Methods for preparing purified polypeptide compositions

• US 9,206,223 B2
• Filed: 09/22/2009
• Issued: 12/08/2015
• Est. Priority Date: 09/22/2008
• Status: Active Grant

• Current Assignee: Aileron Therapeutics Inc.
• Original Assignee: Aileron Therapeutics Inc.
• Inventors: Nash, Huw M., Iadanza, Matthew, Leitheiser, Christopher, Kawahata, Noriyuki
• Primary Examiner(s): Skowronek, Karlheinz R
• Assistant Examiner(s): Lee, Li
• Application Number:
  US12/564,909
• Publication Number:
  US 20100210515A1
• Time in Patent Office: 2,268 Days
• Field of Search: None
• US Class Current: 1/1
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: A61P 1/00 Drugs for disorders of the alimentary tract or the digestive system
• CPC Class Codes: A61P 1/04 for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
• CPC Class Codes: A61P 11/00 Drugs for disorders of the respiratory system
• CPC Class Codes: A61P 11/06 Antiasthmatics
• CPC Class Codes: A61P 13/12 of the kidneys
• CPC Class Codes: A61P 17/00 Drugs for dermatological disorders
• CPC Class Codes: A61P 17/02 for treating wounds, ulcers, burns, scars, keloids, or the like
• CPC Class Codes: A61P 19/00 Drugs for skeletal disorders
• CPC Class Codes: A61P 19/02 for joint disorders, e.g. arthritis, arthrosis
• CPC Class Codes: A61P 21/02 Muscle relaxants, e.g. for tetanus or cramps
• CPC Class Codes: A61P 25/00 Drugs for disorders of the nervous system
• CPC Class Codes: A61P 25/14 for treating abnormal movements, e.g. chorea, dyskinesia
• CPC Class Codes: A61P 25/16 Anti-Parkinson drugs
• CPC Class Codes: A61P 25/28 for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
• CPC Class Codes: A61P 27/02 Ophthalmic agents
• CPC Class Codes: A61P 3/00 Drugs for disorders of the metabolism of the blood or the extracellular fluid A61P7/00
• CPC Class Codes: A61P 3/06 Antihyperlipidemics
• CPC Class Codes: A61P 3/10 for hyperglycaemia, e.g. antidiabetics
• CPC Class Codes: A61P 31/12 Antivirals
• CPC Class Codes: A61P 35/00 : Antineoplastic agents
• CPC Class Codes: A61P 35/02 : specific for leukemia
• CPC Class Codes: A61P 35/04 : specific for metastasis
• CPC Class Codes: A61P 37/06 : Immunosuppressants, e.g. drugs for graft rejection
• CPC Class Codes: A61P 43/00 : Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: A61P 5/00 : Drugs for disorders of the endocrine system
• CPC Class Codes: A61P 7/00 : Drugs for disorders of the blood or the extracellular fluid
• CPC Class Codes: A61P 7/02 : Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
• CPC Class Codes: A61P 7/06 : Antianaemics
• CPC Class Codes: A61P 9/00 : Drugs for disorders of the cardiovascular system
• CPC Class Codes: A61P 9/04 : Inotropic agents, i.e. stimulants of cardiac contraction; Drugs for heart failure
• CPC Class Codes: A61P 9/10 : for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
• CPC Class Codes: A61P 9/12 : Antihypertensives
• CPC Class Codes: C07K 1/107 : by chemical modification of precursor peptides
• CPC Class Codes: C07K 14/00 : Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
• CPC Class Codes: C07K 9/00 : Peptides having up to 20 amino acids, containing saccharide radicals and having a fully defined sequence; Derivatives thereof

Abstract

The present invention relates to purified peptidomimetic macrocycles. The invention additionally provides methods of preparing and using such macrocycles, for example in therapeutic applications.

Claims

1. A method for preparing a pharmaceutical composition comprising a purified peptidomimetic macrocycle comprising an alpha-helix, the method comprising:

a) contacting a peptidomimetic precursor attached to a solid support comprising at least two moieties capable of undergoing a metathesis reaction with a metathesis catalyst comprising a metal to produce a crude peptidomimetic macrocycle comprising a cross-linker connecting a first amino acid and a second amino acid, the cross-linker spanning from 1 turn to 5 turns of the alpha-helix;

b) purifying the crude peptidomimetic macrocycle to result in a purified peptidomimetic macrocycle composition comprising less than 10 ppm by weight of the metal from the metathesis catalyst, wherein said purifying comprises the step of washing said crude peptidomimetic macrocycle with a solvent prior to removing said crude peptidomimetic macrocycle from the solid support; and

c) formulating the purified peptidomimetic macrocycle composition into the pharmaceutical composition for administration to a human subject.

2. The method of claim 1, wherein said purifying step comprises washing with DMSO.

3. The method of claim 2, wherein following said washing, said purifying step further comprises at least two chromatographic purification steps.

4. The method of claim 1, wherein the purification step does not involve crystallization.

5. The method of claim 1 wherein the purified peptidomimetic macrocycle composition comprises less than 5 ppm by weight of the metal.

6. The method of claim 1, wherein the purified peptidomimetic macrocycle comprises an alpha-helical domain of a BCL-2 family member.

7. The method of claim 1, wherein the purified peptidomimetic macrocycle comprises a ring of 18 atoms to 26 atoms, or of 29 atoms to 37 atoms.

8. The method of claim 1, wherein the purified peptidomimetic macrocycle carries a net positive charge at pH 7.4.

9. The method of claim 1, wherein the first amino acid and the second amino acid are separated by three or six amino acids.

