阅读说明：本技术 包含药代动力学增强子的修饰的松弛素多肽及其用途 (Modified relaxin polypeptides comprising pharmacokinetic enhancers and uses thereof ) 是由 G·M·杜波奇克 O·S·古德蒙森 X·韩 R·M·劳伦斯 D·利波夫斯克 C·S·马德森 于 2018-02-08 设计创作，主要内容包括：本公开总体上涉及包含与药代动力学增强子连接的非天然编码氨基酸的修饰的松弛素多肽,如修饰的人松弛素2多肽,以及这样的多肽的治疗用途,例如用于治疗心血管病症(例如心力衰竭)和/或与纤维化相关的病症。(The present disclosure relates generally to modified relaxin polypeptides, such as modified human relaxin 2 polypeptides, comprising a non-naturally encoded amino acid linked to a pharmacokinetic enhancer, and therapeutic uses of such polypeptides, for example for treating cardiovascular disorders (e.g., heart failure) and/or disorders associated with fibrosis.)

1. A modified relaxin polypeptide comprising a non-naturally encoded amino acid, wherein:

(a) the modified relaxin polypeptides comprise a relaxin A chain polypeptide of SEQ ID NO. 4 and a relaxin B chain polypeptide of SEQ ID NO. 5 or SEQ ID NO. 6, and are substituted with a non-naturally encoded amino acid at a position selected from the group consisting of: an a chain residue 1, an a chain residue 2, an a chain residue 5, an a chain residue 13, an a chain residue 18, a B chain residue 5, a B chain residue 7 and a B chain residue 25, and optionally, up to two additional amino acid substitutions, insertions and/or deletions in the relaxin a chain and/or the relaxin B chain;

(b) the non-naturally encoded amino acid has the following structure:

wherein the R group is any substituent other than the side chain found in the following amino acids: alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine; and

(c) the non-naturally encoded amino acid is linked to a pharmacokinetic enhancer comprising a peptide component of 2 to 30 amino acids and a half-life extending moiety.

2. The modified relaxin polypeptide of claim 1, wherein the relaxin A chain polypeptide comprises SEQ ID NO 4 substituted at residue 1 with the non-naturally encoded amino acid, and optionally having up to two additional amino acid substitutions, insertions, and/or deletions.

3. The modified relaxin polypeptide of claim 1, wherein the relaxin A chain polypeptide comprises SEQ ID NO 4 substituted at residue 1 with the non-naturally encoded amino acid, and optionally having one additional amino acid substitution, insertion, and/or deletion.

4. The modified relaxin polypeptide of claim 3, wherein the relaxin B chain polypeptide comprises SEQ ID NO 5 or SEQ ID NO 6, optionally with one additional amino acid substitution, insertion or deletion.

5. The modified relaxin polypeptide of claim 1, wherein the relaxin a chain polypeptide comprises SEQ ID No. 4 substituted at residue 1 with the non-naturally encoded amino acid and the relaxin B chain polypeptide comprises SEQ ID No. 5 or SEQ ID No. 6.

6. The modified relaxin polypeptide of any one of claims 1-5, wherein the non-naturally encoded amino acid comprises a carbonyl group, an aminooxy group, a hydrazide group, a hydrazine group, a semicarbazide group, an azide group, or an alkyne group.

7. The modified relaxin polypeptide of any one of claims 1-5, wherein the non-naturally encoded amino acid comprises a phenylalanine derivative.

8. The modified relaxin polypeptide of any one of claims 1-5, wherein the non-naturally encoded amino acid is selected from the group consisting of a para-substituted, ortho-substituted, or meta-substituted phenylalanine.

9. The modified relaxin polypeptide of any one of claims 1 to 5, wherein the non-naturally encoded amino acid is selected from a para-, ortho-or meta-substituted phenylalanine comprising a carbonyl group, an aminooxy group, a hydrazide group, a hydrazine group, a semicarbazide group, an azide group or an alkyne group.

10. The modified relaxin polypeptide of any one of claims 1-5, wherein the non-naturally encoded amino acid comprises para-acetyl-L-phenylalanine.

11. The modified relaxin polypeptide of any one of claims 1-10, wherein the non-naturally encoded amino acid is linked to the peptide component.

12. The modified relaxin polypeptide of any one of claims 1-11, wherein the non-naturally encoded amino acid is linked to the pharmacokinetic enhancer by an oxime linkage or a triazole linkage.

13. The modified relaxin polypeptide of any one of claims 1-11, wherein the non-naturally encoded amino acid is linked to the pharmacokinetic enhancer by an oxime linkage.

14. The modified relaxin polypeptide of claim 1, wherein the relaxin a chain polypeptide comprises SEQ ID NO 35 and the relaxin B chain polypeptide comprises SEQ ID NO 5 or SEQ ID NO 6, and wherein the relaxin a chain or B chain optionally has up to two additional amino acid substitutions, insertions, or deletions.

15. The modified relaxin polypeptide of claim 13, wherein the modified relaxin polypeptide comprises: comprises a relaxin A chain polypeptide having at least 90% amino acid sequence identity to SEQ ID NO. 35 and a relaxin B chain polypeptide having at least 90% amino acid sequence identity to SEQ ID NO. 5 or SEQ ID NO. 6.

16. The modified relaxin polypeptide of claim 15, wherein the relaxin a chain polypeptide has at least 95% amino acid sequence identity to SEQ ID No. 35.

17. The modified relaxin polypeptide of claim 15, wherein the relaxin B chain polypeptide has at least 95% amino acid sequence identity to SEQ ID No. 5 or SEQ ID No. 6.

18. The modified relaxin polypeptide of claim 15, wherein the relaxin a chain polypeptide comprises SEQ ID NO 35 and the relaxin B chain polypeptide comprises SEQ ID NO 5 or SEQ ID NO 6.

19. The modified relaxin polypeptide of any one of claims 1-18, wherein the peptide component comprises 2 to 25 amino acids.

20. The modified relaxin polypeptide of claim 19, wherein the peptide component comprises 2 to 20 amino acids.

21. The modified relaxin polypeptide of claim 19, wherein the peptide component comprises 3 to 10 amino acids.

22. The modified relaxin polypeptide of claim 19, wherein the peptide component comprises 4 to 8 amino acids.

23. The modified relaxin polypeptide of any one of claims 1-18, wherein the peptide component comprises Glu, Glu^γ、GGGGS-Glu^γ(SEQ ID NO:139)、DRDDRD(SEQ ID NO:102)、KKKKKK-Glu^γ(SEQ ID NO:103)、RGGEEKKKEKEK-Glu^γ(SEQ ID NO:104)、GGGEEE-Glu^γ(SEQ ID NO:105)、EEEGGG-Glu^γ(SEQ ID NO:106)、KKKGGG-Glu^γ(SEQ ID NO:107)、GETGSSGEGT-Glu^γ(SEQ ID NO:108)、GGGKKK-Glu^γ(SEQ ID NO:109)、GSHHHHHGS-Glu^γ(SEQ ID NO:110)、Sar-Sar-Sar-Sar-Ser-Sar-Sar-Sar-Sar-Glu^γ(SEQ ID NO:111)、Sar-Sar-Sar-Sar-Ser-Glu^γ(SEQ ID NO:112)、Sar-Sar-Sar-Glu-Glu-Glu^γ(SEQ ID NO:113)、KKKSGGSGG-Glu^γ(SEQ ID NO:118)、KKSGGSGG-Glu^γ(SEQ ID NO:114)、KKSGGSGG-Glu^α(SEQ ID NO:115)、KKSAGSAG-Glu^γ(SEQ ID NO:116)、KSGGSGG-Glu^γ(SEQ ID NO:117)、KKSGGSGGEE-Glu^γ(SEQ ID NO:119)、dKdKdKdKdKdK-Glu^γ(SEQ ID NO:120)、EESGGSGG-Glu^γ(SEQ ID NO:121)、GSGSGSGS-Glu^γ(SEQ ID NO:123)、EEEGGG-dGlu^γ(SEQ ID NO:128)、EGGGGSK-Glu^γ(SEQ ID NO:130)、EEEEEE-Glu^γ(SEQ ID NO:131)、EEEEPEEEEPEEEEPEEEE-Glu^γ(SEQ ID NO:133)、EEEEPEEEEPEEEEPEEGGG(SEQ ID NO:135)、EEEEGEEEEGEEEEGEEEE-Glu^γ(SEQ ID NO:136)、KGGEEKKKEKEKEPKGGEEKKKEKEK-Glu^γ(SEQ ID NO:137)、EAQKAQAEAQKAQAEAQKAQA-Glu^γ(SEQ ID NO:138)、KK-Glu^γ(SEQ ID NO:140)、Glu^γ、KGPKGP-Glu^γ(SEQ ID NO:146)、SGGGS-Glu^γ(SEQ ID NO:147)、KGGGS-Glu^γ(SEQ ID NO:148)、KGGGSE-Glu^γ(SEQ ID NO:149)、GSPGSP-Glu^γ(SEQ ID NO:150)、GGGGP-Glu^γ(SEQ ID NO:151)、EGGS-Glu^γ(SEQ ID NO:152)、EGGGP-Glu^γ(SEQ ID NO:153)、KGPGSE-Glu^γ(SEQ ID NO:154), spermine-Glu^γOr KKGGS-Glu^γ(SEQ ID NO:156)。

24. The modified relaxin polypeptide of any one of claims 1-18, wherein the peptide component comprises GGGGS-Glu^γ(SEQ ID NO:139)、DRDDRD(SEQ ID NO:102)、KKKKKK-Glu^γ(SEQ ID NO:103)、RGGEEKKKEKEK-Glu^γ(SEQ ID NO:104)、GGGEEE-Glu^γ(SEQ ID NO:105)、EEEGGG-Glu^γ(SEQ ID NO:106)、KKKGGG-Glu^γ(SEQ ID NO:107)、GGGKKK-Glu^γ(SEQ ID NO:109)、GSHHHHHGS-Glu^γ(SEQ ID NO:110)、Sar-Sar-Sar-Sar-Ser-Glu^γ(SEQ ID NO:112)、Sar-Sar-Sar-Glu-Glu-Glu^γ(SEQ ID NO:113) or KSGGSGG-Glu^γ(SEQ ID NO:117)。

25. The modified relaxin polypeptide of any one of claims 1-18, wherein the peptide component comprises Glu^γ。

26. The modified relaxin polypeptide of any one of claims 1-18, wherein the peptide component comprises GGGGS-Glu^γ(SEQ ID NO:139)。

27. The modified relaxin polypeptide of any one of claims 1-18, wherein the relaxin polypeptide comprises a relaxin a chain polypeptide of SEQ ID No. 4 and a relaxin B chain polypeptide of SEQ ID No. 5 or SEQ ID No. 6, and is substituted at residue 1 of the a chain with a non-naturally encoded amino acid, wherein the non-naturally encoded amino acid is linked to the pharmacokinetic enhancer, and the pharmacokinetic enhancer comprises the peptide component GGGGS-Glu^γ(SEQ ID NO:139)。

28. The modified relaxin polypeptide of any one of claims 1-27, wherein the half-life extending moiety comprises a fatty acid or derivative thereof, wherein the fatty acid or derivative thereof is covalently linked to the peptide component.

29. The modified relaxin polypeptide of any one of claims 1-27, wherein the half-life extending moiety comprises a saturated fatty acid or a derivative thereof.

30. The modified relaxin polypeptide of any one of claims 1-29, wherein the half-life extending moiety comprises a fatty acid terminated with a carboxylic acid.

31. The modified relaxin polypeptide of any one of claims 1-30, wherein the half-life extending moiety comprises a fatty acid of formula I:

-Cn-COOH (formula I)

Wherein n is between 10 and 18.

32. The modified relaxin polypeptide of claim 31, wherein n is between 12 and 17.

33. The modified relaxin polypeptide of claim 31, wherein n is 13, 14 or 15.

34. The modified relaxin polypeptide of any one of claims 1-33, wherein the half-life extending module comprises-C14-COOH.

35. The modified relaxin polypeptide of any one of claims 1-34, wherein the pharmacokinetic enhancer comprises:

wherein an aminooxy group is attached to a non-natural amino acid in the modified relaxin polypeptide.

36. The modified relaxin polypeptide of any one of claims 1-35, wherein the half-life extending moiety is conjugated to the peptide component through an amide bond.

37. The modified relaxin polypeptide of claim 1, wherein the relaxin polypeptide comprises a relaxin A chain polypeptide of SEQ ID NO 4 and a relaxin B chain polypeptide of SEQ ID NO 5 or SEQ ID NO 6, and is substituted at residue 1 of the A chain with a non-naturally encoded amino acid.

Wherein the non-naturally encoded amino acid comprises para-acetyl-L-phenylalanine linked to the pharmacokinetic enhancer comprising

Comprising GGGGS-Glu^γ(SEQ ID NO: 139); and

a half-life extending module comprising-C14-COOH.

38. A modified relaxin polypeptide comprising:

wherein said AQ 1-relaxin comprises a relaxin A chain polypeptide having at least 90% amino acid sequence identity to SEQ ID NO. 35 and a relaxin B chain polypeptide having at least 90% amino acid sequence identity to SEQ ID NO. 5 or SEQ ID NO. 6, wherein the modified p-acetyl-phenylalanine depicted in formula III is located at the N-terminus of said relaxin A chain polypeptide.

39. The modified relaxin polypeptide of claim 38, wherein the relaxin a chain polypeptide has at least 95% amino acid sequence identity to SEQ ID No. 35.

40. The modified relaxin polypeptide of claim 38, wherein the relaxin B chain polypeptide has at least 95% amino acid sequence identity to SEQ ID No. 5 or SEQ ID No. 6.

41. The modified relaxin polypeptide of claim 38, wherein AQ 1-relaxin comprises the relaxin a chain polypeptide of SEQ ID No. 35 and the relaxin B chain polypeptide of SEQ ID No. 5 or SEQ ID No. 6.

42. AQ1-GGGGS-Glu^γ(SEQ ID NO:139) -C14-COOH, comprising the structure shown in FIG. 8, comprising the structure shown in FIG. 9, or comprising a relaxin A chain polypeptide of SEQ ID NO:35 and a relaxin B chain polypeptide of SEQ ID NO:6, wherein the modified p-acetyl-L-phenylalanine and PK enhancer GGGGS-Glu, located at the N-terminus of the relaxin A chain polypeptide^γ(SEQ ID NO:139) -C14-COOH linkage as depicted in formula III:

43. the modified relaxin polypeptide of any one of claims 1-42, wherein the modified relaxin polypeptide has biological activity.

44. The modified relaxin polypeptide of any one of claims 1-43, wherein the modified relaxin polypeptide has a therapeutic effect in treating one or more diseases or disorders associated with relaxin.

45. The modified relaxin polypeptide of any one of claims 1-44, wherein the modified relaxin polypeptide results in less renal vacuole formation compared to a PEG of AQ1-20 kDa.

46. The modified relaxin polypeptide of any one of claims 1-45, wherein the modified relaxin polypeptide does not result in, or results in a reduction in, renal function impairment as compared to AQ1-20kDa PEG.

47. The modified relaxin polypeptide of claim 46, wherein the renal function is measured by determining one or more of an estimated glomerular filtration rate, creatine clearance, inulin glomerular filtration rate, or isotopic glomerular filtration rate.

48. The modified relaxin polypeptide of any one of claims 1-47, wherein the modified relaxin polypeptide does not reduce renal blood flow after administration.

49. The modified relaxin polypeptide of any one of claims 1-48, wherein the modified relaxin polypeptide is capable of increasing renal blood flow after administration.

50. The method of claim 48 or 49, wherein said renal blood flow is measured by determining clearance of aminohippurate.

51. The modified relaxin polypeptide of any one of claims 1-50, wherein the modified relaxin polypeptide exhibits increased in vivo half-life.

52. The modified relaxin polypeptide of claim 51, wherein the in vivo half-life is increased at least 5-fold, at least 10-fold, at least 20-fold, at least 30-fold, or at least 50-fold.

53. A pharmaceutical composition comprising the modified relaxin polypeptide of any one of claims 1-52, and a pharmaceutically acceptable carrier.

54. A pharmaceutical composition comprising an effective amount of the modified relaxin polypeptide of any one of claims 1-52 for treating a relaxin-related disorder, and a pharmaceutically acceptable carrier.

55. A method of treating a relaxin-related disease comprising administering to a patient in need thereof an effective amount of the modified relaxin polypeptide of any one of claims 1-52 or the composition according to any one of claims 53-54.

56. A method of treating a cardiovascular disease, comprising administering to a patient in need thereof an effective amount of the modified relaxin polypeptide of any one of claims 1-52 or the composition according to any one of claims 53-54.

57. The method of claim 56, wherein the cardiovascular disease is selected from the group consisting of coronary artery disease, heart attack, cardiac arrhythmia, heart failure, cardiomyopathy, and vascular disease.

58. A method of treating or alleviating a symptom of heart failure comprising administering to a patient in need thereof an effective amount of the modified relaxin polypeptide of any one of claims 1-52 or the composition according to any one of claims 53-54.

59. The method of claim 58, wherein the heart failure is selected from the group consisting of advanced heart failure, cardiorenal syndrome, heart failure with impaired renal function, chronic heart failure, midrange ejection fraction chronic heart failure (HFmEF), acute heart failure, post-acute heart failure, compensated heart failure, decompensated heart failure, right heart failure, left heart failure, whole heart failure, ischemic cardiomyopathy, dilated cardiomyopathy, heart failure associated with congenital heart defects, heart failure associated with heart valve defects, heart failure associated with combined heart valve defects, diabetic heart failure, alcoholic cardiomyopathy, heart failure associated with cardiac storage disorders, diastolic heart failure, systolic heart failure, post-myocardial infarction remodeling, heart failure with preserved ejection fraction (HFpEF), heart failure with reduced ejection fraction (HFrEF), Angina pectoris, hypertension, pulmonary hypertension and pulmonary hypertension.

60. The method of claim 58, wherein the heart failure is selected from the group consisting of chronic heart failure, acute heart failure, post-acute heart failure, chronic heart failure with reduced ejection fraction (HFrEF), chronic heart failure with preserved ejection fraction (HFpEF), mid-range type chronic heart failure with ejection fraction (HFmEF), diastolic heart failure, systolic heart failure, post-myocardial infarction remodeling, angina, hypertension, pulmonary hypertension, and pulmonary hypertension.

61. The method of claim 58, wherein the heart failure is selected from the group consisting of post-acute heart failure, advanced heart failure, cardiorenal syndrome, and heart failure with impaired renal function.

62. The method of any one of claims 55-61, further comprising administering in combination with the modified relaxin polypeptide, simultaneously or sequentially, at least one additional therapeutic agent selected from the group consisting of ACE inhibitors, beta blockers, diuretics, saltsCorticoid receptor antagonists, ryanodine receptor modulators, SERCA2a activators, renin inhibitors, calcium channel blockers, adenosine A1 receptor agonists, partial adenosine A1 receptor, dopamine beta-hydroxylase inhibitors, angiotensin II receptor antagonists having agonist-biased effects on selected cell signaling pathways, combinations of angiotensin II receptor antagonists and neutral endopeptidase inhibitors, soluble guanylate cyclase activators, myosin ATPase activators, rho kinase 1 inhibitors, rho kinase 2 inhibitors, apelin receptor agonists, nitroxyl donor compounds, calcium-dependent kinase II inhibitors, antifibrotic agents, galectin-3 inhibitors, vasopressin receptor antagonists, FPR2 receptor modulators, natriuretic peptide receptor agonists, and pharmaceutically acceptable salts thereof, Transient receptor potential vanilloid-4 channel blockers, antiarrhythmics, I_f"pacemaker current" channel blockers, nitrates, digitalis compounds, inotropic agents, beta-receptor agonists, cell membrane resealing agents such as poloxamer 188, antihyperlipidemic agents, plasma HDL-raising agents, antihypercholesterolemic agents, cholesterol biosynthesis inhibitors (such as HMG CoA reductase inhibitors), LXR agonists, FXR agonists, probucol, raloxifene, nicotinic acid, nicotinamide, cholesterol absorption inhibitors, bile acid sequestrants, anion exchange resins, quaternary amines, cholestyramine, colestipol, low density lipoprotein receptor inducers, clofibrate, fenofibrate, bezafibrate, ciprofibrate, gemfibrozil, vitamin B6, vitamin B12, anti-oxidant vitamins, antidiabetics, platelet aggregation inhibitors, fibrinogen receptor antagonists, aspirin and fibric acid derivatives, PCSK9 inhibitors, aspirin, and P2Y12 inhibitors such as clopidogrel.

63. A method of treating a disease associated with fibrosis, comprising administering to a patient in need thereof a therapeutically effective amount of the modified relaxin polypeptide of any one of claims 1-52 or the composition according to any one of claims 53-54.

64. The method of claim 62, wherein the disease associated with fibrosis comprises fibrosis of the heart, lung, kidney, bone marrow or liver, skin fibrosis, or ocular fibrosis.

65. The method of claim 63, wherein the disease associated with fibrosis is selected from the group consisting of liver fibrosis, nonalcoholic steatohepatitis (NASH), cirrhosis, pre-cirrhosis, diffuse parenchymal lung disease, cystic fibrosis, pulmonary fibrosis, progressive massive fibrosis, idiopathic pulmonary fibrosis, injection fibrosis, renal fibrosis, chronic kidney disease, diabetic nephropathy, focal segmental glomerulosclerosis, membranous nephropathy, IgA nephropathy, myelofibrosis, heart failure, metabolic heart failure, myocardial fibrosis, cataract, ocular scarring, pancreatic fibrosis, skin fibrosis, intestinal stenosis, endocardial fibrosis, atrial fibrosis, mediastinal fibrosis, Crohn's disease, retroperitoneal fibrosis, keloids, nephrogenic systemic fibrosis, scleroderma, systemic sclerosis, joint fibrosis, Peyronie syndrome, Dupuytren's (Dupuytren) contracture, diabetic neuropathy, adhesive capsulitis, alcoholic liver disease, hepatic steatosis, viral hepatitis, biliary tract disease, primary hemochromatosis, drug-related cirrhosis, cryptogenic cirrhosis, Wilson's (Wilson) disease, alpha 1-antitrypsin deficiency, Interstitial Lung Disease (ILD), human fibrotic lung disease, macular degeneration, retinopathies, vitreoretinopathy, myocardial fibrosis, Grave's (Grave) eye disease, drug-induced ergotoxemia, cardiovascular disease, atherosclerosis/restenosis, hypertrophic scarring, primary or idiopathic myelofibrosis, inflammatory bowel disease and collagenous colitis.

66. The method of any one of claims 63-65, further comprising administering to said patient said modified relaxin polypeptide in combination with at least one anti-fibrotic drug, simultaneously or sequentially.

67. The method of claim 66, wherein the at least one anti-fibrotic drug is selected from the group consisting of nintedanib, pirfenidone, LPA1 antagonists, LPA1 receptor antagonists, GLP1 analogs, tralokinumab (IL-13, AstraZeneca), vismodegib (hedgehog antagonist, Roche), PRM-151 (pentraxin-2, TGF β -1, Promedeor), SAR-156597 (bispecific antibody IL-4& IL-13, Sanofi), semuzumab (anti-lysyloxidase-like 2 (anti-LOXL 2) antibody, Gilead), CKD-942, PTL-202(PDE inhalation/pentone base/NAC oral controlled release, Pacific ir.), omega collagen (oral PI3K/mTOR inhibitor, GSIWK), O-001 (oral solution, modified bovine V protein, STmuneks), IMMUX-100. alpha. -V/6, Stromedix/Biogen), Actimmune (IFN γ), PC-SOD (intermediate dismutase; inhalation, LTT Bio-Pharma/CKD Pharm), Lygodiizumab (anti-IL-13 SC humanized monoclonal antibody, Roche), AQX-1125(SHIP1 activator, Aquinox), CC-539(JNK inhibitor, Celgene), FG-3019(FibroGen), SAR-100842(Sanofi) and obeticholic acid (OCA or INT-747, Intercept).

68. A method of treating or preventing renal failure, comprising administering to a patient in need thereof a therapeutically effective amount of the modified relaxin polypeptide of any one of claims 1-52 or the composition according to any one of claims 53-54.

