALYLVCGERGFFYTPKT (SEQ ID NO: 2), FVNQHLCGSHLVEALYLVCGERGFFYTPKTRR (SEQ ID NO: 5) or FVNQHLCGSHLVEALYLVCGERGFFYTPKTRKK (SEQ ID NO: 8).

In various aspects, the insulin a chain peptide comprises sequence GIVEQCCTSICSLYQLENYCG (SEQ ID NO: 3), the insulin B chain peptide comprises sequence FVNQHLCGSHLVEALYLVCGERGFFYTPKTRKK (SEQ ID NO: 8), the B29 lysine residue is unmodified, each of the B33 lysine residue and the B34 lysine residue is directly conjugated to an organoboronate group, and each occurrence of the organoboronate group has a structure represented by the following formula:

in various aspects, the peptide is directly conjugated to two organoboronate groups.

In various aspects, the peptide is directly conjugated to at least one organoboronate group through a lysine residue. In another aspect, the peptide is directly conjugated to one organoboronate group through a lysine residue. In another aspect, the peptide is directly conjugated to two organoboronate groups through two lysine residues.

In various aspects, the disclosed peptides can comprise one or more unnatural amino acids, modified amino acids, or synthetic amino acid analogs. Such amino acids include, but are not limited to, the D-isomers of common amino acids, 2, 4-diaminobutyric acid, alpha-aminoisobutyric acid, 4-aminobutyric acid, 2-aminobutyric acid, 6-aminocaproic acid, 2-aminoisobutyric acid, 3-aminopropionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline, cysteine, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, cyclopentylalanine, beta-alanine, fluoroamino acids, design amino acids such as beta-methyl amino acids, C alpha-methyl amino acids, N alpha-methyl amino acids, and amino acid analogs in general. Also included within the scope are peptides that are differentially modified during or after synthesis by, for example, biotinylation, benzylation, glycosylation, acetylation, phosphorylation, amidation, derivatization of known protecting/blocking groups, proteolytic cleavage, linkage to antibody molecules or other cellular ligands, and the like. Without wishing to be bound by theory, these modifications may be used to improve the stability and/or biological activity of the peptide.

In various aspects, the invention discloses therapeutic proteins having an a chain peptide bonded to a B chain peptide by at least one disulfide bond, wherein the insulin B chain peptide comprises at least 32 amino acid residues, and wherein at least three amino acid residues of the insulin B chain peptide are lysine residues. Without wishing to be bound by theory, it is understood that the disclosed therapeutic proteins may be used in pharmaceutical compositions and in conjunction with the treatment of conditions such as, for example, diabetes.

1. Organic borate group

In one aspect, the disclosed peptides are directly conjugated to at least one organoboronate group. In another aspect, the disclosed peptides are directly conjugated to one organoboronate group. In another aspect, the disclosed peptides are directly conjugated to a plurality of organoboronate groups. In another aspect, the disclosed peptides are directly conjugated to two organoboronate groups.

In various aspects, the organoboronate group has a structure represented by the formula:

wherein Z is selected from C (O) and SO₂(ii) a Wherein Ar is¹Selected from 5-membered aryl, 5-membered heteroaryl, 6-membered aryl and 6-membered heteroaryl, and substituted with 0, 1, 2 or 3 groups independently selected from: halogen, -CN, -NO₂OH, -OH, C1-C4 alkyl, C1-C4 haloalkyl, C1-C4 hydroxyalkyl, C1-C4 alkoxy, C1-C4 alkylaminoA group and (C1-C4) (C1-C4) a dialkylamino group.

In various aspects, the organoboronate group has a structure represented by the formula:

wherein R is^1a、R^1b、R^1c、R^1dAnd R^1eEach of which is independently selected from hydrogen, halogen, -CN, -NO₂OH, -OH, C1-C4 alkyl, C1-C4 haloalkyl, C1-C4 hydroxyalkyl, C1-C4 alkoxy, C1-C4 alkylamino, (C1-C4) (C1-C4) dialkylamino and-B (OH)₂With the proviso that R is^1a、R^1b、R^1c、R^1dAnd R^1eOne and only one of them is-B (OH)₂。

In various aspects, the organoboronate group has a structure represented by the formula:

in various aspects, the organoboronate group has a structure represented by the formula:

in various aspects, the organoboronate group has a structure represented by the formula:

in various aspects, the organoboronate group has a structure represented by the formula:

in various aspects, the organoboronate group has a structure represented by the formula:

in various aspects, the organoboronate group has a structure represented by the formula:

in various aspects, the organoboronate group has a structure represented by the formula:

a.Z radical

In one aspect, Z is selected from C (O) and SO₂. In another aspect, Z is c (o). In another aspect, Z is SO₂。

b.R^1A、R^1B、R^1C、R^1DAnd R^1ERadical (I)

In one aspect, R^1a、R^1b、R^1c、R^1dAnd R^1eEach of which is independently selected from hydrogen, halogen, -CN, -NO₂OH, -OH, C1-C4 alkyl, C1-C4 haloalkyl, C1-C4 hydroxyalkyl, C1-C4 alkoxy, C1-C4 alkylamino, (C1-C4) (C1-C4) dialkylamino and-B (OH)₂With the proviso that R is^1a、R^1b、R^1c、R^1dAnd R^1eOne and only one of them is-B (OH)₂. In another aspect, R^1a、R^1b、R^1c、R^1dAnd R^1eAre independently selected from hydrogen and-B (OH)₂。

In another aspect, R^1a、R^1b、R^1c、R^1dAnd R^1eEach of which is independently selected from hydrogen, -F, -Cl, -CN, -NO₂、-OH、C1-C4 alkyl, C1-C4 haloalkyl, C1-C4 hydroxyalkyl, C1-C4 alkoxy, C1-C4 alkylamino, (C1-C4) (C1-C4) dialkylamino and-B (OH)₂. In another aspect, R^1a、R^1b、R^1c、R^1dAnd R^1eEach of which is independently selected from hydrogen, -F, -Cl, -CN, -NO₂-OH, methyl, ethyl, n-propyl, isopropyl, -CF₃、-CHF₂、-CH₂F、-CH₂CH₂F、-CH(CH₃)CH₂F、-CH₂CH₂CH₂F、-CCl₃、-CHCl₂、-CH₂Cl、-CH₂CH₂Cl、-CH(CH₃)CH₂Cl、-CH₂CH₂CH₂Cl、-CH₂OH、-CH₂CH₂OH、-CH(CH₃)CH₂OH、-CH₂CH₂CH₂OH、-OCH₃、-OCH₂CH₃、-OCH(CH₃)₂、-OCH₂CH₂CH₃、-NHCH₃、-NHCH₂CH₃、-NHCH(CH₃)₂、-NHCH₂CH₂CH₃、-N(CH₃)₂、-N(CH₃)CH₂CH₃、-N(CH₂CH₃)CH₂CH₃、-N(CH₃)CH(CH₃)₂、-N(CH₃)CH₂CH₂CH₃and-B (OH)₂. In another aspect, R^1a、R^1b、R^1c、R^1dAnd R^1eEach of which is independently selected from hydrogen, -F, -Cl, -CN, -NO₂-OH, methyl, ethyl, -CF₃、-CHF₂、-CH₂F、-CH₂CH₂F、-CCl₃、-CHCl₂、-CH₂Cl、-CH₂CH₂Cl、-CH₂OH、-CH₂CH₂OH、-OCH₃、-OCH₂CH₃、-NHCH₃、-NHCH₂CH₃、-N(CH₃)₂、-N(CH₃)CH₂CH₃、-N(CH₂CH₃)CH₂CH₃and-B (OH)₂. In another aspect, R^1a、R^1b、R^1c、R^1dAnd R^1eEach of which is independently selected from hydrogen, -F, -Cl, -CN, -NO₂-OH, methyl, -CF₃、-CHF₂、-CH₂F、-CCl₃、-CHCl₂、-CH₂Cl、-CH₂OH、-OCH₃、-OCH₂CH₃、-NHCH₃、-N(CH₃)₂and-B (OH)₂。

In another aspect, R^1a、R^1b、R^1c、R^1dAnd R^1eAre each independently selected from hydrogen, halogen and-B (OH)₂. In another aspect, R^1a、R^1b、R^1c、R^1dAnd R^1eAre independently selected from hydrogen, -F, -CI, -Br and-B (OH)₂. In another aspect, R^1a、R^1b、R^1c、R^1dAnd R^1eAre independently selected from hydrogen, -F, -CI and-B (OH)₂. In another aspect, R^1a、R^1b、R^1c、R^1dAnd R^1eAre independently selected from hydrogen, -F and-B (OH)₂。

In another aspect, R^1ais-B (OH)₂. In another aspect, R^1bis-B (OH)₂. In another aspect, R^1cis-B (OH)₂. In another aspect, R^1dis-B (OH)₂. In another aspect, R^1eis-B (OH)₂。

In another aspect, R^1a、R^1b、R^1c、R^1dAnd R^1eIs halogen. In another aspect, R^1a、R^1b、R^1c、R^1dAnd R^1eOne of which is-F.

In another aspect, R^1aIs halogen. In another aspect, R^1ais-F.

c.AR¹Radical (I)

In one aspect, Ar¹Selected from 5-membered aromatic hydrocarbonsA group, a 5-membered heteroaryl group, a 6-membered aryl group, and a 6-membered heteroaryl group, and is substituted with 0, 1, 2, or 3 groups independently selected from: halogen, -CN, -NO₂OH, -C1-C4 alkyl, -C1-C4 haloalkyl, -C1-C4 hydroxyalkyl, -C1-C4 alkoxy, -C1-C4 alkylamino and (C1-C4) (C1-C4) dialkylamino. In another aspect, Ar¹Selected from 5-membered aryl, 5-membered heteroaryl, 6-membered aryl and 6-membered heteroaryl, and substituted with 0, 1 or 2 groups independently selected from: halogen, -CN, -NO₂OH, -C1-C4 alkyl, -C1-C4 haloalkyl, -C1-C4 hydroxyalkyl, -C1-C4 alkoxy, -C1-C4 alkylamino and (C1-C4) (C1-C4) dialkylamino. In another aspect, Ar¹Selected from the group consisting of 5-membered aryl, 5-membered heteroaryl, 6-membered aryl and 6-membered heteroaryl, and substituted with 0 or 1 group selected from the group consisting of: halogen, -CN, -NO₂OH, -C1-C4 alkyl, -C1-C4 haloalkyl, -C1-C4 hydroxyalkyl, -C1-C4 alkoxy, -C1-C4 alkylamino and (C1-C4) (C1-C4) dialkylamino. In another aspect, Ar¹Selected from the group consisting of 5-membered aryl, 5-membered heteroaryl, 6-membered aryl and 6-membered heteroaryl, and mono-substituted with a group selected from: halogen, -CN, -NO₂OH, -C1-C4 alkyl, -C1-C4 haloalkyl, -C1-C4 hydroxyalkyl, -C1-C4 alkoxy, -C1-C4 alkylamino and (C1-C4) (C1-C4) dialkylamino. In another aspect, Ar¹Selected from the group consisting of 5-membered aryl, 5-membered heteroaryl, 6-membered aryl and 6-membered heteroaryl and is unsubstituted.

In various aspects, Ar¹Selected from 5-membered aryl and 5-membered heteroaryl, and substituted with 0, 1, 2 or 3 groups independently selected from: halogen, -CN, -NO₂OH, -C1-C4 alkyl, -C1-C4 haloalkyl, -C1-C4 hydroxyalkyl, -C1-C4 alkoxy, -C1-C4 alkylamino and (C1-C4) (C1-C4) dialkylamino. In another aspect, Ar¹Selected from 5-membered aryl and 5-membered heteroaryl, and substituted with 0, 1 or 2 groups independently selected from: halogen, -CN, -NO₂OH, -C1-C4 alkyl, -C1-C4 haloalkyl, -C1-C4 hydroxyalkyl, -C1-C4 alkoxy, -C1-C4 alkylamino and (C1-C4) (C1-C4) dialkylamino. In another aspect, Ar¹Is selected from 5-membered aryl and 5-membered heteroaryl, and is selected fromSubstitution of 0 or 1 of the following groups: halogen, -CN, -NO₂OH, -C1-C4 alkyl, -C1-C4 haloalkyl, -C1-C4 hydroxyalkyl, -C1-C4 alkoxy, -C1-C4 alkylamino and (C1-C4) (C1-C4) dialkylamino. In another aspect, Ar¹Selected from 5-membered aryl and 5-membered heteroaryl, and mono-substituted with a group selected from: halogen, -CN, -NO₂OH, -C1-C4 alkyl, -C1-C4 haloalkyl, -C1-C4 hydroxyalkyl, -C1-C4 alkoxy, -C1-C4 alkylamino and (C1-C4) (C1-C4) dialkylamino. In another aspect, Ar¹Selected from 5-membered aryl and 5-membered heteroaryl and is unsubstituted.

