阅读说明：本技术 肽抑制剂的药物制剂 (Pharmaceutical formulations of peptide inhibitors ) 是由 L·霍坎松 K·阿克维斯特 于 2019-09-27 设计创作，主要内容包括：本文描述了适合用于制备包含免疫调节性肽抑制剂的药物制剂的药物产品。本文描述了包含免疫调节性肽抑制剂的药物制剂。本文也描述了通过施用包含免疫调节性肽抑制剂的药物制剂改善、抑制、治疗癌症或减轻其症状的方法。(Described herein are pharmaceutical products suitable for use in the preparation of pharmaceutical formulations comprising an immunomodulatory peptide inhibitor. Pharmaceutical formulations comprising an immunomodulatory peptide inhibitor are described herein. Also described herein are methods of ameliorating, inhibiting, treating, or alleviating a symptom of cancer by administering a pharmaceutical formulation comprising an immunomodulatory peptide inhibitor.)

1. A pharmaceutical product comprising:

a first solution comprising an isolated peptide comprising the amino acid sequence FFVKLS (SEQ ID NO:1) dissolved in the first solution, the first solution having a pH of less than 7 or about less than 7, and a sub-osmolality; and

a second solution comprising a tonicity agent and a base,

wherein the first and second solutions when combined with each other produce an isotonic gel, wherein the isotonic gel comprises the isolated peptide in a concentration of at least 0.2mg/ml or about at least 0.2mg/ml and a pH of 6.5-7.5 or about 6.5-7.5.

2. The pharmaceutical product of claim 1, wherein the first solution is substantially free of particles greater than 0.2 μ Μ diameter or particles greater than about 0.2 μ Μ diameter, and wherein the second solution is substantially free of particles greater than 0.2 μ Μ or particles greater than about 0.2 μ Μ diameter.

3. The pharmaceutical product of any one of claims 1-2, wherein the first solution comprises less than or equal to 10mM NaCl, or less than or equal to about 10mM NaCl, but non-zero.

4. The pharmaceutical product of any one of claims 1-2, wherein the first solution does not comprise NaCl.

5. The pharmaceutical product of any one of claims 1-4, wherein the first solution further comprises a buffer having a buffer capacity equivalent to 1.5mM or about 1.5mM sodium acetate or less but not zero.

6. The pharmaceutical product of any one of claims 1-4, wherein the first solution further comprises sodium acetate at a concentration of less than or equal to 1.5mM or less than or equal to about 1.5mM or less, but not zero.

7. The pharmaceutical product of any one of claims 1-4, wherein the first solution does not comprise a buffer.

8. The pharmaceutical product of any one of claims 1-7, wherein the tonicity agent is NaCl, and wherein the second solution is in a gel formulation to comprise 100mM-120mM NaCl or about 100mM-120mM NaCl.

9. The pharmaceutical product of any one of claims 1-8, wherein the first solution is substantially free of gel.

10. The drug product of any one of claims 1-9, wherein the isolated peptide dissolved in the first solution is not substantially in a β -sheet conformation.

11. The pharmaceutical product of any one of claims 1-10, wherein the first solution is a gel configured to contain the isolated peptide at a concentration of at least 0.4mg/ml or at least about 0.4 mg/ml.

12. The pharmaceutical product of any one of claims 1-11, wherein the isolated peptide comprises no more than 30 amino acid residues.

13. The pharmaceutical product of any one of claims 1-12, wherein the isolated peptide comprises amino acid sequence KKLDTFFVKLSLFTER (SEQ ID NO: 2).

14. The pharmaceutical product of any one of claims 1-11, wherein the isolated peptide consists of amino acid sequence KKLDTFFVKLSLFTER (SEQ ID NO: 2).

15. The pharmaceutical product of any one of claims 1-11, wherein the first solution is capable of maintaining at least 95% or at least about 95% of the isolated peptide dissolved in the first solution at 5 ℃ for at least 12-25 months or at least about 12-25 months.

16. The pharmaceutical product of any one of claims 1-15, wherein the first solution and the second solution are in a gel configuration to comprise:

at least 0.4mg/ml or at least about 0.4mg/ml of an isolated peptide consisting of amino acid sequence KKLDTFFVKLSLFTER (SEQ ID NO: 2);

30-40mM or at least about 30-40mM acetic acid;

1.2 to 1.6mM or at least about 1.2 to 1.6mM sodium acetate;

less than or equal to 30mM or less than or equal to about 30mM sodium hydroxide; and

100-,

wherein the gel has an osmolality of 280-300mOSmol/L or about 280-300 mOSmol/L.

17. A process for preparing the pharmaceutical product of any one of claims 1-16, the process comprising:

aseptically filtering a precursor solution comprising an isolated peptide comprising the amino acid sequence FFVKLS (SEQ ID NO:1) dissolved in the precursor solution at a concentration of at least 0.2mg/ml or at least about 0.2mg/ml, wherein the precursor solution has a pH of less than 7 or less than about 7 and a sub-osmolality, thereby producing the first solution; and

providing the second solution comprising a tonicity agent and a base.

18. The method of claim 17, wherein the precursor solution is sterile filtered with a filter having a pore size of about 0.2 μ Μ or 0.2 μ Μ.

19. The method of any one of claims 17-18, wherein the precursor solution has a pH of less than 4.5 or less than about 4.5.

20. The method of any one of claims 17-19, wherein the isolated peptide comprises no more than 30 amino acid residues.

21. The method of any one of claims 17-20, wherein the isolated peptide comprises amino acid sequence KKLDTFFVKLSLFTER (SEQ ID NO: 2).

22. The method of any one of claims 17-21, wherein the isolated peptide consists of amino acid sequence KKLDTFFVKLSLFTER (SEQ ID NO: 2).

23. A method of preparing a pharmaceutical formulation from the pharmaceutical product of any one of claims 1-22, the method comprising combining the first solution and the second solution to form the gel.

24. A pharmaceutical formulation comprising a gel, comprising:

at least 0.4mg/ml or at least about 0.4mg/ml of an isolated peptide comprising the amino acid sequence FFVKLS (SEQ ID NO: 1);

a buffer system comprising acetic acid and sodium acetate, the buffer system comprising less than or equal to 1.6mM or less than or equal to about 1.6mM sodium acetate; and

a tonicity agent, a suspending agent,

wherein the gel is isotonic and has a pH of 4.5-7.5 or about 4.5-7.5.

25. The pharmaceutical formulation of claim 24, wherein the gel is substantially free of particles greater than 0.2 μ Μ or greater than about 0.2 μ Μ diameter.

26. The pharmaceutical formulation of any one of claims 24-25, wherein the tonicity agent is sodium chloride at a concentration of 100-120mM or about 100-120 mM.

27. The pharmaceutical formulation of any one of claims 24-26, wherein the buffer system comprises 30-40mM or about 30-40mM acetic acid and 1.2-1.6mM or about 1.2-1.6mM sodium acetate.

28. The pharmaceutical formulation of any one of claims 24-27, further comprising sodium hydroxide at a concentration less than or equal to 30mM or less than or equal to about 30mM but not zero.

29. The pharmaceutical formulation of any one of claims 24-28, wherein the gel has an osmolarity of 280-300mOSmol/L or about 280-300 mOSmol/L.

30. The pharmaceutical formulation of any one of claims 24-29, wherein the isolated peptide comprises no more than 30 amino acid residues.

31. The pharmaceutical formulation of any one of claims 24-30, wherein the isolated peptide comprises amino acid sequence KKLDTFFVKLSLFTER (SEQ ID NO: 2).

32. The pharmaceutical formulation of any one of claims 24-31, wherein the isolated peptide consists of amino acid sequence KKLDTFFVKLSLFTER (SEQ ID NO: 2).

33. A pharmaceutical formulation comprising:

an isolated peptide comprising the amino acid sequence FFVKLS (SEQ ID NO:1), wherein the isolated peptide is dissolved in the pharmaceutical formulation at a concentration of 0.2-20mg/ml or about 0.2-20 mg/ml; and

a non-ionic tonicity agent which is a mixture of,

wherein the pharmaceutical formulation is isotonic and has a pH of 5.0-5.5 or about 5.0-5.5, and

wherein the pharmaceutical formulation is a liquid.

34. The pharmaceutical formulation of claim 33, wherein the pharmaceutical composition is substantially free of particles having a diameter greater than 0.2 μ Μ or greater than about 0.2 μ Μ.

35. The pharmaceutical formulation of any one of claims 33-34, further comprising a weak acid, wherein the peptide and the weak acid constitute a buffer system that maintains the pharmaceutical formulation at a pH of 5.0-5.5 or about 5.0-5.5.

36. The pharmaceutical formulation according to claim 35, wherein the weak acid is acetic acid, which is present at a concentration of 0.01M or about 0.01M.

37. The pharmaceutical formulation of any one of claims 33-36, wherein the non-ionic tonicity agent is glucose, which is present at a concentration of 0.2M-0.4M or about 0.2M-0.4M.

38. The pharmaceutical formulation of any one of claims 33-37, wherein the isolated peptide is at a concentration of 0.2-5mg/ml, about 0.2-5mg/ml, 0.2-10mg/ml, about 0.2-10mg/ml, 1-5mg/ml, about 1-5mg/ml, 1-10mg/ml, or about 1-10 mg/ml.

39. The pharmaceutical formulation of any one of claims 33-38, wherein the isolated peptide comprises no more than 30 amino acid residues.

40. The pharmaceutical formulation of any one of claims 33-39, wherein the isolated peptide comprises amino acid sequence KKLDTFFVKLSLFTER (SEQ ID NO: 2).

41. The pharmaceutical formulation of any one of claims 33-38, wherein the isolated peptide consists of amino acid sequence KKLDTFFVKLSLFTER (SEQ ID NO: 2).

42. The pharmaceutical formulation of any one of claims 33-41, further comprising 0.01M or about 0.01M acetic acid,

wherein the isolated peptide consists of amino acid sequence KKLDTFFVKLSLFTER (SEQ ID NO:2) and the isolated peptide is present at a concentration of 1-10mg/ml or about 1-10mg/ml, and

wherein the non-ionic tonicity agent is glucose, which is present at a concentration of 0.2 to 0.4M or about 0.2 to 0.4M.

43. A method of ameliorating, inhibiting, treating, or alleviating a symptom of cancer in a patient in need thereof, the method comprising administering to the patient an effective amount of the pharmaceutical formulation of any one of claims 24-42.

44. The method of claim 43, wherein the effective amount is at most 1mg/kg or at most 5 mg/kg.

45. The method of any one of claims 43-44, wherein the effective amount of the pharmaceutical formulation comprises 8-800 μ g or about 8-800 μ g of the isolated peptide.

46. The method of any one of claims 43-44, wherein the effective amount of the pharmaceutical formulation comprises 60-100 μ g or about 60-100 μ g of the isolated peptide.

47. The method of any one of claims 43-46, wherein the pharmaceutical formulation is administered intratumorally, subcutaneously, lymphogenically, and/or to the interstitial fluid of the patient.

48. The method of any one of claims 43-47, further comprising repeating the administration of the pharmaceutical formulation.

49. The method of any one of claims 43-48, wherein the cancer comprises a tumor.

50. The method of any one of claims 43-49, wherein the cancer is selected from the group consisting of: head and neck cancer, breast cancer, kidney cancer, colorectal cancer, skin cancer, ovarian cancer, prostate cancer, pancreatic cancer, lung cancer, malignant melanoma, small cell lung cancer, non-small cell lung cancer (adenocarcinoma), squamous cell carcinoma, bladder cancer, osteosarcoma, bronchial cancer, or hematopoietic cell cancer.

51. The method of any one of claims 43-50, further comprising receiving the results of the detection of the presence and/or level of peptide P3028(SEQ ID NO:3) or a P3028 structure in a sample of the patient, such as a sample comprising hematopoietic tissue, a bodily fluid, a blood sample, a tumor biopsy sample, or a biopsy of peritumoral tissue.

52. The method of claim 51, further comprising selecting the patient to receive an effective amount of a pharmaceutical agent if denatured or damaged albumin, such as P3028 structures, is present in the sample or exceeds a predetermined level.

53. The method of any one of claims 43-52, further comprising receiving results of the detection of the presence of immune cells in a sample of the patient, such as a tumor biopsy sample.

54. The method of claim 53, wherein the patient's sample is collected at least 5 days after administration of the pharmaceutical composition.

55. The method of any one of claims 53-54, further comprising, if immune cells are present in the sample, selecting the patient to receive an effective amount of a pharmaceutical agent.

56. The method of any one of claims 43-55, further comprising selecting the patient as comprising the peptide 3028 and/or immune cells in a sample of the patient, such as a sample comprising hematopoietic tissue, a body fluid, a blood sample, or a tumor biopsy sample.

57. The method of any one of claims 43-56, further comprising selecting the patient as comprising a tumor that is substantially free of T-cell infiltrates, a tumor that comprises a majority of T-cell infiltrates in the stroma, or a tumor that is inflamed and infiltrated by inactive T-cells.

58. The method of any one of claims 43-57, further comprising detecting an inflammatory response to the tumor, such as suppressor cell infiltration of effector cells and/or the tumor, after administration of the pharmaceutical formulation.

59. The method of any one of claims 43-58, further comprising detecting death, such as apoptosis or necrosis, of the tumor cell after administration of the pharmaceutical composition.

60. The method of any one of claims 43-59, further comprising administering to the patient an additional therapeutic agent.

61. The method of claim 60, wherein the additional therapeutic agent comprises an antibody that specifically binds PD-1 or PDL-1, or an antibody that is bispecific for PD-1 and PDL-1.

FIELD

Embodiments herein relate to pharmaceutical products and pharmaceutical formulations comprising immunomodulatory peptides and methods of using the same.

Background

The immune system is fine-tuned to detect and eradicate foreign molecules, and at the same time avoid excessive reactions that may cause destruction of normal tissues that cause autoimmune or chronic inflammatory diseases. The initiation of a specific immune response is a coordinated chain of events that culminate in the activation of effector functions (such as the release of cytokines, the production of specific antibodies, and/or the cytotoxic activity of cells).

Although the data indicate that the immune system is very important for cancer control (Dunn GP, et al, Immunity.200421: 137-48, Galon J, et al, science.2006313: 1960-4, KoebelCM, et al, Nature.2007450: 903-7, Clinchy B, et al, cancer.2007109: 1742-9, Teng MW, et al, J Leukoc biol.200884: 988-93), malignant tumors continue to grow and the efficacy of immunotherapy is very poor with an objective remission rate of 10-20%. This apparent conflict may have several reasons, e.g., tumor avoidance of recognition by the immune system (since tumor antigens are weak self-antigens), poor antigen presentation (down-regulation by TAP and MHC I and II), or induction of tolerance or cancer-related immunosuppression. The influence of adverse intratumoral environment was confirmed by results from animal experiments (Perdrizet GA, et al, JExp Med.1990; 171: 1205-20., Yu P, et al, J Exp Med.2005201: 779-91.) and human tumors (Gajewski TF, et al, J immunother.200629: 233-40, Whiteside TL, oncogene.200827: 5904-12).

Different types of immunosuppressive cells, regulatory T-cells, Immature Dendritic Cells (iDC), tumor-associated macrophages (TAMs), and bone marrow-derived suppressor cells (MDSCs) can function substantially in cancer-related immunosuppression. Immune balance generally tends to be dominated by Th2 characterized by cytokines such as IL-4, IL-10 and PGE 2. In addition, other immunosuppressive mechanisms (such as serum blocking factors, circulating immune complexes, enhanced IL-1Ra production and enhanced intratumoral proteolytic activity) may play a role in cancer-related immunosuppression.

In studying the induction mechanism of interleukin-6 (IL-6) in cancer patients, immunomodulatory peptide sequences derived from serum albumin were discovered (see, e.g., U.S. Pat. Nos. 7,960,126, 8,110,347; and 8,110,347; and U.S. publication No. 2010/0323370, and PCT publication No. WO 2016/144650), each of which is hereby expressly incorporated by reference in its entirety). Interleukin-2 (IL-2) plays an important role in the initiation and activation of the immune response and its ability to induce lymphokine activated killer cells (LAK cells), T-cell proliferation and cytotoxicity. Several reports have shown that Peripheral Blood Mononuclear Cells (PBMC) from Cancer patients have reduced ability to synthesize IL-2(Wanebo HJ, et al, cancer.198657: 656-62, Mantovani, G., et al, Diagn. Clin. Immunol.19875: 104-111, Lauerova L, et al, Neopalasma 199946: 141-149) and respond to IL-2 (Tssubono M, et al, J Clin Lab Immunol 199033: 107-115, Pellegrini P, et al, Cancer Immunol 199642: 1-8). Soluble products from tumor explants or serum from cancer patients may inhibit cytokine production, inhibit IL-2 receptor expression (Botti C, et al, Intl J Biol Markers 199813: 51-69, Lauerova L, et al, Neoplama 199946: 141-149) and/or reduce proliferative capacity in normal T lymphocytes (Botti C, et al, Intl J Biol Markers 199813: 51-69).

