What is claimed is:
1. A method of producing a mean plasma concentration of a histone deacetylase (HDAC) inhibitor capable of inhibiting a histone deacetylase in vivo in a subject over a period of at least two hours following administration, which comprises administering to said subject an effective amount of a pharmaceutical composition comprising a HDAC inhibitor or a pharmaceutically acceptable salt or hydrate thereof, and a pharmaceutically acceptable carrier or diluent.
2. The method according to claim 1, wherein the pharmaceutical composition is administered orally.
3. The method according to claim 1, wherein the mean plasma concentration of said HDAC inhibitor is at least about 10 nM.
4. The method according to claim 1, wherein said composition provides a mean plasma concentration of said HDAC inhibitor of at least about 10 nM in vivo for a period of at least 10 hours following administration.
5. The method according to claim 1, wherein said composition provides a mean plasma concentration of said HDAC inhibitor of at least about 2.5 μM over a period of at least 2 hours following administration.
6. The method according to claim 1, wherein said composition is contained within a gelatin capsule.
7. The method according to claim 1, wherein said carrier or diluent is microcrystalline cellulose.
8. The method according to claim 1, further comprising sodium croscarmellose as a disintegrating agent.
9. The method according to claim 1, further comprising magnesium stearate as a lubricant.
10. The method according to claim 1, wherein said composition is administered once-daily, twice-daily or three times-daily.
11. The method of claim 1, wherein said HDAC inhibitor is administered to the subject at a total daily dosage of between about 25-4000 mg/m
12. The method according to claim 1, wherein said composition is administered to the subject at a total daily dose of 200 mg.
13. The method according to claim 1, wherein said composition is administered to the subject at a total daily dose of 400 mg.
14. The method according to claim 1, wherein said HDAC inhibitor is suberoylanilide hydroxamic acid (SAHA):
15. The method according to claim 1, wherein said HDAC inhibitor is pyroxamide, represented by the structure:
16. The method according to claim 1, wherein said HDAC inhibitor is represented by the structure:
17. The method according to claim 1, wherein said HDAC inhibitor is represented by the structure:
18. The method according to claim 1, wherein said HDAC inhibitor is represented by the structure:
19. A method of producing a mean plasma concentration of at least about 10 nM of suberoylanilide hydroxamic acid (SAHA) in vivo in a subject over a period of at least two hours following administration, which comprises administering to said subject an effective amount of a pharmaceutical composition comprising SAHA or a pharmaceutically acceptable salt or hydrate thereof, and a pharmaceutically acceptable carrier or diluent.
20. The method according to claim 19, wherein the pharmaceutical composition is administered orally.
21. The method according to claim 19, wherein said composition provides a mean plasma concentration of SAHA of at least about 10 nM in vivo for a period of at least 10 hours following administration.
22. The method according to claim 19, wherein said composition provides a mean plasma concentration of SAHA of at least about 2.5 μM over a period of at least 2 hours following administration.
23. The method according to claim 19, wherein said composition is contained within a gelatin capsule.
24. The method according to claim 19, wherein said carrier or diluent is microcrystalline cellulose.
25. The method according to claim 19, further comprising sodium croscarmellose as a disintegrating agent.
26. The method according to claim 19, further comprising magnesium stearate as a lubricant.
27. The method according to claim 19, wherein said composition is administered once-daily, twice-daily or three times-daily.
28. The method of claim 19, wherein SAHA is administered to the subject at a total daily dosage of between about 25-4000 mg/m
29. The method according to claim 19, wherein said composition is administered to the subject at a total daily dose of 200 mg.
30. The method according to claim 19, wherein said composition is administered to the subject at a total daily dose of 400 mg.
31. A method of selectively inducing terminal differentiation of neoplastic cells in a subject and thereby inhibiting proliferation of such cells in said subject, said method comprising producing a mean plasma concentration of a histone deacetylase (HDAC) inhibitor capable of inhibiting a histone deacetylase in vivo in a subject over a period of at least two hours following administration by administering to said subject an effective amount of a pharmaceutical composition comprising a HDAC inhibitor or a pharmaceutically acceptable salt or hydrate thereof, and a pharmaceutically acceptable carrier or diluent.
32. The method according to claim 31, wherein the pharmaceutical composition is administered orally.
33. The method according to claim 31, wherein the mean plasma concentration of said HDAC inhibitor is at least about 10 nM.
34. The method according to claim 31, wherein said composition provides a mean plasma concentration of said HDAC inhibitor of at least about 10 nM in vivo for a period of at least 10 hours following administration.
35. The method according to claim 31, wherein said composition provides a mean plasma concentration of said HDAC inhibitor of at least about 2.5 μM over a period of at least 2 hours following administration.
36. The method according to claim 31, wherein said composition is contained within a gelatin capsule.
37. The method according to claim 31, wherein said carrier or diluent is microcrystalline cellulose.
38. The method according to claim 31, further comprising sodium croscarmellose as a disintegrating agent.
39. The method according to claim 31, further comprising magnesium stearate as a lubricant.
40. The method according to claim 31, wherein said composition is administered once-daily, twice-daily or three times-daily.
41. The method of claim 31, wherein said HDAC inhibitor is administered to the subject at a total daily dosage of between 25-4000 mg/m
42. The method according to claim 31, wherein said composition is administered to the subject at a total daily dose of 200 mg.
43. The method according to claim 31, wherein said composition is administered to the subject at a total daily dose of 400 mg.
44. The method according to claim 31, wherein said HDAC inhibitor is suberoylanilide hydroxamic acid (SAHA):
45. The method according to claim 31, wherein said HDAC inhibitor is pyroxamide, represented by the structure:
46. The method according to claim 31, wherein said HDAC inhibitor is represented by the structure:
47. The method according to claim 31, wherein said HDAC inhibitor is represented by the structure:
48. The method according to claim 31, wherein said HDAC inhibitor is represented by the structure:
49. A method of selectively inducing terminal differentiation of neoplastic cells in a subject and thereby inhibiting proliferation of such cells in said subject, said method comprising producing a mean plasma concentration of at least about 10 nM of suberoylanilide hydroxamic acid (SAHA) in vivo in a subject over a period of at least two hours following administration, which comprises administering to said subject an effective amount of a pharmaceutical composition comprising SAHA or a pharmaceutically acceptable salt or hydrate thereof, and a pharmaceutically acceptable carrier or diluent.
50. The method according to claim 49, wherein the pharmaceutical composition is administered orally.
51. The method according to claim 49, wherein said composition provides a mean plasma concentration of SAHA of at least about 10 nM in vivo for a period of at least 10 hours following administration.
52. The method according to claim 49, wherein said composition provides a mean plasma concentration of SAHA of at least about 2.5 μM over a period of at least 2 hours following administration.
