CHEMOTHERAPEUTIC METHODS AND COMPOSITIONS

• US 20100209415A1
• Filed: 01/05/2010
• Published: 08/19/2010
• Est. Priority Date: 01/06/2009
• Status: Active Grant

• Current Assignee: Gilead Biologics, Inc. (Gilead Sciences Inc.)
• Original Assignee: Gilead Biologics, Inc. (Gilead Sciences Inc.)
• Inventors: Holzer, Alison Kay, Smith, Victoria
• Granted Patent: US 9,107,935 B2
• Time in Patent Office: Days
• US Class Current: 514/1.100
• CPC Class Codes: A61K 2300/00 Mixtures or combinations of active ingredients, wherein at least one active ingredient is fully defined in groups A61K31/00 - A61K41/00
• CPC Class Codes: A61K 31/282 Platinum compounds
• CPC Class Codes: A61K 31/337 having four-membered rings, e.g. taxol
• CPC Class Codes: A61K 31/513 having oxo groups directly attached to the heterocyclic ring, e.g. cytosine
• CPC Class Codes: A61K 31/517 ortho- or peri-condensed with carbocyclic ring systems, e.g. quinazoline, perimidine
• CPC Class Codes: A61K 31/519 ortho- or peri-condensed with heterocyclic rings
• CPC Class Codes: A61K 31/704 attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin digitoxin A61K31/7048
• CPC Class Codes: A61K 31/7068 having oxo groups directly attached to the pyrimidine ring, e.g. cytidine, cytidylic acid
• CPC Class Codes: A61K 31/7105 Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
• CPC Class Codes: A61K 33/24 Heavy metals; Compounds thereof
• CPC Class Codes: A61K 33/243 Platinum; Compounds thereof
• 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 35/00 Antineoplastic agents
• CPC Class Codes: C07K 16/40 against enzymes
• CPC Class Codes: C12N 15/1137 against enzymes viral enzymes C12N15/1131; receptors C12N15/1138
• CPC Class Codes: C12N 2310/14 interfering N.A.

Abstract

Disclosed herein are methods and compositions for enhancing the cell-killing activity of anti-neoplastic agents by inhibiting the activity of a lysyl oxidase-type enzyme. Also disclosed are methods for screening for chemotherapeutic agents, and for molecules that enhance the activity of chemotherapeutic agents, using cells grown on an extracellular matrix.

Claims

1. A method for enhancing the cell killing activity of an anti-neoplastic agent the method comprising:

contacting the cell with the anti-neoplastic agent and an inhibitor of a lysyl oxidase-type enzyme;

wherein the cell is (i) growing in the presence of an extracellular matrix and (ii) is resistant to the anti-neoplastic agent.

2. The method of claim 1, wherein the cell is in culture.

3. The method of claim 1, wherein the cell is present in a tumor.

4. The method of claim 1, wherein the anti-neoplastic agent is a chemotherapeutic drug.

5. The method of claim 1, wherein the anti-neoplastic agent is a nucleic acid.

6. The method of claim 5, wherein the nucleic acid is a siRNA.

7. The method of claim 1, wherein the anti-neoplastic agent is a polypeptide.

8. The method of claim 7, wherein the polypeptide is an antibody.

9. The method of claim 1, wherein the extracellular matrix comprises a collagen.

10. The method of claim 9, wherein the collagen is type I collagen.

11. The method of claim 1, wherein the inhibitor of a lysyl oxidase-type enzyme is a low molecular weight (<

1 kD) organic molecule.

12. The method of claim 1, wherein the inhibitor of a lysyl oxidase-type enzyme is a nucleic acid.

13. The method of claim 12, wherein the nucleic acid is a siRNA.

14. The method of claim 1, wherein the inhibitor of a lysyl oxidase-type enzyme is a polypeptide.

15. The method of claim 14, wherein the polypeptide is an antibody.

16. The method of claim 1, wherein the lysyl oxidase-type enzyme is LOXL2.

17. A method for killing a malignant cell, the method comprising:

contacting the malignant cell with an anti-neoplastic agent and an inhibitor of a lysyl oxidase-type enzyme;

wherein the malignant cell is (i) growing in the presence of an extracellular matrix and (ii) is resistant to the anti-neoplastic agent.