Integrins are a superfamily of transmembrane glycoproteins found primarily on leukocytes that mediate cell-cell and cell-matrix interactions. Integrins play an important role in immune regulation, and in particular, α L β 2 (leukocyte function-associated molecule-1, LFA-1) is also critical for initiation and regulation of immune responses, tissue recruitment and migration of inflammatory cells, and cytotoxic activity of lymphocytes (Hogg N, et al, J Cell sci.2003116: 4695-705, Giblin PA, et al, Curr Pharm des.200612: 2771-95, Evans R, et al, Cell sci.2009122: 215-25). In addition, LFA-1 is involved in proliferative responses to interleukin-2 (Vyth-Dreese FA, Eur J Immunol.199312: 3292-9), and some fragments of albumin bind to LFA-1 and/or IL-2 receptors, thereby modulating functional properties mediated through these receptors, including immune cell proliferation (see U.S. publication No. 2011/0262470, which is hereby expressly incorporated by reference in its entirety).

Disclosure of Invention

Option 1 comprises, consists essentially of, or consists of: a pharmaceutical product comprising a first solution comprising an isolated peptide comprising the amino acid sequence FFVKLS (SEQ ID NO:1) dissolved in the first solution, the first solution having a pH of less than 7 or about less than 7 and an osmolarity. The pharmaceutical product may comprise a second solution comprising a tonicity agent and a base, wherein the first solution and the second solution when combined with each other produce an isotonic gel, and wherein the isotonic gel comprises the isolated peptide at a concentration of at least 0.2mg/ml or about at least 0.2mg/ml and a pH of 6.5-7.5 or about 6.5-7.5.

Option 2 comprises, consists essentially of, or consists of: the pharmaceutical product of option 1, wherein the first solution is substantially free of particles greater than 0.2 μ Μ diameter or particles greater than about 0.2 μ Μ diameter, and wherein the second solution is substantially free of particles greater than 0.2 μ Μ or particles greater than about 0.2 μ Μ diameter.

Option 3 comprises, consists essentially of, or consists of: the pharmaceutical product of any one of options 1-2, wherein the first solution comprises less than or equal to 10mM NaCl, or less than or equal to about 10mM NaCl, but is non-zero.

Option 4 comprises, consists essentially of, or consists of: the pharmaceutical product of any one of options 1-2, wherein the first solution does not comprise NaCl.

Option 5 comprises, consists essentially of, or consists of: the pharmaceutical product of any one of options 1-4, wherein the first solution further comprises a buffer having a buffer capacity equivalent to 1.5mM or about 1.5mM sodium acetate or less but not zero.

Option 6 comprises, consists essentially of, or consists of: the pharmaceutical product of any one of options 1-4, wherein the first solution further comprises sodium acetate in a concentration less than or equal to 1.5mM or less than or equal to about 1.5mM or less but not zero.

Option 7 comprises, consists essentially of, or consists of: the pharmaceutical product of any one of options 1-4, wherein the first solution does not comprise a buffer.

Option 8 comprises, consists essentially of, or consists of the following: the pharmaceutical product of any one of options 1-7, wherein the tonicity agent is NaCl, and wherein the second solution is in a gel formulation to comprise 100mM-120mM NaCl or about 100mM-120mM NaCl.

Option 9 comprises, consists essentially of, or consists of the following: the pharmaceutical product of any one of options 1-8, wherein the first solution is substantially free of gel.

Option 10 comprises, consists essentially of, or consists of: the drug product of any one of options 1-9, wherein the isolated peptide dissolved in the first solution is not substantially in a β -sheet conformation.

Option 11 comprises, consists essentially of, or consists of: the pharmaceutical product of any one of options 1-11, wherein the first solution is a gel configured to comprise the isolated peptide at a concentration of at least 0.4mg/ml or at least about 0.4 mg/ml.

Option 12 comprises, consists essentially of, or consists of: the pharmaceutical product of any one of options 1-11, wherein the isolated peptide comprises no more than 30 amino acid residues.

Option 13 comprises, consists essentially of, or consists of: the pharmaceutical product of any one of options 1-12, wherein the isolated peptide comprises amino acid sequence KKLDTFFVKLSLFTER (SEQ ID NO: 2).

Option 14 comprises, consists essentially of, or consists of: the pharmaceutical product of any one of options 1-11, wherein the isolated peptide consists of amino acid sequence KKLDTFFVKLSLFTER (SEQ ID NO: 2).

Option 15 comprises, consists essentially of, or consists of: the pharmaceutical product of any one of options 1-11, wherein the first solution is capable of maintaining at least 95% or at least about 95% of the isolated peptide dissolved in the first solution at 5 ℃ for at least 12-25 months or at least about 12-25 months.

Option 16 comprises, consists essentially of, or consists of: the pharmaceutical product of any one of options 1-15, wherein the first solution and the second solution are in a gel configuration to comprise: at least 0.4mg/ml or at least about 0.4mg/ml of an isolated peptide consisting of amino acid sequence KKLDTFFVKLSLFTER (SEQ ID NO: 2); 30-40mM or at least about 30-40mM acetic acid; 1.2 to 1.6mM or at least about 1.2 to 1.6mM sodium acetate; less than or equal to 30mM or less than or equal to about 30mM sodium hydroxide; and 100-. The gel may have an osmolarity of 280-300mOSmol/L or about 280-300 mOSmol/L.

Option 17 comprises, consists essentially of, or consists of the following: a method of making the pharmaceutical product of any one of options 1-16. The method can include sterile filtering a precursor solution comprising an isolated peptide comprising the amino acid sequence FFVKLS (SEQ ID NO:1) dissolved in the precursor solution at a concentration of at least 0.2mg/ml or at least about 0.2mg/ml, wherein the precursor solution has a pH of less than 7 or less than about 7 and a sub-osmolarity, thereby producing the first solution. The method can include providing a second solution comprising a tonicity agent and a base.

Option 18 comprises, consists essentially of, or consists of: the method of option 17, wherein the precursor solution is sterile filtered with a filter of about 0.2 μ Μ or 0.2 μ Μ pore size.

Option 19 comprises, consists essentially of, or consists of: the method of any of options 17-18, wherein the precursor solution has a pH of less than 4.5 or less than about 4.5.

Option 20 comprises, consists essentially of, or consists of: the method of any one of options 17-19, wherein the isolated peptide comprises no more than 30 amino acid residues.

Option 21 comprises, consists essentially of, or consists of: the method of any one of options 17-20, wherein the isolated peptide comprises amino acid sequence KKLDTFFVKLSLFTER (SEQ ID NO: 2).

Option 22 comprises, consists essentially of, or consists of: the method of any one of options 17-21, wherein the isolated peptide consists of amino acid sequence KKLDTFFVKLSLFTER (SEQ ID NO: 2).

Option 23 comprises, consists essentially of, or consists of: a method of preparing a pharmaceutical formulation from the pharmaceutical product of any one of options 1-22. The method may include combining the first solution and the second solution to form the gel.

Option 24 comprises, consists essentially of, or consists of the following: a pharmaceutical formulation comprising a gel. The gel may comprise at least 0.4mg/ml or at least about 0.4mg/ml of an isolated peptide comprising the amino acid sequence FFVKLS (SEQ ID NO: 1). The gel may comprise a buffer system comprising acetic acid and sodium acetate, the buffer system comprising less than or equal to 1.6mM or less than or equal to about 1.6mM sodium acetate. The gel may comprise a tonicity agent. The gel may be isotonic and have a pH of 4.5-7.5 or about 4.5-7.5.

Option 25 comprises, consists essentially of, or consists of: the pharmaceutical formulation of option 24, wherein the gel is substantially free of particles greater than 0.2 μ Μ or greater than about 0.2 μ Μ diameter.

Option 26 comprises, consists essentially of, or consists of the following: the pharmaceutical formulation of any one of options 25-26, wherein the tonicity agent is sodium chloride at a concentration of 100-120mM or about 100-120 mM.

Option 27 comprises, consists essentially of, or consists of: the pharmaceutical formulation of any one of options 24-26, wherein the buffer system comprises 30-40mM or about 30-40mM acetic acid and 1.2-1.6mM or about 1.2-1.6mM sodium acetate.

Options 28 include, consist essentially of, or consist of: the pharmaceutical formulation of any one of options 24-27, further comprising sodium hydroxide at a concentration less than or equal to 30mM or less than or equal to about 30mM but not zero.

Option 29 comprises, consists essentially of, or consists of the following: the pharmaceutical formulation of any one of options 24-28, wherein the gel has an osmolarity of 280-300mOSmol/L or about 280-300 mOSmol/L.

Option 30 comprises, consists essentially of, or consists of: the pharmaceutical formulation of any one of options 24-29, wherein the isolated peptide comprises no more than 30 amino acid residues.

Option 31 comprises, consists essentially of, or consists of: the pharmaceutical formulation of any one of options 24-30, wherein the isolated peptide comprises amino acid sequence KKLDTFFVKLSLFTER (SEQ ID NO: 2).

Option 32 comprises, consists essentially of, or consists of: the pharmaceutical formulation of any one of options 24-31, wherein the isolated peptide consists of amino acid sequence KKLDTFFVKLSLFTER (SEQ ID NO: 2).

Option 33 comprises, consists essentially of, or consists of: a pharmaceutical formulation. The pharmaceutical formulation may comprise an isolated peptide comprising the amino acid sequence FFVKLS (SEQ ID NO:1), wherein the isolated peptide is dissolved in the pharmaceutical formulation at a concentration of 0.2-20mg/ml or about 0.2-20 mg/ml. The pharmaceutical formulation may comprise a non-ionic tonicity agent. The pharmaceutical formulation may be isotonic and have a pH of 5.0-5.5 or about 5.0-5.5, and the pharmaceutical formulation may be a liquid.

Options 34 include, consist essentially of, or consist of the following: the pharmaceutical formulation of option 33, wherein the pharmaceutical composition is substantially free of particles having a diameter greater than 0.2 μ Μ or greater than about 0.2 μ Μ.

Option 35 comprises, consists essentially of, or consists of the following: the pharmaceutical formulation of any one of options 33-34, further comprising a weak acid. The peptide and the weak acid may constitute a buffer system that maintains the pharmaceutical formulation at a pH of 5.0-5.5 or about 5.0-5.5.

Option 36 comprises, consists essentially of, or consists of the following: the pharmaceutical formulation of option 35, wherein the weak acid is acetic acid, which is present at a concentration of 0.01M or about 0.01M.

Option 37 comprises, consists essentially of, or consists of the following: the pharmaceutical formulation of any one of options 33-36, wherein the non-ionic tonicity agent is glucose, which is present at a concentration of 0.2M-0.4M or about 0.2M-0.4M.

Options 38 include, consist essentially of, or consist of: the pharmaceutical formulation of any one of options 33-37, wherein the isolated peptide concentration is 0.2-5mg/ml, about 0.2-5mg/ml, 0.2-10mg/ml, about 0.2-10mg/ml, 1-5mg/ml, about 1-5mg/ml, 1-10mg/ml, or about 1-10 mg/ml.

Option 39 comprises, consists essentially of, or consists of the following: the pharmaceutical formulation of any one of options 33-38, wherein the isolated peptide comprises no more than 30 amino acid residues.

Option 40 comprises, consists essentially of, or consists of the following: the pharmaceutical formulation of any one of options 33-39, wherein the isolated peptide comprises amino acid sequence KKLDTFFVKLSLFTER (SEQ ID NO: 2).

Option 41 comprises, consists essentially of, or consists of: the pharmaceutical formulation of any one of options 33-38, wherein the isolated peptide consists of amino acid sequence KKLDTFFVKLSLFTER (SEQ ID NO: 2).

Option 42 comprises, consists essentially of, or consists of: the pharmaceutical formulation of any one of options 33-41, further comprising 0.01M or about 0.01M acetic acid. The isolated peptide may consist of amino acid sequence KKLDTFFVKLSLFTER (SEQ ID NO:2), and the isolated peptide may be present at a concentration of 1-10mg/ml or about 1-10 mg/ml. The non-ionic tonicity agent may be glucose, which is present at a concentration of 0.2 to 0.4M or about 0.2 to 0.4M.

Option 43 comprises, consists essentially of, or consists of the following: a method of ameliorating, inhibiting, treating, or alleviating a symptom of a cancer in a patient in need thereof, the method comprising administering to the patient an effective amount of the pharmaceutical formulation of any one of options 24-42.

Options 44 include, consist essentially of, or consist of the following: the method of option 43, wherein the effective amount is at most 1mg/kg or at most 5 mg/kg.

Option 45 comprises, consists essentially of, or consists of the following: the method of any one of options 43-44, wherein the effective amount of the pharmaceutical formulation comprises 8-800 μ g or about 8-800 μ g of the isolated peptide.

Options 46 include, consist essentially of, or consist of the following: the method of any one of options 43-44, wherein the effective amount of the pharmaceutical formulation comprises 60-100 μ g or about 60-100 μ g of the isolated peptide.

Option 47 comprises, consists essentially of, or consists of the following: the method of any one of options 43-46, wherein the pharmaceutical formulation is administered intratumorally, subcutaneously, lymphogenically, and/or to the interstitial fluid of the patient.

Options 48 include, consist essentially of, or consist of: the method of any one of options 43-47, further comprising repeating the administration of the pharmaceutical formulation.

Option 49 comprises, consists essentially of, or consists of: the method of any one of options 43-48, wherein the cancer comprises a tumor.

Options 50 include, consist essentially of, or consist of: the method of any one of options 43-49, wherein the cancer is selected from the group consisting of: head and neck cancer, breast cancer, kidney cancer, colorectal cancer, skin cancer, ovarian cancer, prostate cancer, pancreatic cancer, lung cancer, malignant melanoma, small cell lung cancer, non-small cell lung cancer (adenocarcinoma), squamous cell carcinoma, bladder cancer, osteosarcoma, bronchial cancer, or hematopoietic cell cancer.

Option 51 comprises, consists essentially of, or consists of: the method of any one of options 43-50, further comprising receiving the results of the detection of the presence and/or level of peptide P3028(SEQ ID NO:3) or a P3028 structure in a sample of the patient, such as a sample comprising hematopoietic tissue, a bodily fluid, a blood sample, a tumor biopsy sample, or a biopsy of tissue surrounding the tumor. In some embodiments, the sample comprises a hematopoietic tissue, a blood sample, or a tumor biopsy sample.

Options 52 include, consist essentially of, or consist of: the method of option 51, further comprising selecting the patient to receive an effective amount of the pharmaceutical agent if denatured or damaged albumin, such as P3028 structures, is present in the sample or exceeds a predetermined level.

Option 53 comprises, consists essentially of, or consists of: the method of any one of options 43-52, further comprising receiving results of the detection of the presence of immune cells in a sample (such as a tumor biopsy sample) of the patient.

Options 54 include, consist essentially of, or consist of the following: the method of option 53, wherein the patient's sample is collected at least 5 days after administration of the pharmaceutical composition.

Option 55 comprises, consists essentially of, or consists of the following: the method of any one of options 53-54, further comprising selecting the patient to receive an effective amount of the pharmaceutical agent if immune cells are present in the sample.

Option 56 comprises, consists essentially of, or consists of the following: the method of any one of options 43-55, further comprising selecting the patient as comprising the peptide 3028 and/or immune cells in a sample of the patient, such as a sample comprising hematopoietic tissue, a body fluid, a blood sample, or a tumor biopsy sample. In some embodiments, the sample comprises a hematopoietic tissue, a blood sample, or a tumor biopsy sample.

Option 57 comprises, consists essentially of, or consists of: the method of any one of options 43-56, further comprising selecting the patient as comprising: tumors that contain substantially no T-cell infiltrates, tumors that contain the majority of T-cell infiltrates in the stroma, or tumors that are inflamed and infiltrated by inactive T-cells.

Options 58 include, consist essentially of, or consist of: the method of any of options 43-57, further comprising detecting an inflammatory response to the tumor, such as suppressor cell infiltration of effector cells and/or the tumor, after administration of the pharmaceutical formulation.

Option 59 comprises, consists essentially of, or consists of the following: the method of any one of options 43-58, further comprising detecting death, such as apoptosis or necrosis, of the tumor cell after administration of the pharmaceutical composition.

Option 61 comprises, consists essentially of, or consists of the following: the method of any one of options 43-59, further comprising administering an additional therapeutic agent to the patient.

Options 62 include, consist essentially of, or consist of the following: the method of option 61, wherein the additional therapeutic agent comprises an antibody that specifically binds PD-1 or PDL-1, or an antibody that is bispecific for PD-1 and PDL-1.

Brief description of the drawings

FIGS. 1A-D are a series of graphs illustrating the stimulatory activity of P28R on the suppressed proliferative response to IL-2. Fig. 1A, 1B, 1C and 1D illustrate the stimulatory activity of four different cancer patients, respectively.