53. The method according to claim 49, wherein said composition is contained within a gelatin capsule.
54. The method according to claim 49, wherein said carrier or diluent is microcrystalline cellulose.
55. The method according to claim 49, further comprising sodium croscarmellose as a disintegrating agent.
56. The method according to claim 49, further comprising magnesium stearate as a lubricant.
57. The method according to claim 49, wherein said composition is administered once-daily, twice-daily or three times-daily.
58. The method of claim 49, wherein SAHA is administered to the subject at a total daily dosage of between 25-4000 mg/m
59. The method according to claim 49, wherein said composition is administered to the subject at a total daily dose of 200 mg.
60. The method according to claim 49, wherein said composition is administered to the subject at a total daily dose of 400 mg.
61. A method of inducing differentiation of tumor cells in a subject having a tumor, said method comprising producing a mean plasma concentration of a histone deacetylase (HDAC) inhibitor capable of inhibiting a histone deacetylase in vivo in a subject over a period of at least two hours following administration, which comprises administering to said subject an effective amount of a pharmaceutical composition comprising a HDAC inhibitor or a pharmaceutically acceptable salt or hydrate thereof, and a pharmaceutically acceptable carrier or diluent.
62. The method according to claim 61, wherein the pharmaceutical composition is administered orally.
63. The method according to claim 61, wherein the mean plasma concentration of said HDAC inhibitor is at least about 10 nM.
64. The method according to claim 61, wherein said composition provides a mean plasma concentration of said HDAC inhibitor of at least about 10 nM in vivo for a period of at least 10 hours following administration.
65. The method according to claim 61, wherein said composition provides a mean plasma concentration of said HDAC inhibitor of at least about 2.5 μM over a period of at least 2 hours following administration.
66. The method according to claim 61, wherein said composition is contained within a gelatin capsule.
67. The method according to claim 61, wherein said carrier or diluent is microcrystalline cellulose.
68. The method according to claim 61, further comprising sodium croscarmellose as a disintegrating agent.
69. The method according to claim 61, further comprising magnesium stearate as a lubricant.
70. The method according to claim 61, wherein said composition is administered once-daily, twice-daily or three times-daily.
71. The method of claim 61, wherein said HDAC inhibitor is administered to the subject at a total daily dosage of between about 25-4000 mg/m
72. The method according to claim 61, wherein said composition is administered to the subject at a total daily dose of 200 mg.
73. The method according to claim 61, wherein said composition is administered to the subject at a total daily dose of 400 mg.
74. The method according to claim 61, wherein said HDAC inhibitor is suberoylanilide hydroxamic acid (SAHA):
75. The method according to claim 61, wherein said HDAC inhibitor is pyroxamide, represented by the structure:
76. The method according to claim 61, wherein said HDAC inhibitor is represented by the structure:
77. The method according to claim 61, wherein said HDAC inhibitor is represented by the structure:
78. The method according to claim 61, wherein said HDAC inhibitor is represented by the structure:
79. A method of inducing differentiation of tumor cells in a subject having a tumor, said method comprising producing a mean plasma concentration of at least about 10 nM of suberoylanilide hydroxamic acid (SAHA) in vivo in a subject over a period of at least two hours following administration by administering to said subject an effective amount of a pharmaceutical composition comprising SAHA or a pharmaceutically acceptable salt or hydrate thereof, and a pharmaceutically acceptable carrier or diluent.
80. The method according to claim 79, wherein the pharmaceutical composition is administered orally.
81. The method according to claim 79, wherein said composition provides a mean plasma concentration of SAHA of at least about 10 nM in vivo for a period of at least 10 hours following administration.
82. The method according to claim 79, wherein said composition provides a mean plasma concentration of SAHA of at least about 2.5 μM over a period of at least 2 hours following administration.
83. The method according to claim 79, wherein said composition is contained within a gelatin capsule.
84. The method according to claim 79, wherein said carrier or diluent is microcrystalline cellulose.
85. The method according to claim 79, further comprising sodium croscarmellose as a disintegrating agent.
86. The method according to claim 79, further comprising magnesium stearate as a lubricant.
87. The method according to claim 79, wherein said composition is administered once-daily, twice-daily or three times-daily.
88. The method of claim 79, wherein SAHA is administered to the subject at a total daily dosage of between about 25 to about 4000 mg/m
89. The method according to claim 79, wherein said composition is administered to the subject at a total daily dose of 200 mg.
90. The method according to claim 79, wherein said composition is administered to the subject at a total daily dose of 400 mg.
91. A method of selectively inducing cell growth arrest of neoplastic cells in a subject and thereby inhibiting proliferation of such cells in said subject, said method comprising producing a mean plasma concentration of a histone deacetylase (HDAC) inhibitor capable of inhibiting a histone deacetylase in vivo in a subject over a period of at least two hours following administration by administering to said subject an effective amount of a pharmaceutical composition comprising a HDAC inhibitor or a pharmaceutically acceptable salt or hydrate thereof, and a pharmaceutically acceptable carrier or diluent.
92. The method according to claim 91, wherein the pharmaceutical composition is administered orally.
93. The method according to claim 91, wherein the mean plasma concentration of said HDAC inhibitor is at least about 10 nM.
94. The method according to claim 91, wherein said composition provides a mean plasma concentration of said HDAC inhibitor of at least about 10 nM in vivo for a period of at least 10 hours following administration.
95. The method according to claim 91, wherein said composition provides a mean plasma concentration of said HDAC inhibitor of at least about 2.5 μM over a period of at least 2 hours following administration.
96. The method according to claim 91, wherein said composition is contained within a gelatin capsule.
97. The method according to claim 91, wherein said carrier or diluent is microcrystalline cellulose.
98. The method according to claim 91, further comprising sodium croscarmellose as a disintegrating agent.
99. The method according to claim 91, further comprising magnesium stearate as a lubricant.
100. The method according to claim 91, wherein said composition is administered once-daily, twice-daily or three times-daily.
101. The method of claim 91, wherein said HDAC inhibitor is administered to the subject at a total daily dosage of between about 25-4000 mg/m
102. The method according to claim 91, wherein said composition is administered to the subject at a total daily dose of 200 mg.
103. The method according to claim 91, wherein said composition is administered to the subject at a total daily dose of 400 mg.