69. A method of improving, stabilizing or restoring kidney function in a patient in need thereof, comprising administering a therapeutically effective amount of the modified relaxin polypeptide of any one of claims 1-52 or the composition according to any one of claims 53-54.

70. A method of making a modified relaxin polypeptide according to any one of claims 1-52, comprising:

(a) providing a polypeptide comprising said relaxin a chain and said relaxin B chain, wherein said polypeptide comprises said non-naturally encoded amino acid; and

(b) linking the non-naturally encoded amino acid to the pharmacokinetic enhancer.

71. The method of claim 70, wherein the non-naturally encoded amino acid is linked to the pharmacokinetic enhancer by an oxime linkage.

72. The method of claim 71, wherein the oxime linkage is formed by the reaction of a carbonyl group and an aminooxy group.

73. The method of claim 72, wherein the carbonyl group is a substituent of the non-naturally encoded amino acid and the aminooxy group is a component of the pharmacokinetic enhancer.

74. The method of claim 72, wherein the aminooxy group is a substituent of the non-naturally encoded amino acid and the carbonyl group is a component of the pharmacokinetic enhancer.

75. The method of any one of claims 70-74, wherein the non-naturally encoded amino acid is incorporated into the polypeptide via a ribosome.

Summary of The Invention

Provided herein are modified relaxin polypeptides comprising a non-naturally encoded amino acid, wherein (a) the relaxin polypeptide comprises a relaxin a chain polypeptide SEQ ID NO:4 and a relaxin B chain polypeptide SEQ ID NO:5 or SEQ ID NO:6 substituted with the non-naturally encoded amino acid at a position selected from the group consisting of: an a chain residue 1, an a chain residue 2, an a chain residue 5, an a chain residue 13, an a chain residue 18, a B chain residue 5, a B chain residue 7 and a B chain residue 25, and optionally having up to two additional amino acid substitutions, insertions and/or deletions in the relaxin a chain and/or the relaxin B chain; (b) the non-naturally encoded amino acid has the following structure:

wherein the R group is any substituent other than the side chain found in alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine; and (c) the non-naturally encoded amino acid is linked to a pharmacokinetic enhancer comprising a peptide component of 2 to 30 amino acids and a half-life extending moiety.

Also provided herein are compositions comprising AQ1-GGGGS-Glu^γ(SEQ ID NO:139) -C14-COOH, having the structure:

wherein said AQ 1-relaxin comprises a relaxin A chain polypeptide having at least 90% amino acid sequence identity to SEQ ID NO. 35 and a relaxin B chain polypeptide having at least 90% amino acid sequence identity to SEQ ID NO.5 or SEQ ID NO.6, wherein the modified p-acetyl-phenylalanine depicted in formula III is located at the N-terminus of said relaxin A chain polypeptide.

Also provided herein are compositions comprising AQ 1-relaxin-GGGGS-Glu^γ(SEQ ID NO:139) -C14-COOH, having the structure of formula III as described herein, wherein AQ 1-relaxin comprises the relaxin a chain polypeptide SEQ ID NO:35 and the relaxin B chain polypeptide SEQ ID NO:6, wherein the modified p-acetyl-phenylalanine depicted in formula III is located at the N-terminus of said relaxin a chain polypeptide.

Also provided herein are modified relaxin polypeptides comprising the structure shown in figure 8.

Also provided herein are modified relaxin polypeptides comprising the structure shown in figure 9.

In another aspect, provided herein is a pharmaceutical composition comprising a modified relaxin polypeptide as described herein and a pharmaceutically acceptable carrier.

In another aspect, provided herein are pharmaceutical compositions comprising an effective amount of a modified relaxin polypeptide as described herein for treating a relaxin-related disorder and a pharmaceutically acceptable carrier.

In another aspect, provided herein are methods of treating a relaxin-related disease comprising administering an effective amount of a modified relaxin polypeptide or a composition comprising a modified relaxin polypeptide as described herein.

In another aspect, provided herein is a method of treating a cardiovascular disease comprising administering to a patient in need thereof an effective amount of a modified relaxin polypeptide or a composition comprising a modified relaxin polypeptide as described herein.

In another aspect, provided herein is a method of treating or alleviating a symptom of heart failure comprising administering to a patient in need thereof an effective amount of a modified relaxin polypeptide or a composition comprising a modified relaxin polypeptide as described herein.

In another aspect, provided herein is a method of treating a disease associated with fibrosis, comprising administering to a patient in need thereof a therapeutically effective amount of a modified relaxin polypeptide or a composition comprising a modified relaxin polypeptide as described herein.

In another aspect, provided herein is a method of treating or preventing renal failure, comprising administering to a patient in need thereof a therapeutically effective amount of a modified relaxin polypeptide or a composition comprising a modified relaxin polypeptide as described herein.

In another aspect, provided herein is a method of improving, stabilizing or restoring kidney function in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of a modified relaxin polypeptide or a composition comprising a modified relaxin polypeptide as described herein.

In another aspect, provided herein is a method of making a modified relaxin polypeptide as described herein, comprising: (a) providing a polypeptide comprising said relaxin a chain and said relaxin B chain, wherein said polypeptide comprises said non-naturally encoded amino acid; and (b) linking the non-naturally encoded amino acid to the pharmacokinetic enhancer.

Brief description of several views of the drawings

FIG. 1 is a sequence of a human relaxin 2 polypeptide without a non-naturally encoded amino acid. The A chain (SEQ ID NO:4) and B chain (SEQ ID NO:6) polypeptides are linked by a disulfide bond as depicted in FIG. 5. It has been observed that modification of position 1 (underlined position, shown as substitution of aspartic acid with alanine) in the B chain relative to the human relaxin 2 polypeptide B chain (SEQ ID NO:5) improves manufacturing without significantly adversely affecting potency. In the working examples herein, the polypeptide is referred to as "wild-type" or WT-RLX.

FIG. 2. sequence of a human relaxin 2 polypeptide RLX-AQ1 ("AQ 1") in which position 1 of the A chain is replaced by the non-naturally encoded amino acid pair-acetyl-phenylalanine (pAcF, underlined). The A chain (SEQ ID NO:35) and the B chain (SEQ ID NO:6) are linked by a disulfide bond at the positions depicted in FIG. 5.

FIG. 3. sequence of a human relaxin 2 polypeptide RLX-BM25/AN1 ("BM 25/AN 1"), in which position 25 of the B chain is substituted with the non-naturally encoded amino acid pair acetyl-phenylalanine (pAcF, underlined) and further position 1 of the A chain contains a glutamine to asparagine substitution (substitution underlined). The A chain (SEQ ID NO:36) and B chain (SEQ ID NO:37) are linked by a disulfide bond at the positions depicted in FIG. 5.

FIG. 4 is a schematic representation of the prepro-relaxin polypeptide used to express RLX-AQ 1. The prepro-relaxin polypeptide (SEQ ID NO:38) is expressed in E.coli or another expression system. Non-naturally encoded amino acids such as pAcF can be ribosomally incorporated into the prepro-relaxin polypeptide sequence to facilitate subsequent linkage to a pharmacokinetic enhancer ("PK extender"). Grey circles indicate leader sequences specific for e.coli expression. The black circle indicates the C peptide (linker peptide) which is derived from proinsulin with the addition of a peptide cleavage site and a substitution of Lys to Gly. The leader sequence and C peptide were removed by enzymatic cleavage (cleavage site indicated by unlabeled arrow). The alanine substitution at residue 1 ("B1") of the B chain (present in SEQ ID NO:6) helped to remove the leader sequence. The pAcF residues for attachment of the site-specific PK enhancer are indicated. The A and B chains can be aligned with the HUGO named RLN2, which is derived from the relaxin 2 transcript variant 1cDNA (refseq NM-134441).

FIG. 5 structural depiction of mature RLX-AQ1 comprising an A chain (SEQ ID NO:36) and a B chain (SEQ ID NO:6) linked to a pharmacokinetic enhancer ("PK extender") through the non-naturally encoded amino acid at position 1 of the A chain.

FIGS. 6A-6B.WT-RLX (FIG. 6A) and AQ1-GGGGS-Glu^γ(SEQ ID NO:139) -C14-COOH (FIG. 6B) on changes in smooth muscle α -actin gene expression in TGF- β treated human cardiac fibroblasts, where mRNA concentrations were measured using qRT-PCR. The numbers above the bars indicate the percent reduction relative to TGF- β, with vehicle alone set at baseline. Mean ± SEM; p<0.05. Veh is vehicle. nanomolar nM.

Figure 7A-7d PEG-related renal cortical tubular epithelial vacuole formation in female Sprague Dawley rats. Kidneys from rats treated once daily with vehicle or relaxin analogue Subcutaneously (SQ). PEG-containing pineRelaxin analogs (fig. 7B and 7C) showed significant cortical tubule vacuole formation characterized by the presence of large coalesced vacuoles. No vacuoles were observed in animals treated with vehicle (fig. 7A) or non-pegylated relaxin (fig. 7D). FIG. 7A: treatment was performed subcutaneously with vehicle (150 mM Arg in 10mM citrate buffer pH 5.5) for 10 days. FIG. 7B: the cells were treated subcutaneously with AQ1-PEG36-Glu-C13-COOH (1.5kDPEG) at 40 mg/kg/day for 7 days. FIG. 7C: the cells were treated subcutaneously with AQ1-20-kDa-PEG (20kD PEG) at 15 mg/kg/day for 10 days. FIG. 7D: using AQ1-GGGGS-Glu^γ(SEQ ID NO:139) -C14-COOH was subcutaneously treated at 40mg/kg for 7 days. Magnification of 40 times, H&E。

FIG. 8 AQ1-GGGGS-Glu depicted in formula III^γ(SEQ ID NO:139) -C14-COOH.

FIG. 9 AQ1-GGGGS-Glu depicted in formula III^γ(SEQ ID NO:139) -C14-COOH.

Detailed Description

Definition of

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 "relaxin", "relaxin polypeptide" or "modified relaxin polypeptide" refers to one or more such proteins and includes equivalents thereof known to those of ordinary skill in the art, and so forth.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

The terms "pharmacokinetic extender," "pharmacokinetic enhancer," "PK extender," and "PK enhancer" are used interchangeably and all refer to a pharmaceutically acceptable moiety, domain, or molecule that is covalently linked ("conjugated" or "fused") to a modified relaxin polypeptide described herein, optionally linked by a non-naturally encoded amino acid; it may prevent, delay or mitigate proteolytic degradation or other activity-attenuating chemical modifications of the modified relaxin polypeptide in vivo, increase half-life (serum half-life and/or therapeutic half-life), and/or improve or alter other pharmacokinetic or biophysical properties, including but not limited to increasing the absorption rate of the modified relaxin polypeptide, decreasing toxicity, improving solubility, reducing aggregation, increasing biological activity and/or target selectivity, and/or decreasing immunogenicity of the modified relaxin polypeptide, as compared to a comparison subject, e.g., an unconjugated form of the modified relaxin polypeptide or a wild-type relaxin polypeptide. The PK enhancer described herein may include a peptide component comprising one or more amino acids and a half-life extending moiety.

The term "half-life extending moiety" refers to a pharmaceutically acceptable moiety, domain or molecule that is covalently linked ("conjugated" or "fused") to a modified relaxin polypeptide described herein, either directly or through a linker (e.g., a peptide component or PEG), optionally through a non-naturally encoded amino acid, e.g., as a component of a PK extender; it increases half-life (serum half-life and/or therapeutic half-life) and/or improves or alters other pharmacokinetic or biophysical properties, including but not limited to increasing the rate of absorption of the modified relaxin polypeptide, decreasing toxicity, improving solubility, reducing aggregation, increasing biological activity and/or target selectivity, increasing manufacturability, and/or decreasing immunogenicity of the modified relaxin polypeptide, as compared to a comparison subject (e.g., an unconjugated form of the modified relaxin polypeptide or a wild-type relaxin polypeptide). The term half-life extending module includes, but is not limited to, non-proteinaceous half-life extending modules such as fatty acids or derivatives thereof, water soluble polymers such as polyethylene glycol (PEG) or discrete PEG, hydroxyethyl starch (HES), lipids, branched or unbranched acyl groups, branched or unbranched C8-C30 acyl groups, branched or unbranched alkyl groups and branched or unbranched C8-C30 alkyl groups; proteinaceous half-life extending moieties, such as serum albumin, transferrin, adnectins (e.g., albumin binding or pharmacokinetic extension (PKE) adnectins), Fc domains, and unstructured polypeptides, such as XTEN and PAS polypeptides (e.g., conformationally disordered polypeptide sequences consisting of amino acids Pro, Ala, and/or Ser), and fragments of any of the foregoing. The term "linker" or "spacer" may be any component that links the half-life extending moiety to the modified relaxin polypeptide. Exemplary linkers include, but are not limited to, small organic compounds, water soluble polymers of various lengths, such as poly (ethylene glycol) or polyglucan, and peptides or polypeptides having, for example, up to 50, 40, 30, 25, 20, 15, 10, or up to 6 amino acids in length.

The term "peptide component" refers to a component of a PK extender and comprises peptides up to 50 amino acids in length. Exemplary peptide components include, but are not limited to, peptides up to 40, 30, 25, 20, 15, 10, 9,8, 7, 6, 5,4, 3,2, or 1 amino acid in length. Such a peptide component may covalently link the half-life extending moiety to the modified relaxin polypeptides described herein. In some embodiments, the peptide component may comprise 2-50 amino acids. In some embodiments, the peptide component may comprise 2-30 amino acids. In some embodiments, the peptide component may comprise 3-20 amino acids. In some embodiments, the peptide component may comprise 5-10 amino acids.

The term "stability" or "thermostability" refers to the ability of a polypeptide to resist unfolding when heated. Generally, the higher the thermostability of the molecule, the higher the temperature required for unfolding of the polypeptide. Exemplary methods of determining the thermal stability of a polypeptide are Differential Scanning Calorimetry (DSC) and thermal scanning fluorescence. Thermostability can be determined relative to a subject compound of comparison, e.g., to identify polypeptides having increased thermostability.

The term "aggregation" refers to the tendency of a polypeptide to form a complex with other molecules (e.g., other molecules of the same polypeptide) in a non-covalent linkage, thereby forming a high molecular weight complex. An exemplary method of measuring aggregate formation includes analytical size exclusion chromatography. The relative amount of aggregation can be determined relative to a subject compound of comparison, e.g., to identify polypeptides with reduced aggregation.

The term "deamidation" refers to the tendency of amino acid residues within a polypeptide to spontaneously undergo deamidation reactions thereby altering the chemical structure of the amino acid and possibly affecting the function of the polypeptide. An exemplary method of measuring deamidation includes imaging capillary isoelectric focusing (icIEF). The relative amount of deamidation can be determined relative to a subject compound of comparison, e.g., to identify a polypeptide having reduced deamidation.

The term "in vivo proteolysis" refers to cleavage of a polypeptide when introduced into a living system (e.g., when injected into an organism) that may be caused by proteases present in the organism. Proteolysis can potentially affect the biological activity or half-life of the polypeptide. For example, wild-type relaxin may undergo cleavage, resulting in a truncated inactive polypeptide. An exemplary method of measuring in vivo proteolysis of relaxin is an electrochemiluminescence immunoadsorption assay (ECLIA) based on Meso Scale Discovery (MSD). The relative amount of in vivo proteolysis can be determined relative to a subject compound of comparison, e.g., to identify polypeptides with reduced in vivo proteolysis.

The term "solubility" refers to the amount of one substance that can be dissolved in another substance, e.g., the amount of unmodified or modified relaxin polypeptide that can be dissolved in an aqueous solution. An exemplary method of measuring the solubility of an unmodified or modified relaxin polypeptide is a plug flow solubility test. Relative solubility can be determined relative to a subject compound, e.g., to identify polypeptides with increased solubility.

The term "biological activity" or "biological activity" refers to the ability of a molecule to affect any physical or biochemical characteristic of a biological system, pathway, molecule, or interaction associated with an organism, including but not limited to viruses, bacteria, bacteriophages, transposons, prions, insects, fungi, plants, animals, and humans. For example, in the context of unmodified or modified relaxin, biological activity includes any function performed by relaxin. For example, a "biologically active" modified relaxin polypeptide may exhibit one or more biological activities of wild-type relaxin, including but not limited to: activation of the relaxin 2 receptor RXFP1, including human and non-human RXFP1 orthologs, activation of another relaxin receptor, such as RXFP2, including human and non-human orthologs, anti-fibrotic activity in vitro or in vivo, efficacy in treating heart failure or fibrotic diseases (whether in human, non-human animals and/or model systems), and other biological activities disclosed herein, e.g., reducing Systemic Vascular Resistance (SVR), increasing cardiac output, increasing overall arterial compliance, increasing Renal Blood Flow (RBF) and/or Glomerular Filtration Rate (GFR). Exemplary methods of determining whether a molecule has at least one biological activity of wild-type relaxin (e.g., wild-type relaxin polypeptides of SEQ ID NO:4 and SEQ ID NO:5) can include in vitro activity assays or receptor binding assays, e.g., intracellular cAMP accumulation assays. The relative level of biological activity can be determined relative to a subject compound of comparison, e.g., identifying a polypeptide having biological activity or sufficiently high biological activity for the intended therapeutic use, e.g., having less than 5-fold, 10-fold, less than 20-fold, less than 50-fold, or less than 100-fold higher EC50 in an in vitro or in vivo activity assay relative to the EC50 of a comparator.

The compound of interest for comparison described herein may be another sequence lacking modifications (e.g., the modifications described herein). For example, the comparison subject compound may be the same modified relaxin polypeptide sequence that is not linked to a PK extender. Exemplary comparison subject compounds include, but are not limited to, a wild-type relaxin polypeptide having SEQ ID NO 4 and SEQ ID NO 5, a modified relaxin polypeptide having SEQ ID NO 4 and SEQ ID NO 6, a modified relaxin polypeptide having SEQ ID NO 36 and SEQ ID NO 6, a modified relaxin polypeptide having SEQ ID NO 35 and SEQ ID NO 6, or another comparison subject compound. In some embodiments, the subject compound may contain at least one non-naturally encoded amino acid, which may be linked to a linker, polymer, biologically active molecule, peptide, polypeptide, or half-life extending moiety described herein (e.g., PEG or fatty acid). In some embodiments, the subject compound may contain at least one non-naturally encoded amino acid, which may or may not be linked to a linker, polymer, biologically active molecule, peptide, polypeptide, or half-life extending moiety described herein. In some embodiments, the subject compounds may contain additional amino acid substitutions, deletions, and/or insertions. In some embodiments, the comparison may be made with acylated or non-acylated forms of the polypeptides; in the former case, the comparison may be made with polypeptides that include or do not include the non-naturally encoded amino acid.

The term "corresponding" refers to a position ("corresponding position" or "corresponding position") or region ("corresponding region" or "corresponding region") within a polypeptide or polynucleotide sequence that is identified by comparison to a reference sequence. The reference sequence may be a wild-type or unmodified sequence, such as a wild-type relaxin polypeptide having SEQ ID NO 4 and SEQ ID NO 5. The corresponding position or region can be identified by alignment of the sequence with a reference sequence. For example, "a position corresponding to amino acid 1 in the relaxin A chain" refers to a position in a sequence that is in the same alignment as amino acid 1 in SEQ ID NO:4 when the sequence is aligned with SEQ ID NO: 4. In an alignment, an amino acid or nucleotide may or may not match an amino acid or nucleotide at a corresponding position in a reference sequence.

Alignments for identifying corresponding positions or regions can be obtained using conventional alignment algorithms, such as Blast (Altschul et al, J Mol biol.1990 Oct 5; 215(3):403-10), Smith-Waterman (Smith and Waterman, J Mol biol.1981Mar 25; 147(1):195-7) or Needleman-Wunsch (Needleman and Wunsch, J Mol biol.1970Mar; 48(3): 443-53). To obtain the highest scoring global alignment (i.e., an alignment comprising every residue in both sequences, but which may begin and/or end at a gap), the Needleman-Wunsch algorithm may be used. Whether using Blast, Smith-Waterman or Needleman-Wunsch, the "default" parameters can be used to identify the highest scoring alignment, for example using the BLOSUM62 scoring matrix, the gap opening penalty of 11, the gap extension penalty of 1, and (when aligned using Blast) word size of 3.

The term "diseases associated with fibrosis" includes diseases, disorders and conditions in which fibrosis has been observed to occur, or fibrosis is known or believed to be associated with or contributing to the disease cause, progression or symptom, or fibrosis is known or believed to occur as the disease progresses. Fibrosis can affect organs or tissues such as pancreas, lung, heart, kidney, liver, eye, nervous system, bone marrow, lymph nodes, endocardium or posterior peritoneum. Exemplary diseases associated with fibrosis include, but are not limited to, nonalcoholic steatohepatitis (NASH), liver fibrosis, pre-cirrhosis, diffuse parenchymal lung disease, cystic fibrosis, lung or lung fibrosis, progressive massive fibrosis, idiopathic pulmonary fibrosis, injection fibrosis, fibrosis of the kidney or kidney, chronic kidney disease, diabetic nephropathy, focal segmental glomerulosclerosis, membranous nephropathy, IgA nephropathy, myelofibrosis, heart failure, metabolic heart failure, myocardial fibrosis, cataract, ocular scarring, pancreatic fibrosis, skin fibrosis, intestinal stenosis, endocardial myocardial fibrosis, atrial fibrosis, mediastinal fibrosis, crohn's disease, retroperitoneal fibrosis, keloids, nephrogenic fibrosis, scleroderma, systemic sclerosis, joint fibrosis, liver fibrosis, cystic fibrosis, pulmonary fibrosis, and pulmonary fibrosis, Peyronie syndrome, Dupuytren's (Dupuytren) contracture, diabetic neuropathy, adhesive capsulitis, alcoholic liver disease, hepatic steatosis, viral hepatitis, biliary tract disease, primary hemochromatosis, drug-related cirrhosis, cryptogenic cirrhosis, Wilson's (Wilson) disease, alpha 1-antitrypsin deficiency, Interstitial Lung Disease (ILD), human fibrotic lung disease, macular degeneration, retinopathies, vitreoretinopathy, myocardial fibrosis, Grave's (Grave) eye disease, drug-induced ergotoxemia, cardiovascular disease, atherosclerosis/restenosis, hypertrophic scarring, primary or idiopathic myelofibrosis, and inflammatory bowel disease (including but not limited to collagenous colitis). In some embodiments, the diseases associated with fibrosis may include liver fibrosis, kidney or kidney fibrosis, lung or lung fibrosis, and heart or heart fibrosis. In some embodiments, the disease associated with fibrosis can be liver fibrosis. In some embodiments, the disease associated with fibrosis may be NASH.

The term "substantially purified" refers to an unmodified or modified relaxin polypeptide that may be substantially or essentially free of proteins or components that interact with proteins typically found in the host cell with which it naturally occurs (i.e., naive cells) or in the case of recombinantly produced unmodified or modified relaxin polypeptides. Unmodified or modified relaxin polypeptides that may be substantially free of cellular material include preparations of protein having less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1% (dry or wet) contaminating protein. Thus, a "substantially purified" unmodified or modified relaxin polypeptide produced by a method of the disclosure may have a purity level of at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, specifically at least about 75%, 80%, 85%, and more specifically at least about 90%, at least about 95%, at least about 99% or higher, as determined by suitable methods such as SDS/PAGE analysis, RP-HPLC, SEC, and capillary electrophoresis.

"recombinant host cell" or "host cell" refers to a cell that comprises an exogenous polynucleotide regardless of the method used for insertion, e.g., direct uptake, transduction, f-mating, or other methods known in the art for establishing recombinant host cells. The exogenous polynucleotide may be maintained in the form of a non-integrating vector, such as a plasmid, or may be integrated into the host genome.

As used herein, the term "eukaryote" refers to organisms belonging to the phylogenetic domain eukaryotic (Eucarya), such as animals (including but not limited to mammals, insects, reptiles, birds, etc.), ciliates, plants (including but not limited to monocots, dicots, algae, etc.), fungi, yeasts, flagellates, microsporidia, protists, etc.