FIGS. 2A-B are a series of graphs illustrating the effect of full-length peptide P28R and the 6 amino acid center sequence (32230, FFVKLS, SEQ ID NO:1) in medium containing normal human AB serum. Activation was determined as the percentage of cells with enhanced markers CD69 or CD71 using flow cytometry. PBMCs were incubated with peptide (40. mu.g/mL) in RPMI + 10% human AB serum for 24 hours. Fig. 2A illustrates the results of two experiments (420 and 422) performed for each peptide. Fig. 2B illustrates the results of two experiments (424 and 426) performed for each peptide.

FIG. 3 is a graph illustrating the alignment of full-length peptide P28R and the 6 amino acid "P28 core" sequence (32230, FFVKLS, SEQ ID NO:1) in medium containing sera from two different cancer patients ("human cancer serum 1" 430 and "human cancer serum 2" 432).

Figure 4 is a graph showing the evaluation of P28R treatment in 7 dogs with a breast tumor compared to 5 untreated control dogs according to some embodiments herein. In the representative panel (n ═ 1-5), the total number of tumor cells from the treatment group (dark bars 881 in P28R #4, #7, #8, #11, #13, #16 and # 17) and from the control tumors (dark bars 882 in control #2, #3, #4, #5 and # 6) was counted and compared to the number of inflammatory cells (light gray bars 883).

Fig. 5A-B series of graphs show that subcutaneous P28R treatment of spontaneous canine breast tumors (single dose 80 micrograms) resulted in recruitment of inflammatory cells and tumor cell death (control N-12 and treatment N-5).

Fig. 6A-C are a series of microscope images of three different tongue cancers double stained with antibodies against P3028 (red) and CD3 (brown). Immune desert cancer (fig. 6A) has a strong expression of 3028 and only a few scattered T-cells in the stroma. The immune rejection cancer in the middle (fig. 6B) has a strong expression of 3028 and T-cell infiltration in the stroma, the inflammatory cancer on the right (fig. 6C) only stains weakly with respect to 3028 and has a very strong T-cell infiltration.

Fig. 7A-B are a series of microscopic images of tumor sections from breast cancer patients showing inflammatory cells stained with an antibody against CD11 a. Fresh frozen tumor sections without any fixation were incubated with buffer (fig. 7A) or P28R (fig. 7B) prior to staining.

Detailed Description

Pharmaceutical products comprising, consisting essentially of, or consisting of a peptide inhibitor are described herein, as well as pharmaceutical formulations of the peptide inhibitor, as well as methods of making and using the pharmaceutical products and formulations. The peptide inhibitors interact with immunomodulatory peptides that cause immunosuppression in humans (e.g., humans with cancer), and have been shown to reduce immunosuppression of immune cells in serum of cancer patients in vitro, and to cause degenerative changes and eradication of mammalian tumors in vivo (see examples 1-4 and 8). These results were further confirmed in additional dose escalation studies in dogs (see example 4). It has been observed herein that conventional formulations of peptide inhibitors can affect the health of cells in the vicinity of the injection site, for example due to low osmolarity and/or the presence of acetate (see example 9). Furthermore, it has been observed herein that peptide inhibitors can form gels at high pH and/or in the presence of high sodium, which can interfere with sterile filtration by plugging the pores of the filter (see examples 10 and 11). Thus, described according to some embodiments herein are pharmaceutical products, pharmaceutical formulations, which are sterile, stably maintain a peptide inhibitor, and allow the administration of appropriate doses of peptide inhibitors while avoiding the adverse effects associated with acetate and non-isotonic osmolarity.

In some embodiments, a pharmaceutical product is provided comprising a first solution comprising a peptide inhibitor such as P28R at an acidic pH and a second solution comprising a base. The first and second solutions can be combined prior to use to form a gel comprising the peptide inhibitor, the gel having a pH and osmolality suitable for administration to a cancer patient. The first solution may be sterile, for example by sterile filtration (the acidic pH and low sodium content of the first solution may avoid the formation of gels that may clog the pores of the filter; see example 10). The first solution may have an acidic pH and a sub-osmolality. The second solution may have a basic pH and an osmolarity that exceeds isotonicity. In some embodiments, the peptide inhibitor is a peptide comprising, consisting essentially of, or consisting of the amino acid sequence FFVKLS (SEQ ID NO: 1). The peptide may comprise no more than 50, 45, 40, 35, 30, 25 or 20 amino acids. In some embodiments, the peptide inhibitor is a peptide comprising, consisting essentially of, or consisting of amino acid sequence KKLDTFFVKLSLFTER (SEQ ID NO: 2).

Doubling and quadrupling the initial 80 microgram dose of P28R was observed in the dose escalation study described herein to maintain the anti-tumor effect, while no adverse effects were observed (see example 4). Thus, it is expected that formulations comprising even higher concentrations of peptide inhibitors may be safe and effective. Described according to some embodiments herein are pharmaceutical products comprising, consisting essentially of, or consisting of a pharmaceutical formulation of a peptide inhibitor at a concentration of about 0.2-20mg/ml (e.g., 0.2-5mg/ml, 0.2-10mg/ml, 1-5mg/ml, 1-10mg/ml, or 1-20 mg/ml). The pharmaceutical formulation may comprise a peptide inhibitor and a non-ionic tonicity agent as described herein. The formulation may be isotonic and have a pH of 5.0-5.5 or about 5.0-5.5. The formulation may be a liquid. The peptide inhibitor may remain dissolved in the liquid. Thus, the pharmaceutical formulation may be free or substantially free of gel. Thus, the pharmaceutical formulation may be free or substantially free of precipitate.

As used herein, "substantially free" has its ordinary and customary meaning as would be understood by one of ordinary skill in the art in view of this disclosure. It represents trace amounts with no appreciable effect on the stability and efficacy of the pharmaceutical product or formulation and also includes the absence of the specified substance. If other numerical accuracies are of interest, in some embodiments, a composition (such as a pharmaceutical product or pharmaceutical formulation) is substantially free of a substance when it contains no more than 5% (w/w) of the substance, e.g., no more than 5%, 4%, 3%, 2%, 1%, 0.9%, 0.5%, 0.2%, 0.1%, 0.05%, or 0.01% (including ranges between any two of the listed values) of the substance.

Also described herein are methods of ameliorating, inhibiting, treating, or alleviating a symptom of cancer in a patient in need thereof, comprising administering to the patient an effective amount of a pharmaceutical formulation described herein.

Peptide inhibitors

Some embodiments include peptide inhibitors. It has been shown that albumin-derived peptide P3028 (vfdefkplveeppqnlik-SEQ ID NO:3) is sufficient to inhibit immune cell proliferation and activation, and as a "P3028 structure" causes blockade of the immune system (e.g., damaged and/or denatured albumin, which can be identified by antibodies and/or peptides specific for P3028) (see example 1; see also U.S. patent nos. 9,796,77, and PCT publication nos. WO 2015/035332 and WO2016/144650, each of which is incorporated herein by reference in its entirety). Peptide-based binding partners of P3028 were developed. It is shown that peptides comprising, consisting essentially of, and consisting of the amino acid sequence FFVKLS (SEQ ID NO:1), such as P28R (KKLDTFFVKLSLFTER; SEQ ID NO:2), are sufficient to bind peptide P3028 (see examples 10 and 36 of PCT publication WO 2016/144650). Furthermore, peptide inhibitors comprising, consisting essentially of, and consisting of the motif FFVKLS (SEQ ID NO:1), such as P28R (SEQ ID NO:2), are sufficient to mitigate the immunosuppressive effects of P3028 (see examples 1-2), and can mitigate immunosuppression, thereby causing immune system infiltration and tumor destruction in vivo (see examples 3-5).

The peptide inhibitors of the compositions, pharmaceutical products, pharmaceutical formulations, and methods of some embodiments can bind to and inhibit an immunomodulatory peptide, such as P3028 and/or one or more other albumin-derived immunomodulatory peptides (see, e.g., the blocker peptides identified in tables 1-4 of PCT publication No. WO 2016/144650). Peptide inhibitors of some embodiments may include, but are not limited to: peptides, cyclic peptides, peptidomimetics, and proteins, including, for example, synthetic peptides. The following sections provide more details regarding antibody or antibody fragment-based peptide inhibitors.

In the compositions, pharmaceutical products, pharmaceutical formulations and methods of some embodiments, the peptide inhibitor comprises, consists essentially of or consists of the amino acid sequence FFVKLS (SEQ ID NO: 1). In the compositions, pharmaceutical products, pharmaceutical formulations and methods of some embodiments, the peptide inhibitor comprises the amino acid sequence FFVKLS (SEQ ID NO:1) and has a length of NO more than 100 amino acids, e.g., NO more than 100, 90, 80, 70, 60, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7 or 6 amino acid residues, including ranges between any two of the listed values, e.g., 6-100, 6-50, 6-30, 6-29, 6-25, 6-20, 6-15, 6-16, 10-100, 10-50, 1, and 6-25, 10-30, 10-29, 10-25, 10-20, 10-16, 15-100, 15-50, 15-30, 15-29, 15-25, 15-20, or 15-16 amino acid residues. In the compositions, pharmaceutical products, formulations and methods of some embodiments, the peptide inhibitor comprises, consists essentially of or consists of P28R (KKLDTFFVKLSLFTER; SEQ ID NO: 2).

The peptide inhibitors of some embodiments bind to, consist essentially of, or consist of a peptide comprising the amino acid sequence of P3028 (VFDEFLKPLVEEPQNLIK-SEQ ID NO: 3). The peptide inhibitors of some embodiments inhibit the binding of P3028 to LFA-1 receptor, thereby unblocking LFA-1 receptor. Thus, the peptide inhibitors of some embodiments are sufficient to induce activation of immune cells that have been immunosuppressed by damaged or denatured albumin or albumin peptides such as P3028 structures. Examples of immune cell activation include, but are not limited to, proliferation, enhanced expression of CD69 and/or CD71, secretion of IL-12 or IFN γ, or secretion of perforin or granzyme B, enhanced cytotoxicity, cell migration, or cytokine production, or two or more of the listed items. In some embodiments, a pharmaceutical product, pharmaceutical formulation, composition or method comprises an amount of a peptide inhibitor effective to inhibit the binding of P3028 to LFA-1 receptor thereby unblocking LFA-1 receptor. In some embodiments, a pharmaceutical product, pharmaceutical preparation, composition or method comprises an amount of a peptide inhibitor effective to activate immune cells that have been immunosuppressed by damaged or denatured albumin or albumin peptides such as P3028 structures.

Unless expressly stated otherwise, whenever reference is made herein to an "isolated peptide", for example in the context of a pharmaceutical product, formulation, composition or method of some embodiments, it is understood that it means a "peptide inhibitor" as described herein, unless otherwise specified.

Buffer system

As used herein, "buffer" and "buffer system" have their ordinary and customary meaning as would be understood by one of ordinary skill in the art in view of this disclosure. They represent compositions that resist pH changes in solution. Thus, the buffer may facilitate maintaining the composition, pharmaceutical product, and/or pharmaceutical formulation of some embodiments within a specified pH range.

The buffer or buffer system may comprise, consist essentially of, or consist of a weak acid and its conjugate base. K_aDenotes the dissociation constant of the proton of the acid and can be calculated as K_a＝([H⁺][A^-]/[HA]). Buffer capacity at pK of buffer_a(i.e. K)_aNegative logarithm of) is maximum. Thus, the buffer may be selected to have a pK at or near the desired pH or pH range of the buffered material_aFor example, a pK within. + -. 2 of the desired pH_apK within. + -. 1 of the desired pH_aOr pK within. + -. 0.5 of the desired pH_a. It should be noted that some acids may have more than one proton, and thus, the buffer system may have more than one pK_a. In addition to conventional small molecule buffers (such as phosphate, acetate, citrate and borate systems), amino acids represent weak acids, as do the acidic side chains of amino acids, and thus these amino acids and side chains can also provide buffering. Thus, it is contemplated that the acidic side chains of some embodiments (such as Asp, Glu, and His) may facilitate buffering of the peptide inhibitors as described herein, particularly the pK at the acidic side chain_aAt or near the pH range (note, pK of the side chain of Asp, Glu and His)_a3.7, 4.3 and 6.5, respectively).

"buffer capacity" has its ordinary and customary meaning as would be understood by one of ordinary skill in the art in view of this disclosure. It means the amount of strong acid or strong base required to change the pH by one unit. In generalThe buffer capacity is expressed in units of grams or molar equivalents. The buffer capacity may be determined empirically (e.g., by acid and/or base titration), and may also be calculated. For example, equation (I) below may be used to estimate the acid [ HA ] included]And its conjugate base [ A^-]And has K_aThe buffer capacity of the buffer system of dissociation constant (d):

buffer capacity ═ a^-]+([H⁺][A^-]/K_a)(I)

It will be appreciated that the comparison of the buffer capacities (e.g., between the buffer and a reference buffer such as sodium acetate or sodium citrate) is suitably performed under comparable conditions such as temperature and/or pressure. In some embodiments, the buffer capacity is determined at or near room temperature at a pressure of 1 atmosphere. In some embodiments, the buffer capacity is determined at standard temperature and pressure (0 ℃ and 1 atmosphere).

In some embodiments, the composition, pharmaceutical product or formulation comprises a buffer system selected from the group consisting of: tromethamine, N-bis (2-hydroxyethyl) glycine, Tris (hydroxymethyl) methylglycine, MOPS, MOPSO, MOBS, Tris, Hepes, HEPBS, MES, phosphate, carbonate, acetate, citrate, glycolate, lactate, borate, ACES, ADA, tartrate, AMP, AMPD, AMPSO, BES, CABS, dimethylarsinate, CHES, DIPSO, EPPS, ethanolamine, glycine, HEPPSO, imidazole lactic acid, PIPES, SSC, SSPE, POPSO, TAPS, TABS, TAPSO, TES, and the acidic side chains of peptide inhibitors as described herein, including two or more of the listed items. In some embodiments, the composition, pharmaceutical product or pharmaceutical formulation comprises a buffer system selected from the group consisting of: acetate and the acidic side chain of a peptide inhibitor as described herein, or both. In some embodiments, the composition, pharmaceutical product, or pharmaceutical formulation comprises a buffer system comprising acetate and an acidic side chain of a peptide inhibitor as described herein.

Tonicity agent

In order to provide the compositions, pharmaceutical products, or formulations of some embodiments with a suitable tonicity (so as to minimize pain and tissue damage after administration of the composition, pharmaceutical product, or formulation), the compositions, pharmaceutical products, or pharmaceutical formulations may include a tonicity agent. Tonicity agents may be included in a composition, drug product or formulation as described herein such that the osmolality of the composition, drug product or drug formulation is at or near the physiological range. Generally, the osmolarity of human blood is 275-299mOsm/L or about 275-299mOsm/L, for example 275-295mOsm/L, 275-296mOsm/L, 275-297mOsm/L, 280-295mOsm/L, 280-296mOsm/L, 280-297mOsm/L, 281-295mOsm/L, 281-296mOsm/kg or 281-297 mOsm/L.

While various factors (such as dehydration) may affect the osmolarity of the blood of an individual, it should be understood that according to embodiments herein, isotonic osmolality means a osmolarity at or near or within the expected osmolarity range of the patient's blood that is equal to or close to the osmolarity of the patient's blood so as to allow administration thereto while inhibiting discomfort and adverse physiological effects due to differences in osmolarity. If additional numerical precision is of interest, in some embodiments, the osmolarity is within ± 10% of the osmolarity of the patient's blood, such as within ± 10%, ± 9%, ± 8%, ± 7%, ± 6%, ± 5%, ± 4%, ± 3%, ± 2% or ± 1%, including ranges between any two of the listed values. In some embodiments, the osmolality represents about 280-300mOsmol/L or 280-300 mOsmol/L. Thus, it is also understood that "sub-isotonic" osmolarity refers to osmolarity that is numerically lower than the osmolarity, e.g., less than 280mOsm/L, 270mOsm/L, or 260 mOsm/L. It is also understood that osmolarity greater than tonicity (tonic) means osmolarity that is numerically greater than the isotonic osmolarity, e.g., greater than 300, 310, or 320 mOsm/L.

In some embodiments, the composition, drug product, or formulation has an osmolality of about 280-300mOsmol/L or 280-300 mOsmol/L. In some embodiments, the composition, pharmaceutical product or formulation has an osmolarity of 270-280mOsmol/L, 270-290mOsmol/L, 270-300mOsmol/L, 270-310mOsmol/L, 280-290mOsmol/L, 280-300mOsmol/L, 280-310mOsmol/L, 290-300mOsmol/L, 290-310mOsmol/L, or 300-310 mOsmol/L.