104. The method according to claim 91, wherein said HDAC inhibitor is suberoylanilide hydroxamic acid (SAHA):
105. The method according to claim 91, wherein said HDAC inhibitor is pyroxamide, represented by the structure:
106. The method according to claim 91, wherein said HDAC inhibitor is represented by the structure:
107. The method according to claim 91, wherein said HDAC inhibitor is represented by the structure:
108. The method according to claim 91, wherein said HDAC inhibitor is represented by the structure:
109. A method of selectively inducing cell growth arrest of neoplastic cells in a subject and thereby inhibiting proliferation of such cells in said subject, said method comprising producing a mean plasma concentration of at least about 10 nM of suberoylanilide hydroxamic acid (SAHA) in vivo in a subject over a period of at least two hours following administration by administering to said subject an effective amount of a pharmaceutical composition comprising SAHA or a pharmaceutically acceptable salt or hydrate thereof, and a pharmaceutically acceptable carrier or diluent.
110. The method according to claim 109, wherein the pharmaceutical composition is administered orally.
111. The method according to claim 109, wherein said composition provides a mean plasma concentration of SAHA of at least about 10 nM in vivo for a period of at least 10 hours following administration.
112. The method according to claim 109, wherein said composition provides a mean plasma concentration of SAHA of at least about 2.5 μM over a period of at least 2 hours following administration.
113. The method according to claim 109, wherein said composition is contained within a gelatin capsule.
114. The method according to claim 109, wherein said carrier or diluent is microcrystalline cellulose.
115. The method according to claim 109, further comprising sodium croscarmellose as a disintegrating agent.
116. The method according to claim 109, further comprising magnesium stearate as a lubricant.
117. The method according to claim 109, wherein said composition is administered once-daily, twice-daily or three times-daily.
118. The method of claim 109, wherein SAHA is administered to the subject at a total daily dosage of between about 25-4000 mg/m .
119. The method according to claim 109, wherein said composition is administered to the subject at a total daily dose of 200 mg.
120. The method according to claim 109, wherein said composition is administered to the subject at a total daily dose of 400 mg.
121. A method of selectively inducing apoptosis of neoplastic cells in a subject and thereby inhibiting proliferation of such cells in said subject, said method comprising producing a mean plasma concentration of a histone deacetylase (HDAC) inhibitor capable of inhibiting a histone deacetylase in vivo in a subject over a period of at least two hours following administration by administering to said subject an effective amount of a pharmaceutical composition comprising a HDAC inhibitor or a pharmaceutically acceptable salt or hydrate thereof, and a pharmaceutically acceptable carrier or diluent.
122. The method according to claim 121, wherein the pharmaceutical composition is administered orally.
123. The method according to claim 121, wherein the mean plasma concentration of said HDAC inhibitor is at least about 10 nM.
124. The method according to claim 121, wherein said composition provides a mean plasma concentration of said HDAC inhibitor of at least about 10 nM in vivo for a period of at least 10 hours following administration.
125. The method according to claim 121, wherein said composition provides a mean plasma concentration of said HDAC inhibitor of at least about 2.5 μM over a period of at least 2 hours following administration.
126. The method according to claim 121, wherein said composition is contained within a gelatin capsule.
127. The method according to claim 121, wherein said carrier or diluent is microcrystalline cellulose.
128. The method according to claim 121, further comprising sodium croscarmellose as a disintegrating agent.
129. The method according to claim 121, further comprising magnesium stearate as a lubricant.
130. The method according to claim 121, wherein said composition is administered once-daily, twice-daily or three times-daily.
131. The method of claim 121, wherein said HDAC inhibitor is administered to the subject at a total daily dosage of between about 25-4000 mg/m
132. The method according to claim 121, wherein said composition is administered to the subject at a total daily dose of 200 mg.
133. The method according to claim 121, wherein said composition is administered to the subject at a total daily dose of 400 mg.
134. The method according to claim 121, wherein said HDAC inhibitor is suberoylanilide hydroxamic acid (SAHA):
135. The method according to claim 121, wherein said HDAC inhibitor is pyroxamide, represented by the structure:
136. The method according to claim 121, wherein said HDAC inhibitor is represented by the structure:
137. The method according to claim 121, wherein said HDAC inhibitor is represented by the structure:
138. The method according to claim 121, wherein said HDAC inhibitor is represented by the structure:
139. A method of selectively inducing apoptosis of neoplastic cells in a subject and thereby inhibiting proliferation of such cells in said subject, said method comprising producing a mean plasma concentration of at least about 10 nM of suberoylanilide hydroxamic acid (SAHA) in vivo in a subject over a period of at least two hours following administration by administering to said subject an effective amount of a pharmaceutical composition comprising SAHA or a pharmaceutically acceptable salt or hydrate thereof, and a pharmaceutically acceptable carrier or diluent.
140. The method according to claim 139, wherein the pharmaceutical composition is administered orally.
141. The method according to claim 139, wherein said composition provides a mean plasma concentration of SAHA of at least about 10 nM in vivo for a period of at least 10 hours following administration.
142. The method according to claim 139, wherein said composition provides a mean plasma concentration of SAHA of at least about 2.5 μM over a period of at least 2 hours following administration.
143. The method according to claim 139, wherein said composition is contained within a gelatin capsule.
144. The method according to claim 139, wherein said carrier or diluent is microcrystalline cellulose.
145. The method according to claim 139, further comprising sodium croscarmellose as a disintegrating agent.
146. The method according to claim 139, further comprising magnesium stearate as a lubricant.
147. The method according to claim 139, wherein said composition is administered once-daily, twice-daily or three times-daily.
148. The method of claim 139, wherein SAHA is administered to the subject at a total daily dosage of between about 25-4000 mg/m
149. The method according to claim 139, wherein said composition is administered to the subject at a total daily dose of 200 mg.
150. The method according to claim 139, wherein said composition is administered to the subject at a total daily dose of 400 mg.
151. A pharmaceutical composition for oral administration comprising: a histone deacetylase (HDAC) inhibitor or a pharmaceutically acceptable salt or hydrate thereof; microcrystalline cellulose; croscarmellose sodium; and magnesium stearate.
152. The composition according to claim 151, wherein said HDAC inhibitor is suberoylanilide hydroxamic acid (SAHA).
153. The composition according to claim 151, comprising: 50-70% by weight of said histone deacetylase (HDAC) inhibitor or a pharmaceutically acceptable salt or hydrate thereof; 20-40% by weight microcrystalline cellulose; 5-15% by weight croscarmellose sodium; and 0.1-5% by weight magnesium stearate.
154. The composition according to claim 151, comprising about 50-200 mg of said HDAC inhibitor.
155. The composition according to claim 151 contained within a gelatin capsule.
156. A pharmaceutical composition for oral administration comprising: suberoylanilide hydroxamic acid (SAHA) or a pharmaceutically acceptable salt or hydrate thereof; microcrystalline cellulose; croscarmellose sodium; and magnesium stearate.
157. The composition according to claim 156, comprising: 50-70% by weight of SAHA or a pharmaceutically acceptable salt or hydrate thereof; 20-40% by weight microcrystalline cellulose; 5-15% by weight croscarmellose sodium; and 0.1-5% by weight magnesium stearate.