As used herein, the term "non-eukaryotic organism" refers to a non-eukaryotic organism or a prokaryote. For example, the non-eukaryotic organism may belong to the phylogenetic domain of eubacteria (including, but not limited to, Escherichia coli, Thermus thermophilus, Bacillus stearothermophilus, Pseudomonas fluorescens, Pseudomonas aeruginosa, Pseudomonas putida, etc.) or the phylogenetic domain of archaea (including, but not limited to, Methanococcus jannaschii, Methanobacterium thermoautotrophicum, Halobacterium such as Haloferax volcanii and Halobacterium species NRC-1, Archaeoglobus fulgidus, Pyrococcus furiosus, Pyrococcus horikoshii, Pyrococcus pernici (Aeuropyrromyces pernix, etc.).

As used herein, the term "culture medium" or "medium" includes any medium, solution, solid, semi-solid, or rigid support that can support or contain any host cell, including bacterial host cells, yeast host cells, insect host cells, plant host cells, eukaryotic host cells, mammalian host cells, CHO cells, prokaryotic host cells, escherichia coli, or pseudomonas host cells, as well as the cell contents. Thus, the term may encompass media in which the host cell has been grown, for example media in which the unmodified or modified relaxin polypeptide has been secreted, including media before or after the propagation step. The term may also encompass buffers or reagents containing host cell lysates, for example where unmodified or modified relaxin polypeptides are produced intracellularly and the host cell is lysed or disrupted to release the unmodified or modified relaxin polypeptides.

As used herein, the term "relaxin" refers to human relaxin, particularly human relaxin 2 (also known as H2 relaxin or RLN2), the amino acid sequence and spatial structure of which are well known unless the context indicates otherwise. Human relaxin consists of an a chain of twenty-four amino acids and a B chain of twenty-nine amino acids cross-linked by disulfide bonds (see fig. 5). Correctly cross-linked wild-type relaxin contains three disulfide bridges: one between position 11 of the A chain and position 11 of the B chain, a second between position 24 of the A chain and position 23 of the B chain, and a third between positions 10 and 15 of the A chain.

As used herein, "modified relaxin polypeptides" or "modified relaxin" are used interchangeably and include those polypeptides and proteins that differ from wild-type relaxin (e.g., wild-type human relaxin with SEQ ID NO:4 and SEQ ID NO:5) and generally have at least one biological activity of relaxin 2, as well as relaxin analogs, relaxin isoforms, relaxin mimetics, polymorphisms (e.g., naturally occurring relaxin sequence variants), relaxin fragments, hybrid relaxin proteins, fusion proteins, oligomers and multimers, homologs, glycosylation pattern variants, splice variants, and muteins thereof. The terms "modified relaxin polypeptide" and "modified relaxin" include relaxin polypeptides comprising one or more amino acid substitutions, additions or deletions.

Substitutions may be introduced at numerous amino acid positions in naturally occurring relaxin to produce modified relaxin polypeptides. Substitutions include, but are not limited to, substitutions that modulate solubility or stability, increase agonist activity, increase in vivo or in vitro half-life, increase protease resistance, decrease immunogenicity or toxicity, facilitate purification or manufacturability, and the like, and are encompassed by the term "modified relaxin polypeptide" or "modified relaxin". In some embodiments, the non-naturally encoded amino acid substitutions described herein can be combined with other additions, substitutions, or deletions within the modified relaxin polypeptide to affect the biological trait of the modified relaxin polypeptide relative to another relaxin polypeptide (e.g., a wild-type relaxin polypeptide having SEQ ID nos. 4 and 5, or another relaxin polypeptide such as the same relaxin polypeptide without the additions, substitutions, or deletions, or another unmodified or modified relaxin or unmodified or modified polypeptide). In some embodiments, other additions, substitutions, or deletions may increase the stability (including but not limited to resistance to proteolytic degradation) of the modified relaxin polypeptide or increase the affinity of the modified relaxin polypeptide for its receptor. In some cases, other additions, substitutions, or deletions may increase the drug stability of the modified relaxin polypeptide. In some embodiments, other additions, substitutions, or deletions may increase the solubility of the modified relaxin polypeptide (including, but not limited to, when expressed in e. In some embodiments, the addition, substitution, or deletion can increase the solubility of the polypeptide after expression in E.coli or other recombinant host cells. In some embodiments, sites for substitution with naturally encoded or unnatural amino acids can be selected, in addition to another site for incorporation of an unnatural amino acid that results in increased solubility of the polypeptide after expression in E.coli or other recombinant host cells.

In some embodiments, the modified relaxin polypeptide further comprises an additional insertion, substitution, or deletion that modulates a biological activity of the modified relaxin polypeptide. For example, additions, substitutions, or deletions may modulate one or more properties or activities of the modified relaxin. For example, additions, substitutions or deletions may modulate affinity for a relaxin polypeptide receptor, binding protein or related ligand, modulate signal transduction upon binding to a relaxin receptor, modulate in vivo or circulatory half-life, modulate therapeutic half-life, modulate stability of the polypeptide, modulate cleavage by proteases, modulate dosage, modulate release or bioavailability, facilitate purification, reduce deamidation, reduce aggregation, improve shelf-life or in vitro half-life, or modify or alter a particular route of administration. Similarly, modified relaxin polypeptides may comprise protease cleavage sequences, reactive groups, antibody binding domains (including but not limited to FLAG or polyhistidine (poly-His)) or other affinity-based sequences (including but not limited to FLAG, poly-His, glutathione-S transferase (GST), etc.) or linker molecules (including but not limited to biotin) that improve detection (including but not limited to GFP), purification or other properties of the polypeptide.

The term "modified relaxin polypeptides" also includes biologically active fragments, biologically active variants and stereoisomers of naturally occurring relaxin as well as agonist, mimetic and antagonist variants of naturally occurring relaxin and polypeptide fusions thereof. Fusions comprising additional amino acids at the amino terminus, the carboxy terminus, or both are encompassed by the term "modified relaxin polypeptide". Exemplary fusions include, but are not limited to, for example, methionyl relaxin, in which methionine is linked to the N-terminus of a relaxin a chain and/or B chain produced by recombinant expression of a mature form of relaxin or a portion thereof lacking a leader sequence or signal peptide (e.g., methionine is linked to the N-terminus of a relaxin a chain and/or B chain produced, for example, by recombinant expression in e.coli); fusions for purification purposes (including but not limited to polyhistidine or affinity epitopes); fusions with serum albumin binding peptides (e.g., PKE adnectins) and fusions with serum proteins (e.g., serum albumin); and fusion proteins comprising relaxin and one or more other molecules ("fusion partners") including, but not limited to, serum albumin, Fc domains, immunoglobulin constant regions, unstructured polypeptides, and adnectins and fragments thereof. Any such fragment can be prepared from the protein by standard biochemical methods or by expression of the polynucleotide encoding the fragment.

Unless otherwise indicated, the terms "relaxin polypeptide" and "relaxin" as used herein encompass unmodified (i.e., wild-type) relaxin and modified relaxin polypeptides.

The term "modified relaxin polypeptide" includes polypeptides conjugated to a polymer (e.g., PEG or PK extender), and optionally comprising derivatization of one or more cysteine, lysine, or other residues. In some embodiments, the modified relaxin polypeptide may comprise a linker, polymer, or PK extender, wherein the amino acid conjugated to the linker, polymer, or PK extender may be a non-natural amino acid according to the present disclosure or a naturally encoded amino acid (e.g., lysine or cysteine) using techniques known in the art (e.g., coupling to lysine or cysteine).

The term "modified relaxin polypeptide" also includes relaxin polypeptides conjugated or linked to a PK extender described herein through a naturally encoded amino acid (e.g., cysteine or lysine) in the relaxin polypeptide. In some embodiments, the naturally encoded amino acid may be a substitution or insertion in a relaxin polypeptide.

The term "modified relaxin polypeptide" also includes glycosylation modified relaxins, such as, but not limited to, polypeptides glycosylated at any amino acid position, N-linked or O-linked glycosylated forms of polypeptides. In addition, splice variants are also included.

All references herein to amino acid positions in unmodified or modified relaxin are based on the corresponding positions in the A chain of SEQ ID NO:4 or the B chain of SEQ ID NO:5, unless otherwise indicated. One skilled in the art will appreciate that amino acid positions in any other relaxin molecule (e.g., relaxin fusions, variants, fragments, etc.) that correspond to positions in SEQ id No.4 or 5 or another relaxin sequence can be readily identified. For example, a sequence alignment program (e.g., BLAST) can be used to align and identify specific positions in a protein that correspond to positions in SEQ ID NO:4 or 5 or other relaxin sequences. With reference to SEQ ID NOs 4 or 5 or other relaxin sequences, amino acid substitutions, deletions or additions described herein are also contemplated for substitutions, deletions or additions at corresponding positions in relaxin fusions, variants, fragments, etc., described herein or known in the art, and are expressly encompassed by the present disclosure.

The term "modified relaxin polypeptide" also encompasses homodimers, heterodimers, homomultimers, and heteromultimers formed or linked via a fusion partner (e.g., an Fc domain), including, but not limited to, direct linkage via a non-naturally encoded amino acid side chain (same or different non-naturally encoded amino acid side chain), a naturally encoded amino acid side chain, or indirect linkage via a linker. Exemplary linkers include, but are not limited to, small organic compounds, water soluble polymers of various lengths, such as poly (ethylene glycol) or polyglucan, or peptides or polypeptides having, for example, up to 50, 40, 30, 25, 20, 15, 10, 9,8, 7, 6, 5,4, 3,2, or up to 1 amino acid in length.

As used herein, the term "modified" refers to any change made to a given polypeptide, such as a change in polypeptide length, amino acid sequence, chemical structure, co-translational modification or post-translational modification of the polypeptide. The term "post-translationally modified" refers to any modification that occurs to a natural or unnatural amino acid after that amino acid has been incorporated into a polypeptide chain. Merely by way of example, the term encompasses co-translational in vivo modifications, co-translational in vitro modifications (e.g., in a cell-free translation system), post-translational in vivo modifications, and post-translational in vitro modifications.

By "non-naturally encoded amino acid" is meant "amino acid" is meant an amino acid that is not one of the 20 common amino acids or pyrrolysine or selenocysteine. Other terms that may be used synonymously with the term "non-naturally encoded amino acid" are "non-natural amino acid", "unnatural amino acid", "non-naturally occurring amino acid", and various hyphenated and non-hyphenated forms thereof. The term "non-naturally encoded amino acid" also includes, but is not limited to, amino acids that occur through modification (e.g., post-translational modification) of naturally encoded amino acids (including, but not limited to, the 20 common amino acids or pyrrolysine and selenocysteine), but which are not themselves naturally incorporated into a growing polypeptide chain through translation complexes. Examples of such non-naturally encoded amino acids include, but are not limited to, N-acetylglucosaminyl-L-serine, N-acetylglucosaminyl-L-threonine, and O-phosphotyrosine.

In the context of modified relaxin polypeptides, the terms "para-acetyl-phenylalanine" and "para-acetyl-L-phenylalanine" (including the abbreviations pAcF and pAF) as used herein also include chemical reaction products in which the acetyl group at the para-position is not present, but instead is linked to another molecule. Thus, for example, in a modified relaxin polypeptide comprising p-acetyl-phenylalanine (e.g., p-acetyl-L-phenylalanine), the p-acetyl-phenylalanine may comprise an oxime, triazole, amide or other linkage in the para position, e.g., having an oxime linkage in the para position resulting from the reaction of the carbonyl group of the p-acetyl-L-phenylalanine with an aminooxy group, and no carbonyl carbon. Such a para-acetyl-phenylalanine may be referred to as a "modified para-acetyl-phenylalanine", such as a "modified para-acetyl-L-phenylalanine".

An exemplary non-naturally encoded amino acid is para-acetyl-phenylalanine (pACF). The amino acid may be synthesized using the procedures previously described in Zhang, z., Smith, b.a.c., Wang, l., Brock, a., Cho, C. & Schultz, p.g., Biochemistry, (2003)42, 6735-.

An "amino-terminal modifying group" refers to any molecule that can be attached to the amino terminus of a polypeptide. Similarly, a "carboxy-terminal modifying group" refers to any molecule that can be attached to the carboxy terminus of a polypeptide. Terminal modifying groups include, but are not limited to, various water soluble polymers, methionine, peptides or proteins, such as serum albumin, Fc domains, immunoglobulin constant regions, unstructured polypeptides, adnectins or fragments thereof, fatty acids or derivatives thereof, or other moieties that increase the serum (in vivo) half-life of the polypeptide.

The terms "functional group," "active moiety," "activating group," "leaving group," "reactive site," "chemically reactive group," and "chemically reactive moiety" are used in the art and herein to refer to a clearly distinguishable, definable portion or unit of a molecule. These terms are to some extent synonymous in the chemical arts and are used herein to refer to a moiety in a molecule that performs a function or activity and is capable of reacting with other molecules.

The term "attached" is used herein to refer to a group or bond that is typically formed by a chemical reaction, and is typically a covalent attachment. A hydrolytically stable linkage means that the linkage is substantially stable in water and does not react with water at useful pH values, including but not limited to under physiological conditions for extended periods of time, possibly indefinitely. Hydrolytically unstable or degradable linkages means that the linkages can degrade in water or aqueous solutions, including, for example, blood. An enzymatically labile or degradable linkage means that the linkage can be degraded by one or more enzymes. As understood in the art, PEG, fatty acids, and other polymers may include degradable linkages in the polymer backbone or in the linking group between the polymer backbone and one or more terminal functional groups of the polymer molecule. For example, the ester linkage that may be formed by the reaction of a carboxylic acid or activated carboxylic acid with an alcohol group on a bioactive agent is typically hydrolyzed under physiological conditions to release the bioactive agent. Other hydrolytically degradable linkages include, but are not limited to, carbonate linkages; imine linkages, which may result from the reaction of an amine and an aldehyde; a phosphate ester bond, which can be formed by reacting an alcohol with a phosphate group; a hydrazone linkage, which may be the reaction product of a hydrazide and an aldehyde; an acetal linkage, which may be the reaction product of an aldehyde and an alcohol; ortho ester linkages, which may be the reaction product of a formate ester and an alcohol; peptide bonds, which may be formed from, but are not limited to, amine groups at the termini of the polymer and carboxyl groups of the peptide; oxime or semicarbazone linkages which may be the reaction product of a carbonyl functional group and a hydroxylamine-containing, or semicarbazone-containing reagent; and oligonucleotide linkages, which may be formed from phosphoramidite groups at the polymer termini and the 5' hydroxyl groups of the oligonucleotides.

Where substituents are specified by their conventional formula, written left to right, they also encompass chemically identical substituents resulting from writing the structure from right to left, e.g., structure CH2O is equivalent to structure-OCH 2. Compositions provided herein can include isotopically-labeled compounds in which one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature. The compositions provided herein can include isomers, including but not limited to diastereomers, enantiomers, and mixtures thereof, such as L-amino acids and/or D-amino acids (e.g., p-acetyl-D-phenylalanine and/or p-acetyl-L-phenylalanine).

The term "substituent" includes, but is not limited to, "non-interfering substituents". "non-interfering substituents" are those groups that result in stable compounds. Suitable non-interfering substituents or groups include, but are not limited to, halogen, C1-C10 alkyl, C2-C10 alkenyl, C10-C10 alkynyl, C10-C10 alkoxy, C10-C10 aralkyl, C10-C10 alkaryl, C10-C10 cycloalkyl, C10-C10 cycloalkenyl, phenyl, substituted phenyl, toluoyl, xylyl, biphenyl, C10-C10 alkoxyalkyl, C10-C10 alkoxyaryl, C10-C10 aryloxyalkyl, C10-C10 oxyaryl, C10-C10 alkylsulfinyl, C10-C10 alkylsulfonyl, - - (CH 10) m- -O- - (C10-C10 alkyl), where m is 1-8, aryl, substituted alkoxy, heterocyclyl, substituted heterocyclyl, nitroalkyl, - - (NO- -10), - - (C1-C10 alkyl), - -C (O) - - - (C1-C10 alkyl), C2-C10 alkylthioalkyl, - -C (O) O- - (C1-C10 alkyl), - -OH, - -SO2, - - (S, - - (COOH), - - (NR 2, carbonyl, - -C (O) - - - (C1-C10 alkyl) - -CF3, - -C (O) - -CF3, - - (C (O)) NR2, - - (C1-C10 aryl) - -S- - (C6-C10 aryl), - -C (O) - - (C1-C10 aryl), - - (CH2) m- -O- - (CH2) m- -O- - (C1-C10 alkyl), wherein each m is 1 to 8, - -C (O) NR2, - -C (S) NR2, - -SO2NR2, - -NRC (O) NR2, - -NRC (S) NR2, salts thereof, and the like. As used herein, R is each H, alkyl or substituted alkyl, aryl or substituted aryl, aralkyl or alkaryl.

The term "halogen" includes fluorine, chlorine, iodine and bromine.

Unless otherwise indicated, the term "alkyl" by itself or as part of another substituent means a straight or branched chain or cyclic hydrocarbon group or combination thereof, which may be fully saturated, mono or polyunsaturated, and may include divalent and polyvalent groups having the indicated number of carbon atoms (i.e., C1-C10 represents 1-10 carbons). Examples of saturated hydrocarbon groups include, but are not limited to, groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, sec-butyl, cyclohexyl, (cyclohexyl) methyl, cyclopropylmethyl, homologs and isomers of, for example, n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like. Unsaturated alkyl is alkyl having one or more double or triple bonds. Examples of unsaturated alkyl groups include, but are not limited to, ethenyl, 2-propenyl, crotyl, 2-isopentenyl, 2- (butadienyl), 2, 4-pentadienyl, 3- (1, 4-pentadienyl), ethynyl, 1-and 3-propynyl, 3-butynyl, and higher homologs and isomers. Unless otherwise indicated, the term "alkyl" is also meant to include those alkyl derivatives defined in more detail below, such as "heteroalkyl". Alkyl groups limited to hydrocarbyl groups are referred to as "homoalkyl groups".

The term "alkylene" by itself or as part of another substituent means a divalent radical derived from an alkane, such as, but not limited to, those groups of the structures-CH 2CH2 and-CH 2CH2-, and further including those groups described below as "heteroalkylene". Typically, alkyl (or alkylene) groups have 1 to 24 carbon atoms, with those groups having 10 or fewer carbon atoms being particular embodiments of the methods and compositions described herein. "lower alkyl" or "lower alkylene" is a relatively short chain alkyl or alkylene group, typically having 8 or fewer carbon atoms.

The terms "alkoxy", "alkylamino" and "alkylthio" (or thioalkoxy) are used in their conventional sense and refer to those alkyl groups attached to the rest of the molecule through an oxygen atom, an amino group or a sulfur atom, respectively.

Unless otherwise specified, the term "heteroalkyl," alone or in combination with another term, refers to a stable straight or branched chain or cyclic hydrocarbon radical, or combinations thereof, consisting of the stated number of carbon atoms and at least one heteroatom selected from O, N, Si and S, wherein the nitrogen and sulfur atoms are optionally oxidized, and the nitrogen heteroatom is optionally quaternized. The heteroatoms O, N and S and Si can be located at any internal position of the heteroalkyl group or at a position where the alkyl group is attached to the remainder of the molecule. Examples include, but are not limited to, -CH2-CH2-O-CH3, -CH2-CH2-NH-CH3, -CH2-CH2-N (CH3) -CH3, -CH2-S-CH2-CH3, -CH2-CH2, -S (O) -CH3, -CH2-CH2-S (O)2-CH3, -CH ═ CH-O-CH3, -Si (CH3)3, -CH2-CH ═ N-OCH3, and-CH ═ CH-N (CH3) -CH 3. Up to two heteroatoms may be consecutive, such as, for example, -CH2-NH-OCH3 and-CH 2-O-Si (CH3) 3. Similarly, the term "heteroalkylene" by itself or as part of another substituent means a divalent radical derived from a heteroalkyl group, such as, but not limited to, -CH2-CH2-S-CH2CH 2-and-CH 2-S-CH2-CH2-NH-CH 2-. For heteroalkylene groups, the same or different heteroatoms can also occupy either or both of the chain ends (including, but not limited to, alkyleneoxy, alkylenedioxy, alkyleneamino, alkylenediamino, aminooxyalkylene, and the like). Still further, for alkylene and heteroalkylene linking groups, the orientation of the linking group is not implied by the direction in which the chemical formula of the linking group is written. For example, the formula-C (O)2R ' represents both-C (O)2R ' and R ' C (O) 2.

Unless otherwise indicated, the terms "cycloalkyl" and "heterocycloalkyl" by themselves or in combination with other terms denote the cyclic forms of "alkyl" and "heteroalkyl", respectively. Thus, cycloalkyl or heterocycloalkyl includes saturated, partially unsaturated, and fully unsaturated ring linkages. In addition, for heterocycloalkyl, a heteroatom may occupy the position at which the heterocycle is attached to the rest of the molecule. Examples of cycloalkyl groups include, but are not limited to, cyclopentyl, cyclohexyl, 1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, and the like. Examples of heterocycloalkyl include, but are not limited to, 1- (1,2,5, 6-tetrahydropyridinyl), 1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl, tetrahydrothiophen-2-yl, tetrahydrothiophen-3-yl, 1-piperazinyl, 2-piperazinyl, and the like. In addition, the term includes bicyclic and tricyclic structures. Similarly, the term "heterocycloalkylene" by itself or as part of another substituent means a divalent radical derived from a heterocycloalkyl group, and the term "cycloalkylene" by itself or as part of another substituent means a divalent radical derived from a cycloalkyl group.

Unless otherwise indicated, the term "aryl" means a polyunsaturated aromatic hydrocarbon substituent which may be a single ring or multiple rings (including but not limited to 1 to 3 rings) which are fused together or linked covalently. The term "heteroaryl" refers to an aryl (or ring) containing 1 to 4 heteroatoms selected from N, O and S, wherein the nitrogen and sulfur atoms are optionally oxidized, and the nitrogen atoms are optionally quaternized. The heteroaryl group may be attached to the rest of the molecule through a heteroatom. Non-limiting examples of aryl and heteroaryl groups include phenyl, 1-naphthyl, 2-naphthyl, 4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrazolyl, 2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl, 2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidinyl, 4-pyrimidinyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl, 1-isoquinolyl, 5-isoquinolyl, 2-quinoxalyl, 5-quinoxalyl, 3-quinolyl and 6-quinolyl. The substituents for each of the above aryl and heteroaryl ring systems are selected from the group of acceptable substituents described below.

For the sake of brevity, the term "aryl" when used in combination with other terms (including, but not limited to, aryloxy, arylthioxy, aralkyl) includes aryl and heteroaryl rings as defined above. Thus, the term "aralkyl" is meant to include those groups in which an aryl group is attached to an alkyl group (including but not limited to benzyl, phenethyl, pyridylmethyl and the like), including those alkyl groups in which a carbon atom (including but not limited to methylene) has been replaced by, for example, an oxygen atom (including but not limited to phenoxymethyl, 2-pyridyloxymethyl, 3- (1-naphthyloxy) propyl and the like).

Each of the above terms (including but not limited to "alkyl", "heteroalkyl", "aryl" and "heteroaryl") is intended to include both substituted and unsubstituted forms of the indicated group. Exemplary substituents for each group type are provided below.

Substituents for alkyl and heteroalkyl groups (including those groups commonly referred to as alkylene, alkenyl, heteroalkylene, heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, and heterocycloalkenyl) can be one or more of a variety of groups selected from, but not limited to, the following groups: -OR ', -O, ═ NR ', -N-OR ', -NR ' R ", -SR ', -halogen, -SiR ' R", R ' ", oc (O) R ', -C (O) R ', -CO2R ', -CONR ' R", -oc (O) NR ' R ", -NR" C (O) R ', NR ' C (O) NR "R '", -NR "C (O)2R ', -NR-C (NR ' R" R ' ") -NR '", NR C (NR ' R ") -NR '", -s (O) R ', -s (O)2NR ' R ', NRSO2R ', -CN and-NO 2 in a number range of 0 to (2m ' +1), where m ' is the total number of carbon atoms in such a group. R ', R ", R'" and R "" each independently refer to hydrogen, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, including but not limited to aryl substituted with 1-3 halogens, substituted or unsubstituted alkyl, alkoxy or thioalkoxy or aralkyl. When a compound of the invention includes more than one R group, for example, when more than one of these groups is present, each R group is independently selected from the respective R ', R ", R'" and R "" groups. When R' and R "are attached to the same nitrogen atom, they may combine with the nitrogen atom to form a 5-, 6-or 7-membered ring. For example, -NR' R "is meant to include, but is not limited to, 1-pyrrolidinyl and 4-morpholinyl. From the above discussion of substituents, those skilled in the art will understand that the term "alkyl" is intended to include groups including carbon atoms bonded to groups other than hydrogen radicals, such as haloalkyl (including, but not limited to, -CF3 and-CH 2CF3) and acyl (including, but not limited to, -C (O) CH3, -C (O) CF3, -C (O) CH2OCH3, and the like).