Examples of suitable tonicity agents for the compositions, pharmaceutical products or formulations of some embodiments include, but are not limited to, sodium chloride, potassium chloride, glucose, sucrose, dextrose, mannitol, sorbitol, trehalose, glycerol, or a combination of two or more of these. Examples of tonicity agents can also be found in: remington's The Science and Practice of Pharmacy, 21 st edition, Lippincott Williams & Wilkins (2005), and Gilman et al (1990); goodman and Gilman: the pharmaceutical Basis of Therapeutics, 8 th edition, Pergamon Press, each of which is incorporated herein by reference in its entirety.

In some embodiments, the composition, pharmaceutical product, or formulation (e.g., a formulation comprising at least 10mg/ml of a peptide inhibitor, such as formulation C) comprises a non-ionic tonicity agent, such as glucose, sucrose, dextrose, mannitol, glycerol, or a combination of two or more of the listed items. In some embodiments, the tonicity agent of the composition, pharmaceutical product or formulation does not contain sodium. Thus, in some embodiments, the composition, pharmaceutical product or formulation does not contain or substantially does not contain sodium. In some embodiments, the tonicity agent of the composition, pharmaceutical product or formulation does not comprise sodium chloride. Thus, in some embodiments, the composition, pharmaceutical product or formulation is free or substantially free of sodium chloride.

Carrier

The composition, pharmaceutical product or pharmaceutical formulation may be formulated in a suitable carrier. The carrier may be a solvent for some or all of the components of the composition, pharmaceutical product or pharmaceutical formulation. Examples of suitable carriers include, but are not limited to, syrups, elixirs, emulsions and/or suspensions, for example containing, consisting essentially of or consisting of ethanol, glycerol, propylene glycol, polyethylene glycol, liquid sucrose, sorbitol and/or water. In some embodiments, the composition, pharmaceutical product, or formulation is an aqueous solution. In some embodiments, the composition, pharmaceutical product, or pharmaceutical formulation is formulated in a carrier that comprises, consists essentially of, or consists of water. In some embodiments, the composition, pharmaceutical product, or pharmaceutical formulation is formulated in a carrier that is water. In some embodiments, the composition, pharmaceutical product, or pharmaceutical formulation comprises, consists essentially of, or consists of an aqueous formulation.

Pharmaceutical product

In some embodiments, the pharmaceutical product comprises, consists essentially of, or consists of: a formulation of a peptide inhibitor, or a component of a formulation of a peptide inhibitor. The pharmaceutical product of some embodiments comprises a first solution comprising a peptide inhibitor at a pH of less than 7 or about less than 7 (e.g., less than or equal to 7, 6, 5, 4.5, 4, 3.5, or 3) and having a sub-isotonic osmolality. The first solution may be sterile, e.g., by sterile filtration. It has been shown herein that at relatively high pH (such as pH 4.5 or greater), peptide inhibitors can form gels that interfere with sterile filtration and can precipitate out of solution (see examples 10-11). Thus, it is contemplated that a specified acidic pH may be advantageous for maintaining the peptide inhibitor in a sterile filterable solution. The pharmaceutical product may further comprise a second solution comprising a tonicity agent. The first solution and the second solution, when combined, can produce an isotonic gel comprising the peptide inhibitor having a concentration of at least 0.2mg/ml or about at least 0.2mg/ml and a pH of 6 to 8 or about 6 to 8. The peptide inhibitor may comprise, consist essentially of, or consist of a P28 core (FFVKLS; SEQ ID NO: 1). In some embodiments, the first solution is substantially free of particles greater than 0.2 μ M or greater than about 0.2 μ M in diameter. The second solution may be substantially free of particles having a diameter greater than 0.2 μ M or greater than about 0.2 μ M. In some embodiments, the first solution and the second solution, when combined, produce an isotonic gel having a pH of 4.5-7.5, about 4.5-7.5, 6.5-7.5, or about 6.5-7.5. In some embodiments, the first solution and the second solution, when combined, produce an isotonic gel comprising the peptide inhibitor in a concentration of at least 0.2mg/ml or at least about 0.2mg/ml and a pH of 4.5-7.5 or about 4.5-7.5. In some embodiments, the first solution and the second solution, when combined, produce an isotonic gel comprising the peptide inhibitor in a concentration of at least 0.2mg/ml or about at least 0.2mg/ml and a pH of 6.5-7.5 or about 6.5-7.5.

It has been shown herein that increasing the concentration of NaCl can cause the peptide inhibitors as described herein to aggregate and form a gel (example 11). The gel may interfere with sterile filtration. Thus, in some embodiments, the peptide inhibitor is in a liquid, such as a first solution. The first solution can be sterile, such as by sterile filtration through a filter having a pore size of no greater than 0.2 μ M (e.g., 0.2 μ M or less or 0.1 μ M or less). Thus, the gel formed by the combination of the first solution and the second solution may be substantially free of particles having a diameter greater than or equal to the diameter of the filter pores. Thus, in some embodiments, the first solution and the second solution each do not contain particles greater than 0.2 μ M diameter or do not contain particles greater than about 0.2 μ M diameter. In some embodiments, the first solution and the second solution are each substantially free of particles greater than 0.2 μ M in diameter or substantially free of particles greater than about 0.2 μ M in diameter.

In some embodiments, the peptide inhibitor comprises, consists essentially of, or consists of P28R (KKLDTFFVKLSLFTER; SEQ ID NO: 2). In some embodiments, the first solution comprises a peptide inhibitor at a concentration of at least 0.4mg/ml, or at least about 0.4 mg/ml. In some embodiments, the first solution comprises a buffer having a buffer capacity of about 1.5mM sodium acetate or less, or 1.5mM sodium acetate or less, e.g., no more than a buffer capacity of 1.5mM, 1.3mM, 1.2mM, 1mM, 0.9mM, 0.7mM, or 0.5mM sodium acetate. In some embodiments, the buffer comprises, consists essentially of, or consists of sodium acetate (and its conjugate acid). It should be noted that the buffer capacity can be compared to a reference (such as sodium acetate) under the same conditions (such as temperature and pressure). In some embodiments, the buffer capacity is determined at standard temperature and pressure. It is shown herein that sodium (such as sodium acetate, sodium hydroxide and/or sodium chloride) can cause the peptide to form a gel, which can interfere with solubility and sterile filtration (see example 11). Thus, minimizing the sodium content in the peptide-containing solution is expected to facilitate solubility and sterile filtration. In some embodiments, the first solution does not comprise sodium acetate. In some embodiments, the first solution does not comprise a buffer. In some embodiments, the first solution does not comprise NaCl. In some embodiments, the first solution is substantially free of NaCl. In some embodiments, the first solution comprises, consists essentially of, or consists of: less than or equal to 10mM NaCl or less than or equal to about 10mM NaCl, but not zero. In some embodiments, the first solution has the same volume or about the same volume as the second solution.

In some embodiments, the pharmaceutical product comprises, consists essentially of, or consists of the first solution of table 1A and the second solution of table 1B. The first solution and the second solution may be separated from each other. The first solution and the second solution, when combined with each other, can produce a pharmaceutical formulation comprising, consisting essentially of, or consisting of the gel of table 1C. Thus, it is understood that according to some embodiments herein, a pharmaceutical formulation comprises, consists essentially of, or consists of a gel of table 1C.

Table 1A: first solution of some embodiments

Table 1B: second solution of some embodiments

Tonicity agents, such as sodium chloride
	Bases, such as sodium hydroxide
Osmolarity in excess of isotonic
	Optionally, no or substantially no particles greater than 0.2 μ M diameter

Table 1C: gels of some embodiments

In some embodiments, the first solution comprises, consists essentially of, or consists of a solution of table 1A comprising a buffer having a buffer capacity equivalent to about 1.5mM or less sodium acetate buffer. The peptide inhibitor may comprise, consist essentially of, or consist of the amino acid sequence FFVKLS (SEQ ID NO: 1). The first solution may have a pH of less than about 7. The second solution can comprise, consist essentially of, or consist of the solution of table 1B. The first solution and the second solution, when combined with each other, can be configured to produce a gel of Table 1C comprising the peptide inhibitor at a concentration of at least about 0.2mg/ml and having a pH of about 6.5-7.5 and an osmolality of about isotonic, such as about 270-310mOsmol/L or about 280-300 mOsmol/L. In some embodiments, the peptide inhibitor comprises, consists essentially of, or consists of the amino acid sequence of P28R (KKLDTFFVKLSLFTER; SEQ ID NO: 2). In some embodiments, the first solution and the second solution do not contain particles greater than 0.2 μ Μ diameter or do not contain particles greater than about 0.2 μ Μ diameter.

In some embodiments, the first solution comprises, consists essentially of, or consists of a solution of table 1A comprising a buffer having a buffer capacity equivalent to 1.5mM or less sodium acetate buffer. The peptide inhibitor may comprise, consist essentially of, or consist of the amino acid sequence FFVKLS (SEQ ID NO: 1). The first solution may have a pH of less than 7. The second solution can comprise, consist essentially of, or consist of the solution of table 1B. The first solution and the second solution, when combined with each other, can form a gel of Table 1C comprising the peptide inhibitor at a concentration of at least 0.2mg/ml and having a pH of 6.5-7.5 and an osmolality such as 270-310mOsmol/L or 280-300 mOsmol/L. In some embodiments, the peptide inhibitor comprises, consists essentially of, or consists of the amino acid sequence of P28R (KKLDTFFVKLSLFTER; SEQ ID NO: 2). In some embodiments, the first solution and the second solution are free or substantially free of particles greater than 0.2 μ Μ diameter.

In some embodiments, the first solution comprises, consists essentially of, or consists of a solution of table 1A comprising a buffer having a buffer capacity equivalent to about 1.5mM or less sodium acetate buffer. The peptide inhibitor may comprise, consist essentially of, or consist of the amino acid sequence FFVKLS (SEQ ID NO: 1). The first solution may have a pH of less than about 5. The second solution can comprise, consist essentially of, or consist of the solution of table 1B. The first solution and the second solution, when combined with each other, can form a gel of Table 1C comprising the peptide inhibitor at a concentration of at least about 0.2mg/ml and having a pH of about 6.5-7.5 and an osmolality of about, for example, 270-310mOsmol/L or 280-300 mOsmol/L. In some embodiments, the peptide inhibitor comprises, consists essentially of, or consists of the amino acid sequence of P28R (KKLDTFFVKLSLFTER; SEQ ID NO: 2). In some embodiments, the first solution and the second solution are free or substantially free of particles greater than 0.2 μ Μ diameter.

In some embodiments, the first solution comprises, consists essentially of, or consists of a solution of table 1A comprising a buffer having a buffer capacity equivalent to 1.5mM or less sodium acetate buffer. The peptide inhibitor may comprise, consist essentially of, or consist of the amino acid sequence FFVKLS (SEQ ID NO: 1). The first solution may have a pH of less than 5. The second solution can comprise, consist essentially of, or consist of the solution of table 1B. The first solution and the second solution, when combined with each other, can form a gel of Table 1C comprising the peptide inhibitor at a concentration of at least 0.2mg/ml and having a pH of 6.5-7.5 and an osmolality such as 270-310mOsmol/L or 280-300 mOsmol/L. In some embodiments, the peptide inhibitor comprises, consists essentially of, or consists of the amino acid sequence of P28R (KKLDTFFVKLSLFTER; SEQ ID NO: 2). In some embodiments, the first solution and the second solution do not contain particles greater than 0.2 μ Μ diameter or do not contain particles greater than about 0.2 μ Μ diameter.

In some embodiments, the first solution comprises, consists essentially of, or consists of a solution of table 1A comprising a buffer having a buffer capacity equivalent to about 1.5mM or less sodium acetate buffer. The peptide inhibitor may comprise, consist essentially of, or consist of the amino acid sequence FFVKLS (SEQ ID NO: 1). The first solution may have a pH of about 3 to about 4.5. The second solution can comprise, consist essentially of, or consist of the solution of table 1B. The first solution and the second solution, when combined with each other, can form a gel of Table 1C comprising the peptide inhibitor at a concentration of at least about 0.2mg/ml and having a pH of about 6.5-7.5 and an osmolality of about isotonic, such as about 270-310mOsmol/L or about 280-300 mOsmol/L. In some embodiments, the peptide inhibitor comprises, consists essentially of, or consists of the amino acid sequence of P28R (KKLDTFFVKLSLFTER; SEQ ID NO: 2). In some embodiments, the first solution and the second solution do not contain particles greater than 0.2 μ Μ diameter or do not contain particles greater than about 0.2 μ Μ diameter.

In some embodiments, the first solution comprises, consists essentially of, or consists of a solution of table 1A comprising a buffer having a buffer capacity equivalent to 1.5mM or less sodium acetate buffer. The peptide inhibitor may comprise, consist essentially of, or consist of the amino acid sequence FFVKLS (SEQ ID NO: 1). The first solution may have a pH of 3-4.5. The second solution can comprise, consist essentially of, or consist of the solution of table 1B. The first solution and the second solution, when combined with each other, can form a gel of Table 1C comprising the peptide inhibitor at a concentration of at least 0.2mg/ml and having a pH of 6.5-7.5 and an osmolality such as 270-310mOsmol/L or 280-300 mOsmol/L. In some embodiments, the peptide inhibitor comprises, consists essentially of, or consists of the amino acid sequence of P28R (KKLDTFFVKLSLFTER; SEQ ID NO: 2). In some embodiments, the first solution and the second solution do not contain particles greater than 0.2 μ Μ diameter or do not contain particles greater than about 0.2 μ Μ diameter.

In some embodiments, the first solution comprises, consists essentially of, or consists of a solution of table 1A comprising about 1.5mM or less sodium acetate buffer. The peptide inhibitor may comprise, consist essentially of, or consist of the amino acid sequence FFVKLS (SEQ ID NO: 1). The first solution may have a pH of less than about 7. The second solution can comprise, consist essentially of, or consist of the solution of table 1B. The first solution and the second solution, when combined with each other, can form a gel of Table 1C comprising the peptide inhibitor at a concentration of at least about 0.2mg/ml and having a pH of about 6.5-7.5 and an osmolality of about, for example, 270-310mOsmol/L or 280-300 mOsmol/L. In some embodiments, the peptide inhibitor comprises, consists essentially of, or consists of the amino acid sequence of P28R (KKLDTFFVKLSLFTER; SEQ ID NO: 2). In some embodiments, the first solution and the second solution do not contain particles greater than 0.2 μ Μ diameter or do not contain particles greater than about 0.2 μ Μ diameter.

In some embodiments, the first solution comprises, consists essentially of, or consists of a solution of table 1A comprising 1.5mM or less sodium acetate buffer. The peptide inhibitor may comprise, consist essentially of, or consist of the amino acid sequence FFVKLS (SEQ ID NO: 1). The first solution may have a pH of less than 7. The second solution can comprise, consist essentially of, or consist of the solution of table 1B. The first solution and the second solution, when combined with each other, can form a gel of Table 1C comprising the peptide inhibitor at a concentration of at least 0.2mg/ml and having a pH of 6.5-7.5 and an osmolality such as 270-310mOsmol/L or 280-300 mOsmol/L. In some embodiments, the peptide inhibitor comprises, consists essentially of, or consists of the amino acid sequence of P28R (KKLDTFFVKLSLFTER; SEQ ID NO: 2). In some embodiments, the first solution and the second solution do not contain particles greater than 0.2 μ Μ diameter or do not contain particles greater than about 0.2 μ Μ diameter.

In some embodiments, the first solution comprises, consists essentially of, or consists of a solution of table 1A comprising about 1.5mM or less sodium acetate buffer. The peptide inhibitor may comprise, consist essentially of, or consist of the amino acid sequence FFVKLS (SEQ ID NO: 1). The first solution may have a pH of less than about 5. The second solution can comprise, consist essentially of, or consist of the solution of table 1B. The first solution and the second solution, when combined with each other, can form a gel of Table 1C comprising the peptide inhibitor at a concentration of at least about 0.2mg/ml and having a pH of about 6.5-7.5 and an osmolality of about isotonic, such as about 270-310mOsmol/L or about 280-300 mOsmol/L. In some embodiments, the peptide inhibitor comprises, consists essentially of, or consists of the amino acid sequence of P28R (KKLDTFFVKLSLFTER; SEQ ID NO: 2). In some embodiments, the first solution and the second solution do not contain particles greater than 0.2 μ Μ diameter or do not contain particles greater than about 0.2 μ Μ diameter.

In some embodiments, the first solution comprises, consists essentially of, or consists of a solution of table 1A comprising 1.5mM or less sodium acetate buffer. The peptide inhibitor may comprise, consist essentially of, or consist of the amino acid sequence FFVKLS (SEQ ID NO: 1). The first solution may have a pH of less than 5. The second solution can comprise, consist essentially of, or consist of the solution of table 1B. The first solution and the second solution, when combined with each other, can form a gel of Table 1C comprising the peptide inhibitor at a concentration of at least 0.2mg/ml and having a pH of 6.5-7.5 and an osmolality such as 270-310mOsmol/L or 280-300 mOsmol/L. In some embodiments, the peptide inhibitor comprises, consists essentially of, or consists of the amino acid sequence of P28R (KKLDTFFVKLSLFTER; SEQ ID NO: 2). In some embodiments, the first solution and the second solution do not contain particles greater than 0.2 μ Μ diameter or do not contain particles greater than about 0.2 μ Μ diameter.