18. The method of claim 17, wherein the malignant cell is in culture.

19. The method of claim 17, wherein the malignant cell is present in a tumor.

20. The method of claim 17, wherein the anti-neoplastic agent is a chemotherapeutic drug.

21. The method of claim 17, wherein the anti-neoplastic agent is a nucleic acid.

22. The method of claim 21, wherein the nucleic acid is a siRNA.

23. The method of claim 17, wherein the anti-neoplastic agent is a polypeptide.

24. The method of claim 23, wherein the polypeptide is an antibody.

25. The method of claim 17, wherein the extracellular matrix comprises a collagen.

26. The method of claim 25, wherein the collagen is type I collagen.

27. The method of claim 17, wherein the inhibitor of a lysyl oxidase-type enzyme is a low molecular weight (<

1 kD) organic molecule.

28. The method of claim 17, wherein the inhibitor of a lysyl oxidase-type enzyme is a nucleic acid.

29. The method of claim 28, wherein the nucleic acid is a siRNA.

30. The method of claim 17, wherein the inhibitor of a lysyl oxidase-type enzyme is a polypeptide.

31. The method of claim 30, wherein the polypeptide is an antibody.

32. The method of claim 17, wherein the lysyl oxidase-type enzyme is LOXL2.

33. A method for reversing resistance of a malignant cell to an anti-neoplastic agent, the method comprising:

contacting the malignant cell with the anti-neoplastic agent and an inhibitor of a lysyl oxidase-type enzyme,

wherein the malignant cells is (i) growing in the presence of an extracellular matrix and (ii) is resistant to the anti-neoplastic agent.

34. The method of claim 33, wherein the malignant cell is in culture.

35. The method of claim 33, wherein the malignant cell is present in a tumor.

36. The method of claim 33, wherein the anti-neoplastic agent is a chemotherapeutic drug.

37. The method of claim 33, wherein the anti-neoplastic agent is a nucleic acid.

38. The method of claim 37, wherein the nucleic acid is a siRNA.

39. The method of claim 33, wherein the anti-neoplastic agent is a polypeptide.

40. The method of claim 39, wherein the polypeptide is an antibody.

41. The method of claim 33, wherein the extracellular matrix comprises a collagen.

42. The method of claim 41, wherein the collagen is type I collagen.

43. The method of claim 33, wherein the inhibitor of a lysyl oxidase-type enzyme is a low molecular weight (<

1 kD) organic molecule.

44. The method of claim 33, wherein the inhibitor of a lysyl oxidase-type enzyme is a nucleic acid.

45. The method of claim 44, wherein the nucleic acid is a siRNA.

46. The method of claim 33, wherein the inhibitor of a lysyl oxidase-type enzyme is a polypeptide.

47. The method of claim 46, wherein the polypeptide is an antibody.

48. The method of claim 33, wherein the lysyl oxidase-type enzyme is LOXL2.

49. A method for identifying a molecule that enhances the cell killing activity of an anti-neoplastic agent, the method comprising:

providing a cell culture comprising one or more cells, wherein the cells are;

(i) growing in the presence of an extracellular matrix, and

(ii) resistant to the anti-neoplastic agent;

contacting the cell culture with the anti-neoplastic agent;

optionally contacting the cell culture with a test molecule; and

measuring cell viability of the cell culture;

wherein lower cell viability in the presence of the test molecule and the anti-neoplastic agent, compared to cell viability in the presence of the anti-neoplastic agent alone, indicates that the test molecule is a molecule that enhances the cell killing activity of the anti-neoplastic agent.

50. The method of claim 49, wherein the anti-neoplastic agent is a chemotherapeutic drug.

51. The method of claim 50, wherein the chemotherapeutic drug is selected from the group consisting of methotrexate, cisplatin, doxorubicin, docetaxel, erlotinib, taxol, paraclitaxel, taxotere, 5-fluorouracil and gemcitibine.

52. The method of claim 49, wherein the anti-neoplastic agent is a nucleic acid.

53. The method of claim 52, wherein the nucleic acid is a siRNA.

54. The method of claim 49, wherein the anti-neoplastic agent is a polypeptide.

55. The method of claim 54, wherein the polypeptide is an antibody.

56. The method of claim 49, wherein the extracellular matrix comprises a collagen.

57. The method of claim 56, wherein the collagen is type I collagen.

58. The method of claim 49, wherein the test molecule is a low molecular weight (<

1 kD) organic molecule.

59. The method of claim 49, wherein the test molecule is a nucleic acid.

60. The method of claim 59, wherein the nucleic acid is a siRNA.

61. The method of claim 49, wherein the test molecule is a polypeptide.

62. The method of claim 61, wherein the polypeptide is an antibody.

63. The method of claim 49, wherein the test molecule is an inhibitor of a lysyl oxidase-type enzyme.

64. The method of claim 63, wherein the lysyl oxidase-type enzyme is LOXL2.

65. The method of claim 49, further comprising administering the anti-neoplastic agent and the molecule that enhances the cell killing activity of the anti-neoplastic to a subject with a malignancy or neoplastic disorder.

