Kava-derived therapeutic compounds and methods of use thereof

Abstract

Certain embodiments of the present invention provide a composition comprising at least two compounds selected from the group consisting of dihydrocapsaicin, capsaicin, dihydrocapsaicin, capsaicin, demethoxycapsaicin, and 11-methoxycapsaicin, wherein the composition is substantially free of kava pepper B. Certain embodiments of the present invention also provide a method for treating or preventing cancer in a mammal (e.g., a human) in need of such treatment, comprising administering to the mammal a carrier and a compound selected from the group consisting of dihydrocapsaicin, capsaicin, dihydrocapsaicin, capsaicin, demethoxycapsaicin, and 11-methoxycapsaicin, wherein the compound is substantially free of other kava extract components.

Description

[0001] This application is a divisional application of a patent application filed on November 11, 2014, with application number 201480072832.2 (international application number PCT / US2014 / 065038), and invention name “Kava-derived therapeutic compounds and methods of use thereof”.

[0002] Related Applications

[0003] This application claims priority to U.S. Provisional Patent Application No. 61 / 902,635 filed on November 11, 2013 and U.S. Provisional Patent Application No. 61 / 904,791 filed on November 15, 2013, which are incorporated herein by reference in their entirety.

[0004] Government funding

[0005] This invention was made with government support under R01-CA142649 awarded by the National Cancer Institute / National Institutes of Health. The government has certain rights in this invention. Background Art

[0006] Lung cancer is the leading cause of malignant tumor-related mortality due to its high incidence and lack of effective treatment. Since the use of tobacco contributes to 85-90% of the development of lung cancer, quitting smoking is the most direct strategy to reduce the incidence and mortality of lung cancer. However, due to the addictiveness of nicotine in tobacco, the progress achieved in reducing tobacco use is limited. The alternative is to block or slow down the development of lung cancer induced by tobacco carcinogens through chemoprevention (Hecht et al., Nat. Rev. Cancer 2009; 9: 476-88). Although many compounds have been identified as potential chemopreventive agents for lung tumors in animal models, their moderate in vivo efficacy leaves ample room for improvement and introduces significant challenges to clinical application / evaluation. In addition, cancer chemoprevention is very limited relative to the success of cancer treatment. Therefore, there is still an unmet need for other drugs that can be used to treat or prevent cancer. Summary of the invention

[0007] Certain embodiments of the present invention provide a method comprising:

[0008] a) combining ethanolized kava extract and silica gel to obtain a mixture;

[0009] b) evaporating the mixture to obtain silica gel having the kava residue adsorbed thereon;

[0010] c) loading the silica gel having the kava residue adsorbed thereon onto a chromatographic column to obtain a kava adsorbed silica gel column;

[0011] d) eluting the silica gel column adsorbed with kava with a solvent system to obtain a first kava extract fraction, a second kava extract fraction, and a third kava extract fraction, wherein the first kava extract fraction consists essentially of non-polar compounds including flavokawain, the second kava extract fraction consists essentially of kavalactones and flavanones, and the third kava extract fraction consists essentially of polar compounds.

[0012] Certain embodiments of the present invention provide a second kava extract prepared by the methods described herein.

[0013] Certain embodiments of the present invention provide a composition comprising 11-methoxyyangonin and / or flavanone:

[0014]

[0015] and at least one compound selected from the group consisting of dihydromethysticin, methysticin, dihydrokavain, kavain and desmethoxyyangonin, wherein the composition is substantially free of kava piperine B and / or kava piperine A.

[0016] Certain embodiments of the present invention provide a composition comprising at least one compound selected from the group consisting of: dihydrocapsaicin, wherein the weight percentage of dihydrocapsaicin in the composition is about 20 to 99%; capsaicin, wherein the weight percentage of capsaicin in the composition is about 10 to 99%; dihydrocapsaicin, wherein the weight percentage of dihydrocapsaicin in the composition is about 40 to 99%; capsaicin, wherein the weight percentage of capsaicin in the composition is about 40 to 99%; demethoxycapsaicin, wherein the weight percentage of demethoxycapsaicin in the composition is about 30 to 99%; and 11-methoxycapsaicin, wherein the weight percentage of 11-methoxycapsaicin in the composition is about 20 to 99%.

[0017] Certain embodiments of the present invention provide a kava extract comprising 11-methoxycapsaicin and / or flavanones:

[0018]

[0019] and at least one compound selected from the group consisting of dihydrocapsaicin, capsaicin, dihydrocapsaicin, capsaicin and demethoxycapsaicin, wherein the extract is substantially free of kavapetin B and / or kavapetin A.

[0020] Certain embodiments of the present invention provide a kava extract consisting essentially of dihydrocapsaicin, 11-methoxycapsaicin, demethoxycapsaicin, dihydrocapsaicin, capsaicin, capsaicin, and flavanones:

[0021]

[0022] Certain embodiments of the present invention provide a pharmaceutical composition comprising a composition or kava extract as described herein and a pharmaceutically acceptable carrier.

[0023] Certain embodiments of the present invention provide a method for treating or preventing cancer in a mammal in need of such treatment, comprising administering to the mammal dihydrocapsamone and a carrier, wherein the dihydrocapsamone is substantially free of other kava extract components.

[0024] Certain embodiments of the present invention provide a method for treating or preventing cancer in a mammal in need of such treatment, comprising administering to the mammal anthemis nobilis and a carrier, wherein the anthemis nobilis is substantially free of other kava extract components.

[0025] Certain embodiments of the present invention provide a method for treating or preventing cancer in a mammal in need of such treatment, comprising administering to the mammal dihydrocapsaicin and a carrier, wherein the dihydrocapsaicin is substantially free of other kava extract components.

[0026] Certain embodiments of the present invention provide a method for treating or preventing cancer in a mammal in need of such treatment, comprising administering to the mammal kava extract and a carrier, wherein the kava extract is substantially free of other kava extract components.

[0027] Certain embodiments of the present invention provide a method for treating or preventing cancer in a mammal in need of such treatment, comprising administering to the mammal demethoxycapsaicin and a carrier, wherein the demethoxycapsaicin is substantially free of other kava extract components.

[0028] Certain embodiments of the present invention provide a method for treating or preventing cancer in a mammal in need of such treatment, comprising administering to the mammal 11-methoxycapsaicin and a carrier, wherein the 11-methoxycapsaicin is substantially free of other kava extract components.

[0029] Certain embodiments of the present invention provide a method for treating or preventing cancer in a mammal in need of such treatment, comprising administering to the mammal a composition or kava extract as described herein.

[0030] Certain embodiments of the present invention provide methods for preventing tumorigenesis, reducing DNA damage, reducing protein damage and / or detoxifying physical or chemical carcinogens in a mammal in need of such treatment, comprising administering to the mammal dihydrocapsamone and a carrier, wherein the dihydrocapsamone is substantially free of other kava extract components.

[0031] Certain embodiments of the present invention provide methods for preventing tumorigenesis, reducing DNA damage, reducing protein damage and / or detoxifying physical or chemical carcinogens in a mammal in need of such treatment, comprising administering to the mammal anthemis nobilis and a carrier, wherein the anthemis nobilis is substantially free of other kava extract components.

[0032] Certain embodiments of the present invention provide methods for preventing tumorigenesis, reducing DNA damage, reducing protein damage, and / or detoxifying physical or chemical carcinogens in a mammal in need of such treatment, comprising administering to the mammal dihydrocapsaicin and a carrier, wherein the dihydrocapsaicin is substantially free of other kava extract components.

[0033] Certain embodiments of the present invention provide methods for preventing tumorigenesis, reducing DNA damage, reducing protein damage, and / or detoxifying physical or chemical carcinogens in a mammal in need of such treatment, comprising administering to the mammal a kava extract and a carrier, wherein the kava extract is substantially free of other kava extract components.

[0034] Certain embodiments of the present invention provide methods for preventing tumorigenesis, reducing DNA damage, reducing protein damage, and / or detoxifying physical or chemical carcinogens in a mammal in need of such treatment, comprising administering to the mammal demethoxycapsaicin and a carrier, wherein the demethoxycapsaicin is substantially free of other kava extract components.

[0035] Certain embodiments of the present invention provide methods for preventing tumorigenesis, reducing DNA damage, reducing protein damage and / or detoxifying physical or chemical carcinogens in a mammal in need of such treatment, comprising administering to the mammal 11-methoxy capsaicin and a carrier, wherein the 11-methoxy capsaicin is substantially free of other kava extract components.

[0036] Certain embodiments of the invention provide methods for preventing tumorigenesis, reducing DNA damage, reducing protein damage, and / or detoxifying physical or chemical carcinogens in a mammal in need of such treatment, comprising administering to the mammal a composition or kava extract as described herein.

[0037] The present invention also includes the following items:

[0038] 1. A method comprising:

[0039] a) combining ethanolated kava extract and silica gel to obtain a mixture;

[0040] b) evaporating the mixture to obtain silica gel having the kava residue adsorbed thereon;

[0041] c) loading the silica gel having the kava residue adsorbed thereon onto a chromatographic column to obtain a kava adsorbed silica gel column;

[0042] d) eluting the silica gel column adsorbed with kava with a solvent system to obtain a first kava extract fraction, a second kava extract fraction, and a third kava extract fraction, wherein the first kava extract fraction consists essentially of non-polar compounds including kava piperine, the second kava extract fraction consists essentially of kavalactones and flavanones, and the third kava extract fraction consists essentially of polar compounds.

[0043] 2. The method of item 1, wherein step d) comprises eluting with 28% ethyl acetate (EA) and 72% hexane (Hex) for 5 column volumes (CV), followed by eluting with 90% EA and 10% Hex for 4.1 CV, and then eluting with 35% MeOH and 65% EA for 5.5 CV.

[0044] 3. The method of claim 1 , wherein the weight ratio of the kava residue to the total weight of the silica gel having the kava residue adsorbed thereon is 0.3±0.2.

[0045] 4. A second kava extract fraction prepared by the method of any one of items 1-3.

[0046] 5. A composition comprising 11-methoxy capsaicin and / or flavanone:

[0047]

[0048] and at least one compound selected from the group consisting of dihydrocapsaicin, capsaicin, dihydrocapsaicin, capsaicin and demethoxycapsaicin, wherein the composition is substantially free of kava piperine B and / or kava piperine A.

[0049] 6. The composition of claim 5, wherein the at least one compound is dihydrocapsaicin.

[0050] 7. The composition of claim 5, wherein at least two compounds are selected from the group consisting of dihydrocapsaicin, capsaicin, dihydrocapsaicin, capsaicin and demethoxycapsaicin, wherein the composition is substantially free of kava piperine B and / or kava piperine A.

[0051] 8. The composition of claim 5, wherein at least three compounds are selected from the group consisting of dihydrocapsaicin, capsaicin, dihydrocapsaicin, capsaicin, and demethoxycapsaicin, wherein the composition is substantially free of kava pepper B and / or kava pepper A.

[0052] 9. The composition of claim 5, wherein at least four compounds are selected from the group consisting of dihydrocapsaicin, capsaicin, dihydrocapsaicin, capsaicin, and demethoxycapsaicin, wherein the composition is substantially free of kava pepper B and / or kava pepper A.

[0053] 10. The composition of any one of items 5-9, wherein the composition comprises 11-methoxycapsaicin.

[0054] 11. The composition of any one of items 5-9, wherein the composition comprises flavanone:

[0055]

[0056] 12. The composition of any one of items 5-9, wherein the composition comprises 11-methoxycapsaicin and flavanone:

[0057]

[0058] 13. The composition of any one of items 5-12, which is substantially free of kava piperine A.

[0059] 14. The composition of any one of items 5 to 13, which is substantially free of bornyl ester of 3,4-methylenedioxycinnamic acid:

[0060]

[0061] 15. The composition of any one of items 5 to 14, which is substantially free of bornyl ester of cinnamic acid:

[0062]

[0063] 16. The composition of any one of items 5 to 15, which is substantially free of flavanones:

[0064]

[0065] 17. The composition of any one of items 5 to 16, which is substantially free of chloranone.

[0066] 18. The composition of any one of items 5-17, which is substantially free of kava B.

[0067] 19. A composition comprising at least one compound selected from the group consisting of: dihydrocapsaicin, wherein the weight percentage of dihydrocapsaicin in the composition is about 20 to 99%; capsaicin, wherein the weight percentage of capsaicin in the composition is about 10 to 99%; dihydrocapsaicin, wherein the weight percentage of dihydrocapsaicin in the composition is about 40 to 99%; capsaicin, wherein the weight percentage of capsaicin in the composition is about 40 to 99%; demethoxycapsaicin, wherein the weight percentage of demethoxycapsaicin in the composition is about 30 to 99%; and 11-methoxycapsaicin, wherein the weight percentage of 11-methoxycapsaicin in the composition is about 20 to 99%.

[0068] 20. The composition of claim 19, wherein the at least one compound is dihydrocapsaicin, and wherein the weight percentage of dihydrocapsaicin in the composition is about 20 to 99%.

[0069] 21. The composition of claim 19, wherein the at least one compound is capsaicin, and wherein the weight percentage of capsaicin in the composition is 40 to 99%.

[0070] 22. The composition of claim 19, wherein the at least one compound is capsaicin, and wherein the weight percentage of capsaicin in the composition is 10 to 99%.

[0071] 23. The composition of claim 19, wherein the at least one compound is dihydrocapsaicin, and wherein the weight percentage of dihydrocapsaicin in the composition is 40 to 99%.

[0072] 24. The composition of claim 19, wherein the at least one compound is demethoxycapsaicin, and wherein the weight percentage of demethoxycapsaicin in the composition is 30 to 99%.

[0073] 25. The composition of claim 19, wherein the at least one compound is 11-methoxy capsaicin, and wherein the weight percentage of 11-methoxy capsaicin in the composition is 20 to 99%.

[0074] 26. The composition of claim 19, which is substantially free of capsaicin.

[0075] 27. The composition of any one of items 19-26, further comprising flavanone:

[0076] and

[0077] The weight percentage of flavanone in the composition is about 20 to 99%.

[0078] 28. The composition of any one of items 19-27, which is substantially free of kava pepper B.

[0079] 29. The composition of any one of items 19-28, which is substantially free of kava piperine A.

[0080] 30. The composition of any one of items 19-29, which is substantially free of bornyl ester of 3,4-methylenedioxycinnamic acid:

[0081]

[0082] 31. The composition of any one of items 19-30, which is substantially free of bornyl ester of cinnamic acid:

[0083]

[0084] 32. The composition of any one of items 19-31, which is substantially free of flavanones:

[0085]

[0086] 33. The composition of any one of items 19-32, which is substantially free of chloranone.

[0087] 34. The composition of any one of items 19-33, wherein the composition is a kava extract.

[0088] 35. A kava extract comprising 11-methoxycapsaicin and / or flavanones:

[0089]

[0090] and at least one compound selected from the group consisting of dihydrocapsaicin, capsaicin, dihydrocapsaicin, capsaicin and demethoxycapsaicin, wherein the extract is substantially free of kava piperine B and / or kava piperine A.

[0091] 36. The kava extract of claim 35, wherein the at least one compound is dihydrocapsaicin.

[0092] 37. The kava extract of claim 35, wherein the at least one compound is capsaicin.

[0093] 38. The kava extract of claim 35, wherein the at least one compound is capsaicin.

[0094] 39. The kava extract of claim 35, wherein the at least one compound is dihydrocapsara.

[0095] 40. The kava extract of claim 35, wherein the at least one compound is demethoxycapsaicin.

[0096] 41. The kava extract of any one of items 35-40, which is substantially free of kava piperine B.

[0097] 42. The kava extract of any one of items 35-41, which is substantially free of kavapenin A.

[0098] 43. The kava extract of any one of items 35-42, which is substantially free of bornyl ester of 3,4-methylenedioxycinnamic acid:

[0099]

[0100] 44. The kava extract of any one of items 33-40, which is substantially free of bornyl esters of cinnamic acid:

[0101]

[0102] 45. The kava extract of any one of items 35-44, which is substantially free of flavanones:

[0103]

[0104] 46. ​​The kava extract of any one of items 35-45, which is substantially free of chloranone.

[0105] 47. A kava extract consisting essentially of dihydrocapsaicin, 11-methoxycapsaicin, demethoxycapsaicin, dihydrocapsaicin, capsaicin, capsaicin, and flavanones:

[0106]

[0107] 48. The composition or kava extract of any one of items 4-47, wherein the composition or extract is suitable for ingestion by a mammal (e.g., a human).

[0108] 49. The composition or kava extract of any one of items 4-48, wherein the composition or extract is formulated as a tablet, capsule, powder, spray, chewing gum, cream, gel, stent, inhalable, patch, nanoemulsion or liquid.

[0109] 50. A pharmaceutical composition comprising the composition or kava extract of any one of items 4-49 and a pharmaceutically acceptable carrier.

[0110] 51. A method for treating or preventing cancer in a mammal in need of such treatment, comprising administering to said mammal dihydrocapsamone and a carrier, wherein said dihydrocapsamone is substantially free of other kava extract components.

[0111] 52. A composition for the prophylactic or therapeutic treatment of cancer in a mammal, the composition comprising dihydrocapsaicin and a carrier, wherein the dihydrocapsaicin is substantially free of other kava extract components.

[0112] 53. The method of claim 51 or the composition of claim 52, wherein the other kava extract components are selected from the group consisting of: 11-methoxy capsaicin, demethoxy capsaicin, dihydrocapsaicin, capsaicin, capsaicin, chloranone, kava piperine B, kava piperine A,

[0113]

[0114] 54. A method for treating or preventing cancer in a mammal in need of such treatment comprising administering to said mammal anthemis nobilis and a carrier, wherein said anthemis nobilis is substantially free of other kava extract components.

[0115] 55. A composition for the prophylactic or therapeutic treatment of cancer in a mammal, the composition comprising capsaicin and a carrier, wherein the capsaicin is substantially free of other kava extract components.

[0116] 56. The method of claim 54 or the composition of claim 55, wherein the other kava extract components are selected from the group consisting of: 11-methoxy capsaicin, demethoxy capsaicin, dihydrocapsaicin, capsaicin, dihydrocapsaicin, chloranone, kava piperine B, kava piperine A,

[0117]

[0118] 57. A method for treating or preventing cancer in a mammal in need of such treatment comprising administering to said mammal dihydrocapsaicin and a carrier, wherein said dihydrocapsaicin is substantially free of other kava extract components.

[0119] 58. A composition for the prophylactic or therapeutic treatment of cancer in a mammal, said composition comprising dihydrocapsaicin and a carrier, wherein said dihydrocapsaicin is substantially free of other kava extract components.

[0120] 59. The method of claim 57 or the composition of claim 58, wherein the other kava extract components are selected from the group consisting of: 11-methoxy capsaicin, demethoxy capsaicin, dihydro capsaicin, capsaicin, capsaicin, chloranone, kava pepper B, kava pepper A,

[0121]

[0122] 60. A method for treating or preventing cancer in a mammal in need of such treatment comprising administering to said mammal zapotein and a carrier, wherein said zapotein is substantially free of other kava extract components.

[0123] 61. A composition for the prophylactic or therapeutic treatment of cancer in a mammal, said composition comprising capsaicin and a carrier, wherein said capsaicin is substantially free of other kava extract components.

[0124] 62. The method of claim 60 or the composition of claim 61, wherein the other kava extract components are selected from the group consisting of: 11-methoxy capsaicin, demethoxy capsaicin, dihydrocapsaicin, dihydrocapsaicin, capsaicin, chloranone, kava piperine B, kava piperine A,

[0125]

[0126]

[0127] 63. A method for treating or preventing cancer in a mammal in need of such treatment, comprising administering to said mammal demethoxycapsaicin and a carrier, wherein said demethoxycapsaicin is substantially free of other kava extract components.

[0128] 64. A composition for the prophylactic or therapeutic treatment of cancer in a mammal, said composition comprising demethoxycapsaicin and a carrier, wherein said demethoxycapsaicin is substantially free of other kava extract components.

[0129] 65. The method of claim 63 or the composition of claim 64, wherein the other kava extract components are selected from the group consisting of: 11-methoxy capsaicin, dihydrocapsaicin, dihydrocapsaicin, capsaicin, capsaicin, bromocapsaicin, kava piperine B, kava piperine A,

[0130]

[0131] 66. A method for treating or preventing cancer in a mammal in need of such treatment, comprising administering to said mammal 11-methoxy capsaicin and a carrier, wherein said 11-methoxy capsaicin is substantially free of other kava extract components.

[0132] 67. A composition for the prophylactic or therapeutic treatment of cancer in a mammal, said composition comprising 11-methoxy capsaicin and a carrier, wherein said 11-methoxy capsaicin is substantially free of other kava extract components.

[0133] 68. The method of claim 66 or the composition of claim 67, wherein the other kava extract components are selected from the group consisting of: demethoxy capsaicin, dihydrocapsaicin, dihydrocapsaicin, capsaicin, capsaicin, chloranone, kava piperine B, kava piperine A,

[0134]

[0135] 69. The method of any one of items 51, 54, 57, 60, 63 and 66 or the composition of any one of items 52, 55, 58, 61, 64 and 67, wherein the other kava extract components are selected from the group consisting of: bromocriptone, kava piperin B, kava piperin A,

[0136]

[0137] 70. A method for treating or preventing cancer in a mammal in need of such treatment, comprising administering to the mammal the composition or kava extract of any one of items 4-50.

[0138] 71. The composition or kava extract of any one of items 4-50, for use in the prophylactic or therapeutic treatment of cancer in a mammal.

[0139] 72. Use of a composition or kava extract as described in any one of items 4 to 50 in the preparation of a medicament for treating or preventing cancer in a mammal.

[0140] 73. The method, composition or use of any one of items 51-72, wherein the cancer is lung cancer, prostate cancer, skin cancer, melanoma, genitourinary cancer, colorectal cancer, breast cancer, ovarian cancer, esophageal cancer, pancreatic cancer, bladder cancer, cervical cancer, liver cancer, kidney cancer or renal cancer, head and neck cancer, brain cancer or various hematological cancers.

[0141] 74. A method for preventing tumorigenesis, reducing DNA damage, reducing protein damage and / or detoxifying physical or chemical carcinogens in a mammal in need of such treatment, comprising administering to said mammal dihydrocapsamone and a carrier, wherein said dihydrocapsamone is substantially free of other kava extract components.

[0142] 75. A composition for preventing tumorigenesis, reducing DNA damage, reducing protein damage and / or detoxifying physical or chemical carcinogens in a mammal, the composition comprising dihydrocapsaicin and a carrier, wherein the dihydrocapsaicin is substantially free of other kava extract components.

[0143] 76. The method of claim 74 or the composition of claim 75, wherein the other kava extract components are selected from the group consisting of: 11-methoxy capsaicin, demethoxy capsaicin, dihydrocapsaicin, capsaicin, capsaicin, chloranone, kava piperine B, kava piperine A,

[0144]

[0145] 77. A method for preventing tumorigenesis, reducing DNA damage, reducing protein damage and / or detoxifying physical or chemical carcinogens in a mammal in need of such treatment, comprising administering to said mammal anthemis nobilis and a carrier, wherein said anthemis nobilis is substantially free of other kava extract components.

[0146] 78. A composition for preventing tumorigenesis, reducing DNA damage, reducing protein damage and / or detoxifying physical or chemical carcinogens in a mammal, the composition comprising capsaicin and a carrier, wherein the capsaicin is substantially free of other kava extract components.

[0147] 79. The method of claim 77 or the composition of claim 78, wherein the other kava extract components are selected from the group consisting of: 11-methoxy capsaicin, demethoxy capsaicin, dihydrocapsaicin, capsaicin, dihydrocapsaicin, chloranone, kava piperine B, kava piperine A,

[0148]

[0149] 80. A method for preventing tumorigenesis, reducing DNA damage, reducing protein damage and / or detoxifying physical or chemical carcinogens in a mammal in need of such treatment, comprising administering to said mammal dihydrocapsaicin and a carrier, wherein said dihydrocapsaicin is substantially free of other kava extract components.

[0150] 81. A composition for preventing tumorigenesis, reducing DNA damage, reducing protein damage and / or detoxifying physical or chemical carcinogens in a mammal, the composition comprising dihydrocapsaicin and a carrier, wherein the dihydrocapsaicin is substantially free of other kava extract components.