In some embodiments, the first solution comprises, consists essentially of, or consists of a solution of table 1A comprising about 1.5mM or less sodium acetate buffer. The peptide inhibitor may comprise, consist essentially of, or consist of the amino acid sequence FFVKLS (SEQ ID NO: 1). The first solution may have a pH of about 3.5-4.5. The second solution can comprise, consist essentially of, or consist of the solution of table 1B. The first solution and the second solution, when combined with each other, can form a gel of Table 1C comprising the peptide inhibitor at a concentration of at least about 0.2mg/ml and having a pH of about 6.5-7.5 and an osmolality of about, for example, 270-310mOsmol/L or 280-300 mOsmol/L. In some embodiments, the peptide inhibitor comprises, consists essentially of, or consists of the amino acid sequence of P28R (KKLDTFFVKLSLFTER; SEQ ID NO: 2). In some embodiments, the first solution and the second solution do not contain particles greater than 0.2 μ Μ diameter or do not contain particles greater than about 0.2 μ Μ diameter.

In some embodiments, the first solution comprises, consists essentially of, or consists of the solution of table 1A, and comprises 1.5mM or less sodium acetate buffer. The peptide inhibitor may comprise, consist essentially of, or consist of the amino acid sequence FFVKLS (SEQ ID NO: 1). The first solution may have a pH of 3.5-4.5. The second solution can comprise, consist essentially of, or consist of the solution of table 1B. The first solution and the second solution, when combined with each other, can form a gel of Table 1C comprising the peptide inhibitor at a concentration of at least 0.2mg/ml and having a pH of 6.5-7.5 and an osmolality such as 270-310mOsmol/L or 280-300 mOsmol/L. In some embodiments, the peptide inhibitor comprises, consists essentially of, or consists of the amino acid sequence of P28R (KKLDTFFVKLSLFTER; SEQ ID NO: 2). In some embodiments, the first solution and the second solution do not contain particles greater than 0.2 μ Μ diameter or do not contain particles greater than about 0.2 μ Μ diameter.

In some embodiments, the first solution comprises, consists essentially of, or consists of a solution of table 1A and comprises a buffer having a buffer capacity equivalent to about 1.5mM or less sodium acetate buffer. The peptide inhibitor may comprise, consist essentially of, or consist of the amino acid sequence of P28R (KKLDTFFVKLSLFTER; SEQ ID NO: 2). The first solution may have a pH of less than about 5, such as less than about 5, 4.5, 4, 3.5, or 3, including ranges between any two of the listed values, such as a pH of about 3-5, about 3.5-5, about 4-5, about 4.5-5, about 3-4.5, about 3.5-4.5, about 4-4.5, about 3-4, or about 3.5-4. The second solution can comprise, consist essentially of, or consist of the solution of table 1B. The first solution and the second solution, when combined with each other, can form a gel of Table 1C comprising the peptide inhibitor at a concentration of at least 0.2mg/ml (such as at least 0.4mg/ml) and having a pH of about 6.5-7.5 and an osmolarity such as 270-310mOsmol/L or 280-300 mOsmol/L. In some embodiments, the first solution and the second solution do not contain particles greater than 0.2 μ Μ diameter or do not contain particles greater than about 0.2 μ Μ diameter.

In some embodiments, the first solution comprises, consists essentially of, or consists of a solution of table 1A and comprises a buffer having a buffer capacity equivalent to 1.5mM or less sodium acetate buffer. The peptide inhibitor may comprise, consist essentially of, or consist of the amino acid sequence of P28R (KKLDTFFVKLSLFTER; SEQ ID NO: 2). The first solution may have a pH of less than about 5, such as less than 5, 4.5, 4, 3.5, or 3, including ranges between any two of the listed values, such as a pH of 3-5, 3.5-5, 4-5, 4.5-5, 3-4.5, 3.5-4.5, 4-4.5, 3-4, or 3.5-4. The second solution can comprise, consist essentially of, or consist of the solution of table 1B. The first solution and the second solution, when combined with each other, can form a gel of Table 1C comprising the peptide inhibitor at a concentration of at least 0.2mg/ml (such as at least 0.4mg/ml) and having a pH of 6.5-7.5 and an osmolality such as 270-310mOsmol/L or 280-300 mOsmol/L. In some embodiments, the first solution and the second solution do not contain particles greater than 0.2 μ Μ diameter or do not contain particles greater than about 0.2 μ Μ diameter.

In some embodiments, the first solution comprises, consists essentially of, or consists of the solution of table 1A and comprises about 1.5mM or less sodium acetate buffer. The peptide inhibitor may comprise, consist essentially of, or consist of the amino acid sequence of P28R (KKLDTFFVKLSLFTER; SEQ ID NO: 2). The first solution may have a pH of less than about 5, such as less than about 5, 4.5, 4, 3.5, or 3, including ranges between any two of the listed values, such as a pH of about 3-5, about 3.5-5, about 4-5, about 4.5-5, about 3-4.5, about 3.5-4.5, about 4-4.5, about 3-4, or about 3.5-4. The second solution can comprise, consist essentially of, or consist of the solution of table 1B. The first solution and the second solution, when combined with each other, can form a gel of Table 1C comprising the peptide inhibitor at a concentration of at least about 0.2mg/ml (such as at least about 0.4mg/ml) and having a pH of about 6.5-7.5 and an osmolarity such as 270-310mOsmol/L or 280-300 mOsmol/L. In some embodiments, the first solution and the second solution do not contain particles greater than 0.2 μ Μ diameter or do not contain particles greater than about 0.2 μ Μ diameter.

In some embodiments, the first solution comprises, consists essentially of, or consists of a solution of table 1A and comprises 1-1.5mM sodium acetate buffer. The peptide inhibitor may comprise, consist essentially of, or consist of the amino acid sequence of P28R (KKLDTFFVKLSLFTER; SEQ ID NO: 2). The first solution may have a pH of less than about 5, such as less than 5, 4.5, 4, 3.5, or 3, including ranges between any two of the listed values, such as a pH of 3-5, 3.5-5, 4-5, 4.5-5, 3-4.5, 3.5-4.5, 4-4.5, 3-4, or 3.5-4. The second solution can comprise, consist essentially of, or consist of the solution of table 1B. The first solution and the second solution, when combined with each other, can form a gel of Table 1C comprising the peptide inhibitor at a concentration of at least 0.2mg/ml (such as at least 0.4mg/ml) and having a pH of 6.5-7.5 and an osmolality such as 270-310mOsmol/L or 280-300 mOsmol/L. In some embodiments, the first solution and the second solution do not contain particles greater than 0.2 μ Μ diameter or do not contain particles greater than about 0.2 μ Μ diameter.

In some embodiments, the first solution comprises, consists essentially of, or consists of a solution of table 1A and comprises a buffer having a buffer capacity equivalent to about 1.5mM sodium acetate buffer. The peptide inhibitor may have the amino acid sequence of P28R (KKLDTFFVKLSLFTER; SEQ ID NO: 2). The first solution may have a pH of 3-4.5. The second solution can comprise, consist essentially of, or consist of the solution of table 1B. The first solution and the second solution, when combined with each other, can form a gel of Table 1C comprising the peptide inhibitor at a concentration of at least 0.2mg/ml (such as at least about 0.4mg/ml) and having a pH of 6.5-7.5 and an osmolality such as 270-310mOsmol/L or 280-300 mOsmol/L. In some embodiments, the first solution may comprise no more than about 10mM NaCl. The tonicity agent of the second solution may comprise NaCl. Without being limited by theory, it is contemplated that by providing NaCl in the second solution (for tonicity and/or other purposes), sodium in the first solution may be minimized, thereby minimizing gel formation of the peptide inhibitor in the first solution. In some embodiments, the tonicity agent of the second solution is NaCl, and the gel of table 1C comprises 100mM to 120mM NaCl, or about 100mM to 120mM NaCl. In some embodiments, the first solution is free or substantially free of gel. In some embodiments, the peptide inhibitor in the first solution is not in a beta-sheet conformation, or is substantially free of peptide inhibitor in a beta-sheet conformation. In some embodiments, the first solution and the second solution do not contain particles greater than 0.2 μ Μ diameter or do not contain particles greater than about 0.2 μ Μ diameter.

In some embodiments, for any of the pharmaceutical products described herein, the first solution comprises less than or equal to about 10mM NaCl, e.g., less than or equal to about 10mM NaCl, but non-zero, e.g., less than or equal to about 10, 9, 8, 7, 56, 5, 4, 3, 2, 1, or 0.1mM NaCl, including ranges between any two of the listed values, e.g., about 0.1-10mM NaCl, about 0.1-7mM NaCl, about 1-10mM NaCl, about 5-7mM NaCl, or about 5-10mM NaCl. In some embodiments, for any of the pharmaceutical products described herein, the first solution comprises less than or equal to 10mM NaCl, e.g., less than or equal to 10, 9, 8, 7, 56, 5, 4, 3, 2, 1, or 0.1mM NaCl, including ranges between any two of the listed values, e.g., 0.1-10mM NaCl, 0.1-7mM NaCl, 1-10mM NaCl, 5-7mM NaCl, or 5-10mM NaCl. In some embodiments, for any of the pharmaceutical products described herein, the first solution is substantially free of NaCl. In some embodiments, for any of the pharmaceutical products described herein, the first solution does not comprise NaCl.

In some embodiments, for any of the pharmaceutical products described herein, the first solution comprises a buffer having a buffer capacity equivalent to 1.5mM or less, or about 1.5mM sodium acetate or less but non-zero, e.g., less than or equal to 1.5mM, 1.3mM, 1.1mM, 1mM, 0.7mM, or 0.5mM sodium acetate (but non-zero), including ranges between any two of the listed values. In some embodiments, for any of the pharmaceutical products described herein, the first solution does not comprise a buffer. For example, in some embodiments, the first solution of table 1A does not comprise a buffer or is substantially free of a buffer. In some embodiments, for any of the pharmaceutical products described herein, the first solution is substantially free of buffer. It should be noted that a pharmaceutical product that does not contain or substantially does not contain a buffer may have a buffering capacity attributable to the peptide inhibitor itself, but does not contain or substantially does not contain a small molecule buffering agent such as sodium acetate.

In some embodiments, for any of the pharmaceutical products described herein, the first solution comprises sodium acetate at a concentration of 1.5mM or less or about 1.5mM or less but not zero, e.g., less than or equal to 1.5mM, 1.3mM, 1.1mM, 1mM, 0.7mM, or 0.5mM sodium acetate (but not zero), including ranges between any two of the listed values. In some embodiments, for any of the pharmaceutical products described herein, the first solution does not comprise sodium acetate. In some embodiments, for any of the pharmaceutical products described herein, the first solution is substantially free of sodium acetate.

In some embodiments, for any of the pharmaceutical products or formulations described herein, the tonicity agent comprises, consists essentially of, or consists of NaCl. In some embodiments, the second solution is in a gel configuration to comprise 100mM-120mM NaCl or about 100mM-120mM NaCl. In some embodiments, the second solution is in a gel formulation to comprise 90mM-130mM NaCl, about 90mM-130mM NaCl, 110mM-120mM NaCl, about 110mM-120mM NaCl, 100mM-110mM NaCl, or about 100mM-110mM NaCl. In some embodiments, for any of the pharmaceutical products described herein, the first solution does not comprise NaCl. In some embodiments, a pharmaceutical product or pharmaceutical formulation as described herein is substantially free of NaCl. In some embodiments, the pharmaceutical product or pharmaceutical formulation does not comprise NaCl. Thus, the tonicity agent of the second solution may be a tonicity agent other than NaCl, such as a non-ionic toxic agent.

In some embodiments, for any of the pharmaceutical products described herein, the first solution is free of a gel, or substantially free of a gel. In some embodiments, less than 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1% of the peptide inhibitor of the first solution is in a gel. In some embodiments, for any of the pharmaceutical products described herein, the peptide inhibitor is dissolved in the first solution. In some embodiments, for any of the pharmaceutical products described herein, the peptide inhibitor is dissolved in the first solution and is not substantially in a β -sheet conformation or in a β -sheet conformation. In some embodiments, for any of the pharmaceutical products described herein, the peptide inhibitor is dissolved in the first solution and less than 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1% of the peptide inhibitor of the first solution is in a beta-sheet conformation.

In some embodiments, for any of the pharmaceutical products described herein, the first solution is a gel formulation of a pharmaceutical formulation to comprise a peptide inhibitor at a concentration of at least 0.4mg/ml or at least about 0.4 mg/ml. In some embodiments, for any of the pharmaceutical products described herein, the first solution is a gel configured to contain at least 0.4mg/ml, such as at least 0.4, 0.5, 0.6, 0.8, or 1mg/ml, of the peptide inhibitor at a concentration that includes a range between any two of the listed values.

In some embodiments, for any of the pharmaceutical products or pharmaceutical formulations described herein, the peptide inhibitor comprises no more than 30 amino acid residues, e.g., no more than 30, 29, 28, 27, 25, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, or 10 amino acid residues, including ranges between any two of the listed values.

In some embodiments, for any of the pharmaceutical products or pharmaceutical formulations described herein, the peptide inhibitor comprises amino acid sequence KKLDTFFVKLSLFTER (SEQ ID NO: 2). In some embodiments, for any of the pharmaceutical products described herein, the peptide inhibitor consists of amino acid sequence KKLDTFFVKLSLFTER (SEQ ID NO: 2).

In some embodiments, the first solution is capable of maintaining at least 95% or at least about 95% of the peptide inhibitor dissolved in the first solution at 5 ℃ for at least 12-25 months or at least about 12-25 months, for example when at least 95%, 96%, 97%, 98% or 99% of the peptide inhibitor is dissolved. In some embodiments, the first solution is capable of solubilizing at least 95% or at least about 95% of the peptide inhibitor solubilized in the first solution for at least 12-19 months or at least about 12-19 months, e.g., within at least 95%, 96%, 97%, 98%, or 99% of the peptide inhibitor solubilized at 5 ℃.

In some embodiments, the first solution and the second solution are gel configured to comprise at least 0.4mg/ml or at least about 0.4mg/ml of a peptide inhibitor consisting of amino acid sequence KKLDTFFVKLSLFTER (SEQ ID NO: 2). The first solution and the second solution may further be in a gel configuration to comprise: 30-40mM or at least about 30-40mM acetic acid; 1.2 to 1.6mM or at least about 1.2 to 1.6mM sodium acetate; less than or equal to 30mM or less than or equal to about 30mM sodium hydroxide; and 100-. The gel may have an osmolarity of 280-300mOSmol/L or about 280-300 mOSmol/L.

Pharmaceutical preparation

Gel formulations

In some embodiments, pharmaceutical formulations are described. The pharmaceutical formulation may comprise, consist essentially of, or consist of a gel (e.g., the gel of table 1C). The gel may comprise, consist essentially of, or consist of at least 0.4mg/ml or at least about 0.4mg/ml of an isolated peptide comprising, consisting essentially of the amino acid sequence FFVKLS (SEQ ID NO: 1). The gel may comprise a buffer system comprising acetic acid and sodium acetate, the buffer system comprising less than or equal to 1.6mM or less than or equal to about 1.6mM sodium acetate. The gel may comprise a tonicity agent. The gel may be isotonic and/or may have a pH of 4.5-7.5 or about 4.5-7.5. In some embodiments, the pharmaceutical formulation is for medical use. In some embodiments, the pharmaceutical formulation is for ameliorating, inhibiting, treating, or alleviating a symptom of a cancer as described herein.

As noted herein, although the gel may interfere with sterile filtration, the precursor solution may be sterile filtered, for example, through a filter having a pore size of 0.2 μm or less, e.g., 0.2 μm or less, or 0.1 μm or less. Thus, in some embodiments, the gel is substantially free of particles greater than 0.2 μ M in diameter or greater than about 0.2 μ M in diameter. In some embodiments, the gel does not contain particles greater than 0.2 μ M or greater than about 0.2 μ M in diameter. In some embodiments, the gel does not contain particles greater than 0.1 μ M or greater than about 0.1 μ M in diameter.

In some embodiments, for any pharmaceutical formulation comprising a gel as described herein, the tonicity agent of the gel comprises, consists essentially of, or consists of NaCl. The gel may comprise a tonicity agent (NaCl) at a concentration of 100-120mM or about 100-120 mM. In some embodiments, the gel comprises NaCl at a concentration of no more than 50, 60, 70, 80, 90, 100, 105, 110, 115, 120, 130, 140, or 150mM, including ranges between any two of the listed values, e.g., NaCl at a concentration of 50-150 mM. In some embodiments, the gel comprises NaCl at a concentration of 105-115mM or about 105-115 mM. In some embodiments, the gel comprises NaCl at a concentration of about 110 mM.

