METHODS FOR PREPARING PURIFIED POLYPEPTIDE COMPOSITIONS

• US 20100210515A1
• Filed: 09/22/2009
• Published: 08/19/2010
• Est. Priority Date: 09/22/2008
• Status: Active Grant

• Current Assignee: Aileron Therapeutics Inc.
• Original Assignee: Aileron Therapeutics Inc.
• Inventors: Kawahata, Noriyuki, Iadanza, Matthew, Leitheiser, Christopher, Nash, Huw M.
• Granted Patent: US 9,206,223 B2
• Time in Patent Office: Days
• US Class Current: 514/1.100
• 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 purified peptidomimetic macrocycle composition comprising:

a) contacting a peptidomimetic precursor comprising at least two moieties capable of undergoing a metathesis reaction with a metathesis catalyst comprising a metal to produce a crude peptidomimetic macrocycle; and

b) purifying the crude peptidomimetic macrocycle to result in a purified peptidomimetic macrocycle composition comprising less than 30 ppm by weight of the metal.

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

3. The method of claim 2, wherein said purifying step further comprises at least two chromatographic purification steps, for example by HPLC.

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

5. The method of claim 1 wherein the peptidomimetic macrocycle composition comprises less than 20, 10, 5, or 1 ppm by weight of the metal.

6. The method of claim 1, wherein the peptidomimetic macrocycle comprises a helix, for example wherein the helix is an alpha-helix.

7. The method of claim 1, wherein the peptidomimetic macrocycle comprises an alpha-helical domain of a BCL-2 family member, for example wherein the peptidomimetic macrocycle comprises a BH3 domain.

8. The method of claim 1, wherein the peptidomimetic macrocycle comprises at least 60%, 70%, 80%, or 90% of a sequence in Table 1, 2, 3 or 4.

9. The method of claim 1, wherein the peptidomimetic macrocycle comprises a crosslinker connecting a first amino acid to a second amino acid.

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

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

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

13. The method of claim 9, wherein the crosslinker comprises between 6 and 14 consecutive bonds, or between 8 and 16 consecutive bonds.

14. The method of claim 9, wherein the crosslinker spans from 1 turn to 5 turns of an alpha-helix.

15. The method of claim 14, wherein the crosslinker comprises a length of about 5 Å

to about 9 Å

per turn of the alpha-helix.

16. The method of claim 9, wherein an alpha position of the first or second amino acid is additionally substituted.

17. 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.

18. The method of claim 1 wherein the peptidomimetic macrocycle composition comprises a metal at a concentration between about 0.5 ppm and about 10 ppm by weight.

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

20. 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.

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

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

23. The method of claim 1 wherein the peptidomimetic macrocycle composition comprises a pharmaceutically acceptable salt of the peptidomimetic macrocycle, for example a hydrochloric acid or an acetate salt.

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

25. A α

-helical peptidomimetic macrocycle composition comprising;

a) an α

-helical peptidomimetic macrocycle comprising a cross-linker connecting a first amino acid and a second amino acid; and

b) ruthenium or osmium at a concentration between about 0.5 ppm and about 30 ppm by weight.

26. A composition according to claim 25, further wherein at least one of said amino acids is an α

,α

-disubstituted amino acid, for example wherein the first or second amino acid is α

,α

-disubstituted.

27. The composition of claim 25, wherein the first amino acid and the second amino acid are separated by three amino acids.

28. The composition of claim 27, wherein the cross-linker comprises between 6 and 14 consecutive bonds, or between 8 and 12 consecutive bonds.

29. The composition of claim 25, wherein the first amino acid and the second amino acid are separated by six amino acids.

30. The composition of claim 29, wherein the cross-linker comprises between 8 and 16 consecutive bonds, or between 10 and 13 consecutive bonds.

31. The composition of claim 25, wherein the cross-linker spans from 1 turn to 5 turns of the alpha-helix, for example 1 or 2 turns of the alpha helix.

32. The composition of claim 31, wherein the length of the cross-linker is about 5 Å

to about 9 Å

per turn of the alpha-helix.

33. The composition of claim 32, wherein the peptidomimetic macrocycle carries a net positive charge at pH 7.4.

34. The composition of claim 25, wherein the peptidomimetic macrocycle comprises one or more of a therapeutic agent, halogen, alkyl group, a fluorescent moiety, affinity label, targeting moiety, or a radioisotope.

35. The composition of claim 25, wherein the metal comprises a metathesis catalyst or a derivative of a metathesis catalyst.

36. The composition of claim 35, wherein the metathesis catalyst comprises iodine, bromine, chlorine, phenyl, carbene, cyclohexyl, phosphine, tricyclohexylphosphine, imidazole, or benzylidene.

37. The composition of claim 25, comprising a total concentration of metals of less than about 30, 20, 10, 5 or 1 ppm by weight.

38. The composition of claim 25, wherein the peptidomimetic macrocycle comprises a pharmaceutically acceptable salt thereof, for example a hydrochloric acid salt or acetate salt.