158. The composition according to claim 156, comprising about 50-200 mg of SAHA.
159. The composition according to claim 156 contained within a gelatin capsule.
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional Application No. 60/361,759, filed Mar. 4, 2002. The entire teachings of this provisional application are incorporated herein by reference.
GOVERNMENT INTEREST STATEMENT
FIELD OF THE INVENTION
[0003] The present invention provides methods of selectively inducing terminal differentiation, cell growth arrest and/or apoptosis of neoplastic cells, and/or inhibiting histone deacetylases (HDAC) administration of pharmaceutical compositions comprising HDAC inhibitors. The oral formulations of the pharmaceutical compositions have favorable pharmacokinetic profiles such as high bioavailability and surprisingly give rise to high blood levels of the active compounds over an extended period of time.
BACKGROUND OF THE INVENTION
[0004] Throughout this application various publications are referenced by arabic numerals within parentheses. Full citations for these publications may be found at the end of the specification immediately preceding the claims. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains.
[0005] Cancer is a disorder in which a population of cells has become, in varying degrees, unresponsive to the control mechanisms that normally govern proliferation and differentiation. For many years there have been two principal strategies for chemotherapeutic treatment of cancer: a) blocking hormone-dependent tumor cell proliferation by interference with the production or peripheral action of sex hormones; and b) killing cancer cells directly by exposing them to cytotoxic substances, which injure both neoplastic and normal cell populations.
[0006] Cancer therapy is also being attempted by the induction of terminal differentiation of the neoplastic cells (1). In cell culture models differentiation has been reported by exposure of cells to a variety of stimuli, including: cyclic AMP and retinoic acid (2,3), aclarubicin and other anthracyclines (4).
[0007] Despite many advances in the field of oncology, the majority of solid tumors remain incurable in the advanced stages. Cytotoxic therapy is used in most cases, however, it often causes significant morbidity to the patient without significant clinical benefit. Less toxic and more specific agents to treat and control advanced malignancies are being explored.
[0008] There is abundant evidence that neoplastic transformation does not necessarily destroy the potential of cancer cells to differentiate (1,5,6). There are many examples of tumor cells which do not respond to the normal regulators of proliferation and appear to be blocked in the expression of their differentiation program, and yet can be induced to differentiate and cease replicating. A variety of agents, including some relatively simple polar compounds (5,7-9), derivatives of vitamin D and retinoic acid (10-12), steroid hormones (13), growth factors (6,14), proteases (15,16), tumor promoters (17,18), and inhibitors of DNA or RNA synthesis (4,19-24), can induce various transformed cell lines and primary human tumor explants to express more differentiated characteristics.
[0009] Early studies identified a series of polar compounds that were effective inducers of differentiation in a number of transformed cell lines (8,9). Of these, the most effective inducer was the hybrid polar/apolar compound N,N′-hexamethylene bisacetamide (HMBA) (9). The use of this polar/apolar compound to induce murine erythroleukemia cells (MELC) to undergo erythroid differentiation with suppression of oncogenicity has proved a useful model to study inducer-mediated differentiation of transformed cells (5,7-9). HMBA-induced MELC terminal erythroid differentiation is a multi-step process. Upon addition of HMBA to MELC (745A-DS19) in culture, there is a latent period of 10 to 12 hours before commitment to terminal differentiation is detected. Commitment is defined as the capacity of cells to express terminal differentiation despite removal of inducer (25). Upon continued exposure to HMBA there is progressive recruitment of cells to differentiate. The present inventors have reported that MELC cell lines made resistant to relatively low levels of vincristine become markedly more sensitive to the inducing action of HMBA and can be induced to differentiate with little or no latent period (26).
[0010] HMBA is capable of inducing phenotypic changes consistent with differentiation in a broad variety of cells lines (5). The characteristics of the drug-induced effect have been most extensively studied in the murine erythroleukemia cell system (MELC) (5,25,27,28). MELC induction of differentiation is both time and concentration dependent. The minimum concentration required to demonstrate an effect in vitro in most strains is 2 to 3 mM; the minimum duration of continuous exposure generally required to induce differentiation in a substantial portion (>20%) of the population without continuing drug exposure is about 36 hours.
[0011] The primary target of action of HMBA is not known. There is evidence that protein kinase C is involved in the pathway of inducer-mediated differentiation (29). The in vitro studies provided a basis for evaluating the potential of HMBA as a cytodifferentiation agent in the treatment of human cancers (30). Several phase I clinical trials with HMBA have been completed (31-36). Clinical trials have shown that this compound can induce a therapeutic response in patients with cancer (35,36). However, these phase I clinical trials also have demonstrated that the potential efficacy of HMBA is limited, in part, by dose-related toxicity which prevents achieving optimal blood levels and by the need for intravenous administration of large quantities of the agent, over prolonged periods.
[0012] It has been reported that a number of compounds related to HMBA with polar groups separated by apolar linkages that, on a molar basis, are as active (37) or 100 times more active than HMBA (38). As a class, however, it has been found that the symmetrical dimers such as HMBA and related compounds are not the best cytodifferentiating agents.
[0013] It has unexpectedly been found that the best compounds comprise two polar end groups separated by a flexible chain of methylene groups, wherein one or both of the polar end groups is a large hydrophobic group. Preferably, the polar end groups are different and only one is a large hydrophobic group. These compounds are unexpectedly a thousand times more active than HMBA and ten times more active than HMBA related compounds.
[0014] Histone deacetylase inhibitors such as suberoylanilide hydroxamide acid (SAHA), belong to this class of agents that have the ability to induce tumor cell growth arrest, differentiation and/or apoptosis (39). These compounds are targeted towards mechanisms inherent to the ability of a neoplastic cell to become malignant, as they do not appear to have toxicity in doses effective for inhibition of tumor growth in animals (40). There are several lines of evidence that histone acetylation and deacetylation are mechanisms by which transcriptional regulation in a cell is achieved (41). These effects are thought to occur through changes in the structure of chromatin by altering the affinity of histone proteins for coiled DNA in the nucleosome. There are five types of histones that have been identified in nucleosomes (designated H1, H2A, H2B, H3 and H4). Each nucleosome contains two of each histone type within its core, except for H1, which is present singly in the outer portion of the nucleosome structure. It is believed that when the histone proteins are hypoacetylated, there is a greater affinity of the histone to the DNA phosphate backbone This affinity causes DNA to be tightly bound to the histone and renders the DNA inaccessible to transcriptional regulatory elements and machinery. The regulation of acetylated states occurs through the balance of activity between two enzyme complexes, histone acetyl transferase (HAT) and histone deacetylase (HDAC). The hypoacetylated state is thought to inhibit transcription of associated DNA. This hypoacetylated state is catalyzed by large multiprotein complexes that include HDAC enzymes. In particular, HDACs have been shown to catalyze the removal of acetyl groups from the chromatin core histones.