Similar to the substituents described for alkyl, aryl and heteroaryl substituents are diverse and are selected from, but not limited to: halogen, OR ', -O, ═ NR', -N-OR ', -NR' R ", -SR ', -halogen, -SiR' R" R '", oc (O) R', -c (O) R ', CO 2R', -CONR 'R", -oc (O) NR' R ", -NR "C (o) R ', NR' C (o) NR" R '", -NR" C (o) 2R', NR-C (NR 'R "R'") ═ NR "", NR C (NR 'R ") ═ NR'", -s (o) R ', -s (o)2 NR' R ", NRSO2R ', -CN and-NO 2, -R', -N3, -ch (ph)2, fluoro (C1-C4) alkoxy and fluoro (C1-C4) alkyl in amounts ranging from 0 to the total number of open valences on the aromatic ring system; wherein R ', R ", R'" and R "" are independently selected from the group consisting of hydrogen, alkyl, heteroalkyl, aryl and heteroaryl. When a compound of the invention includes more than one R group, for example, when more than one of these groups is present, each R group is independently selected from the respective R ', R ", R'" and R "" groups.

The term "fatty acid" refers to a saturated or unsaturated acyl chain having 6 to 20 carbon atoms or a derivative thereof. In some embodiments, the fatty acid may be linked to the peptide component of the PK enhancer described herein and end capped with a carboxylic acid or a methyl group. In some embodiments, the fatty acid linked to the peptide component of the PK enhancer may be capped with a carboxylic acid. In some embodiments, the fatty acid can be linked to the peptide component through an amide bond. In some embodiments, the fatty acid may have from 10 to 18 carbon atoms. In some embodiments, the fatty acid may have from 12 to 17 carbon atoms. In some embodiments, the fatty acid may have 14, 15, or 16 carbon atoms. In some embodiments, the fatty acid may have 15 carbon atoms.

As used herein, the term "water-soluble polymer" refers to any polymer that is soluble in an aqueous solvent. Attachment of a water-soluble polymer to a modified relaxin polypeptide can result in a variety of changes, including but not limited to: increased or modulated serum (in vivo) half-life or increased or modulated therapeutic half-life relative to an unmodified form, reduced or modulated immunogenicity or toxicity, modulated physical association characteristics (e.g., reduced aggregation and multimer formation), altered receptor binding, altered binding to one or more binding partners, altered receptor dimerization or multimerization. The water-soluble polymer may or may not have its own biological activity and may serve as a linker for attaching the modified relaxin to other substances, including but not limited to one or more unmodified or modified relaxin polypeptides or one or more biologically active molecules. Suitable polymers include, but are not limited to, polyethylene glycol propionaldehyde, mono-C1-C10 alkoxy or aryloxy derivatives thereof (described in U.S. Pat. No.5,252,714, incorporated herein by reference), monomethoxy-polyethylene glycol, discrete PEG, polyvinylpyrrolidone, polyvinyl alcohol, polyamino acids, divinyl ether maleic anhydride, N- (2-hydroxypropyl) -methacrylamide, dextran derivatives (including dextran sulfate), polypropylene glycol, polyoxypropylene/ethylene oxide copolymers, polyoxyethylated polyols, heparin fragments, polysaccharides, oligosaccharides, polysaccharides, cellulose and cellulose derivatives (including, but not limited to, methyl cellulose and carboxymethyl cellulose), starch and starch derivatives, polypeptides of two or more amino acids, polyalkylene glycols and derivatives thereof, Copolymers of polyalkylene glycols and derivatives thereof, polyvinyl ethyl ethers and α - β -poly [ (2-hydroxyethyl) -DL-asparagine, and the like, or mixtures thereof. Examples of such water-soluble polymers include, but are not limited to, polyethylene glycol and serum albumin.

As used herein, the term "polyalkylene glycol" (PEG) or "poly (alkylene glycol)" refers to polyethylene glycol (poly (ethylene glycol)), polypropylene glycol, polybutylene glycol, and derivatives thereof. The term "polyalkylene glycol" includes linear and branched polymers having an average molecular weight of from 0.1kDa to 100 kDa. Other exemplary embodiments are listed, for example, in a catalog of commercial suppliers, such as the catalog "Polyethylene glycols and deviations for Biomedical Applications" by Shearwater Corporation (2001).

As used herein, the term "modulated serum half-life" or "modulated in vivo half-life" and similar terms refer to a positive or negative change in the circulatory half-life of modified relaxin relative to a comparative subject (e.g., its unmodified form or wild-type relaxin). Serum half-life can be measured by taking blood samples at different time points after administration of the modified relaxin, and determining the concentration of the molecule in each sample. Correlation of serum concentration with time allows calculation of serum half-life. The increase in serum (in vivo) half-life may desirably be at least about 2-fold, but a smaller increase may be useful, for example where it enables a satisfactory dosing regimen or avoids toxic effects. In some embodiments, the increase may be at least about three times, at least about five times, at least about ten times, at least about twenty times, or at least about fifty times.

The term "modulated therapeutic half-life" as used herein means a positive or negative change in serum or in vivo half-life of a therapeutically effective amount of a modified relaxin polypeptide described herein, relative to a comparative subject (e.g., an unmodified form or wild-type relaxin thereof). Therapeutic half-life is measured by measuring the pharmacokinetic and/or pharmacodynamic properties of the molecule at various time points after administration. Increased therapeutic half-life desirably provides the opportunity to achieve a particular beneficial dosing regimen, a particular beneficial total dose, or to avoid undesirable effects. In some embodiments, what results in increased therapeutic half-life is: increased potency, increased or decreased binding of the modified molecule to its target, increased or decreased breakdown of the molecule by an enzyme (e.g., a protease), or an increase or decrease in another parameter or mechanism of action of the non-modified molecule, or an increase or decrease in receptor-mediated clearance of the molecule. In some embodiments, the increase in therapeutic half-life may be at least about two-fold, at least about three-fold, at least about five-fold, at least about ten-fold, at least about twenty-fold, or at least about fifty-fold.

The term "isolated", when applied to a nucleic acid or protein, means that the nucleic acid or protein is free of at least some cellular components with which it is naturally associated, or that the nucleic acid or protein has been concentrated to a level greater than that at which it is produced in vivo or in vitro. It may be in a homogenous state. The isolated material may be in a dry or semi-dry state, or in solution, including but not limited to aqueous solutions. It may be a component of a pharmaceutical composition comprising further pharmaceutically acceptable carriers and/or excipients. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. The protein, which is the major material present in the preparation, is substantially purified. In particular, the isolated gene is separated from open reading frames that flank the gene and encode proteins other than the gene of interest. The term "purified" means that the nucleic acid or protein produces a substantial band in the electrophoresis gel. Specifically, it may mean that the nucleic acid or protein is at least 85% pure, at least 90% pure, at least 95% pure, at least 99% pure, or purer.

The term "nucleic acid" refers to deoxyribonucleotides, deoxyribonucleosides, ribonucleosides, or ribonucleotides and polymers thereof, in either single-or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise specifically limited, the term also refers to oligonucleotide analogs, including PNA (peptide nucleic acid), DNA analogs used in antisense technology (phosphorothioate, phosphoramidate, etc.). Unless otherwise indicated, a particular nucleic acid sequence also implicitly includes conservatively modified variants thereof (including but not limited to degenerate codon substitutions) and complementary sequences as well as the sequence explicitly indicated.

The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. That is, the description for polypeptides applies equally to the description for peptides and to the description for proteins, and vice versa. The terms apply to naturally occurring amino acid polymers as well as amino acid polymers in which one or more amino acid residues are a non-naturally encoded amino acid. As used herein, the term encompasses amino acid chains of any length, including full length proteins, in which the amino acid residues are linked by covalent peptide bonds.

"conservatively modified variants" refers to amino acid sequences that contain conservative substitutions. Exemplary conservatively modified variants include substitutions, deletions or insertions of nucleic acids, peptides, polypeptides or protein sequences which alter, add or delete a single amino acid or a small percentage of amino acids in a polypeptide sequence or encoded polypeptide sequence, for example, up to 1,2,3,4 or 5 amino acids, or up to 0.5%, 1%, 1.5%, 2%, 2.5% or 3.5% amino acids in a polypeptide sequence or encoded polypeptide sequence, optionally amino acid substitutions which may be or may include chemically similar amino acids. Conservative substitution tables providing functionally similar amino acids are known to those of ordinary skill in the art. Such conservatively modified variants are in addition to, but not exclusively, polymorphic variants, interspecies homologs, and alleles of the disclosed modified relaxin polypeptides.

Conservative substitution tables providing functionally similar amino acids are known to those of ordinary skill in the art. The following eight groups each contain amino acids that are conservative substitutions between each other:

1) alanine (a or Ala), glycine (G or Gly);

2) aspartic acid (D or Asp), glutamic acid (E or Glu);

3) asparagine (N or Asn), glutamine (Q or Gln);

4) arginine (R or Arg), lysine (K or Lys), histidine (H or His);

5) isoleucine (I or Ile), leucine (L or Leu), methionine (M or Met), valine (V or Val);

6) phenylalanine (F or Phe), tyrosine (Y or Tyr), tryptophan (W or Trp);

7) serine (S or Ser), threonine (T or Thr); and

8) cysteine (C or Cys), methionine (M or Met)

(see, e.g., Creighton, Proteins: Structures and Molecular Properties (W H Freeman & Co.; 2 nd edition (12 months 1993))

The term "identical" or percent "identity," in the context of two or more nucleic acid or polypeptide sequences, refers to two or more identical sequences or subsequences. Sequences are "substantially identical" if they have a percentage of amino acid residues or nucleotides that are identical (i.e., about 60% identical, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% identical over the specified region) when compared and aligned for maximum correspondence over a comparison window, or the specified region is measured as using one of the following sequence comparison algorithms (or other algorithms available to one of ordinary skill in the art) or by manual alignment and visual inspection. Identity may exist over a region or comparison window of at least about 20 amino acids or nucleotides in length, or over a region of at least about 25 amino acids or nucleotides in length, or, where not specified, over the entire sequence of the polynucleotide or polypeptide. As used herein, a "comparison window" includes reference to any segment having contiguous positions, e.g., at least or about 10, 15, 20, 25, or 30 amino acids, wherein a sequence can be compared after optimal alignment of the two sequences to a reference sequence having the same number of contiguous positions. Examples of algorithms that may be suitable for determining sequence identity and percent sequence similarity are the BLAST and BLAST 2.0 algorithms described in Altschul et al (1997) Nuc. acids Res.25: 3389-.

The term "subject" as used herein refers to an animal, in some embodiments a mammal, and in other embodiments a human, who is the subject of treatment, observation or experiment. The animal can be a companion animal (e.g., dog, cat, etc.), a farm animal (e.g., cow, sheep, pig, horse, etc.), or a laboratory animal (e.g., rat, mouse, guinea pig, etc.).

The term "effective amount" as used herein means an amount of a compound (e.g., a modified relaxin polypeptide described herein) administered that prevents, cures, alleviates, delays onset of, reduces severity of, or reduces to some extent one or more symptoms of the disease, disorder, or condition being treated. Compositions containing the modified relaxin polypeptides described herein may be administered for prophylactic, booster, and/or therapeutic treatment.

The term "enhance" means to increase or prolong the efficacy or duration of a desired effect. Thus, for enhancing the effect of a therapeutic agent, the term "enhance" refers to the ability to increase or prolong the efficacy or duration of the effect of other therapeutic agents on the system.

In prophylactic applications, compositions containing modified relaxin polypeptides are administered to a patient susceptible to or otherwise at risk of a particular disease, disorder or condition. Such an amount is defined as a "prophylactically effective amount".

In therapeutic applications, a composition comprising a modified unnatural amino acid polypeptide is administered to a patient already suffering from a disease, disorder, or condition in an amount sufficient to cure or at least partially arrest or alleviate symptoms of the disease, disorder, or condition. Such an amount is defined as a "therapeutically effective amount" and may depend on the severity and course of the disease, disorder or condition, previous treatments, the patient's health and response to the drug, and the judgment of the treating physician. Determination of such therapeutically effective amounts by routine experimentation (e.g., dose escalation clinical trials) is considered well known to those skilled in the art.

The term "treatment" is used to refer to prophylactic and/or therapeutic treatment.

"reducing agent" as used herein with respect to protein refolding is defined as any compound or substance that maintains sulfhydryl groups in a reduced state and reduces intramolecular or intermolecular disulfide bonds. Suitable reducing agents include, but are not limited to, Dithiothreitol (DTT), 2-mercaptoethanol, cysteine, cysteamine (2-aminoethanethiol), and reduced glutathione. It will be apparent to those of ordinary skill in the art that a variety of reducing agents are suitable for use in the methods and compositions of the present invention.

An "oxidizing agent" as used herein with respect to protein refolding is defined as any compound or substance capable of removing an electron from an oxidized compound. Suitable oxidizing agents include, but are not limited to, oxidized glutathione, cysteine, cystamine, oxidized dithiothreitol, oxidized erythritol, and oxygen. It will be apparent to those of ordinary skill in the art that a variety of oxidizing agents are suitable for use in the methods of the present invention.

As used herein, a "Denaturing agent" or "denaturant" is defined as any compound or substance that will cause reversible unfolding of a protein. The strength of the denaturant will be determined by the nature and concentration of the particular denaturant. Suitable denaturants may be chaotropic agents, detergents, organic solvents, water miscible solvents, phospholipids or combinations of two or more such agents. Suitable chaotropic agents include, but are not limited to, urea, guanidine, and sodium thiocyanate. Useful detergents may include, but are not limited to, strong detergents such as sodium lauryl sulfate, or polyoxyethylene ethers (e.g., tween or Triton detergents), sodium lauryl sarcosinate (Sarkosyl), mild nonionic detergents (e.g., digitonin), mild cationic detergents such as N- >2,3- (dioleoxy) -propyl-N, N-trimethylammonium, mild ionic detergents (e.g., sodium cholate or sodium deoxycholate), or zwitterionic detergents including, but not limited to, sulfobetaine (Zwittergent), 3- (3-cholamidopropyl) dimethylamino-1-propane sulfate (CHAPS), and 3- (3-cholamidopropyl) dimethylamino-2-hydroxy-1-propane sulfonate (CHAPSO). Organic water-miscible solvents such as acetonitrile, lower alkanols (in particular C2-C4 alkanols such as ethanol or isopropanol), or lower alkanediols (in particular C2-C4 alkanediols such as ethylene glycol) may be used as denaturants. The phospholipids useful in the present invention may be naturally occurring phospholipids, such as phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine and phosphatidylinositol, or synthetic phospholipid derivatives or variants, such as dihexanylphosphatidylcholine or diheptanylphosphatidylcholine.

"refolding" as used herein describes any process, reaction or method that converts a disulfide-containing polypeptide from an incorrectly folded or unfolded state into a native or correctly folded conformation relative to the disulfide bond.

"Co-folding" as used herein refers in particular to a refolding process, reaction or process employing at least two polypeptides that interact with each other and result in the conversion of an unfolded or incorrectly folded polypeptide into a native correctly folded polypeptide.

The term "protected" refers to a "protecting group" or the presence of a moiety that prevents the reaction of a chemically reactive functional group under certain reaction conditions. The protecting group will vary depending on the type of chemically reactive group being protected. For example, if the chemically reactive group is an amine or hydrazide, the protecting group may be selected from t-butyloxycarbonyl (t-Boc) and 9-fluorenylmethoxycarbonyl (Fmoc). If the chemically reactive group is a thiol, the protecting group may be an n-pyridyl disulfide. If the chemically reactive group is a carboxylic acid, such as butyric or propionic acid, or a hydroxyl group, the protecting group may be a benzyl group or an alkyl group, such as methyl, ethyl or tert-butyl. Other protecting groups known in the art may also be used in or with the methods and compositions described herein, including photolabile groups such as Nvoc and MeNvoc. Other protecting groups known in the art may also be used in or with the methods and compositions described herein.

By way of example only, the blocking/protecting group may be selected from:

other protecting Groups are described in Greene and Wuts, Protective Groups in Organic Synthesis,3rd Ed., John Wiley & Sons, New York, NY,1999, which is incorporated herein in its entirety by reference.

The term "disorder of the cardiovascular system" or "cardiovascular disorder" includes, for example, the following disorders: hypertension (elevated blood pressure), peripheral and cardiovascular disorders, coronary heart disease, stable and unstable angina, heart attacks, myocardial insufficiency, abnormal heart rhythm (or arrhythmias), persistent ischemic dysfunction ("hibernating myocardium"), transient post-ischemic dysfunction ("stunting myocardium"), heart failure, peripheral blood flow disorder, acute coronary syndrome, heart failure, myocardial disease (cardiomyopathy), myocardial infarction, and vascular disease (vascular disease).

The term "heart failure" includes acute and chronic manifestations of heart failure, and more specifically or related disease types, such as advanced heart failure, post-acute heart failure, cardiorenal syndrome, heart failure with impaired renal function, chronic heart failure, midrange-ejection fraction chronic heart failure (hfmeef), compensated heart failure, decompensated heart failure, right heart failure, left heart failure, total heart failure, ischemic cardiomyopathy, dilated cardiomyopathy, heart failure associated with congenital heart defects, heart valve defects, heart failure associated with heart valve defects, mitral stenosis, mitral insufficiency, aortic stenosis, aortic insufficiency, tricuspid stenosis, tricuspid insufficiency, pulmonary stenosis, pulmonary insufficiency, heart failure associated with combined heart valve defects, mitral insufficiency, and combinations thereof, Myocardial inflammation (myocarditis), chronic myocarditis, acute myocarditis, viral myocarditis, diabetic heart failure, alcoholic cardiomyopathy, heart failure associated with cardiac storage disorders, diastolic heart failure, systolic heart failure, a period of acute exacerbation of heart failure, heart failure with preserved ejection fraction (HFpEF), heart failure with reduced ejection fraction (HFrEF), chronic heart failure with preserved ejection fraction (HFpEF), post-myocardial infarction remodeling, angina, hypertension, pulmonary hypertension, and pulmonary hypertension.

The term "fibrotic conditions" includes diseases and conditions characterized by fibrosis, and in particular includes the following diseases and conditions: fibrosis of the liver or liver, cirrhosis, NASH, pulmonary or pulmonary fibrosis, cardiac fibrosis, endomyocardial fibrosis, nephropathy, glomerulonephritis, interstitial renal fibrosis, fibrotic damage caused by diabetes, myelofibrosis and similar fibrotic disorders, scleroderma, morphea, keloids, hypertrophic scarring (also after surgery), nevi, diabetic retinopathy, proliferative vitreoretinopathy and connective tissue disorders (e.g. sarcoidosis).

The terms "relaxin-related disorders" and "relaxin-related diseases" encompass diseases and disorders that can be prevented, treated, alleviated, or otherwise affected by modulating relaxin levels in vivo (e.g., increasing serum relaxin protein levels). Relaxin-related disorders include, but are not limited to, disorders of the cardiovascular system and fibrotic disorders described herein.

Polypeptides comprising a non-naturally encoded amino acid provided herein can include isotopically labeled compounds, in which one or more atoms 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, fluorine and chlorine, such as 2H, 3H, 13C, 14C, 15N, 18O, 17O, 35S, 18F, 36Cl, respectively. Certain isotopically-labeled compounds described herein (e.g., those into which radioactive isotopes such as 3H and 14C are incorporated) are useful in drug and/or substrate tissue distribution assays. In addition, substitution with isotopes such as deuterium (i.e., 2H) may afford certain therapeutic advantages resulting from greater metabolic stability, for example increased in vivo half-life or reduced dosage requirements.

I. Overview

The present disclosure provides modified relaxin molecules linked to a PK enhancer. The exemplary embodiments demonstrate at least one advantageous property selected from the group consisting of increased in vivo half-life, reduced renal vacuole formation, increased renal blood flow, higher solubility, reduced aggregation, reduced viscosity, increased manufacturability, as compared to other modified or wild-type relaxin polypeptides.

In one aspect, the present disclosure provides a modified relaxin polypeptide comprising a non-naturally encoded amino acid, wherein:

(a) the modified relaxin polypeptides comprise a relaxin a chain polypeptide SEQ ID No.4 and a relaxin B chain polypeptide SEQ ID No.5 or SEQ ID No.6 substituted with a non-naturally encoded amino acid at a position selected from the group consisting of: an a chain residue 1, an a chain residue 2, an a chain residue 5, an a chain residue 13, an a chain residue 18, a B chain residue 5, a B chain residue 7 and a B chain residue 25, and optionally having up to two additional amino acid substitutions, insertions and/or deletions in the relaxin a chain and/or the relaxin B chain;

(b) the non-naturally encoded amino acid has the following structure:

wherein the R group is any substituent other than the side chain found in the following amino acids: alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine;

(c) the non-naturally encoded amino acid is linked to a pharmacokinetic enhancer comprising a peptide component of 2 to 30 amino acids and a half-life extending moiety.

In some embodiments, the relaxin a chain polypeptide may comprise SEQ ID NO 4 substituted at residue 1 with the non-naturally encoded amino acid, and optionally with up to two additional amino acid substitutions, insertions, and/or deletions. The relaxin a chain polypeptide may comprise SEQ ID NO 4 substituted at residue 1 with the non-naturally encoded amino acid, and optionally with one additional amino acid substitution, insertion or deletion. The relaxin B chain polypeptide may comprise SEQ ID NO 5 or SEQ ID NO 6 optionally with up to two additional amino acid substitutions, insertions or deletions. The relaxin B chain polypeptide may comprise SEQ ID NO 5 or SEQ ID NO 6 optionally with one additional amino acid substitution, insertion or deletion. The relaxin a chain polypeptide may comprise SEQ ID No.4 substituted at residue 1 with the non-naturally encoded amino acid, and the relaxin B chain polypeptide may comprise SEQ ID No.5 or SEQ ID No. 6.

In some embodiments, the at least one non-naturally encoded amino acid can comprise a carbonyl group, an aminooxy group, a hydrazide group, a hydrazine group, a semicarbazide group, an azide group, or an alkyne group. The non-naturally encoded amino acid can include a phenylalanine derivative. The non-naturally encoded amino acid may be selected from para-substituted, ortho-substituted, or meta-substituted phenylalanine. The non-naturally encoded amino acid can be selected from para-, ortho-or meta-substituted phenylalanine comprising a carbonyl group, an aminooxy group, a hydrazide group, a hydrazine group, a semicarbazide group, an azide group, or an alkyne group. The non-naturally encoded amino acid can include p-acetyl-L-phenylalanine. The non-naturally encoded amino acid may be linked to the pharmacokinetic enhancer. For example, the non-naturally encoded amino acid can be linked to the pharmacokinetic enhancer by an oxime linkage or a triazole linkage (e.g., an oxime linkage).

In some embodiments, the relaxin a chain polypeptide may comprise SEQ ID NO 35 and the relaxin B chain polypeptide may comprise SEQ ID NO 5 or SEQ ID NO 6, and wherein the relaxin a chain or B chain optionally may have up to two additional amino acid substitutions, insertions, or deletions. The modified relaxin polypeptide may comprise a relaxin a chain polypeptide having at least 90% amino acid sequence identity to SEQ ID No. 35 and a relaxin B chain polypeptide having at least 90% amino acid sequence identity to SEQ ID No.5 or SEQ ID No. 6. The relaxin a chain polypeptide can have at least 95% amino acid sequence identity to SEQ ID No. 35. The relaxin B chain polypeptide may have at least 95% amino acid sequence identity to SEQ ID NO 5 or SEQ ID NO 6. The relaxin A chain polypeptide can comprise SEQ ID NO 35 and the relaxin B chain polypeptide can comprise SEQ ID NO 5 or SEQ ID NO 6. In some embodiments, the relaxin a chain polypeptide can comprise SEQ ID No. 35 and the relaxin B chain polypeptide can comprise SEQ ID No. 6.