In various aspects, Ar¹Is a 5-membered aryl group substituted with 0, 1, 2 or 3 groups independently selected from: halogen, -CN, -NO₂OH, -C1-C4 alkyl, -C1-C4 haloalkyl, -C1-C4 hydroxyalkyl, -C1-C4 alkoxy, -C1-C4 alkylamino and (C1-C4) (C1-C4) dialkylamino. In another aspect, Ar¹Is a 5-membered aryl group substituted with 0, 1 or 2 groups independently selected from: halogen, -CN, -NO₂OH, -C1-C4 alkyl, -C1-C4 haloalkyl, -C1-C4 hydroxyalkyl, -C1-C4 alkoxy, -C1-C4 alkylamino and (C1-C4) (C1-C4) dialkylamino. In another aspect, Ar¹Is a 5-membered aryl group substituted by 0 or 1 group selected from: halogen, -CN, -NO₂OH, -C1-C4 alkyl, -C1-C4 haloalkyl, -C1-C4 hydroxyalkyl, -C1-C4 alkoxy, -C1-C4 alkylamino and (C1-C4) (C1-C4) dialkylamino. In another aspect, Ar¹Is a 5-membered aryl group monosubstituted with a group selected from: halogen, -CN, -NO₂OH, -C1-C4 alkyl, -C1-C4 haloalkyl, -C1-C4 hydroxyalkyl, -C1-C4 alkoxy, -C1-C4 alkylamino and (C1-C4) (C1-C4) dialkylamino. In another aspect, Ar¹Is an unsubstituted 5-membered aryl group.

In various aspects, Ar¹Is a 5-membered heteroaryl group substituted with 0, 1, 2 or 3 groups independently selected from: halogen, -CN, -NO₂OH, -C1-C4 alkyl, -C1-C4 haloalkyl, -C1-C4 hydroxyalkyl, -C1-C4 alkoxy, -C1-C4 alkylamino and (C1-C4) (C1-C4) dialkylamino. Examples of 5-membered heteroaryl groups include, but are not limited toFuryl, pyrrolyl, thienyl, imidazolyl, pyrrolyl, oxazolyl, isoxazolyl and thiazolyl. In another aspect, Ar¹Is a 5-membered heteroaryl group substituted with 0, 1 or 2 groups independently selected from: halogen, -CN, -NO₂OH, -C1-C4 alkyl, -C1-C4 haloalkyl, -C1-C4 hydroxyalkyl, -C1-C4 alkoxy, -C1-C4 alkylamino and (C1-C4) (C1-C4) dialkylamino. In another aspect, Ar¹Is a 5-membered heteroaryl group substituted with 0 or 1 group selected from: halogen, -CN, -NO₂OH, -C1-C4 alkyl, -C1-C4 haloalkyl, -C1-C4 hydroxyalkyl, -C1-C4 alkoxy, -C1-C4 alkylamino and (C1-C4) (C1-C4) dialkylamino. In another aspect, Ar¹Is a 5 membered heteroaryl group monosubstituted with a group selected from: halogen, -CN, -NO₂OH, -C1-C4 alkyl, -C1-C4 haloalkyl, -C1-C4 hydroxyalkyl, -C1-C4 alkoxy, -C1-C4 alkylamino and (C1-C4) (C1-C4) dialkylamino. In another aspect, Ar¹Is an unsubstituted 5 membered heteroaryl.

In various aspects, Ar¹Selected from 6-membered aryl and 6-membered heteroaryl, and substituted with 0, 1, 2 or 3 groups independently selected from: halogen, -CN, -NO₂OH, -C1-C4 alkyl, -C1-C4 haloalkyl, -C1-C4 hydroxyalkyl, -C1-C4 alkoxy, -C1-C4 alkylamino and (C1-C4) (C1-C4) dialkylamino. In another aspect, Ar¹Selected from 6-membered aryl and 6-membered heteroaryl, and substituted with 0, 1 or 2 groups independently selected from: halogen, -CN, -NO₂OH, -C1-C4 alkyl, -C1-C4 haloalkyl, -C1-C4 hydroxyalkyl, -C1-C4 alkoxy, -C1-C4 alkylamino and (C1-C4) (C1-C4) dialkylamino. In another aspect, Ar¹Selected from 6-membered aryl and 6-membered heteroaryl, and substituted with 0 or 1 group selected from: halogen, -CN, -NO₂OH, -C1-C4 alkyl, -C1-C4 haloalkyl, -C1-C4 hydroxyalkyl, -C1-C4 alkoxy, -C1-C4 alkylamino and (C1-C4) (C1-C4) dialkylamino. In another aspect, Ar¹Selected from 6-membered aryl and 6-membered heteroaryl, and mono-substituted with a group selected from: halogen, -CN, -NO₂OH, -OH, C1-C4 alkyl, C1-C4 haloalkyl, C1-C4 hydroxyalkylA C1-C4 alkoxy group, a C1-C4 alkylamino group and a (C1-C4) (C1-C4) dialkylamino group. In another aspect, Ar¹Selected from 6-membered aryl and 6-membered heteroaryl and is unsubstituted.

In various aspects, Ar¹Is a 6-membered aryl group substituted with 0, 1, 2 or 3 groups independently selected from: halogen, -CN, -NO₂OH, -C1-C4 alkyl, -C1-C4 haloalkyl, -C1-C4 hydroxyalkyl, -C1-C4 alkoxy, -C1-C4 alkylamino and (C1-C4) (C1-C4) dialkylamino. In another aspect, Ar¹Is a 6-membered aryl group substituted with 0, 1 or 2 groups independently selected from: halogen, -CN, -NO₂OH, -C1-C4 alkyl, -C1-C4 haloalkyl, -C1-C4 hydroxyalkyl, -C1-C4 alkoxy, -C1-C4 alkylamino and (C1-C4) (C1-C4) dialkylamino. In another aspect, Ar¹Is a 6-membered aryl group substituted by 0 or 1 group selected from: halogen, -CN, -NO₂OH, -C1-C4 alkyl, -C1-C4 haloalkyl, -C1-C4 hydroxyalkyl, -C1-C4 alkoxy, -C1-C4 alkylamino and (C1-C4) (C1-C4) dialkylamino. In another aspect, Ar¹Is a 6-membered aryl group monosubstituted with a group selected from: halogen, -CN, -NO₂OH, -C1-C4 alkyl, -C1-C4 haloalkyl, -C1-C4 hydroxyalkyl, -C1-C4 alkoxy, -C1-C4 alkylamino and (C1-C4) (C1-C4) dialkylamino. In another aspect, Ar¹Is an unsubstituted 6-membered aryl group.

In various aspects, Ar¹Is a6 membered heteroaryl group substituted with 0, 1, 2 or 3 groups independently selected from: halogen, -CN, -NO₂OH, -C1-C4 alkyl, -C1-C4 haloalkyl, -C1-C4 hydroxyalkyl, -C1-C4 alkoxy, -C1-C4 alkylamino and (C1-C4) (C1-C4) dialkylamino. Examples of 6-membered heteroaryl groups include, but are not limited to: pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, 1, 2, 3-triazinyl, 1, 2, 4-triazinyl, and 1, 3, 5-triazinyl. In another aspect, Ar¹Is a6 membered heteroaryl group substituted with 0, 1 or 2 groups independently selected from: halogen, -CN, -NO₂OH, -C1-C4 alkyl, -C1-C4 haloalkyl, -C1-C4 hydroxyalkyl, -C1-C4 alkoxy, -C1-C4 alkylamino and (C1-C4) (C1-C4) dialkylamino.In another aspect, Ar¹Is a6 membered heteroaryl group substituted with 0 or 1 group selected from: halogen, -CN, -NO₂OH, -C1-C4 alkyl, -C1-C4 haloalkyl, -C1-C4 hydroxyalkyl, -C1-C4 alkoxy, -C1-C4 alkylamino and (C1-C4) (C1-C4) dialkylamino. In another aspect, Ar¹Is a6 membered heteroaryl group monosubstituted with a group selected from: halogen, -CN, -NO₂OH, -C1-C4 alkyl, -C1-C4 haloalkyl, -C1-C4 hydroxyalkyl, -C1-C4 alkoxy, -C1-C4 alkylamino and (C1-C4) (C1-C4) dialkylamino. In another aspect, Ar₁Is unsubstituted 6 membered heteroaryl.

C. Pharmaceutical composition

In one aspect, the invention discloses pharmaceutical compositions comprising a therapeutically effective amount of one or more of the disclosed peptides and a pharmaceutically acceptable carrier. Thus, in various aspects, the invention discloses pharmaceutical compositions comprising a therapeutically effective amount of peptides, including insulin a chain peptides and insulin B chain peptides, wherein the insulin B chain peptides comprise at least 32 amino acid residues, and wherein at least three amino acid residues of the insulin B chain peptides are lysine residues. In various other aspects, the present invention discloses pharmaceutical compositions comprising a therapeutically effective amount of peptides, including insulin a chain peptides and insulin B chain peptides, wherein the peptides are directly conjugated to at least one organoboronate group.

In various aspects, a composition comprising an insulin derivative having glucose-dependent solubility is disclosed. On the other hand, the isoelectric point (pI) of the insulin derivative composition decreases due to the generation of negative charges upon glucose binding. Thus, during hypoglycemic states, insulin remains microcrystalline like insulin glargine; however, as blood glucose levels rise, solubility increases, which causes insulin to become monomeric, thereby increasing bioavailability.

In various aspects, the disclosed peptides can be formulated or administered in and/or with a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" as used herein refers to sterile aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions just prior to use. Examples of suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), carboxymethylcellulose and suitable mixtures thereof, vegetable oils (such as olive oil), and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of a coating material, such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants. These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersions. Prevention of the action of microorganisms can be ensured by including various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like. Prolonged absorption of the injectable pharmaceutical form can be brought about by the inclusion of agents which delay absorption, such as aluminum monostearate and gelatin. Injectable depot forms are made by forming microcapsule matrices of the drug in biodegradable polymers such as polylactide-polyglycolide, poly (orthoester) and poly (anhydride). Depending on the ratio of drug to polymer and the nature of the particular polymer used, the rate of drug release can be controlled. Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissues. The injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium just prior to use. Suitable inert carriers may include sugars such as lactose. It is desirable that at least 95% by weight of the active ingredient particles have an effective particle size in the range of 0.01 to 10 microns.

Thus, the compositions disclosed herein can comprise lipids, such as liposomes, such as cationic liposomes (e.g., DOTMA, DOPE, DC cholesterol) or anionic liposomes. Liposomes can also contain proteins that help target specific cells, if desired. Administration of a composition comprising a peptide and a cationic liposome can be administered to the blood, target organ, or inhaled into the respiratory tract to target respiratory tract cells. For example, a composition comprising a peptide or nucleic acid sequence described herein and a cationic liposome can be administered to a subject's lung cells. For liposomes, see, e.g., Brigham et al am.J.Resp.cell.mol.biol.1: 95100 (1989); felgner et al proc.natl.acad.sci USA 84: 74137417 (1987); U.S. patent No. 4,897,355. In addition, the compounds may be administered as components of microcapsules that can target specific cell types (such as macrophages), or where diffusion of the compound or delivery of the compound from the microcapsules is designed for a specific rate or dose.

In various aspects, the invention discloses pharmaceutical compositions comprising any of the disclosed peptides described herein, or a pharmaceutically acceptable salt or solvate thereof, and a pharmaceutically acceptable carrier, buffer or diluent. In various aspects, the peptide of the pharmaceutical composition is encapsulated in a delivery vehicle. In another aspect, the delivery vehicle is a liposome, a microcapsule, or a nanoparticle. In another aspect, the delivery vehicle is pegylated.

In the methods described herein, the composition can be delivered to the cell by a variety of mechanisms. As defined above, disclosed herein are compositions comprising any one or more of the peptides described herein, and may further include a carrier, such as a pharmaceutically acceptable carrier. For example, the present invention discloses pharmaceutical compositions comprising a peptide disclosed herein and a pharmaceutically acceptable carrier.

In various aspects, the invention discloses pharmaceutical compositions comprising the disclosed peptides. That is, the pharmaceutical composition can be provided as a composition comprising a therapeutically effective amount of at least one disclosed peptide or at least one product of the disclosed methods and a pharmaceutically acceptable carrier.