In some embodiments, for any pharmaceutical formulation comprising a gel as described herein, the buffer capacity of the buffer system does not exceed the buffer capacity of a 20mM sodium acetate buffer, e.g., does not exceed 20, 15, 10, 5, 4, 3, 2, or 1mM sodium acetate, including ranges between any two of the listed values, e.g., the buffer capacity of a 1-2mM, 1-5mM, 1-10mM, 1-20mM, 2-5mM, 2-10mM, 2-20mM, 5-10mM, or 5-20mM sodium acetate buffer. In some embodiments, the buffer system comprises 30-40mM acetic acid and 1.2-1.6mM sodium acetate, or about 30-40mM acetic acid and about 1.2-1.6mM sodium acetate. In some embodiments, the buffer system does not comprise sodium acetate. In some embodiments, the buffer system is substantially free of sodium acetate. In some embodiments, any of the pharmaceutical formulations as described herein are substantially free of buffer. In some embodiments, any pharmaceutical formulation as described herein does not comprise a buffer.

In some embodiments, any of the pharmaceutical formulations described herein comprise sodium hydroxide. The sodium hydroxide can be at a concentration of less than or equal to 30mM or less than or equal to about 30mM but non-zero, e.g., less than or equal to 30mM, 25mM, 20mM, 15mM, 10mM, or 1mM, including ranges between any two of the listed values.

In some embodiments, for any pharmaceutical formulation comprising a gel as described herein, the gel has a tonicity osmolality (tonic osmolarity). In some embodiments, the gel has an osmolarity of 270-.

In some embodiments, for any pharmaceutical formulation comprising a gel as described herein, the peptide inhibitor comprises no more than 30 amino acid residues, e.g., no more than 30, 29, 28, 27, 25, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, or 10 amino acid residues, including ranges between any two of the listed values.

In some embodiments, for any pharmaceutical formulation comprising a gel as described herein, the peptide inhibitor comprises amino acid sequence KKLDTFFVKLSLFTER (SEQ ID NO: 2). In some embodiments, for any of the pharmaceutical products described herein, the peptide inhibitor consists of amino acid sequence KKLDTFFVKLSLFTER (SEQ ID NO: 2).

Single solution pharmaceutical formulation

In some embodiments, the pharmaceutical formulation comprises, consists essentially of, or consists of a liquid. It has been shown herein that peptide inhibitors can be formulated at relatively high concentrations (e.g., up to about 10mg/ml) (see example 14). Without being limited by theory, it is expected that although sodium and/or a pH above 4.5 may cause precipitation of the peptide inhibitor (see examples 10-11), in higher concentration formulations the peptide inhibitor itself may act as a weak buffer (e.g., due to interactions between acid side chains on the peptide inhibitor), thus allowing for low levels of sodium. The isolated peptide of the pharmaceutical formulation may comprise the amino acid sequence FFVKLS (SEQ ID NO: 1). The isolated peptide may be dissolved in the pharmaceutical formulation at a concentration of 0.2-20mg/ml or about 0.2-20 mg/ml. The pharmaceutical formulation may further comprise a non-ionic tonicity agent. The pharmaceutical formulation may be isotonic. In some embodiments, the pharmaceutical formulation is isotonic and has a pH of 5.0-5.5 or about 5.0-5.5. In some embodiments, the pharmaceutical formulation is for medical use. In some embodiments, the pharmaceutical formulation is for ameliorating, inhibiting, treating, or alleviating a symptom of a cancer as described herein.

In some embodiments, for any pharmaceutical formulation described herein, the pharmaceutical composition is free or substantially free of particles having a diameter greater than 0.2 μ Μ or greater than about 0.2 μ Μ, e.g., particles having a diameter greater than 0.1 μ Μ.

In some embodiments, for any of the pharmaceutical formulations described herein, the pharmaceutical formulation further comprises a weak acid. The peptide and weak acid may constitute a buffer system that maintains the pharmaceutical formulation at a pH of 4.5-5, e.g., a pH of about 4.5-5, 4.5-5.5, 4.5-6, 4.5-6.5, 4.5-7, 5-5.5, 5-6, 5-6.5, 5-7, 5.5-6, 5.5-6.5, 5.5-7, 6-6.5, or 6-7.

In some embodiments, for any pharmaceutical formulation described herein, the weak acid is acetic acid, which is present at a concentration of no more than 0.02M or about 0.02M, e.g., no more than 0.02M, about 0.02M, 0.01M, about 0.01M, 0.005M, or about 0.005M.

In some embodiments, for any pharmaceutical formulation described herein, the non-ionic tonicity agent is dextrose. In some embodiments, the non-ionic tonicity agent is glucose and is present in a concentration effective to provide tonicity to the pharmaceutical formulation. In some embodiments, the non-ionic tonicity agent is glucose and is present at a concentration of 0.1 to 0.5M, about 0.1 to 0.5M, 0.2 to 0.4M, about 0.2 to 0.4M, 0.2 to 0.3M, about 0.2 to 0.3M, 0.3 to 0.4M, or about 0.3 to 0.4M.

In some embodiments, for any pharmaceutical formulation comprising, consisting essentially of, or consisting of a liquid as described herein, the isolated peptide has a concentration of 0.2-5mg/ml, about 0.2-5mg/ml, 0.2-10mg/ml, about 0.2-10mg/ml, 0.2-20mg/ml, about 0.2-20mg/ml, 1-5mg/ml, about 1-5mg/ml, 1-10mg/ml, about 1-10mg/ml, 1-20mg/ml, about 1-20mg/ml, 5-10mg/ml, about 5-10mg/ml, 5-20mg/ml, or about 5-20 mg/ml. In some embodiments, the isolated peptide is at a concentration of 10mg/ml or less, e.g., at a concentration of no more than 10mg/ml, about 10mg/ml, 5mg/ml, about 5mg/ml, 1mg/ml, or about 1 mg/ml.

In some embodiments, for any pharmaceutical formulation as described herein, the peptide inhibitor comprises no more than 30 amino acid residues, e.g., no more than 30, 29, 28, 27, 25, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, or 10 amino acid residues, including ranges between any two of the listed values.

In some embodiments, for any pharmaceutical formulation comprising, consisting essentially of, or consisting of a liquid as described herein, the peptide inhibitor comprises amino acid sequence KKLDTFFVKLSLFTER (SEQ ID NO: 2). In some embodiments, for any pharmaceutical formulation comprising, consisting essentially of, or consisting of a liquid as described herein, the peptide inhibitor consists of amino acid sequence KKLDTFFVKLSLFTER (SEQ ID NO: 2).

In some embodiments, any pharmaceutical formulation comprising, consisting essentially of, or consisting of a liquid as described herein further comprises 0.01M or about 0.01M acetic acid. The isolated peptide consists of amino acid sequence KKLDTFFVKLSLFTER (SEQ ID NO: 2). The peptide inhibitor may be present at a concentration of 1-10mg/ml or about 1-10 mg/ml. The non-ionic tonicity agent may be glucose, which may be present at a concentration of 0.2 to 0.4M or about 0.2 to 0.4M. The pharmaceutical preparation may be isotonic as described herein, for example having an osmolarity of about 280-300mOsm/L or 280-300 mOsm/L.

Method for producing pharmaceutical preparations

Some embodiments include methods of preparing a pharmaceutical formulation from a pharmaceutical product as described herein. The method can include combining a first solution as described herein (e.g., the first solution of table 1A) with a second solution as described herein (e.g., the second solution of table 1B) to form a gel (such as the gel of table 1C). The pharmaceutical formulation may comprise, consist essentially of, or consist of a gel.

Method for preparing a pharmaceutical product

In some embodiments, methods of making a pharmaceutical product are described. The method can include sterile filtering a solution comprising a peptide inhibitor comprising the amino acid sequence FFVKLS (SEQ ID NO:1) as described herein. The peptide inhibitor may be dissolved in the precursor solution at a concentration of at least 0.2mg/ml, 0.3mg/ml, 0.4mg/ml, 0.5mg/ml, 0.6mg/ml, 0.7ml/ml, 0.8mg/ml, 0.9mg/ml or 1mg/ml (including ranges between any two of the listed values) or at least about 0.2mg/ml, 0.3mg/ml, 0.4mg/ml, 0.5mg/ml, 0.6mg/ml, 0.7ml/ml, 0.8mg/ml, 0.9mg/ml or 1mg/ml (including ranges between any two of the listed values). It will be appreciated that high pH and/or high sodium may cause the peptide inhibitors as described herein to form a gel (see examples 10-11). Thus, it will be understood that in some embodiments herein, the peptide inhibitor is dissolved in a precursor solution (such as the solution of table 1A) selected to avoid gel formation prior to sterile filtration, e.g., such a solution: comprising an acidic pH such as less than about 5, 4.5, 4, 3.5, 3, 2.5, or 2 or a range between any two of the listed values, e.g., 2-3, 2-3.5, 2-4, 2-4.5, 2-5, 3-3.5, 3-4, 3-4.5, 3-5, 3.5-4, 3.5-4.5, 3.5-5, 4-4.5, or 4-5; and less than or equal to 10mM NaCl or less than or equal to about 10mM NaCl; and less than or equal to 1.5mM NaCl or less than or equal to about 1.5mM NaCl or less but non-zero. In some embodiments, the pH, NaCl, and sodium acetate content of the solution is such that the peptide inhibitor remains dissolved in the precursor solution, and the precursor solution is free or substantially free of gel.

In some embodiments, the precursor solution comprising the peptide inhibitor is sterile filtered with a filter having a pore size of about 0.2 μ Μ or 0.2 μ Μ. It will be appreciated that at a pH greater than 4.5, or in the presence of sodium chloride levels (see examples 10-11), the peptide inhibitor may form a gel which interferes with sterile filtration. It is further understood that the sterile-filtered precursor solution will be free or substantially free of particles having a diameter of at least about the pore size of the filter. For example, it will be appreciated that a precursor solution comprising a peptide inhibitor sterile filtered with a filter having a pore size of 0.2 μ M will be free or substantially free of particles having a diameter greater than 0.2 μ M. In some embodiments, a precursor solution comprising a peptide inhibitor is sterile filtered with a filter having a pore size of no more than 1 μ M, about 1 μ M, 0.5 μ M, about 0.5 μ M, 0.4 μ M, about 0.4 μ M, 0.3 μ M, about 0.3 μ M, 0.2 μ M, about 0.2 μ M, 0.1 μ M, or about 0.1 μ M (including ranges between any two of the listed values).

It has been shown that at high pH, peptide inhibitors can form gels (example 11). In the method of some embodiments, the precursor solution has an acidic pH. The pH may be sufficient to inhibit gel formation of the peptide inhibitor. In the method of some embodiments, the precursor solution has a pH of less than about 5, 4.5, 4, 3.5, 3, 2.5, or 2, including ranges between any two of the listed values, e.g., a pH of 2-3, 2-3.5, 2-4, 2-4.5, 2-5, 3-3.5, 3-4, 3-4.5, 3-5, 3.5-4, 3.5-4.5, 3.5-5, 4-4.5, or 4-5.

In the methods of some embodiments, the peptide inhibitor comprises no more than 30 amino acid residues, e.g., no more than 30, 29, 28, 27, 25, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, or 10 amino acid residues, including ranges between any two of the listed values. In the methods of some embodiments, the peptide inhibitor comprises amino acid sequence KKLDTFFVKLSLFTER (SEQ ID NO: 2). In the methods of some embodiments, the peptide inhibitor consists of amino acid sequence KKLDTFFVKLSLFTER (SEQ ID NO: 2).

Methods of ameliorating, inhibiting, treating, or alleviating symptoms of cancer

In some embodiments, methods of ameliorating, inhibiting, treating, or alleviating a symptom of cancer in a patient in need thereof are described. The method may comprise administering to the patient an effective amount of a pharmaceutical formulation as described herein. It is to be understood that whenever a method of ameliorating, inhibiting, treating, or alleviating a symptom of cancer (and the method includes a pharmaceutical formulation) is described herein, the corresponding pharmaceutical formulation for ameliorating, inhibiting, treating, or alleviating a symptom of cancer is also expressly contemplated. A "patient" in need of amelioration, inhibition, treatment, or symptom relief from cancer may also be referred to herein as a "subject".

In the methods of some embodiments, the effective amount of the pharmaceutical formulation refers to the amount of the pharmaceutical formulation that comprises a sufficient amount of the peptide inhibitor to ameliorate, inhibit, treat or reduce a symptom of the cancer in the patient. In the methods of some embodiments, the effective amount of the pharmaceutical formulation comprises an amount of the peptide inhibitor sufficient to cause immune cell infiltration of the tumor. In the methods of some embodiments, the effective amount of the pharmaceutical formulation comprises an amount of the peptide inhibitor sufficient to cause cell death in the tumor. In the methods of some embodiments, the effective amount of the pharmaceutical formulation comprises at most 5mg/kg of the peptide inhibitor, e.g., at most 0.01, 0.02, 0.05, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, or 5mg/kg, including ranges between any two of the listed values. In the methods of some embodiments, the effective amount of the pharmaceutical formulation comprises up to about 5mg/kg or 1mg/kg of the peptide inhibitor. In methods of some embodiments, the effective amount comprises up to 2000 μ g or up to about 2000 μ g of the peptide inhibitor, e.g., up to 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, or 2000 μ g, including ranges between any two of the listed values, e.g., 1-50 μ g, 1-80 μ g, 1-100 μ g, 1-500 μ g, 1-800 μ g, 1-1000 μ g, 1-2000 μ g, 8-50 μ g, 8-80 μ g, 8-100 μ g, 8-500 μ g, 8-800 μ g, 8-1000 μ g, 8-2000 μ g, 10-50 μ g, 10-80 μ g, 10-100 μ g, 10-500 μ g, 10-800 μ g, 10-1000 μ g, 10-2000 μ g, 50-80 μ g, 50-100 μ g, 50-500 μ g, 50-800 μ g, 50-1000 μ g, 50-2000 μ g, 100-800 μ g, 100-1000 μ g, 100-2000 μ g, 500-800 μ g, 500-1000 μ g or 500-2000 μ g peptide inhibitors. In the methods of some embodiments, the effective amount of the pharmaceutical formulation comprises 8-800 μ g or about 8-800 μ g of the peptide inhibitor. In the methods of some embodiments, the effective amount of the pharmaceutical formulation comprises 60-100 μ g or about 60-100 μ g of the peptide inhibitor.

Any number of suitable routes of administration may be used in the methods of some embodiments herein. In some embodiments, the pharmaceutical formulation is administered intratumorally, subcutaneously, lymphomically, and/or to the interstitial fluid of a patient.

In the methods of some embodiments, the administration of the pharmaceutical formulation is repeated. The administration can be performed at least 2, 3, 4, 5, 6,7, 8, 9, 10, 15, 20, 30, 40, or 50 times, including ranges between any two of the listed values. In some embodiments, administration of the pharmaceutical formulation is repeated over a period of time, e.g., at least every 1, 2, 3, 4, 5, 6,7, 8, 9, or 10 weeks.

In the methods of some embodiments, the cancer comprises, consists essentially of, or consists of a tumor. In the methods of some embodiments, the cancer is selected from: head and neck cancer, breast cancer, kidney cancer, colorectal cancer, skin cancer, ovarian cancer, prostate cancer, pancreatic cancer, lung cancer, malignant melanoma, small cell lung cancer, non-small cell lung cancer (adenocarcinoma), squamous cell carcinoma, bladder cancer, osteosarcoma, bronchial cancer and/or hematopoietic cell cancer. In some embodiments, the patient has two or more of the listed cancers.

It is understood that the peptide inhibitors of some embodiments may alleviate immunosuppression associated with P3028 structures (see examples 1-3). Thus, in the methods of some embodiments, the patient is selected to have P3028 structure, and/or immunosuppression of P3028 structure. It is envisioned that this selection may identify the patient as a candidate for treatment with a pharmaceutical composition comprising a peptide inhibitor as described herein (see example 7). In some embodiments, the methods comprise detecting the presence and/or level of peptide P3028(SEQ ID NO:3) or a P3028 structure such as damaged or denatured albumin in a sample (such as a biopsy of hematopoietic tissue, body fluid, blood sample, tumor biopsy, or peritumoral tissue) of a patient. In some embodiments, the methods comprise detecting the presence and/or level of a P3028 structure, such as damaged or denatured albumin, in a sample (such as a hematopoietic tissue, a bodily fluid, a blood sample, a tumor biopsy sample, or a biopsy of peri-tumor tissue) of the patient. If the patient comprises the presence and/or level of peptide P3028(SEQ ID NO:3) or the P3028 structure that is greater than the corresponding value of a healthy (non-immunosuppressive) control, the patient may be selected for treatment with a pharmaceutical composition comprising a peptide inhibitor. In some embodiments, the method comprises receiving the results of a detection of the presence and/or level of peptide P3028(SEQ ID NO:3) or a P3028 structure such as damaged or denatured albumin in a sample of the patient, such as a sample comprising hematopoietic tissue, a bodily fluid, a blood sample, a tumor biopsy sample, or a biopsy of peritumoral tissue. In some embodiments, the method comprises receiving results of a detection of the presence and/or level of a P3028 structure, such as damaged or denatured albumin, in a sample of the patient (such as a sample comprising hematopoietic tissue, a bodily fluid, a blood sample, a tumor biopsy, or a biopsy of peritumoral tissue). If the patient comprises the presence and/or level of peptide P3028(SEQ ID NO:3) or the P3028 structure that is greater than the corresponding value of a healthy (non-immunosuppressive) control, the patient may be selected for treatment with a pharmaceutical composition comprising a peptide inhibitor. In some embodiments, the sample comprises a hematopoietic tissue, a blood sample, a tumor biopsy sample, or a biopsy of peritumoral tissue.