In some embodiments, the peptide component may comprise 1 to 25 amino acids, such as 2 to 20 amino acids, 3 to 10 amino acids, or 4 to 8 amino acids. The peptide component may comprise Glu, Glu^γ、GGGGS-Glu^γ(SEQ ID NO:139)、DRDDRD(SEQ ID NO:102)、KKKKKK-Glu^γ(SEQ ID NO:103)、RGGEEKKKEKEK-Glu^γ(SEQ ID NO:104)、GGGEEE-Glu^γ(SEQ ID NO:105)、EEEGGG-Glu^γ(SEQ ID NO:106)、KKKGGG-Glu^γ(SEQ ID NO:107)、GETGSSGEGT-Glu^γ(SEQ ID NO:108)、GGGKKK-Glu^γ(SEQ ID NO:109)、GSHHHHHGS-Glu^γ(SEQ ID NO:110)、Sar-Sar-Sar-Sar-Ser-Sar-Sar-Sar-Sar-Glu^γ(SEQ ID NO:111)、Sar-Sar-Sar-Sar-Ser-Glu^γ(SEQ ID NO:112)、Sar-Sar-Sar-Glu-Glu-Glu^γ(SEQ ID NO:113)、KKKSGGSGG-Glu^γ(SEQ ID NO:118)、KKSGGSGG-Glu^γ(SEQ ID NO:114)、KKSGGSGG-Glu^α(SEQ ID NO:115)、KKSAGSAG-Glu^γ(SEQ ID NO:116)、KSGGSGG-Glu^γ(SEQ ID NO:117)、KKSGGSGGEE-Glu^γ(SEQ ID NO:119)、dKdKdKdKdKdK-Glu^γ(SEQ ID NO:120)、EESGGSGG-Glu^γ(SEQ ID NO:121)、GSGSGSGS-Glu^γ(SEQ ID NO:123)、EEEGGG-dGlu^γ(SEQ ID NO:128)、EGGGGSK-Glu^γ(SEQ ID NO:130)、EEEEEE-Glu^γ(SEQ ID NO:131)、EEEEPEEEEPEEEEPEEEE-Glu^γ(SEQ ID NO:133)、EEEEPEEEEPEEEEPEEGGG(SEQ ID NO:135)、EEEEGEEEEGEEEEGEEEE-Glu^γ(SEQ ID NO:136)、KGGEEKKKEKEKEPKGGEEKKKEKEK-Glu^γ(SEQ ID NO:137)、EAQKAQAEAQKAQAEAQKAQA-Glu^γ(SEQ ID NO:138)、KK-Glu^γ(SEQ ID NO:140)、Glu^γ、KGPKGP-Glu^γ(SEQ ID NO:146)、SGGGS-Glu^γ(SEQ ID NO:147)、KGGGS-Glu^γ(SEQ ID NO:148)、KGGGSE-Glu^γ(SEQ ID NO:149)、GSPGSP-Glu^γ(SEQ ID NO:150)、GGGGP-Glu^γ(SEQ ID NO:151)、EGGS-Glu^γ(SEQ ID NO:152)、EGGGP-Glu^γ(SEQ ID NO:153)、KGPGSE-Glu^γ(SEQ ID NO:154), spermine-Glu^γOr KKGGS-Glu^γ(SEQ ID NO:156)。

The peptide component may comprise GGGGS-Glu^γ(SEQ ID NO:139)、DRDDRD(SEQ ID NO:102)、KKKKKK-Glu^γ(SEQ ID NO:103)、RGGEEKKKEKEK-Glu^γ(SEQ ID NO:104)、GGGEEE-Glu^γ(SEQ ID NO:105)、EEEGGG-Glu^γ(SEQ ID NO:106)、KKKGGG-Glu^γ(SEQ ID NO:107)、GGGKKK-Glu^γ(SEQ ID NO:109)、GSHHHHHGS-Glu^γ(SEQ ID NO:110)、Sar-Sar-Sar-Sar-Ser-Glu^γ(SEQ ID NO:112)、Sar-Sar-Sar-Glu-Glu-Glu^γ(SEQ ID NO:113) or KSGGSGG-Glu^γ(SEQ ID NO:117)。

The peptide component may comprise Glu^γ. For example, the peptide component may comprise GGGGS-Glu^γ(SEQ ID NO:139)。

In exemplary embodiments, the modified relaxin polypeptide may comprise a relaxin A chain polypeptide SEQ ID NO 4 and a relaxin B chain polypeptide SEQ ID NO 5 or SEQ ID NO 6 substituted at residue 1 of the A chain with a non-naturally encoded amino acid, wherein the non-naturally encoded amino acid is linked to the pharmacokinetic enhancer and the pharmacokinetic enhancer comprises the peptide component GGGGS-Glu^γ(SEQ ID NO: 139). In some embodiments, the peptide component can be covalently linked to the non-naturally encoded amino acid, for example, through an oxime.

In exemplary embodiments, the half-life extending module may comprise a fatty acid or derivative thereof, wherein the fatty acid or derivative thereof may be covalently linked to the peptide component. The half-life extending module may comprise a saturated fatty acid or derivative thereof. The half-life extending module may comprise a fatty acid capped with a carboxylic acid. The half-life extending module may comprise a fatty acid of formula I: -Cn-COOH (formula I) wherein n may be 10-18, such as 12-17, or 13, 14, 15 or 16. In some embodiments, the-Cn-of formula I may comprise the first carbonyl carbon and- (CH)₂)_n-1-. For example, -C13-may be- (C ═ O) - (CH)₂)₁₂-; -C14-may be- (C ═ O) - (CH)₂)₁₃-; -C15-may be- (C ═ O) - (CH)₂)₁₄-. In some embodiments, the first carbonyl carbon of the fatty acid may be bonded to the (γ) Glu residue, optionally through an amide bond. In some embodiments, the first carbonyl carbon of the fatty acid can be bonded to the gammaglu residue through an amide bond.

In further exemplary embodiments, the half-life extending module may comprise-C14-COOH.

In further exemplary embodiments, the pharmacokinetic enhancer may comprise the structure:

wherein the aminooxy group of formula II is linked to an unnatural amino acid in the modified relaxin polypeptide, e.g., via an oxime.

In further exemplary embodiments, the half-life extending moiety may be conjugated to the peptide component through an amide bond.

In further exemplary embodiments, the relaxin polypeptide may comprise a relaxin a chain polypeptide SEQ ID No.4 and a relaxin B chain polypeptide SEQ ID No.5 or SEQ ID No.6 substituted at a chain residue 1 with a non-naturally encoded amino acid; wherein the non-naturally encoded amino acid comprises para-acetyl-L-phenylalanine; wherein the non-naturally encoded amino acid is linked to the pharmacokinetic enhancer comprising: contains GGGGS-Glu^γ(SEQ ID NO: 139); and contain-C14-COOH, e.g., - (C ═ O) - (CH)₂)₁₃-a half-life extending module of-COOH.

In further embodiments, the present disclosure provides modified relaxin polypeptides comprising the structure:

wherein said AQ 1-relaxin comprises a relaxin A chain polypeptide having at least 90% amino acid sequence identity to SEQ ID NO. 35 and a relaxin B chain polypeptide having at least 90% amino acid sequence identity to SEQ ID NO.5 or SEQ ID NO.6, wherein the para-acetyl-L-phenylalanine depicted in formula III is located at the N-terminus of said relaxin A chain polypeptide. The relaxin a chain polypeptide can have at least 95% amino acid sequence identity to SEQ ID No. 35. The relaxin B chain polypeptide may have at least 95% amino acid sequence identity to SEQ ID NO 5 or SEQ ID NO 6. The AQ 1-relaxin may comprise a relaxin A chain polypeptide SEQ ID NO:35 and a relaxin B chain polypeptide SEQ ID NO:5 or SEQ ID NO: 6.

In a further embodiment, the present disclosure provides a composition comprising the relaxin conjugate "AQ 1-GGGGS-Glu^γ(SEQ ID NO:139) -C14-COOH "comprising a relaxin A chain polypeptidePeptides SEQ ID NO 35 and relaxin B chain polypeptide SEQ ID NO 6 wherein the p-acetyl-L-phenylalanine at the N-terminus of the relaxin A chain polypeptide may be combined with the PK enhancer GGGGS-Glu^γ(SEQ ID NO:139) -C14-COOH linkage as depicted in formula III:

in a further embodiment, the modified relaxin polypeptide comprises the structure shown in figure 8.

In a further embodiment, the modified relaxin polypeptide comprises the structure shown in figure 9.

In exemplary embodiments, the modified relaxin polypeptide may have biological activity. The modified relaxin polypeptides may have a therapeutic effect for treating one or more diseases or disorders associated with relaxin. The modified relaxin polypeptide can exhibit reduced renal vacuole formation compared to a subject compound of comparison (e.g., AQ1-20kDa PEG (AQ 1 relaxin conjugated to PEG, with an average MW of 20 kDa.) the modified relaxin polypeptide can exhibit no impairment of renal function or reduced impairment of renal function compared to a subject compound of comparison, e.g., AQ1-20kDa PEG.

In exemplary embodiments, the modified relaxin polypeptides do not exhibit reduced renal blood flow after administration, and/or are capable of increasing renal blood flow. Renal blood flow can be measured by determining the clearance of aminohippurate.

The modified relaxin polypeptide may exhibit an extended half-life in vivo, e.g., at least 5-fold, at least 10-fold, at least 20-fold, at least 30-fold, or at least 50-fold, as compared to a subject compound of comparison (e.g., wild-type relaxin).

In another aspect, the present disclosure provides a pharmaceutical composition comprising a modified relaxin polypeptide as described herein and a pharmaceutically acceptable carrier.

In another aspect, the disclosure provides a pharmaceutical composition comprising an effective amount of a modified relaxin polypeptide as described herein for treating or preventing a relaxin-related disorder and a pharmaceutically acceptable carrier.

In another aspect, the present disclosure provides a method of treating or preventing a relaxin-related disease comprising administering to a patient in need thereof an effective amount of a modified relaxin polypeptide or a composition comprising a modified relaxin polypeptide as described herein.

In another aspect, the present disclosure provides a method of treating or preventing a cardiovascular disease comprising administering to a patient in need thereof an effective amount of a modified relaxin polypeptide or a composition comprising a modified relaxin polypeptide as described herein. The cardiovascular disease may be selected from coronary artery disease, heart attack, arrhythmia, heart failure, cardiomyopathy and vascular disease.

In another aspect, the present disclosure provides a method of treating, preventing, or alleviating a symptom of heart failure, comprising administering to a patient in need thereof an effective amount of a modified relaxin polypeptide or a composition comprising a modified relaxin polypeptide as described herein. The heart failure may be selected from the group consisting of advanced heart failure, cardiorenal syndrome, heart failure with impaired renal function, chronic heart failure of the midrange ejection fraction type (HFmEF), acute heart failure, post-acute heart failure, compensated heart failure, decompensated heart failure, right heart failure, left heart failure, whole heart failure, ischemic cardiomyopathy, dilated cardiomyopathy, heart failure associated with congenital heart defects, heart failure associated with heart valve defects, heart failure associated with combined heart valve defects, diabetic heart failure, alcoholic cardiomyopathy, heart failure associated with cardiac storage disorders, diastolic heart failure, systolic heart failure, remodeling after myocardial infarction, heart failure with preserved ejection fraction (HFpEF), heart failure with reduced ejection fraction (HFrEF), angina pectoris, myocardial infarction, hypertension, pulmonary hypertension and pulmonary hypertension.

In some embodiments, the heart failure may be selected from chronic heart failure, acute heart failure, post-acute heart failure, chronic heart failure with reduced ejection fraction (HFrEF), chronic heart failure with preserved ejection fraction (HFpEF), chronic heart failure with mid-range ejection fraction (hfmeef), diastolic heart failure, systolic heart failure, post-myocardial infarction remodeling, angina, hypertension, pulmonary hypertension, and pulmonary hypertension.

In some embodiments, the heart failure may be selected from post-acute heart failure, advanced heart failure, cardiorenal syndrome, and heart failure with impaired renal function.

The method of treatment may further comprise administering, simultaneously or sequentially, in combination with the modified relaxin polypeptide, at least one additional therapeutic agent selected from the group consisting of ACE inhibitors, beta-blockers, diuretics, mineralocorticoid receptor antagonists, ryanodine receptor modulators, SERCA2a activators, renin inhibitors, calcium channel blockers, adenosine a1 receptor agonists, partial adenosine a1 receptor, dopamine beta-hydroxylase inhibitors, angiotensin II receptor antagonists having a biased agonistic effect on selected cellular signaling pathways, combinations of angiotensin II receptor antagonists and neutral endopeptidase inhibitors, soluble guanylate cyclase activators, myosin atpase activators, rho kinase 1 inhibitors, rho kinase 2 inhibitors, apelin receptor agonists, renin inhibitors, Nitroxyl donor compounds, calcium-dependent kinase II inhibitors, antifibrotic agents, galectin-3 inhibitors, vasopressin receptor antagonists, FPR2 receptor modulators, natriuretic peptide receptor agonists, transient receptor potential vanilloid-4 channel blockers, antiarrhythmics, I_f"pacing current" ("funny current") channel blockers, nitrates, digitalis compounds, inotropic and beta-receptor agonists, cell membrane resealing agents such as poloxamer 188, antihyperlipidemic agents, plasma HDL-raising agents, antihypercholesterolemic agents, cholesterol biosynthesis inhibitors (e.g., HMG CoA reductase inhibitors), LXR agonists, FXR agonists, probucol, raloxifene, nicotinic acid, nicotinamide, cholesterol absorption inhibitors, bile acid sequestrants, anion exchange resins, quaternary amines, cholestyramine, colestyramineTipoxil, low density lipoprotein receptor inducer, clofibrate, fenofibrate, bezafibrate, ciprofibrate, gemfibrozil (gemfibrazol), vitamin B6, vitamin B12, antioxidant vitamins, antidiabetics, platelet aggregation inhibitors, fibrinogen receptor antagonists, aspirin and fibric acid derivatives, PCSK9 inhibitors, aspirin and P2Y12 inhibitors such as clopidogrel.

In another aspect, the present disclosure provides a method of treating, preventing, or alleviating a symptom of a disease associated with fibrosis, comprising administering to a patient in need thereof a therapeutically effective amount of a modified relaxin polypeptide or a composition comprising a modified relaxin polypeptide as described herein. The diseases associated with fibrosis may include fibrosis of the heart, lung, kidney, bone marrow or liver, skin fibrosis or fibrotic eye disease. The fibrosis-associated disease may be selected from liver fibrosis, non-alcoholic steatohepatitis (NASH), cirrhosis, pre-cirrhosis, diffuse parenchymal lung disease, cystic fibrosis, lung fibrosis, progressive massive fibrosis, idiopathic pulmonary fibrosis, injection fibrosis, kidney fibrosis, chronic kidney disease, diabetic nephropathy, focal segmental glomerulosclerosis, membranous nephropathy, IgA nephropathy, myelofibrosis, heart failure, metabolic heart failure, myocardial fibrosis, cataract, ocular scarring, pancreatic fibrosis, skin fibrosis, intestinal stenosis, endomyocardial fibrosis, atrial fibrosis, mediastinal fibrosis, crohn's disease, retroperitoneal fibrosis, keloid, nephrogenic systemic fibrosis, scleroderma, systemic sclerosis, joint fibrosis, peonie syndrome, Dupuytren's (duytren's) contracture, crohn's disease, progressive massive fibrosis, and/or acute fibrosis Diabetic neuropathy, adhesive capsulitis, alcoholic liver disease, hepatic steatosis, viral hepatitis, biliary tract diseases, primary hemochromatosis, drug-related cirrhosis, cryptogenic cirrhosis, Wilson's disease, alpha 1-antitrypsin deficiency, Interstitial Lung Disease (ILD), human fibrotic lung disease, macular degeneration, retinal retinopathy, vitreoretinopathy, myocardial fibrosis, Grave's (Grave) eye disease, drug-induced ergotoxemia, cardiovascular disease, atherosclerosis/restenosis, hypertrophic scars, primary or idiopathic myelofibrosis, inflammatory bowel disease and collagen colitis.

The method of treating a fibrosis-associated disease may further comprise administering to the patient the modified relaxin polypeptide in combination, simultaneously or sequentially with at least one anti-fibrotic drug. The anti-fibrotic agent may be selected from nintedanib, pirfenidone, LPA1 antagonists, LPA1 receptor antagonists, GLP1 analogs, tralokinumab (tralokinumab) (IL-13, AstraZeneca), vismodegib (vismodegib) (hedgehog antagonists, Roche), PRM-151 (pentameric protein-2, TGF β -1, Promedior), SAR-156597 (bispecific Mab IL-4& IL-13, Sanofi), simuzumab (simtuzumab) ((anti-lysyloxidase-like 2 (anti-LOXL 2) antibody, Gilead), CKD-942, PTL-202(PDE inhalation/pentoxifylline/naclone theobromine/nacle oral controlled release, pacic Ther's plug), imiri (oral ipolisib) (PI 3K/mTOR inhibitor, GSK), IW-001, IW-modified bovine serum albumin, beta-100 collagen/100 alpha (stv), Stromedix/Biogen), Actimmune (IFN γ), PC-SOD (intermediate dismutase; inhalation, LTT Bio-Pharma/CKD Pharm), Lebrikizumab (Lebrikizumab) (anti-IL-13 SC humanized mAb, Roche), AQX-1125(SHIP1 activator, Aquinox), CC-539(JNK inhibitor, Celgene), FG-3019(FibroGen), SAR-100842(Sanofi) and obeticholic acid (OCA or INT-747, Intercept).

In another aspect, the present disclosure provides a method of treating or preventing renal failure, comprising administering to a patient in need thereof a therapeutically effective amount of a modified relaxin polypeptide or a composition comprising a modified relaxin polypeptide as described herein.

In another aspect, the present disclosure provides a method of improving, stabilizing or restoring kidney function in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of a modified relaxin polypeptide or a composition comprising a modified relaxin polypeptide as described herein.

In another aspect, the present disclosure provides a method of making a modified relaxin polypeptide as described herein, comprising: (a) providing a polypeptide comprising a relaxin a chain and a relaxin B chain, wherein the polypeptide comprises a non-naturally encoded amino acid; and (b) linking the non-naturally encoded amino acid to the pharmacokinetic enhancer. The non-naturally encoded amino acid may be linked to the pharmacokinetic enhancer by an oxime linkage, e.g., linking the non-naturally encoded amino acid to a peptide component of the pharmacokinetic enhancer. The oxime linkage may be formed by the reaction of a carbonyl group and an aminooxy group. The carbonyl group can be a substituent of the non-naturally encoded amino acid, and the aminooxy group can be a component of the pharmacokinetic enhancer. The aminooxy group may be a substituent of the non-naturally encoded amino acid, and the carbonyl group may be a component of the pharmacokinetic enhancer, for example a component of a peptide component of the pharmacokinetic enhancer. The non-naturally encoded amino acid can be ribosomally incorporated into the polypeptide.

Universal recombinant nucleic acids and methods for use with the disclosed modified relaxin polypeptides

In embodiments of the present disclosure, nucleic acids encoding modified relaxin polypeptides of interest may be isolated, cloned, and often altered using recombinant methods. Such embodiments may be used, including but not limited to, for protein expression or in the production of variants, derivatives or other sequences derived from modified relaxin polypeptides. In some embodiments, the sequence encoding the modified relaxin polypeptides of the disclosure is operably linked to a heterologous promoter. In some embodiments, the codon usage of DNA in the polynucleotide sequence encoding the modified relaxin polypeptide may be optimized for e.coli or mammalian cell (e.g., CHO) expression using techniques well known in the art.

Exemplary polynucleotides encoding the relaxin A chain (SEQ ID NO:10), relaxin B chain (SEQ ID NO:11), relaxin A and B chains (SEQ ID NO:12), and leader sequences (SEQ ID NO:13-14) are provided herein.

The nucleotide sequence encoding the modified relaxin polypeptides described herein may be synthesized based on the amino acid sequence of a parent polypeptide, including but not limited to, a parent polypeptide having the amino acid sequence set forth in SEQ ID NOs 4,5, 6, 35, 36, or 37, and then the nucleotide sequence is altered to effect the introduction (i.e., incorporation or substitution) or removal (i.e., deletion or substitution) of the relevant amino acid residue. The nucleotide sequence may conveniently be modified by site-directed mutagenesis in accordance with conventional procedures. Alternatively, the nucleotide sequence may be prepared by chemical synthesis (including but not limited to by using an oligonucleotide synthesizer), wherein the oligonucleotide is designed based on the amino acid sequence of the desired polypeptide and preferably those codons are selected which are advantageous in the host cell in which the recombinant polypeptide is to be produced.

In addition, selector codons that encode non-naturally encoded amino acids can be incorporated into a polynucleotide sequence, as further described herein.

The disclosure also relates to eukaryotic host cells, non-eukaryotic host cells, and organisms for in vivo incorporation of an unnatural amino acid by orthogonal tRNA/RS pairs. Host cells are genetically engineered (including but not limited to, transformed, transduced or transfected) with a polynucleotide of the present disclosure or a construct comprising a polynucleotide of the present disclosure, including but not limited to a vector of the present disclosure, which may be, for example, a cloning vector or an expression vector. For example, the coding regions for the orthogonal tRNA, the orthogonal tRNA synthetase, and the protein to be derivatized are operably linked to gene expression control elements that function in the desired host cell. The vector may be in the form of, for example, a plasmid, cosmid, phage, bacterium, virus, naked polynucleotide, or conjugated polynucleotide. The vector may be introduced into the cells and/or microorganisms by standard methods.

Selector codon (selector codon)

Selector codons of the present disclosure extend the genetic codon framework of the protein biosynthetic machinery. For example, selector codons include, but are not limited to, unique three base codons, nonsense codons, such as stop codons, including, but not limited to, amber codons (UAG), ochre codons, or opal codons (UGA); unnatural codons, codons of four or more bases, rare codons, and the like. It will be apparent to one of ordinary skill in the art that the number of selector codons that can be introduced into a desired gene or polynucleotide is wide ranging, including, but not limited to, one or more, two or more, three or more, 4,5, 6, 7, 8, 9, 10 or more in a single polynucleotide encoding at least a portion of a modified relaxin polypeptide.

In one embodiment, the method involves the use of a selector codon, which is a stop codon, for the in vivo incorporation of one or more non-natural (i.e., non-naturally encoded) amino acids. For example, an O-tRNA is generated that recognizes a stop codon (including but not limited to UAG) and aminoacylated with an O-RS that carries the desired unnatural amino acid. This O-tRNA is not recognized by the naturally occurring host aminoacyltRNA synthetases. Conventional site-directed mutagenesis can be used to introduce a stop codon, including but not limited to TAG, at a site of interest in the polypeptide of interest. See, e.g., Sayers, J.R. et al (1988),Nucleic Acids Res,16:791-802. When the O-RS, O-tRNA and nucleic acid encoding the polypeptide of interest are combined in vivo, the unnatural amino acid is incorporated in response to the UAG codon, thereby producing a polypeptide that contains the unnatural amino acid at the specified position.

The in vivo incorporation of the unnatural amino acid can be performed under conditions that do not significantly disturb the eukaryotic host cell. For example, because the suppression efficiency of the UAG codon depends on the competition between the O-tRNA (including but not limited to the amber suppressor tRNA) and a eukaryotic release factor (including but not limited to eRF) (which binds to a stop codon and initiates release of the growing peptide from the ribosome), the suppression efficiency can be modulated by, including but not limited to, increasing the expression level of the O-tRNA and/or the suppressor tRNA.