[0015] The inhibition of HDAC by SAHA is thought occur through direct interaction with the catalytic site of the enzyme as demonstrated by X-ray crystallography studies (42). The result of HDAC inhibition is not believed to have a generalized effect on the genome, but rather, only affects a small subset of the genome (43). Evidence provided by DNA microarrays using malignant cell lines cultured with a HDAC inhibitor shows that there are a finite (1-2%) number of genes whose products are altered. For example, cells treated in culture with HDAC inhibitors show a consistent induction of the cyclin-dependent kinase inhibitor p21 (44). This protein plays an important role in cell cycle arrest. HDAC inhibitors are thought to increase the rate of transcription of p21 by propagating the hyperacetylated state of histones in the region of the p21 gene, thereby making the gene accessible to transcriptional machinery. Genes whose expression is not affected by HDAC inhibitors do not display changes in the acetylation of regional associated histones (45).
[0016] It has been shown in several instances that the disruption of HAT or HDAC activity is implicated in the development of a malignant phenotype. For instance, in acute promyelocytic leukemia, the oncoprotein produced by the fusion of PML and RAR alpha appears to suppress specific gene transcription through the recruitment of HDACs (46). In this manner, the neoplastic cell is unable to complete differentiation and leads to excess proliferation of the leukemic cell line.
[0017] U.S. Pat. Nos. 5,369,108, 5,932,616, 5,700,811, 6,087,367 and 6,511,990, issued to some of the present inventors, disclose compounds useful for selectively inducing terminal differentiation of neoplastic cells, which compounds have two polar end groups separated by a flexible chain of methylene groups or a by a rigid phenyl group, wherein one or both of the polar end groups is a large hydrophobic group. Some of the compounds have an additional large hydrophobic group at the same end of the molecule as the first hydrophobic group which further increases differentiation activity about 100 fold in an enzymatic assay and about 50 fold in a cell differentiation assay. Methods of synthesizing the compounds used in the methods and pharmaceutical compositions of this invention are fully described the aforementioned patents, the entire contents of which are incorporated herein by reference.
[0018] The aforementioned patents do not disclose specific oral formulations of the HDAC inhibitors or specific dosages and dosing schedules of the recited compounds. Importantly, the aforementioned patents do not disclose oral formulations that have favorable pharmacokinetic profiles such as high bioavailability which gives rise to high blood levels of the active compounds over an extended period of time.
[0019] The class of compounds of the present invention may be useful for selectively inducing terminal differentiation of neoplastic cells and therefore aid in treatment of tumors in patients. Thus there is an urgent need to discover suitable dosages and dosing schedules of these compounds, and to develop formulations, preferably oral formulations, which give rise to steady, therapeutically effective blood levels of the active compounds over an extended period of time.
SUMMARY OF THE INVENTION
[0020] The present invention provides methods of producing a mean plasma concentration of a histone deacetylase (HDAC) inhibitor capable of inhibiting a histone deacetylase in vivo in a subject over a period of at least two hours following administration, which comprises administering to said subject an effective amount of a pharmaceutical composition comprising a HDAC inhibitor or a pharmaceutically acceptable salt or hydrate thereof, and a pharmaceutically acceptable carrier or diluent.
[0021] The present invention also provides methods for selectively inducing terminal differentiation, cell growth arrest and/or apoptosis of neoplastic cells, thereby inhibiting proliferation of such cells, and methods for inducing differentiation of tumor cells by producing a mean plasma concentration of a histone deacetylase (HDAC) inhibitor capable of inhibiting a histone deacetylase in vivo in a subject over a period of at least two hours following administration, by administering to said subject an effective amount of a pharmaceutical composition comprising a HDAC inhibitor or a pharmaceutically acceptable salt or hydrate thereof, and a pharmaceutically acceptable carrier or diluent.
[0022] The present invention further provides methods of producing a mean plasma concentration of at least about 10 nM of suberoylanilide hydroxamic acid (SAHA) in vivo in a subject over a period of at least two hours following administration, which comprises administering to said subject an effective amount of a pharmaceutical composition comprising SAHA or a pharmaceutically acceptable salt or hydrate thereof, and a pharmaceutically acceptable carrier or diluent.
[0023] The present invention also provides methods for selectively inducing terminal differentiation, cell growth arrest and/or apoptosis of neoplastic cells, thereby inhibiting proliferation of such cells, and methods for inducing differentiation of tumor cells by producing a mean plasma concentration of at least 10 nM of SAHA in vivo in a subject over a period of at least two hours following administration, by administering to said subject an effective amount of a pharmaceutical composition comprising SAHA or a pharmaceutically acceptable salt or hydrate thereof, and a pharmaceutically acceptable carrier or diluent.
[0024] The present invention provides pharmaceutical compositions suitable for oral administration, which comprise a compound useful for selectively inducing terminal differentiation, cell growth arrest and/or apoptosis of neoplastic cells, and/or which is a potent inhibitor of histone deacetylase (HDAC). The pharmaceutical compositions are further comprised of microcrystalline cellulose, croscarmellose sodium and magnesium stearate. The present invention also provides for pharmaceutical compositions for oral administration comprising SAHA, microcrystalline cellulose, croscarmellose sodium and magnesium stearate. The oral bioavailability of the active compounds in the formulations of the present invention is surprisingly high. Moreover, the formulations unexpectedly give rise to high, therapeutically effective blood levels of the active compounds over an extended period of time. The present invention further provides a safe, daily dosing regimen of these formulations, which is easy to follow and to adhere to.
[0025] As demonstrated herein, it has been surprisingly and unexpectedly found that oral formulations comprising HDAC inhibitors, particularly suberoylanilide hydroxamic acid (SAHA), have very high overall oral bioavailability of the active compound in vivo. Furthermore, the formulations give rise to high blood levels of the active compound, which remain unexpectedly high over an extended period of time, for example, up to 10-12 hours. The oral formulations of the present invention have many advantages, especially when compared to parenteral formulations, since on the one end they provide high, stable and prolonged therapeutically effective blood levels of HDAC inhibitors, and on the other hand are easy to administer to patients by any conventional mode of oral administration.
[0026] Accordingly, the present invention provides a pharmaceutical composition for oral administration comprising a histone deacetylase (HDAC) inhibitor or a pharmaceutically acceptable salt or hydrate thereof, and a pharmaceutically acceptable carrier or diluent; wherein the composition provides a mean plasma concentration of the HDAC inhibitor effective to inhibit a histone deacetylase (HDAC) in vivo for a period of at least 2 hours following administration. In a preferred embodiment, the concentration of the HDAC inhibitor is effective to inhibit the HDAC for a period of at least 10 hours following administration.