In various aspects, the disclosed pharmaceutical compositions comprise the disclosed peptides (including one or more pharmaceutically acceptable salts thereof) as an active ingredient, a pharmaceutically acceptable carrier, and, optionally, other therapeutic ingredients or adjuvants. The present compositions include those suitable for oral, rectal, topical and parenteral (including subcutaneous, intramuscular and intravenous) administration, although the most suitable route in any given case will depend on the particular host and the nature and severity of the condition for which the active ingredient is being administered. The pharmaceutical compositions may be conveniently presented in unit dosage form and prepared by any of the methods well known in the art of pharmacy.

In various aspects, the present invention discloses pharmaceutical compositions comprising a pharmaceutically acceptable carrier or diluent and, as an active ingredient, a therapeutically effective amount of the disclosed peptides, products of the disclosed methods of preparation, pharmaceutically acceptable salts, solvates, or polymorphs thereof, hydrates thereof, solvates thereof, polymorphs thereof, or stereochemically isomeric forms thereof. In another aspect, the disclosed peptides, products of the disclosed methods of preparation, pharmaceutically acceptable salts, solvates, or polymorphs thereof, hydrates thereof, solvates thereof, polymorphs thereof, or stereochemically isomeric forms thereof, or any subgroup or combination thereof, may be formulated into various pharmaceutical forms for administration purposes.

The term "pharmaceutically acceptable salt" as used herein refers to salts prepared from pharmaceutically acceptable non-toxic bases or acids. When the compounds of the present invention are acidic, their corresponding salts may be conveniently prepared from pharmaceutically acceptable non-toxic bases, including inorganic and organic bases. Salts derived from such inorganic bases include aluminum, ammonium, calcium, copper (cupric and cupric), iron, ferrous, lithium, magnesium, manganese (manganous and manganous), potassium, sodium, zinc and the like salts. Particularly preferred are ammonium, calcium, magnesium, potassium and sodium salts. Organic non-toxic bases derived from pharmaceutically acceptable sources include salts of primary, secondary and tertiary amines, as well as cyclic and substituted amines, such as naturally occurring and synthetic substituted amines. Other pharmaceutically acceptable organic non-toxic bases from which salts can be formed include ion exchange resins such as, for example, arginine, betaine, caffeine, choline, N' -dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine, ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, glucamine, glucosamine, histidine, hydrabamine, isopropylamine, lysine, meglumine, morpholine, piperazine, piperidine, polyamine resins, procaine, purines, theobromine, triethylamine, trimethylamine, tripropylamine, tromethamine and the like.

As used herein, the term "pharmaceutically acceptable non-toxic acid" includes inorganic acids, organic acids and salts prepared therefrom, for example, acetic acid, benzenesulfonic acid, benzoic acid, camphorsulfonic acid, citric acid, ethanesulfonic acid, fumaric acid, gluconic acid, glutamic acid, hydrobromic acid, hydrochloric acid, isethionic acid, lactic acid, maleic acid, malic acid, mandelic acid, methanesulfonic acid, mucic acid, nitric acid, pamoic acid, pantothenic acid, phosphoric acid, succinic acid, sulfuric acid, tartaric acid, p-toluenesulfonic acid and the like. Preferred are citric, hydrobromic, hydrochloric, maleic, phosphoric, sulfuric and tartaric acids.

For therapeutic use, salts of the disclosed compounds are those in which the counterion is pharmaceutically acceptable. However, salts of non-pharmaceutically acceptable acids and bases may also be used, for example, in the preparation or purification of pharmaceutically acceptable compounds. All salts, pharmaceutically acceptable or not, are included within the scope of the present invention.

The pharmaceutically acceptable acid and base addition salts as mentioned above or below are intended to comprise the therapeutically active non-toxic acid and base addition salt forms which the disclosed compounds are capable of forming. Pharmaceutically acceptable acid addition salts may conveniently be obtained by treating the base form with such a suitable acid. Suitable acids include, for example, inorganic acids such as hydrohalic acids (e.g., hydrochloric or hydrobromic acid), sulfuric acid, nitric acid, phosphoric acid, and the like; or organic acids such as, for example, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid (i.e., oxalic acid), malonic acid, succinic acid (i.e., succinic acid), maleic acid, fumaric acid, malic acid, tartaric acid, citric acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, cyclamic acid, salicylic acid, p-aminosalicylic acid, pamoic acid, and the like. Conversely, the salt form may be converted to the free base form by treatment with a suitable base.

The disclosed compounds containing acidic protons may also be converted into their non-toxic metal or amine addition salt forms by treatment with appropriate organic and inorganic bases. Suitable base salt forms include, for example, the ammonium salts, the alkali and alkaline earth metal salts (e.g., lithium, sodium, potassium, magnesium, calcium salts, and the like), salts with organic bases such as primary, secondary and tertiary aliphatic and aromatic amines (such as methylamine, ethylamine, propylamine, isopropylamine, the four butylamine isomers, dimethylamine, diethylamine, diethanolamine, dipropylamine, diisopropylamine, di-n-butylamine, pyrrolidine, piperidine, morpholine, trimethylamine, triethylamine, tripropylamine, quinuclidine, pyridine, quinoline amine, and isoquinoline); benzathine salts, N-methyl-D-glucamine salts, hydrabamine salts, and salts with amino acids such as, for example, arginine, lysine, and the like. Conversely, the salt form can be converted to the free acid form by treatment with an acid.

In practice, the peptides described herein or pharmaceutically acceptable salts thereof of the present invention can be combined as the active ingredient in an intimate mixture with a pharmaceutical carrier according to conventional pharmaceutical compounding techniques. The carrier can take a wide variety of forms depending on the form of the article of manufacture desired for administration, e.g., oral or parenteral (including intravenous). Thus, the pharmaceutical compositions of the present invention may be provided in discrete units suitable for oral administration, such as capsules, cachets or tablets, each containing a predetermined amount of the active ingredient. In addition, the compositions may be provided as a powder, as a granule, as a solution, as a suspension in an aqueous liquid, as a non-aqueous liquid, as an oil-in-water emulsion, or as a water-in-oil liquid emulsion. In addition to the conventional dosage forms listed above, the compounds of the present invention and/or one or more pharmaceutically acceptable salts thereof may also be administered by controlled release means and/or delivery devices. The compositions may be prepared by any method of pharmacy. Generally, such methods include the step of bringing into association the active ingredient with the carrier which constitutes one or more required ingredients. Generally, compositions are prepared by uniformly and intimately admixing the active ingredient with liquid carriers or finely divided solid carriers or both. The product may then be conveniently shaped into the desired presentation.

The foregoing pharmaceutical compositions are formulated in unit dosage form, which is particularly advantageous for ease of administration and maintenance of dosage uniformity. A unit dosage form, as used herein, refers to physically discrete units suitable as unitary dosages, each unit containing a predetermined quantity of active ingredient calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. Examples of such unit dosage forms are tablets (including scored or coated tablets), capsules, pills, powder packets (powder packets), wafers, suppositories, injectable solutions or suspensions, and the like, and segregated multiples thereof (segregated multiples).

Thus, the pharmaceutical compositions of the invention may include a pharmaceutically acceptable carrier and a compound or a pharmaceutically acceptable salt of a compound of the invention. By "pharmaceutically acceptable" is meant a material or carrier that will be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject, as will be well known to those skilled in the art. The compounds of the present invention or pharmaceutically acceptable salts thereof may also be included in pharmaceutical compositions in combination with one or more other therapeutically active compounds.

The pharmaceutical carrier used may be, for example, a solid, liquid or gas. Examples of solid carriers include lactose, terra alba, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate and stearic acid. Examples of liquid carriers are syrup, peanut oil, olive oil and water. Examples of gas carriers include carbon dioxide and nitrogen. Other examples of carriers include Dimyristoylphosphatidyl (DMPC), phosphate buffered saline, or multivesicular liposomes. For example, PG: PC: cholesterol: peptide or PC: peptides may be used as carriers in the present invention. Other suitable pharmaceutically acceptable carriers and formulations thereof are described in Remington: the Science and Practice of Pharmacy (19 th edition) eds A.R. Gennaro, Mack Publishing Company, Easton, PA 1995. Typically, an appropriate amount of a pharmaceutically acceptable salt is used in the formulation to render the formulation isotonic. Other examples of pharmaceutically acceptable carriers include, but are not limited to, saline, Ringer's solution, and dextrose solution. The pH of the solution may be from about 5 to about 8, or from about 7 to about 7.5. Other carriers include sustained release articles such as semipermeable matrices of solid hydrophobic polymers containing the composition, which matrices are in the form of shaped articles, e.g., films, stents (which are implanted in blood vessels during angioplasty), liposomes or microparticles. It will be apparent to those skilled in the art that certain carriers may be more preferred depending on, for example, the route of administration and the concentration of the composition being administered. These will most typically be standard carriers for administering drugs to humans, including solutions such as sterile water, saline, and buffered solutions at physiological pH.

To enhance the solubility and/or stability of the disclosed peptides in pharmaceutical compositions, it may be advantageous to use α -, β -or γ -cyclodextrins or derivatives thereof (in particular hydroxyalkyl-substituted cyclodextrins, such as 2-hydroxypropyl- β -cyclodextrin or sulfobutyl- β -cyclodextrin). Co-solvents (such as alcohols) may also improve the solubility and/or stability of the compounds according to the invention in pharmaceutical compositions.

The pharmaceutical compositions may also include carriers, thickeners, diluents, buffers, preservatives and the like, as long as the desired activity of the polypeptides, peptides, nucleic acids, vectors of the invention is not impaired. The pharmaceutical compositions may also contain one or more active ingredients (in addition to the compositions of the present invention), such as antibacterial agents, anti-inflammatory agents, anesthetic agents, and the like. The pharmaceutical composition may be administered in a variety of ways depending on whether local or systemic treatment is desired and depending on the area to be treated.

Oral administration is preferred because of its ease of administration, and tablets and capsules represent the most advantageous oral dosage unit form, in which case solid pharmaceutical carriers are obviously employed. In preparing the compositions for oral dosage form, any convenient pharmaceutical medium may be employed. For example, water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents, and the like may be used to form oral liquid preparations such as suspensions, elixirs, and solutions; and carriers such as starches, sugars, microcrystalline cellulose, diluents, granulating agents, lubricants, binders, disintegrating agents and the like may be used to form oral solid articles such as powders, capsules and tablets. Because of their ease of administration, tablets and capsules are the preferred oral dosage units, whereby solid pharmaceutical carriers are employed. Optionally, the tablets may be coated by standard aqueous or non-aqueous techniques.

Compositions for oral administration include powders or granules, suspensions or solutions in aqueous or non-aqueous media, capsules, granules or tablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders may be desirable. Some compositions can potentially be administered as pharmaceutically acceptable acid or base addition salts formed by reaction with inorganic acids (such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid) and organic acids (such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, and fumaric acid) or by reaction with inorganic bases (such as sodium hydroxide, ammonium hydroxide, potassium hydroxide) and organic bases (such as monoalkyl, dialkyl, trialkyl, and aryl amines and substituted ethanolamines).

Tablets containing the composition of the invention may be prepared by compression or moulding, optionally together with one or more accessory ingredients or adjuvants. Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as a powder or granules, optionally mixed with a binder, lubricant, inert diluent, surface active or dispersing agent. Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.

The pharmaceutical compositions of the invention comprise a peptide such as an sPRR (or a pharmaceutically acceptable salt thereof) as the active ingredient, a pharmaceutically acceptable carrier, and optionally one or more additional therapeutic agents or adjuvants. Although the present compositions include compositions suitable for oral, rectal, topical and parenteral (including subcutaneous, intramuscular and intravenous) administration, the most suitable route in any given case will depend on the particular host and the nature and severity of the condition for which the active ingredient is being administered. The pharmaceutical compositions may be conveniently presented in unit dosage form and prepared by any of the methods well known in the art of pharmacy.

Pharmaceutical compositions of the invention suitable for parenteral administration may be prepared as solutions or suspensions of the active compound in water. Suitable surfactants may be included, such as, for example, hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols and mixtures thereof in oils. In addition, preservatives may be included to prevent the unwanted growth of microorganisms.

Pharmaceutical compositions of the invention suitable for injectable use include sterile aqueous solutions or dispersions. In addition, the compositions may be in the form of sterile powders for the extemporaneous preparation of such sterile injectable solutions or dispersions. Generally, the final injectable form should be sterile and should effectively be a fluid that is easy to inject. The pharmaceutical compositions should be stable under the conditions of manufacture and storage; therefore, it should preferably be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol), vegetable oils, and suitable mixtures thereof.