[0151] 82. The method of claim 80 or the composition of claim 81, wherein the other kava extract components are selected from the group consisting of: 11-methoxy capsaicin, demethoxy capsaicin, dihydrocapsaicin, capsaicin, capsaicin, chloranone, kava piperine B, kava piperine A,

[0152]

[0153] 83. A method for preventing tumorigenesis, reducing DNA damage, reducing protein damage and / or detoxifying physical or chemical carcinogens in a mammal in need of such treatment, comprising administering to said mammal a zebubble and a carrier, wherein said zebubble is substantially free of other kava extract components.

[0154] 84. A composition for preventing tumorigenesis, reducing DNA damage, reducing protein damage and / or detoxifying physical or chemical carcinogens in a mammal, the composition comprising capsaicin and a carrier, wherein the capsaicin is substantially free of other kava extract components.

[0155] 85. The method of claim 83 or the composition of claim 84, wherein the other kava extract components are selected from the group consisting of: 11-methoxy capsaicin, demethoxy capsaicin, dihydrocapsaicin, dihydrocapsaicin, capsaicin, chloranone, kava pepper B, kava pepper A,

[0156]

[0157] 86. A method for preventing tumorigenesis, reducing DNA damage, reducing protein damage and / or detoxifying physical or chemical carcinogens in a mammal in need of such treatment, comprising administering to said mammal demethoxycapsaicin and a carrier, wherein said demethoxycapsaicin is substantially free of other kava extract components.

[0158] 87. A composition for preventing tumorigenesis, reducing DNA damage, reducing protein damage and / or detoxifying physical or chemical carcinogens in a mammal, the composition comprising demethoxy capsaicin and a carrier, wherein the demethoxy capsaicin is substantially free of other kava extract components.

[0159] 88. The method of claim 86 or the composition of claim 87, wherein the other kava extract components are selected from the group consisting of: 11-methoxy capsaicin, capsaicin, dihydro capsaicin, dihydro capsaicin, capsaicin, chloranone, kava pepper B, kava pepper A,

[0160]

[0161]

[0162] 89. A method for preventing tumorigenesis, reducing DNA damage, reducing protein damage and / or detoxifying physical or chemical carcinogens in a mammal in need of such treatment, comprising administering to said mammal 11-methoxy capsaicin and a carrier, wherein said 11-methoxy capsaicin is substantially free of other kava extract components.

[0163] 90. A composition for preventing tumorigenesis, reducing DNA damage, reducing protein damage and / or detoxifying physical or chemical carcinogens in a mammal, the composition comprising 11-methoxy capsaicin and a carrier, wherein the 11-methoxy capsaicin is substantially free of other kava extract components.

[0164] 91. The method of claim 89 or the composition of claim 90, wherein the other kava extract components are selected from the group consisting of: demethoxy capsaicin, capsaicin, dihydro capsaicin, dihydro capsaicin, capsaicin, chloranone, kava pepper B, kava pepper A,

[0165]

[0166] 92. The method of any one of items 74, 77, 80, 83, 86 and 89 or the composition of any one of items 75, 78, 81, 84, 87 and 90, wherein the other kava extract components are selected from the group consisting of: bromocriptone, kava piperin B, kava piperin A,

[0167]

[0168]

[0169] 93. A method for preventing tumorigenesis, reducing DNA damage, reducing protein damage and / or detoxifying physical or chemical carcinogens in a mammal in need of such treatment, comprising administering to the mammal a composition or kava extract as described in any one of items 4-50.

[0170] 94. The composition or kava extract of any one of items 4-50, for use in preventing tumorigenesis, reducing DNA damage, reducing protein damage and / or detoxifying physical or chemical carcinogens in a mammal.

[0171] 95. Use of a composition or kava extract as described in any one of items 4 to 50 for the preparation of a medicament for preventing tumorigenesis, reducing DNA damage, reducing protein damage and / or detoxifying physical or chemical carcinogens in a mammal.

[0172] 96. The method, composition or use of any one of items 74-95, wherein the DNA damage is a DNA adduct.

[0173] 97. The method of claim 96, wherein the DNA adduct is selected from the group consisting of BaP, PhIP, POB and PHB adducts.

[0174] 98. The method of claim 96, wherein the DNA adduct is 6 -Methylguanine DNA adducts.

[0175] Description of Figures and Tables

[0176] Figure 1 Characterization of the effects of kava and kava fractions on DNA adducts induced by NNK in the lungs of A / J mice (n=3 per group): *p<0.05; **p<0.01; ***p<0.001. A. Amounts of DNA adducts at different time points after NNK treatment; NNK alone: NNK+ Kava: ). B. Relative amounts of DNA adducts in the NNK+kava treated group at different time points after NNK treatment (amount of kava treatment normalized to the amount induced by NNK alone at the same time point). C. Amount of DNA adducts treated with different kava fractions 24h after NNK treatment. D. Relative amounts of DNA adducts from different kava fractions normalized to the amount induced by NNK alone at the 24h time point.

[0177] Figure 2 Two metabolic activation pathways of NNK leading to differently methylated DNA adducts and 4-(3-pyridyl)-4-oxobutan-1-yl (pob)-DNA adducts. Solid blocks indicate the measured reduction of different types of DNA adducts, such as 6 -mG to 7-pobG, O 2 -pobdT and O 6 Dashed blocks represent hypothetical points of action by kava chemicals or their metabolites: (i) differential inhibition of cytochrome P450 isoform-mediated metabolic activation of NNK or (ii) direct reaction with NNK methylene hydroxylated metabolites acting as chemical traps.

[0178] Figure 3 . Compounds in fraction B, including dihydrocapsaicin and capsaicin.

[0179] Figure 4.Designed to evaluate the chemopreventive efficacy of different kava treatment regimens for NNK-induced lung adenoma formation in A / J mice. Starting at 6-7 weeks of age, each group of female A / J mice (Groups 2-9) was treated with two doses of NNK (100 and 67 mg / kg body weight, respectively, on the 7th and 14th days) injected intraperitoneally (ip) in 0.1 mL saline. Two doses of saline (each 0.1 mL) were given to Group 1 mice on the 7th and 14th days. Groups 1 and 2 mice were kept on standard diet treatment for the entire experimental period. Groups 3-9 of mice were given a diet supplemented with kava at a dose of 5 mg / g diet according to the following schedule: Group 3 (Day 1-14); Group 4 (Day 1-21); Group 5 (Day 1-119); Group 6 (Day 15-119); Group 7 (Day 15-28); Group 8 (Day 22-119) and Group 9 (Day 29-119). The study was terminated on Day 119.

[0180] Figure 5 Representative photomicrographs of H&E stained lung sections from negative control mice (A), mice treated with NNK alone (B), and mice treated with NNK + kava at a dose of 5 mg / g diet (C) (n = 4 in each group).

[0181] Figure 6 .Different Kava fractions 1 H-NMR spectra and mass balance of each fraction. I. Kavas; II. Fraction A; III. Kavas reconstructed from fractions A, B and C; IV. Fraction B; V, comparison of kava (bottom line) with kava reconstructed from fractions A, B and C (top line); VI. Fraction C; VII. Mass balance of each fraction.

[0182] Figure 7 HPLC traces of traditional kava, kava from Gaia Herbs, reconstituted kava, and fractions A, B, and C.

[0183] Figure 8 Chemical substances isolated from kava and their natural abundance.

[0184] Fig. 9 . Genitourinary tract weights (16 weeks of age) (excluding 1 tumor-bearing mouse). For each group, the bars on the left represent wild-type mice and the bars on the right represent TRAMP mice.

[0185] Fig.10 . Prostate weight (16 weeks of age). For each group, the bars on the left represent wild-type mice and the bars on the right represent TRAMP mice.

[0186] Fig.11.GU weight (28 weeks of age) (excluding tumor-bearing mice). For each group, the bars on the left represent wild-type mice and the bars on the right represent TRAMP mice.

[0187] Fig.12 . Prostate weight (28 weeks of age) (excluding tumor-bearing mice). For each group, the bars on the left represent wild-type mice and the bars on the right represent TRAMP mice.

[0188] Fig.13 .Effects of dihydrocapsaicin and capsaicin on reducing lung tumor volume inoculated with human lung H2009 cancer cell line.

[0189] Fig.14 Effect of fractions A, B and C on reducing the tumor volume of lung tumors inoculated with human lung A549 cancer cell line. In the bottom panel (B), the bars represent from left to right: vehicle control, Kava fraction A, Kava fraction B and Kava fraction C.

[0190] Fig.15 Effect and safety of Kava and fraction B on reducing tumor volume of lung tumors inoculated with human lung A549 cancer cell line.

[0191] Fig.16 Metabolic pathways of NNK leading to the formation of DNA adducts and putative sites of action of DHM for selectively reducing a subset of NNK-induced DNA lesions in the lungs of A / J mice—preferential inhibition of NNAL activation via hydroxylation (A) or enhancement of NNAL detoxification via glucuronidation (B).

[0192] Fig.17 Characterization of natural kavalactones against NNK-induced DNA damage in lung tissue of A / J mice. A, Chemical structures of five natural kavalactones isolated from fraction B. B, Their effects on NNK-induced POB adducts and O 6 Effect of -mG adduct. When ONE-WAY ANOVA was statistically significant, comparison with NNK-treated group was performed by Dunnett's test. n=3 per group. *p<0.05 and **p<0.01.

[0193] Fig.18 . Characterization of the effects of different drugs on NNK-induced DNA damage in lung tissue of A / J mice. A, Natural (+)-DHM has a stronger effect on NNK-induced O compared with kava, (+)-DHK and synthetic (±)-DHM. 6Dose-response effects of 7-mG (a) and three POB adducts (bd). B, Dose-response effects of natural (+)-DHM on NNK-induced 7-mG (a) and three PHB adducts (bd) compared with kava, (+)-DHK and synthetic (±)-DHM. C, Percentage of different DNA adducts from mice treated with (+)-DHM (1 mg / g diet) relative to those from control mice treated with NNK. For A and B, when ONE-WAY ANOVA was statistically significant, comparisons were made to the NNK-treated group by Dunnett's test. n = 3 per group. *p<0.05 and **p<0.01. For C, ONE-WAY ANOVA was not statistically significant.

[0194] Fig.19 Body weight and major organs from control mice and mice exposed to (+)-DHM at a dose of 0.5 mg / g diet at week 17. The control group (n=5) and (+)-DHM treated group (n=10) were compared using a two-sided Student t-test and p values ​​are given.

[0195] Fig. 20 . Body weight and food consumption data from A / J mice in control and (+)-DHM (0.5 mg / g diet) treated groups. A, Weekly time course of body weight change. B, Weekly estimated average daily food consumption (disappearance). Weekly body weights of control and (+)-DHM treated mice were analyzed by two-sided Student's t-test, and neither control was statistically significant.

[0196] Fig.21 Clinical chemistry results of serum samples from control mice and mice exposed to (+)-DHM at a dose of 0.5 mg / g diet. A, 8 weeks. B, 17 weeks. P values ​​are given when two-sided Student t-tests can be used to compare the control group (n=5) and the (+)-DHM treatment group (n=10).

[0197] Fig. 22 Hematological results of blood samples from control mice and mice exposed to (+)-DHM at a dose of 0.5 mg / g diet for 17 weeks. P values ​​are given when two-sided Student's t-test can be used to compare the control group (n=4) and the treatment group (n=10).

[0198] Fig.23 Effect of daily kava treatment (500 mg / kg body weight) via gavage for 14 weeks on serum ALT (A) and AST (B) in mice. The control group (n=4) and the kava-treated group (n=4) were compared using a two-sided Student's t-test, and p values ​​are given.

[0199] Fig.24 Effects of daily kava treatment (500 mg / kg body weight) via gavage for 3 days on serum ALT and AST in mice and liver injury with / without APAP treatment (800 mg / kg body weight). A. Serum ALT and AST. B. Number of mice with different grades of liver injury. C. Relationship between plasma ALT, AST and grade of liver injury. For A, comparison with APAP-treated group was performed by Dunnett's test when ONE-WAY ANOVA was statistically significant (n=8-15).

[0200] Fig.25 .Chemical structures of kava pepper A, B and dihydrocapsaicin.

[0201] Fig.26 Effects of daily administration of DHM (37.5 mg / kg) or FKB (11.5 mg / kg) via gavage for 3 days on serum ALT and AST in mice with / without APAP treatment. When ONE-WAY ANOVA was statistically significant (n=6-15), comparisons with the APAP-treated group were made by Dunnett's test.

[0202] Fig. 27 .Dose-response effects of daily administration of FKA and FKA via gavage for 3 days on serum ALT and AST in mice and liver treated with / without APAP. A&B. Serum ALT and AST. Comparison with APAP-treated group was performed by Dunnett's test when ONE-WAY ANOVA was statistically significant (n=5). C. Serum levels of ALT and AST from mice that died in the high-dose FKA and FKB groups co-treated with APAP. D. Micrographs of H&E-stained livers from control mice (Panel A) and mice treated with FKA and FKB+APAP (Panel B). Note: Extensive nuclear fragmentation (arrows) reflects acute necrosis of hepatocytes (eosinophilia) in mice treated with FKA, FKB, and APAP (Panel B).

[0203] Fig.28 Dose-response effect of dihydrocapsara in reducing DNA damage induced by 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (A) and benzo(a)pyrene (B) in Hepe1c1c7 cells.

[0204] Fig.29 Effects of different kava treatment regimens on the incidence and multiplicity of NNK-induced lung tumors in A / J mice at the day 119 endpoint.

[0205] Fig.30 Effects of kava on the incidence and multiplicity of NNK-induced lung tumors in A / J mice at the 25-week (175-day) and 34-week (238-day) endpoints.

[0206] Fig.31 Effects of daily bolus kava gavage on the incidence and multiplicity of NNK-induced lung tumors in A / J mice.

[0207] Fig.32 Dose-response effects of kava and different kava fractions on the incidence and multiplicity of NNK-induced lung tumors in A / J mice.

[0208] Fig.33 . Experimental design for preventing prostate cancer using the TRAMP model.

[0209] Fig.34 . Prostate tumor at 28 weeks of age.

[0210] Fig.35 . Prostate tumor weight (g) and lobe location (VP = ventral prostate lobe).

[0211] Fig.36 Effects of different drugs on the incidence and multiplicity of NNK-induced lung tumors in A / J mice. DETAILED DESCRIPTION

[0212] Certain embodiments of the present invention provide a composition comprising at least two compounds selected from the group consisting of dihydrocapsaicin, capsaicin, dihydrocapsaicin, capsaicin, demethoxycapsaicin, and 11-methoxycapsaicin, wherein the composition is substantially free of kava piperine B and / or kava piperine A.

[0213] In certain embodiments, the at least two compounds are dihydrocapsaicin and capsaicin.

[0214] In certain embodiments, the at least two compounds are dihydrocapsaicin and dihydrocapsaicin.

[0215] In certain embodiments, the at least two compounds are dihydrocapsaicin and capsaicin.

[0216] In certain embodiments, the at least two compounds are capsaicin and dihydrocapsaicin.

[0217] In certain embodiments, the at least two compounds are capsaicin and sonocapsaicin.

[0218] In certain embodiments, the at least two compounds are dihydrocapsaicin and capsaicin.

[0219] In certain embodiments, the composition comprises at least three compounds selected from the group consisting of dihydrocapsaicin, capsaicin, dihydrocapsaicin, capsaicin, demethoxycapsaicin, and 11-methoxycapsaicin, wherein the composition is substantially free of kava piperine B and / or kava piperine A.

[0220] In certain embodiments, the at least three compounds are dihydrocapsaicin, capsaicin, and dihydrocapsaicin.

[0221] In certain embodiments, the at least three compounds are dihydrocapsaicin, capsaicin, and capsaicin.

[0222] In certain embodiments, the at least three compounds are dihydrocapsaicin, dihydrocapsaicin, and capsaicin.

[0223] In certain embodiments, the at least three compounds are capsaicin, dihydrocapsaicin, and capsaicin.

[0224] In certain embodiments, the composition comprises at least four compounds selected from the group consisting of dihydrocapsaicin, capsaicin, dihydrocapsaicin, capsaicin, demethoxycapsaicin, and 11-methoxycapsaicin, wherein the composition is substantially free of kavapetin B and / or kavapetin A.

[0225] In certain embodiments, the composition comprises at least five compounds selected from the group consisting of dihydrocapsaicin, capsaicin, dihydrocapsaicin, capsaicin, demethoxycapsaicin, and 11-methoxycapsaicin, wherein the composition is substantially free of kava piperine B and / or kava piperine A.

[0226] In certain embodiments, the composition comprises dihydrocapsaicin, capsaicin, dihydrocapsaicin, capsaicin, demethoxycapsaicin, and 11-methoxycapsaicin, wherein the composition is substantially free of kavapetin B and / or kavapetin A.

[0227] In certain embodiments, the composition further comprises a flavanone:

[0228]

[0229] In certain embodiments, the composition is substantially free of kava B.

[0230] In certain embodiments, the composition is substantially free of kava A.

[0231] In certain embodiments, the composition is substantially free of bornyl ester of 3,4-methylenedioxycinnamic acid:

[0232]

[0233] In certain embodiments, the composition is substantially free of bornyl esters of cinnamic acid:

[0234]

[0235] In certain embodiments, the composition is substantially free of flavanones:

[0236]

[0237] In certain embodiments, the composition is substantially free of pinostrobin.

[0238] Certain embodiments of the present invention provide a composition comprising 11-methoxy kavachianine and / or flavanones:

[0239]

[0240] and at least one compound selected from the group consisting of dihydrocapsaicin, capsaicin, dihydrocapsaicin, capsaicin and demethoxycapsaicin, wherein the composition is substantially free of kava piperine B and / or kava piperine A.

[0241] In certain embodiments, the at least one compound is dihydrocapsaicin.

[0242] In certain embodiments, the composition comprises at least two compounds selected from the group consisting of dihydrocapsaicin, capsaicin, dihydrocapsaicin, capsaicin, and demethoxycapsaicin, wherein the composition is substantially free of kavapetin B and / or kavapetin A.

[0243] In certain embodiments, the composition comprises at least three compounds selected from the group consisting of dihydrocapsaicin, capsaicin, dihydrocapsaicin, capsaicin, and demethoxycapsaicin, wherein the composition is substantially free of kavapetin B and / or kavapetin A.

[0244] In certain embodiments, the composition comprises at least four compounds selected from the group consisting of dihydrocapsaicin, capsaicin, dihydrocapsaicin, capsaicin, and demethoxycapsaicin, wherein the composition is substantially free of kavapetin B and / or kavapetin A.

[0245] In certain embodiments, the composition comprises 11-methoxycapsaicin.

[0246] In certain embodiments, the composition comprises a flavanone:

[0247]

[0248] In certain embodiments, the composition comprises 11-methoxycapsaicin and a flavanone:

[0249]

[0250] In certain embodiments, the composition is substantially free of kava B.

[0251] In certain embodiments, the composition is substantially free of kava A.

[0252] In certain embodiments, the composition is substantially free of bornyl ester of 3,4-methylenedioxycinnamic acid:

[0253]

[0254] In certain embodiments, the composition is substantially free of bornyl esters of cinnamic acid:

[0255]

[0256] In certain embodiments, the composition is substantially free of flavanones:

[0257]

[0258] In certain embodiments, the composition is substantially free of chloranone.

[0259] In certain embodiments, the composition is substantially free of capsaicin.

[0260] Certain embodiments of the present invention provide a composition comprising at least one compound selected from the group consisting of: dihydrocapsaicin, wherein the weight percentage of dihydrocapsaicin in the composition is 15±5%; capsaicin, wherein the weight percentage of capsaicin in the composition is 6±5%; dihydrocapsaicin, wherein the weight percentage of dihydrocapsaicin in the composition is 30±5%; capsaicin, wherein the weight percentage of capsaicin in the composition is 29±5%; demethoxycapsaicin, wherein the weight percentage of demethoxycapsaicin in the composition is 12±5%; and 11-methoxycapsaicin, wherein the weight percentage of 11-methoxycapsaicin in the composition is 0.8±0.5%.

[0261] In certain embodiments, the at least one compound is dihydrocapsaicin, and the weight percentage of dihydrocapsaicin in the composition is 15±5%.

[0262] In certain embodiments, the at least one compound is capsaicin, and the weight percentage of capsaicin in the composition is 29±5%.

[0263] In certain embodiments, the at least one compound is capsaicin, and the weight percentage of capsaicin in the composition is 6±5%.

[0264] In certain embodiments, the at least one compound is dihydrocapsaicin, and the weight percentage of dihydrocapsaicin in the composition is 30±5%.

[0265] In certain embodiments, the at least one compound is demethoxycapsaicin, and the weight percentage of demethoxycapsaicin in the composition is 12±5%.

[0266] In certain embodiments, the at least one compound is 11-methoxy capsaicin, and the weight percentage of 11-methoxy capsaicin in the composition is 0.8±0.5%.

[0267] In certain embodiments of the present invention, the composition comprises at least two compounds selected from the following: dihydrocapsaicin, wherein the weight percentage of dihydrocapsaicin in the composition is 15±5%; capsaicin, wherein the weight percentage of capsaicin in the composition is 6±5%; dihydrocapsaicin, wherein the weight percentage of dihydrocapsaicin in the composition is 30±5%; capsaicin, wherein the weight percentage of capsaicin in the composition is 29±5%; demethoxycapsaicin, wherein the weight percentage of demethoxycapsaicin in the composition is 12±5%; and 11-methoxycapsaicin, wherein the weight percentage of 11-methoxycapsaicin in the composition is 0.8±0.5%.

[0268] In certain embodiments of the present invention, the composition comprises at least three compounds selected from the following: dihydrocapsaicin, wherein the weight percentage of dihydrocapsaicin in the composition is 15±5%; capsaicin, wherein the weight percentage of capsaicin in the composition is 6±5%; dihydrocapsaicin, wherein the weight percentage of dihydrocapsaicin in the composition is 30±5%; capsaicin, wherein the weight percentage of capsaicin in the composition is 29±5%; demethoxycapsaicin, wherein the weight percentage of demethoxycapsaicin in the composition is 12±5%; and 11-methoxycapsaicin, wherein the weight percentage of 11-methoxycapsaicin in the composition is 0.8±0.5%.

[0269] In certain embodiments of the present invention, the composition comprises at least four compounds selected from the following: dihydrocapsaicin, wherein the weight percentage of dihydrocapsaicin in the composition is 15±5%; capsaicin, wherein the weight percentage of capsaicin in the composition is 6±5%; dihydrocapsaicin, wherein the weight percentage of dihydrocapsaicin in the composition is 30±5%; capsaicin, wherein the weight percentage of capsaicin in the composition is 29±5%; demethoxycapsaicin, wherein the weight percentage of demethoxycapsaicin in the composition is 12±5%; and 11-methoxycapsaicin, wherein the weight percentage of 11-methoxycapsaicin in the composition is 0.8±0.5%.

[0270] In certain embodiments of the present invention, the composition comprises at least five compounds selected from the following: dihydrocapsaicin, wherein the weight percentage of dihydrocapsaicin in the composition is 15±5%; capsaicin, wherein the weight percentage of capsaicin in the composition is 6±5%; dihydrocapsaicin, wherein the weight percentage of dihydrocapsaicin in the composition is 30±5%; capsaicin, wherein the weight percentage of capsaicin in the composition is 29±5%; demethoxycapsaicin, wherein the weight percentage of demethoxycapsaicin in the composition is 12±5%; and 11-methoxycapsaicin, wherein the weight percentage of 11-methoxycapsaicin in the composition is 0.8±0.5%.

[0271] In certain embodiments of the present invention, the composition comprises dihydrocapsaicin, wherein the weight percentage of dihydrocapsaicin in the composition is 15±5%; capsaicin, wherein the weight percentage of capsaicin in the composition is 6±5%; dihydrocapsaicin, wherein the weight percentage of dihydrocapsaicin in the composition is 30±5%; capsaicin, wherein the weight percentage of capsaicin in the composition is 29±5%; demethoxycapsaicin, wherein the weight percentage of demethoxycapsaicin in the composition is 12±5%; and 11-methoxycapsaicin, wherein the weight percentage of 11-methoxycapsaicin in the composition is 0.8±0.5%.

[0272] In certain embodiments, the composition further comprises a flavanone:

[0273] and

[0274] The weight percentage of flavanone in the composition is 1±0.5%.

[0275] In certain embodiments, the composition is substantially free of kava B.

[0276] In certain embodiments, the composition is substantially free of kava A.

