METHODS AND COMPOSITIONS FOR TREATMENT AND DIAGNOSIS OF FIBROSIS, TUMOR INVASION, ANGIOGENESIS, AND METASTASIS

• US 20090104201A1
• Filed: 08/01/2008
• Published: 04/23/2009
• Est. Priority Date: 08/02/2007
• Status: Active Grant

• Current Assignee: Gilead Biologics, Inc. (Gilead Sciences Inc.)
• Original Assignee: Gilead Biologics, Inc. (Gilead Sciences Inc.)
• Inventors: SMITH, VICTORIA, BARRY, VIVIAN E., OYASU, MIHO, OGG, SCOTT, RODRIGUEZ, HECTOR, MARSHALL, DEREK, GARCIA, CARLOS AURELIO, HOLZER, ALISON KAY, BIERMANN, DONNA HIROKO TOKUOKA, MCCAULEY, SCOTT ALAN, VLASSELAER, PETER VAN
• Granted Patent: US 8,679,485 B2
• Time in Patent Office: Days
• US Class Current: 424/139.100
• CPC Class Codes: A61K 2039/505 comprising antibodies
• CPC Class Codes: A61K 39/3955 against proteinaceous materials, e.g. enzymes, hormones, lymphokines
• CPC Class Codes: A61K 45/06 Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
• CPC Class Codes: A61P 1/04 for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
• CPC Class Codes: A61P 1/16 for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
• CPC Class Codes: A61P 1/18 for pancreatic disorders, e.g. pancreatic enzymes
• CPC Class Codes: A61P 11/00 Drugs for disorders of the respiratory system
• CPC Class Codes: A61P 13/08 of the prostate
• CPC Class Codes: A61P 13/10 of the bladder
• CPC Class Codes: A61P 13/12 of the kidneys
• CPC Class Codes: A61P 15/00 Drugs for genital or sexual disorders for disorders of sex hormones A61P5/24; Contraceptives
• CPC Class Codes: A61P 17/00 Drugs for dermatological disorders
• CPC Class Codes: A61P 17/02 for treating wounds, ulcers, burns, scars, keloids, or the like
• CPC Class Codes: A61P 19/00 Drugs for skeletal disorders
• CPC Class Codes: A61P 19/04 for non-specific disorders of the connective tissue
• CPC Class Codes: A61P 21/00 Drugs for disorders of the muscular or neuromuscular system
• CPC Class Codes: A61P 25/00 Drugs for disorders of the nervous system
• CPC Class Codes: A61P 25/28 for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
• CPC Class Codes: A61P 3/10 for hyperglycaemia, e.g. antidiabetics
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 35/04 : specific for metastasis
• CPC Class Codes: A61P 43/00 : Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: A61P 9/00 : Drugs for disorders of the cardiovascular system
• CPC Class Codes: A61P 9/10 : for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
• CPC Class Codes: A61P 9/12 : Antihypertensives
• CPC Class Codes: C07K 16/40 : against enzymes
• CPC Class Codes: C07K 2317/24 : containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
• CPC Class Codes: C07K 2317/30 : characterized by aspects of specificity or valency
• CPC Class Codes: C07K 2317/33 : Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
• CPC Class Codes: C07K 2317/34 : Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
• CPC Class Codes: C07K 2317/565 : Complementarity determining region [CDR]
• CPC Class Codes: C07K 2317/76 : Antagonist effect on antigen, e.g. neutralization or inhibition of binding
• CPC Class Codes: C07K 2317/77 : Internalization into the cell
• CPC Class Codes: C07K 2317/92 : Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
• CPC Class Codes: C12Q 1/6886 : for cancer immunoassay for cancer G01N33/574
• CPC Class Codes: G01N 2333/90633 : with oxygen as acceptor (1.4.3) in general
• CPC Class Codes: G01N 33/57423 : of lung
• CPC Class Codes: G01N 33/57438 : of liver, pancreas or kidney
• CPC Class Codes: G01N 33/57484 : involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites

Abstract

The present disclosure provides innovative methodology and related compositions and kits for preventing and treating various diseases associated with abnormal cell proliferation, angiogenesis and fibrosis, by using an inhibitor of processed forms of lysyl oxidase or lysyl oxidase-like proteins, an inhibitor of LOX and an inhibitor of a LOXL, or a synergistic combination of an inhibitor of LOX or LOXL combined with other therapeutic agents. Also provided are innovative methods for selecting agents that prevent or inhibit tumor invasion, angiogenesis and metastasis, by contacting cells that are in an epithelial-mesenchymal transition (EMT) state with a candidate agent and detecting a change in the EMT state of the cells. Methods and related compositions and kits for diagnosing or monitoring various diseases associated with abnormal cell proliferation, angiogenesis and fibrosis, by using molecules or agents that specifically recognize processed forms of LOX or LOXL are also provided. Also provided herein are methods and related compositions, medical devices, systems and kits for preventing or treating various diseases and conditions associated with fibrosis with compositions comprising inhibitors of LOX or LOXL. Such diseases or conditions include pathological cardiovascular conditions and diseases such as hypertension, hypertensive heart disease, myocardial infarction, atherosclerosis, and restenosis, liver fibrosis, kidney fibrosis, lung fibrosis, dermal scaring, keloid formation, and Alzheimer'"'"'s disease.

Claims

1. A method for treating or preventing tumor invasion or metastasis in a subject in vivo, comprising:

administering to the subject an effective amount of an inhibitor of active lysyl oxidase or lysyl oxidase-like protein.

5. The method of claim 1, wherein the tumor is metastatic tumor.

8. The method of claim 1, 2, 3, 4, or 6, wherein the LOX or LOXL inhibitor is an antibody against LOX or LOXL, a small molecule inhibitor, siRNA, shRNA or an antisense polynucleotide against LOX or LOXL.

9. The method of claim 1, 2, 3, 4, or 6, wherein the LOX or LOXL inhibitor is an antibody that specifically binds to a region of LOX or LOXL having an amino acid sequence selected from SEQ ID NOs:

1-18.

10. The method of claim 1, 2, 3, 4, or 6, wherein the LOX or LOXL inhibitor is an inhibitor of active LOX, LOXL2 or LOXL4.

11. The method of claim 1, 2, 3, 4, or 6, wherein the LOX or LOXL inhibitor inhibits both active LOX and active LOXL2.

12. The method of claim 1, 2, 3, 4, or 6 wherein the active LOX or LOXL is a mature form of LOX or LOXL after proteolytic processing of the preproprotein.

2. A method for treating or preventing angiogensis in a subject in vivo, comprising:

administering to the subject an effective amount of an inhibitor of active lysyl oxidase or lysyl oxidase-like protein.

3. A method for treating or preventing fibrosis in a subject in vivo, comprising:

administering to the subject an effective amount of an inhibitor of active lysyl oxidase or lysyl oxidase-like protein.

4. A method for reducing tumor growth in a subject in vivo, comprising:

administering to the subject an effective amount of an inhibitor of active lysyl oxidase or lysyl oxidase-like protein such that the tumor growth is reduced by at least 25%, 50%, 75%, 90%, or 95%.

6. A method for increasing or enhancing the chances of survival of a subject with metastatic tumor, comprising:

administering to a subject in need thereof an effective amount of an inhibitor of active lysyl oxidase or lysyl oxidase-like protein,

thereby increasing or enhancing the chances of survival of the subject treated by a certain period of time.

7. The method of claim 6, wherein the survival of the subject is increased by at least 10 days, 1 month, 3 months, 6 months, 1 year, 1.5 years, 2 years, 3 years, 4 years, 5 years, 8 years, or 10 years.

13. A method for treating or preventing tumor invasion or metastasis in a subject in vivo, comprising:

administering to the subject an effective amount of an inhibitor of lysyl oxidase and an inhibitor of a lysyl oxidase-like protein.

18. The method of claim 13, 14, or 16, wherein the LOX or LOXL inhibitor is an antibody against LOX or LOXL, a small molecule inhibitor, siRNA, shRNA or an antisense polynucleotide against LOX or LOXL.

19. The method of claim 13, 14, or 16, wherein the LOX inhibitor and the LOXL inhibitor are different.

20. The method of claim 13, 14, or 16, wherein the LOX inhibitor and the LOXL inhibitor are different and specifically bind to the LOX and the LOXL, respectively.

21. The method of claim 13, 14, or 16, wherein the LOX inhibitor or the LOXL inhibitor is an antibody that specifically binds to a region of the LOX or the LOXL having an amino acid sequence selected from SEQ ID NOs:

1-18.

22. The method of claim 13, 14, or 16, wherein the LOX inhibitor and the LOXL inhibitor is a single antibody, wherein the antibody has binding affinity for both the LOX and the LOXL.