Injectable solutions may be prepared, for example, where the carrier comprises saline solution, glucose solution, or a mixture of saline and glucose solution. Injectable suspensions may also be prepared in which case appropriate liquid carriers, suspending agents and the like may be employed. Also included are solid form articles which are intended to be converted to liquid form articles shortly before use.

Preparations for parenteral administration include sterile aqueous or undesired solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils (such as olive oil), and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcohol/desired solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, ringer's dextrose, dextrose and sodium chloride, lactated ringer's solution or fixed oils. Intravenous vehicles include liquid and nutritional supplements, electrolyte supplements (such as ringer's dextrose-based supplements), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, antioxidants, chelating agents, and inert gases and the like.

The pharmaceutical compositions of the present invention may be in a form suitable for topical use, such as, for example, aerosols, creams, ointments, lotions, dusting powders, mouthwashes, gargles, and the like. Further, the composition may be in a form suitable for use in a transdermal device. These formulations can be prepared by conventional processing methods using the compounds of the present invention or pharmaceutically acceptable salts thereof. For example, a cream or ointment may be prepared by mixing a hydrophilic material and water along with about 5% to about 10% by weight of the compound to produce a cream or ointment having a desired consistency.

In compositions suitable for transdermal administration, the carrier optionally includes a penetration enhancer and/or a suitable humectant, optionally in combination with minor proportions of any suitable natural additive that does not significantly adversely affect the skin. The additives may facilitate application to the skin, and/or may aid in the preparation of the desired composition. These compositions can be administered in various ways, for example as a transdermal patch, as a spot-on, as an ointment.

The pharmaceutical compositions of the invention may be in a form suitable for rectal administration, wherein the carrier is a solid. The mixture is preferably formed into unit dose suppositories. Suitable carriers include cocoa butter and other materials commonly used in the art. Suppositories can be conveniently formed by first mixing the composition with one or more carriers which soften or melt, followed by cooling and shaping in a mould.

Formulations for topical administration may include ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be desirable.

In addition to the foregoing diluents, buffers, flavoring agents, binders, surfactants, thickeners, lubricants, preservatives (including antioxidants), and the like. In addition, other adjuvants may be included to render the formulation isotonic with the blood of the intended recipient. Compositions containing the disclosed peptides, and/or pharmaceutically acceptable salts thereof, can also be prepared in powder or liquid concentrated form.

The exact dosage and frequency of administration depends on the particular disclosed peptide, the product of the disclosed methods of preparation, a pharmaceutically acceptable salt, solvate, or polymorph thereof, a hydrate thereof, a solvate thereof, a polymorph thereof, or a stereochemically isomeric form thereof; the particular condition being treated and the severity of the condition being treated; various factors specific to the history of the subject to whom the dose is administered, such as age; the weight, sex, extent of disorder and general physical condition of the particular subject, as well as other medications that the individual may take; as is well known to those skilled in the art. Furthermore, it will be apparent that the effective daily dose may be reduced or increased according to the response of the subject being treated and/or according to the evaluation of the physician prescribing the composition.

Depending on the mode of administration, the pharmaceutical composition will comprise from 0.05% to 99%, preferably from 0.1% to 70%, more preferably from 0.1% to 50% by weight of the active ingredient, and from 1% to 99.95%, preferably from 30% to 99.9%, more preferably from 50% to 99.9% by weight of a pharmaceutically acceptable carrier, all percentages being based on the total weight of the composition.

In the treatment of conditions where increased insulin receptor activity is required, the appropriate dosage level will generally be from about 0.01 to 1000mg/kg of patient body weight per day and may be administered in single or multiple doses. In various aspects, the dosage level will be about 0.1 to about 500mg/kg per day, about 0.1 to 250mg/kg per day, or about 0.5 to 100mg/kg per day. Suitable dosage levels may be about 0.01 to 1000mg/kg per day, about 0.01 to 500mg/kg per day, about 0.01 to 250mg/kg per day, about 0.05 to 100mg/kg per day, or about 0.1 to 50mg/kg per day. Dosages within this range may be 0.05 to 0.5, 0.5 to 5.0, or 5.0 to 50mg/kg per day. For oral administration, the compositions are preferably provided in the form of tablets containing from 1.0 to 1000mg of active ingredient, in particular 1.0, 5.0, 10, 15, 20, 25, 50, 75, 100, 150, 200, 250, 300, 400, 500, 600, 750, 800, 900 and 1000mg of active ingredient, for the symptomatic adjustment of the dosage to the patient to be treated. The composition may be administered on a regimen of 1 to 4 times per day, preferably once or twice per day. This dosing regimen may be adjusted to provide the optimal therapeutic response.

Such unit doses as described above and below may be administered more than once per day, for example 2, 3, 4, 5 or 6 times a day. In various aspects, such unit doses may be administered 1 or 2 times per day, such that the total dose per administration for a 70kg adult ranges from 0.001 to about 15mg/kg body weight subject. In another aspect, the dose per administration is from 0.01 to about 1.5mg/kg body weight of the subject, and such therapy may extend over weeks or months, and in some cases, years. It will be understood, however, that the specific dosage level for any particular patient will depend upon a variety of factors including the activity of the specific composition employed; the age, weight, general health, sex and diet of the individual to be treated; time and route of administration; the rate of excretion; other drugs that have been previously administered; and the severity of the particular disease being treated, as is well understood by those skilled in the art.

Typical doses may be in the form of a tablet of 1mg to about 100mg or 1mg to about 300mg taken once a day or more than once a day, or a time-release capsule or tablet taken once a day and containing a proportionally higher content of active ingredient. The time-delay effect may be obtained by a capsule material that dissolves at different pH values, by a capsule that slowly releases under osmotic pressure, or by any other known means of controlled release.

In another aspect, the dose may be 100U to 300U vials, e.g., 100U to 200U vials, 200U to 300U vials, or 150U to 250U vials. It can be administered once a day or more than once a day. In various aspects, it may be administered daily, weekly, or monthly.

As will be apparent to those skilled in the art, it may be necessary in some instances to use dosages outside these ranges. Further, it should be noted that the clinician or treating physician will know how and when to initiate, interrupt, adjust or terminate therapy in connection with individual patient responses.

The invention also relates to a method for the manufacture of a medicament for modulating insulin receptor activity (e.g., treating type 1 diabetes) in a mammal (e.g., a human), comprising combining one or more of the disclosed peptides or compositions with a pharmaceutically acceptable carrier or diluent. Thus, in one aspect, the invention relates to a method for the manufacture of a medicament, said method comprising combining at least one disclosed peptide with a pharmaceutically acceptable carrier or diluent.

The disclosed pharmaceutical compositions may also comprise other therapeutically active compounds that are typically used to treat insulin-related conditions.

It is understood that the disclosed compositions can be prepared from the disclosed peptides. It is also understood that the disclosed compositions can be used in the disclosed methods of use.

As already mentioned, the present invention relates to a pharmaceutical composition comprising a therapeutically effective amount of the disclosed peptide, a pharmaceutically acceptable salt, hydrate, solvate or polymorph thereof, a hydrate thereof, a solvate thereof, a polymorph thereof and a pharmaceutically acceptable carrier. In addition, the present invention relates to a method for preparing a pharmaceutical composition characterized by intimately mixing a pharmaceutically acceptable carrier with a therapeutically effective amount of the disclosed peptide.

As already mentioned, the present invention also relates to a pharmaceutical composition comprising the disclosed peptide, a pharmaceutically acceptable salt, solvate or polymorph thereof and one or more other drugs in the treatment, prevention, control, amelioration or reduction of risk of a disease or condition for which the disclosed peptide or other drug may have utility, and to the use of such a composition in the manufacture of a medicament. The invention also relates to a combination of the disclosed peptide, a pharmaceutically acceptable salt, solvate, or polymorph thereof, and an anti-cancer therapeutic. In various other aspects, the invention also relates to a combination of the disclosed peptides, pharmaceutically acceptable salts, solvates, or polymorphs thereof. The invention also relates to such a combination for use as a medicament. The different drugs of such compositions or products may be combined in a single preparation with a pharmaceutically acceptable carrier or diluent, or they may each be present in separate preparations with a pharmaceutically acceptable carrier or diluent.

In various aspects, the disclosed peptides can be administered in an amount of 10 μ g/kg/day to 300 μ g/kg/day. In another aspect, a dosing regimen can comprise a single administration of one or more of the disclosed peptides. In another aspect, a dosing regimen can include administering one or more of the disclosed peptides once a week, twice a week, three times a week, four times a week, five times a week, six times a week, or seven times a week for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, or 52 weeks.

In various aspects, the disclosed peptides can be administered by needle and syringe, pen, pump, inhaler, injection port, or jet injector.

D. Method for producing peptide

In one aspect, the invention discloses methods of making the disclosed peptides. Accordingly, in various aspects, the present invention discloses a method of preparing an insulin B-chain peptide, wherein the insulin B-chain peptide is directly conjugated to an organoboronate group, the method comprising: a step of reacting the peptide-bound insulin B chain resin with a phenylboronic acid having a structure represented by the following formula:

wherein Z is selected from C (O) and SO₂(ii) a Wherein Ar is¹Selected from 5-membered aryl, 5-membered heteroaryl, 6-membered aryl and 6-membered heteroaryl, and substituted with 0, 1, 2 or 3 groups independently selected from: halogen, -CN, -NO₂OH, -OH, C1-C4 alkyl, C1-C4 haloalkyl, C1-C4 hydroxyalkyl, C1-C4 alkoxy, C1-C4 alkylamino and (C1-C4) (C1-C4) dialkylamino; and cleaving the resin, thereby preparing the insulin B chain peptide.

In various aspects, the amino acid at position B29 is a lysine residue. In another aspect, the B29 lysine residue is modified. In another aspect, the B29 lysine residue is unmodified.

In various aspects, the amino acid at position B33 is a lysine residue. In another aspect, the B33 lysine residue is modified. In another aspect, the B33 lysine residue is unmodified.

In various aspects, the amino acid at position B34 is a lysine residue. In another aspect, the B34 lysine residue is modified. In another aspect, the B34 lysine residue is unmodified.

In various aspects, the amino acid at position B29 and the amino acid at position B33 are lysine residues. In another aspect, the amino acid at position B29 and the amino acid at position B34 are lysine residues. In another aspect, the amino acid at position B33 and the amino acid at position B34 are lysine residues. In another aspect, the amino acid at position B29, the amino acid at position B33, and the amino acid at position B34 are lysine residues.

In various aspects, the B29 lysine residue is unmodified, and each of the B33 and B34 lysine residues are modified. In another aspect, the B33 lysine residue is unmodified, and each of the B29 and B34 lysine residues are modified. In another aspect, the B34 lysine residue is unmodified, and each of the B29 and B33 lysine residues are modified. In another aspect, each of the B29, B33, and B34 lysine residues is modified. In another aspect, each of the B29, B33, and B34 lysine residues is unmodified.

In various aspects, the insulin B chain peptide comprises sequence FVNQHLCGSHLVEALYLVCGERGFFYTPKTRKK (SEQ ID NO: 8).

In various aspects, the insulin a chain peptide comprises sequence GIVEQCCTSICSLYQLENYCG (SEQ ID NO: 3), the insulin B chain peptide comprises sequence FVNQHLCGSHLVEALYLVCGERGFFYTPKTRKK (SEQ ID NO: 8), the B29 lysine residue is unmodified, each of the B33 lysine residue and the B34 lysine residue is directly conjugated to an organoboronate group, and each occurrence of the organoboronate group has a structure represented by the following formula:

in various aspects, the method further comprises the step of coupling the insulin B chain peptide to the insulin a chain peptide.

E. Methods of modifying insulin receptor activation

In one aspect, the present invention discloses a method of modifying insulin receptor activation in a subject, the method comprising administering to a subject in need thereof an effective amount of any of the disclosed peptides or pharmaceutical compositions. In another aspect, the subject in need thereof can be a subject known to have reduced insulin receptor activation compared to a standard activation level. In another aspect, the standard level of activation of insulin receptor activation can be based on a determined level in a healthy individual. In another aspect, the standard level of activation of insulin receptor activation can be based on a level determined in a subject treated prior to determining whether increased insulin receptor activation is required.