[0277] In certain embodiments, the composition is substantially free of bornyl ester of 3,4-methylenedioxycinnamic acid:

[0278]

[0279] In certain embodiments, the composition is substantially free of bornyl esters of cinnamic acid:

[0280]

[0281] In certain embodiments, the composition is substantially free of flavanones:

[0282]

[0283] In certain embodiments, the composition is substantially free of chloranone.

[0284] Certain embodiments of the present invention provide a composition comprising at least one compound selected from the group consisting of: dihydrocapsaicin, wherein the weight percentage of dihydrocapsaicin in the composition is about 20 to 99%; capsaicin, wherein the weight percentage of capsaicin in the composition is about 10 to 99%; dihydrocapsaicin, wherein the weight percentage of dihydrocapsaicin in the composition is about 40 to 99%; capsaicin, wherein the weight percentage of capsaicin in the composition is about 40 to 99%; demethoxycapsaicin, wherein the weight percentage of demethoxycapsaicin in the composition is about 30 to 99%; and 11-methoxycapsaicin, wherein the weight percentage of 11-methoxycapsaicin in the composition is about 20 to 99%.

[0285] In certain embodiments, the at least one compound is dihydrocapsaicin, wherein the weight percentage of dihydrocapsaicin in the composition is about 20 to 99% (e.g., about 25 to 95%, about 30 to 90%, about 35 to 85%, about 40 to 80%, about 45 to 75%, about 50 to 70%, or about 55 to 65%).

[0286] In certain embodiments, the at least one compound is capsaicin, wherein the weight percentage of capsaicin in the composition is about 40 to 99% (e.g., about 45 to 95%, about 50 to 90%, about 55 to 85%, about 60 to 80%, or about 65 to 75%).

[0287] In certain embodiments, the at least one compound is anisopiconol, wherein the weight percentage of anisopiconol in the composition is about 10 to 99% (e.g., about 15 to 95%, about 20 to 90%, about 25 to 85%, about 30 to 80%, about 35 to 75%, about 40 to 70%, about 45 to 65%, or about 50 to 60%).

[0288] In certain embodiments, the at least one compound is dihydrocapsaicin, wherein the weight percentage of dihydrocapsaicin in the composition is about 40 to 99% (e.g., about 45 to 95%, about 50 to 90%, about 55 to 85%, about 60 to 80%, or about 65 to 75%).

[0289] In certain embodiments, the at least one compound is demethoxycapsaicin, wherein the weight percentage of demethoxycapsaicin in the composition is 30 to 99% (e.g., about 35 to 95%, about 40 to 90%, about 45 to 85%, about 50 to 80%, about 55 to 75%, or about 60 to 70%).

[0290] In certain embodiments, the at least one compound is 11-methoxy capsaicin, wherein the weight percentage of 11-methoxy capsaicin in the composition is 20 to 99% (e.g., about 25 to 95%, about 30 to 90%, about 35 to 85%, about 40 to 80%, about 45 to 75%, about 50 to 70%, or about 55 to 65%).

[0291] In certain embodiments, the composition is substantially free of capsaicin.

[0292] In certain embodiments, the composition further comprises a flavanone:

[0293] and

[0294] The weight percentage of flavanones in the composition is about 20 to 99% (e.g., about 25 to 95%, about 30 to 90%, about 35 to 85%, about 40 to 80%, about 45 to 75%, about 50 to 70% or about 55 to 65%).

[0295] In certain embodiments, the composition is substantially free of kava B.

[0296] In certain embodiments, the composition is substantially free of kava A.

[0297] In certain embodiments, the composition is substantially free of bornyl ester of 3,4-methylenedioxycinnamic acid:

[0298]

[0299] In certain embodiments, the composition is substantially free of bornyl esters of cinnamic acid:

[0300]

[0301] In certain embodiments, the composition is substantially free of flavanones:

[0302]

[0303] In certain embodiments, the composition is substantially free of chloranone.

[0304] In certain embodiments, the composition is a kava extract.

[0305] Certain embodiments of the present invention provide a kava extract comprising at least one compound selected from the group consisting of dihydrocapsaicin, capsaicin, dihydrocapsaicin, capsaicin, demethoxycapsaicin, and 11-methoxycapsaicin, wherein the extract is substantially free of kava piperine B and / or kava piperine A.

[0306] In certain embodiments, the at least one compound is dihydrocapsaicin.

[0307] In certain embodiments, the at least one compound is capsaicin.

[0308] In certain embodiments, the at least one compound is capsaicin.

[0309] In certain embodiments, the at least one compound is dihydrocapsaicin.

[0310] In certain embodiments, the at least one compound is demethoxycapsaicin.

[0311] In certain embodiments, the at least one compound is 11-methoxycapsaicin.

[0312] In certain embodiments of the present invention, the kava extract comprises at least two compounds selected from the group consisting of dihydrocapsaicin, capsaicin, dihydrocapsaicin, capsaicin, demethoxycapsaicin, and 11-methoxycapsaicin, wherein the extract is substantially free of kava piperine B and / or kava piperine A.

[0313] In certain embodiments of the present invention, the kava extract comprises at least three compounds selected from the group consisting of dihydrocapsaicin, capsaicin, dihydrocapsaicin, capsaicin, demethoxycapsaicin, and 11-methoxycapsaicin, wherein the extract is substantially free of kava piperine B and / or kava piperine A.

[0314] In certain embodiments of the present invention, the kava extract comprises at least four compounds selected from the group consisting of dihydrocapsaicin, capsaicin, dihydrocapsaicin, capsaicin, demethoxycapsaicin, and 11-methoxycapsaicin, wherein the extract is substantially free of kava piperine B and / or kava piperine A.

[0315] In certain embodiments of the present invention, the kava extract comprises at least five compounds selected from the group consisting of dihydrocapsaicin, capsaicin, dihydrocapsaicin, capsaicin, demethoxycapsaicin, and 11-methoxycapsaicin, wherein the extract is substantially free of kava piperine B and / or kava piperine A.

[0316] In certain embodiments of the present invention, the kava extract comprises dihydrocapsaicin, capsaicin, dihydrocapsaicin, capsaicin, demethoxycapsaicin, and 11-methoxycapsaicin, wherein the extract is substantially free of kava piperine B and / or kava piperine A.

[0317] In certain embodiments, the kava extract further comprises flavanones:

[0318]

[0319] In certain embodiments, the kava extract is substantially free of kava B.

[0320] In certain embodiments, the kava extract is substantially free of kavapetroleum sulfoxide.

[0321] In certain embodiments, the kava extract is substantially free of bornyl ester of 3,4-methylenedioxycinnamic acid:

[0322]

[0323] In certain embodiments, the kava extract is substantially free of bornyl esters of cinnamic acid:

[0324]

[0325] In certain embodiments, the kava extract is substantially free of flavanones:

[0326]

[0327] In certain embodiments, the kava extract is substantially free of chloranthone.

[0328] Certain embodiments of the present invention provide a kava extract comprising 11-methoxycapsaicin and / or flavanones:

[0329]

[0330] and at least one compound selected from the group consisting of dihydrocapsaicin, capsaicin, dihydrocapsaicin, capsaicin and demethoxycapsaicin, wherein the extract is substantially free of kava piperine B and / or kava piperine A.

[0331] In certain embodiments, the at least one compound is dihydrocapsaicin.

[0332] In certain embodiments, the at least one compound is capsaicin.

[0333] In certain embodiments, the at least one compound is capsaicin.

[0334] In certain embodiments, the at least one compound is dihydrocapsaicin.

[0335] In certain embodiments, the at least one compound is demethoxycapsaicin.

[0336] In certain embodiments, the kava extract is substantially free of capsaicin.

[0337] In certain embodiments, the kava extract is substantially free of kava B.

[0338] In certain embodiments, the kava extract is substantially free of kavapetroleum sulfoxide.

[0339] In certain embodiments, the kava extract is substantially free of bornyl ester of 3,4-methylenedioxycinnamic acid:

[0340]

[0341] In certain embodiments, the kava extract is substantially free of bornyl esters of cinnamic acid:

[0342]

[0343] In certain embodiments, the kava extract is substantially free of flavanones:

[0344]

[0345] In certain embodiments, the kava extract is substantially free of chloranthone.

[0346] Certain embodiments of the present invention provide a kava extract consisting essentially of dihydrocapsaicin, 11-methoxycapsaicin, demethoxycapsaicin, dihydrocapsaicin, capsaicin, capsaicin, and flavanones:

[0347]

[0348] Certain embodiments of the present invention provide a kava extract consisting essentially of dihydrocapsaicin, 11-methoxycapsaicin, demethoxycapsaicin, dihydrocapsaicin, capsaicin, and capsaicin.

[0349] Certain embodiments of the present invention provide a kava extract consisting essentially of dihydrocapsaicin, dihydrocapsaicin, capsaicin, and capsaicin.

[0350] Certain embodiments of the present invention provide a composition or kava extract as described herein, wherein the composition or extract is suitable for ingestion by a mammal (eg, a human or a dog).

[0351] Certain embodiments of the present invention provide a composition or kava extract formulated as a tablet, capsule, powder, spray, chewing gum, inhalable, patch, nanoemulsion, cream, gel, stent, or liquid.

[0352] Certain embodiments of the present invention provide a pharmaceutical composition comprising a composition or kava extract as described herein and a pharmaceutically acceptable carrier.

[0353] Certain embodiments of the present invention provide a method comprising:

[0354] a) combining ethanolated kava extract and silica gel to obtain a mixture;

[0355] b) evaporating the mixture to obtain silica gel having the kava residue adsorbed thereon;

[0356] c) loading the silica gel having the kava residue adsorbed thereon onto a chromatographic column to obtain a kava adsorbed silica gel column;

[0357] d) eluting the silica gel column adsorbed with kava with a solvent system to obtain a first kava extract fraction, a second kava extract fraction, and a third kava extract fraction, wherein the first kava extract fraction consists essentially of non-polar compounds including kava piperine, the second kava extract fraction consists essentially of kavalactones and flavanones, and the third kava extract fraction consists essentially of polar compounds.

[0358] In certain embodiments, step d) comprises eluting with 28% ethyl acetate (EA) and 72% hexane (Hex) for 5 column volumes (CV), followed by eluting with 90% EA and 10% Hex for 4.1 CV, and then eluting with 35% MeOH and 65% EA for 5.5 CV.

[0359] In certain embodiments, the weight ratio of the kava residue to the total weight of the silica gel having the kava residue adsorbed thereon is 0.3 ± 0.2.

[0360] In certain embodiments, the weight ratio of the kava residue to the total weight of the silica gel having the kava residue adsorbed thereon is 0.3 ± 0.15.

[0361] In certain embodiments, the weight ratio of the kava residue to the total weight of the silica gel having the kava residue adsorbed thereon is 0.3 ± 0.1.

[0362] Certain embodiments of the present invention provide a second kava extract fraction prepared as described herein.

[0363] Certain embodiments of the present invention provide methods for treating or preventing cancer in a mammal (e.g., a human or dog) in need of such treatment, comprising administering to the mammal a carrier and a compound selected from the group consisting of dihydrocapsaicin, capsaicin, dihydrocapsaicin, capsaicin, demethoxycapsaicin, and 11-methoxycapsaicin, wherein the compound is substantially free of other kava extract components.

[0364] Certain embodiments of the present invention provide a composition comprising a carrier and a compound selected from the group consisting of dihydrocapsaicin, capsaicin, dihydrocapsaicin, capsaicin, demethoxycapsaicin and 11-methoxycapsaicin for use in medical therapy, wherein the compound is substantially free of other kava extract components.

[0365] Certain embodiments of the present invention provide a composition comprising a carrier and a compound selected from the group consisting of dihydrocapsaicin, capsaicin, dihydrocapsaicin, capsaicin, demethoxycapsaicin and 11-methoxycapsaicin for use in the preventive or therapeutic treatment of cancer, wherein the compound is substantially free of other kava extract components.

[0366] Certain embodiments of the present invention provide the use of a composition for the preparation of a medicament for preventing or treating cancer in a mammal, the composition comprising a carrier and a compound selected from the group consisting of dihydrocapsaicin, capsaicin, dihydrocapsaicin, capsaicin, demethoxycapsaicin and 11-methoxycapsaicin, wherein the compound is substantially free of other kava extract components.

[0367] Certain embodiments of the present invention provide methods for treating or preventing cancer in a mammal (e.g., a human or a dog) in need of such treatment, comprising administering to the mammal dihydrocapsamone and a carrier, wherein the dihydrocapsamone is substantially free of other kava extract components.

[0368] Certain embodiments of the present invention provide a composition comprising dihydrocapsaicin and a carrier for use in the prophylactic or therapeutic treatment of cancer in a mammal, wherein the dihydrocapsaicin is substantially free of other kava extract components.

[0369] Certain embodiments of the present invention provide a use of a composition for the preparation of a medicament for treating or preventing cancer in a mammal, the composition comprising dihydrocapsaicin and a carrier, wherein the dihydrocapsaicin is substantially free of other kava extract components.

[0370] Certain embodiments of the present invention provide for use of a composition for medical therapy, the composition comprising dihydrocapsaicin and a carrier, wherein the dihydrocapsaicin is substantially free of other kava extract components.

[0371] In certain embodiments, the dihydrocapsaicin is (+)-dihydrocapsaicin.

[0372] In certain embodiments, the dihydrocapsaicin is (±)-dihydrocapsaicin.

[0373] In certain embodiments, the dihydrocapsaicin is enriched in (+)-dihydrocapsaicin (e.g., at least about 51%, 60%, 70%, 80%, 90%, 95%, or 99% (+)-dihydrocapsaicin).

[0374] In certain embodiments, the dihydrocapsaicin is enriched in (-)-dihydrocapsaicin (eg, at least about 51%, 60%, 70%, 80%, 90%, 95%, or 99% (-)-dihydrocapsaicin).

[0375] In certain embodiments, the dihydrocapsaicin is (-)-dihydrocapsaicin.

[0376] In certain embodiments, the other kava extract components are selected from: 11-methoxy capsaicin, demethoxy capsaicin, dihydrocapsaicin, capsaicin, capsaicin, chloranone, kava piperine B, kava piperine A,

[0377]

[0378] Certain embodiments of the present invention provide a method for treating or preventing cancer in a mammal in need of such treatment, comprising administering to the mammal anthemis nobilis and a carrier, wherein the anthemis nobilis is substantially free of other kava extract components.

[0379] Certain embodiments of the present invention provide a composition for the prophylactic or therapeutic treatment of cancer in a mammal, the composition comprising capsaicin and a carrier, wherein the capsaicin is substantially free of other kava extract components.

[0380] Certain embodiments of the present invention provide a use of a composition for the preparation of a medicament for treating or preventing cancer in a mammal, the composition comprising anthemis nobilis and a carrier, wherein the anthemis nobilis is substantially free of other kava extract components.

[0381] Certain embodiments of the present invention provide for use of a composition for medical therapy, the composition comprising capsaicin and a carrier, wherein the capsaicin is substantially free of other kava extract components.

[0382] In certain embodiments, the other kava extract components are selected from: 11-methoxy capsaicin, demethoxy capsaicin, dihydrocapsaicin, capsaicin, dihydrocapsaicin, chloranone, kava piperine B, kava piperine A,

[0383]

[0384] Certain embodiments of the present invention provide a method for treating or preventing cancer in a mammal in need of such treatment, comprising administering to the mammal dihydrocapsaicin and a carrier, wherein the dihydrocapsaicin is substantially free of other kava extract components.

[0385] Certain embodiments of the present invention provide a composition for the prophylactic or therapeutic treatment of cancer in a mammal, the composition comprising dihydrocapsaicin and a carrier, wherein the dihydrocapsaicin is substantially free of other kava extract components.

[0386] Certain embodiments of the present invention provide for use of a composition comprising dihydrocapsaicin and a carrier, wherein the dihydrocapsaicin is substantially free of other kava extract components, in the preparation of a medicament for treating or preventing cancer in a mammal.

[0387] Certain embodiments of the present invention provide for use of a composition for medical therapy, the composition comprising dihydrocapsaicin and a carrier, wherein the dihydrocapsaicin is substantially free of other kava extract components.

[0388] In certain embodiments, the other kava extract components are selected from: 11-methoxy capsaicin, demethoxy capsaicin, dihydro capsaicin, capsaicin, capsaicin, chloranone, kava pepper B, kava pepper A,

[0389]

[0390] Certain embodiments of the present invention provide a method for treating or preventing cancer in a mammal in need of such treatment, comprising administering to the mammal kava extract and a carrier, wherein the kava extract is substantially free of other kava extract components.

[0391] Certain embodiments of the present invention provide a composition for the prophylactic or therapeutic treatment of cancer in a mammal, the composition comprising capsaicin and a carrier, wherein the capsaicin is substantially free of other kava extract components.

[0392] Certain embodiments of the present invention provide a use of a composition comprising capsaicin and a carrier for the preparation of a medicament for treating or preventing cancer in a mammal, wherein the capsaicin is substantially free of other kava extract components.

[0393] Certain embodiments of the present invention provide for use of a composition for medical therapy, the composition comprising capsaicin and a carrier, wherein the capsaicin is substantially free of other kava extract components.

[0394] In certain embodiments, the other kava extract components are selected from: 11-methoxy capsaicin, demethoxy capsaicin, dihydrocapsaicin, dihydrocapsaicin, capsaicin, chloranone, kava pepper B, kava pepper A,

[0395]

[0396] Certain embodiments of the present invention provide a method for treating or preventing cancer in a mammal in need of such treatment, comprising administering to the mammal demethoxycapsaicin and a carrier, wherein the demethoxycapsaicin is substantially free of other kava extract components.

[0397] Certain embodiments of the present invention provide a composition for the prophylactic or therapeutic treatment of cancer in a mammal, the composition comprising demethoxycapsaicin and a carrier, wherein the demethoxycapsaicin is substantially free of other kava extract components.

[0398] Certain embodiments of the present invention provide for use of a composition comprising demethoxycapsaicin and a carrier, wherein the demethoxycapsaicin is substantially free of other kava extract components, in the preparation of a medicament for treating or preventing cancer in a mammal.

[0399] Certain embodiments of the present invention provide for use of a composition for medical therapy, the composition comprising demethoxycapsaicin and a carrier, wherein the demethoxycapsaicin is substantially free of other kava extract components.

[0400] In certain embodiments, the other kava extract components are selected from: 11-methoxy capsaicin, dihydrocapsaicin, dihydrocapsaicin, capsaicin, capsaicin, chloranone, kava pepper B, kava pepper A,

[0401]

[0402] Certain embodiments of the present invention provide a method for treating or preventing cancer in a mammal in need of such treatment, comprising administering to the mammal 11-methoxycapsaicin and a carrier, wherein the 11-methoxycapsaicin is substantially free of other kava extract components.

[0403] Certain embodiments of the present invention provide a composition for the prophylactic or therapeutic treatment of cancer in a mammal, the composition comprising 11-methoxycapsaicin and a carrier, wherein the 11-methoxycapsaicin is substantially free of other kava extract components.

[0404] Certain embodiments of the present invention provide use of a composition for the preparation of a medicament for treating or preventing cancer in a mammal, the composition comprising 11-methoxycapsaicin and a carrier, wherein the 11-methoxycapsaicin is substantially free of other kava extract components.

[0405] Certain embodiments of the present invention provide for use of a composition for medical therapy, the composition comprising 11-methoxycapsaicin and a carrier, wherein the 11-methoxycapsaicin is substantially free of other kava extract components.

[0406] In certain embodiments, the other kava extract components are selected from the group consisting of: demethoxy capsaicin, dihydrocapsaicin, dihydrocapsaicin, capsaicin, capsaicin, chloranone, kava piperine B, kava piperine A,

[0407]

[0408] In certain embodiments, the other kava extract components are selected from the group consisting of: chloranthone, kava piperine B, kava piperine A,

[0409]

[0410] Certain embodiments of the present invention provide a method for treating or preventing cancer in a mammal in need of such treatment, comprising administering to the mammal a composition or kava extract as described herein.

[0411] Certain embodiments of the present invention provide a composition or kava extract as described herein for use in the prophylactic or therapeutic treatment of cancer in a mammal.

[0412] Certain embodiments of the present invention provide use of a composition or kava extract as described herein in the preparation of a medicament for treating or preventing cancer in a mammal.

[0413] Certain embodiments of the present invention provide a composition or kava extract as described herein for use in medical therapy.

[0414] In certain embodiments, the cancer is lung cancer, prostate cancer, skin cancer, melanoma, genitourinary cancer, colorectal cancer, breast cancer, ovarian cancer, esophageal cancer, pancreatic cancer, bladder cancer, cervical cancer, liver cancer, kidney cancer, head and neck cancer, brain cancer, or various hematological cancers.

[0415] Certain embodiments of the invention provide methods for preventing tumorigenesis, reducing DNA damage, reducing protein damage and / or detoxifying physical or chemical carcinogens in a mammal in need of such treatment, comprising administering to the mammal a carrier and a compound selected from the group consisting of dihydrocapsaicin, capsaicin, dihydrocapsaicin, capsaicin, demethoxycapsaicin and 11-methoxycapsaicin, wherein the compound is substantially free of other kava extract components.

[0416] Certain embodiments of the invention provide methods for preventing tumorigenesis, reducing DNA damage, reducing protein damage, and / or detoxifying physical or chemical carcinogens in a mammal in need of such treatment, comprising administering to the mammal a composition or kava extract as described herein.

[0417] Certain embodiments of the present invention provide a method for preventing tumorigenesis, reducing DNA damage, reducing protein damage and / or detoxifying physical or chemical carcinogens in a mammal (e.g., a human) in need of such treatment, comprising administering to the mammal dihydrocapsamone and a carrier, wherein the dihydrocapsamone is substantially free of other kava extract components.

[0418] Certain embodiments of the present invention provide a composition for preventing tumorigenesis, reducing DNA damage, reducing protein damage and / or detoxifying physical or chemical carcinogens in a mammal, the composition comprising dihydrocapsaicin and a carrier, wherein the dihydrocapsaicin is substantially free of other kava extract components.

[0419] Certain embodiments of the present invention provide a use of a composition for the preparation of a medicament for preventing tumorigenesis, reducing DNA damage, reducing protein damage and / or detoxifying physical or chemical carcinogens in a mammal, the composition comprising dihydrocapsaicin and a carrier, wherein the dihydrocapsaicin is substantially free of other kava extract components.

[0420] In certain embodiments, the dihydrocapsaicin is (+)-dihydrocapsaicin.

[0421] In certain embodiments, the dihydrocapsaicin is (±)-dihydrocapsaicin.

[0422] In certain embodiments, the dihydrocapsaicin is enriched in (+)-dihydrocapsaicin (e.g., at least about 51%, 60%, 70%, 80%, 90%, 95%, or 99% (+)-dihydrocapsaicin).

[0423] In certain embodiments, the dihydrocapsaicin is enriched in (-)-dihydrocapsaicin (eg, at least about 51%, 60%, 70%, 80%, 90%, 95%, or 99% (-)-dihydrocapsaicin).

[0424] In certain embodiments, the dihydrocapsaicin is (-)-dihydrocapsaicin.

[0425] In certain embodiments, the other kava extract components are selected from: 11-methoxy capsaicin, demethoxy capsaicin, dihydrocapsaicin, capsaicin, capsaicin, chloranone, kava piperine B, kava piperine A,

[0426]

[0427] Certain embodiments of the present invention provide a method for preventing tumorigenesis, reducing DNA damage, reducing protein damage and / or detoxifying physical or chemical carcinogens in a mammal in need of such treatment, comprising administering to the mammal anthemis nobilis and a carrier, wherein the anthemis nobilis is substantially free of other kava extract components.

[0428] Certain embodiments of the present invention provide a composition for preventing tumorigenesis, reducing DNA damage, reducing protein damage and / or detoxifying physical or chemical carcinogens in a mammal, the composition comprising anthemis nobilis and a carrier, wherein the anthemis nobilis is substantially free of other kava extract components.

[0429] Certain embodiments of the present invention provide a use of a composition for the preparation of a medicament for preventing tumorigenesis, reducing DNA damage, reducing protein damage and / or detoxifying physical or chemical carcinogens in a mammal, the composition comprising anthemis nobilis and a carrier, wherein the anthemis nobilis is substantially free of other kava extract components.

[0430] In certain embodiments, the other kava extract components are selected from: 11-methoxy capsaicin, demethoxy capsaicin, dihydrocapsaicin, capsaicin, dihydrocapsaicin, chloranone, kava piperine B, kava piperine A,

[0431]

[0432] Certain embodiments of the present invention provide a method for preventing tumorigenesis, reducing DNA damage, reducing protein damage and / or detoxifying physical or chemical carcinogens in a mammal in need of such treatment, comprising administering to the mammal dihydrocapsaicin and a carrier, wherein the dihydrocapsaicin is substantially free of other kava extract components.