66. A composition comprising:

(a) cells growing in the presence of an extracellular matrix, wherein the cells are resistant to an anti-neoplastic agent;

(b) the anti-neoplastic agent; and

(c) an inhibitor of a lysyl oxidase-type enzyme.

67. The composition of claim 66, wherein the cells are in culture.

68. The composition of claim 66, wherein the cells are present in a tumor.

69. The composition of claim 66, wherein the anti-neoplastic agent is a chemotherapeutic drug.

70. The composition of claim 69, wherein the chemotherapeutic drug is selected from the group consisting of methotrexate, cisplatin, doxorubicin, docetaxel, erlotinib, taxol, paraclitaxel, taxotere, 5-fluorouracil and gemcitibine.

71. The composition of claim 66, wherein the anti-neoplastic agent is a nucleic acid.

72. The composition of claim 71, wherein the nucleic acid is a siRNA.

73. The composition of claim 66, wherein the anti-neoplastic agent is a polypeptide.

74. The composition of claim 73, wherein the polypeptide is an antibody.

75. The composition of claim 66, wherein the extracellular matrix comprises a collagen.

76. The composition of claim 75, wherein the collagen is type I collagen.

77. The composition of claim 66, wherein the inhibitor of a lysyl oxidase-type enzyme is a low molecular weight (<

1 kD) organic molecule.

78. The composition of claim 66, wherein the inhibitor of a lysyl oxidase-type enzyme is a nucleic acid.

79. The composition of claim 78, wherein the nucleic acid is a siRNA.

80. The composition of claim 66, wherein the inhibitor of a lysyl oxidase-type enzyme is a polypeptide.

81. The composition of claim 80, wherein the polypeptide is an antibody.

82. The composition of claim 66, wherein the lysyl oxidase-type enzyme is LOXL2.

83. A method for assessing the sensitivity of a malignant cell to a test molecule, the method comprising:

growing the malignant cell in the presence of an extracellular matrix;

optionally contacting the cell with the test molecule; and

assaying for cell survival;

wherein, if cell survival is lower in the presence of the test molecule than in its absence, the malignant cell is sensitive to the test molecule.

84. The method of claim 83, wherein the malignant cell is in culture.

85. The method of claim 83, wherein the malignant cell is present in a tumor.

86. The method of claim 83, wherein the extracellular matrix comprises a collagen.

87. The method of claim 86, wherein the collagen is type I collagen.

88. The method of claim 83, wherein the test molecule is a low molecular weight (<

1 kD) organic molecule.

89. The method of claim 83, wherein the test molecule is a nucleic acid.

90. The method of claim 89, wherein the nucleic acid is a siRNA.

91. The method of claim 83, wherein the test molecule is a polypeptide.

92. The method of claim 91, wherein the polypeptide is an antibody.

93. The method of claim 83, wherein the test molecule is an inhibitor of a lysyl oxidase-type enzyme.

94. The method of claim 93, wherein the lysyl oxidase-type enzyme is LOXL2.

95. The method of claim 83, further comprising growing the malignant cell in the presence of an anti-neoplastic agent.

96. The method of claim 95, wherein the anti-neoplastic agent is a chemotherapeutic drug.

97. The method of claim 96, wherein the chemotherapeutic drug is selected from the group consisting of methotrexate, cisplatin, doxorubicin, docetaxel, erlotinib, taxol, paraclitaxel, taxotere, 5-fluorouracil and gemcitibine.

98. The method of claim 95, wherein the anti-neoplastic agent is a nucleic acid.

99. The method of claim 98, wherein the nucleic acid is a siRNA.

100. The method of claim 95, wherein the anti-neoplastic agent is a polypeptide.

101. The method of claim 100, wherein the polypeptide is an antibody.

102. The method of claim 93, wherein the inhibitor of a lysyl oxidase-type enzyme is a low molecular weight (<

1 kD) organic molecule.

103. The method of claim 93, wherein the inhibitor of a lysyl oxidase-type enzyme is a nucleic acid.

104. The method of claim 103, wherein the nucleic acid is a siRNA.

105. The method of claim 93, wherein the inhibitor of a lysyl oxidase-type enzyme is a polypeptide.

106. The method of claim 105, wherein the polypeptide is an antibody.