Unnatural amino acids can also be encoded with rare codons. For example, it has been demonstrated that the rare arginine codon, AGG, efficiently intercalates Ala with alanine-acylated synthetic tRNA when arginine concentration is reduced in an in vitro protein synthesis reaction. See for example Ma et al,Biochemistry,32:7939(1993). In this case, the synthetic tRNA competes with the naturally occurring tRNA Arg in E.coli, which is present as a minor species.

Selector codons also include extended codons, including, but not limited to, four or more base codons, e.g., four, five, six, or more base codons. Examples of four base codons include, but are not limited to, AGGA, CUAG, UAGA, CCCU, and the like. Examples of five base codons include, but are not limited to, AGGAC, CCCCU, CCCUC, CUAGA, CUACU, UAGGC, and the like.

In one embodiment, extended codons based on rare codons or nonsense codons can be used in the present disclosure, which can reduce missense readthrough and frameshift suppression at otherwise undesirable sites.

For a given system, where an endogenous system does not use (or rarely uses) a native three base codon, a selector codon can also include that codon. For example, this includes systems lacking a tRNA that recognizes the three base codon in nature, and/or systems in which the three base codon is a rare codon.

Genes encoding proteins or polypeptides of interest (e.g., modified relaxin polypeptides) can be mutagenized using methods known to those of ordinary skill in the art and described herein to incorporate, for example, one or more selector codons for incorporation of unnatural amino acids. For example, the nucleic acid of the protein of interest is mutagenized to include one or more selector codons to provide for the incorporation of one or more unnatural amino acids. The present disclosure includes any such variant, including but not limited to a mutant version of any protein, e.g., one that includes at least one unnatural amino acid.

Nucleic acid molecules encoding proteins of interest, such as modified relaxin polypeptides, can be readily mutated to introduce a cysteine at any desired position of the polypeptide. Cysteine is widely used to introduce active molecules, water-soluble polymers, proteins or a variety of other molecules onto a protein of interest.

Non-naturally encoded amino acids

A wide variety of non-naturally encoded amino acids are suitable for use in the present disclosure. Any number of non-naturally encoded amino acids can be introduced into the modified relaxin polypeptide. In general, the introduced non-naturally encoded amino acids are substantially chemically inert to the 20 common genetically encoded amino acids (i.e., alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine). In some embodiments, the non-naturally encoded amino acid includes side chain functional groups (including, but not limited to, azide, keto, aldehyde, and aminoxy groups) that efficiently and selectively react with functional groups found in the 20 common amino acids to form stable conjugates. For example, a modified relaxin polypeptide comprising a non-naturally encoded amino acid comprising an azido functional group can be reacted with a polymer or PK extender, or can be reacted with a second polypeptide comprising an alkyne moiety to form a stable conjugate, resulting in selective reaction of the azide and alkyne functional groups to form a Huisgen [3+2] cycloaddition product.

The general structure of the α -amino acids is shown below (formula IV):

IV

the non-naturally encoded amino acid is generally any structure having the formula listed above and may be suitable for use in the modified relaxin polypeptides of the present disclosure, wherein the R group is any substituent other than the substituents used in the twenty natural amino acids. Because the non-naturally encoded amino acids of the present disclosure typically differ from natural amino acids only in side chain structure, the non-naturally encoded amino acids form amide bonds with other amino acids (including, but not limited to, naturally or non-naturally encoded) in the same manner in which they are formed in naturally occurring polypeptides. However, non-naturally encoded amino acids have side chain groups that distinguish them from natural amino acids. For example, R optionally includes alkyl-, aryl-, acyl-, ketone-, azide-, hydroxyl-, hydrazine, cyano-, halo-, hydrazide, alkenyl, alkynyl, ether, thiol, seleno-, sulfonyl-, borate, phosphate, phosphonyl, phosphine, heterocycle, enone, imine, aldehyde, ester, thioacid, hydroxylamine, amino, and the like, or any combination thereof.

Exemplary non-naturally encoded amino acids that may be suitable for use in the present disclosure and that may be used to react with PK extenders and polymers include, but are not limited to, those having carbonyl, aminoxy, hydrazine, hydrazide, semicarbazide, azide, and alkyne reactive groups. In some embodiments, the non-naturally encoded amino acid comprises a sugar moiety. Examples of such amino acids include N-acetyl-L-glucosaminyl-L-serine, N-acetyl-L-galactosaminyl-L-serine, N-acetyl-L-glucosaminyl-L-threonine, N-acetyl-L-glucosaminyl-L-asparagine, and O-mannosaminyl-L-serine.

Many of the non-naturally encoded amino acids provided herein are commercially available, for example, from Sigma-Aldrich (St. Louis, MO, USA), Novabiochem (a division of EMD Biosciences, Darmstadt, Germany), or Peptech (Burlington, MA, USA). Those amino acids that are not commercially available are optionally synthesized as provided herein, or using standard methods known to those of ordinary skill in the art. For organic synthesis techniques see, for example, Fessendon and FessendonOrganic Chemistry(second edition, 1982, Willad Grant Press, Boston Mass.); of MarchAdvanced Organic Chemistry(third edition, 1985, Wiley and Sons, New York); and of Carey and SundbergAdvanced Organic Chemistry(third edition, parts A and B, 1990, Plenum Press, New York). See also U.S. Pat. Nos. 7,045,337 and 7,083,970, which are incorporated herein by reference. In addition to unnatural amino acids containing novel side chains, unnatural amino acids useful in the disclosure optionally comprise modified backbone structures, including, but not limited to, those shown in the structures of formulas V and VI:

V

VI

wherein Z typically comprises OH, NH₂SH, NH-R 'or S-R'; x and Y may be the same or different, typically include S or O, and R' (optionally the same or different) are typically selected from the same list of components for the R group described above for the unnatural amino acid of formula I and hydrogen. For example, the unnatural amino acids of the disclosure optionally comprise substitutions in the amino or carboxyl group, as shown in formulas V and VI. This type of unnatural amino acid includes, but is not limited to, alpha-hydroxy acids, alpha-thio acids, alpha-aminothiocarboxylates, including, but not limited to, those having side chains or unnatural side chains corresponding to the common twenty natural amino acids. In addition, substitutions on the α -carbon optionally include, but are not limited to, L, D or α - α -disubstituted amino acids, such as D-glutamic acid, D-alanine, D-methyl-O-tyrosine, aminobutyric acid, and the like. Other structural substitutions include cyclic amino acids such as proline analogs and 3,4, 6, 7, 8 and 9 membered ring proline analogs, beta and gamma amino acids such as substituted beta-alanine and gamma-aminobutyric acid.

Many unnatural amino acids are based on natural amino acids, e.g., tyrosine, glutamine, phenylalanine, and the like, and are suitable for use in the present disclosure. Tyrosine analogs include, but are not limited to, para-substituted tyrosines, ortho-substituted tyrosines, and meta-substituted tyrosines, where the substituted tyrosines comprise, but are not limited to, keto groups (including, but not limited to, acetyl groups), benzoyl groups, amino groups, hydrazine, hydroxylamine, thiol groups, carboxyl groups, isopropyl groups, methyl groups, C groups₆-C₂₀Straight or branched chain hydrocarbons, saturated or unsaturated hydrocarbons, O-methyl, polyether groups, nitro groups, alkynyl groups, and the like. Additionally, polysubstituted aromatic rings are also contemplated. Glutamine analogs that may be suitable for use in the present disclosure include, but are not limited to, alpha-hydroxy derivatives, gamma-substituted derivatives, cyclic derivatives, and amide-substituted glutamine derivatives. Exemplary phenylalanine analogs that may be suitable for use in the present disclosure include, but are not limited to, para-substituted phenylalanines, ortho-substituted phenylalanines, and meta-substituted phenylalanines, wherein the substituents include, but are not limited to, hydroxy, methoxy, methyl, allyl, aldehyde, azido, iodo, bromo, keto (including, but not limited to, acetyl), benzyl, and the likeAcyl, alkynyl, and the like. Specific examples of unnatural amino acids that can be suitable for use in the present disclosure include, but are not limited to, p-acetyl-L-phenylalanine, O-methyl-L-tyrosine, L-3- (2-naphthyl) alanine, 3-methyl-phenylalanine, O-4-allyl-L-tyrosine, 4-propyl-L-tyrosine, tri-O-acetyl-GlcNAc β -serine, L-dopa, fluorinated phenylalanine, isopropyl-L-phenylalanine, p-azido-L-phenylalanine, p-acyl-L-phenylalanine, p-benzoyl-L-phenylalanine, L-phosphoserine, phosphonoserine, phosphonotyrosine, beta-tyrosine, p-iodophenylalanine, p-bromophenylalanine, p-amino-L-phenylalanine, isopropyl-L-phenylalanine, and p-propargyloxy-phenylalanine, etc.

In one embodiment, a composition is provided comprising a modified relaxin polypeptide of an unnatural amino acid (e.g., p-acetyl-L-phenylalanine). Also provided are various compositions comprising p-acetyl-L-phenylalanine and including, but not limited to, proteins and/or cells. In one aspect, a composition comprising a p-acetyl-L-phenylalanine unnatural amino acid further comprises an orthogonal tRNA. The unnatural amino acid can be bonded (including but not limited to covalently bonded) to the orthogonal tRNA, including but not limited to covalently bonded to the orthogonal tRNA through an amino-acyl bond, covalently bonded to a 3 'OH or a 2' OH of a terminal ribose sugar of the orthogonal tRNA, and the like.

The modified relaxin polypeptides described herein may comprise a non-naturally encoded amino acid described in U.S. patent No.8,735,539, entitled "relaxin polypeptides comprising a non-naturally encoded amino acid," which is incorporated herein by reference in its entirety.

Structure and Synthesis of unnatural amino acids

In some embodiments, the disclosure provides a modified relaxin polypeptide linked to a PK prolonging agent (e.g., comprising a fatty acid) by an oxime bond or linkage. Many types of non-naturally encoded amino acids are suitable for forming oxime linkages. These include, but are not limited to, non-naturally encoded amino acids containing carbonyl, dicarbonyl, carbonyl-like groups, masked carbonyl, protected carbonyl, or hydroxylamine groups. Such amino acids, their structures and syntheses are described in U.S. Pat. nos. 8,012,931 and 8,735,539, which are incorporated herein by reference in their entirety.

Exemplary structures and synthetic methods for non-naturally encoded amino acids, including hydroxylamine-containing amino acids, are known in the art, for example, as disclosed in U.S. patent No.7,332,571, U.S. patent publication nos. 2006/0194256, 2006/0217532, and 2006/0217289, and WO 2006/069246, each of which is incorporated herein in its entirety.

Chemical synthesis of non-naturally encoded amino acids

Many unnatural amino acids suitable for use in the present disclosure are commercially available, e.g., from sigma (USA) or Aldrich (Milwaukee, WI, USA). Those amino acids that are not commercially available are optionally synthesized as provided herein or as provided in various publications, or using standard methods known to those of ordinary skill in the art.

A. Carbonyl reactive groups

Amino acids having a carbonyl-reactive group particularly allow for a variety of reactions to attach molecules, including but not limited to PEG or other water-soluble molecules, such as peptide components, via nucleophilic addition or aldol condensation reactions.

The synthesis of acetyl- (+/-) -phenylalanine and meta-acetyl- (+/-) -phenylalanine is described in Zhang, Z et al, Biochemistry 42:6735-6746(2003), which is incorporated herein by reference. Other carbonyl-containing amino acids can be similarly prepared by one of ordinary skill in the art.

In some embodiments, a modified relaxin polypeptide comprising a non-naturally encoded amino acid may be chemically modified to generate a reactive carbonyl functional group. For example, aldehyde functionality useful for conjugation reactions can be generated from functional groups having adjacent amino and hydroxyl groups.

In the present disclosure, non-naturally encoded amino acids with adjacent hydroxyl and amino groups can be incorporated into polypeptides as "masked" aldehyde functionalities. For example, 5-hydroxylysine bears a hydroxyl group adjacent to the epsilon amine. The reaction conditions for the generation of aldehydes typically involve the addition of a molar excess of sodium metaperiodate under mild conditions to avoid oxidation at other sites within the polypeptide. The pH of the oxidation reaction is typically about 7.0. A typical reaction involves the addition of about a 1.5 molar excess of sodium metaperiodate to a buffered solution of the polypeptide, followed by incubation in the dark for about 10 minutes. See, for example, U.S. patent No.6,423,685, which is incorporated herein by reference.

The carbonyl function can selectively react in aqueous solution under mild conditions with a hydrazine, hydrazide, hydroxylamine or semicarbazide containing reagent to form the corresponding hydrazone, oxime or semicarbazone linkage, respectively, that is stable under physiological conditions. See, e.g., Jencks, w.p., j.am.chem.soc.81,475-481 (1959); shao, J.and Tam, J.P., J.Am.chem.Soc.117: 3893-. In addition, the unique reactivity of the carbonyl group allows for selective modification in the presence of other amino acid side chains. See, e.g., Cornish, V.W., et al, J.Am.chem.Soc.118: 8150-; geoghegan, K.F. & Stroh, J.G., bioconjugate. chem.3:138-146 (1992); mahal, L.K. et al, Science 276:1125-1128 (1997).

B. Hydrazine, hydrazide or semicarbazide reactive groups

Non-naturally encoded amino acids containing nucleophilic groups such as hydrazine, hydrazide, or semicarbazide allow for the reaction with various electrophilic groups to form conjugates.

Amino acids containing hydrazide, hydrazine and semicarbazide are available from commercial sources. For example, L-glutamic acid γ -hydrazide can be obtained from Sigma Chemical (st. louis, MO). Other non-commercially available amino acids can be prepared by one of ordinary skill in the art. See, for example, U.S. patent No.6,281,211, which is incorporated herein by reference.

Modified relaxin polypeptides containing non-naturally encoded amino acids with hydrazide, hydrazine, or semicarbazide functional groups can react efficiently and selectively with a variety of molecules containing aldehydes or other functional groups with similar chemical reactivity. See, e.g., Shao, J. and Tam, J., J.Am.chem.Soc.117: 3893-. The unique reactivity of hydrazide, hydrazine, and semicarbazide functional groups makes them significantly more reactive toward aldehydes, ketones, and other electrophilic groups than nucleophilic groups present on the 20 common amino acids (including but not limited to the hydroxyl groups of serine or threonine or the amino and N-termini of lysine).

C. Amino acids containing an amino group

Non-naturally encoded amino acids containing an aminooxy (also referred to as hydroxylamine) group allow for reaction with various electrophilic groups to form conjugates, including but not limited to reaction with PEG or peptide components. Like hydrazine, hydrazides, and semicarbazides, the enhanced nucleophilicity of aminoxy groups enables them to react efficiently and selectively with a variety of molecules containing aldehydes or other functional groups with similar chemical reactivity. See, e.g., Shao, J. and Tam, J., J.Am.chem.Soc.117: 3893-; H.Hang and C.Bertozzi, Acc.chem.Res.34:727-736 (2001). Although the reaction with a hydrazine group results in the corresponding hydrazone, the reaction of an aminoxy group with a carbonyl-containing group (e.g., a ketone) typically produces an oxime.

Amino acids containing an amino acid group can be prepared from readily available amino acid precursors (homoserine, serine and threonine). See, e.g., M.Carrasco and R.Brown, J.org.chem.68:8853-8858 (2003). Certain amino acid containing an aminooxy group have been isolated from natural sources, such as L-2-amino-4- (aminooxy) butanoic acid (Rosenthal, G., Life Sci.60:1635-1641 (1997). other amino acid containing an aminooxy group can be prepared by one of ordinary skill in the art.

D. Azide and alkyne reactive groups

The unique reactivity of azide and alkyne functional groups makes them useful for the selective modification of polypeptides and other biomolecules. Organic azides (particularly aliphatic azides) and alkynes are generally stable to common reactive chemical conditions. In particular, both the azide and alkyne functional groups are inert to the side chains (i.e., R groups) of the 20 common amino acids found in naturally occurring polypeptides. However, when the azide group and the alkyne group are brought into close proximity, their "spring-loaded" nature is revealed, reacting selectively and efficiently via the Huisgen [3+2] cycloaddition reaction to produce the corresponding triazole. See, e.g., Chin J. et al, Science 301:964-7 (2003); wang, q, et al, j.am.chem.soc.125,3192-3193 (2003); chin, J.W. et al, J.Am.chem.Soc.124: 9026-.

Because the Huisgen cycloaddition reaction involves a selective cycloaddition reaction (see, e.g., Padwa, A., COMPREHENSIVE ORGANIC SYNTHESIS, Vol.4, (Trost, B.M. Ed., 1991),pages 1069 and 1109; huisgen, r.1,3-dipola cycloaddion chemist (Padwa, editors at a.19, 1984), pages 1-176), rather than nucleophilic substitution, incorporation of a non-naturally encoded amino acid bearing an azide and an alkyne-containing side chain allows the resulting polypeptide to be selectively modified at the position of the non-naturally encoded amino acid. Cycloaddition reactions involving azide-or alkyne-containing modified relaxin polypeptides can be carried out at room temperature under aqueous conditions with catalytic amounts of reducing agent [ catalyst for in situ reduction of Cu (II) to Cu (I)]In the presence of Cu (II) (including but not limited to catalytic amounts of CuSO)₄Of the form(s) can be used. See, e.g., Wang, q, et al, j.am.chem.soc.125,3192-3193 (2003); tornoe, C.W. et al, J.org.chem.67:3057-3064 (2002); rostovtsev et al, Angew. chem. Int. Ed.41: 2596-. Exemplary reducing agents include, but are not limited to, ascorbate, copper metal, quinine (quinine), hydroquinone, vitamin K, glutathione, cysteine, Fe²⁺、Co²⁺And an applied potential.

In some cases, where a Huisgen [3+2] cycloaddition reaction between an azide and an alkyne is desired, the modified relaxin polypeptide may comprise a non-naturally encoded amino acid comprising an alkyne module, and the PK enhancer to be attached to the amino acid may comprise an azide module. Alternatively, the reverse reaction may be performed (i.e., an azide moiety on the amino acid and an alkyne moiety on the water-soluble polymer).

The azide functionality may also be selectively reacted with the aryl ester-containing water-soluble polymer and appropriately functionalized via the arylphosphine module to form an amide linkage. The arylphosphine group reduces the azide in situ and the resulting amine then reacts efficiently with a proximal ester linkage to yield the corresponding amide. See, e.g., e.saxon and c.bertozzi, sciences 287,2007-2010 (2000). The azide-containing amino acid may be an alkyl azide (including but not limited to 2-amino-6-azido-1-hexanoic acid) or an aryl azide (p-azido-phenylalanine).

The azide functionality can also be selectively reacted with a thioester-containing water-soluble polymer and appropriately functionalized via an arylphosphine moiety to form an amide linkage. The arylphosphine group reduces the azide in situ and the resulting amine is then reacted efficiently with a thioester linkage to yield the corresponding amide.

Alkyne-containing amino acids are commercially available. Alternatively, the alkyne-containing amino acids can be prepared according to standard methods. For example, the propargyloxyphenylalanine may be synthesized as described in Deiters, A. et al, J.Am.chem.Soc.125:11782-11783(2003) and the 4-alkynyl-L-phenylalanine may be synthesized as described in Kayser, B. et al, Tetrahedron 53(7) 2475-2484 (1997). Other alkyne-containing amino acids can be prepared by one of ordinary skill in the art.

The azide-containing amino acids are available from commercial sources. For those azide-containing amino acids that are not commercially available, the azide group can be prepared relatively easily using standard methods known to those of ordinary skill in the art, including but not limited to by displacement of a suitable leaving group (including but not limited to halide, mesylate, tosylate) or by opening an appropriately protected lactone. See, e.g., March' sAdvanced Organic Chemistry(third edition, 1985, Wiley and Sons, New York).

Molecules that can be added to the proteins of the present disclosure by [3+2] cycloaddition include virtually any molecule with an azide or alkynyl derivative. Such molecules include, but are not limited to, dyes, fluorophores, cross-linkers, sugar derivatives, polymers (including, but not limited to, polymers comprising polyethylene glycol), photocrosslinkers, cytotoxic compounds, affinity tags, biotin derivatives, resins, beads, peptides, second protein or polypeptide (or more), polynucleotides (including, but not limited to, DNA, RNA, etc.), metal chelators, cofactors, fatty acids, carbohydrates, and the like. These molecules can be added to unnatural amino acids with an alkynyl group, including but not limited to p-propargyloxyphenylalanine, or to unnatural amino acids with an azido group, including but not limited to p-azido-phenylalanine.

E. Aminothiol reactive groups

The unique reactivity of the β -substituted aminothiol functional groups makes them useful for selectively modifying polypeptides and other biomolecules containing aldehyde groups by forming thiazolidines. See, e.g., J.Shao and J.Tam, J.Am.chem.Soc.1995,117(14) 3893-3899. In some embodiments, a β -substituted aminothiol amino acid may be incorporated into a modified relaxin polypeptide and then reacted with a PK enhancer comprising an aldehyde functional group. In some embodiments, a PK enhancer, drug conjugate, or other payload may be coupled to a modified relaxin polypeptide comprising a β -substituted aminothiol amino acid by formation of thiazolidine.

F. Additional reactive groups

Additional reactive groups and non-naturally encoded amino acids that can be incorporated into the modified relaxin polypeptides of the present disclosure are described in the following patent applications, all of which are incorporated herein in their entirety by reference: U.S. patent publication No. 2006/0194256, U.S. patent publication No. 2006/0217532, U.S. patent publication No. 2006/0217289, and International patent application publication No. WO/2007/070659.

In vivo production of modified relaxin polypeptides comprising non-naturally encoded amino acids

The modified relaxin polypeptides of the disclosure can be produced in vivo by using modified trnas and tRNA synthetases to add or replace amino acids that are not encoded in naturally occurring systems. Such a process is described in U.S. patent No.8,735,539, which is incorporated herein in its entirety by reference.

Methods of producing trnas and tRNA synthetases that utilize amino acids that are not encoded in naturally occurring systems are described, for example, in U.S. Pat. nos. 7,045,337 and 7,083,970, which are incorporated herein by reference. These methods involve the generation of a suite of translation machinery that functions independently of endogenous synthetases and tRNAs of the translation system (hence sometimes referred to as "orthogonal"). Typically, such a translation system comprises an orthogonal tRNA (O-tRNA) and an orthogonal aminoacyl tRNA synthetase (O-RS). Typically, the O-RS preferentially aminoacylates the O-tRNA with at least one non-naturally occurring amino acid in the translation system, and the O-tRNA recognizes at least one selector codon that is not recognized by other tRNAs in the system. Thus, the translation system inserts a non-naturally encoded amino acid into a protein produced in the system in response to the encoded selector codon, thereby "replacing" the amino acid in a position that encodes a polypeptide.

The use of O-tRNA/aminoacyl-tRNA synthetases involves the selection of specific codons that encode non-naturally encoded amino acids. Although any codon can be used, it is generally desirable to select codons that are rarely or never used in cells expressing O-tRNA/aminoacyl-tRNA synthetases. For example, exemplary codons include nonsense codons, such as stop codons (amber, ochre, and opal), four or more base codons, and other natural three base codons that are rarely or never used.

Specific selector codons can be introduced into appropriate positions of the modified relaxin polynucleotide coding sequence using mutagenesis methods known in the art (including, but not limited to, site-specific mutagenesis, cassette mutagenesis, restriction-selection mutagenesis, and the like).

Expression in non-eukaryotes and eukaryotes

Expression systems, culture and isolation

Unmodified or modified relaxin polypeptides may be expressed in any number of suitable expression systems, including, for example, yeast, insect cells (e.g., baculovirus-infected insect cells), mammalian cells, and bacteria. The following provides a description of an exemplary expression system.

Yeast: as used herein, the term "yeast" includes any of a variety of yeasts capable of expressing a gene encoding a modified relaxin polypeptide, including, but not limited to, pichia pastoris, saccharomyces glumae (p.guilliermondii), saccharomyces cerevisiae, saccharomyces carlsbergensis (s.carlsbergensis), saccharomyces diastaticus (s.diastaticus), kluyveromyces douglas (s.douglaii), kluyveromyces (s.kluyveri), saccharomyces norbensis (s.norbensis), ovula (s.oviformis), kluyveromyces lactis (k.lactis), kluyveromyces fragilis (k.fragilis), candida albicans (c.albicans), candida maltosa (c.maltosa), and hansenula polymorpha (h.polymorpha). WO 2005/091944, incorporated herein by reference, describes the expression of relaxin in yeast.