[0433] Certain embodiments of the present invention provide a composition for preventing tumorigenesis, reducing DNA damage, reducing protein damage and / or detoxifying physical or chemical carcinogens in a mammal, the composition comprising dihydrocapsaicin and a carrier, wherein the dihydrocapsaicin is substantially free of other kava extract components.

[0434] Certain embodiments of the present invention provide a use of a composition for the preparation of a medicament for preventing tumorigenesis, reducing DNA damage, reducing protein damage and / or detoxifying physical or chemical carcinogens in a mammal, the composition comprising dihydrocapsaicin and a carrier, wherein the dihydrocapsaicin is substantially free of other kava extract components.

[0435] In certain embodiments, the other kava extract components are selected from: 11-methoxy capsaicin, demethoxy capsaicin, dihydro capsaicin, capsaicin, capsaicin, chloranone, kava pepper B, kava pepper A,

[0436]

[0437]

[0438] Certain embodiments of the present invention provide methods for preventing tumorigenesis, reducing DNA damage, reducing protein damage, and / or detoxifying physical or chemical carcinogens in a mammal in need of such treatment, comprising administering to the mammal a kava extract and a carrier, wherein the kava extract is substantially free of other kava extract components.

[0439] Certain embodiments of the present invention provide a composition for preventing tumorigenesis, reducing DNA damage, reducing protein damage and / or detoxifying physical or chemical carcinogens in a mammal, the composition comprising capsaicin and a carrier, wherein the capsaicin is substantially free of other kava extract components.

[0440] Certain embodiments of the present invention provide a use of a composition for the preparation of a medicament for preventing tumorigenesis, reducing DNA damage, reducing protein damage and / or detoxifying physical or chemical carcinogens in a mammal, the composition comprising capsaicin and a carrier, wherein the capsaicin is substantially free of other kava extract components.

[0441] In certain embodiments, the other kava extract components are selected from: 11-methoxy capsaicin, demethoxy capsaicin, dihydrocapsaicin, dihydrocapsaicin, capsaicin, chloranone, kava pepper B, kava pepper A,

[0442]

[0443] Certain embodiments of the present invention provide methods for preventing tumorigenesis, reducing DNA damage, reducing protein damage, and / or detoxifying physical or chemical carcinogens in a mammal in need of such treatment, comprising administering to the mammal demethoxycapsaicin and a carrier, wherein the demethoxycapsaicin is substantially free of other kava extract components.

[0444] Certain embodiments of the present invention provide a composition for preventing tumorigenesis, reducing DNA damage, reducing protein damage and / or detoxifying physical or chemical carcinogens in a mammal, the composition comprising demethoxycapsaicin and a carrier, wherein the demethoxycapsaicin is substantially free of other kava extract components.

[0445] Certain embodiments of the present invention provide a use of a composition for the preparation of a medicament for preventing tumorigenesis, reducing DNA damage, reducing protein damage and / or detoxifying physical or chemical carcinogens in a mammal, the composition comprising demethoxycapsaicin and a carrier, wherein the demethoxycapsaicin is substantially free of other kava extract components.

[0446] In certain embodiments, the other kava extract components are selected from: 11-methoxy capsaicin, capsaicin, dihydro capsaicin, dihydro capsaicin, capsaicin, chloranone, kava pepper B, kava pepper A,

[0447]

[0448] Certain embodiments of the present invention provide methods for preventing tumorigenesis, reducing DNA damage, reducing protein damage and / or detoxifying physical or chemical carcinogens in a mammal in need of such treatment, comprising administering to the mammal 11-methoxy capsaicin and a carrier, wherein the 11-methoxy capsaicin is substantially free of other kava extract components.

[0449] Certain embodiments of the present invention provide a composition for preventing tumorigenesis, reducing DNA damage, reducing protein damage and / or detoxifying physical or chemical carcinogens in a mammal, the composition comprising 11-methoxy capsaicin and a carrier, wherein the 11-methoxy capsaicin is substantially free of other kava extract components.

[0450] Certain embodiments of the present invention provide a use of a composition for the preparation of a medicament for preventing tumorigenesis, reducing DNA damage, reducing protein damage and / or detoxifying physical or chemical carcinogens in a mammal, the composition comprising 11-methoxy capsaicin and a carrier, wherein the 11-methoxy capsaicin is substantially free of other kava extract components.

[0451] In certain embodiments, the other kava extract components are selected from the group consisting of: demethoxy capsaicin, capsaicin, dihydro capsaicin, dihydro capsaicin, capsaicin, chloranone, kava piperine B, kava piperine A,

[0452]

[0453] In certain embodiments, the other kava extract components are selected from the group consisting of: chloranthone, kava piperine B, kava piperine A,

[0454]

[0455] Certain embodiments of the invention provide methods for preventing tumorigenesis, reducing DNA damage, reducing protein damage, and / or detoxifying physical or chemical carcinogens in a mammal in need of such treatment, comprising administering to the mammal a composition or kava extract as described herein.

[0456] Certain embodiments of the present invention provide a composition or kava extract as described herein for use in preventing tumorigenesis, reducing DNA damage, reducing protein damage, and / or detoxifying physical or chemical carcinogens in a mammal.

[0457] Certain embodiments of the present invention provide for the use of a composition or kava extract as described herein in the preparation of a medicament for preventing tumorigenesis, reducing DNA damage, reducing protein damage, and / or detoxifying physical or chemical carcinogens in a mammal.

[0458] In certain embodiments, the DNA damage is reduced by about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% compared to a mammal not administered a composition or kava extract as described herein.

[0459] In certain embodiments, the protein damage is reduced by about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% compared to a mammal not administered a composition or kava extract as described herein.

[0460] In certain embodiments, the DNA damage is a DNA adduct caused by a physical or chemical carcinogen.

[0461] In certain embodiments, the DNA adduct is 6 -Methylguanine DNA adducts.

[0462] In certain embodiments, the DNA adduct is a 7-methylguanine DNA adduct.

[0463] In certain embodiments, the DNA adduct is a BaP, PhIP, POB and / or PHB adduct.

[0464] In certain embodiments, the DNA adducts are POB and PHB DNA adducts (eg, 7-pobG, 7-[4-(3-pyridinyl)-4-oxobutan-1-yl]guanine; 2 -pobdT,O 2 -[4-(3-pyridinyl)-4-oxobutan-1-yl]thymidine; O 6 -pobdG,O 6 -[4-(3-pyridyl)-4-oxobutan-1-yl]-2′-deoxyguanosine; O 2 -pobC,O 2 -[4-(3-pyridinyl)-4-oxobutan-1-yl]cytidine).

[0465] Certain embodiments of the present invention provide a composition for use in medical therapy, comprising a carrier and a compound selected from the group consisting of dihydrocapsaicin, capsaicin, dihydrocapsaicin, capsaicin, demethoxycapsaicin, and 11-methoxycapsaicin, wherein the compound is substantially free of other kava extract components.

[0466] Certain embodiments of the present invention provide a composition for the prophylactic or therapeutic treatment of cancer, comprising a carrier and a compound selected from the group consisting of dihydrocapsaicin, capsaicin, dihydrocapsaicin, capsaicin, demethoxycapsaicin and 11-methoxycapsaicin, wherein the compound is substantially free of other kava extract components.

[0467] Certain embodiments of the present invention provide a composition for preparing a medicament for preventing or treating cancer in a mammal, the composition comprising a carrier and a compound selected from the group consisting of dihydrocapsaicin, capsaicin, dihydrocapsaicin, capsaicin, demethoxycapsaicin and 11-methoxycapsaicin, wherein the compound is substantially free of other kava extract components.

[0468] In certain embodiments, the compound is dihydrocapsaicin.

[0469] In certain embodiments, the compound is capsaicin.

[0470] In certain embodiments, the compound is dihydrocapsaicin.

[0471] In certain embodiments, the compound is capsaicin.

[0472] In certain embodiments, the compound is demethoxycapsaicin.

[0473] In certain embodiments, the compound is 11-methoxycapsaicin.

[0474] The invention also provides methods and intermediates described herein for preparing the compositions and extracts described herein (see, eg, the Examples).

[0475] As used herein, the phrase "substantially free" means that the composition or kava extract contains less than about 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1% or 0.05% by weight of one or more specified compounds and / or components.

[0476] As used herein, the phrase "consisting essentially of" means that the composition or kava extract contains less than about 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1% or 0.05% by weight of other compounds and / or components.

[0477] The terms "treat" and "treatment" refer to therapeutic treatments and prophylactic or preventive measures, wherein the purpose is to prevent or slow down (mitigate) the development or spread of undesirable physiological changes or disorders such as cancer. For the purposes of the present invention, beneficial or desired clinical results include, but are not limited to, relief of symptoms, reduction of lesion extent, stabilization (i.e., not worsening) of the disease state, delay or slowing of disease progression, improvement or alleviation of the disease state, and improvement (partial improvement or complete improvement), whether these results are detectable or undetectable. "Treatment" can also mean prolonged survival compared to the expected survival without treatment. Subjects in need of treatment include subjects who already have a disease or condition and subjects who are susceptible to the disease or condition or subjects in which the disease or condition is to be prevented. As described in the Examples, the compositions, kava extracts, and compounds described herein (e.g., such as dihydrocapsaicin, capsaicin, dihydrocapsaicin, capsaicin, demethoxycapsaicin, and 11-methoxycapsaicin) have chemopreventive properties and are therefore useful for the treatment and prevention of cancer.

[0478] The terms "cancer" and "cancerous" refer to or describe a physiological condition in mammals that is typically characterized by unregulated cell aggregation. A "tumor" comprises one or more cancerous cells. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancy. Specific examples of cancer include, but are not limited to, lung cancer, prostate cancer, skin cancer, melanoma, genitourinary cancer, colorectal cancer, breast cancer, ovarian cancer, esophageal cancer, pancreatic cancer, bladder cancer, cervical cancer, liver cancer, kidney cancer, head and neck cancer, brain cancer, or various hematological cancers. Examples of cancer also include, but are not limited to, cancerous lesions in the above tissues, such as familial adenomatous polyps, hyperplasia, dysplasia, aberrant crypt foci, adenoma, etc.

[0479] The term "detoxifying a physical or chemical carcinogen" refers to enhancing the elimination / inactivation of toxic species produced by a physical or chemical carcinogen, reducing the generation of toxic species from a physical or chemical carcinogen, and / or activating the immune system to improve self-defense against a physical or chemical carcinogen.

[0480] As used herein, the term "protein damage" refers to a native protein (such as hemoglobin) modified by reactive metabolites / species produced by chemical or physical carcinogens (Murphy et al., Chemico-Biological Interactions 1997; 103: 153-166). Methods for measuring protein damage are known in the art, for example, as described in Murphy et al., Chemico-Biological Interactions 1997; 103: 153-166.

[0481] As used herein, the term "mammal" includes, but is not limited to, humans, mice, rats, guinea pigs, monkeys, dogs, cats, horses, cows, pigs, as well as sheep and poultry.

[0482] It will be appreciated by those skilled in the art that compounds of the invention having one chiral center may exist and be separated in optically active and racemic forms. Some compounds may exhibit polymorphism. It will be appreciated that the present invention includes any racemic, optically active, polymorphic or stereoisomeric forms of compounds of the invention, or mixtures thereof, having the useful properties described herein, and it is well known in the art how to prepare optically active forms (e.g., by resolving racemic forms via recrystallization techniques, by synthesis from optically active starting materials, by chiral synthesis, or by chromatographic separation using a chiral stationary phase).

[0483] When a bond in a general formula of a compound herein is depicted in a non-stereochemical manner (e.g., planar), the atom to which the bond is attached includes all stereochemical possibilities. For example, in certain embodiments, all stereochemical possibilities include for the following compounds:

[0484]

[0485]

[0486]

[0487] When bonds in the formulas of compounds herein are depicted in a clear stereochemical manner (e.g., bold, bold wedges, dashed lines, or dashed wedges), it is understood that the atoms to which the stereochemical bonds are attached are enriched in the absolute stereoisomers depicted, unless otherwise indicated. In one embodiment, the compound may be at least 51% of the absolute stereoisomers depicted. In another embodiment, the compound may be at least 60% of the absolute stereoisomers depicted. In another embodiment, the compound may be at least 80% of the absolute stereoisomers depicted. In another embodiment, the compound may be at least 90% of the absolute stereoisomers depicted. In another embodiment, the compound may be at least 95% of the absolute stereoisomers depicted. In another embodiment, the compound may be at least 99% of the absolute stereoisomers depicted. For example, in certain embodiments, the atoms to which the stereochemical bonds are attached are enriched in the absolute stereoisomers depicted for the following compounds:

[0488]

[0489]

[0490] In certain embodiments, the present invention provides a composition enriched in unnatural (-)-dihydrocapsaicin:

[0491]

[0492] The compositions, kava extracts, and compounds described herein (e.g., such as dihydrocapsaicin, capsaicin, dihydrocapsaicin, capsaicin, demethoxycapsaicin, and 11-methoxycapsaicin) can be formulated into compositions (e.g., medicaments) and administered to a mammalian host (such as a human patient) in a variety of forms suitable for the chosen route of administration, i.e., orally or parenterally, by intravenous, intramuscular, topical, or subcutaneous routes.

[0493] Thus, the compositions, kava extracts and compounds of the present invention may be combined with a pharmaceutical vehicle (such as an inert diluent or an assimilable edible carrier) for systemic (e.g., oral) administration. They may be enclosed in hard or soft shell gelatin capsules, may be compressed into tablets, or may be incorporated directly into the food and beverages of the patient's diet. For oral therapeutic administration, the compositions, kava extracts and compounds of the present invention may be combined with one or more excipients and used in the form of ingestible tablets, buccal tablets, lozenges, capsules, elixirs, suspensions, syrups, sprays, nanoemulsions, patches, creams, jelly, gels, stents, wafers, beverages, and the like. The compositions and preparations should contain at least 0.01% of the compositions, kava extracts and compounds of the present invention. Of course, the percentages of the compositions and preparations are variable and may conveniently be from about 0.1 to about 60% by weight of a given unit dosage form. The amount of the compositions, kava extracts and compounds of the present invention in the therapeutically useful compositions is such that an effective dosage level is obtained.

[0494] The tablets, lozenges, pills, capsules, etc. may also include the following materials that can be added: adhesives, such as tragacanth, gum arabic, corn starch or gelatin; excipients, such as dicalcium phosphate; disintegrants, such as corn starch, potato starch, alginic acid, etc.; lubricants, such as magnesium stearate; and sweeteners, such as sucrose, fructose, lactose or aspartame, or flavoring agents, such as peppermint, wintergreen oil or cherry flavoring agents. When the unit dosage form is a capsule, in addition to the above-mentioned types of materials, it may include a liquid carrier, such as vegetable oil or polyethylene glycol. Various other materials may exist in a coated form or otherwise change the physical form of the solid unit dosage form. For example, tablets, pills or capsules may be coated with gelatin, wax, shellac or sugar, etc. A syrup or elixir may contain the composition, kava extract or compound of the present invention; sucrose or fructose as a sweetener; methyl and propyl hydroxybenzoates as preservatives; dyes and flavorings, such as cherry flavor or orange flavor. Of course, any material used in preparing any unit dosage form should be pharmaceutically acceptable and substantially nontoxic in the amounts employed. In addition, the composition, kava extract and compound of the present invention may be incorporated into sustained-release preparations and devices.

[0495] The compositions, kava extracts and compounds of the present invention may also be administered intravenously or intraperitoneally by infusion or injection. Solutions of the compositions, kava extracts and compounds of the present invention may be prepared in water, optionally mixed with a nontoxic surfactant. Dispersions may also be prepared in glycerol, liquid polyethylene glycols, triacetin and mixtures thereof, and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

[0496] Pharmaceutical dosage forms suitable for injection or infusion may include sterile aqueous solutions or dispersions or sterile powders containing the compositions of the invention, kava extracts and compounds, which are suitable for the ready-to-use preparation of sterile injectable or infusible solutions or dispersions, optionally encapsulated in liposomes. In all cases, the final dosage form should be sterile, liquid and stable under the conditions of manufacture and storage. The liquid carrier or vehicle may be a solvent or liquid dispersion medium, which includes, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), vegetable oils, non-toxic glycerides and suitable mixtures thereof. For example, by forming liposomes, in the case of dispersions, by maintaining the desired particle size or by using a surfactant, appropriate fluidity can be maintained. Prevention of microbial action can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc. In many cases, it is preferred to include isotonic agents, such as sugars, buffers or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0497] Sterile injectable solutions are prepared by incorporating the compositions, kava extracts or compounds of the present invention in the required amount in an appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze drying techniques which yield powders of the compositions, kava extracts and compounds of the present invention and any additional desired ingredient present in the previously sterile-filtered solution.

[0498] For topical administration, the compositions, kava extracts and compounds of the present invention may be applied in pure form, i.e., when they are liquids. However, it will generally be desirable to apply them to the skin as a composition or formulation in combination with a dermatologically acceptable carrier, which may be a solid or a liquid.

[0499] Useful solid carriers include finely divided solids such as talc, clay, microcrystalline cellulose, silica, alumina, and the like. Useful liquid carriers include water, alcohols or glycols or water-alcohol / glycol blends, in which the compositions, kava extracts, and compounds of the present invention may be dissolved or dispersed at effective levels, optionally with the aid of nontoxic surfactants. Adjuvants such as fragrances and additional antimicrobial agents may be added to optimize properties for a given use. The resulting liquid compositions may be applied from absorbent pads, used to impregnate bandages and other dressings, or sprayed onto infected areas using pump-type or aerosol sprayers.

[0500] Thickening agents such as synthetic polymers, fatty acids, fatty acid salts and esters, fatty alcohols, modified celluloses or modified mineral materials can also be employed with the liquid carrier to form spreadable pastes, gels, ointments, soaps and the like for application directly to the user's skin.

[0501] Examples of useful dermatological compositions that can be used to deliver the compositions, kava extracts, and compounds of the present invention to the skin are known in the art; see, e.g., Jacquet et al. (U.S. Pat. No. 4,608,392), Geria (U.S. Pat. No. 4,992,478), Smith et al. (U.S. Pat. No. 4,559,157), and Wortzman (U.S. Pat. No. 4,820,508).

[0502] Useful dosages of the compositions, kava extracts, and compounds of the invention can be determined by comparing their in vitro activity and in vivo activity in animal models. The extrapolation of effective doses in mice and other animals to humans is known in the art; for example, see U.S. Pat. No. 4,938,949.

[0503] The amount of the compositions, kava extracts or compounds of the present invention required to treat or prevent cancer, prevent tumorigenesis, reduce DNA and protein damage and / or detoxify physical or chemical carcinogens will vary not only with the particular form selected, but also with the route of administration, the nature of the condition being treated and the age and condition of the patient, and will ultimately be at the discretion of the attending physician or clinician.

[0504] However, in general, a suitable dosage range is from about 0.05 to about 100 mg / kg, for example, from about 1 to about 75 mg / kg body weight / day, such as 3 to about 50 mg / kg of recipient body weight / day, preferably in the range of 0.2 to 90 mg / kg / day, and most preferably in the range of 1 to 15 mg / kg / day. For example, in certain embodiments, dihydrocapsaicin can be administered in the following ranges: from about 0.2 to 1.0 mg / kg / day, or from about 0.4 to about 0.8 mg / kg / day, or 0.6 mg / kg / day.

[0505] The compositions, kava extracts and compounds of the invention are conveniently formulated in unit dosage form; for example, each unit dosage form contains 5 to 1000 mg, conveniently 10 to 750 mg, most conveniently 50 to 500 mg of the composition, kava extract or compound of the invention. In one embodiment, the invention provides a composition comprising a composition, kava extract or compound of the invention formulated in said unit dosage form.

[0506] The desired dose may conveniently be presented in a single dose or as divided doses administered at appropriate intervals, for example, as two, three, four or more sub-doses per day. The sub-doses themselves may be further divided, for example, into a number of discrete, loosely spaced administrations.

[0507] The invention will now be illustrated by the following non-limiting examples.

[0508] Example 1

[0509] As described herein, the tumorigenic stage specificity of kava, potential active compounds, and potential mechanisms of NNK-induced lung tumorigenesis in A / J mice have been studied. In the first experiment, a diet containing kava was given to mice treated with NNK at a dose of 5 mg / g of diet at different time periods. Kava therapy covering the initial stage reduced the diversity of lung adenomas by about 99%. The minimum effective dose has not yet been determined, because kava at two lower doses (2.5 and 1.25 mg / g diet) was as effective as the 5 mg / g diet in completely inhibiting lung adenoma formation. Daily gavage of kava (one day before, during, and after NNK treatment) also completely blocked lung adenoma formation. Fraction B, which is rich in kava lactones, fully reproduced the chemopreventive efficacy of kava, while fractions A and C, which do not contain kava lactones, were much less effective. Mechanistically, kava and fraction B reduce NNK-induced DNA in lung tissue, accompanied by a unique and preferred reduction in O 6 -methylguanine (O 6 -mG), highly tumorigenic DNA damage by NNK, and the associated and predictive efficacy in blocking lung adenoma formation. In conclusion, these results demonstrate the outstanding efficacy of kava in preventing NNK-induced lung tumorigenesis in A / J mice, accompanied by high selectivity at the initiation stage and O 6 They also established a knowledge base for identifying the active compound or compounds in kava.

[0510] Therefore, based on the experiments described herein, the compositions, kava extracts and compounds of the present invention (e.g., dihydrocapsaicin, capsaicin, dihydrocapsaicin, capsaicin, demethoxycapsaicin and 11-methoxycapsaicin) can be used to treat and prevent cancer. They are also expected to have a high safety profile relative to typical cancer therapies and show excellent efficacy and pharmaceutical properties (e.g., oral bioavailability).

[0511] introduction

[0512] Lung cancer is the leading cause of malignant tumor-related mortality due to its high incidence and lack of effective treatment. Since the use of tobacco contributes to 85-90% of the development of lung cancer, quitting smoking is the most direct strategy to reduce lung cancer morbidity and mortality. However, due to the addictiveness of tobacco, limited progress has been made in reducing tobacco use. An alternative approach is to block or slow down the development of lung cancer induced by tobacco carcinogens through chemoprevention (Hecht et al., Nat. Rev. Cancer 2009; 9: 476-88). Although many compounds have been identified as potential chemopreventive agents for lung tumors in animal models, their in vivo efficacy leaves ample room for improvement. Therefore, it is necessary to identify additional candidates with novel chemical structures, unique mechanisms and better efficacy.

[0513] A / J mice carry the lung adenoma susceptibility 1 (Pas1) gene that is closely linked to the Kras oncogene (O'Donnell et al., Cancer Lett 2006; 241: 197-202), making them highly sensitive to lung tumor development. Aging A / J mice develop lung tumors even without tobacco carcinogen treatment, with high tumor incidence and low tumor multiplicity. With appropriate tobacco carcinogen treatment, A / J mice develop lung tumors with 100% incidence and high multiplicity in a relatively short period of time (Hecht et al., Nat. Rev. Cancer 2009; 9: 476-88). The induced tumors also have morphological, histological, and molecular features similar to human lung adenocarcinoma (Malkinson, Lung Cancer 2001; 32: 265-79). Therefore, the tobacco carcinogen-treated A / J mouse model is the most commonly used model of lung tumorigenesis for evaluating chemopreventive agents, where tumor multiplicity is the most useful endpoint.

[0514] Kava is an aqueous extract of the root of Piper methysticum and is traditionally used as a beverage by South Pacific Islanders. Kava has also been used to treat anxiety (Boerner et al., Phytomedicine 2003; 10 Suppl 4:38-49; Sarris J, et al., J Clin Psychopharmacol 2013; 33(5):643-648), in which case it is prepared as an organic extract. In several South Pacific countries, epidemiological surveillance detected very low rates of cancer, including lung cancer (Henderson et al., Fourth symposium on epidemiology and cancer registries in the pacific basin 1984:73-81; Henderson et al., Natl. Cancer Inst. Monogr. 1985;69:73-81), and traditional kava use may be a risk-reducing factor (Steiner GG., Hawaii Med. J. 2000;59:420-2). Kava contains a unique class of chemicals, kavalactones (Rowe et al., Mini Rev Med Chem 2011;11:79-83), which have not been reported to prevent tumorigenesis. Kava, especially anxiolytic preparations, also contain chalcone-based kava piperin, flavanones, and bornyl esters, which may inhibit cancer development.