In one aspect, the present invention discloses a method of modifying insulin receptor activation in at least one cell, the method comprising contacting the at least one cell with an effective amount of any of the disclosed peptides or pharmaceutical compositions, thereby increasing insulin receptor activation in the at least one cell.

In various aspects, modifications are increasing.

For example, disclosed herein are methods of modifying insulin receptor activation in a subject, the methods comprising administering to the subject an effective amount of a peptide comprising an insulin a chain peptide and an insulin B chain peptide, wherein the insulin B chain peptide comprises at least 32 amino acid residues, and wherein at least three amino acid residues of the insulin B chain peptide are lysine residues, thereby modifying insulin receptor activation in the subject. Additionally, the present invention discloses a method of modifying insulin receptor activation in a subject, the method comprising administering to the subject an effective amount of a peptide comprising an insulin a chain peptide and an insulin B chain peptide, wherein the peptide is directly conjugated to at least one organoboronate group, thereby modifying insulin receptor activation in the subject.

For example, a method of modifying insulin receptor activation in at least one cell is disclosed, the method comprising contacting the at least one cell with an effective amount of a peptide comprising an insulin a chain peptide and an insulin B chain peptide, wherein the insulin B chain peptide comprises at least 32 amino acid residues, and wherein at least three amino acid residues of the insulin B chain peptide are lysine residues, thereby modifying insulin receptor activation in the at least one cell. Additionally, a method of modifying insulin receptor activation in at least one cell is disclosed, the method comprising contacting the at least one cell with an effective amount of a peptide comprising an insulin a chain peptide and an insulin B chain peptide, wherein the peptide is directly conjugated to at least one organoboronate group, thereby modifying insulin receptor activation in the at least one cell.

In various aspects, the insulin A chain peptide and the insulin B chain peptide are bonded by at least one disulfide bond, the insulin A chain peptide comprises sequence GIVEQCCHRICSLYQLENYCN (SEQ ID NO: 1), and the insulin B chain peptide comprises sequence FVNQHLCGSHLVEALYLVCGERGFFYTPKTRKK (SEQ ID NO: 8). In another aspect, one or both of B33 lysine residues and B34 lysine residues are modified. In another aspect, one of the B33 lysine residue and B34 lysine residue is modified. In another aspect, the B33 lysine residue is modified. In another aspect, the B34 lysine residue is modified. In another aspect, both of the B33 lysine residue and the B34 lysine residue are modified.

In various aspects, the insulin a chain peptide comprises sequence GIVEQCCTSICSLYQLENYCG (SEQ ID NO: 3), the insulin B chain peptide comprises sequence FVNQHLCGSHLVEALYLVCGERGFFYTPKTRKK (SEQ ID NO: 8), the B29 lysine residue is unmodified, each of the B33 lysine residue and the B34 lysine residue is directly conjugated to an organoboronate group, and each occurrence of the organoboronate group has a structure represented by the following formula:

in various aspects, the cell is a mammal. In another aspect, the cell is human.

In various aspects, the contacting is by administration to the subject. In another aspect, prior to the administering step, the subject has been diagnosed as in need of treatment for diabetes. In another aspect, the method further comprises the step of identifying a subject in need of treatment for diabetes.

In various aspects, the diabetes is type 1 diabetes. In another aspect, the diabetes is type 2 diabetes. In another aspect, the diabetes is gestational diabetes.

In various aspects, the subject is a mammal. In another aspect, the mammal is a human.

F. Method for lowering blood sugar

In one aspect, the invention discloses a method of reducing blood glucose in a subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of any of the disclosed peptides or pharmaceutical compositions. In various aspects, the subject in need thereof can be a subject known to have increased blood glucose as compared to a standard blood glucose level. In another aspect, the standard level of activation of insulin receptor activation can be based on a determined level in a healthy individual. In another aspect, the standard level of activation of insulin receptor activation can be based on a level determined in a subject treated prior to determining whether increased insulin receptor activation is required.

For example, the present invention discloses a method of reducing blood glucose in a subject, the method comprising administering to the subject a therapeutically effective amount of a peptide comprising an insulin a chain peptide and an insulin B chain peptide, wherein the insulin B chain peptide comprises at least 32 amino acid residues, and wherein at least three amino acid residues of the insulin B chain peptide are lysine residues, thereby reducing blood glucose in the subject. In addition, the present invention discloses a method of reducing blood glucose in a subject, the method comprising administering to the subject a therapeutically effective amount of a peptide comprising an insulin a chain peptide and an insulin B chain peptide, wherein the peptide is directly conjugated to at least one organoboronate group, thereby reducing blood glucose in the subject.

In various aspects, the insulin A chain peptide and the insulin B chain peptide are bonded by at least one disulfide bond, the insulin A chain peptide comprises sequence GIVEQCCHRICSLYQLENYCN (SEQ ID NO: 1), and the insulin B chain peptide comprises sequence FVNQHLCGSHLVEALYLVCGERGFFYTPKTRKK (SEQ ID NO: 8). In another aspect, one or both of B33 lysine residues and B34 lysine residues are modified. In another aspect, one of the B33 lysine residue and B34 lysine residue is modified. In another aspect, the B33 lysine residue is modified. In another aspect, the B34 lysine residue is modified. In another aspect, both of the B33 lysine residue and the B34 lysine residue are modified.

In various aspects, the insulin a chain peptide comprises sequence GIVEQCCTSICSLYQLENYCG (SEQ ID NO: 3), the insulin B chain peptide comprises sequence FVNQHLCGSHLVEALYLVCGERGFFYTPKTRKK (SEQ ID NO: 8), the B29 lysine residue is unmodified, each of the B33 lysine residue and the B34 lysine residue is directly conjugated to an organoboronate group, and each occurrence of the organoboronate group has a structure represented by the following formula:

in various aspects, the subject is a mammal. In another aspect, the mammal is a human.

In another aspect, prior to the administering step, the subject has been diagnosed as in need of reducing blood glucose. In another aspect, the method further comprises the step of identifying a subject in need of having their blood glucose lowered.

In another aspect, the subject has been diagnosed with a condition associated with hypertension, such as, for example, diabetes and hyperglycemia. In another aspect, the method further comprises the step of identifying a subject in need of treatment for a condition associated with hypertension (such as, for example, diabetes and hyperglycemia).

In another aspect, prior to the administering step, the subject has been diagnosed as in need of treatment for diabetes. In another aspect, the method further comprises the step of identifying a subject in need of treatment for diabetes.

In various aspects, the diabetes is type 1 diabetes. In another aspect, the diabetes is type 2 diabetes. In another aspect, the diabetes is gestational diabetes.

G. Methods of use of peptides

Herein, improved insulin (i.e., smart insulin) with increased solubility and elevated glucose concentration is described. Without wishing to be bound by theory, two advantages of injecting smart insulin, the activity of which is regulated in vivo by circulating blood glucose levels, include: (1) errors in insulin underdosing are significantly reduced because glucose-responsive insulin (GRI) derivatives are released from the subcutaneous depot whenever glucose levels are high; and (2) errors of insulin overdose will be significantly reduced, as the GRI analog will be inactivated when glucose levels begin to drop, thereby reducing the risk of hypoglycemia. Because of the high content of glycated hemoglobin with complications such as cardiovascular disease, nephropathy and retinopathy as a result of chronic hyperglycemia, the resulting hyperglycemia produced by treatment with glucose-responsive insulin analogues has improved therapeutic value. The GRI analogs can reduce hypoglycemic disturbances in diabetic populations.

As disclosed, in one aspect, smart insulin has similar activity as insulin glargine under high glucose conditions. According to one aspect of the technology, smart insulin incorporates phenylboronic acid (PBA) on the insulin molecule. For example, negatively charged PBA-glucose complexes can decrease the isoelectric point (pI) of insulin by binding to glucose, thereby increasing the solubility of insulin at high glucose concentrations to allow faster access to the blood.

Thus, in various aspects, the peptides and pharmaceutical compositions of the invention are useful for treating or controlling diabetes. For the treatment or control of disorders, the peptides and pharmaceutical compositions comprising the peptides are administered to a subject in need thereof, such as a vertebrate, e.g., a mammal, fish, bird, reptile, or amphibian. The subject may be a human, non-human primate, horse, pig, rabbit, dog, sheep, goat, cow, cat, guinea pig, or rodent. The term does not denote a particular age or gender. Thus, adult and newborn subjects, as well as fetuses, whether male or female, are intended to be encompassed. The subject is preferably a mammal, such as a human. Prior to administration of the compound or composition, the subject may be diagnosed as in need of treatment for diabetes.

The peptide or composition may be administered to the subject according to any method. Such methods are well known to those of skill in the art and include, but are not limited to, oral administration, transdermal administration, administration by inhalation, nasal administration, topical administration, intravaginal administration, ocular administration, otic administration, intracranial administration, rectal administration, sublingual administration, buccal administration, and parenteral administration, including injectable, such as intravenous administration, intraarterial administration, intramuscular administration, and subcutaneous administration. The administration may be continuous or intermittent. The article of manufacture can be administered therapeutically; i.e., administered to treat an existing disease or condition. The article can also be applied prophylactically; i.e., administration to prevent a disease or condition.

The therapeutically effective amount or dosage of the peptide may vary widely. In each particular case, such dosages are adjusted to the individual requirements, including the particular peptide or peptides being administered, the route of administration, the condition being treated, and the patient being treated. Generally, in the case of oral or parenteral administration to an adult human weighing about 70Kg or more, a daily dose of about 10mg to about 10000mg, preferably about 200mg to about 1000mg, should be appropriate, but the upper limit may be exceeded. The daily dose may be administered in a single or divided dose, or for parenteral administration as a continuous infusion. A single dose composition may contain the peptide or composition in such an amount or submultiples thereof to make up a daily dose. In the case of any contraindication, the dosage may be adjusted by the individual physician. The dosage may vary and may be administered in one or more doses per day for one or more days.

1. Method of treatment

The peptides disclosed herein are useful for treating or controlling diabetes. Accordingly, a method is provided comprising administering to a subject a therapeutically effective amount of a composition comprising a disclosed compound.

a. Treating diabetes

In one aspect, the invention discloses a method of treating diabetes in a subject, the method comprising the step of administering to the subject an effective amount of at least one disclosed peptide.

Accordingly, in various aspects, the invention discloses a method of treating diabetes in a subject, the method comprising administering to the subject a therapeutically effective amount of a peptide comprising an insulin a chain peptide and an insulin B chain peptide, wherein the insulin B chain peptide comprises at least 32 amino acid residues, and wherein at least three amino acid residues of the insulin B chain peptide are lysine residues, thereby treating diabetes in the subject.

In various aspects, the invention discloses methods of treating diabetes in a subject, the methods comprising administering to the subject a therapeutically effective amount of a peptide comprising an insulin a chain peptide and an insulin B chain peptide, wherein the peptide is directly conjugated to at least one organoboronate group, thereby treating diabetes in the subject.

In another aspect, prior to the administering step, the subject has been diagnosed as in need of treatment for diabetes.

In another aspect, the subject is a mammal. In another aspect, the mammal is a human.

In another aspect, the method further comprises the step of identifying a subject in need of treatment for diabetes.

In another aspect, the method further comprises the step of administering a therapeutically effective amount of at least one agent known to treat or control diabetes. Examples of agents known to treat or control diabetes include, but are not limited to, rapid acting insulin, short acting insulin, intermediate acting insulin, long acting insulin, metformin, amylin analogs, and GLP-1 receptor agonists (e.g., abiglutide, dulaglutide, exenatide delay agents, and liraglutide).

In another aspect, the at least one compound or the at least one agent is administered sequentially. In another aspect, the at least one compound or the at least one agent is administered simultaneously.

In another aspect, the at least one compound or the at least one agent is co-formulated. In another aspect, the at least one compound or the at least one agent is co-packaged.

In various aspects, the diabetes is type 1 diabetes. In another aspect, the diabetes is type 2 diabetes. In another aspect, the diabetes is gestational diabetes.

2. Use of compounds

In one aspect, the invention relates to the use of the disclosed peptides or products of the disclosed methods. In another aspect, the use relates to the manufacture of a medicament for treating diabetes in a mammal.

Uses of the disclosed peptides and products are also provided. In one aspect, the invention relates to the use of at least one of the disclosed peptides. In another aspect, the peptides used are the products of the disclosed methods of preparation.

In another aspect, the use relates to a method for the manufacture of a pharmaceutical composition comprising a therapeutically effective amount of the disclosed peptide or a product of the disclosed manufacturing process for use as a medicament.