Escherichia coli, pseudomonas species and other prokaryotes:

the term "bacterial host" or "bacterial host cell" refers to a bacterium that can be, or has been, used as a recipient for recombinant vectors or other transferred DNA. The term includes progeny of the original bacterial host cell that has been transfected. It is understood that, due to accidental or deliberate mutation, progeny of a single parent cell may not necessarily be identical in morphology or in genomic or total DNA complementary to the original parent. Progeny of a parent cell that is sufficiently similar to the parent to be characterized by the relevant property (e.g., the presence of a nucleotide sequence encoding an unmodified or modified relaxin polypeptide) is included in the progeny intended by this definition.

In selecting a bacterial host for expression, suitable hosts may include those that exhibit, inter alia, good inclusion body formation ability, low proteolytic activity, and overall robustness. Industrial/pharmaceutical fermentation generally uses bacteria derived from the K strain (e.g., W3110) or bacteria derived from the B strain (e.g., BL 21). Other examples of suitable E.coli hosts include, but are not limited to, strains of BL21, DH10B, or derivatives thereof. In another embodiment of the disclosed method, the E.coli host is a protease deficient strain, including but not limited to OMP-and LON-. The host cell strain may be a Pseudomonas species, including but not limited to Pseudomonas fluorescens, Pseudomonas aeruginosa, and Pseudomonas putida. Pseudomonas fluorescens biotype 1, designated strain MB101, is known to be useful for recombinant production and in therapeutic protein production processes.

Once a recombinant host cell strain has been established (i.e., the expression construct has been introduced into the host cell and the host cell with the appropriate expression construct isolated), the recombinant host cell strain is cultured under conditions suitable for the production of the modified relaxin polypeptide.

The recombinant host cells may be cultured in batch or continuous form, with cell harvesting (in the case of intracellular accumulation of the modified relaxin polypeptide) or harvesting of the culture supernatant being performed in batch or continuous form.

The modified relaxin polypeptide produced in the bacterial host cell may be poorly soluble or insoluble (in inclusion body form). In one embodiment of the present disclosure, amino acid substitutions selected to increase the solubility of a recombinantly produced protein may be readily made in modified relaxin polypeptides. The modified relaxin polypeptide may be solubilized with, for example, urea or guanidine hydrochloride.

In the case of soluble modified relaxin proteins, relaxin can be secreted into the periplasmic space or into the culture medium. For example, the modified relaxin is secreted into the periplasmic space of W3110-B2 cells by transforming these plasmids into W3110-B2 cells using plasmids encoding constructs containing eight different leader sequences (including those listed in SEQ ID NOS: 39-44), and then growing the cells at 37 ℃ until the OD reaches about 0.8, at which time expression is induced with 0.01% arabinose. After five hours, a periplasmic release sample may be prepared from the culture. In addition, soluble modified relaxin may be present in the cytoplasm of the host cell. It may be desirable to concentrate the soluble modified relaxin before performing the purification step.

When the modified relaxin polypeptide is produced as a fusion protein, the fusion sequence may be removed. Removal of the fusion sequence can be accomplished by enzymatic or chemical cleavage. Enzymatic removal of the fusion sequence can be accomplished using methods known to those of ordinary skill in the art. As will be apparent to one of ordinary skill in the art, the choice of enzyme used to remove the fusion sequence may be determined by the nature of the fusion, and the reaction conditions may be dictated by the choice of enzyme. Chemical cleavage can be accomplished using reagents known to those of ordinary skill in the art, including but not limited to cyanogen bromide, TEV protease, and other reagents. The cleaved modified relaxin polypeptide can be purified from the cleaved fusion sequence by methods known to one of ordinary skill in the art.

In general, it is sometimes desirable to denature and reduce the expressed polypeptide and then refold the polypeptide into a preferred conformation. For example, guanidine, urea, DTT, DTE, and/or chaperone proteins may be added to the translation product of interest. The protein may be refolded in a redox buffer containing, including but not limited to, oxidized glutathione and L-arginine. The refolding agent can be flowed or otherwise moved to contact one or more polypeptides or other expression products, or vice versa.

In the case of prokaryotic production of modified relaxin polypeptides, the modified relaxin polypeptides so produced may be misfolded and, thus, lack or have reduced biological activity. The biological activity of the protein can be restored by "refolding". In general, misfolded unmodified or modified relaxin polypeptides are refolded by solubilizing (in the case where the modified relaxin polypeptide is also insoluble), unfolding and reducing the polypeptide chain, using, for example, one or more chaotropic agents (e.g., urea and/or guanidine) and a reducing agent capable of reducing disulfide bonds (e.g., dithiothreitol, DTT, or 2-mercaptoethanol (2-ME)). At moderate concentrations of chaotropic agents, oxidizing agents (e.g., oxygen, cystine, or cystamine) are then added to allow disulfide bond reformation. The modified relaxin polypeptides may be refolded using standard methods known in the art, such as those described in U.S. Pat. nos. 4,511,502, 4,511,503 and 4,512,922, which are incorporated herein by reference. The modified relaxin polypeptides may also be co-folded with other proteins to form heterodimers or heteromultimers.

After refolding, the modified relaxin may be further purified. Purification of the modified relaxin may be accomplished using a variety of techniques known to those of ordinary skill in the art, including hydrophobic interaction chromatography, size exclusion chromatography, ion exchange chromatography, reverse phase high performance liquid chromatography, affinity chromatography, and the like, or any combination thereof. Additional purification may also include a step of drying or precipitating the purified protein.

After purification, the modified relaxin may be exchanged into a different buffer and/or concentrated by any of a variety of methods known in the art, including, but not limited to, diafiltration and dialysis. The modified relaxin provided in the form of a single purified protein may be subjected to aggregation and precipitation.

The purified modified relaxin may be at least 90% pure (as measured by reverse phase high performance liquid chromatography RP-HPLC, or sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)), or at least 95% pure, or at least 98% pure, or at least 99% pure or purer. Regardless of the exact numerical value of the purity of the modified relaxin, the modified relaxin is sufficiently pure for use as a pharmaceutical product or for further processing, e.g., conjugation with a PK enhancer.

Certain modified relaxin molecules may be used as therapeutic agents in the absence of other active ingredients or proteins (other than excipients, carriers and stabilizers, serum albumin, etc.), or they may be complexed with another protein or polymer.

In some embodiments of the present disclosure, the yield of modified relaxin after each purification step may be at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.9%, or at least about 99.99% of unmodified or modified relaxin the starting material of each purification step.

Expression in alternative systems

The modified relaxin polypeptides of the disclosure may be expressed using a cell-free (e.g., in vitro) translation system. The translation system may be a cellular system or a cell-free system, and may be prokaryotic or eukaryotic. Cellular translation systems include, but are not limited to, whole cell preparations, such as permeabilized cells or cell cultures, in which a desired nucleic acid sequence can be transcribed into mRNA and the mRNA can be translated. Cell-free translation systems are commercially available and many different types and systems are well known. Examples of cell-free systems include, but are not limited to, prokaryotic lysates, such as e.coli lysates; and eukaryotic lysates such as wheat germ extract, insect cell lysate, rabbit reticulocyte lysate, rabbit oocyte lysate, and human cell lysate. Membrane extracts, such as canine pancreas extract containing microsomal membranes, can also be obtained and used to translate secreted proteins.

IX. fusion proteins containing modified relaxin polypeptides

The disclosure also provides a modified relaxin polypeptide or fragment thereof comprising a modified relaxin polypeptide sequence and a fusion partner. The fusion partner may confer a functional property, including but not limited to, an extended half-life, facilitating protein purification and/or manufacture, enhanced biophysical properties (e.g., increased solubility or stability), and reduced immunogenicity or toxicity, or any other purpose. For example, the fusion protein can exhibit an extended in vivo half-life, thereby facilitating less frequent dosing in a treatment regimen (e.g., twice weekly, or every other week, etc.). Exemplary fusion proteins comprise a modified relaxin fused to a fusion partner, such as albumin (e.g., human serum albumin), PK extension (PKE) adnectin, XTEN, Fc domain, immunoglobulin constant region, or a fragment of any of the foregoing, or a combination of any of the foregoing. Fusion proteins may be produced by expressing in the same reading frame a nucleic acid encoding a modified relaxin polypeptide sequence and a fusion partner sequence, optionally separated by a sequence encoding a linker peptide. The fusion protein may comprise the modified relaxin polypeptide and the fusion partner in any order, for example, one or more fusion partners attached to the N-terminus and/or C-terminus of the modified relaxin polypeptide sequence, or one or more fusion partners attached to both the N-terminus and C-terminus.

Glycosylation of modified and unmodified relaxin polypeptides

The present disclosure includes modified relaxin polypeptides comprising one or more non-naturally encoded amino acids with sugar residues. The sugar residues may be natural (including but not limited to N-acetylglucosamine) or non-natural (including but not limited to 3-fluorogalactose). The sugar may be linked to the non-naturally encoded amino acid by an N-or O-linked glycosidic linkage (including but not limited to N-acetylgalactosamine-L-serine) or a non-natural linkage (including but not limited to an oxime or a corresponding C-or S-linked glycoside).

Sugar (including but not limited to glycosyl) moieties can be added to the modified relaxin polypeptides in vivo or in vitro. In some embodiments of the present disclosure, a modified relaxin polypeptide comprising a carbonyl-containing non-naturally encoded amino acid can be modified with an aminooxy-derivatized sugar to generate a corresponding glycosylated polypeptide linked by an oxime linkage. Once attached to the non-naturally encoded amino acid, saccharin may be further elaborated by treatment with glycosyltransferases and other enzymes to produce oligosaccharides that bind to the modified relaxin polypeptide. See, e.g., H.Liu et al, J.am.chem.Soc.125:1702-1703 (2003).

Xi. administration and pharmaceutical compositions

Also provided herein are compositions comprising a therapeutically effective amount of a modified relaxin polypeptide described herein and a pharmaceutically acceptable carrier or excipient. Such carriers or excipients include, but are not limited to, saline, buffered saline, dextrose, sucrose, histidine, water, glycerol, PS80 (polysorbate 80), ethanol, and/or combinations thereof. The formulation is formulated to be suitable for administration.

For example, a modified relaxin polypeptide as described herein may be administered to a subject at a concentration of about 0.1 to 100mg/kg of body weight of the subject patient. In one embodiment, a modified relaxin polypeptide as described herein may be administered to a subject at a concentration of about 0.5-5mg/kg of body weight of the subject patient. In another embodiment, a modified relaxin polypeptide as described herein may be administered to a recipient patient at a frequency of from once a day to once every two, three or four weeks, e.g., once a week, twice a week, once every two days, once every three days, once every four days, once every five days, or once every six days. In another embodiment, the modified relaxin polypeptide described herein is administered to the patient once per week.

It will be appreciated that the concentration of the modified relaxin polypeptide administered to a given patient may be higher or lower than the exemplary dosing concentrations listed above.

Based on the information provided in this disclosure, one skilled in the art will be able to determine effective dosages and dosing frequency by routine experimentation, for example, in light of the disclosure herein and Goodman et al (2006), Goodman & Gilman's the pharmaceutical basic of therapeutics. Or Howland et al, (2006) Pharmacology. Lippincott's industrialized reviews. Philadelphia: Lippincott Williams & Wilkins as a guide.

The average amount of modified relaxin may vary, in particular should be based on recommendations and prescriptions of qualified physicians. The exact amount of modified relaxin is a matter of preference, depending on factors such as the exact type of condition being treated, the condition of the patient being treated, and other ingredients in the composition. The present disclosure also provides for the administration of a therapeutically effective amount of another active agent. The amount to be administered can be readily determined by one of ordinary skill in the art.

"pharmaceutical composition" refers to a chemical or biological composition suitable for administration to a mammal. Such compositions may be specifically formulated for administration by one or more of a variety of routes including, but not limited to, oral, epidermal, epidural, inhalation, intra-arterial, intracardiac (intracardiac), intracerebroventricular, intradermal, intramuscular, intranasal, intraocular, intraperitoneal, intraspinal, intrathecal, intravenous, oral, parenteral, rectal (via enema or suppository), subcutaneous, subdermal, sublingual, transdermal and transmucosal. Alternatively, administration may be by injection, powder, liquid, gel, drop or other mode of administration.

As used herein, "pharmaceutically acceptable carrier" or "excipient" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents that are physiologically compatible. In one embodiment, the carrier is suitable for parenteral administration. In another embodiment, the carrier is suitable for subcutaneous administration. Alternatively, the carrier may be adapted for intravenous, intraperitoneal, intramuscular or sublingual administration. Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.

In some embodiments, the pharmaceutical composition may be in a lyophilized form. The compositions may be formulated as solutions, microemulsions, liposomes or other ordered structures suitable for high drug concentrations. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. In some embodiments, the pharmaceutical composition comprises a stabilizing agent. Proper fluidity can be maintained, for example, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants.

In some embodiments, the pharmaceutical composition comprises an isotonic agent, for example, a sugar in the composition (e.g., sucrose), a polyol such as mannitol, sorbitol, or sodium chloride. By including agents such as monostearate salts and gelatin, the absorption of the injectable composition can be prolonged. Furthermore, the polypeptide may be formulated in a time release formulation, for example in a composition comprising a slow release polymer. The active compounds may be formulated with carriers that protect the compound from rapid release, such as controlled release formulations, including implants and microencapsulated delivery systems. Biodegradable biocompatible polymers such as vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, polylactic acid, and polylactic and polyglycolic copolymers (PLG) may be used.

The modified relaxin polypeptides and compositions of the present disclosure may be administered by any conventional route suitable for proteins or peptides, including but not limited to parenterally, such as by injection, including but not limited to subcutaneous or intravenous injection or any other form of injection or infusion. The polypeptide compositions can be administered by a number of routes including, but not limited to, oral, intravenous, intraperitoneal, intramuscular, transdermal, subcutaneous, topical, sublingual, or rectal means. Compositions comprising modified relaxin may also be administered via liposomes. The modified relaxin polypeptides may be used alone or in combination with other suitable components, such as pharmaceutical carriers. The modified relaxin polypeptides may be used in combination with other agents described herein.

Formulations suitable for parenteral administration, e.g., by intraarticular (in the joints), intravenous, intramuscular, intradermal, intraperitoneal, and subcutaneous routes, include aqueous and non-aqueous isotonic sterile injection solutions, which can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions, which can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. The formulations of the modified relaxin may be provided in unit-dose or multi-dose sealed containers such as ampoules and vials.

In the context of the present disclosure, a dose is administered to a patient that is sufficient to have a beneficial therapeutic response in the patient over time or other suitable activity, depending on the application. The dosage depends on the efficacy of the formulation, the activity, stability or serum half-life of the modified relaxin polypeptide employed and the condition of the patient as well as the weight or surface area of the patient to be treated.

For example, the dose administered to a 70 kg patient is generally within a range equivalent to the dose of therapeutic protein currently used adjusted for altered activity or serum half-life of the relevant composition.

For administration, the formulations of the present disclosure are administered at a rate (including but not limited to, the body weight and general health of the patient) determined by observation of any side effects of the LD-50 or ED-50 and/or various concentrations of the modified relaxin polypeptide of the relevant formulation. Administration can be accomplished in a single dose or in divided doses.

The modified relaxin polypeptides of the present disclosure may be administered directly to a mammalian subject. The modified relaxin polypeptides of the present disclosure may be prepared as a mixture of unit dose injectable forms (including but not limited to solutions, suspensions or emulsions) using a pharmaceutically acceptable carrier. The modified relaxin polypeptides of the present disclosure may also be administered by continuous infusion (using, including but not limited to, micropumps, such as osmotic pumps), single bolus injection, or sustained release depot formulations.

Formulations suitable for administration include aqueous and non-aqueous solutions, isotonic sterile solutions (which may contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic), and aqueous and non-aqueous sterile suspensions (which may contain suspending agents, solubilizers, thickeners, stabilizers, and preservatives). Solutions and suspensions may be prepared from sterile powders, granules, and tablets of the type previously described.

Pharmaceutically acceptable carriers include, but are not limited to, buffers containing succinate, phosphate, borate, HEPES, citrate, histidine, imidazole, acetate, bicarbonate and other organic acids; antioxidants, including but not limited to ascorbic acid; low molecular weight polypeptides, including but not limited to polypeptides of less than about 10 residues; proteins, including but not limited to serum albumin, gelatin, or immunoglobulins; hydrophilic polymers including, but not limited to, polyvinylpyrrolidone; amino acids, including but not limited to glycine, glutamine, asparagine, arginine, histidine or histidine derivatives, methionine, glutamic acid or lysine; monosaccharides, disaccharides, and other carbohydrates including, but not limited to, trehalose, sucrose, glucose, mannose, or dextrins; chelating agents include, but are not limited to, EDTA and disodium ethylenediaminetetraacetate; divalent metal ions including, but not limited to, zinc, cobalt, or copper; sugar alcohols, including but not limited to mannitol or sorbitol; salt-forming counterions including, but not limited to, sodium and sodium chloride; and/or nonionic surfactants, including but not limited to Tween^TM(including but not limited to Tween 80 (polysorbate 80 or PS80) and Tween 20 (polysorbate 20), Pluronics^TMAnd other pluronic acids, including but not limited to, other pluronic acids, including but not limited to pluronic acid F68 (poloxamer 188) or PEG. Suitable surfactants include, for example, but are not limited to, polyethers based on poly (ethylene oxide) -poly (propylene oxide) -poly (ethylene oxide), i.e. (PEO-PPO-PEO) or poly (propylene oxide) -poly (ethylene oxide) -poly (propylene oxide), i.e. (PPO-PEO-PPO) or combinations thereof. PEO-PPO-PEO and PPO-PEO-PPO can be produced by the trade name Pluronics^TM、R-Pluronics^TM、Tetronics^TMAnd R-Tetronics^TM(BASF Wyandotte Corp., Wyandotte, Mich.) is commercially available and is further described in U.S. Pat. No.4,820,352, which is incorporated herein by reference in its entirety. Other ethylene/polypropylene block polymers may be suitable surfactants. Surfactants or surface-active substances may be usedThe combination of agents stabilizes the modified relaxin against one or more stresses, including but not limited to stresses resulting from agitation. Some of the above carriers may be referred to as "fillers". Some may also be referred to as "tonicity modifiers". Antimicrobial preservatives may also be employed for product stability and antimicrobial efficacy; suitable preservatives include, but are not limited to, benzyl alcohol, benzalkonium chloride, m-cresol, methyl/propyl hydroxybenzoates, cresols, and phenols, or combinations thereof. In some embodiments, the pharmaceutical composition may comprise a buffer comprising succinate, phosphate, borate, HEPES, citrate, histidine, imidazole, acetate, bicarbonate, and/or other organic acids; carbohydrates (e.g., monosaccharides, disaccharides, and other carbohydrates), such as trehalose, sucrose, glucose, mannose, or dextrins; and surfactants, e.g. Tween^TM80 (polysorbate 80 or PS80) or Tween 20 (polysorbate 20). In some embodiments, the pharmaceutical composition may comprise a buffer comprising histidine, a carbohydrate (e.g., sucrose); and a surfactant (e.g., PS 80).

The modified relaxin polypeptides of the present disclosure (including those linked to the PK enhancer) may also be administered by or as part of a sustained release system. Sustained release compositions include, but are not limited to, semipermeable polymeric matrices in the form of shaped articles, including, but not limited to, films, or microcapsules. The sustained release matrix includes a biocompatible material such as poly (2-hydroxyethyl methacrylate), ethylene vinyl acetate (or poly-D- (-) -3-hydroxybutyric acid, polylactide (polylactic acid), polyglycolide (glycolic acid polymer), polylactide-glycolide copolymer (copolymer of lactic acid and glycolic acid) polyanhydride, copolymer of L-glutamic acid and γ -ethyl-L-glutamic acid, poly (ortho) ester, polypeptide, hyaluronic acid, collagen, chondroitin sulfate, carboxylic acid, fatty acid, phospholipid, polysaccharide, nucleic acid, polyamino acid, amino acid (e.g., phenylalanine, tyrosine, isoleucine), polynucleotide, polyvinyl propylene, polyvinyl pyrrolidone and silicone.

In the context of the present disclosure, the dose administered to a patient should be sufficient to elicit a beneficial response in the subject over time. Generally, the total pharmaceutically effective amount of a modified relaxin polypeptide of the disclosure per dose administered parenterally (e.g., intravenously or subcutaneously) may range from about 0.01 μ g/kg to about 500mg/kg, from about 0.01 μ g/kg to about 100mg/kg, from about 0.05mg/kg to about 50mg/kg, from about 100 μ g/kg to about 40mg/kg, from about 0.2mg/kg to about 20mg/kg, or from about 0.5mg/kg to about 10mg/kg, or from about 0.5mg/kg, about 1mg/kg, about 2mg/kg, about 3mg/kg, about 4mg/kg, about 5mg/kg, about 6mg/kg, about 7mg/kg, about 8mg/kg, about 9mg/kg, about 10mg/kg, about 12mg/kg, about 15mg/kg, about 20mg/kg, about 10mg/kg, About 30mg/kg, about 40mg/kg, about 50mg/kg, or about 100mg/kg of patient body weight, although this depends on the treatment decision. In some embodiments, the modified relaxin polypeptides of the disclosure may range from about 0.2mg/kg to about 5mg/kg, 0.5mg/kg to about 3mg/kg, or 1mg/kg to about 3mg/kg, or about 0.5mg/kg, 1mg/kg, 2mg/kg, 3mg/kg, 4mg/kg, 5mg/kg, 10mg/kg, 20mg/kg, 30mg/kg, 50mg/kg, or 80mg/kg per dose of parenteral (e.g., intravenous or subcutaneous) administration. In some embodiments, each dose of a modified relaxin polypeptide of the present disclosure administered parenterally (e.g., intravenously or subcutaneously) may be about 1 mg/kg. In some embodiments, the modified relaxin polypeptide of the disclosure administered parenterally (e.g., intravenously or subcutaneously) per dose may be a single dose in the range of about 0.1mg to about 10,000mg, about 1mg to about 5,000mg, about 10mg to about 2,500mg, about 100mg to about 1,000mg, about 200mg to about 800mg, about 400mg to about 600mg, about 1mg to about 10mg, about 10mg to about 50mg, about 50mg to about 100mg, about 100mg to about 300mg, about 300mg to about 500mg, about 500mg to about 700mg, about 700mg to about 900mg, 900mg to about 1,200mg, about 1,000mg to about 2,000mg, about 2,000mg to about 3,000mg, about 3,000mg to about 4,000mg, about 4,000mg to about 5,000mg, about 5,000mg to about 6,000mg, about 6,000mg to about 7,000mg, about 7,000mg to about 8,000mg, about 9,000mg to about 9,000mg, or about 9,000 mg.

Therapeutic use of modified relaxin polypeptides

The present disclosure provides for the use of modified relaxin polypeptides in the treatment of a disease, including a cardiovascular disease or a fibrotic disease, such as heart failure, pancreatitis, inflammation, cancer, scleroderma, pulmonary fibrosis, kidney fibrosis, liver fibrosis or heart fibrosis.

The cardiovascular disease may include, but is not limited to, coronary artery disease, heart attack, arrhythmia or arrhythmia, heart failure, heart valve disease, congenital heart disease, cardiomyopathy (heart disease), pericardial disease, aortic disease, Marfan's syndrome, or vascular disease (vascular disease).