[0515] Recently, dietary supplementation of ethanolic kava extract at a dose of 10 mg / g diet during the initiation or post-initiation phase has been shown to effectively reduce the multiplicity of lung adenomas induced by eight gavages of a mixture of the known tobacco carcinogens 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and benzo(a)pyrene (BaP) in A / J mice without adverse side effects (Johnson et al., Am J Chin Med 2011; 39:727-42; Johnson et al., Cancer Prev Res (Phila) 2008; 1:430-8). Because NNK and BaP induce adenoma formation through different mechanisms, the two-carcinogen model does not provide a viable system to address questions about the underlying mechanism and authentic chemistry of kava. The studies described herein are intended to address these issues by using the NNK-induced lung tumorigenesis A / J mouse model. Similar studies using the BaP-induced lung tumorigenesis model can also be performed.

[0516] Materials and methods

[0517] abbreviation

[0518] NNK, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone; BaP, benzo(a)pyrene; O 6 -mG,O 6 - Methylguanine; 7-pobG, 7-[4-(3-pyridinyl)-4-oxobutan-1-yl]guanine; O 2 -pobdT, O 2 -[4-(3-pyridinyl)-4-oxobutan-1-yl]thymidine; O 6 -pobdG,O 6 -[4-(3-pyridyl)-4-oxobutan-1-yl]-2′-deoxyguanosine; O 2 -pobC,O 2 -[4-(3-pyridinyl)-4-oxobutan-1-yl]cytidine; 7-mG, 7-methylguanine; O 4 -mT,O 4 -methylthymidine; CV, column volume; TLC, thin layer chromatography; H&E, hematoxylin and eosin; Pas1, lung adenoma susceptibility 1; ANOVA, analysis of variance; PEITC, phenylethyl isothiocyanate.

[0519] Chemicals, reagents and animal diets

[0520] NNK is synthetic (Hecht et al., Carcinogenesis 1983; 4: 305-10). Kava products were purchased from Gaia Herbs, Inc. (Brevard, NC). It is an ethanol extract of wild, hand-made lateral roots from Vanuatu (standardized to 150 mg / mL total kavalactones). AIN-93 purified diet from Harlan Teklad (Madison, WI) was used herein. The AIN-93G powdered diet was started one week before the first NNK administration and ended one week after the second NNK administration; thereafter, it was replaced with the AIN-93M powdered diet. 6 -methylguanine (O 6 -mG) was purchased from Midwest Research Institute (Kansas City, MO). [CD3]O 6 -mG was purchased from Toronto Research Chemicals (Toronto, Ontario, Canada). 7-[4-(3-pyridinyl)-4-oxobutan-1-yl]guanine (7-pobG), O 2-[4-(3-pyridinyl)-4-oxobutan-1-yl]thymidine (O 2 -pobdT),O 6 -[4-(3-pyridyl)-4-oxobutan-1-yl]-2′-deoxyguanosine (O 6 -pobdG), and the corresponding [pyridine-D4] analogs were synthesized (Lao et al., Chem Res Toxicol 2006; 19: 674-82; Sturla et al., Chem Res Toxicol 2005; 18: 1048-55). Micrococcal nuclease and phosphodiesterase II were from Worthington Biochemical Corporation (Lakewood, NJ). Alkaline phosphatase was from Roche Molecular Biochemicals (Indianapolis, IN).

[0521] Kava fraction preparation and characterization

[0522] Previous studies of traditional kava and kava from Gaia Herbs found that the Gaia Herbs preparation contained some minor non-polar components that were highly toxic (Shaik et al., Bioorg Med Chem Lett 2009; 19: 5732-6). A fractionation protocol for kava from Gaia Herbs using silica gel chromatography was developed and optimized, which yielded three modalities - fractions A, B and C. Briefly, 350 mL of ethanolic kava extract was mixed with 350 g of silica gel. The solvent was evaporated in vacuo. Based on mass balance, 100 g of kava adsorbed silica gel contained 28 g of kava residue. The kava adsorbed silica gel (350 g) was loaded onto the sample loading chamber and separated by a Biotage semi-preparative system. The elution method was eluted with 28% ethyl acetate (EA) and 72% hexane (Hex) for 5 column volumes (CV), followed by elution with 90% EA and 10% Hex for 4.1 CV, and then elution with 35% MeOH and 65% EA for 5.5 CV. The different eluates were analyzed by thin layer chromatography (TLC) and the desired eluates were combined and the solvent was removed to obtain fractions A, B and C. The amount of each fraction was measured and compared with their 1The integrity of each fraction was characterized by fingerprints of H-NMR spectra. The fractions were also characterized by HPLC on a Beckman Coulter System Gold 126 solvent module with a 168 detector in comparison with conventional Kava. A Clipeus C-18 column (5 μm, 250 x 4.6 mm) was used for HPLC analysis. The flow rate used was 0.5 mL / min. Mobile phase A was water and mobile phase B was acetonitrile. The time program used for the analysis was 70% B (0-5 min), 70-95% B (5-30 min) and 95% B (30-35 min). The compounds in fractions B and C were further separated by normal phase silica gel chromatography and by 1 H-NMR and mass spectrometry characterization.

[0523] Diet preparation

[0524] Appropriate amounts of different kava forms were reconstituted in anhydrous ethanol (50 mL) and then mixed with AIN-93 powdered diet (150 g). Anhydrous ethanol (50 mL) was also mixed with AIN-93 powdered diet (150 g) to prepare a control diet. The reconstituted diet was vacuum dried to remove the ethanol, ground into a fine powder and mixed with additional AIN-93 powdered diet to the desired dose. The initial dose of kava (5 mg / g diet) was selected based on previous findings showing that this dose of kava was well tolerated in A / J mice and its lung cancer chemoprevention efficacy was similar to that at higher doses (Johnson et al., Am J Chin Med 2011; 39:727-42).

[0525] Experiments to evaluate the efficacy of different kava administration regimens on NNK-induced lung adenocarcinoma formation in A / J mice

[0526] According to the animal welfare protocol (animal welfareprotocols) approved by the IACUC of University of Minnesota, 5-6 week-old female A / J mice from Jackson Laboratory (Bar Harbor, ME) were processed. After arrival, mice were placed in the animal facility without specific pathogens of Research Animal Resources, University of Minnesota. After one week of adaptation, mice were weighed, randomly divided into different groups, and converted to AIN-93G powdered diet, which was defined as the 1st day. The number of mice in each group is described in the results section. On the 7th and 14th days, by intraperitoneal (ip) injection, the mice of the negative control group received 0.1mL saline solution, and the mice of other groups received NNK (100 and 67mg / kg respectively in 0.1mL saline solution). At the end of the 21st day, mice were converted to AIN-93M-powdered diet until the end of the study. Dietary consumption was measured twice a week and body weight was monitored weekly. All mice were euthanized with excessive carbon dioxide. Lungs were harvested and tumors on the lung surface were counted.

[0527] For Experiment 1, mice were fed a diet supplemented with or without kava at a dose of 5 mg / g diet for the indicated periods ( Figure 4 and Fig.29 ) to determine the stage specificity of tumorigenesis. This study was terminated at the end of day 119. For experiment 2, mice were fed a diet supplemented with / without kava at a dose of 5 mg / g diet from day 1 to day 14 (initiation phase only, Fig.30 Half of the mice were terminated at the end of week 25 (day 175) and the other half were terminated at the end of week 34 (day 238). For Experiment 3, mice were given vehicle (PEG400:EtOH 9:1, 200 μL) or kava (20 mg / mouse / day) in the same vehicle by daily gavage using the dosing regimen described in the Results section. The study was terminated at the end of day 119 ( Fig.31 For Experiment 4, mice were fed a diet supplemented with kava at lower doses to determine the dose-response pattern, or were fed a diet supplemented with different kava fractions at a dose of 5 mg / g diet from Day 1 to Day 14. The study was terminated at the end of Day 119 ( Fig.32 ).

[0528] Experiments to evaluate the efficacy of kava on acute DNA adduct formation induced by NNK in A / J mice

[0529] After one week of adaptation, A / J mice were weighed and randomly divided into 16 groups (3 mice per group) and switched to AIN-93G powdered diet, which was defined as day 1. Groups 1, 2, 4, 6, 8, 10 and 12 of mice were given a designated AIN-93G diet. Groups 3, 5, 7, 9, 11 and 13 of mice were given an AIN-93G diet supplemented with kava at a dose of 5 mg / g diet, except for groups 9, 11 and 13 of mice, which were switched to a normal AIN-93G diet 1 day after NNK injection to simulate the cessation of kava treatment 1 day after the last NNK treatment in a long-term lung tumorigenesis study. Groups 14-16 of mice were given an AIN-93G diet supplemented with fractions A, B and C at a dose of 2.5 mg / g diet, respectively, in the study. On the 7th day, the 1st group of mice received 0.1 mL of saline solution via intraperitoneal injection, while the other groups of mice received NNK (100 mg / kg in 0.1 mL of saline solution). Groups 1, 2, and 3 of mice were euthanized 4 h after NNK injection. Groups 4 and 5 of mice were euthanized 8 h after NNK injection. Groups 6, 7, 14, 15, and 16 of mice were euthanized 24 h after NNK injection. Groups 8 and 9 of mice were euthanized 48 h after NNK injection. Groups 10 and 11 of mice were euthanized 96 h after NNK injection. Groups 12 and 13 of mice were euthanized 2 weeks after NNK injection (336 h). All mice were euthanized with excess carbon dioxide. Lungs were collected, quickly frozen in liquid nitrogen, and stored at -80 ° C until DNA separation.

[0530] Separation and Quantification of DNA Adducts in Lung Tissue by LC-ESI-MS / MS

[0531] DNA was isolated from whole lung tissue of each individual mouse following the Puregene DNA isolation protocol (Qiagen Corp, Valencia, CA) (Urban et al., Chem Res Toxicol 2012; 25: 2167-78). 7-pobG, O 2 -pobdT、O 6 -pobdG and O 6 -mG quantification was performed following established protocols (Urban et al., Chem Res Toxicol 2012; 25: 2167-78; Peterson et al., Cancer Res 1991; 51: 5557-64).

[0532] Lung tumor histopathology

[0533] 4-μm-thick sections made from formalin-fixed and paraffin-embedded lung tissue were stained with hematoxylin and eosin (H&E).

[0534] Statistical analysis

[0535] Lung adenoma multiplicity data are reported as mean ± SD (n = 5-40). One-way analysis of variance (ANOVA) was used to compare the mean values ​​of NNK and NNK + kava form groups for experiments 1, 3 and 4. The number of tumors on the lung surface between the NNK control and kava form treatment groups was compared using the Dunnett test. p-values ​​≤ 0.05 were considered statistically significant. For experiment 2, an unpaired t-test was used to compare the NNK control and kava form treatment groups. A bilateral p-value ≤ 0.05 was considered statistically significant. Data for DNA adducts are reported as mean ± SD (n = 3). For the time course study, an unpaired t-test was used to compare the NNK control and kava form treatment groups. A bilateral p-value ≤ 0.05 was considered statistically significant: *p < 0.05, **p < 0.01, and ***p < 0.001. For the 24-h time point study, a one-way ANOVA was used to compare the mean values. The NNK control and kava form treatment groups were compared using the Dunnett test. p-values ​​≤ 0.05 were considered statistically significant.All analyses were performed in GraphPad Prism 4 (GraphPad Software, Inc. La Jolla, CA).

[0536] result

[0537] Effects of Kava Treatment Regimens on NNK Exposure on Lung Adenoma Formation in A / J Mice – Experiment 1

[0538] To test whether kava inhibits specific stages of NNK-induced lung tumorigenesis, A / J mice were given two doses of NNK (100 and 67 mg / kg body weight by intraperitoneal injection on days 7 and 14, respectively). At different time periods for NNK exposure, A / J mice treated with NNK were given a diet supplemented with kava at a dose of 5 mg / g diet. Both adenoma incidence (presence of one detectable surface adenoma) and number of adenomas on the lung surface were quantified at the end of 119 days ( Fig.29 ).

[0539] As expected, A / J mice without NNK treatment had low adenoma incidence (10%) and low adenoma multiplicity (0.1±0.3 lung adenomas / mouse), while A / J mice treated with NNK had 100% adenoma incidence and high adenoma multiplicity (17.5±4.8 lung adenomas / mouse). The kava treatment regimen that began after the last NNK treatment (Groups 6-9, i.e., after initiation) had no effect on adenoma incidence. The treatment also had only a small effect on adenoma multiplicity, with the exception of the 15-119 day regimen (Group 6), which reduced adenoma multiplicity by 24% (13.3±4.3 lung adenomas / mouse, p<0.05). On the other hand, kava treatment prior to and covering the NNK exposure period (Groups 3-5, i.e., the initiation phase) not only reduced adenoma incidence by 67-87%, but also reduced adenoma multiplicity by approximately 99%, reaching levels similar to those of mice without NNK treatment, which was unexpected for its outstanding efficacy. None of the long-term kava treatment regimens (Groups 5, 6, 8, and 9) caused >10% weight loss, and the short-term treatment regimens (Groups 3, 4, and 7) did not reduce body weight relative to NNK-treated mice (Group 2). None of the kava treatment regimens caused significant changes in liver weight compared to NNK-treated mice (Group 2). These data suggest that kava has a complete blockade of the initiation of NNK-induced lung tumorigenesis, with a modest post-initiation inhibitory efficacy.

[0540] Effects of Kava on Long-term Lung Tumorigenesis in A / J Mice – Experiment 2

[0541] To verify the anti-initiation efficacy of short-term kava treatment during NNK treatment (Day 1-14) and to determine whether the inhibition would persist through later stages of tumorigenesis, the initial kava and NNK treatment experiments were repeated and tumor status was analyzed at Week 25 (Day 175) and Week 34 (Day 238). Fig.30As shown in, at week 25, A / J mice without NNK treatment had no adenoma, while A / J mice treated with NNK had 100% adenoma incidence and high adenoma multiplicity (18.1 ± 5.1 lung adenomas / mouse). Kava given at a dose of 5 mg / g diet during days 1-14 reduced adenoma incidence by 73% and reduced adenoma multiplicity by 98.5%. As expected for the longer duration of tumor growth, A / J mice at week 34 had a higher adenoma multiplicity (26.5 ± 7.8 lung adenomas / mouse) than mice at week 25. A / J mice without NNK treatment also had a higher spontaneous tumor incidence (25%) and multiplicity (0.5 ± 1.0 lung adenomas / mouse) than mice at week 25. Kava given during days 1-14 did not reduce adenoma incidence, but significantly reduced adenoma multiplicity by 97.7% (1.1 ± 0.6 lung adenomas / mouse). Figure 5 Representative micrographs of lung sections from mice not treated with NNK ( Figure 5 A) Mice treated with NNK ( Figure 5 B) and NNK-treated and kava-treated mice ( Figure 5 C), which demonstrated that the internal lung tumors treated with kava were reduced to the same magnitude as enumerated by counting visible lung surface lesions. The kava treatment regimen did not cause changes in mouse body and liver weights relative to the NNK control group. The data from this experiment not only demonstrated the initial specific inhibitory efficacy of kava on NNK-induced lung tumorigenesis, but also demonstrated the long-lasting protective nature of such a brief treatment.

[0542] Effect of daily gavage of kava on lung adenoma formation in A / J mice – Experiment 3

[0543] Given the potential pharmacokinetic differences between kava consumption to date in humans (most practically via bolus dosing of dietary supplement pills / drinks) and kava consumption by continuous rodent food intake in experiments, experiments were conducted to explore whether once-daily gavage of kava would be as effective in preventing NNK-induced adenoma formation in A / J mice. The dose of kava was selected to be 20 mg / mouse / day based on the fact that A / J mice consume 3-4 g diet / day and the kava dose in the diet is 5 mg / g diet. In one protocol, once-daily kava gavages were initiated the day before the first NNK treatment and continued until the day after the second NNK treatment - Group 3 ( Fig.31 In the second protocol, once-daily kava gavage was started one day before the first NNK treatment, ended one day after the first NNK treatment, restarted one day before the second NNK treatment and ended one day after the second NNK treatment - Group 4 ( Fig.31). When the incidence and number of adenomas on the lung surface were quantified at the end of day 119 ( Fig.31 ), A / J mice not treated with NNK had low adenoma incidence (20%) and low adenoma multiplicity (0.2±0.4 lung adenomas / mouse), while A / J mice treated with NNK had 100% adenoma incidence and high adenoma multiplicity (16.6±3.1 lung adenomas / mouse). Both kava gavage regimens reduced adenoma incidence (60-100%) and reduced adenoma multiplicity by approximately 99%. Neither of these regimens caused significant changes in body weight or liver weight compared to mice in Group 2. Therefore, these data convincingly establish the feasibility of using kava to block NNK-induced adenoma initiation with as few as 3 once-daily bolus treatments (i.e., one dose each before, during, and after NNK injection). Similar experiments can be designed and implemented to test whether a single dose given shortly before or concurrently with NNK is sufficient.

[0544] Preparation and characterization of three kava fractions

[0545] Kava from Gaia Herbs was separated into three fractions – Fractions A, B and C – over 19 replicates. 1 H-NMR characterization of the chemical identity of each fraction ensures reproducible integrity of each form ( Figure 6 ). Determine the mass of each fraction ( Figure 6 ). Fractions A, B and C accounted for 36.3%, 51.4% and 9.9% of the kava mass balance, respectively. The quantitative mass balance (97.7%) indicated that most, if not all, of the components were recovered. 1 H-NMR and HPLC analysis also showed that the composition of kava reconstituted from fractions A, B and C was no different from the original kava preparation ( Figure 6 and 7 HPLC analysis also showed that fraction C contained chemicals not detected in traditional kava ( Figure 7 ).

[0546] The main chemical substances in fractions B and C were separated by 1 H-NMR and mass spectrometry characterization and determination of abundance ( Figure 8All of these chemicals have been previously identified in kava products (Duke J, Dr. Duke's phytochemical and ethnobotanical databases, in, 2013 pp. http: / / www.ars-grin.gov / duke / .). Fraction B contained six kavalactones and one flavanone. Fraction C included two additional flavanones, two bornyl esters, and two chalcone-based kava esters, A and B. Since the chemopreventive efficacy data showed that fraction A was the least effective and the chemopreventive potential of Gaia Herbs kava could be reproduced by fraction B, the chemicals in fraction A were not characterized ( Fig.32 ).

[0547] Evaluation of the minimum effective dose and search for active fractions against NNK-induced lung adenoma formation in A / J mice – Experiment 4

[0548] To determine the minimum kava dose that is effective in inhibiting NNK-induced lung adenoma formation in A / J mice, the same carcinogen protocol as described above was used to initiate tumorigenesis. A / J mice treated with NNK were given a diet supplemented with kava at doses of 5, 2.5, and 1.25 mg / g diet during days 1-14. The incidence and number of adenomas on the lung surface at the end of day 119 were quantified ( Fig.32 ). Similar to previous results, A / J mice not treated with NNK had low adenoma incidence (20%) and multiplicity (0.2±0.4 lung adenomas / mouse), whereas A / J mice treated with NNK had 100% adenoma incidence and high adenoma multiplicity (16.0±5.2 lung adenomas / mouse). Kava treatment at all doses reduced adenoma incidence by 73-87% and reduced adenoma multiplicity by approximately 99%. These data suggest that future experiments are needed to explore lower doses to determine the minimal effective dose of kava to block tumor initiation in this model.

[0549] In this study, the efficacy of three kava fractions at a dose of 2.5 mg / g as diet was also evaluated to rank their anti-initiation efficacy ( Fig.32, Groups 6-8). Fraction A, which based on its abundance in kava was equivalent to a dose of 6.9 mg / g diet of kava, did not cause a reduction in adenoma incidence and only slightly reduced adenoma diversity by 25% (12.0±5.0 lung adenomas / mouse, p<0.01). Fraction B, which was equivalent to a dose of 4.9 mg / g diet of kava, reduced adenoma incidence by 93% and reduced adenoma diversity to baseline levels (0.1±0.5 lung adenomas / mouse, p<0.01). Fraction C, which was equivalent to a dose of 25.2 mg / g diet of kava, did not reduce adenoma incidence but reduced adenoma diversity by 70% (3.5±2.5 lung adenomas / mouse, p<0.01). None of these regimens caused significant changes in body or liver weights compared to Group 2 mice. These data indicate that fraction B contains most, if not all, of the active compounds, fraction C contains the smallest amount of active compounds and fraction A contains virtually no active compounds.

[0550] Effects of kava and its fractions on NNK-induced DNA damage in lung tissue of A / J mice

[0551] Since the data convincingly establish the highly selective anti-initiation efficacy of kava and its fraction B against NNK-induced lung adenoma formation, the reduction of NNK-induced DNA damage as a plausible chemopreventive mechanism is focused on and described below. Additional experiments were designed to collect lung tissue to characterize the expression of four NNK-derived DNA adducts (7-pobG, O2-pobdT, O2-pobdT) in lung tissue of A / J mice after exposure to kava at a dose of 5 mg / g diet. 6 -pobdG and O 6 As expected, no NNK-derived DNA adducts were detected in negative control mice (data not shown), whereas significant amounts of all four DNA adducts were detected in NNK-treated mice ( Figure 1 A). Kava treatment reduced the amount of all four DNA adducts ( Figure 1 A). When the abundance of each DNA adduct was normalized to its time control NNK-treated group ( Figure 1 B), 7-pobG, O2-pobdT and O 2 -pobdG was reduced to a similar extent (30-40%), especially during the first 24 h after NNK treatment, when the contribution of DNA repair and the inherent instability of these adducts are less important. 6 -mG, reduced by 70-80% ( Figure 1 B). Since the relative abundance of any of the four DNA adducts did not differ at different time points after NNK treatment ( Figure 1B), the kava-induced reduction of these DNA adducts is most likely mediated by inhibition of their formation rather than activation of DNA repair mechanisms.

[0552] Next, the effects of fractions A, B, and C at a dose of 2.5 mg / g diet 24 h after NNK treatment on NNK-induced DNA adducts were evaluated. Figure 1 As shown in C, all kava fractions reduced NNK-induced DNA damage. However, 7-pobG, O 2 -pobdT and O 6 -pobdG reduction was very similar ( Figure 1 D) and has nothing to do with its different ability to block lung adenoma formation. 6 The degree of reduction of -mG is quite different. Fraction B greatly reduces O 6 -mG (72%), while fractions A and C were positive for O 6 -mG No effect. 6 The degree of reduction in -mG correlated with its ability to reduce lung adenoma multiplicity ( Fig.32 These data suggest that blocking of O in lung DNA by active compounds in kava fraction B may be a promising target for the treatment of lung cancer. 6 The formation of -mG (and possibly other methylated adducts) is a possible mechanism for its efficacy in NNK-induced lung tumorigenesis in this model.

[0553] discuss

[0554] The results of this study clearly show that kava is very effective in preventing lung adenoma formation in A / J mice when given before and during NNK treatment (initiation phase), reducing adenoma multiplicity by about 99% at doses as low as 1.25 mg / g diet. The minimum effective dose remains to be determined. The treatment also reduces lung tumor incidence. A similar degree of chemoprevention, even when the study was terminated at a later stage, remained, indicating that kava blocks lung tumorigenesis, rather than slowing down the process. In addition, the data convincingly establish the feasibility of using kava to block the adenoma initiation activity of NNK with as little as 3 daily bolus treatments (i.e., before, during, and after NNK injection). While these data further confirm the chemopreventive potential of kava for tobacco-induced lung cancer, they also demonstrate a new paradigm of kava's extremely effective initiation phase specificity for this carcinogen. This significant efficacy has not been previously reported in the literature, except for several synthetic derivatives of phenylethyl isothiocyanate (PEITC) (Alworth et al., Carcinogenesis 1993; 14: 1711-3).

