Protease inhibitor-containing compositions, compositions comprising same, and methods for producing and using same

• US 10,342,764 B2
• Filed: 01/31/2013
• Issued: 07/09/2019
• Est. Priority Date: 02/01/2012
• Status: Active Grant

• Current Assignee: Oramed Ltd. (Oramed Pharmaceuticals, Inc.)
• Original Assignee: Oramed Ltd. (Oramed Pharmaceuticals, Inc.)
• Inventors: Hershko, Avraham, Kidron, Miriam
• Primary Examiner(s): Carlson, Karen Cochrane
• Application Number:
  US14/376,292
• Publication Number:
  US 20140377344A1
• Time in Patent Office: 2,350 Days
• CPC Class Codes: A61K 38/17 from animals; from humans enzyme inhibitors A61K38/005
• CPC Class Codes: A61K 38/26 Glucagons
• CPC Class Codes: A61K 38/28 Insulins
• CPC Class Codes: A61K 38/56 from plants
• CPC Class Codes: A61K 47/42 Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein oligopeptides having up to five amino acids A61K47/183; polyamino acids A61K47/34
• CPC Class Codes: A61K 9/19 lyophilised , i.e. freeze-dried, solutions or dispersions lyophilised products with subsequent particle size reduction A61K9/14; granules or pellets made by lyphilisation A61K9/1682; solid oral dosage forms made by lyophilisation A61K9/2095; lyophilisation additives A61K47/00
• CPC Class Codes: A61K 9/4858 Organic compounds
• CPC Class Codes: A61K 9/4866 Organic macromolecular compounds
• CPC Class Codes: A61K 9/4875 Compounds of unknown constitution, e.g. material from plants or animals oils, fats, waxes, shellac A61K9/4858
• CPC Class Codes: A61K 9/4891 Coated capsules; Multilayered drug free capsule shells with drug coating for immediate release A61K9/4808; osmotic devices A61K9/0004
• CPC Class Codes: A61P 3/08 for glucose homeostasis pancreatic hormones A61P5/48
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Abstract

Provided herein are methods and compositions for oral administration of therapeutic proteins, improved protease inhibitor preparations, methods for producing same, and compositions comprising same.

Claims

1. An oral pharmaceutical composition produced by a process comprising

(a) subjecting soybean trypsin inhibitor (SBTI) to column chromatography under conditions in which fractions containing the SBTI'"'"'s Bowman-Birk Inhibitor (BBI) activity elute separately from fractions containing the SBTI'"'"'s Kunitz Trypsin Inhibitor (KTI) activity;

(b) eluting and combining fractions from (a) that contain the BBI activity;

(c) filtering the combined fractions from (b) that contain the BBI activity under conditions that reduce the contaminants having a molecular weight of greater than 30 KDa to be less than 0.1% of the BBI preparation, thus producing a purified BBI product characterized in that;

(i) contaminants having a molecular weight greater than 30 KDa are less than 0.1% of the preparation; and

(ii) 1 mg of the purified BBI product has an activity of about 40 BTEE units per mg of protein;

(d) eluting and combining the fractions from (a) that contain the KTI activity and in which contaminants having a molecular weight greater than 30 KDa are less than 0.1% of the preparation thus producing a purified KTI product characterized in that;

(i) contaminants having a molecular weight greater than 30 KDa are less than 0.1% of the preparation; and

(ii) 1 mg of the purified KTI product has an activity of about 10,000 BAEE units per mg of protein;

(e) combining

(i) an oil-based liquid formulation,

(ii) a therapeutic protein having a molecular weight of up to 100 kilodalton,

(iii) a chelator of divalent cations,

(iv) the purified BBI product of part (c); and

(v) the purified KTI product of part (d),

such that the ratio of anti-chymotrypsin activity to anti-trypsin activity present in the pharmaceutical composition is between 1.5;

1 and 1;

1 inclusive.

2. The oral pharmaceutical composition of claim 1, wherein said oil-based liquid formulation further comprises a trypsin inhibitor other than said BBI and said KTI.

3. The oral pharmaceutical composition of claim 1, wherein said KTI is Kunitz trypsin inhibitor 3 (KTI3).

4. The oral pharmaceutical composition of claim 3, wherein said KTI3 has been purified to at least 85% purity as measured by SDS-PAGE.

5. The oral pharmaceutical composition of claim 3, wherein said KTI3 has been purified to a protein content of greater than 95% as measured by BCA assay.