[0027] In a preferred embodiment, the present invention provides a pharmaceutical composition for oral administration comprising SAHA or a pharmaceutically acceptable salt or hydrate thereof, and a pharmaceutically acceptable carrier or diluent; wherein the composition provides a mean plasma concentration of SAHA effective to inhibit a histone deacetylase (HDAC) in vivo for a period of at least 2 hours following administration. In a preferred embodiment, the concentration of SAHA is effective to inhibit the HDAC for a period of at least 10 hours following administration.
[0028] The formulations of the present invention are useful for selectively inducing terminal differentiation, cell growth arrest and/or apoptosis of neoplastic cells and therefore aid in treatment of tumors in patients.
[0029] Accordingly, the present invention also provides a method of selectively inducing terminal differentiation of neoplastic cells in a subject and thereby inhibiting proliferation of such cells in the subject, comprising the step of orally administering to the subject an effective amount of a pharmaceutical composition comprising a histone deacetylase (HDAC) inhibitor or a pharmaceutically acceptable salt or hydrate thereof, and a pharmaceutically acceptable carrier or diluent; wherein the composition provides a mean plasma concentration of the HDAC inhibitor effective to inhibit a histone deacetylase (HDAC) in vivo for a period of at least 2 hours following administration.
[0030] Furthermore, the present invention also provides a method of selectively inducing cell growth arrest of neoplastic cells in a subject and thereby inhibiting proliferation of such cells in the subject, comprising the step of orally administering to the subject an effective amount of a pharmaceutical composition comprising a histone deacetylase (HDAC) inhibitor or a pharmaceutically acceptable salt or hydrate thereof, and a pharmaceutically acceptable carrier or diluent; wherein the composition provides a mean plasma concentration of the HDAC inhibitor effective to inhibit a histone deacetylase (HDAC) in vivo for a period of at least 2 hours following administration.
[0031] Furthermore, the present invention also provides a method of selectively inducing apoptosis of neoplastic cells in a subject and thereby inhibiting proliferation of such cells in the subject, comprising the step of orally administering to the subject an effective amount of a pharmaceutical composition comprising a histone deacetylase (HDAC) inhibitor or a pharmaceutically acceptable salt or hydrate thereof, and a pharmaceutically acceptable carrier or diluent; wherein the composition provides a mean plasma concentration of the HDAC inhibitor effective to inhibit a histone deacetylase (HDAC) in vivo for a period of at least 2 hours following administration.
[0032] Furthermore, the present invention also provides a method of inducing differentiation of tumor cells in a subject having a tumor, comprising the step of orally administering to the subject an effective amount of a pharmaceutical composition comprising a histone deacetylase (HDAC) inhibitor or a pharmaceutically acceptable salt or hydrate thereof, and a pharmaceutically acceptable carrier or diluent; wherein the composition provides a mean plasma concentration of the HDAC inhibitor effective to inhibit a histone deacetylase (HDAC) in vivo for a period of at least 2 hours following administration.
[0033] Furthermore, the present invention provides a method of inhibiting the activity of a histone deacetylase in a subject, comprising the step of orally administering to the subject an effective amount of a pharmaceutical composition comprising a histone deacetylase (HDAC) inhibitor or a pharmaceutically acceptable salt or hydrate thereof, and a pharmaceutically acceptable carrier or diluent; wherein the composition provides a mean plasma concentration of the HDAC inhibitor effective to inhibit a histone deacetylase (HDAC) in vivo for a period of at least 2 hours following administration.
[0034] Furthermore, the present invention also provides a method of selectively inducing terminal differentiation, cell growth arrest and/or apoptosis of neoplastic cells in a subject and thereby inhibiting proliferation of such cells in the subject, comprising the step of administering to the subject an effective amount of a pharmaceutical composition comprising SAHA or a pharmaceutically acceptable salt or hydrate thereof; wherein the composition provides a mean plasma concentration of SAHA effective to inhibit a histone deacetylase (HDAC) in vivo for a period of at least 2 hours following administration.
[0035] Furthermore, the present invention also provides a method of inducing differentiation of tumor cells in a subject having a tumor comprising the step of administering to the subject an effective amount of a pharmaceutical composition comprising SAHA or a pharmaceutically acceptable salt or hydrate thereof; wherein the composition provides a mean plasma concentration of SAHA effective to inhibit a histone deacetylase (HDAC) in vivo for a period of at least 2 hours following administration.
[0036] Furthermore, the present invention provides a method of inhibiting the activity of a histone deacetylase in a subject, comprising the step of orally administering to the subject an effective amount of a pharmaceutical composition comprising SAHA or a pharmaceutically acceptable salt or hydrate thereof, and a pharmaceutically acceptable carrier or diluent; wherein the composition provides a mean plasma concentration of SAHA effective to inhibit a histone deacetylase (HDAC) in vivo for a period of at least 2 hours following administration.
[0037] In a preferred embodiment, SAHA or any of the HDAC inhibitors are administered to the patient at a total daily dosage of between 25-4000 mg/m
[0038] In one preferred embodiment, the composition provides a mean plasma concentration of the HDAC inhibitor (e.g., SAHA) capable of inhibiting histone deacetylase over a period of at least 2 hours following administration, which is preferably at a concentration of at least about 10 nM. In yet another preferred embodiment, the composition provides a mean plasma concentration of the HDAC inhibitor of at least about 10 nM over a period of at least 10 hours following administration.
[0039] In one preferred embodiment, the composition provides a mean plasma concentration of the HDAC inhibitor (e.g., SAHA) capable of selectively inducing terminal differentiation, cell growth arrest and/or apoptosis of neoplastic cells, or capable of inducing differentiation of tumor cells in a tumor, wherein the concentration is maintained over a period of at least 2 hours following administration, which is preferably at a concentration of at least about 2.5 μM. In yet another preferred embodiment, the composition provides a mean plasma concentration of the HDAC inhibitor of at least about 2.5 μM over a period of at least 10 hours following administration.
[0040] The compositions of the present invention may be formulated in any unit dosage form (liquid or solid) suited for oral administration, for example, in the form of a pellet, a tablet, a coated tablet, a capsule, a gelatin capsule, a solution, a suspension, or a dispersion. In a preferred embodiment, the composition is in the form of a gelatin capsule.