[0555] On the other hand, when kava treatment was started after the second NNK administration, it showed lower efficacy, indicating that kava at this dose and form mainly blocked the initiation of lung tumorigenesis induced by NNK. These results are different from the results of previous studies, which demonstrated that kava at a dose of 10 mg / g diet reduced the adenoma formation induced by NNK and BaP in A / J mice in the post-initiation stage and in the initial stage (i.e., kava treatment was started one day after the last dose of carcinogen treatment) (Johnson et al., Cancer Prev Res (Phila) 2008; 1: 430-8). There are several differences in these studies, which may explain the above obvious differences. First, the route and dosage intensity of carcinogen administration are different. In this study, NNK was given by intraperitoneal injection (2 injections per week), while previous work involved NNK and BaP mixture gavage (8 gavages per week). These may lead to different metabolic processing of one or more carcinogens and reactive metabolites to DNA, thereby leading to different pathogenic changes in target tissues in these models. Second, the kava treatment regimen in this study (Group 6), which most closely mimicked the regimen used in the previous study, showed a modest reduction in lung adenoma multiplicity ( Fig.29 ), although to a lesser extent than in previous studies. This may be explained by the differences in doses in these studies. Finally, kava did not completely block NNK and BAP-induced lung adenoma formation when given even at a dose of 10 mg / g diet during the initiation phase (Johnson et al., Cancer Prev Res (Phila) 2008; 1:430-8). Kava may be less effective in blocking BaP-induced lung tumor initiation compared to its high efficacy against NNK-induced initiation in the current study.

[0556] In the search for one or more active compounds, a highly reproducible fractionation protocol was developed to separate kava into three fractions. Fraction A contained polar chemicals, fraction B contained chemicals with intermediate polarity, and fraction C contained non-polar chemicals that were not detected in traditional kava. When evaluated at a dose of 2.5 mg / g diet, fractions A and C, at an equivalent dose higher than 5 mg / g diet of kava, only slightly reduced lung adenoma multiplicity without reducing tumor incidence. On the other hand, fraction B completely blocked NNK-induced lung adenoma formation at a dose equivalent to 5 mg / g diet of kava. These data clearly indicate that fraction B fully reproduces the lung chemopreventive efficacy of kava and contains active compounds, while fractions A and C contain no or very few active compounds. Six kavalactones have been identified in fraction B, accounting for 94% of its mass balance. Although their efficacy has not been reported in any in vivo tumorigenesis model, these kavalactones may be responsible for the efficacy of kava in blocking NNK-induced lung tumorigenesis in A / J mice. It is noteworthy that fraction B does not contain kava piperine A and B, which may contribute to the hepatotoxicity risk of kava (Zhou P, et al., Faseb J. 2010; 24: 4722-32). Although both kava piperine A and B have shown anticancer activity in several xenograft models (Zi et al., Cancer Res 2005; 65: 3479-86; Tang Y et al., Int J Cancer 2010; 127: 1758-68; Lin et al., J Nutr Biochem 2012; 23: 368-78), the results of the current study indicate that they are not active compounds against the initiation of NNK-induced lung tumorigenesis, which is consistent with previous research results (Johnson et al., Am J Chin Med 2011; 39: 727-42).

[0557] Given the highly selective anti-NNK-initiating effects of kava and its fraction B, their effects on NNK-induced DNA damage in lung tissue were characterized as a possible mechanism. NNK is an asymmetric nitrosamine that can be activated into two types of DNA-reactive species ( Figure 2 ). The methyl group is hydroxylated to generate 4-oxo-4-(3-pyridyl)-1-butanediazonium hydroxide, which gives a group of DNA adducts, including 7-pobG, O 2 -[4-(3-pyridinyl)-4-oxobutan-1-yl]cytidine (O 2 -pobC), O 2 -pobdT and O 6-pobdG (Urban et al., Chem Res Toxicol 2012; 25: 2167-78). Methylene hydroxylation produces methane diazonium hydroxide, which gives another group of DNA adducts, including 7-methylguanine (7-mG), O 6 -mG and O 4 -methylthymidine (O 4 -mT). The abundance of methylated DNA adducts is generally 10-20 times that of pob DNA adducts, which may be due to a combination of preferred methylene hydroxylation by NNK and the high reactivity of the methanediazonium hydroxide intermediate (Peterson et al., Cancer Res 1991; 51: 5495-500). The four DNA adducts in the lung, 7-pobG, O 2 -pobdT、O 6 -pobdG and O 6 -mG, because of its better stability, represents two NNK-activated pathways as well as potential tumorigenicity (Urban et al., Chem Res Toxicol 2012; 25: 2167-78; Peterson et al., Cancer Res 1991; 51: 5557-64; Loechler et al., Proc Natl Acad Sci US A 1984; 81: 6271-5).

[0558] Surprisingly, kava treatment resulted in different reductions of these four DNA adducts, with the most significant reductions for O 6 -mG is a high priority. To our knowledge, kava is the first candidate to demonstrate the unique mechanism. 6 -mG is produced by methylene hydroxylation ( Figure 2 ), the Kava fraction B chemical preferentially inhibits NNK methyl hydroxylation relative to methyl hydroxylation. The fraction B chemical better reacts with and captures methane diazonium hydroxide relative to 4-oxo-4-(3-pyridyl)-1-butane diazonium hydroxide, leading to the observed O 6 -mG is preferentially reduced. However, work from Peterson et al. showed that O 6 -mG has a strong positive correlation with lung tumor multiplicity in A / J mice (Peterson et al., Cancer Res 1991; 51: 5557-64). 6-mG DNA repair capacity of A / J mice is also less susceptible to NNK-induced lung tumorigenesis (Liu et al., Carcinogenesis 1999; 20: 279-84). 6 -mG's error coding properties have been established (Loechler et al., Proc Natl Acad Sci USA 1984; 81: 6271-5). These results suggest that 6 The high tumorigenicity of -mG relative to POB adducts, and the impressive anti-initiation efficacy of kava and fraction B chemicals may have caused their reduced effects.

[0559] In summary, kava blocked NNK-induced lung tumorigenesis in A / J mice with high selectivity targeting the initiation stage. Mechanistically, kava fraction B chemicals preferentially reduced NNK-induced O in lung tissue. 6 -mG DNA adducts. These results also suggest that the kavalactones in fraction B may be active compounds.

[0560] Example 2

[0561] Effect of kava fraction B supplementation on carcinogenesis in transgenic adenocarcinoma of the mouse prostate (TRAMP) mice.

[0562] Model Introduction

[0563] In the TRAMP model, the rat Probasin promoter drives transgenic "prostate-specific" expression of simian virus 40 (SV40) T-antigen (T-Ag) and small t-antigen; T-Ag binds and inactivates the retinoblastoma (Rb) and p53 tumor suppressor genes; and small t-antigen also drives cell proliferation (Greenberg et al, PNAS, 1995).

[0564] There are at least 2 different carcinogenic lineages in this model (Chiaverotti et al, Am J Pathol, 2008; Huss et al, Neoplasia, 2007, Wang et al, Prostate, 2011). One lineage is androgen receptor (AR)-dependent glandular epithelial damage, which usually involves the dorsolateral prostate (DLP) lobe, less for the anterior (AP) and ventral (VP) lobes. The other lineage is AR-independent neuroendocrine (NE)-like poorly differentiated (PD) carcinoma: Lifetime NE-Ca incidence = 1 / 3 in C57BL / 6 mice, usually arising from the ventral prostate (VP) lobe, which is the smallest of all lobes. The weight of the urogenital (GU) tract (bladder, seminal vesicle, prostate lobe) is usually used as a measure of tumor burden. The weight of the DLP lobe is more specifically extended for epithelial damage.

[0565] Study Design and Safety Monitoring

[0566] The detailed experimental design is listed in Fig.33 The experimental protocol is included below. Specifically, the AIN 93M purified powdered diet was supplemented with 0.4% Kava extract fraction B (see, Example 1 and Figures). Fresh diet was prepared once a week, made into wet biscuits and air-dried, and stored at 4°C. To assess food intake through food disappearance data, the biscuits were weighed before and after. Fresh diet was provided twice a week. Food intake = approximately 3g / mouse per day. The health of the mice was initially monitored 2-3 times; however, monitoring was performed more frequently when the mice were over 16 weeks old. The weight of the mice was measured once a week initially; then once every two weeks.

[0567] Results and Conclusions

[0568] like Figure 9-12 and Fig.34 and 35 As shown in Figure 2, feeding 0.4% Kava extract fraction B in TRAMP mice starting at 8 weeks of age (early PIN lesions) significantly reduced genito-urinary tract weights and prostate weights at 16 and 28 weeks of age, respectively. The dietary therapy reduced the incidence of NE-cancer from 50% to 14.3% at 28 weeks (X 2 Test, p<0.05). Kava fraction B inhibited both epithelial injury lineage and NE-oncogenic lineage.

[0569] Example 3

[0570] Effects of dihydrocapsaicin and kava fraction B on lung cancer treatment in xenograft models

[0571] Model Introduction

[0572] Cancer xenograft models inoculated with human cancer cell lines have been widely accepted as animal models to evaluate and develop cancer therapeutics. Human H2009 and A549 lung cancer cell lines are two common tumor cell lines used to establish xenograft models. In this example, the potential of kava and its forms (including fractions A, B, C, dihydrocapsamone and capsamone) to inhibit tumor growth has been evaluated, which supports the potential use of these compounds / compositions as therapeutic agents for human cancer (see, Example 1 for the description of fractions A, B and C).

[0573] Study Design and Safety Monitoring

[0574] Human lung cancer cells are usually cultured and mixed with Metrigel. 2-5x10 6 When the tumor size reaches about 50mm 3 The mice were randomly divided into different groups. The treated mice received the drug candidate at a dose of 40 mg / kg body weight by intraperitoneal injection or oral gavage. Tumor size was monitored once or twice a week until 5 weeks after treatment or when the tumor size was >1000 mm 3 The mice were subjected to CO 2 Following euthanasia, the tumors were harvested and weighed. Major organs were grossly inspected and similarly weighed.

[0575] Results and Conclusions

[0576] like Fig.13 As shown in , dihydrocapsaicin treatment significantly reduced the tumor volume induced by H2009 human lung cancer cell line relative to the control group, indicating that dihydrocapsaicin is a promising anticancer agent. Fig.14 and 15 As shown in , fraction B containing dihydrocapsaicin also significantly reduced the tumor volume induced by A549 human cancer cell line relative to the control group, suggesting that fraction B and dihydrocapsaicin may be possible anticancer agents against different malignancies.

[0577] Example 4

[0578] Dihydrocapsammonium (DHM) from kava blocks tobacco carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced lung tumorigenesis and uniquely reduces DNA damage in A / J mice

[0579] summary

[0580] As discussed herein, kava and its kava-free fraction B completely blocked 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced lung tumorigenesis in A / J mice, with a preferential reduction in NNK-induced O 6 -methylguanine (O 6 In this study, the effects of five major compounds in fraction B on reducing O 6 -mG in vivo efficacy, natural (+)-dihydroanesthenic pepper bitter (DHM) was identified as the lead compound. (+)-DHM showed outstanding chemopreventive activity against NNK-induced lung tumorigenesis in A / J mice, where adenoma diversity was reduced by 97% at a dose of 0.05mg / g diet (50ppm). In these bioassays, synthetic (±)-DHM is equivalent to natural (+)-DHM and may be more effective, while the structurally similar analog (+)-dihydroanesthenic pepper bitter (DHK) is completely inactive, which shows a sharp in vivo structure-activity relationship (SAR). Analysis of NNK-induced DNA adducts in the extended group showed that DHM reduced a subset of DNA adducts in lung tissue derived from 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL, an active metabolite of NNK). A preliminary 17-week safety study of DHM in A / J mice at a dose of 0.5 mg / g diet (at least 10 times its minimum effective dose) showed no adverse effects, suggesting that DHM may not carry the hepatotoxicity risk of kava. These results demonstrate the outstanding efficacy and promising safety profile of DHM in preventing NNK-induced lung tumorigenesis in A / J mice, with a unique mechanism of action and high target specificity.

[0581] introduction

[0582] Lung cancer causes approximately 160,000 deaths in the United States and 1.4 million deaths worldwide each year. Prevention is extremely important due to the limited success of treatment (Cohen, V et al. (2004) Curr. Opin. Pulm. Med. 10: 279-283). There is no doubt that smoking cessation would be the ideal strategy and should be highly promoted because smoking causes 90% of lung cancer. However, smoking cessation is very challenging due to the addictive nature of nicotine in tobacco products; many smokers will not succeed even after multiple attempts and the best smoking cessation support. These individuals contribute significantly to an estimated 200,000 cases of lung cancer in the United States each year, with an estimated annual medical cost of $12 billion. Therefore, in addition to further optimizing smoking cessation methods, there is a need to develop chemopreventive agents for these high-risk populations. In the past few decades, many compounds have been identified as potential lung cancer chemopreventive agents and several of them have been evaluated in the clinic, but unfortunately without success (Hecht, SS et al. (2009) Nat. Rev. Cancer. 9: 476-488). The lack of clinical effectiveness is at least in part due to the modest in vivo efficacy of some lead compounds. Therefore, the development of novel substances with superior efficacy and unique mechanisms is needed.

[0583] 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a tobacco-specific and highly potent lung carcinogen that selectively induces lung adenomas and adenocarcinomas in different species (Hecht, SS. (1998) Chem. Res. Toxicol. 11: 559-603). A large amount of evidence also shows that NNK in cigarette smoke contributes to lung adenocarcinoma in American smokers. In the United States, the incidence of lung adenocarcinoma has increased over the past few decades. This change has only been observed in smokers, suggesting that it is associated with cigarette-related factors. One study pointed out that the level of NNK in the mainstream smoke of major American non-filtered cigarettes increased between 1978 and 1992, while the level of benzo[a]pyrene (BaP), another well-characterized lung carcinogen, decreased in the same cigarettes between 1959 and 1992 (Hoffmann, D et al. (1993) Journal of Smoking-related disorders. 4: 165-189). Further evidence supporting a role for NNK in human lung adenocarcinoma comes from a comparison of NNK levels in cigarette smoke and adenocarcinoma rates in the United States, United Kingdom, Canada, and Australia (Burns, DM et al. (2011) Cancer Causes Control. 22:13-22).

[0584] Mechanistically, NNK is metabolically activated via α-hydroxylation to generate two reactive species, resulting in two types of DNA modifications – methylation and pyridyloxybutylation (Hecht, SS. (2012) Int. J. Cancer. 131: 2724-2732; Hecht, SS. (2008) Chem. Res. Toxicol. 21: 160-171) (Figure 16). The extent of DNA damage can be characterized by quantifying different DNA adducts – for methylation, O 6 -methylguanine (O 6 -mG) and 7-methylguanine (7-mG), and for pyridyl oxobutylation 7-[4-(3-pyridyl)-4-oxobutan-1-yl]guanine (7-pobG), O 6 -[4-(3-pyridyl)-4-oxobutan-1-yl]-2′-deoxyguanosine (O 6 -pobdG) and O 2 -[4-(3-pyridinyl)-4-oxobutan-1-yl]thymidine (O 2 -pobdT). NNK is also metabolized to 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) ( Fig.16 ). NNAL can also be activated via α-hydroxylation to generate two reactive species, resulting in two types of DNA modifications – methylation and pyridyl hydroxybutylation. The extent of pyridyl hydroxybutylation can be determined by quantifying 7-[4-(3-pyridyl)-4-hydroxybutan-1-yl]guanine (7-phbG), O 6 -[4-(3-pyridyl)-4-hydroxybutan-1-yl]-2′-deoxyguanosine (O 6 -phbdG) and O 2 -[4-(3-pyridyl)-4-hydroxybutan-1-yl]thymidine (O 2 -phbdT). DNA damage caused by NNK and NNAL has been established as a major potential mechanism for NNK-induced lung tumorigenesis (Hecht, SS. (1998) Chem. Res. Toxicol. 11: 559-603). Therefore, reducing the DNA damage is a plausible strategy for chemoprevention of lung cancer.

[0585] The A / J mouse NNK-induced lung tumorigenesis model has been widely used to evaluate lung cancer preventive agents (Hecht, SS et al. (2009) Nat. Rev. Cancer. 9: 476-488), because A / J mice are prone to lung tumorigenesis (O'Donnell, EP et al. (2006) Cancer Lett. 241: 197-202) and the tumors have similar morphological, histological and molecular characteristics to human lung adenocarcinoma (Malkinson, AM. (2001) Lung Cancer. 32: 265-279). After NNK treatment, A / J mice develop lung tumors with a 100% incidence and high multiplicity (Hecht, SS et al. (2009) Nat. Rev. Cancer. 9: 476-488; Malkinson, AM. (2001) Lung Cancer. 32: 265-279). In NNK-induced DNA adducts, O 6 -mG has a strong positive correlation with lung tumor multiplicity (Peterson, LA et al. (1991) Cancer Res. 51: 5557-5564) and has an increased 6 -mG-specific DNA repair ability of A / J mice is less susceptible to NNK-induced lung tumorigenesis (Liu, L et al. (1999) Carcinogenesis. 20: 279-284). 6 -mG(19-22) high error coding nature, NNK-induced O 6 -mG is considered to be required for initiation of lung tumorigenesis in A / J mice.

[0586] As described herein, a commercial kava product effectively blocks NNK-induced lung tumorigenesis in A / J mice at a dose of 1.25 mg / g diet (Leitzman, P et al. (2014) Cancer. Prev. Res., 7: 86-96). Fraction B of this product (containing most of the kavalactones ( Fig.17 A) fully reproduced its chemopreventive efficacy. Kava and fraction B reduced NNK-induced DNA damage in the lung, with a preferential reduction of O over POB adducts 6 -mG adducts, while the ineffective fractions 6 -mG had no effect (Leitzman, P et al. (2014) Cancer. Prev. Res., 7: 86-96). Therefore, the effect of single chemical substances in fraction B on O 6The effect of -mG will be an economical strategy to identify one or more active lead compounds. 6 The preferential reduction of -mG adducts is also mechanistically interesting.

[0587] Therefore, the identification and determination of dihydroanecarbomer (DHM) as an active compound is described herein. DHM potently and effectively blocks NNK-induced lung tumorigenesis in A / J mice. As described below, the mechanism leading to its unique reduction of NNK-induced DNA damage has been studied and preliminary long-term safety of DHM in A / J mice exposed to high doses is provided.

[0588] abbreviation

[0589] DHM, dihydrocapsaicin; DHK, dihydrocapsaicin; NNK, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone; NNAL, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol; BaP, benzo[a]pyrene; O 6 -mG,O 6 -methylguanine; 7-mG, 7-methylguanine; 7-pobG, 7-[4-(3-pyridyl)-4-oxobutan-1-yl]guanine; O 6 -pobdG,O 6 -[4-(3-pyridyl)-4-oxobutan-1-yl]-2′-deoxyguanosine; O 2 -pobdT,O 2 -[4-(3-pyridinyl)-4-oxobutan-1-yl]thymidine; 7-phbG, 7-[4-(3-pyridinyl)-4-hydroxybutan-1-yl]guanine; O 6 -phbdG,O 6 -[4-(3-pyridinyl)-4-hydroxybutan-1-yl]-2′-deoxyguanosine; O 2 -phbdT,O 2 -[4-(3-pyridyl)-4-hydroxybut-1-yl]thymidine; AGT, O6-alkylguanine-DNA alkyltransferase; ANOVA, analysis of variance; ALP, alkaline phosphatase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; BUN, blood urea nitrogen; CK, creatine kinase; H&E, hematoxylin and eosin; MTD, maximum tolerated dose; PEITC, phenylethyl isothiocyanate; I3C, indole-3-carbinol.

[0590] Materials and methods

[0591] Chemicals, reagents and animal diets

[0592] NNK was prepared by following the procedures reported previously (Hecht, SS et al. (1983) Carcinogenesis. 4:305-310). [CD3] 6 -mG was purchased from Toronto Research Chemicals (Toronto, Ontario, Canada). [ 13 CD3] 7-methylguanine was prepared by following a reported procedure (Peterson, LA et al. (2013) Chem. Res. Toxicol. 26: 1464-1473). 7-PobG, O 6 -pobdG,O 2 -pobdT, 7-phbG, O 6 -phbdG,O 2 -phbdT and the corresponding [pyridine-D4] analogs were synthesized by following reported procedures (Lao, Y et al. (2006) Res. Toxicol. 19: 674-682; Sturla, SJ et al. (2005) Chem. Res. Toxicol. 18: 1048-1055; Upadhyaya, P et al. (2008) Chem. Res. Toxicol. 21: 1468-1476). Micrococcal nuclease and phosphodiesterase II were purchased from Worthington Biochemical Corporation (Lakewood, NJ). Alkaline phosphatase was purchased from Roche Molecular Biochemicals (Indianapolis, IN).

[0593] Kava was obtained from Gaia Herbs, Inc. (Brevard, NC), an ethanol extract of wild, handcrafted lateral roots from Vanuatu. It was standardized to 150 mg / mL total kavalactones. AIN-93 G and M powdered diets were purchased from Harlan Teklad (Madison, WI). For short-term DNA damage studies, the AIN-93G diet was used during the experiment. For long-term lung tumorigenesis studies, the AIN-93G diet was started one week before the first NNK administration and ended one week after the second NNK administration. The AIN-93M diet was used during the remaining experimental period.

[0594] Separation of Chemical Substances in Fraction B

[0595] Five kavalactones (demethoxycapsaicin, (+)-dihydrocapsaicin (DHK), (+)-capsaicin, (+)-DHM and (+)-capsaicin) were separated from fraction B by normal phase silica gel chromatography using a gradient mixture of hexane and ethyl acetate as the eluent. Fig.17 A) By 1 H-NMR and mass spectrometry characterize their chemical structures. Purity>90% was assessed by HPLC on a Beckman Coulter System Gold 126 solvent module with 168 detectors. Analysis was performed using a Clipeus C-18 column (5 μm, 250 x 4.6 mm). The flow rate was 1 mL / min. Mobile phase A was 10 mM NH4OAc aqueous solution and mobile phase B was acetonitrile. The time program for analysis was 50% B for 15 min.

[0596] Synthesis of (±)-DHM

[0597] (±)-DHM was synthesized by a procedure similar to that reported previously (Shaik, AA, Tan, J., Lu, J., Xing, C. (2012) ARKIVOC. viii: 137-145). 1 Its structure and purity were characterized by H-NMR, mass spectrometry and HPLC.

[0598] Diet preparation

[0599] Kava was reconstituted in anhydrous ethanol (50 mL) and then mixed with the AIN-93 diet (150 g). Similarly, anhydrous ethanol (50 mL each) reconstituted with kavalactones was mixed with the AIN-93 diet (150 g each) as the kavalactone diet. Anhydrous ethanol (50 mL) was mixed with the AIN-93 diet (150 g) as the control diet. The reconstituted diet was vacuum dried to remove the ethanol and then ground into a fine powder. All diets were mixed with additional AIN-93 diets to the desired dose.

[0600] General experimental protocols for animal studies

[0601] Female A / J mice (5-6 weeks old) were purchased from Jackson Laboratory (Bar Harbor, ME) and handled according to University of Minnesota, IACUC-approved animal welfare protocols. Upon arrival, mice were housed in a specific pathogen-free animal facility at Research Animal Resources, University of Minnesota.

[0602] General Protocol for Isolating DNA from Lung and Liver Tissue

[0603] DNA was isolated and purified from approximately 100 mg of lung and liver tissue from each mouse following the Genomic-tip 100 / G protocol from Qiagen Corp (Valencia, CA).

[0604] Evaluation of the effects of five natural kavalactones in fraction B on NNK-induced O in lung tissue of A / J mice 6 Effects of -mG and three POB adducts

[0605] After one week of adaptation, mice were weighed, randomly divided into seven groups (3 mice per group), and switched to AIN-93G diet on a date defined as day 1. Mice in groups 1 and 2 were given AIN-93G diet during days 1 to 7. Mice in groups 3-7 were given AIN-93G diet supplemented with demethoxycapsaicin, (+)-DHK, (+)-capsaicin, (+)-DHM or (+)-capsaicin, respectively, at a dose of 1 mg / g diet from day 1 to day 7. A single dose of NNK at 100 mg / kg body weight in saline (0.1 ml) was given to mice in groups 2-7 via intraperitoneal (ip) injection at the beginning of day 7, while saline (0.1 ml) was given to mice in group 1. Mice were euthanized with excess CO2 24 h after NNK treatment, and lung tissues were collected and stored at -80°C until DNA isolation. Many NNK-induced DNA modifications (O 6 -mG, 7-pobG, O 6 -pobdG and O 2 -pobdT) was quantified by following a reported procedure (Leitzman, P et al. (2014) Cancer. Prev. Res., 7:86-96).