6. The oral pharmaceutical composition of claim 1, wherein the oil-based liquid formulation comprises BBI that has an anti-chymotrypsin activity of at least 40 mg chymotrypsin inhibited per ml of the liquid formulation.

7. The oral pharmaceutical composition of claim 6, wherein said oil-based liquid formulation further comprises an anti-trypsin activity of at least 20 mg trypsin inhibited per ml of the liquid formulation.

8. The oral pharmaceutical composition of claim 1, wherein said therapeutic protein is selected from the group consisting of insulin, influenza hemagglutinin, influenza neuraminidase, a glucagon, interferon gamma, interferon beta, interferon alpha, growth hormone, erythropoietin, GLP-1, a GLP-1 analogue, granulocyte colony stimulating factor (G-CSF), renin, growth hormone releasing factor, parathyroid hormone, thyroid stimulating hormone, follicle stimulating hormone, calcitonin, luteinizing hormone, glucagon, a clotting factor, an anti-clotting factor, atrial natriuretic factor, surfactant protein A (SP-A), surfactant protein B (SP-B), surfactant protein C (SP-C), surfactant protein D (SP-D), a plasminogen activator, bombesin, hemopoietic growth factor (colony-stimulating factor, multiple), a tumor necrosis factor (TNF) protein, enkephalinase, RANTES (regulated on activation normally T-cell expressed and secreted), human macrophage inflammatory protein (MIP-1-alpha), serum albumin, Mullerian-inhibiting substance, relaxin, mouse gonadotropin-releasing hormone, DNase, inhibin, activin, vascular endothelial growth factor (VEGF), a neurotrophic factor, neurotrophin-3, -4, -5, or -6 (NT-3, NT-4, NT-5, or NT-6), nerve growth factor, platelet-derived growth factor (PDGF), a fibroblast growth factor, a transforming growth factor (TGF), insulin-like growth factor-I and -II (IGF-1 and IGF-II), des (1-3)-IGF-1 (brain IGF-1), insulin-like growth factor binding protein 1 (IGFBP-1), IGFBP-2, IGFBP-3, IGFBP-4, IGFBP-5, IGFBP-6, a keratinocyte growth factor, an osteoinductive factor, bone morphogenetic protein (BMP)-2, BMP-7, a colony stimulating factor (CSF), an interleukin (IL), superoxide dismutase, decay accelerating factor, a chemokine family member, and a complement factor.

9. The oral pharmaceutical composition of claim 8, wherein said therapeutic protein is selected from the group consisting of insulin and a GLP-1 analogue.

10. The oral pharmaceutical composition of claim 1, wherein said chelator is EDTA.

11. The oral pharmaceutical composition of claim 1, wherein said oil-based liquid formulation further comprises a polyethylene glycol (PEG) ester of a monoglyceride, a diglyceride, a triglyceride, or a mixture thereof.

12. The oral pharmaceutical composition of claim 11, wherein said oil-based liquid formulation further comprises a free PEG.

13. The oral pharmaceutical composition of claim 11, wherein said PEG ester is provided as a mixture of (a) a monoacylglycerol, a diacylglycerol, a triacylglycerol, or a mixture thereof;

and (b) a polyethylene glycol (PEG) ester of a fatty acid.

14. The oral pharmaceutical composition of claim 13, wherein part (a) of said mixture comprises C₈-C₁₈ monoacylglycerols, diacylglycerols, and triacyl glycerols.

15. The oral pharmaceutical composition of claim 13, wherein part (b) of said mixture comprises PEG monoesters and diesters of a mixture of C₈-C₁₈ fatty acids.

16. The oral pharmaceutical composition of claim 13, wherein part (a) of said mixture comprises C₈-C₁₈ monoacylglycerols, diacylglycerols, and triacylglycerols;

part (b) of said mixture comprises PEG-32 monoesters and diesters of a mixture of C₈-C₁₈ fatty acids;

said oil-based liquid formulation further comprises (c) free PEG-32; and

the weight/weight ratio of part (a) of said mixture to the sum of part (b) of said mixture and (c) is between 10;

90 and 30;

70 inclusive.

17. The oral pharmaceutical composition of claim 16, wherein (a), (b), and (c) together constitute 8-16% weight/weight inclusive of said oil-based liquid formulation.