In another aspect, the use relates to a method for preparing a pharmaceutical composition comprising a therapeutically effective amount of a disclosed peptide or a product of a disclosed method of preparation, wherein a pharmaceutically acceptable carrier is intimately mixed with a therapeutically effective amount of a peptide or a product of a disclosed method of preparation.

In various aspects, the use relates to treating diabetes in a mammal. In one aspect, the use is characterized in that the mammal is a human. In one aspect, the use is characterized in that the diabetes is type 1 diabetes.

In another aspect, the use relates to the manufacture of a medicament for treating diabetes in a mammal.

It is to be understood that the disclosed uses can be used in conjunction with the disclosed peptides, products of the disclosed methods of preparation, methods, compositions, and kits. In another aspect, the invention relates to the use of the disclosed peptides or the disclosed products in the manufacture of a medicament for treating diabetes in a mammal.

3. Manufacture of medicaments

In one aspect, the invention relates to a method for the manufacture of a medicament for treating diabetes in a mammal, the method comprising combining a therapeutically effective amount of the disclosed peptide or a product of the disclosed method with a pharmaceutically acceptable formulation or diluent.

For these uses, the method comprises administering to an animal, particularly a mammal, and more particularly a human, a therapeutically effective amount of a peptide effective to treat diabetes. In the context of the present invention, the dose administered to an animal, particularly a human, should be sufficient to affect the therapeutic response in the animal within a reasonable time frame. One skilled in the art will recognize that the dosage will depend on a variety of factors, including the condition of the animal and the weight of the animal.

The total amount of the peptides of the present disclosure administered in a typical treatment is preferably between about 10mg/kg and about 1000mg/kg body weight (for mice), and between about 100mg/kg and about 500mg/kg body weight, and more preferably between 200mg/kg and about 400mg/kg body weight (for humans). This total amount is typically, but not necessarily, administered in a series of smaller doses from about once daily to about three times daily for a period of about 24 months, and preferably twice daily for a period of about 12 months.

The size of the dose will also be determined by the route, time and frequency of administration, and the presence, nature and extent of any adverse side effects that may accompany the administration of the peptide, as well as the desired physiological effect. One skilled in the art will appreciate that various conditions or disease states, particularly chronic conditions or disease states, may require long-term treatment involving multiple administrations.

Thus, in one aspect, the invention relates to the manufacture of a medicament comprising combining the disclosed peptide, or the product of the disclosed method of preparation, or a pharmaceutically acceptable salt, solvate or polymorph thereof, with a pharmaceutically acceptable carrier or diluent.

4. Reagent kit

In one aspect, the present invention discloses a kit comprising peptides comprising an insulin a chain peptide and an insulin B chain peptide, wherein the insulin B chain peptide comprises at least 32 amino acid residues, and wherein at least three amino acid residues of the insulin B chain peptide are lysine residues; and one or more of the following: (a) agents known to treat diabetes; (b) instructions for administering a peptide for treating diabetes; (b) instructions for treating diabetes; (c) instructions for lowering blood glucose.

In one aspect, the present invention discloses a kit comprising a peptide comprising an insulin a chain peptide and an insulin B chain peptide, wherein the peptide is directly conjugated to at least one organoboronate group; and one or more of the following: (a) agents known to treat diabetes; (b) instructions for administering a peptide for treating diabetes; (b) instructions for treating diabetes; (c) instructions for lowering blood glucose.

Examples of agents known to treat diabetes include, but are not limited to, agents known to increase insulin production, agents known to improve insulin use in humans, and agents known to partially block starch digestion.

In another aspect, the peptide and the agent are co-formulated. In another aspect, the peptide and the agent are co-packaged.

In another aspect, the peptide and the agent are administered sequentially. In another aspect, the peptide and the agent are administered simultaneously.

The kit may also comprise compounds and/or products co-packaged, co-formulated and/or co-delivered with other components. For example, a pharmaceutical manufacturer, pharmaceutical distributor, physician, pharmacy (compounding shop), or pharmacist may provide a kit comprising the disclosed compounds and/or products for delivery to a patient, as well as another component.

It is understood that the disclosed kits can be prepared from the disclosed compounds, products, and pharmaceutical compositions. It is also to be understood that the disclosed kits can be used in conjunction with the disclosed methods of use.

The foregoing description illustrates and describes the present disclosure. Additionally, the disclosure shows and describes only the preferred embodiments, but, as mentioned above, it is to be understood that it is capable of use in various other combinations, modifications, and environments and is capable of changes or modifications within the scope of the inventive concept as expressed herein, commensurate with the above teachings and/or the skill or knowledge of the relevant art. The embodiments described herein above are further intended to explain best modes known to the applicants and to enable others skilled in the art to utilize the disclosure in such embodiments or other embodiments and with various modifications required by the particular applications or uses thereof. Accordingly, the description is not intended to limit the invention to the form disclosed herein. Furthermore, it is intended that the appended claims be construed to include alternative embodiments.

All publications and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference for all and all purposes. In the event of a discrepancy between the present disclosure and any publication or patent application incorporated by reference herein, the present disclosure controls.

G. Examples of the embodiments

Insulin glargine has two additional arginine residues at the C-terminus of the B chain, and has an increased isoelectric point (pI) and decreased solubility at physiological pH compared to native insulin. In one aspect, insulin derivatives are synthesized in which a PBA group and a positively charged group are introduced into insulin, thereby preserving biological activity. According to an embodiment, these modified insulin molecules will remain largely insoluble under hypoglycemic conditions. However, as blood glucose levels increase, the equilibrium changes and free glucose will bind to the negative charges forming PBA, thereby decreasing pI. On the other hand, the introduction of the PBA group itself reduced the solubility by a factor of 5. In another aspect, other intelligent insulin glargine with similar solubility curves may be used. In another aspect, different PBA groups can be used. Different PBA and amino acid combinations will result in different glucose response characteristics and solubilities. In another aspect, a combination of amino acids such as Arg-Glu can be used to increase solubility. Arg-Glu has zero net charge that does not affect pI. In addition, polar functional groups from the side chains may increase overall solubility. In another aspect, PBA groups (one or two) can be introduced into the B chain. On the other hand, for simplicity, a solubility group (Arg-Glu) n, n: 1 to 3, is attached to the C-terminus of the A chain. It is important to note that other derivatives with initial solubilities similar to insulin glargine and with > 5-fold solubility difference between 100mg/dL and 400mg/dL glucose can be used.

In various aspects, the synthesized analogs are tested in a receptor activation assay that identifies analogs that retain biological activity. In another aspect, an Insulin Receptor (IR) binding assay using radiolabeled insulin is used to confirm binding between the analog and the IR. Insulin analogs having at least 85% potency relative to insulin glargine with respect to receptor activation and binding can be selected for further characterization.

In various aspects, structural or sequence modifications of the insulin molecule can result in altered binding affinity and activity to Insulin Receptor (IR) and/or insulin-like growth factor 1 receptor (IGF 1R). In addition, activation of insulin signaling can also lead to metabolism (induction of glucose uptake) and mitogenesis (growth and proliferation). In another aspect, insulin derivatives may have altered metabolic and mitogenic effects compared to human insulin. For example, insulin X10(B10 Asp human insulin) induces cell proliferation in vitro and tumor formation in vivo.

Herein, the design and synthesis of "smart glargine" and its glucose response characteristics are described. Intelligent insulin glargine has in vitro biological activity similar to insulin glargine. It is further demonstrated that smart glargine exhibits nearly 3-fold difference in vivo activity compared to glargine at different glucose concentrations and significantly reduces the incidence of hypoglycemia. Thus, without wishing to be bound by theory, smart insulin glargine represents a new design to achieve glucose-mediated insulin control based on protein solubility.

The following articles and examples are given to enable those skilled in the art to more clearly understand and practice the present invention. They should not be considered as limiting the scope of the invention, but merely as being illustrative and representative thereof.

1. General information

All Fmoc amino acids, reagents and solvents were used without purification. Fmoc amino acids, coupling reagents, and 2-chlorotrityl chloride resin (cat. No. 03498) were purchased from Chem imprex international inc. Rink Amide MBHA resin HL (catalog number 855118) and Novasyn TGA resin (catalog number 855005) were obtained from Novabiochem, and Rink Amide ChemMatrix resin (catalog number 7-600-1310) was purchased from Biotage. N, N' -Dimethylformamide (DMF), Dichloromethane (DCM), Acetonitrile (ACN), methanol (MeOH), diethyl ether (Et2O), acetic acid (AcOH) and trifluoroacetic acid (TFA) were obtained from Fisher Scientific. Piperidine, Triisopropylsilane (TIS), hydroxybenzotriazole (HoBt), N' -Diisopropylcarbodiimide (DIC), 2-dithiodipyridine (DTDP), 2-dithiobis (5-nitropyridine) (DTNP) were obtained from Sigma Aldrich. 1- [ bis (dimethylamino) methylene ] -1H-1, 2, 3-triazolo [4, 5-b ] pyridine 3-oxide Hexafluorophosphate (HATU) was purchased from Chem Impex healthcare. The isoacyl dipeptide Boc-Ser [ Fmoc-Thr (tBu) ] -OH was purchased from Novabiochem.

LC-MS system: agilent 6120 Quadrupole LC/MS system on an Xbridge C185- μm (50X 2.1mm) column with a linear gradient from 0 to 95% aqueous acetonitrile (0.1% formic acid) at 0.4 mL/min.

General RP-HPLC conditions: passed through a Phenomenax Luna C18 column (5u,250x 21.2mm) at a flow rate of 5mL/min from 15% to 50% aqueous acetonitrile (0.1% trifluoroacetic acid) over 60min or a Phenomenax Jupitor C18 column (5u, 250x 10mm) was purified with a linear gradient from 15% to 50% aqueous acetonitrile (0.1% trifluoroacetic acid) over 40min at a flow rate of 3 mL/min.

All B chains were purified by a similar method except that the gradient was from 30% to 65% aqueous acetonitrile (0.1% trifluoroacetic acid) at a flow rate of 5mL/min over 60min or at a flow rate of 3mL/min over 40 min.

Using Fmoc solid phase Synthesis, by Biotage automatic microwave peptide synthesizer (initiator + Alstra)^TM) Synthesizing the A chain. Peptide synthesis was performed on a 0.1mmol scale using the standard HATU/DIEA protocol. For Fmoc deprotection, 20% piperidine in DMF was added and mixed twice for 5min at 25 ℃. For amino acid coupling, 0.2M Fmoc-protected amino acid, 0.2M HATU (coupling reagent) and 1.0M DIEA (base) were prepared in DMF. In each cycle, 5 equivalents of amino acid, 5 equivalents of coupling agent and 10 equivalents of base were added to the reaction vessel and mixed for 5min at 75 ℃ (for cysts)Mixing amino acid and histidine at 50 deg.C for 10 min; for arginine, mix 15min at 50 ℃ and couple twice). After completion of the peptide chain, the resin was washed with DCM and dried using vacuum. The peptide was then cleaved by TFA and further precipitated with cold ether before HPLC purification and lyophilization.

The B chain was synthesized by Prelude X peptide synthesizer without heating. The synthesis scheme is the same as for the a chain except for the coupling time. For amino acid coupling, the reaction was mixed at 25 ℃ for 30min under nitrogen sparge. Detailed intelligent insulin glargine synthesis protocols are described elsewhere herein.

2. Peptide synthesis

The synthesis of the A chain (InsA (G)) was carried out using a Biotage automatic microwave peptide synthesizer on 0.1mmol Rink amide ChemMatrix resin (0.54 mmol/g). The C-terminal amino acid Asn is attached to the resin as Fmoc-Asp-OtBu via its side chain carboxyl group. The isoacyl dipeptide Boc-Ser [ Fmoc-Thr (tBu) ] -OH was used as a single residue and coupled as with the other residues by standard protocols. The peptide bound resin was treated twice with fresh 25% β -mercaptoethanol (15mL) for 1.5h and washed thoroughly with DMF (15mL x 3) and DCM (15mL x 3). DTNP in 10mL DCM (310mg, 10 equiv.) was then added to the resin and shaken for 1 h. The resin was washed again with DMF (15mL x 3) and DCM (15mL x 3) and treated with 1% TFA, 5% TIS in DCM (10mL) for 5 times 2 min. Finally, the resin was shaken in DCM (10mL) for 1H and then cleaved by TFA/TIS/H2O (9 mL/500. mu.L) for 2H.

The synthesis of B chain R2 was carried out on 0.1mmol of 2-chlorotrityl chloride resin (0.4mmol/g) using a Prelude X peptide synthesizer without heating. The C-terminal Arg was manually loaded onto the resin using Fmoc-Arg (pbf) -OH/DIEA (4/4 equivalents relative to the resin). Chain assembly was performed as described in the general information section.