[0606] Evaluation of the dose-response effects of kava and (+)-DHM on (+)-DHK and synthetic (±)-DHM on NNK-induced DNA adducts in lung and liver tissues of A / J mice

[0607] After one week of adaptation, A / J mice were weighed, randomly divided into 10 groups (3 mice per group, except for Group 1 which had 1 mouse as a negative control), and switched to the AIN-93G diet on a date defined as Day 1. Mice in Groups 1 and 2 were given the AIN-93G diet during Days 1 to 7. Mice in Groups 3-10 were given the AIN-93G diet supplemented with kava, (+)-DHK, (+)-DHM, and synthetic (±)-DHM, respectively, at specific doses from Days 1 to 7. Mice in Groups 2-10 were given a single dose of NNK at 100 mg / kg body weight in saline (0.1 ml) via intraperitoneal injection at the beginning of Day 7, while mice in Group 1 were given saline (0.1 ml). Mice were euthanized with excess CO2 24 h after NNK treatment, and lung tissues were collected and stored at -80°C until DNA isolation. A set of NNK-induced DNA adducts (NNK) was quantified by the following reported procedures ( 6 -mG, 7-mG, 7-pobG, O 6 -pobdG,O 2 -pobdT, 7-phbG, O 6 -phbdG and O 2 -phbdT) (Leitzman, P et al. (2014) Cancer. Prev. Res., 7:86-96; Peterson, LA et al. (2013) Chem. Res. Toxicol. 26:1464-1473; Upadhyaya, P et al. (2008) Chem. Res. Toxicol. 21:1468-1476). For 7-mG separation, DNA (30 μg) was dissolved in 10 mM sodium phosphate buffer pH 7 (500 μL) and added. 13 C1 2 H3 7-mG (10 pmol). The sample was heated to 80°C for 30 minutes. An aliquot (100 μL) was removed for 7-mG analysis.

[0608] Evaluation of the efficacy of (+)-DHM versus (+)-DHK and synthetic (±)-DHM against NNK-induced lung adenoma formation in A / J mice

[0609] After one week of acclimation, mice were weighed, randomized into 8 groups and switched to the AIN-93G diet on a day defined as day 1. The number of mice in each group is specified in the Results section ( Fig.36). During the 1st day to the 14th day, mice were fed a diet supplemented with different compounds at the dosage specified in the results section. On the 7th and 14th days, mice in the negative control group received 0.1mL saline solution via intraperitoneal injection, while mice in other groups received NNK (respectively 100 and 67mg / kg, in 0.1mL saline solution). At the end of the 21st day, mice were switched to the AIN-93M diet until the end of the study. Supplementary diet twice a week. Dietary consumption was measured twice a week and body weight was monitored weekly. This study terminated at the end of the 119th day. All mice were euthanized with excessive CO2. Lungs were collected and tumors on the lung surface were counted by a pathologist (MGO'S.) certified by the ACVP committee under blind conditions.

[0610] Evaluation of the safety of A / J mice exposed to (+)-DHM at a dose of 0.5 mg / g diet for 17 weeks

[0611] After one week of adaptation, A / J mice were weighed and randomly divided into two groups (5 mice in the control group and 10 mice in the (+)-DHM treatment group). The mice in the control group maintained the AIN-93G diet for 3 weeks and then switched to the AIN-93G diet until the end of the 17th week. The mice in the (+)-DHM treatment group maintained the same diet as the control group supplemented with the (+)-DHM of the 0.5mg / g diet. Supplementary diet twice a week. Monitor food intake twice a week and monitor mouse weight once a week. At the end of the 8th week, serum samples of each mouse were prepared by blood collected through the facial vein and analyzed for a group of clinical chemistry analytes. At the end of the 17th week, mice were euthanized via excessive CO2. Blood was collected, one of which was used to analyze hematology, and the other was processed for serum and analyzed for the whole group of clinical chemistry analytes. Record individual animal weights and major organ weights (lungs, liver, heart, spleen and kidney). Lungs, liver, heart, spleen, kidney and pancreas were fixed in 10% neutral buffered formalin solution. Appropriately fixed tissues were processed into paraffin blocks using standard histological techniques and cut into 5μ sections and stained with hematoxylin and eosin (H&E). Histological slides were examined using light microscopy by an experienced ACVP board-certified pathologist (MGO'S.).

[0612] Statistical analysis

[0613] Lung adenoma diversity data are reported as mean ± SD (n = 5-9). One-way analysis of variance (ANOVA) was used to compare the mean values ​​of NNK and NNK / treatment groups. When the one-way ANOVA analysis was statistically significant, the number of tumors between the NNK control and treatment groups was compared using Dunnett's test. p-value ≤ 0.05 was considered statistically significant. For DNA damage studies, one-way ANOVA was used to compare mean ± SD (n = 3). When the one-way ANOVA analysis was statistically significant, the NNK control and treatment groups were compared using Dunnett's test. p-value ≤ 0.05 was considered statistically significant. For safety studies, unpaired Student's t-test was used to compare the control and (+)-DHM treatment groups. Bilateral p-value ≤ 0.05 was considered statistically significant. All analyses were performed using GraphPad Prism 4 (GraphPad Software, Inc. La Jolla, CA).

[0614] result

[0615] Effects of five kavalactones in fraction B on NNK-induced O in lung tissue of A / J mice 6 -mG, 7-pobG, O 6 -pobdG and O 2 -The role of pobdT

[0616] As previously described in this article, data have shown that different kava substances reduce lung tissue O 6 -mG is positively correlated with its lung tumor prevention efficacy, and fraction B reproduces the chemopreventive effect of kava on NNK-induced lung tumorigenesis in A / J mice (Leitzman, P et al. (2014) Cancer. Prev. Res., 7: 86-96). This experiment was designed to identify the O-mG in fraction B that reduces NNK-induced 6 -One or more key compounds of mG, which may be active chemopreventive agents.

[0617] Five known kavalactones have been isolated from fraction B as its major components ( Fig.17 A), accounting for 93% of its mass balance (Leitzman, P et al. (2014) Cancer. Prev. Res., 7:86-96). The effects of these five compounds on NNK-induced DNA damage in lung tissue were studied, including O 6 -mG and three POB adducts (7-pobG, O 6 -pobdG and O 2 -pobdT)( Fig.17B). Two compounds (+)-DHM and (+)-pespermum parkinsonian significantly reduced NNK-induced O 6 -mG, while the other compounds did not, indicating the importance of (+)-DHM and (+)-pespermol as potential active compounds and the 1,3-dioxole functional group. All five compounds had a weaker effect on reducing POB adducts ( Fig.17 B), which is consistent with the effect of fraction B (Leitzman, P et al. (2014) Cancer. Prev. Res., 7: 86-96). Although not statistically significant, the O 6 The extent of reduction in -mG was greater than that by (+)-capsaicin. Therefore, (+)-DHM was selected as a lead compound for further study, with DHK as a negative control, due to its higher structural similarity to (+)-DHM relative to demethoxycapsaicin and (+)-capsaicin.

[0618] Dose-response effects of (+)-DHM on an expanded set of NNK-induced DNA damage in lung tissues of A / J mice compared with kava, (+)-DHK, and synthetic (±)-DHM

[0619] This study was designed to address three questions. First, what dose of (+)-DHM would induce O to a similar extent as that achieved by kava at a dose of 1.25 mg / g diet? 6 -mG reduction (which effectively blocks NNK-induced lung tumor formation) (Leitzman, P et al. (2014) Cancer. Prev. Res., 7: 86-96)? Kava-5 and 1.25 mg / g diet were evaluated at two doses, while (+)-DHM-1, 0.25, 0.1 and 0.01 mg / g diet were evaluated at four doses, with (+)-DHK evaluated at a dose of 1 mg / g diet as a control. Secondly, given that the isolated (+)-DHM contains impurities, how can the possibility of impurities as active compounds be ruled out? Therefore, synthetic (±)-DHM was prepared and evaluated at a dose of 1 mg / g diet. Finally, (+)-DHM preferentially reduced O compared to POB adducts in lung tissue. 6 What is the mechanism of -mG? Based on the molecular basis of NNK metabolism and DNA damage ( Fig.16 ) (Hecht, SS. (1998) Chem. Res. Toxicol. 11: 559-603), four possible mechanisms have been identified: 1) preferential inhibition of NNK methylene hydroxylation over NNK methyl hydroxylation; 2) increased O 6-alkylguanine-DNA alkyltransferase (AGT)-mediated O 6 -mG repair (Belinsky, SA et al. (1988) Carcinogenesis.9:2053-2058; Peterson, LA et al. (1993) Cancer Res.53:2780-2785); 3) preferential inhibition of NNAL hydroxylation relative to NNK hydroxylation; or 4) increased detoxification of NNAL. These mechanisms can be distinguished by characterizing the effects of DHM on an expanded set of DNA adducts, including POB DNA adducts (7-pobG, O 6 -pobdG and O 2 -pobdT), PHB DNA adducts (7-phbG, O 6 -phbdG and O 2 -phbdT) and methyl DNA adducts (O 6 -mG and 7-mG).

[0620] Consistent with previous results, kava preferentially and dose-dependently reduced O in lung tissue. 6 -mG (Figure 18A.a), while it had minimal effect on POB adducts ( Fig.18 Ab-d). (+)-DHM also preferentially and dose-dependently reduced O 6 -mG( Fig.18 Aa), with a weaker effect on POB adducts (Fig. 18A.bd), while (+)-DHK did not reduce any of these adducts even at a dose of 1 mg / g diet. 6 The reduction in -mG was comparable to that induced by kava at a dose of 1.25 mg / g diet ( Fig.18 Aa). Since (+)-DHM at a dose of 0.01 mg / g diet has an effect on 6 -mG has no effect, the minimum chemopreventive dose of DHM would be 0.01-0.1 mg / g diet. (+)-DHM also dose-dependently reduced 7-mG ( Fig.18 Ba), where the reduction degree is the same as O 6 - Those in mG are similar to ( Fig.18 These results suggest that AGT-mediated O 6 -mG repair was not involved in the reduction of DNA damage by DHM. Surprisingly, (+)-DHM also dose-dependently reduced PHB adducts in lung tissue ( Fig.18 Bb-d), where the reduction degree is similar to that of O 6-mG and 7-mG are similar to those in ( Fig.18 Aa and 18B.a). Since (+)-DHM had minimal effect on POB adducts, these results suggest that the reduction of NNK-induced DNA damage may be mediated through the NNAL pathway, either by inhibiting its activation or enhancing its detoxification effect ( Fig.16 A and B in Figure 1). Synthetic (±)-DHM at 1 mg / g diet had a similar effect as natural (+)-DHM in reducing DNA damage ( Fig.18 A and B), which demonstrated DHM as the active compound.

[0621] Effects of Kava, (+)-DHM, (+)-DHK, and synthetic (±)-DHM deficiency on NNK-induced DNA damage in liver tissue of A / J mice

[0622] Since the liver is the main metabolic organ of the body, we also characterized the effects of kava, (+)-DHM, (+)-DHK and (±)-DHM on NNK-induced DNA damage in liver tissue. Surprisingly, none of the treatments had a significant effect on NNK-induced DNA damage in liver tissue (data not shown).

[0623] Efficacy of Kava, (+)-DHM, (+)-DHK, and synthetic (±)-DHM against NNK-induced lung adenoma formation in A / J mice

[0624] To verify the chemopreventive potential of DHM, we conducted Fig.36NNK-induced lung adenoma assay as shown in . A / J mice not treated with NNK had low adenoma incidence (20%) and low adenoma multiplicity (0.2±0.4 lung adenomas / mouse, Group 1), while A / J mice treated with NNK had 100% adenoma incidence and high adenoma multiplicity (13.9±6.9 lung adenomas / mouse, Group 2). Kava at a dose of 1.25 mg / g diet slightly reduced adenoma incidence by 20%, but significantly reduced adenoma multiplicity by 95.6% (to 0.8±0.4, Group 3). Based on the estimated minimum effective dose of (+)-DHM to be 0.01 to 0.1 mg / g diet, we tested 0.5 and 0.05 mg / g diet (Groups 4 and 5). Both doses of (+)-DHM reduced adenoma incidence by 40% and significantly reduced adenoma multiplicity by 97.1% (to 0.6 ± 0.5, Groups 4 and 5), indicating that its minimum effective dose has not yet been reached. All mice treated with (±)-DHM at a dose of 0.5 mg / g diet were adenoma-free (Group 8). (+)-DHK, as a negative control, showed no reduction at all at 0.5 or 0.05 mg / g diet (Groups 6 and 7). Overall, these data establish DHM as a lung cancer chemopreventive agent with a minimum effective dose below 0.05 mg / g (50 ppm).

[0625] Safety evaluation of A / J mice after 17-week continuous exposure to (+)-DHM at a dose of 0.5 mg / g diet

[0626] Recognizing the concerns about hepatotoxicity associated with kava (Rowe, A et al. (2012) Res. 26: 1768-1770; Teschke, R et al. (2013) Phytother Res. 27: 472-474), we characterized a number of parameters for evaluating the safety of (+)-DHM. Mice fed with (+)-DHM started with slightly higher mean body weights and remained heavier than control mice in this study, but none of the differences were statistically significant (Figure 20). Food intake was also similar ( Fig. 20 The clinical chemistry results of serum samples collected at the end of week 8 are summarized in Fig.21A, and they include alkaline phosphatase (ALP), alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), albumin, total bilirubin, creatine kinase (CK), glucose, cholesterol and amylase. All of these parameters reflect liver function, except amylase (a marker of the pancreas). No significant differences were detected in any of these parameters between the control group and the (+)-DHM group. The clinical chemistry results of the serum samples collected at the end of the 17th week are summarized in Figure 21B. As with the results of 8 weeks, no significant differences were detected in any of these parameters between the control group and the (+)-DHM group. The time course changes of these parameters for each mouse also showed no significant differences between the control and (+)-DHM groups (data not shown). At the 17th week, the concentrations of various salts in the serum were also quantified; serum osmotic pressure and anion gap were calculated using these data, which showed no difference between the control and (+)-DHM groups (data not shown). Blood samples from week 17 were also analyzed for whole group hematology (one blood sample from the control mice clotted and could not be analyzed, resulting in four data points for the control group, Fig. 22 There were no significant differences in these parameters between the control and (+)-DHM groups. Finally, the final body weight and relative weights of the five organs ( Fig.19 ) were not significantly different - except for lung tissue. The mean relative lung weight of the control group was significantly higher than that of the (+)-DHM group (p = 0.05). Examination of the data from each mouse showed that one mouse in the control group had increased lung weight, which was attributed to multifocal hemorrhage in the lung at the time of euthanasia. Aside from this animal, there were no significant differences between the control and (+)-DHM groups (p = 0.10). Histopathological examination of liver, lung, heart, kidney, spleen, and pancreatic tissues showed no significant differences between the control and (+)-DHM groups (data not shown).

[0627] Discussion and Conclusion

[0628] The results described herein clearly identify DHM as an effective chemopreventive agent that safely and completely blocks NNK-induced lung tumorigenesis in A / J mice at a dietary dose of 0.05 mg / g (50 ppm). 6 -mG, (+)-DHM was identified as the lead compound among five structurally similar compounds ( Fig.17B). In subsequent dose response experiments using DNA adduct reduction as a readout, its minimum effective dose was estimated to be 0.01-0.1 mg / g diet (Figure 18). (+)-DHM was therefore tested at doses of 0.5 mg / g (500 ppm) and 0.05 mg / g (50 ppm) diet, with a 2-week exposure window only during NNK initiation ( Fig.36 , Groups 4 and 5). Since 0.05 mg / g of (+)-DHM reduced the multiplicity of NNK-induced lung adenomas by 97% and was as effective as 0.5 mg / g of (+)-DHM, its minimum effective dose is likely to be lower than 0.05 mg / g. According to the body surface area normalization method (Reagan-Shaw, S et al. (2008) FASEB J. 22: 659-661), the extrapolation of the effective dose of 0.05 mg / g diet for 20 g mice (3 g food intake per day) would be equivalent to a human dose of 47 mg / day (based on a 75 kg human body weight). Therefore, in terms of dosage, (+)-DHM is highly feasible for human use. The results of synthetic (±)-DHM also provide useful information. First, they confirm that DHM acts as an active chemopreventive agent. Second, the synthetic racemic form appears to be superior to natural (+)-DHM in blocking NNK-induced DNA adducts and lung adenoma formation at the doses tested at 1 mg / g and 0.5 mg / g, respectively, although the difference was not statistically significant. Whether synthetic (-)-DHM may be more effective than natural (+)-DHM in a dose range close to its minimum effective dose remains to be determined.

[0629] Interestingly, it was noted that the structural analog of (+)-DHM, (+)-DHK ( Fig.17 A) In blocking DNA damage ( Fig.17 and 18 ) or pulmonary adenoma formation ( Fig.36 ) is completely inactive in terms of (+)-DHM and (+)-DHK. Such a sudden change in the in vivo structure-activity relationship (SAR) between (+)-DHM and (+)-DHK indicates the high target specificity of (+)-DHM. This also highlights the importance of the 1,3-dioxole functional group in (+)-DHM for its efficacy. The five-membered ring may be critical for the specific interaction of (+)-DHM with its molecular target, which remains to be determined, leading to its excellent chemopreventive efficacy. Alternatively, (+)-DHM can be metabolized to an in vivo active form via the 1,3-dioxole functional group. Further studies are needed to distinguish these possibilities.

[0630] Mechanistically, (+)-DHM reduced O in lung tissue in a dose-dependent manner with a similar reduction. 6 -mG, 7-mG, 7-phbG, O 6-phbdG and O 2 -phbdT( Fig.18 Aa and 18B.ad), while it has no effect on 7-pobG, O 6 -pobdG and O 2 -pobdT has a weaker effect ( Fig.18 Ab-d). These data suggest that DHM may inhibit NNAL activation ( Fig.16 A) or increase NNAL detoxification ( Fig.16 B) can preferentially block NNAL-mediated DNA damage. Closer inspection of the reduction of methyl and PHB adducts revealed that methyl adducts were slightly more reduced, although the difference was not statistically significant ( Fig.18 C shows a representative set of data for treatment with (+)-DHM at a dose of 1 mg / g diet). Since the reduction in POB and PHB adducts was less than that in methyl adducts, DHM may also preferentially inhibit methylene hydroxylation compared to methyl hydroxylation of NNK and NNAL. The effect of DHM deficiency on NNK-induced DNA damage in the liver is of interest, which may be mediated by potential differences in DHM abundance in lung and liver tissues. Alternatively, lung tissue may contain biomolecules that interact with DHM that are involved in NNAL activation / detoxification that are not present in liver tissue.

[0631] Reports characterizing the various DNA adducts in lung and liver tissues of NNK-treated A / J mice are limited. An early study by Morse et al. showed that phenylethyl isothiocyanate (PETIC) reduced O in lung tissue. 6 -mG, but other DNA adducts and liver tissue were not analyzed (Morse, MA et al. (1989) Cancer Res. 49:2894-2897). A later study by Prokopczyk et al. showed that synthetic 1,4-phenylenebis(methylene)selenocyanate (an analog of PEITC) preferentially reduced O compared to 7-mG. 6 -mG (Prokopczyk, B et al. (1996) Carcinogenesis. 17: 749-753). Crampsie et al. recently reported that phenylbutyl isoselenocynate (ISC-4) (also a synthetic selenium analog of PEITC) reduced methyl and POB DNA damage in lung and liver tissues of A / J mice (Crampsie, MA et al. (2011) Cancer Prev. Res. 4: 1884-1894). Another promising lung cancer chemopreventive agent indole-3-carbinol (I3C) was discovered, which reduces O in lung tissue. 6-mG, but may increase liver tissue O by increasing liver NNK metabolism 6 -mG (Morse, MA et al. (1990) Cancer Res. 50:2613-2617). The studies described herein are the first to characterize DNA damage in all three types of A / J mice and DHM appears to have a different mechanism relative to PEITC and I3C-based lung cancer chemoprevention agents.

[0632] Further characterization has been performed on NNK-induced DNA damage in lung and liver tissues of F344 rats, and several studies have also analyzed the effect of the chirality of NNAL (Lao, Y et al. (2006) Chem. Res. Toxicol. 19: 674-682; Upadhyaya, P et al. (2008) Chem. Res. Toxicol. 21: 1468-1476; Lao, Y et al. (2007) Chem. Res. Toxicol. 20: 235-245; Upadhyaya, P et al. (2009) Drug Metab. Dispos. 37: 1147-1151). Upadhyaya et al. demonstrated that (R)-NNAL preferentially produces PHB adducts, while (S)-NNAL and NNK primarily produce POB adducts. Although the DNA damage induced by (R)- and (S)-NNAL in A / J mice has not been characterized, the two isomers appear to have different metabolism and tumorigenicity (Upadhyaya, P et al. (1999) Carcinogenesis. 20: 1577-1582). It remains to be determined whether PHB DNA damage in the lung tissue of A / J mice is mainly derived from (R)-NNAL and whether DHM selectively inhibits the activation of (R)-NNAL, which leads to a preferential reduction of PHB DNA adducts in lung tissue. Alternatively, DHM can selectively enhance the detoxification effect of (R)-NNAL.

[0633] The safety of (+)-DHM is of great importance due to the purported risk of hepatotoxicity of kava. A / J mice continuously exposed to (+)-DHM at a dose of 0.5 mg / g diet (at least ten times its minimum effective dose) for 17 weeks did not present any adverse effects characterized by the following parameters: mean weekly body weight gain; mean weekly food intake; clinical chemistry analysis of serum samples collected at weeks 8 and 17; hematology analysis at week 17; final body weight; relative weights of heart, lung, liver, kidney, and spleen at week 17; and pathology of heart, lung, liver, kidney, spleen, and pancreas at week 17. Since the dose of 0.5 mg / g appears to be still below the maximum tolerated dose (MTD, which remains to be determined), it is expected that (+)-DHM will have a wide safety margin as a lung cancer chemopreventive agent. A non-kavalactone compound has been identified in kava that reproduces the hepatotoxic risk of kava, whereas (+)-DHM showed no evidence of hepatotoxic risk under the same conditions, suggesting that (+)-DHM may not have the hepatotoxic concerns associated with kava (see, Example 5).

[0634] In summary, the data described herein support DHM as a powerful and effective chemopreventive agent for NNK-induced lung tumorigenesis in A / J mice, which has a unique mechanism of action relative to several well-known lung cancer chemopreventive agents. Its sudden change in vivo SAR shows high target specificity, which is very necessary to minimize the adverse effects reflected by its impressive safety profile. Therefore, DHM is a promising lung cancer chemopreventive agent.

[0635] Example 5

[0636] Kavapigenins A and B, but not dihydropiperazine, from kava enhance acetaminophen-induced hepatotoxicity in C57BL / 6 mice.

[0637] summary

[0638] Anxiolytic kava products have been associated with rare but severe hepatotoxicity in humans. This adverse potential has never been captured in animal models and the responsible compound or compounds remain to be identified. Lack of this knowledge greatly impedes the preparation of safer kava products and limits their beneficial applications. In this study, the toxicity of kava as a single substance or in combination with acetaminophen (APAP) was evaluated in C57BL / 6 mice. Kava alone showed no adverse effects with long-term use even at a dose of 500 mg / kg body weight. In contrast, three days of kava pretreatment enhanced APAP-induced hepatotoxicity, resulting in increases in serum ALT and AST and increased severity of liver injury. The chalcone-based kava kavalactones A (FKA) and B (FKB) in kava reproduced their hepatotoxic synergy with APAP, while dihydroanecarotene (DHM, a representative kavalactone and potential lung cancer chemopreventive agent) did not have such an effect. These results are the first to demonstrate the hepatotoxicity risk of kava and its chalcones-based FKA and FKB in vivo and suggest that herb-drug interactions may lead to the rare hepatotoxicity associated with anxiolytic kava use in humans. Kava preparations, such as Fraction B described herein, which do not contain kava piperin A and B, would have minimized risk and enriched beneficial components.