[0041] Any inert excipient that is commonly used as a carrier or diluent may be used in the formulations of the present invention, such as for example, a gum, a starch, a sugar, a cellulosic material, an acrylate, or mixtures thereof. A preferred diluent is microcrystalline cellulose. The compositions may further comprise a disintegrating agent (e.g., sodium croscarmellose) and a lubricant (e.g., magnesium stearate), and in addition may comprise one or more additives selected from a binder, a buffer, a protease inhibitor, a surfactant, a solubilizing agent, a plasticizer, an emulsifier, a stabilizing agent, a viscosity increasing agent, a sweetener, a film forming agent, or any combination thereof. Furthermore, the compositions of the present invention may be in the form of controlled release or immediate release formulations.
[0042] A wide variety of HDAC inhibitors are suitable for use in the compositions of the present invention. In a preferred embodiment, the HDAC inhibitor is suberoylanilide hydroxamic acid (SAHA).
[0043] Other non-limiting examples of HDAC inhibitors that are suitable for use in the compositions of the present invention are:
[0044] Pyroxamide, represented by the structure:
[0045] A compound represented by the structure:
[0046] wherein R
[0047] A compound represented by the structure:
[0048] wherein R is a substituted or unsubstituted phenyl, piperidine, thiazole, 2-pyridine, 3-pyridine or 4-pyridine and n is an integer from about 4 to about 8.
[0049] A compound represented by the structure:
[0050] wherein A is an amide moiety, R
[0051] Furthermore, in accordance with specific embodiments of the present invention, there is provided a pharmaceutical composition for oral administration comprising a histone deacetylase (HDAC) inhibitor or a pharmaceutically acceptable salt or hydrate thereof, microcrystalline cellulose as a carrier or diluent; croscarmellose sodium as a disintegrant; and magnesium stearate as a lubricant; wherein the composition provides a mean plasma concentration of the HDAC inhibitor effective to inhibit a histone deacetylase in vivo for a period of at least 2 hours following administration. In a preferred embodiment, the HDAC inhibitor is suberoylanilide hydroxamic acid (SAHA).
[0052] Furthermore, in accordance with specific embodiments of the present invention, there is provided a pharmaceutical composition for oral administration comprising suberoylanilide hydroxamic acid (SAHA) or a pharmaceutically acceptable salt or hydrate thereof; microcrystalline cellulose as a carrier or diluent; croscarmellose sodium as a disintegrant; and magnesium stearate as a lubricant; wherein the composition provides a mean plasma concentration of the HDAC inhibitor effective to inhibit a histone deacetylase in vivo for a period of at least 2 hours following administration. In another preferred embodiment, the composition comprises 50-70% by weight of SAHA or a pharmaceutically acceptable salt or hydrate thereof; 20-40% by weight microcrystalline cellulose as a carrier or diluent; 5-15% by weight croscarmellose sodium as a disintegrant; and 0.1-5% by weight magnesium stearate as a lubricant. In another preferred embodiment, the composition comprises about 50-200 mg of SAHA. In a particularly preferred embodiment, the composition is in the form of a gelatin capsule.
[0053] The present invention further provides a safe, daily dosing regimen of these formulations, which is easy to follow and to adhere to. The formulations of the present invention are useful for selectively inducing terminal differentiation, cell growth arrest and/or apoptosis of neoplastic cells and therefore aid in treatment of tumors in patients.
BRIEF DESCRIPTION OF THE DRAWINGS
[0054] The foregoing and other objects, features and advantages of the invention will be apparent from the following more particular description of preferred embodiments of the invention, as illustrated in the accompanying drawings in which like reference characters refer to the same parts throughout the different views. The drawings are not necessarily to scale, emphasis instead being placed upon illustrating the principles of the invention.
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[0063] FIGS.
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DETAILED DESCRIPTION OF THE INVENTION
[0067] The present invention provides methods of producing a mean plasma concentration of a histone deacetylase (HDAC) inhibitor capable of inhibiting a histone deacetylase in vivo in a subject over a period of at least two hours following administration, which comprises administering to said subject an effective amount of a pharmaceutical composition comprising a HDAC inhibitor or a pharmaceutically acceptable salt or hydrate thereof, and a pharmaceutically acceptable carrier or diluent.
[0068] The present invention also provides methods for selectively inducing terminal differentiation, cell growth arrest and/or apoptosis of neoplastic cells, thereby inhibiting porliferation of such cells, and methods for inducing differentiation of tumor cells by producing a mean plasma concentration of a histone deacetylase (HDAC) inhibitor capable of inhibiting a histone deacetylase in vivo in a subject over a period of at least two hours following administration, by administering to said subject an effective amount of a pharmaceutical composition comprising a HDAC inhibitor or a pharmaceutically acceptable salt or hydrate thereof, and a pharmaceutically acceptable carrier or diluent.
[0069] The present invention further provides methods of producing a mean plasma concentration of at least about 10 nM of suberoylanilide hydroxamic acid (SAHA) in vivo in a subject over a period of at least two hours following administration, which comprises administering to said subject an effective amount of a pharmaceutical composition comprising SAHA or a pharmaceutically acceptable salt or hydrate thereof, and a pharmaceutically acceptable carrier or diluent.
[0070] The present invention also provides methods for selectively inducing terminal differentiation, cell growth arrest and/or apoptosis of neoplastic cells, thereby inhibiting proliferation of such cells, and methods for inducing differentiation of tumor cells by producing a mean plasma concentration of at least 10 nM of SAHA in vivo in a subject over a period of at least two hours following administration, by administering to said subject an effective amount of a pharmaceutical composition comprising SAHA or a pharmaceutically acceptable salt or hydrate thereof, and a pharmaceutically acceptable carrier or diluent.
[0071] The present invention provides pharmaceutical compositions suitable for oral administration, which comprise a compound useful for selectively inducing terminal differentiation, cell growth arrest and/or apoptosis of neoplastic cells, and/or which is a potent inhibitor of histone deacetylase (HDAC). The pharmaceutical compositions are further comprised of microcrystalline cellulose, croscarmellose sodium and magnesium stearate. The present invention also provides for pharmaceutical compositions for oral administration comprising SAHA, microcrystalline cellulose, croscarmellose sodium and magnesium stearate. The oral bioavailability of the active compounds in the formulations of the present invention is surprisingly high. Moreover, the formulations unexpectedly give rise to high, therapeutically effective blood levels of the active compounds over an extended period of time. The present invention further provides a safe, daily dosing regimen of these formulations, which is easy to follow and to adhere to.
[0072] The oral bioavailability of the active compounds in the formulations of the present invention is surprisingly high. Moreover, the formulations unexpectedly give rise to high, therapeutically effective blood levels of the active compounds over an extended period of time. The present invention further provides a safe, daily dosing regimen of these formulations, which is easy to follow, and which gives rise to a therapeutically effective amount of the recited compounds in vivo. The formulations of the present invention are useful for selectively inducing terminal differentiation, cell growth arrest and/or apoptosis of neoplastic cells, and therefore aid in treatment of tumors in patients.