23. The method of claim 13, 14, or 16, wherein the LOXL inhibitor is an inhibitor of LOXL1, LOXL2, or LOXL4.

24. The method of claim 13, 14, or 16, wherein the LOXL inhibitor is an inhibitor of LOXL2 or LOXL4.

25. The method of claim 13, 14, or 16, wherein the LOX or LOXL inhibitor is an inhibitor of active LOX, LOXL2 or LOXL4.

26. The method of claim 13, 14, or 16, wherein the LOX or LOXL inhibitor inhibits both active LOX and active LOXL2.

14. A method for reducing tumor growth in a subject in vivo, comprising:

administering to the subject an effective amount of an inhibitor of lysyl oxidase and an inhibitor of a lysyl oxidase-like protein such that the tumor growth is reduced by at least 25%, 50%, 75%, 90%, or 95%.

15. The method of claim 14, wherein the tumor is metastatic tumor.

16. A method for increasing or enhancing the chances of survival of a subject with metastatic tumor, comprising:

administering to a subject in need thereof an effective amount of an inhibitor of lysyl oxidase and an inhibitor of a lysyl oxidase-like protein,

thereby increasing or enhancing the chances of survival of the subject treated by a certain period of time.

17. The method of claim 16, wherein the survival of the subject is increased by at least 10 days, 1 month, 3 months, 6 months, 1 year, 1.5 years, 2 years, 3 years, 4 years, 5 years, 8 years, or 10 years.

27. A composition comprising:

an inhibitor of lysyl oxidase,

an inhibitor of a lysyl oxidase-like protein, and

a pharmaceutically acceptable carrier.

29. The method of claim 27, wherein the LOX or LOXL inhibitor is an antibody against LOX or LOXL, a small molecule inhibitor, siRNA, shRNA or an antisense polynucleotide against LOX or LOXL.

30. The method of claim 27, wherein the LOX or LOXL inhibitor is an antibody that specifically binds to a region of LOX or LOXL having an amino acid sequence selected from the group consisting of SEQ ID NOs:

1-18.

31. The method of claim 27, wherein the LOX or LOXL inhibitor is an inhibitor of active LOX, LOXL2 or LOXL4.

32. The method of claim 27, wherein the LOX or LOXL inhibitor inhibits both active LOX and active LOXL2.

33. The method of claim 30, wherein the active LOX or LOXL is a mature form of LOX or LOXL after proteolytic processing of the preproprotein.

34. The method of claim 27, wherein the second therapeutic agent is a chemotherapeutic agent.

35. The method of claim 27, wherein the second therapeutic agent is a therapeutic biologic.

36. The method of claim 27, wherein the second therapeutic agent is radiation.

37. The method of claim 27, wherein the LOX or LOXL inhibitor and the second therapeutic agent treat the disease synergistically through inhibition of LOX or LOXL which renders the diseased cells in the host more susceptible to the anti-proliferative or pro-apoptotic effects of the therapeutic agent and reduces the viability of the diseased cells.

38. The method of claim 27, wherein the disease associated with abnormal cell proliferation is cancer, tumors, or restenosis.

39. The method of claim 27, wherein the fibrotic disease is selected from dermal scar formation, keloids, liver fibrosis, lung fibrosis, kidney fibrosis, diabetic nephropathy, scleroderma, and glomerulosclerosis.

28. A method for treating a host with a disease associated with abnormal cell proliferation, abnormal angiogenesis, abnormal cell invasion or migration in local tissues and/or at distant sites, or a fibrotic disease, comprising:

administering to the host a therapeutically effective amount of an inhibitor of at least one lysyl oxidase or lysyl oxidase-like protein in combination with a second therapeutic agent that is not the LOX or LOXL inhibitor.

40. A composition comprising:

an inhibitor of at least one lysyl oxidase or a lysyl oxidase-like protein,

a second therapeutic agent that is not the at least one lysyl oxidase or a lysyl oxidase-like protein, and

a pharmaceutically acceptable carrier.

41. A method for selecting an inhibitor of tumor invasion, angiogenesis or metastasis, comprising:

contacting cells that are in an epithelial-mesenchymal transition (EMT) state with an inhibitor of lysyl oxidase or a lysyl oxidase-like protein; and

detecting a change in the EMT state of the cells,

wherein reduction of the EMT state or a shift from the EMT to a mesenchymal-epithelial transition (MET) indicates that the LOX or LOXL inhibitor is an inhibitor of tumor invasion, angiogenesis or metastasis.

42. The method of claim 41, wherein the EMT state of cells has a characteristic selected from positive vimentin, positive fibronectin staining, low or non-detectable levels of E-cadherin staining, an elongated and remodeled actin cytoskeleton as revealed by phalloidin staining of F-actin.