[0639] introduction

[0640] Traditionally, kava is an aqueous extract of the root of Piper methysticum and is used as a ritual and daily drink or herbal remedy by South Pacific islanders (Gounder, R. (2006) Pac. Health Dialog 13, 131-135). Kava has also been used to treat mild and moderate anxiety based on a large number of clinical trials (LaPorte, et al. (2011) Hum. Psychopharmacol. 26, 102-111; Sarris, et al. (2009) Hum. Psychopharmacol. 24, 41-48; Sarris, et al. (2009) Psychopharmacology (Berl) 205, 399-407; Pittler, MH, and Ernst, E. (2000) J. Clin. Psychopharmacol. 20, 84-89). Anxiolytic kava is usually prepared as an organic extract of kava root extracted with ethanol or acetone, rather than the traditional aqueous preparation. Anxiolytic kava has been banned in Europe and some other countries since 2002 because it induces the risk of hepatotoxicity, and it has been included in the US FDA Advisory Committee (Teschke, R., and Wolff, A. (2009) Dig. Liver Dis. 41, 891-901; Teschke, et al. (2013) Phytother. Res. 27, 472-474), but the German Federal Administrative Court denied the ban in June 2014 (Carreno, et al. (2014) FRATINIVERGANO-European Lawyers 13, 2-5).

[0641] Various causes of kava's hepatotoxic risk have been proposed, but none have been verified to date. First, in response to high demand, antianxiety kava may include non-root toxic plant parts (Schulze, et al. (2003) Phytomedicine 10, 68-73). It has also been hypothesized that some kava roots are not properly dried, leading to hepatotoxin contamination (Anke, J., and Ramzan, I. (2004) Planta Med. 70, 193-196). The use of non-traditional varieties may be another reason; different kava varieties have different chemical profiles, and traditional kava is prepared from only a few of them (Anke, J., and Ramzan, I. (2004) Planta Med. 70, 193-196; Lebot, et al. (2014) Food Chem. 151, 554-560). Due to preparation differences, traditional and anxiolytic kava have different composition profiles (Shaik, et al. (2009) Bioorg. Med. Chem. Lett. 19, 5732-5736; Leitzman, et al. (2014) Cancer Prev. Res. (Phila) 7, 86-96), which may also impose different hepatotoxicity risks. In addition, approximately 90% of the reported hepatotoxicity cases associated with kava usage involve concomitant consumption of other drugs or dietary supplements (WH Organization (2007) Assessments of the risk of hepatotoxicity with kava products. WHO Document Production Service; Teschke, R., et al. (2008) Eur. J. Gastroenterol. Hepatol. 20, 1182-1193), suggesting that the hepatotoxicity risk of kava may be mediated through herb-herb or herb-drug interactions.In addition to kava's anxiolytic benefits, an epidemiological survey suggests that traditional kava use may be able to reduce cancer risk (Steiner, GG (2000) Hawaii Med. J. 59, 420-422), which is supported by results from several laboratory animal tumorigenesis models (Leitzman, P., et al. (2014) Cancer Prev. Res. (Phila) 7, 86-96; Johnson, et al. (2011) Am. J. Chin. Med. 39, 727-742; Johnson, et al. (2008) Cancer Prev. Res. (Phila) 1, 430-438; Triolet, et al. (2012) Nutr. Cancer 64, 838-846; Zi, X., and Simoneau, AR (2005) Cancer Res. 65, 3479-3486; Narayanapillai, et al. (2008) Cancer Prev. Res. (Phila) 1, 430-438. al. (2014) Carcinogenesis 35(10), 2365-72). Moreover, despite the ban and listing on the USA FDA advisory list, kava consumption has experienced a global resurgence based on the amount of kava exported by the major kava producing countries (Republic of Vanuatu, Fiji and Tonga) between 2008 and 2013 (Martin, et al. (2014) Measuring the chemical and cytotoxic variability of commercially available kava. Plos One, Accepted). With the recent overturning of the kava ban in Germany, its global use is expected to increase further. Our recent metabolic and cytotoxic analysis of a series of currently commercialized kava products showed that they are diverse in terms of chemical profile and cytotoxicity (Martin, et al. (2014) Plos One, 9(11):e111572.doi:10.1371 / journal.pone.0111572) and may differ in their health benefits and risks.

[0642] Considering the increased human exposure and diverse chemical composition of current kava products, the hepatotoxicity risk of kava needs to be clarified and the responsible chemicals need to be identified, which was the focus of this study. Results showed that kava was safe when given alone but significantly enhanced acetaminophen (APAP)-induced hepatotoxicity in C57BL / 6 mice. Chalcone-based fumaric acid fumaric acid A (FKA) and fumaric acid B (FKB) reproduced kava-enhanced APAP-induced hepatotoxicity, whereas dihydroanapoxanthin (DHM) lacked such risk.

[0643] abbreviation

[0644] Acetaminophen, APAP; Kava piperine A, FKA; Kava piperine B, FKB; Dihydroanecarboxymethyl, DHM; Analysis of variance, ANOVA; Alanine aminotransferase, ALT; Aspartate aminotransferase, AST; Polyethylene glycol-400, PEG-400.

[0645] Materials and methods

[0646] Chemicals and reagents

[0647] An ethanol extract of wild hand-processed kava root from Vanuatu was purchased from Gaia Herbs, Inc (Brevard, NC, standardized to 150 mg / mL total kavalactones). DHM was purified from the kava product using normal phase silica gel chromatography as described earlier (Narayanapillai, et al. (2014) Carcinogenesis 35(10), 2365-72). FKA and FKB were synthesized and characterized following established procedures (Johnson, et al. (2011) Am. J. Chin. Med. 39, 727-742). Kava and all compounds were completely vacuum dried to remove any solvent residues. APAP was purchased from Sigma Aldrich (St Louis, MO). The desired pharmaceutical formulations were prepared by mixing kava or pure compounds with PEG-400 and stored at 4°C until use.

[0648] Animal study design

[0649] All animal studies were in accordance with the approval and guidance of the University of Minnesota Institutional Animal Care and Use Committee. Six-week-old female C57BL / 6J mice (Jackson Laboratories, ME) were housed in a specific pathogen-free animal facility at Research Animal Resources, University of Minnesota with free access to standard rodent chow and water. All mice were acclimated for one week before use in experiments. Mice were fed the dose formulation at the indicated dose and number of times, euthanized with an overdose of CO2, and necropsy was performed by experienced researchers.

[0650] A long-term study was designed to evaluate the hepatotoxicity of kava alone. C57BL / 6 mice were randomized (n=4). Mice in the control group were given PEG-400 (200 μL) daily via gavage, 6 days a week, for 14 weeks. Mice in the kava-treated group were given kava at a dose of 500 mg / kg body weight daily via gavage, 6 days a week, for 14 weeks. The selected kava dose was based on a recent safety study of another kava product conducted by the National Toxicology Program (National Toxicology, P. (2012) Natl. Toxicol. Program Tech. Rep. Ser. 1-186). The weight of the mice was measured once a week. Upon autopsy, the final body weight was measured and the serum of each mouse was analyzed for two major biomarkers of liver function, alanine aminotransferase (ALT) and aspartate aminotransferase (AST).

[0651] A short-term combination study was designed to evaluate the potential synergistic effects of kava and its chemicals with APAP-induced hepatotoxicity. C57BL / 6 mice were randomized (8-15 mice per group) and given PEG-400 (200 μL), kava (500 mg / kg body weight), DHM or FKA and FKB at the designated doses daily via oral gavage for two days. On the third day, APAP (800 mg / kg body weight) in PEG-400 (200 μL) was co-administered to each group of mice. Body weights were recorded daily. Necropsy was performed by experienced researchers 24 hours after the last gavage. Serum from each mouse was analyzed for ALT and AST. Liver was collected and stored in 10% neutral buffered formalin. Appropriately fixed tissue was processed into paraffin blocks using standard histological techniques and cut into 5μ sections and stained with hematoxylin and eosin (H&E). Histology slides were examined under blinded conditions by an experienced ACVP board-certified pathologist (MGO'S.) using light microscopy, where liver lesions were graded from 0 to 4 (0 = absent, 1 = minimal, 2 = mild, 3 = moderate, 4 = severe) based on the extent of necrosis.

[0652] Statistical analysis

[0653] Clinical chemistry data are reported as mean ± SD (n = 4-15). For the long-term kava alone study, the mean values ​​between the control and treatment groups were compared using a two-sided Student t-test. p-values ​​≤ 0.05 were considered statistically significant. One-way analysis of variance (ANOVA) was used to compare the mean values ​​between different groups in the short-term combination study. When the one-way ANOVA analysis was statistically significant, Dunnett's test was used to compare APAP and other treatment groups. p-values ​​≤ 0.05 were considered statistically significant. All analyses were performed in GraphPad Prism 4 (GraphPad Software, Inc. La Jolla, CA).

[0654] result

[0655] Kava alone did not affect mouse growth and did not induce signs of hepatotoxicity.

[0656] At the dose tested (500 mg / kg body weight), daily kava treatment did not affect mouse growth (data not shown). There were also no statistically or biologically significant differences between control and kava-treated mice for ALT and AST ( Fig.23 A and 23B).

[0657] Kava enhances APAP-induced hepatotoxicity in C57BL / 6 mice.

[0658] Since approximately 90% of human kava hepatotoxicity cases involve concurrent consumption of other drugs or dietary supplements (WHO rganization (2007) Assessments of the risk of hepatotoxicity with kava products. WHO Document Production Service; Teschke, R., et al. (2008) Eur. J. Gastroenterol. Hepatol. 20, 1182-1193), herb-drug interactions may contribute to the hepatotoxicity risk of kava. On this basis and based on recent reports that kava enhances the toxicity of APAP in vitro (Yang, X., and Salminen, WF (2011) Phytomedicine 18, 592-600), the present study was designed to evaluate the effect of kava on APAP-induced hepatotoxicity in vivo. The treatment regimen was designed to mimic a potential situation in humans - daily consumption of kava with occasional use of APAP. As expected, kava treatment alone had no effect on ALT and AST, whereas APAP treatment significantly increased serum ALT and AST activities ( Fig.24A). The combination of kava and APAP caused a further increase in serum ALT and AST activities (approximately 3-fold relative to APAP alone, Fig.24 A), and these increases were statistically significant compared to APAP treatment alone. Histopathological analysis of liver tissue showed no lesions in control and kava-treated mice ( Fig.24 B), which confirms the lack of hepatotoxicity of kava treatment alone. Lesions from APAP-treated mice were evenly distributed between the different severity categories (0 is no injury and 4 is the highest grade of injury), while the combination of kava and APAP significantly increased the number of mice with the highest level of liver injury ( Fig.24 B), which supports the concept that the increase in ALT and AST activity is biologically significant. These clinical chemistry data and histopathological findings demonstrate for the first time that kava enhances APAP-induced hepatotoxicity in vivo and may mirror the cases of kava hepatotoxicity reported in humans. The severity of histopathological lesions also correlated well with clinical chemistry results ( Fig.24 C). Therefore, only clinical chemistry was performed in subsequent studies.

[0659] DHM did not enhance APAP-induced ALT and AST, while FKB increased both.

[0660] This study was designed to investigate the potential of DHM and FKB to synergize with APAP hepatotoxicity following the same kava and APAP co-treatment regimen ( Fig.25). Thirteen chemicals have been isolated and quantified from the kava product used in this study, of which pipermethystine was not detected (Leitzman, P., et al. (2014) Cancer Prev. Res. (Phila) 7, 86-96). DHM and FKB were chosen for initial evaluation because they are representative of the two major classes of chemicals in kava, kavalactones and chalcones, respectively. In addition, DHM has recently been shown to potently and effectively block NNK-induced lung tumorigenesis in mice (Leitzman, P., et al. Cancer Prev. Res. (Phila) 7, 86-96), while FKB has been identified as the most cytotoxic compound in kava against various cancer cells (Shaik, et al. (2009) Bioorg. Med. Chem. Lett. 19, 5732-5736; Jhoo, et al. (2006) J. Agric. Food Chem. 54, 3157-3162). The doses of DHM (37.5 mg / kg) and FKB (11.5 mg / kg) were based on their abundance in the kava product at a dose of 500 mg / kg (7.5% and 2.3%, respectively) (Leitzman, et al. (2014) Cancer Prev. Res. (Phila) 7, 86-96). DHM and FKB alone had no effect on serum ALT and AST ( Fig.26 A and 26B). When combined with APAP, DHM also had no effect on serum ALT and AST ( Fig.26 On the other hand, when combined with APAP, FKB modestly increased serum levels of ALT and AST, and the increase in AST was statistically significant ( Fig.26 A and 26B), suggesting that FKB contributes to kava-enhanced APAP-induced hepatotoxicity.

[0661] The combination of fava protein A (FKA) and FKB dose-dependently enhanced APAP-induced hepatotoxicity.

[0662] Given that the kava product used in this study contains kavapetroleum A in similar abundance to FKB (Figure 25), this experiment was designed to evaluate the dose-response effect of FKA and FKB together on APAP hepatotoxicity following the same treatment regimen. The final doses of FKA and FKB were 1, 2, and 4 times their abundance in the 500 mg / kg body weight dose of kava (1.6% and 2.3%, respectively). FKA and FKB together did not cause any changes in serum ALT and AST at the three tested doses ( Fig. 27A and 27B). When combined with APAP, FKA and FKB dose-dependently enhanced the increase in ALT and AST induced by APAP ( Fig. 27 A and 27B). Of note, one mouse treated with the highest dose of FKA and FKB combined with APAP died approximately 0.5-2 hours before necropsy (i.e., 22 to 23.5 hours after APAP combined with FKA and FKB). This was the only mouse in the study that died before necropsy. Its serum ALT and AST levels were the highest of all mice ( Fig. 27 C), and was 2-3 times the next highest value. Histopathological examination showed multifocal and combined acute centrilobular necrosis in the liver of the mice (Figure 27D, Group B), while the livers of the control mice ( Fig. 27 The livers of mice treated with FKA and FKB alone (D, Panel A) were within the histologically normal range. These data suggest that severe hepatotoxicity may have contributed to their premature death.

[0663] discuss

[0664] Kava has been shown to have anxiolytic activity clinically and to potentially reduce cancer risk in humans. On the other hand, kava use is hypothesized to be associated with rare but severe hepatotoxicity. Various mechanisms have been proposed and different chemicals have been postulated, but not yet confirmed. Given the global resurgence of kava and the diverse chemical composition of current kava products, it is urgent and important to reproduce kava hepatotoxicity in in vivo models, which can help identify the responsible chemicals and guide the development of strategies to minimize and ideally eliminate the adverse potential.

[0665] The results of the present study demonstrated that kava, when administered alone in a chronic manner via gavage in C57BL / 6 mice, did not induce adverse effects even at a fairly high dose (500 mg / kg body weight / day), as reflected in the growth of mice and the serum levels of ALT and AST ( Fig.23 ). These results are consistent with those of many earlier studies (DiSilvestro, et al. (2007) Food Chem. Toxicol. 45, 1293-1300; Guo, L., (2009) Food Chem. Toxicol. 47, 433-442; Guo, et al. (2010) Food Chem. Toxicol. 48, 686-696; Sorrentino, et al. (2006) Phytomedicine 13, 542-549). On the other hand, kava significantly enhanced the hepatotoxicity of APAP in C57BL / 6 mice, as indicated by increased serum ALT and AST and increased severity of liver damage ( Fig.24The treatment regimen was designed to mimic a potential scenario in human kava users: daily consumption of kava, while other medications (in this case APAP) were used occasionally when needed. Since most cases of kava-associated hepatotoxicity were accompanied by concomitant consumption of other medications or dietary supplements, the results of this study may have direct implications for the hepatotoxicity observed in kava users. It remains to be determined whether kava use could enhance the hepatotoxicity risk of other medications or hepatotoxins, such as alcohol consumption. It also remains to be determined whether other kava treatment regimens, such as chronic kava use or use during fasting phases (as recommended for traditional kava use), could enhance the risk of hepatotoxicity even at lower kava doses.

[0666] Using a C57BL / 6 mouse model that captures the hepatotoxicity risk of kava in vivo, the potentially responsible compound or compounds were investigated. Results demonstrated that FKB (a chalcone-based compound in kava) modestly enhanced APAP hepatotoxicity when evaluated at a dose equivalent to 500 mg / kg body weight of kava, whereas DHM (a representative of the kavalactones in kava) lacked such risk ( Fig.26 Because kava products contain FKA (an analog of FKB) in similar abundance, the combination of FKA and FKB was evaluated, which dose-dependently enhanced APAP-induced hepatotoxicity ( Fig. 27 In fact, early death and histological examinations reflect extensive acute hepatocellular necrosis ( Fig. 27 The highest ALT and AST levels in groups D and B ( Fig. 27 C) One mouse was in the APAP co-treatment group with the highest dose of FKA and FKB. These data overall suggest that FKA and FKB are the responsible compounds in kava that enhance APAP-induced hepatotoxicity, while DHM does not have this risk. In addition to FKA and FKB, kava caulis citrate C (FKC) has been reported in other kava products (Lebot, et al. (2014) Food Chem. 151, 554-560), but was not detected in the kava products used in this study. FKC may be another compound responsible for hepatotoxicity.

[0667] A recent analysis of a panel of kava products currently on the market demonstrated that the abundance of FKA and FKB can vary approximately 20-fold (Martin, et al. (2014) Plos One, 9(11):e111572. doi:10.1371 / journal.pone.0111572). Similarly, a recent study analyzed the abundance of FKA, FKB, and FKC in different kava varieties (Lebot, et al. (2014) Food Chem. 151, 554-560). Varieties not recommended for traditional use were found to contain higher abundances of FKA, FKB, and FKC than traditionally consumed varieties (Lebot, et al. (2014) Food Chem. 151, 554-560). Therefore, further studies are warranted to evaluate whether varieties or kava products with higher levels of FKA, FKB, and FKC impose a higher risk of hepatotoxicity. Further studies are needed to elucidate the molecular mechanisms of the observed enhanced liver toxicity, such as glutathione depletion (Zhou, et al. (2010) FASEB J. 24, 4722-4732). Such knowledge will help guide the preparation of kava products with greater health benefits and minimal adverse effects for human use.

[0668] Example 6

[0669] Dihydrocapsaicin effectively blocks DNA damage induced by 2-amino-1-methyl-6-phenylimidazo(4,5-b)pyridine (PhIP) and benzo(a)pyrene (BaP)

[0670] introduction

[0671] The aim of this study was to investigate the potential scope of dihydro-anesthetic capsaicin in blocking / reducing DNA damage induced by different carcinogens and detoxifying said carcinogens. 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is one of the most abundant heterocyclic amines in cooked meats (Ni W, (2008) Journal of agricultural and food chemistry. 56(1): 68-78.) and may be the most potent carcinogen among them, especially for colon, prostate and breast cancer (Nishikawa A, (2005). Toxicological sciences: an official journal of the Society of Toxicology. 84(2): 243-8.). Benzo(a)pyrene (BaP) is a representative polyaromatic hydrocarbon-based carcinogen in cigarette smoke and is present in a variety of industrial / engine exhaust / waste gases. BaP is a human carcinogen to the lung, stomach and other tissues. Rodent hepatoma cell lines were used in these studies. Briefly, hepe1c1c7 cells were co-treated with dihydrocapsaicin (1, 5, and 25 μM) and PhIP (10 μM) for 24 h, and the isolated DNA and PhIP-based DNA damage quantification ( Fig.28 A). It should be clear that dihydrocapsaicin effectively and dose-dependently reduced PhIP-induced DNA damage, which demonstrated its efficacy in blocking carcinogen-induced DNA damage and indicated its detoxification function and cancer chemoprevention potential against colon cancer, prostate cancer and breast cancer. Similarly, cells were co-treated with dihydrocapsaicin (1, 5 and 25 μM) and BaP (0.3 μM) for 3 hours, where the isolated DNA and BaP-based DNA damage quantification ( Fig.28 B). Dihydrocapsaicin effectively and dose-dependently reduced BaP-induced DNA damage, demonstrating its efficacy in blocking carcinogen-induced DNA damage and suggesting its detoxification function and cancer chemopreventive potential against lung and gastric cancers.

[0672] Example 7

[0673] Representative pharmaceutical dosage forms containing 'Compound X' for therapeutic or prophylactic use in humans are exemplified below. As described herein, Compound X can represent a compound described herein, such as, for example, dihydrocapsaicin, capsaicin, dihydrocapsaicin, capsaicin, demethoxycapsaicin, and 11-methoxycapsaicin.

[0674]

[0675]

[0676]

[0677]

[0678]

[0679]

[0680]

[0681]

[0682]

[0683]

[0684]

[0685] The above-mentioned preparations can be obtained by conventional methods well known in the pharmaceutical field.

[0686] All publications, patents and patent documents are incorporated herein by reference, just as if they were individually incorporated by reference. The present invention has been described with reference to various specific and preferred embodiments and techniques. However, it should be understood that many variations and modifications can be made while still being within the spirit and scope of the present invention.

Claims

1. A composition formulated as a unit dosage form, wherein the composition comprises dihydrocapsaicin, less than 0.3% by weight of kava piperine B, and less than 0.3% by weight of kava piperine A.

2. A composition formulated as a unit dosage form, wherein the composition comprises dihydrocapsaicin, capsaicin, dihydrocapsaicin, demethoxycapsaicin and capsaicin, less than 0.3% by weight of kava piperine B, and less than 0.3% by weight of kava piperine A.

3. The composition according to claim 2, wherein the composition comprises 15±5 wt% of dihydrocapsaicin, 6±5 wt% of capsaicin, 30±5 wt% of dihydrocapsaicin, 29±5 wt% of capsaicin and 12±5 wt% of demethoxycapsaicin.

4. The composition according to claim 1 or 2, comprising 11-methoxy capsaicin and flavanone: wherein the composition comprises less than 0.3% by weight of a bornyl ester of cinnamic acid of the formula:

5. The composition according to claim 1, further comprising at least one compound selected from the group consisting of capsaicin, dihydrocapsaicin and demethoxycapsaicin.

6. The composition according to claim 1 or 2, wherein the weight percentage of dihydrocapsara in the composition is 15±5%.

7. The composition of claim 1, wherein the composition comprises capsaicin.

8. The composition according to claim 1 or 2, wherein the weight percentage of anesthesia capsaicin in the composition is 6±5%.

9. The composition of claim 1, wherein the composition comprises dihydrocapsaicin.

10. The composition according to claim 1 or 2, wherein the weight percentage of dihydrocapsaicin in the composition is 30±5%.

11. The composition of claim 1, wherein the composition comprises demethoxycapsaicin.

12. The composition according to claim 1 or 2, wherein the weight percentage of demethoxy capsaicin in the composition is 12±5%.

13. The composition according to claim 4, wherein the flavanones in the composition are: The weight percentage is 1.0±0.5%.

14. The composition according to claim 4, wherein the weight percentage of 11-methoxy capsaicin in the composition is 0.8±0.5%.

15. The composition of claim 4, wherein the composition comprises less than 5% by weight of bornyl ester of 3,4-methylenedioxycinnamic acid:

16. The composition of claim 4, wherein the composition comprises less than 5% by weight of flavanones:

17. The composition of claim 4, wherein the composition comprises less than 5% by weight of bromocriptone.

18. The composition of claim 4, wherein the composition comprises less than 5% by weight of chloranone, bornyl ester of 3,4-methylenedioxycinnamic acid: And flavanones:

19. The composition according to claim 1 or 2, wherein the composition is formulated into a tablet, a capsule or a sandwich tablet.

20. The composition of claim 19, wherein the composition is administered orally.

21. The composition of claim 4, wherein the sum of the 11-methoxycapsaicin, flavanones and dihydrocapsaicin is present in an amount of 5 mg to 1000 mg.

22. The composition of claim 4, wherein the 11-methoxycapsaicin is present in an amount of 10 mg to 100 mg.

23. The composition of claim 1 or 2, wherein the dihydrocapsara is present in an amount of 10 mg to 100 mg.

24. Use of a composition according to any one of claims 1 to 23 in the preparation of a medicament for preventing cancer and / or reducing DNA damage in a mammal, wherein the cancer is lung cancer, prostate cancer, colorectal cancer or breast cancer.

25. The use according to claim 24, wherein the cancer is lung cancer.

26. The use according to claim 24, wherein the cancer is breast cancer.

27. The use according to claim 24, wherein the cancer is prostate cancer.

28. The use according to claim 24, wherein the cancer is colorectal cancer.

29. The use according to claim 24, wherein the DNA damage is a DNA adduct.

30. The use according to claim 29, wherein the DNA adduct is selected from BaP, PhIP, POB and PHB adducts.

31. The use according to claim 29, wherein the DNA adduct is 6 -Methylguanine DNA adducts.

32. Use of a composition according to any one of claims 1-23 in the preparation of a medicament for treating cancer in a mammal, wherein the cancer is lung cancer.

33. Use of a composition according to claim 2 or 3 in the preparation of a medicament for treating cancer in a mammal, wherein the cancer is prostate cancer.

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