The synthesis of B chain RP2 was carried out using a Prelude X peptide synthesizer on 0.1mmol 2-chlorotrityl chloride resin (0.4mmol/g) without heating. Chain assembly was performed as described in the general information section. Phenyl boronic acids were assembled via Dde protected Lys. Phenylboronic acids were coupled to standard amino acids. The N-terminal amino acid Phe was coupled using Boc-Phe-OH. After synthesis, the peptide-bound resin was treated twice with 2% hydrazine for 5min to remove the Dde group on Lys. After washing with DMF and DCM, phenylboronic acid was assembled to lys using 4-carboxyphenylboronic acid (332mg, 20 equivalents), HoBt (270mg, 20 equivalents) and DIC (313ul, 20 equivalents) 12h or 4-carboxyphenylboronic acid (66mg, 4 equivalents), HATU (152mg, 4 equivalents) and DIEA (70ul, 4 equivalents) twice for 45 min. Finally, the resin was cleaved for 2H by DTDP (330mg, 15 equiv.) in TFA/TIS/H2O (4.5 mL/250. mu.L). Synthesis of B chain RF2 was synthesized in the same manner except that 4-carboxy-3-fluorophenylboronic acid was used.

3. Preparation of insulin analogues

Insulin analogs are prepared by combining the a and B chains using a two-step process. In-strand ligation buffer (6M Urea, 0.2M NH)₄OAc, pH 4.5, 0.8mL) was mixed with chain A (4mg, 1.63. mu. mol) and chain B (7.2mg, 1.62. mu. mol). The combined reaction was allowed to proceed at 25 ℃ for 4h and then purified by RP-HPLC. The combined fractions were then passed through 1M NH₄HCO₃Adjusted to pH 8 and lyophilized.

The lyophilized powder (10mg, 1.47. mu. mol) was dissolved in a mixed solvent of AcOH (200. mu.L) and H2O (800. mu.L) at 25 ℃ and treated with a freshly prepared solution of iodine (11.2mg, 44.1. mu. mol) in AcOH (3mL) under gentle stirring for 10 min.

The oxidation was quenched by the addition of 1M ascorbic acid until the iodine color (purple) disappeared. Passing the final solution through H₂O (16mL) diluted and purified as described in the general information section.

4. In vitro receptor binding assays

The His-tagged IR (isoform B) extracellular domain was immobilized in a 96-well plate. Instead of using radiolabeled insulin, Eu-modified insulin was used. Time resolved fluorescence was measured with 340-nm excitation and 612-nm emission filters. This assay has recently been reported (Menting et al (2016) Nat Struct Mol Biol). Using a binding assay, smart glargine was found to have about a 2-fold reduction in binding affinity compared to native insulin. This is consistent with literature data on insulin glargine IR affinity (fig. 5A).

5. In vitro biological activity assay

To measure the biological activity of human insulin, insulin glargine and smart insulin glargine, pAkt Ser473 levels were measured in the mouse fibroblast cell line NIH 3T3 (gift from a. morrione, Thomas Jefferson University) overexpressing human IR-B. Cells were identified by western blotting to assess their IR expression level compared to that of the parental 3T3 cells: NIH 3T3 cells showed expression levels about ten-fold higher than that of the parent. NIH 3T3 cell lines were cultured in DMEM (Thermo Fisher Scientific) containing 10% FBS, 100U/mL penicillin streptomycin (Thermo Fisher Scientific) and 2. mu.g/mL puromycin (Thermo Fisher Scientific) and shown to be free of mycoplasma contamination. For the assay 40000 cells per well were seeded in 96-well plates with medium containing 1% FBS. After 24h, 50 μ L of insulin solution was pipetted into each well after the original medium was removed. After 30min of treatment, the insulin solution was removed and the intracellular levels of pAkt Ser473 were measured using the HTRF pAkt Ser473 kit (Cisbio, 64 AKSPEH).

Briefly, cells were first treated with cell lysis buffer (50 μ L per well) for 1h under gentle shaking. Then 16 μ L of cell lysate was added to 4 μ L of detection reagent in a white 384 well plate. After 4-h incubation, plates were read in a Synergy Neo plate reader (BioTek) and the data were processed according to the manufacturer's protocol. The assay was repeated four times in total (biological replicates). After curve fitting by nonlinear regression (single-point) analysis, the average EC was calculated₅₀Values and their 95% confidence intervals (using Prism 8).

6. Additional predictive biological Activity assays

The selected insulin derivatives will be further tested for their effect on other key proteins in the insulin signaling pathway to confirm their full activation. First, phosphorylation of IR will be measured using western blotting to confirm receptor phosphorylation. Second, phosphorylation of insulin receptor substrate 1(IRS-1) will be measured using Western blotting. Third, phosphorylation of glycogen synthase kinase 3(GSK-3) will also be measured, since pGSK-3 levels are expected to decrease when insulin activates IR. Due to the well-known effects of insulin on muscle and liver cells, the GRI derivatives will also be evaluated for insulin signaling activation in both the C2C12 and HepG2 cell lines.

7. Circular dichroism

All CD spectra were recorded in water at 25 ℃ on an AVIV type 410 spectrophotometer (AVIV) in 1mm QS quartz cuvettes (Starna). The wavelength scan was performed with a resolution of 1nm and an average time of 1 s. The data from the two scans were averaged, subtracted by the blank, and normalized to the average residual ellipticity by the following equation: [ θ ] ═ 100 × θ/C × l × (n-1), where C is the protein concentration in mM, l is the path length in centimeters, and n is the number of residues in the protein. The concentration of the protein sample used for the CD experiment was 100. mu.M.

8. Solubility determination

To Eppendorf tubes 1mg of peptide was added and suspended in 100. mu.l PBS buffer (pH 5, 7 and 9) containing different concentrations of glucose (0-400 mg/dl). The peptide was added in excess to make a saturated solution with incompletely dissolved peptide at the bottom. The sample was vortexed for 5min and gently shaken overnight. They were then centrifuged at 12000rpm for 10 min. The concentration of the saturated peptide solution was determined by Nanodrop based on the absorbance at 280nm and the calculated extinction coefficient.

9. Animal(s) production

Male Sprague-Dawley rats (SASCO SD, strain code: 400; Charles River Laboratories, Inc., Wilmington, Mass.) weighing 250g to 300g were housed at the University of Utah USA (University of Utah) in polypropylene cages and maintained under standard housing conditions (room temperature 22 ℃ to 24 ℃, light/dark cycle 12 h). Animals were free to obtain food and water and were acclimatized to treatment 1 week prior to the experimental procedure. All procedures were performed according to the National Institutes of Health (NIH) guidelines for the Care and Use of Laboratory Animals, and were approved by the Institutional Animal Care and Use Committee of his university, IACUC.

10. Preparation of insulin glargine sample

Commercial Lantus (100U/ml) was purified by HPLC to obtain insulin glargine. Both lyophilized and smart insulin glargine were dissolved in (3.63mg/ml) dilution buffer pH 4 containing similar ingredients to a commercial Lantus diluent (30 μ g zinc, 2.7mg m-cresol, 20mg glycerol 85%, 20 μ g polysorbate 20).

11. Vascular surgery

Rats were anesthetized by intraperitoneal injection of ketamine/xylazine (75mg/kg ketamine and 5mg/kg xylazine) and were incised at the midline of the ventral side of the neck under sterile conditions for implantation of vascular catheters. A micro-renathane catheter (MRE 025, Braintree Scientific inc., Braintree, MA) was inserted into the right jugular vein and another catheter (MRE 033) was implanted into the left carotid artery. To maintain patency, all catheters were filled with 40% polyvinylpyrrolidone (Sigma, MO) in heparin (1000 units/ml; USP) and tunneled subcutaneously to be placed in the back of the neck. The animals were then allowed to recover in home cages before being placed in the animal facility.

12. Improved euglycemic and hyperglycemic forceps

To evaluate the effects of smart glargine and commercially available glargine, modified euglycemic and hyperglycemic clamp were performed in non-diabetic and diabetic rats, respectively. In these modified glucose clamps, the absorption characteristics of insulin were studied after administration of a single dose subcutaneous injection (as opposed to the intravenous infusion that occurs in traditional glucose clamps). For the euglycemic clamp, non-diabetic control rats were fasted overnight one week after vascular surgery and arterial and venous catheters were exteriorized under isoflurane anesthesia and separately extended through a connector for blood sampling and connection to an infusion pump. After resting for 90 minutes, the basal glucose level was measured from arterial blood samples obtained from conscious, unconstrained rats using a glucometer (Ascensia content BG monitors, Bayer health, IN). After baseline blood glucose measurements, all rats were injected subcutaneously with either commercial insulin glargine (i.e., 0.5mg/kg) or synthetic smart insulin glargine (0.5 mg/kg). Blood glucose was measured at 10 minute intervals throughout the clamp period and normoglycemia (90mg/dl to 110mg/dl) was maintained for 4 hours using a constant variable intravenous infusion of dextrose (50% w/v).

For the hyperglycemic clamp, four days after vascular surgery, rats were injected intraperitoneally with streptozotocin (STZ; 65mg/kg) to induce diabetes. Diabetic rats were selected for these studies because they have the advantage of increasing blood and interstitial glucose concentrations and are the ideal choice for studying the effect of high glucose levels on insulin bioavailability. Three days after STZ injection and after overnight fasting, all diabetic rats were injected subcutaneously with either commercial insulin glargine (i.e., 0.5mg/kg) or synthetic smart insulin glargine (0.5mg/kg) and subjected to a similar clamp regimen, except that the rat was clamped at high blood glucose levels (about 400mg/dl) for 4 hours.

13. Insulin resistance test (ITT)

After fasting for 4 to 5h, STZ diabetic rats were subjected to the insulin resistance test (ITT). After baseline blood glucose levels were obtained, rats were injected subcutaneously with commercial insulin glargine (1mg/kg) or synthetic smart insulin glargine (1 mg/kg). Tail vein samples were taken every 15 minutes over four hours using a blood glucose meter (Ascensia content BG monitors, Bayer health care, IN) to assess blood glucose levels.

14. Statistical analysis

Results are expressed as mean ± Standard Error of Mean (SEM). Data were analyzed by student (unpaired) "t" test. Repeated measures ANOVA (two-way) were performed to analyze data for glucose clamp and ITT over a 4h period. Post hoc analysis was performed by multiple comparative tests on graph bases (Tukey). The 5% probability level is considered statistically significant.

15. Development of insulin analogs

Chemical synthesis of smart glargine was achieved by using solid phase peptide synthesis (figure 3). A chain and B chain were synthesized separately. To avoid degradation of PBA under harsh peptide coupling conditions, PBA was introduced late after synthesis of the entire B chain using lys (dde) residues. To form all three disulfide bonds in a controlled manner, four different Cys protecting groups were used, as in Liu et al (2014) angelwan Chemie iht. ed.53 (15): 3983-. First, A6 Cys (S-tBu) was deprotected using mercaptoethanol, followed by activation with 2, 2' -dithiobis (5-nitropyridine) (DTNP). Next, a11 cys (mmt) was deprotected using 1% TFA to obtain thiol. An A6-A11 intramolecular disulfide bond is then formed by a disulfide bond substitution reaction. The a chain was then cleaved from the resin to give A7Cys (Acm) and a20 free Cys (Trt deprotected). The A and B chains are then joined by a similar disulfide bond substitution reaction. After HPLC purification (> 98% purity), iodine was used to form the final disulfide bond to obtain intelligent insulin glargine. 2-fluorophenyl boronic acids were used because their pKa was similar to physiological pH (FIG. 4A) (Matsumoto et al (2012) Angewandte Chemi51 (9): 2124-8).

Referring to fig. 3, smart glargine is synthesized in two chains, followed by chain assembly using orthogonal protecting groups.

Referring to fig. 4A, under high glucose conditions, when glucose is bound to a neutral boronic acid group, the equilibrium shifts to the negatively charged boronic ester complex. Referring to fig. 4B, while insulin glargine is slowly and continuously released from the subcutaneous depot, smart insulin glargine is released in response to elevated glucose levels.