It has further been observed that the presence of immune cells in a tumor can indicate a reduction in immunosuppression and an immune response to the tumor (see, e.g., examples 4-5). Thus, detection of the presence and/or level of immune cells in a tumor of a patient in the methods of some embodiments can indicate that the pharmaceutical formulation is effective to ameliorate, inhibit, treat, or reduce a symptom of cancer in the patient. In some embodiments, the method comprises detecting the presence of immune cells in a sample (such as a tumor biopsy sample) of the patient. In some embodiments, the method includes receiving results of a detection of the presence of immune cells in a sample (such as a tumor biopsy sample) of the patient. A sample of a patient, such as a tumor biopsy sample, may have been collected from the patient at least 5 days (e.g., at least 5, 6,7, 8, 9, 10, 15, or 20 days after administration of a pharmaceutical composition as described herein.

It has been observed that some tumor microenvironments contain a substantial presence of P3028 structures and a loss of immune cells (e.g., "immune deserts" and "immune-depleted" cancers; see example 7). Furthermore, it has been observed that P3028 levels in tumors are inversely correlated with T cell infiltration. It is contemplated that such "immune desert" and "immune-depleted" profiles may indicate patients eligible for treatment with a pharmaceutical composition comprising a peptide inhibitor as described herein. In some embodiments, the method comprises selecting the patient as comprising the presence or level of a P3028 structure (such as peptide P3028, denatured albumin, and/or damaged albumin), and/or the presence or absence of immune cells in a sample of the patient. The sample may comprise a hematopoietic tissue, a body fluid, a blood sample, or a tumor biopsy sample. Such patients may be selected to receive an effective amount of the pharmaceutical formulation. In some embodiments, the method comprises selecting the patient to receive an effective amount of the pharmaceutical agent if immune cells are absent from a tumor in the patient's sample. The sample may comprise a hematopoietic tissue, a bodily fluid, a blood sample, and/or a tumor biopsy sample. In some embodiments, the method comprises selecting the patient to receive an effective amount of the pharmaceutical agent if immune cells are absent from a sample of the patient comprising the tumor and the P3028 structure is in the sample of the patient. The sample may comprise a hematopoietic tissue, a body fluid, a blood sample, and/or a tumor biopsy sample. In some embodiments, the method comprises selecting a patient to receive an effective amount of a pharmaceutical agent if immune cells, such as T cells, are absent from a sample of the patient comprising the tumor. The sample may comprise a hematopoietic tissue, a body fluid, a blood sample, and/or a tumor biopsy sample. In some embodiments, the method comprises selecting the patient to receive an effective amount of the pharmaceutical formulation if the P3028 structure is in a sample of the patient. The sample may comprise a hematopoietic tissue, a body fluid, a blood sample, and/or a tumor biopsy sample. In some embodiments, the method comprises selecting the patient to receive an effective amount of the pharmaceutical formulation if the patient has a tumor that is substantially free of T-cell infiltrates, a tumor that comprises a majority of T-cell infiltrates in the stroma, or a tumor that is inflamed and infiltrated by inactive T-cells. In some embodiments, the method further comprises detecting an inflammatory response to the tumor, such as suppressor cell infiltration of effector cells and/or the tumor, after administration of the pharmaceutical formulation. In some embodiments, the sample comprises a hematopoietic tissue, a blood sample, and/or a tumor biopsy sample.

Death of tumor cells can also be indicative of a reduction in immunosuppression and inhibition of tumors in response to a pharmaceutical composition as described herein (see, e.g., example 6). Thus, in some embodiments, the method further comprises detecting death, such as apoptosis or necrosis, of the tumor cell after administration of the pharmaceutical composition.

It is further contemplated that the pharmaceutical composition as described herein may act synergistically with additional therapeutic agents. For example, it is envisioned that tumors comprising an inflamed moiety that is infiltrated by T cells with low expression of P3028 may be suitable for treatment with checkpoint inhibitors (see example 7). For example, it is contemplated that antibodies to PD-1 or PD-L1 may inhibit PD-L1-mediated immunosuppression, and thus may act synergistically with peptide inhibitors as described herein. Thus, in some embodiments, the method further comprises administering to the patient an additional therapeutic agent. In some embodiments, the additional therapeutic agent comprises, consists essentially of, or consists of: an antibody specific for PD-1, and/or an antibody specific for PD-L1. In some embodiments, the additional therapeutic agent is an antibody specific for PD-L1 selected from the group consisting of bevacizumab, astuzumab, and avizumab. In some embodiments, the additional therapeutic agent is an antibody specific for PD-1 selected from the group consisting of nivolumab and pembrolizumab. In some embodiments, the additional therapeutic agent is an antibody or antibody fragment that is bispecific for PD-1 and PDL-1.

Example 1: low of P3028Effect of molecular weight inhibitors on lymphocyte activation

The analysis of inhibitor P28R of P3028 was performed in a human ex vivo model. The stimulatory activity on PBMCs measured using MTS or CFSE techniques was studied in 7 healthy control samples and 7 variously diagnosed cancer patients. Interestingly, even in the absence of other types of stimulation, P28R had significant stimulatory activity in 6 of 7 cancer patients, while PBMCs from control samples showed only weak or no stimulation.

As shown in fig. 1A-D, the stimulatory activity of P28R on the suppressed proliferative response to IL-2. PBMC were cultured with IL-2 for 7 days and the proliferation rate was determined by incorporation of BrdU. Each bar represents the average of triplicates. Similar to the study on the efficacy of antibodies against P3028 (see figure 22 of PCT publication No. WO 2016/144650) to reverse cancer-associated immunosuppression, which was determined as a poor proliferative response to IL-2 by PBMCs from cancer patients, the efficacy of the low molecular weight inhibitor P28R to reverse inhibited IL-2-induced proliferation was studied. Culture results of PBMCs from four different naive patients are shown in FIGS. 1A-D. For each amount of added P28R, IL-2 stimulated cells 240 are shown on the left and unstimulated 242 are shown on the right. P28R clearly stimulated PBMCs with low initial proliferation (see fig. 1A and 1B), while high initial proliferation was essentially unaffected by the drug (see fig. 1C and 1D). As expected, systemic immunosuppression is not present in all patients, and only those with immunosuppression will be stimulated.

Example 2: effect of P28R and P28 core peptides on PBMC activation

P28R (SEQ ID NO:2) can stimulate PBMCs from healthy controls in short-term culture when RPMI + 10% normal human AB serum was used as the culture medium. The truncation of P28R was also evaluated with respect to its ability to activate PBMCs. PBMC were incubated with the peptide (40. mu.g/mL) in RPMI + 10% human AB serum for 24 hours. PBMC activation was measured as the percentage of cells with enhanced expression of CD69 (fig. 2A) or CD71 (fig. 2B) using flow cytometry. Two experiments were performed for each peptide.

Tests were performed for the direct stimulatory effect of immune cells. As shown in FIGS. 2A and 2B, peptide P28R (SEQ ID NO:2) efficiently activated healthy PBMC in this model, but peptides 32251(SEQ ID NO: 4) and 32230 ("P28 core") (FFVKLS) (SEQ ID NO:1) did not activate healthy PBMC in this model.

In addition, P28R and P28 cores were tested for their ability to reduce immunosuppression in cancer cells. In PBMC cultures, P28R (SEQ ID NO:2) and P28 core (peptide 32230(FFVKLS) (SEQ ID NO:1) each activated PBMCs, measured as enhanced CD69 expression (see FIG. 3) in the presence of normal human AB-serum in medium replaced with serum from dogs with cancer or human patients with cancer FIG. 3 shows the comparison between full-length peptide P28R (SEQ ID NO:2) and 6 amino acid P28 core sequence (peptide 32230) (FFVKLS) (SEQ ID NO:1) in medium containing serum from two different cancer patients (human cancer serum 1430 and human cancer serum 2432). P28R (SEQ ID NO:2) and P28 core (SEQ ID NO:1) both activated PBMCs in the presence of cancer serum.

In addition, biotinylated P28R has been shown to bind directly to PBMCs as demonstrated by immunocytochemistry or rosetting (binding of beads to cells) of P28R coated beads.

Taken together, these results show that P28R (SEQ ID NO:2) can bind P3028 and unblock cellular receptors, and can also have direct stimulatory activity on immune cells. In addition, the P28 core (SEQ ID NO:1) can bind P3028 and unblock cellular receptors.

Example 3: P28R activates the immune system in immunocompetent mice

The ability of P28R to activate the immune system and thereby induce tumor cell lysis was studied in immunocompetent mice C57Bl vaccinated with B16 melanoma. 20nM P28R in 100 microliters was injected intratumorally and tumors were removed after 3-5 days. Regional lymph node responses are often found in animals treated intratumorally with P28R as shown in example 49 of PCT publication No. WO2016/144 (incorporated herein by reference in its entirety). Intratumoral injection of P28R caused significant tumor regression not only in P28R treated tumors but also in contralateral tumors that were not injected or injected with saline only (figures 55A-D of PCT publication No. WO 2016/144). The effect on untreated distal/contralateral tumors increased over time after P28R was injected into the treated tumors. Similar results were obtained in the Lewis lung cancer model in B57Bl mice. Thus, it was shown that administration of the peptide inhibitor P28R according to some embodiments herein can induce degenerative changes in tumors, including tumors that receive the peptide inhibitor within the tumor as well as tumors at other sites in the patient (e.g., tumors on the contralateral side of the tumor that received the peptide inhibitor).

Example 4: intratumoral treatment of spontaneous tumors in dogs with P28R

Spontaneous canine tumors of different tissues have been treated by intratumoral injection of 40nmol P28R in 200 microliters. Treatment of tumors in dogs with P28R is described in detail in examples 50-60 of PCT publication WO2016/144650, which is incorporated herein by reference in its entirety. Exemplary results of quantifying tumor cells (881 for treated dogs and 882 for untreated control dogs) and lymphocytes (883 for both treated dogs and untreated control dogs) for 7 dogs with treated breast tumors and 5 untreated control dogs are shown in fig. 4. In these dogs with breast tumors, a clear inflammatory infiltrate was found in all treated tumors, and the number of degenerative tumor cells increased compared to 14 untreated tumors. Even if the drug was injected at 200. mu.L only, an antitumor effect was observed in large tumors. In two dogs with multiple tumors, the same response to P28R was found not only in the injected tumor but also in the non-injected tumor.

Spontaneous tumors in dogs were treated in a single dose escalation study using subcutaneous injection of P28R and tumors were excised 3-5 days later. As shown in fig. 5A-B, subcutaneous P28R treatment of spontaneous canine breast tumors (single dose 80 micrograms) resulted in recruitment of inflammatory cells and tumor cell death (control N-12 and treatment N-5). Dogs were also dosed with 160 micrograms and 320 micrograms. No adverse events were observed in any dog at any dose level. In addition, (A) in dogs with systemic high grade B cell lymphoma with multiple lymph node enlargement, a single injection of 200 micrograms of P28R in 500 microliters was administered into a 2cm diameter popliteal lymph node. After 5 days, no injected tumor could be found clinically. (B) In dogs with progressive squamous cell carcinoma of the alveolar ridge (extending right into the hard palate/palate and up under the right eye, protruding below and laterally of the eye, tumor size 3x 4cm), ten weekly intratumoral injections of 80 micrograms of P28R in 200 microliters were administered. After 3 months, a complete histopathological remission of the injected tumor was achieved. (C) In dogs with multiple recurrent breast tumors (with sizes up to 4x4 mm), histopathological examination showed a strong inflammatory response after 9 injections, usually weekly, of 80 micrograms in 20 microliters, but also residual tumor cells. In summary, it can be concluded that the peptide inhibitor P28R of some embodiments is well tolerated and effective in ameliorating, inhibiting and treating various types of tumors.

Example 5: systemic effects of P28R

The effect of systemic subcutaneous administration of P28R was investigated. This study is described in example 62 of PCT publication No. WO 2016/144650. Mice vaccinated with CT26 colon cancer were treated with 12 micrograms of P28R 2 times a week for two weeks. Apoptosis is induced in most tumor cells. These results show that in addition to direct tumor injection, systemic administration of a peptide inhibitor such as P28R according to some embodiments herein can also induce inflammatory infiltration, tumor cell degeneration, and/or eradication of tumor tissue in a patient, and can induce programmed cell death in tumor cells.

Example 6: synergy between P28R and antibodies to PD-1 and _ PD-L1

In the development of cancer drug P28R, antibodies against CD45 were selected solely for the evaluation of enhanced inflammatory responses. The marker does not allow to distinguish between the functional features of different subsets of the intra-tumoral inflammatory subset. In a number of studies, particularly in dog studies, the short-term phase following injection of P28R, i.e. activation of primarily the innate immune system, was studied. In mouse experiments, a strong inflammatory response was induced within 3-5 days, in some experiments already after 24 hours. Interestingly, an impressive eradication of tumor cells was observed within this short period of time. Tumor cell death was assessed based on morphological criteria, TUNEL and Feulgen staining. Apparently, a strong antitumor activity was elicited. In addition, a statistically significant synergistic effect between P28R and antibodies to PD-1 and PD-L1 was demonstrated, evaluated as growth retardation. Without being limited by theory, in view of this observation, the interest in dissecting inflammatory responses is more directed against the tumor effector mechanisms than the possible occurrence of inhibitor mechanisms. Furthermore, without being limited by theory, based on early responses, morphology of major inflammatory cells and macrophage specific markers such as F4/80, it can be concluded that: macrophages are the major type of effector cells. After an extended treatment period of 2 weeks, a significantly enhanced recruitment of CD8+ cells occurred, which is very consistent with the early innate induction of immunogenic cell death and the subsequent adaptive immune response.

Example 7: presence of P3028 structure in human cancers

Currently, treatment disorders in cancer immunotherapy can be described by the following immune blocking phenotypes: immune desert tumors (no infiltration of T-cells), immune-depleted tumors (T-cells restricted to the surrounding interstitial regions) and inflamed tumors (infiltration of non-functional T-cells). As shown in breast and head and neck cancer studies, the emergence of immunosuppressive 3028 is associated with poor recruitment of T cells to tumors. This is very consistent with 3028 blocking LFA-1 and thereby inhibiting the ability of lymphocytes to enter the tumor and migrate within the tumor in the vicinity of the tumor cells.

Three different tongue cancers double stained with antibodies against 3028 (red) and CD3 (brown) are shown. The immune desert cancer on the left (fig. 6A) has a strong expression of 3028 and only a few scattered T cells in the stroma, the immune rejection cancer in the middle (fig. 6B) has a strong expression of 3028 and T cell infiltration in the stroma, the inflammatory cancer on the right (fig. 6C) is only weakly stained for 3028 and has a very strong T cell infiltration.

Without being limited by theory, it is expected that conventional immunotherapy may be challenging in cancers with immune blockages shown in fig. 6A and 6B. Without being limited by theory, in cancers characterized by inflammation, there may still be a migration block to a large extent due to LFA-1 blocking, thereby inhibiting migration of T cells near tumor cells. It is further envisioned that these types of cancers may respond to pharmaceutical formulations comprising peptide inhibitors as described herein. In the inflammatory cancer types shown in fig. 6C, which are infiltrated by T cells and have a low expression of 3028, other inhibitory mechanisms, such as checkpoint molecules, are playing a role. These are the tumors most likely to respond to checkpoint inhibitors.

Further study on human head and neck cancer

The expression of immunosuppressant 3028 in oral cancer was studied. Of the 18 tongue, larynx and tonsil samples evaluated, 16 showed moderate to high expression of 3028 in tumor nodules. The remaining two samples also showed 3028 expression, but to a lesser extent.

Statistical analysis showed that the presence of CD3+ Tumor Infiltrating Lymphocytes (TILs) correlated with a p-value of 0.024, indicating that the amount of CD3+ lymphocytes significantly correlated with 3028 presence in tumor nodules.