18. The oral pharmaceutical composition of claim 11, further comprising a non-ionic detergent.

19. The oral pharmaceutical composition of claim 18, wherein said non-ionic detergent is a polysorbate-based detergent.

20. The oral pharmaceutical composition of claim 19, wherein said polysorbate-based detergent is polysorbate 80.

21. The oral pharmaceutical composition of claim 20, wherein said polysorbate 80 constitutes 3-10% weight/weight inclusive of said oil-based liquid formulation.

22. The oral pharmaceutical composition of claim 1, wherein said oil is a fish oil.

23. The oral pharmaceutical composition of claim 1, wherein said oil-based liquid formulation is water-free.

24. The oral pharmaceutical composition of claim 1, further comprising a coating that resists degradation in the stomach.

25. The oral pharmaceutical composition of claim 24, wherein said coating is a pH-sensitive capsule.

26. The oral pharmaceutical composition of claim 24, wherein said coating is a soft gelatin capsule.

27. The oral pharmaceutical composition of claim 1, wherein the BBI is isolated from a soybean product.

28. The oral pharmaceutical composition of claim 1, wherein the SBTI is recombinant.

29. The oral pharmaceutical composition of claim 1, wherein the SBTI is synthetic.

30. The oral pharmaceutical composition of claim 1, wherein the ratio of the anti-chymotrypsin activity to the anti-trypsin activity present in pharmaceutical composition is between 1.4:

1 and 1.1;

1 inclusive.

31. The oral pharmaceutical composition of claim 1, wherein the ratio of the anti-chymotrypsin activity to the anti-trypsin activity present in the pharmaceutical composition is between 1.35:

1-1.2;

1 inclusive.

32. The oral pharmaceutical composition of claim 1, wherein the BBI has been purified to at least 85% purity, as measured by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).

33. The oral pharmaceutical composition of claim 1, wherein the BBI has been purified to a protein content of greater than 95% by BCA (bicinchoninic acid) assay.

39. A method of preparing a medicament for orally administering a therapeutic protein to a subject, the method comprising coating the pharmaceutical composition of claim 1 with a coating that resists degradation in the stomach.

46. A method for orally administering a therapeutic protein to a subject, said method comprising the step of administering to a subject the oral pharmaceutical composition of claim 1, thereby orally administering a therapeutic protein to a subject.

34. An oral pharmaceutical composition comprising an oil-based liquid formulation, wherein said oil-based liquid formulation comprises a therapeutic protein of up to 100 kilodalton, a chelator of divalent cations, and a Bowman-Birk Inhibitor (BBI), wherein the BBI has a ratio of anti-chymotrypsin activity to anti-trypsin activity present of between 1.5:

1 and 1;

1 inclusive, and wherein the BBI has been produced by a process comprising

(a) subjecting soybean trypsin inhibitor (SBTI) to column chromatography under conditions in which fractions containing the SBTI'"'"'s Bowman-Birk Inhibitor (BBI) activity elute separately from fractions containing the SBTI'"'"'s Kunitz Trypsin Inhibitor (KTI) activity;

(b) eluting and combining the fractions from (a) that contain the BBI activity; and

(c) filtering the combined fractions from (b) that contain the BBI activity under conditions that reduce contaminants having a molecular weight of greater than 30 KDa to be less than 0.1% of the BBI preparation, thus producing a purified BBI product characterized in that;

(i) contaminants having a molecular weight greater than 30 KDa are less than 0.1% of the preparation; and

(ii) 1 mg of the purified BBI product has an activity of about 40 BTEE units per mg of protein.

35. An oral pharmaceutical composition comprising

an oil-based liquid formulation, wherein said oil-based liquid formulation comprises

(a) a mixture of C₈-C₁₈ monoacylglycerols, diacylglycerols, and triacylglycerols;

(b) PEG-32 monoesters and diesters of a mixture of C₈-C₁₈ fatty acids;

(c) free PEG-32;

(d) a therapeutic protein having a molecular weight of up to 100 kilodalton,

(e) a chelator of divalent cations,

(f) a purified BBI product characterized in that;

(i) contaminants having a molecular weight greater than 30 KDa are less than 0.1% of the BBI preparation; and

(ii) 1 mg of the purified BBI product has an activity of about 40 BTEE units per mg of protein;

(g) a purified KTI product characterized in that;

(i) contaminants having a molecular weight greater than 30 KDa are less than 0.1% of the KTI preparation; and

(ii) 1 mg of the purified KTI product has an activity of about 10,000 BAEE units per mg of protein;

wherein the weight/weight ratio of component (a) of said mixture to the sum of components (b) and (c) is between 10;

90 and 30;

70 inclusive; and

wherein the ratio of anti-chymotrypsin activity to anti-trypsin activity in the pharmaceutical composition is between 1.5;

1 and 1;

1 inclusive.