43. The method of claim 41, wherein the MET state of cells has a characteristic selected from low or non-detectable levels of vimentin, low or non-detectable levels of fibronectin staining, positive or high levels of E-cadherin staining, physiologically normal actin cytoskeleton as revealed by phalloidin staining of F-actin.

44. The method of claim 41, wherein the reduction of the EMT state or a shift from the EMT to a MET state is detected by measuring or detecting a decrease in vimentin or fibronectin staining and/or an increase in E-cadherin staining as compared with the cells prior to being contacted with the LOX or LOXL inhibitor.

45. The method of claim 41, wherein the step of detecting includes detecting the invasiveness or migratory capacity of the cells in a scratch assay in vitro.

46. The method of claim 41, wherein the LOX or LOXL inhibitor is an antibody against LOX or LOXL, a small molecule inhibitor, siRNA, shRNA or an antisense polynucleotide against LOX or LOXL.

47. The method of claim 41, wherein the cells are tumor cell lines selected from BT-549, Hs5788t, MBA-MD231, NCI-H226, MCF-7 and SW620 cell lines.

48. The method of claim 41, further comprising:

contacting the cells with a therapeutic agent that is not the LOX or LOXL inhibitor in the presence of the LOX or LOXL inhibitor; and

detecting a change in the viability of the cells.

49. A method for diagnosing cancer metastasis in a subject, comprising:

assessing active LOX or LOXL levels or activity in the blood,

wherein a change in active LOX or LOXL levels or activity in the blood in comparison with a reference sample indicates the presence of metastatic tumor growth.

50. The method of claim 49, wherein active LOX or LOXL levels or activities in the blood is lower than those in a reference sample indicates the presence or increase of cancer metastasis.

52. The method of claim 49 or 51, wherein active LOX or LOXL levels or activities in the tumor is higher than those in a reference sample indicates the presence or increase of cancer metastasis.

53. The method of claim 49 or 51, wherein the step of assessing includes assessing the levels of the active LOX or LOXL using an antibody that binds to the active LOX or LOXL.

54. The method of claim 49 or 51, wherein the step of assessing includes assessing the levels of the active LOX or LOXL using an antibody that binds to the active LOX, LOXL2 or LOXL4.

55. The method of claim 49 or 51, wherein assessing comprises assessing the levels of the active LOX or LOXL using an antibody that binds to both active LOX and active LOXL2.

56. The method of claim 49 or 51, wherein the active LOX or LOXL is a mature form of LOX or LOXL after proteolytic processing of the preproprotein.

51. A method for diagnosing cancer metastasis in a subject having a tumor, comprising:

assessing active LOX or LOXL levels or activity in the tumor,

wherein a change in active LOX or LOXL levels or activity in the tumor in comparison with a reference sample indicates the presence of metastatic tumor growth.

57. A method for monitoring a subject'"'"'s response to a therapy including a modulator of LOX/LOXL in the treatment of a disease, comprising:

detecting a change in the level C-reactive protein in the subject after administration of a modulator of LOX or LOXL to the subject,

wherein the change indicates that the LOX or LOXL modulator has a therapeutic effect on the subject.

58. The method of claim 57, wherein the modulator of LOX or LOXL is an inhibitor of LOX or LOXL.

59. The method of claim 58, wherein a reduced level of C-reactive protein in a blood sample of the subject as compared to that prior to the administration of the LOX or LOXL inhibitor is indicative of the subject'"'"'s response to the treatment with the LOX or LOXL inhibitor.

60. The method of claim 58, wherein the disease is a disease associated with abnormal cell proliferation.

61. The method of claim 60, wherein the disease associated with abnormal cell proliferation is cancer, tumors, or restenosis.

62. The method of claim 58, wherein the disease is a fibrotic disease.

63. The method of claim 62, wherein the fibrotic disease is selected from dermal scar formation, keloids, liver fibrosis, lung fibrosis, kidney fibrosis, diabetic nephropathy, scleroderma, and glomerulosclerosis.

64. A method for treating a pathological cardiovascular condition or disease in a subject, comprising administering to the subject an effective amount of an inhibitor of lysyl oxidase (LOX) or lysyl oxidase-like protein (LOXL).

65. The method of claim 64, wherein the inhibitor of LOX or LOXL inhibits an active form of LOX or LOXL.

66. The method of claim 64, wherein the pathological cardiovascular condition or disease is hypertension, hypertensive heart disease, myocardial infarction, atherosclerosis, or restenosis.

67. The method of claim 66, wherein the pathological cardiovascular condition or disease is associated with cardiovascular fibrosis and the inhibitor of LOX or LOXL is administered locally to the site of cardiovascular fibrosis.