In some embodiments, the heart failure may include one or more of: advanced heart failure, cardiorenal syndrome, heart failure with impaired renal function, chronic heart failure of the midrange ejection fraction type (HFmEF), acute heart failure, post-acute heart failure such as post-acute decompensated heart failure, compensated heart failure, decompensated heart failure, right heart failure, left heart failure, total heart failure, ischemic cardiomyopathy, dilated cardiomyopathy, heart failure associated with congenital heart defects, heart failure associated with heart valve defects, mitral stenosis, mitral insufficiency, aortic stenosis, aortic insufficiency, tricuspid stenosis, tricuspid insufficiency, pulmonary stenosis, pulmonary insufficiency, angina pectoris, hypertension, pulmonary hypertension or pulmonary hypertension, heart failure associated with combined heart valve defects, myocardial inflammation (myocarditis), Chronic myocarditis, acute myocarditis, viral myocarditis, diabetic heart failure, alcoholic cardiomyopathy, heart failure associated with cardiac storage disorders, diastolic heart failure, systolic heart failure, diastolic dysfunction, remodeling following myocardial infarction, chronic heart failure with preserved ejection fraction (HFpEF), or chronic heart failure with reduced ejection fraction (HFrEF).

In some embodiments, the heart failure is selected from the group consisting of chronic heart failure, acute heart failure, post-acute heart failure, chronic heart failure with reduced ejection fraction (HFrEF), chronic heart failure with preserved ejection fraction (HFpEF), chronic heart failure with mid-range ejection fraction (hfmeef), diastolic heart failure, systolic heart failure, post-myocardial infarction remodeling, angina, hypertension, pulmonary hypertension, and pulmonary hypertension. In some embodiments, the heart failure is selected from post-acute heart failure, advanced heart failure, cardiorenal syndrome, and heart failure with impaired renal function.

Fibrosis is the formation of excess fibrous connective tissue in an organ or tissue. Excessive deposition of fibrous tissue is associated with pathological conditions that can lead to impaired organ or tissue function. Affected organs may include the lung (lung or fibrosis of the lung), liver (liver or fibrosis of the liver), kidney (kidney or fibrosis of the kidney), and heart (cardiac fibrosis). Fibrosis may also affect other tissues and organs, including joints, skin, intestine, bone marrow, and the like. Exemplary fibrotic conditions or diseases include, but are not limited to, nonalcoholic steatohepatitis (NASH) affecting the liver; diabetic kidney disease and diabetic nephropathy affecting the kidney; and metabolic heart failure affecting the heart. For example, NASH is characterized by fat, inflammation, and damage in the liver of people who consume little or no alcohol, and can lead to cirrhosis. NASH tends to be diagnosed in overweight or obese middle-aged people, often with elevated blood lipid levels, diabetes or pre-diabetes.

The biological activity of the modified relaxin polypeptides for potential therapeutic use may be determined using standard or known in vitro or in vivo assays. The biological activity of the relaxin polypeptide can be assayed by suitable methods known in the art. Such assays include, but are not limited to, receptor binding assays.

With respect to diseases such as heart failure and fibrotic diseases, the activity of modified relaxin may be determined using one or more in vivo assays. The assays typically involve administering relaxin in an animal model of the disease and determining the effect on disease progression, severity, or other indicators of effective treatment. Such assays can be used to determine effective dosages and treatment regimens in model systems, and predict dosing regimens for clinical use, e.g., for human patients, based thereon. Exemplary HF assays that can be used include: 1) mouse Left Anterior Descending (LAD) coronary ligation model, which mimics the cardiac changes of patients with myocardial infarction and progression to HF (Samuel et al, Lab. immunization 91:675-690, 2011); 2) a limited AngII infusion model in which minimal concentrations of AngII are used to induce cardiac fibrosis (Xu et al, J.Cardiovasc.Pharmacol.51: 62-70,2008); 3) dahl salt-sensitive rat models characterized by hypertension, renal damage and blood volume overload (Sakata et al Circulation 109: 2143-; 4) the senile Spontaneous Hypertensive Rat (SHR) model of cardiac and renal fibrosis, previously used to demonstrate WT-RLX efficacy (Lekgabe et al, Hypertension 46: 412-; and 5) the pressure overload rat thoracic aortic contraction model (Kuster et al Circulation 111:420-427, 2005). In addition to these models, the activity of modified relaxin can also be determined in dog models, for example in normal and rapid pacing (tachysing) induced HF dogs. In addition, these assays can be performed to determine whether it is effective when the modified relaxin is administered not only in a prophylactic mode but also in a therapeutic manner. In addition, modified relaxins can be tested in fibrosis models, including renal fibrosis (Yoshida et al, Nephrol. analysis Transplant 27: 2190-. For example, the efficacy of the modified relaxin can be compared to the efficacy of wild-type relaxin (e.g., wild-type human relaxin).

The amount of relaxin, relaxin polypeptide and/or relaxin analogue administered according to the invention may vary, and in particular should be based on recommendations and prescriptions of a qualified physician. The exact amount of relaxin, relaxin polypeptide and/or relaxin analogue of the invention is a matter of preference depending on factors such as the exact type and/or severity of the condition being treated, the condition of the patient being treated and other ingredients in the composition. The invention may further comprise the administration of a therapeutically effective amount of another active agent. The amount to be administered can be readily determined by one of ordinary skill in the art based on relaxin therapy, available relaxin therapy, and/or other relaxin analogs.

The compounds of the present disclosure are useful for reducing the area of the myocardium affected by an infarction, and for preventing secondary infarctions. Further provided is the use of a compound of the present disclosure for the prevention and/or treatment of thromboembolic disorders, post-ischemic reperfusion injuries, micro-and macrovascular pathologies (vasculitis), arterial and venous thrombosis, edema, ischemia (e.g., myocardial infarction, stroke, and transient ischemic attacks), for cardioprotection in connection with Coronary Artery Bypass Grafting (CABG), direct Percutaneous Transluminal Coronary Angioplasty (PTCA), post-thrombolytic PTCA, remedial PTCA, cardiac transplantation, and open-heart surgery, and for organ protection in connection with transplantation, bypass surgery, catheter examinations, and other surgical procedures. Further provided is the use of a compound of the disclosure for the prevention and/or treatment of respiratory disorders, such as for example chronic obstructive pulmonary disease (chronic bronchitis, COPD), asthma, emphysema, bronchiectasis, cystic fibrosis (mucoviscidosis) and pulmonary hypertension, in particular pulmonary hypertension.

Exemplary embodiments of the present disclosure provide for the use of the modified relaxin of the present disclosure as a medicament for the prevention and/or treatment of renal diseases including, but not limited to, acute and chronic renal diseases and acute and chronic renal insufficiency, as well as acute and chronic renal failure (including acute and chronic renal failure stages with and without dialysis requirements), as well as potential or related renal diseases such as renal hypoperfusion, dialysis-induced hypotension, glomerulopathy, glomeruloproteinuria and tubulointersuria, renal edema, hematuria; primary, secondary, and acute and chronic glomerulonephritis; membranous and membranoproliferative glomerulonephritis, Alport-syndrome, glomerulosclerosis, tubulointerstitial diseases; renal diseases (nephropathic diseases), such as primary and congenital kidney diseases, kidney inflammation; immune kidney diseases such as kidney transplant rejection, immune complex-induced kidney diseases, and the like; and intoxication-induced nephropathies, diabetic and non-diabetic nephropathies, pyelonephritis, cystic kidney, nephrosclerosis, hypertensive nephrosclerosis, nephrotic syndrome, characterized by and diagnostically associated with: abnormal decrease in creatinine clearance and/or water discharge, abnormal increase in blood concentration of urea, nitrogen, potassium, and/or creatinine, changes in renal enzyme, such as glutamyl synthase (glutamyl synthase) activity, urine osmolality, and urine volume; microalbuminuria, macroalbuminuria, glomerular and arteriolar lesions, tubular dilation, hyperphosphatemia, and/or increased dialysis needs.

In addition, the modified relaxin of the present disclosure may be used as a medicament for the prevention and/or treatment of the following diseases: renal cancer, incomplete kidney resection, dehydration after overuse of diuretics, uncontrolled elevation of blood pressure with malignant hypertension, urinary tract obstruction and infections, amyloidosis, and systemic diseases associated with glomerular damage (e.g., lupus erythematosus, rheumatic immune system diseases), as well as renal artery stenosis, renal artery thrombosis, renal vein thrombosis, analgesic-induced renal disease, and renal tubular acidosis.

In addition, the present disclosure provides the use of the modified relaxin of the present disclosure as a medicament for the prevention and/or treatment of contrast-induced and drug-induced acute and chronic interstitial kidney disease, metabolic syndrome and dyslipidemia.

In addition, the present disclosure provides the use of the modified relaxin of the present disclosure as a medicament for preventing, alleviating and/or treating sequelae or symptoms associated with acute and/or chronic kidney disease, such as pulmonary edema, heart failure, uremia, anemia, electrolyte disorders (e.g., hyperkalemia, hyponatremia), and disorders of bone and carbohydrate metabolism.

In addition, the present disclosure provides the use of the modified relaxin of the present disclosure as a medicament for improving, stabilizing or restoring renal function in a patient in need thereof (e.g., a patient having renal disease as described above).

Further exemplary embodiments provide the use of modified relaxin for the treatment and/or prevention of pulmonary diseases, in particular asthma, Pulmonary Arterial Hypertension (PAH) and other forms of pulmonary arterial hypertension (PH), including left heart disease, HIV, sickle cell anemia, thromboembolism (CTEPH), sarcoidosis, COPD or pulmonary fibrosis related pulmonary arterial hypertension, Chronic Obstructive Pulmonary Disease (COPD), Acute Respiratory Distress Syndrome (ARDS), Acute Lung Injury (ALI), alpha-1-antitrypsin deficiency (AATD), pulmonary fibrosis, emphysema, e.g. emphysema caused by cigarette smoke, and Cystic Fibrosis (CF).

Further exemplary embodiments provide the use of the modified relaxin for the treatment and/or prevention of fibrotic disorders, including fibrotic disorders of internal organs such as the lung, heart, kidney, bone marrow, particularly the liver, and also skin fibrosis and fibrotic eye diseases.

Furthermore, the present disclosure provides the use of a modified relaxin for the preparation of a medicament for the treatment and/or prevention of a disorder, in particular a disorder as described above.

In addition, the present disclosure provides methods of treating and/or preventing a condition, particularly a condition described above, using an effective amount of at least one modified relaxin of the present disclosure.

In addition, the present disclosure provides modified relaxin for use in methods of treating and/or preventing coronary heart disease, acute coronary syndrome, heart failure (e.g., acute heart failure, chronic heart failure, or late stage heart failure), and myocardial infarction.

Pharmaceutical compositions suitable for use in the methods and compositions of the present disclosure include compositions which contain an effective amount of the active ingredient to achieve the intended purpose (e.g., treating heart failure). Determination of an effective dose is well within the ability of those skilled in the art in view of this disclosure.

For any compound, the therapeutically effective dose can be estimated initially in an in vitro assay (e.g., RXFP1 receptor activation, ex vivo in an ex vivo perfused rat heart) or in an animal model (typically mouse, rat, rabbit, dog, or pig). Animal models are also used to achieve the desired concentration range and route of administration. Such information can then be used to determine useful doses and routes of administration in humans.

A therapeutically effective dose refers to an amount of modified relaxin that ameliorates a symptom or condition. The therapeutic efficacy and toxicity of such compounds can be determined by standard pharmaceutical procedures in vitro or in experimental animals, e.g., ED50 (the dose therapeutically effective in 50% of the population) and LD50 (the dose lethal to 50% of the population). The dose ratio between therapeutic and toxic effects is the therapeutic index, and it can be expressed as the ratio ED50/LD 50. Exemplary pharmaceutical compositions exhibit a large therapeutic index. Data obtained from in vitro assays and animal studies are used to formulate dosage ranges for human use. The dosage of such compounds is, for example, within the circulating concentration range, which includes ED50 with little or no toxicity. The dosage will vary within this range depending upon the dosage form employed, the sensitivity of the patient, and the route of administration.

The normal dosage may vary from 0.1 to 100,000 mg total dose depending on the route of administration. Guidelines for specific dosages and delivery methods are provided in the literature. See U.S. patent nos. 4,657,760; 5,206,344, respectively; or 5,225,212.

Xiii. combination preparation

The modified relaxin of the present disclosure may be administered alone or in combination with other active compounds. In this context, the term combination encompasses any mode of simultaneous or sequential administration, whether the modified relaxin and the other agent are contained in the same composition or administered separately, and may be administered by the same or different modes of administration. The present disclosure provides a medicament comprising at least one modified relaxin and one or more other active ingredients in a pharmaceutical composition for use in the treatment and/or prevention of the above conditions, wherein the pharmaceutical composition is in admixture with an excipient or a pharmaceutically acceptable carrier.

Suitable active ingredients for combination may include, for example: active ingredients regulating lipid metabolism, antidiabetics, hypotensives, perfusion-enhancing and/or antithrombotic agents, antioxidants, chemokine receptor antagonists, p 38-kinase inhibitors, NPY agonists, orexin agonists, anorectic agents (anorexics), PAF-AH inhibitors, anti-inflammatory agents (COX inhibitors, LTB inhibitors)₄Receptor antagonists), analgesics such as aspirin, P2Y12 inhibitors such as clopidogrel, PCSK9 inhibitors such as elouitumumab (reta) or aleuzumab (prallent), antidepressants, and other psychotropic drugs.

The present disclosure further provides combinations of at least one modified relaxin with at least one active ingredient that alters lipid metabolism, an antidiabetic, a hypotensive active ingredient and/or an agent with an antithrombotic effect.

The modified relaxin may be combined with one or more active ingredients that modulate lipid metabolism, such as HMG-CoA reductase inhibitors, HMG-CoA reductase expression inhibitors, squalene synthesis inhibitors, ACAT inhibitors, LDL receptor inducers, cholesterol absorption inhibitors, polymeric bile acid adsorbents, bile acid reabsorption inhibitors, MTP inhibitors, lipase inhibitors, LpL activators, fibrates, nicotinic acid receptor agonists, CETP inhibitors, PPAR-a, PPAR-gamma and/or PPAR-delta agonists, RXR modulators, FXR modulators, LXR modulators, thyroid hormones and/or thyroid mimetics (thyroid mimetics), ATP citrate lyase inhibitors, lp (a) antagonists, cannabinoid receptor 1 antagonists, leptin receptor agonists, bombesin receptor agonists, PCSK9 inhibitors, Histamine receptor agonists and antioxidants/free radical scavengers.

In some embodiments, the modified relaxin is administered in combination with an HMG-CoA reductase inhibitor from a statin, such as lovastatin, simvastatin, pravastatin, fluvastatin, atorvastatin, rosuvastatin, or pitavastatin. In another embodiment, the modified relaxin is administered in combination with a squalene synthesis inhibitor, e.g., BMS-188494 or TAK-475. In another embodiment, the modified relaxin is administered in combination with an ACAT inhibitor such as avasimibe, melinamide, petetimibe, ibrutin or SMP-797. In another embodiment, the other modified relaxin is administered in combination with a cholesterol absorption inhibitor, such as ezetimibe, tiquinan or pamabrin. In another embodiment, the modified relaxin is administered in combination with an MTP inhibitor such as, for example, inputap, BMS-201038, R-103757, or JTT-130. In another embodiment, the modified relaxin is administered in combination with a lipase inhibitor, e.g. orlistat. In another embodiment, the modified relaxin is administered in combination with a thyroid hormone and/or a thyroid mimetic such as D-thyroxine or 3,5,3' -triiodothyronine (T3). In another embodiment, the modified relaxin is administered in combination with a nicotinic acid receptor agonist, such as nicotinic acid, acipimox, acevulan, or nicotinyl tartrate (radecol). In another embodiment, the modified relaxin is administered in combination with a CETP inhibitor, such as Dacetrapib (dalcetrapib), BAY 60-5521, Anacetrapib (anacetrapib), or CETP vaccine (CETi-1). In another embodiment, the modified relaxin is administered in combination with a PPAR-gamma agonist, such as a thiazolinedione (e.g., pioglitazone or rosiglitazone). In another embodiment, the modified relaxin is administered in combination with a PPAR-delta agonist, such as GW-501516 or BAY 68-5042. In another embodiment, the modified relaxin is administered in combination with a polymeric bile acid adsorbent such as cholestyramine, colestipol, colesevelam, colestyril (CholestaGel), or colestipol (colestimide). In another embodiment, the modified relaxin is administered in combination with a bile acid resorption inhibitor, e.g., an ASBT (IBAT) inhibitor, e.g., AZD-7806, S-8921, AK-105, bali-1741, SC-435, or SC-635. In another embodiment, the modified relaxin is administered in combination with an antioxidant/free radical scavenger such as probucol, AGI-1067, BO-653, or AEOL-10150. In another embodiment, the modified relaxin is administered in combination with a cannabinoid receptor 1 antagonist, such as rimonabant or SR-147778.

The modified relaxin may be combined with one or more antidiabetic agents such as insulin and insulin derivatives, sulfonylureas, biguanides, meglitinide derivatives, glucosidase inhibitors, PPAR-gamma agonists, dipeptidyl peptidase IV inhibitors (DPP-IV inhibitors), oxadiazolidinediones (oxadizidinones), thiazolinediones, GLP1 receptor agonists, glucagon antagonists, insulin sensitizers, CCK1 receptor agonists, leptin receptor agonists, inhibitors of liver enzymes involved in gluconeogenesis and/or glycogenolysis stimulation, glucose uptake modulators, and potassium channel openers.

In some embodiments, the modified relaxin is administered in combination with insulin or an insulin derivative. In another embodiment, the modified relaxin is administered in combination with a sulfonylurea, such as tolbutamide, glyburide, glimepiride, glipizide or gliclazide. In another embodiment, the modified relaxin is administered in combination with a biguanide (e.g., metformin). In another embodiment, the modified relaxin is administered in combination with a meglitinide derivative, such as repaglinide or nateglinide. In another embodiment, the modified relaxin is administered in combination with a glucosidase inhibitor, such as miglitol or acarbose. In another embodiment, the modified relaxin is administered in combination with a DPP-IV inhibitor, such as sitagliptin and vildagliptin. In another embodiment, the modified relaxin is administered in combination with a PPAR-gamma agonist, such as a thiazolidinedione (e.g., pioglitazone or rosiglitazone).

The modified relaxin may be combined with one or more blood pressure lowering active ingredients such as calcium antagonists, angiotensin II antagonists, ACE inhibitors, renin inhibitors, beta-blockers, alpha-blockers, diuretics, aldosterone antagonists, mineralocorticoid receptor antagonists, ECE inhibitors, ACE/NEP inhibitors and vasopeptidase inhibitors.

In some embodiments, the modified relaxin is administered in combination with a calcium antagonist, such as nifedipine, amlodipine, verapamil or diltiazem. In another embodiment, the modified relaxin is administered in combination with an angiotensin II antagonist such as losartan, valsartan, candesartan, emblosartan, olmesartan or telmisartan. In another embodiment, the modified relaxin is administered in combination with an ACE inhibitor, such as enalapril, captopril, lisinopril, ramipril, delapril, fosinopril, quinol (quinopril), perindopril, or trandolapril (trandopril). In another embodiment, the modified relaxin is administered in combination with a beta-receptor blocker such as propranolol, atenolol, timolol, pindolol, alprenolol, penbutolol, blanalol, metiprolol, nadolol, mepindolol, caramolol (carazalol), sotalol, metoprolol, betaxolol, celiprolol (celiprolol), bisoprolol, carteolol, esmolol, labetalol, carvedilol (carvedilol), adaprolol, landiolol, nebivolol, epanolol (epanolol), or bucindolol (bucindolol). In another embodiment, the modified relaxin is administered in combination with an alpha-receptor blocker, such as prazosin. In another embodiment, the modified relaxin is administered in combination with a diuretic such as furosemide, bumetanide, torasemide, bendroflumethiazide, chlorothiazide, hydrochlorothiazide, hydroflumethiazide, mechlorethazine, polythiazide, trichlormethiazide, chlorthalidone, indapamide, metolazone, quinethazone, acetazolamide, dichloramine, methazolamide, glycerol, isosorbide, mannitol, amiloride or triamterene (triamteren). In another embodiment, the modified relaxin is administered in combination with an aldosterone or mineralocorticoid receptor antagonist, such as spironolactone or eplerenone. In another embodiment, the modified relaxin is administered in combination with a vasopressin receptor antagonist such as conivaptan, tolvaptan, rivastigmine, or SR-121463.

The modified relaxin may be combined with one or more of the following: antithrombotic agents, such as platelet aggregation inhibitors or anticoagulants; a diuretic; a vasopressin receptor antagonist; organic nitrates and NO donors; a compound having positive inotropic activity; compounds which inhibit the degradation of cyclic guanosine monophosphate (cGMP) and/or cyclic adenosine monophosphate (cAMP), for example inhibitors of Phosphodiesterase (PDE)1,2,3,4 and/or 5, in particular PDE 5 inhibitors, such as sildenafil, vardenafil and tadalafil, and PDE 3 inhibitors, such as milrinone; natriuretic peptides, such as "atrial natriuretic peptide" (ANP, anaritide), "B-type natriuretic peptide" or "brain natriuretic peptide" (BNP, nesiritide), "C-type natriuretic peptide" (CNP) and also natriuretic peptides; prostacyclin receptor (IP receptor) agonists, such as iloprost, beraprost, cicaprost; inhibitors of the If (pacing channel) channel, such as ivabradine; calcium sensitizers, such as levosimendan; a potassium supplement; NO-dependent but heme-dependent guanylate cyclase stimulators, such as the compounds described in WO 00/06568, WO 00/06569, WO 02/42301 and WO 03/095451; NO-and heme-independent guanylate cyclase activators, such as the compounds described in WO 01/19355, WO 01/19776, WO 01/19778, WO 01/197780, WO 02/070462 and WO 02/070510; human Neutrophil Elastase (HNE) inhibitors, such as cevelstat and DX-890 (relan); compounds that inhibit the signal transduction cascade, such as tyrosine kinase inhibitors, e.g., sorafenib, imatinib, gefitinib and erlotinib; and/or compounds that modulate cardiac energy metabolism, such as etolimus, dichloroacetate, ranolazine, and trimetazidine.

In some embodiments, the modified relaxin is administered in combination with an organic nitrate or NO donor such as sodium nitroprusside, nitroglycerin, isosorbide mononitrate, isosorbide dinitrate, molsidomin, or SIN-1, or in combination with inhaled NO. In another embodiment, the modified relaxin is administered in combination with an inotropic compound, such as a cardiac glycoside (digoxin), a β -adrenergic, and a dopaminergic agonist, such as isoproterenol, epinephrine, norepinephrine, dopamine, or dopbutamine. In another embodiment, the modified relaxin is administered in combination with an anti-sympathetic agent such as reserpine, clonidine or alpha-methyldopa, or with a potassium channel agonist such as minoxidil, diazoxide, dihydralazine or hydralazine, or with a nitric oxide releasing substance such as glycerol nitrate or sodium nitroprusside. In another embodiment, the modified relaxin is administered in combination with a platelet aggregation inhibitor, such as aspirin, clopidogrel, ticlopidine, or dipyridamole. In another embodiment, the modified relaxin is administered in combination with a thrombin inhibitor, such as ximelagatran, melagatran, dabigatran, bivalirudin or cricetera. In another embodiment, the modified relaxin is administered in combination with a GPIIb/IIIa antagonist, such as tirofiban or abciximab. In another embodiment, the modified relaxin is administered in combination with a factor Xa inhibitor, such as rivaroxaban (BAY59-7939), DU-176b, apixaban, omixaban, febrifalaban, rasxaban, fondaparinux (fondaparinux), epidoparin (idraparinux), PMD-3112, YM-150, KFA-1982, EMD-503982, MCM-17, MLN-1021, DX9065a, DPC 906, JTV 803, SSR-126512, or SSR-128428. In another embodiment, the modified relaxin is administered in combination with heparin or a Low Molecular Weight (LMW) heparin derivative. In another embodiment, the modified relaxin is administered in combination with a vitamin K antagonist, such as coumarin (warfarin).

In addition, the present disclosure provides medicaments comprising at least one modified relaxin, typically together with one or more inert, non-toxic, pharmaceutically suitable auxiliaries, and their use for the above-mentioned purposes. Optionally, the medicament may comprise the modified relaxin and another active ingredient (e.g., those identified above). For example, the medicament may comprise an effective amount of the modified relaxin and another active ingredient for the treatment or prevention of heart failure and/or a fibrosis related disease.

Examples