To determine if PBA introduction has an effect on the secondary structure of insulin, insulin was evaluated using near UV Circular Dichroism (CD). All insulin molecules were observed to have CD spectra largely consistent with alpha-helical secondary structure (fig. 5B). To measure in vitro biological activity, a cell-based insulin receptor activation assay was performed using pAkt levels as an indicator of biological activity (fig. 5C). Insulin glargine and Intelligent insulin glargine have similar signal activated EC₅₀(12 nM). The biological activity of human native insulin is 2-fold higher than that of insulin glargine, consistent with literature reports (Varewijck and Janssen (2012) Endocrine-related cancer 19 (5): F63-F75). Next, the solubility curves of the two insulin glargine molecules were measured. Insulin glargine and smart insulin glargine have high solubility at pH 5 and pH 9, with much lower solubility at pH 7, consistent with the biochemical design of insulin glargine (fig. 5D). It should be noted, however, that at pH 7, the solubility of smart glargine is less than one-fourth that of glargine (0.06mg/mL vs./mL)At 0.28 mg/mL). Without wishing to be bound by theory, this is likely due to the hydrophobic nature of the PBA. Next, solubility curves for different glucose concentrations were measured at pH 7 (fig. 5E). While insulin glargine has the same solubility at 0 to 400mg/mL glucose, the solubility of smart insulin glargine increases by about 2.5-fold over the same range. Without wishing to be bound by theory, this result supports the following hypothesis: the negative charge of the boronate complex is attached to the hydrophilic sugar, and the intelligent glycine has increased solubility under high glucose conditions, thus demonstrating the biochemical basis of glucose reactivity.

Referring to fig. 5B, circular dichroism spectra of human insulin, insulin glargine, and smart insulin glargine are shown. Referring to fig. 5C, the in vitro activity of insulin analogs in activating insulin receptors using pAkt levels as a measure is shown. The solubility curves of insulin glargine and smart insulin glargine at pH 5, 7 and 9 are shown in fig. 5D. Solubility curves for insulin glargine and smart insulin glargine at pH 7 are shown in fig. 5E, with glucose concentrations from 0 to 400 mg/dL. While insulin glargine has similar solubility under all conditions, smart insulin glargine has increased solubility under high glucose conditions (increased by about 2-fold between 100mg/dl and 400 mg/dl).

To establish the in vivo glucose responsiveness of smart glargine, a normoglycemic and hyperglycemic clamp study was conducted to compare and contrast the in vivo biological activities of smart glargine and commercially available glargine. Following subcutaneous injection of insulin (0.5mg/kg), the blood glucose levels of both insulin glargine and smart insulin glargine treated rats matched well (by experimental design) during the normoglycemic and hyperglycemic clamp regimens (fig. 6A). During the hyperglycemic clamp (approximately 400mg/dL glucose), the glucose infusion rate required to maintain hyperglycemia for the smart insulin glargine-treated rats was 88% of that of insulin glargine-treated rats (fig. 6B). However, during euglycemic clamp (about 100mg/dL glucose), the rate of exogenous glucose infusion in smart insulin glargine-treated rats was significantly reduced (down to about 30% of insulin glargine-treated rats) (fig. 6B), and this difference was highly significant (P < 0.01). Without wishing to be bound by theory, this result indicates that smart glargine has similar in vivo biological activity as insulin glargine under hyperglycemic conditions, but greatly reduced activity under normoglycemic conditions. This 2.9 fold difference in relative biological activity demonstrates the glucose responsiveness of smart glargine in vivo.

Referring to FIG. 6A, blood glucose levels (mg/dl) during normoglycemic (about 100mg/dl) and hyperglycemic (about 400mg/dl) clamps in non-diabetic control and Streptozotocin (STZ) -diabetic rats, respectively, are shown. In the normoglycemic and hyperglycemic clamp, rats were injected subcutaneously with insulin glargine (0.5mg/kg) or smart insulin glargine (0.5mg/kg) after a baseline reading of t ═ 0. Data are presented as mean ± SEM (n-5-6/panel). ANOVA (two-way) measurements were repeated, followed by post-hoc testing for graph-based comparisons. Dextrose: 50 percent.

Referring to FIG. 6B, the average glucose infusion rate (m/kg) during the last hour of the normoglycemic (about 100mg/dl) and hyperglycemic (about 400mg/dl) jaws is shown^*min). Rats were injected subcutaneously with insulin glargine (0.5mg/kg) or smart insulin glargine (0.5mg/kg) in both the normoglycemic and hyperglycemic clamp. Data are presented as mean ± SEM (n-5-6/panel).^*P＜0.05，^**P < 0.01 for insulin glargine; student "t" (unpaired) test.

Referring to fig. 6C and 6D, GIR (mg/kg/min) during normoglycemia (fig. 6C, about 100mg/dl) and hyperglycemia (fig. 6D, about 400mg/dl) clamping in non-diabetic control and Streptozotocin (STZ) -diabetic rats, respectively, is shown. In the normoglycemic and hyperglycemic clamp, rats were injected subcutaneously with insulin glargine (0.5mg/kg) or smart insulin glargine (0.5mg/kg) after a baseline reading of t ═ 0. Dextrose: 50 percent.

From the results of the clamp study, it was assumed that smart glargine is unlikely to cause hypoglycemia. To assess the potential for insulin-induced hypoglycemia, a high dose (1mg/kg) insulin resistance test (ITT) was performed in STZ-induced diabetic rats. Subcutaneous administration of insulin glargine and smart insulin glargine (1mg/kg) reduced blood glucose levels in the absence of glycemic clamp conditions (fig. 7A). The minimum blood glucose level achieved by insulin glargine treated rats was about 40 mg/dl. Evidence of sustained insulin absorption/action is observed by maintaining hypoglycemia for more than 2 hours. This sustained insulin action is particularly impressive as it is maintained in an environment of (possibly) counter-regulatory response to hypoglycemia. In contrast, equal doses of smart insulin glargine resulted in a gradual decrease in blood glucose with a minimum blood glucose level of about 102 mg/dl. To quantify the hypoglycemic efficacy of these insulins, the duration of time that blood glucose is maintained at hypoglycemia (< 70mg/dl) was quantified. Rats with smart glargine maintained hypoglycemia for significantly shorter duration than rats with commercial glargine (fig. 7B). Without wishing to be bound by theory, this 15-fold reduction in hypoglycemic potency demonstrates that smart insulin glargine predicts a reduced risk of causing hypoglycemia as compared to insulin glargine.

Referring to FIG. 7A, the blood glucose levels (mg/dl) during the insulin resistance test (ITT) performed in STZ-diabetic rats are shown. After a baseline blood glucose reading was obtained, rats were injected subcutaneously with insulin glargine (1mg/kg) or smart insulin glargine (1 mg/kg). Data are presented as mean ± SEM (n-5-6/panel). ANOVA (two-way) measurements were repeated, followed by post-hoc testing for graph-based comparisons.

Referring to FIG. 7B, the time (min) for which blood glucose levels remained below 70mg/dl during ITT is shown for rats injected with insulin glargine (1mg/kg) or smart insulin glargine (1 mg/kg). Data are presented as mean ± SEM (n-5-6/panel).^**P < 0.01 for insulin glargine; student "t" (unpaired) test.

The aim of this study was to increase the therapeutic index of insulin. This innovation is based on the conversion of insulin glargine to smart insulin glargine using phenylboronic acid, which has glucose-dependent solubility at physiological pH. Unlike the stable release of commercially available insulin glargine from subcutaneous depots into the blood; the in vivo bioactivity curve (fig. 7B) indicates that smart glargine exhibits relatively higher absorption under high glucose conditions and significantly less absorption under low glucose conditions. Interestingly, a lower rate of insulin absorption from the subcutaneous depot (under normoglycemic conditions) would give the intelligent insulin glargine the theoretical advantage of extending the duration of insulin action. Another unique advantage of this GRI derivative is compatibility with other published GRI designs. For example, smart insulin glargine can potentially be modified to generate smart insulin glargine-carbohydrate conjugates based on the recent Merck strategy for mannose receptors (Kaarsholm et al (2018) Diabetes 67 (2): 299-. Since these two designs have completely different mechanisms of action, this combination has the potential to further enhance the glucose reactivity.

Insulin-induced hypoglycemia is the most serious acute complication of insulin therapy. Although the introduction of fast-acting and long-acting insulin analogues reduces the risk of hypoglycemia compared to native insulin, these insulin analogues still have a narrow therapeutic window because, once injected, the absorption of insulin in the bloodstream is continuous and not affected by the ambient glucose concentration (Berenson et al (2011) Annals of the New York Academy of Sciences 1243: E40-E54) (fig. 4B). Consistent with this notion of sustained insulin action, it should be noted that pharmacological doses of commercially available insulin glargine caused long-term hypoglycemia (fig. 7B). However, equimolar doses of glucose-responsive smart insulin glargine showed lower hypoglycemic efficacy as shown below: (1) blood glucose level dropped to the lowest point of 102 mg/dl; and (2) a 15-fold reduction in the duration of hypoglycemia. Without wishing to be bound by theory, several factors may contribute to this beneficial finding. First, regardless of the relative bioactivity measurements determined during the steady-state portion of the glycemic clamp experiment, the gradual decline in blood glucose levels during the insulin resistance test using smart insulin glargine indicates (compared to insulin glargine) slower absorption following subcutaneous injection (fig. 7A). The decrease in the absorption rate of smart glargine at physiological pH was consistent with the in vitro findings (fig. 5D) and probably represents the hydrophobic nature of PBA. Second, the biological activity of smart glargine is reduced by 12% under hyperglycemic conditions, which may contribute little to this observed effect. Third, as blood glucose approaches normoglycemia, it has been suggested that a significant decrease in solubility of smart glargine at normoglycemia (fig. 5E and 7B) prevents the development of hypoglycemia. Of particular note, it was suggested that this slower onset and lower in vivo activity of smart glargine was not due to a decrease in intrinsic insulin stimulatory activity, as smart glargine and commercially available glargine showed the same biological activity in activating insulin receptor signaling in vitro (fig. 5C). In summary, smart glargine insulin showed an overall decrease in solubility, as shown in vitro (fig. 5D), and also as shown by a slowing in the onset of action shown in vivo (fig. 7A); however, smart insulin did exhibit increased relative solubility under high glucose conditions in vitro (fig. 5E) and in vivo (fig. 6B), demonstrating glucose response characteristics. Overall, these new findings suggest that the bioactivity profile of insulin glargine can be manipulated by altering its overall solubility in a glucose responsive manner.

In summary, evidence for the synthesis of glucose-responsive smart glargine is provided. It has an advantage in preventing hypoglycemia compared to insulin glargine. Rapid clinical development of insulin derivatives with glucose response characteristics can help insulin-treated diabetic populations achieve glycemic goals while minimizing the risk of hypoglycemia.

16. Prophetic examples: design and chemical synthesis of glucose-reactive insulin (GRI) derivatives to maximize glucose reactivity

Insulin glargine has two additional arginine residues at the C-terminus of the B chain, which results in increased pI and decreased solubility at physiological pH compared to native insulin. Here, the aim is to synthesize insulin derivatives in which PBA groups and positively charged groups are introduced into insulin while retaining the biological activity. Under hypoglycemic conditions, these modified insulin molecules will remain largely insoluble. However, as blood glucose levels rise, the balance shifts, andand free glucose will bind to the negative charge forming PBA, thereby reducing pI (see fig. 4B). To take full advantage of the long-lasting properties of insulin glargine and maximize glucose reactivity, insulin derivatives with glucose-sensitive PBA groups will be synthesized while maintaining relatively low solubility at pH 7. In this case, different PBA and amino acid combinations will result in different glucose response characteristics and solubilities (PBA is aromatic and hydrophobic and will therefore result in a decrease in baseline solubility). The baseline solubility of smart glargine is already 4 times lower than that of glargine. To overcome this limitation, amino acid combinations such as Arg-Glu will be explored to increase solubility. The net charge of Arg-Glu is zero, so it does not affect pI. In addition, polar functional groups from the side chains will increase overall solubility. Since the PBA group (3, 4, 5) will be introduced into the B chain, the solubility group (Arg-Glu) n, n ═ 1-2, will be introduced at the C-terminus of the a chain (fig. 8). In the case of enhanced glucose reactivity, the glucose sensing properties of various PBAs will be evaluated. Since glucose binding is highly dependent on pK of PBA_aTherefore, pK is used_aPBA in the range of 7.0 to 7.8 (fig. 8). Thus, a total of 18 insulin derivatives will be synthesized.

It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the scope or spirit of the invention. Other aspects of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the invention being indicated by the following claims.

Sequence listing

<110> week, Danny Hong Jie (Chou, Danny Hung-Chieh)

<120> glucose-responsive insulin

<130> 21101.0369P1

<150> 62/658372

<151> 2018-04-16

<160> 8

<170> PatentIn version 3.5

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