36. The oral pharmaceutical composition of claim 35, wherein the oil is fish oil.

37. The oral pharmaceutical composition of claim 35, wherein the composition is water-free.

38. The oral pharmaceutical composition of claim 35, wherein the therapeutic protein is insulin or a GLP-1 analogue.

40. A method for making a pharmaceutical composition, the method comprising

(a) providing a preparation of Bowman-Birk Inhibitor (BBI), wherein the BBI has been produced by a process comprising

(i) subjecting soybean trypsin inhibitor (SBTI) to column chromatography under conditions in which fractions containing the SBTI'"'"'s Bowman-Birk Inhibitor (BBI) activity elute separately from fractions containing the SBTI'"'"'s Kunitz Trypsin Inhibitor (KTI) activity;

(ii) eluting and combining the fractions from (i) that contain the BBI activity

(iii) filtering the combined fractions from (ii) that contain the BBI activity under conditions that reduce contaminants having a molecular weight of greater than 30 KDa to be less than 0.1% of the BBI preparation, thus producing a purified BBI product characterized in that;

contaminants having a molecular weight greater than 30 KDa are less than 0.1% of the preparation; and

1 mg of the purified BBI product has an activity of about 40 BTEE units per mg of protein;

and

(b) mixing the preparation purified BBI product of part (iii) and a therapeutic protein of up to 100 kilodaltons into an oil-based liquid formulation;

wherein the preparation of BBI that is mixed with the therapeutic protein is of a purity of at least 85% as measured by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE); and

wherein the ratio of anti-chymotrypsin activity to anti-trypsin activity present in the pharmaceutical composition is between 1.5;

1 and 1;

1 inclusive.

41. The method of claim 40, wherein the BBI is isolated from a soybean product.

42. The method of claim 40, wherein the BBI is recombinant.

43. The method of claim 40, wherein the BBI is synthetic.

44. A method for making an oral pharmaceutical composition comprising

(a) subjecting soybean trypsin inhibitor (SBTI) to column chromatography under conditions in which fractions containing the SBTI'"'"'s Bowman-Birk Inhibitor (BBI) activity elute separately from fractions containing the SBTI'"'"'s Kunitz Trypsin Inhibitor (KTI) activity;

(b) eluting and combining the fractions from (a) that contain the BBI activity;

(c) filtering the combined fractions from (a) that contain the BBI activity under conditions that reduce contaminants having a molecular weight of greater than 30 KDa to be less than 0.1% of the BBI preparation, thus producing a purified BBI product characterized in that;

(i) contaminants having a molecular weight greater than 30 KDa are less than 0.1% of the preparation; and

(ii) 1 mg of the purified BBI product has an activity of about 40 BTEE units per mg of protein;

(d) eluting and combining the fractions from (a) that contain the KTI activity and in which contaminants having a molecular weight greater than 30 KDa are less than 0.1% of the preparation thus producing a purified KTI product characterized in that;

(i) contaminants having a molecular weight greater than 30 KDa are less than 0.1% of the preparation; and

(ii) 1 mg of the purified KTI product has an activity of about 10,000 BAEE units per mg of protein;

(e) combining

(i) an oil-based liquid formulation,

(ii) a therapeutic protein having a molecular weight of up to 100 kilodalton,

(iii) a chelator of divalent cations,

(iv) the purified BBI product of part (b); and

(v) the purified KTI product of part (c),

such that the ratio of anti-chymotrypsin activity to anti-trypsin activity present in the pharmaceutical composition is between 1.5;

1 and 1;

1 inclusive.

45. The method of claim 44, wherein said oil-based liquid formulation further comprises

(a) a mixture of C₈-C₁₈ monoacylglycerols, diacylglycerols, and triacylglycerols;

(b) PEG-32 monoesters and diesters of a mixture of C₈-C₁₈ fatty acids; and

(c) free PEG-32;

and wherein the weight/weight ratio of component (a) of said mixture to the sum of components (b) and (c) is between 10;

90 and 30;

70 inclusive.