Example 8: in vitro treatment of human breast cancer

To further demonstrate the biological relevance of albumin structure expressing 3028 in tumors to LFA-1 blockade, fresh frozen tumor sections from human breast cancer were incubated with P28R and then stained for LFA-1(CD11 a). For comparison, tumor sections were incubated with phosphate buffered saline only. P28R reversed LFA-1 blockade, resulting in increased staining intensity, and LFA-1 receptor function may also be enhanced, which is important for lymphocyte migration and cytotoxic activity (FIG. 7A-B).

Fig. 7A-B show tumor sections from breast cancer patients showing inflammatory cells stained with an antibody against CD11 a. Fresh frozen tumor sections without any fixation were incubated with buffer (fig. 7A) or P28R (fig. 7B) and then stained. Inflammatory cells were observed to infiltrate the tumor sections treated with P28R.

In addition, sections from human breast and tongue cancers were treated ex vivo with P28R. In cancers with a strong spontaneous inflammatory response close to the tumor cells, immune cells show strong membrane staining of LFA-1, whereas in tumors with only moderate infiltration of inflammatory cells, membrane staining is blocked, but may be unblocked by ex vivo P28R treatment.

Example 9: preparation of "preparation A" and "preparation B

P28R is a short peptide that tends to form beta-sheets at neutral pH. It has been successfully formulated in acetate buffer with pH 5.2 ("formulation a"; see table 2A) and used as a vehicle for P28R in a study (study CIG-1301) of dogs with spontaneous tumors (see example 4; see also PCT publication No. WO 2016/144650). When the effect of P28R after intratumoral administration was analyzed, a significant therapeutic effect could be detected, but cells of the tumor near the injection site appeared to be slightly affected by the injected vehicle. Without being limited by theory, it is believed that this results from the acetate salt and/or low osmolality of formulation a. Thus, acetate levels were dramatically reduced by the addition of sodium chloride and osmolality was adjusted to an isotonic level to yield "formulation B" (see table 2B).

Table 2A: preparation A

Table 2B: preparation B

For production technical reasons, P28R in formulation B was split into two vials, which should be mixed before use to yield P28R in formulation B (see example 10).

P28R was produced by cs Bio co.ca, USA according to GMP standards. The chemicals used for the production of the vehicle were glacial acetic acid (Fisher Scientific, UK), sodium chloride (S3014-5kg, Sigma, Steinheim, Germany), sodium hydroxide (Sigma, 221465-500G, Sigma, Steinheim, Germany), sodium acetate (71183-250G, Sigma, Steinheim, Germany). The sterile filters used were: 25mm sterile syringe filters with 0,2 μm Polyethersulfone (PES) membranes (European catalog number 514-. Protein concentration was determined using the "peptide" setting in the DeNovix DS-11 spectrophotometer, with the relevant blank as a control. The pH was measured using a new calibration WTW Inolab pH 720.

Example 10: the tendency of P28R to form a gel is sodium hydroxide and/or pH dependent.

It was observed that P28R in formulation B could not be sterile filtered due to clogging of the filter, and that the limited amount of flow-through that could pass had almost no P28R content (data not shown). Without being limited by theory, it is suspected that the clogging of the filter results from the possibility of short peptides such as P28R forming beta-sheets and/or gels. The P28R peptide formed a clear, transparent liquid in this state, which was in the form of a very thin gel when viewed under a microscope.

Five different batches of formulation B were produced, with different amounts of sodium hydroxide added to create a pH gradient. Small beads were added to the solution to visualize the presence of any gel formation (table 3).

Table 3: gel forming ability of formulation B at different pH. The tendency of P28R to form gels in formulations with different pH ranging from pH 5.35 up to pH 10.97 was identified by the addition of small beads. (. precipitated peptides tend to adhere to the tube wall, resulting in varying concentrations.)

TABLE 3

Gel formation was observed to be affected by the addition of sodium hydroxide. Without being limited by theory, gel formation may be due to sodium addition and/or may be pH dependent. However, there is a high pH threshold around pH 10 where no gel is formed but the formulation changes from a clear solution to a white granular appearance.

Thus, it is expected that the presence of sodium and/or a pH of at least 5.35 may cause the P28R peptide to form a gel, which may interfere with sterile filtration. On the other hand, at lower pH such as 3.6, the P28R peptide remained dissolved in the liquid solution (see, e.g., table 4).

Example 11: sodium chloride induced aggregation of P28R

When sodium hydroxide was added to formulation B, sterile filtration could not be performed due to clogging of the filter, but in the absence of sodium hydroxide and sodium chloride, filtration was possible and no gel formation was detected (data not shown).

To assess whether the dissolved peptide was bound to the membrane during filtration, formulation B was sterilized through a 0.2 μm PES filter without NaOH and NaCl to give a pH of 3.6(10mg P28R (CS8040), 471.7 μ l 10% acetic acid and 23, 16ml 1, 6mM sodium acetate, 0, 27mg/ml P28R when measured with a DeNovix DS-11 spectrophotometer) and the flowthrough was dispensed in 1ml volumes. The P28R concentration of the flow-through was determined using a DeNovixDS-11 spectrophotometer (table 4). It should be noted that P28R has an average molecular mass of 1972.3 Da.

Table 4 shows the concentration of P28R in the flow-through after sterilization using a 25mm PES filter. Formulation B, free of NaOH and NaCl, was sterilized through a 0.2 μm PES filter to give pH3.6 (10mg P28R (CS8040), 471.7 μ l 10% acetic acid and 23, 16ml 1, 6mM sodium acetate). The P28R concentration of the flow-through was determined using a DeNovixDS-11 spectrophotometer. (limited sample selection was measured and P28R concentration was consistently between 0.26-0.28mg/ml, data not shown).

TABLE 4

The same batch was then filtered through a 50mm Baxter filter from Pharma-Skan and the protein content in the flow-through was determined spectrophotometrically (table 5).

Table 5 shows the concentration of P28R in the flow-through after sterilization using a 50mm Baxter filter. Formulation B, free of NaOH and NaCl, was sterilized through a 50mm Baxter filter and the flow-through was dispensed in 1ml volumes. The P28R concentration of the first 24ml flow-through was determined using a DeNovix DS-11 spectrophotometer. (during this time the liquid filled the previously dried areas on the membrane).

TABLE 5

To further evaluate the effect of pH on P28R behavior in the as-formulated state, samples in tubes 3-12 and 15-25 were combined and pH adjusted to 4.8 using sodium hydroxide, determining a protein concentration of 0.26mg/ml, and the solution was then passed through the same Baxter filter as used in table 5. Protein content was determined for six first milliflowings (1.0 ml/tube) (table 6).

Table 6 shows that elevated pH induced binding of P28R to a 50mm Baxter filter. Formulation B, pH 4.8, without NaCl was filtered through a filter saturated with P28R from formulation B, previously filtered at pH 3.6. The P28R concentration of the six first millilitres of flow-through was determined using a DeNovix DS-11 spectrophotometer.

TABLE 6

The first ml flow-through contained a slightly lower concentration than the following samples, indicating that the increased pH (4.8) induced peptide binding to the filter (compared to a pH of 3.6). The filter should have been saturated from the filtration done in the experiments shown in table 5, but elevated pH may change the filter characteristics or the peptides in the formulation, contributing to the increased P28R binding capacity.

To investigate whether sterile filtration of higher P28R concentrations was possible in formulation B without NaCl and NaOH, a new batch with a protein content of 0.37mg/ml was produced and filtered through a PES filter (table 7).

Table 7 shows the concentration of P28R in the flow-through after sterilization using PES filters. Formulation B, having a P28R content of pH3.6 and 0.37mg/ml, was filtered. The P28R concentration of the 12 first millilitre flowthrough was determined using a DeNovix DS-11 spectrophotometer.

TABLE 7

The ability of P28R to form a gel was observed when suspended in formulation B instead of formulation a. Without being limited by theory, it is believed that this phenomenon is the most likely reason behind the following possibilities: formulation a was sterile filtered without clogging, but not formulation B. Formulation B is considered more suitable for clinical use due to the high acetate concentration and low osmolality of formulation a.

To investigate the reason behind gel formation, several forms of formulation B with lower pH and/or without NaCl were investigated. The NaCl-containing formulation failed to pass through a sterile filter, indicating that NaCl or the resulting increase in osmolality was inducing gel formation of the P28R peptide.

After filtration of formulation B without NaCl, it is clear that a small fraction of P28R could be bound to the filter before saturation was reached. As shown in table 5, the amount of P28R that could be bound to the filter before saturation was pH dependent.

The amount of P28R that can be combined with the filter depends on the type of material and the size of the filter. The total amount of peptide required to saturate a 50mm Baxter filter was on the order of 0.1mg of P28R calculated from the peptide loss after filtration in Table 6. The Baxter filter is the same filter used by Pharma-Skan ApS in the P28R sterilization process manufactured for clinical use. Most often, more than 100mg of P28R was used in the production of clinical batches, and the loss of 0.1mg (relative to less than 1/1000) was therefore considered negligible.

Example 12: P28R formulated for clinical use according to GMP

Formulation B was produced by Pharma-Skan ApS in batch No. 611170 (using API CS8040 batch GH1008) using two vials according to current GMP standards. The P28R concentrate (P28R vial-a) contained all P28R in 0.76mL of formulation B buffer without NaCl and NaOH. The diluent (P28R vial-B) contained all the NaCl and NaOH in 0.24mL of solution. Formulation B was later formed by combining "P28R vial-a" and "P28R vial-B" in the clinic just prior to injection.

Formulation B is isotonic and has less acetate than formulation a, making formulation B the first choice for clinical use. The gel-inducing ability of formulation B caused production problems in the filter sterilization step due to NaCl content. To circumvent this problem, P28R can be sterile filtered without NaCl at low pH. Thus, production of P28R for clinical use can be accomplished by dispensing formulation B into a two vial system (P28R concentrate in "P28R vial-a" and diluent in "P28R vial-B"). Vial-a will consist of P28R dissolved in acetic acid and sodium acetate. vial-B will contain sodium chloride and sodium hydroxide. To avoid subjecting P28R to elevated pH during the two vial blending, vial-B of sodium hydroxide had to be added to vial-a containing P28R. When vial a and vial B are mixed, formulation B will be produced ready for clinical use.

Example 13 stability testing of GMP-produced batch P28R

Batch 611170 was tested for stability in connection with monitored storage conditions in cooperation with BirkBioStorage AB and HPLC purity analysis was performed by Q & Q-Labs AB. Purity analysis was performed immediately before storage, and the results of 97.0% purity were used as reference values for comparison for later analysis. After 19 months of storage at +5 ℃, the purity dropped to 94.8%. All stability results are presented in table 8.

Table 8 shows the P28R stability results from batch 611170. From Q&Q-Labs AB(Sweden) the purity of the P28R concentrate (P28R vial-B) stored by a certified storage facility Birka Biostorage AB (Lund, sweden) at monitored +5 ℃ was tested using HPLC at the indicated time points according to current GMP standards. The purity of the P28R peptide, the largest single related species and more than 0.1% of the other single related species are presented in the table.

TABLE 8

Example 14 vehicle of high concentration P28R

For future use, an optimized "formulation C" was developed with the possibility of significantly increasing the concentration of P28R.

Formulation C was produced by dissolving P28R in 0,01M acetic acid to a concentration of 13.3mg/mL, and adding 1.12M glucose thereto to give an isotonic solution with a P28R concentration of 10 mg/mL. The acidity of the 0.01M acetic acid is counteracted by the peptide counterion (being acetate), thus yielding a pH of about 5.2. 10mg/mL of this formulation C can be sterile filtered without clogging the filter, but is sticky and foaming significantly. The important part is that it does not form a gel and is therefore an alternative if a high P28R concentration is required in the future. Formulation C, with a lower P28R concentration, can be easily produced by dilution with isotonic glucose. Preliminary studies with formulation C having P28R showed that concentrations below 5mg/mL were very practical.

Formulation C has high potential in several respects, including production cost, production technology, modest buffer capacity, the possibility of increasing the concentration of P28R, and is more practical in clinics using a single vial system rather than a two vial system.

In at least some of the above embodiments, one or more elements used in one embodiment may be used interchangeably in another embodiment unless such an alternative is not technically feasible. Those skilled in the art will appreciate that various other omissions, additions and modifications may be made to the methods, compositions, pharmaceutical products, pharmaceutical formulations and uses described herein without departing from the scope of the claimed subject matter. All such modifications and variations are intended to fall within the scope of the subject matter defined by the appended claims.

With respect to the use of substantially any plural and/or singular terms herein, those having skill in the art can translate from the plural to the singular and/or from the singular to the plural as is appropriate to the context and/or application. Various singular/plural permutations may be expressly set forth herein for the sake of clarity.

It will be understood by those within the art that, in general, terms used herein, and especially in the appended claims (e.g., bodies of the appended claims) are generally intended as "open" terms (e.g., the term "including" should be interpreted as "including, but not limited to," the term "having" should be interpreted as "having at least," the term "includes" should be interpreted as "includes, but is not limited to," etc.). It will be further understood by those within the art that if a specific number of an introduced claim recitation is intended, such an intent will be explicitly recited in the claim, and in the absence of such recitation no such intent is present. For example, as an aid to understanding, the following appended claims may contain usage of the introductory phrases "at least one" and "one or more" to introduce claim recitations. However, the use of such phrases should not be construed to imply that: the introduction of a claim recitation by the indefinite article "a" or "an" limits any particular claim containing such introduced claim recitation to embodiments containing only one such recitation, even when the same claim includes the introductory phrases "one or more" or "at least one" and indefinite articles such as "a" or "an" (e.g., "a" and/or "an" should be interpreted to mean "at least one" or "one or more"); the use of definite articles to guide the description of the claims is equally applicable. In addition, even if a specific number of an introduced claim recitation is explicitly recited, those skilled in the art will recognize that such recitation should be interpreted to mean at least the recited number (e.g., the bare recitation of "two recitations," without other modifiers, means at least two recitations, or two or more recitations). Further, in those instances where a convention analogous to "at least one of A, B and C, etc." is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (e.g., "a device or substrate having at least one of A, B and C" would include, but not be limited to, devices or substrates having a alone, B alone, C, A and B together, a and C together, B and C together, and/or A, B and C together, etc.). In those instances where a convention analogous to "A, B or at least one of the like" is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (e.g., "a device or substrate having at least one of A, B or C" would include, but not be limited to, devices or substrates having a alone, B alone, C, A alone and B together, a and C together, B and C together, and/or A, B and C together, etc.). It will be further understood by those within the art that substantially any disjunctive word and/or phrase presenting two or more alternative terms, whether in the description, claims, or drawings, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase "a or B" should be understood to include the possibility of "a" or "B" or "a and B". In addition, when methods comprising the compositions, pharmaceutical formulations, or pharmaceutical products are described herein, the corresponding compositions, pharmaceutical formulations, or pharmaceutical products are also contemplated for use. For example, a method of treating cancer comprising administering a pharmaceutical formulation comprising a peptide inhibitor specifically also encompasses a pharmaceutical formulation comprising a peptide inhibitor for treating cancer.

In addition, where features or aspects of the disclosure are described in terms of markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any single member or subgroup of members of the markush group.

Those skilled in the art will appreciate that for any and all purposes, such as in terms of providing a written description, all ranges disclosed herein also encompass any and all possible subranges and combinations of subranges thereof. It can be readily appreciated that any listed range is sufficiently descriptive and enables the same range to be divided into at least equal two, three, four, five, ten, etc. parts. As a non-limiting example, each range discussed herein can be readily divided into a lower third, a middle third, an upper third, and the like. Those skilled in the art will also appreciate that all language such as "at most," "at least," "greater than," "less than," and the like, includes the number recited and indicates the range that can subsequently be subdivided into the subranges discussed above. Finally, as understood by those of skill in the art, a range includes each individual member. Thus, for example, a group having 1-3 items represents a group having 1, 2, or 3 items. Similarly, a group having 1-5 items means a group having 1, 2, 3, 4, or 5 items, and so on.

While various aspects and embodiments have been disclosed herein, other aspects and embodiments will be apparent to those skilled in the art. The various aspects and embodiments disclosed herein are for purposes of illustration and are not intended to be limiting, with the true scope and spirit being indicated by the following claims.

Sequence listing

<110> Cannem Geder therapeutics

<120> pharmaceutical formulations of peptide inhibitors

<130> CANIG.009WO

<150> 62/738859

<151> 2018-09-28

<160> 4

<170> PatentIn 3.5 edition

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<213> Artificial sequence

<220>

<223> peptide 32230

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Phe Phe Val Lys Leu Ser

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<212> PRT

<213> Artificial sequence

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<223> peptide P28R

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Lys Lys Leu Asp Thr Phe Phe Val Lys Leu Ser Leu Phe Thr Glu Arg

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<210> 3

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<213> Intelligent people

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Val Phe Asp Glu Phe Lys Pro Leu Val Glu Glu Pro Gln Asn Leu Ile

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Lys

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<220>

<223> peptide 32251

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Lys Lys Leu Asp Thr Phe Phe Pro Lys Leu Ser Leu Phe Thr Glu Arg

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