10. The method of claim 1, wherein the crosslinker comprises between 6 and 14 consecutive bonds, or between 8 and 16 consecutive bonds.

11. The method of claim 1, wherein the crosslinker comprises a length of about 5 Å

to about 9 Å

per turn of the alpha-helix.

12. The method of claim 1, wherein an alpha position of the first or second amino acid is additionally substituted.

13. The method of claim 1 wherein the peptidomimetic macrocycle comprises moieties comprising one or more of a therapeutic agent, halogen, alkyl group, a fluorescent moiety, affinity label, targeting moiety, or a radioisotope.

14. The method of claim 1 wherein the purified peptidomimetic macrocycle composition comprises a metal at a concentration greater than or equal to 0.5 ppm by weight.

15. The method of claim 1 wherein the metathesis catalyst comprises a ruthenium or osmium catalyst.

16. The method of claim 1 wherein the metathesis catalyst comprises one or more substituents selected from the group consisting of iodine, bromine, chlorine, phenyl, carbene, cyclohexyl, phosphine, tricyclohexylphosphine, imidazole, or a benzylidene.

17. The method of claim 1 wherein the peptidomimetic precursor is bound to a solid support.

18. The method of claim 1 wherein the metathesis catalyst is bound to a solid support.

19. The method of claim 1 wherein the peptidomimetic macrocycle composition comprises a pharmaceutically acceptable salt of the peptidomimetic macrocycle.

20. The method of claim 1 wherein the purified peptidomimetic macrocycle composition can be safely administered to a subject.

21. The method of claim 3, wherein at least one of said two chromatographic purification steps is HPLC.

22. The method of claim 1, wherein the purified peptidomimetic macrocycle comprises a BH3 domain.

23. The method of claim 1, wherein the purified peptidomimetic macrocycle composition comprises less than 1 ppm by weight of the metal.

46. The method of claim 1 or 24, wherein the purified peptidomimetic macrocycle composition is a therapeutic agent.

47. The method of claim 19 or 41, wherein the pharmaceutically acceptable salt of the peptidomimetic macrocycle is a hydrochloric acid or an acetate salt.

24. A method for preparing a pharmaceutical composition comprising a purified peptidomimetic macrocycle comprising an alpha-helix, the method comprising:

a) contacting a peptidomimetic precursor attached to a solid support comprising at least two moieties capable of undergoing a metathesis reaction with a metathesis catalyst comprising ruthenium to produce a crude peptidomimetic macrocycle comprising a cross-linker connecting a first amino acid and a second amino acid, the cross-linker spanning from 1 turn to 5 turns of the alpha-helix;

b) purifying the crude peptidomimetic macrocycle to result in a purified peptidomimetic macrocycle composition comprising less than 10 ppm by weight of ruthenium from the metathesis catalyst, wherein said purifying comprises the step of washing said crude peptidomimetic macrocycle with a solvent prior to removing said crude peptidomimetic macrocycle from the solid support; and

c) formulating the purified peptidomimetic macrocycle composition into the pharmaceutical composition for administration to a human subject.

25. The method of claim 24, wherein said purifying step comprises washing with DMSO.

26. The method of claim 25 wherein following said washing, said purifying step further comprises at least two chromatographic purification steps.

27. The method of claim 24, wherein the purification step does not involve crystallization.

28. The method of claim 24 wherein the purified peptidomimetic macrocycle composition comprises less than 5 ppm by weight of ruthenium.

29. The method of claim 24, wherein the purified peptidomimetic macrocycle comprises an alpha-helical domain of a BCL-2 family member.

30. The method of claim 24, wherein the purified peptidomimetic macrocycle comprises a ring of 18 atoms to 26 atoms, or of 29 atoms to 37 atoms.

31. The method of claim 24, wherein the purified peptidomimetic macrocycle carries a net positive charge at pH 7.4.

32. The method of claim 24, wherein the first amino acid and the second amino acid are separated by three or six amino acids.

33. The method of claim 24, wherein the crosslinker comprises between 6 and 14 consecutive bonds, or between 8 and 16 consecutive bonds.

34. The method of claim 24, wherein the crosslinker comprises a length of about 5 Å

to about 9 Å

per turn of the alpha-helix.

35. The method of claim 24, wherein an alpha position of the first or second amino acid is additionally substituted.

36. The method of claim 24 wherein the peptidomimetic macrocycle comprises moieties comprising one or more of a therapeutic agent, halogen, alkyl group, a fluorescent moiety, affinity label, targeting moiety, or a radioisotope.

37. The method of claim 24 wherein the purified peptidomimetic macrocycle composition comprises ruthenium at a concentration greater than or equal to 0.5 ppm by weight.

38. The method of claim 24 wherein the metathesis catalyst comprises one or more substituents selected from the group consisting of iodine, bromine, chlorine, phenyl, carbene, cyclohexyl, phosphine, tricyclohexylphosphine, imidazole, or a benzylidene.

39. The method of claim 24 wherein the peptidomimetic precursor is bound to a solid support.

40. The method of claim 24 wherein the metathesis catalyst is bound to a solid support.

41. The method of claim 24 wherein the peptidomimetic macrocycle composition comprises a pharmaceutically acceptable salt of the peptidomimetic macrocycle.

42. The method of claim 24 wherein the purified peptidomimetic macrocycle composition can be safely administered to a subject.

43. The method of claim 26, wherein at least one of said two chromatographic purification steps is HPLC.

44. The method of claim 24, wherein the purified peptidomimetic macrocycle comprises a BH3 domain.

45. The method of claim 24, wherein the purified peptidomimetic macrocycle composition comprises less than 1 ppm by weight of ruthenium.