68. The method of claim 67, wherein the inhibitor of LOX or LOXL is administered via a stent.

69. The method of claim 68, wherein the inhibitor of LOX or LOXL is coated on the stent.

70. The method of claim 67, wherein the inhibitor of LOX or LOXL is administered locally to the site of cardiovascular fibrosis via a catheter.

71. The method of claim 64, wherein the inhibitor of LOX or LOXL is administered prior to the onset of the pathological cardiovascular condition.

72. The method of claim 64, wherein the inhibitor of LOX or LOXL is administered after the onset of the pathological cardiovascular condition.

73. The method of claim 72, wherein the pathological cardiovascular condition is myocardial infarction, and the inhibitor of LOX or LOXL is administered at least 1, 2, 3, 5, or 10 hours after the myocardial infarction or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days after the myocardial infarction.

74. The method of claim 64, wherein the inhibitor of LOX or LOXL is an antibody or antigen-binding fragment thereof against LOX or LOXL, a small molecule inhibitor, siRNA, shRNA or an antisense polynucleotide against LOX or LOXL.

75. The method of claim 64, wherein the inhibitor of LOX or LOXL is an antibody or antigen-binding fragment thereof that specifically binds to a region of LOX or LOXL having an amino acid sequence selected from SEQ ID NOs:

1-18.

76. The method of claim 64, wherein the inhibitor of LOX or LOXL is an inhibitor of at least one of active LOX, LOXL2 or LOXL4.

77. The method of claim 64, wherein the inhibitor of LOX or LOXL inhibits both active LOX and active LOXL2.

78. The method of claim 64, wherein the active LOX or LOXL is a mature form of LOX or LOXL after proteolytic processing of a preproprotein form of LOX or LOXL.

79. A method for preventing a pathological cardiovascular condition or disease in a subject, comprising administering to the subject an effective amount of an inhibitor of lysyl oxidase (LOX) or lysyl oxidase-like protein (LOXL) prior to, concurrently, or after an adverse cardiovascular event.

80. The method of claim 79, wherein the adverse cardiovascular event is myocardial infarction.

81. The method of claim 79, wherein the adverse cardiovascular event is acute myocardial infarction.

82. The method of claim 79, wherein the inhibitor of LOX or LOXL is administered at least 1, 2, 3, 5, or 10 hours after the myocardial infarction, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days after the myocardial infarction.

83. A device for preventing or treating a pathological cardiovascular condition or disease in a subject, comprising a component comprising an inhibitor of lysyl oxidase (LOX) or lysyl oxidase-like protein (LOXL).

84. The device of claim 83, wherein the component is a stent.

85. The device of claim 83, wherein the inhibitor of LOX or LOXL has a molecular weight below 500 Daltons.

86. The device of claim 85, wherein the inhibitor of LOX or LOXL is β

-aminoproprionitrile (BAPN) coated on the stent.

87. The device of claim 83, further comprising a catheter for delivering the inhibitor of LOX or LOXL locally to the site of cardiovascular fibrosis caused by an adverse cardiovascular event.

88. A kit comprising a pharmaceutical composition comprising an inhibitor of LOX or LOXL and a pharmaceutically acceptable excipient;

and instructions for how to treat or prevent a pathological cardiovascular condition or disease using the pharmaceutical composition.

89. A method for treating a disease associated with fibrosis in a subject, comprising administering to the subject an effective amount of an inhibitor of lysyl oxidase (LOX) or a lysyl oxidase-like protein (LOXL), wherein the fibrosis is selected from liver fibrosis, kidney fibrosis, lung fibrosis, dermal scaring, keloid formation, and Alzheimer'"'"'s disease.

90. The method of claim 89, wherein the inhibitor of LOX or LOXL inhibits an active form of LOX or LOXL.

91. The method of claim 89, wherein the inhibitor of LOX or LOXL is an antibody or antigen-binding fragment thereof against LOX or LOXL, a small molecule inhibitor of LOX or LOXL, siRNA, shRNA or an antisense polynucleotide against LOX or LOXL.

92. The method of claim 89, wherein the LOX or LOXL inhibitor is an antibody or antigen-binding fragment thereof that specifically binds to a region of LOX or LOXL having an amino acid sequence selected from SEQ ID NOs:

1-18.

93. The method of claim 89, wherein the inhibitor of LOX or LOXL inhibits both active LOX and active LOXL2.

94. The method of claim 89, wherein the LOX or LOXL is a mature form of LOX or LOXL after proteolytic processing of a preproprotein.