[0073] As demonstrated herein, the pharmaceutical compositions provided in the present invention give rise to an initial mean plasma concentration (i.e., the concentration that is obtained immediately after administration of the formulation), which remains unexpectedly high over an extended period of time. As compared with parenteral formulations (such as IV formulations) having the same dosage, in which the active compounds clear almost immediately, the oral compositions retain a high mean plasma concentration of the active compound over an extended period of time, for at least 2 hours, but more typically at least, 10 or 12 hours. Typically, the mean plasma concentration of the oral dosage formulations, does not drop below 50% of the initial mean plasma concentration for a period of time of up to 12 hours or even longer.
[0074] Up until the findings of the present invention, intravenous administration of the HDAC inhibitors described herein has proven to be the most effective. The intravenous administration of the compound must be performed continuously, i.e., daily, for a prolonged period of time, such as for at least 3 days and preferably more than 5 days. This obviously provides a heavy burden on the patient receiving this treatment. The unexpected and surprising findings of the present invention make it possible to formulate oral dosage forms that give rise to high and steady levels of the active compounds in-vivo, without the need to continuously administer the drugs, by IV infusions, which provides a tremendous advantage for the patient receiving the treatment.
[0075] Accordingly, the present invention provides a pharmaceutical composition for oral administration comprising a histone deacetylase (HDAC) inhibitor or a pharmaceutically acceptable salt or hydrate thereof, and a pharmaceutically acceptable carrier or diluent; wherein the composition provides a mean plasma concentration of the HDAC inhibitor effective to inhibit a histone deacetylase (HDAC) in vivo for a period of at least 2 hours following administration. In a preferred embodiment, the concentration of the HDAC inhibitor is effective to inhibit the HDAC for a period of at least 8 hours following administration. In another preferred embodiment, the concentration of the HDAC inhibitor is effective to inhibit the HDAC for a period of at least 10 hours following administration. In another preferred embodiment, the concentration of the HDAC inhibitor is effective to inhibit the HDAC for a period of at least 12 hours following administration.
[0076] In a preferred embodiment, the present invention provides a pharmaceutical composition for oral administration comprising SAHA or a pharmaceutically acceptable salt or hydrate thereof, and a pharmaceutically acceptable carrier or diluent; wherein the composition provides a mean plasma concentration of SAHA effective to inhibit a histone deacetylase (HDAC) in vivo for a period of at least 2 hours following administration. In a preferred embodiment, the concentration of SAHA is effective to inhibit the HDAC for a period of at least 8 hours following administration. In another preferred embodiment, the concentration of SAHA is effective to inhibit the HDAC for a period of at least 10 hours following administration. In another preferred embodiment, the concentration of SAHA is effective to inhibit the HDAC for a period of at least 12 hours following administration.
[0077] The formulations of the present invention are useful for selectively inducing terminal differentiation, cell growth arrest and/or apoptosis of neoplastic cells and therefore aid in treatment of tumors in patients.
[0078] Accordingly, the present invention also provides a method of selectively inducing terminal differentiation of neoplastic cells in a subject and thereby inhibiting proliferation of such cells in the subject, comprising producing a mean plasma concentration of a HDAC inhibitor capable of inhibiting a histone deacetylase in vivo in a subject over a period of at least two hours following administration by administering to said subject an effective amount of a pharmaceutical composition comprising a HDAC inhibitor or a pharmaceutically acceptable salt or hydrate thereof, and a pharmaceutically acceptable carrier or diluent.
[0079] Furthermore, the present invention also provides a method of selectively inducing cell growth arrest of neoplastic cells in a subject and thereby inhibiting proliferation of such cells in the subject, comprising producing a mean plasma concentration of a HDAC inhibitor capable of inhibiting a histone deacetylase in vivo in a subject over a period of at least two hours following administration by administering to said subject an effective amount of a pharmaceutical composition comprising a HDAC inhibitor or a pharmaceutically acceptable salt or hydrate thereof, and a pharmaceutically acceptable carrier or diluent.
[0080] Furthermore, the present invention also provides a method of selectively inducing apoptosis of neoplastic cells in a subject and thereby inhibiting proliferation of such cells in the subject, comprising producing a mean plasma concentration of a HDAC inhibitor capable of inhibiting a histone deacetylase in vivo in a subject over a period of at least two hours following administration by administering to said subject an effective amount of a pharmaceutical composition comprising a HDAC inhibitor or a pharmaceutically acceptable salt or hydrate thereof, and a pharmaceutically acceptable carrier or diluent.
[0081] Furthermore, the present invention also provides a method of inducing differentiation of tumor cells in a subject having a tumor, producing a mean plasma concentration of a HDAC inhibitor capable of inhibiting a histone deacetylase in vivo in a subject over a period of at least two hours following administration by administering to said subject an effective amount of a pharmaceutical composition comprising a HDAC inhibitor or a pharmaceutically acceptable salt or hydrate thereof, and a pharmaceutically acceptable carrier or diluent.
[0082] Furthermore, the present invention provides a method of inhibiting the activity of a histone deacetylase in a subject, comprising producing a mean plasma concentration of a HDAC inhibitor capable of inhibiting a histone deacetylase in vivo in a subject over a period of at least two hours following administration by administering to said subject an effective amount of a pharmaceutical composition comprising a HDAC inhibitor or a pharmaceutically acceptable salt or hydrate thereof, and a pharmaceutically acceptable carrier or diluent.
[0083] Furthermore, the present invention also provides a method of selectively inducing terminal differentiation, cell growth arrest and/or apoptosis of neoplastic cells in a subject and thereby inhibiting proliferation of such cells in the subject, comprising producing a mean plasma concentration of SAHA capable of inhibiting a histone deacetylase in vivo in a subject over a period of at least two hours following administration by administering to said subject an effective amount of a pharmaceutical composition comprising SAHA or a pharmaceutically acceptable salt or hydrate thereof, and a pharmaceutically acceptable carrier or diluent.
[0084] Furthermore, the present invention also provides a method of selectively inducing terminal differentiation, cell growth arrest and/or apoptosis of neoplastic cells in a subject and thereby inhibiting proliferation of such cells in the subject, comprising producing a mean plasma concentration of at least 10 nM of SAHA in vivo in a subject over a period of at least two hours following administration by administering to said subject an effective amount of a pharmaceutical composition comprising SAHA or a pharmaceutically acceptable salt or hydrate thereof, and a pharmaceutically acceptable carrier or diluent.
[0085] Furthermore, the present invention also provides a method of inducing differentiation of tumor cells in a subject having a tumor comprising producing a mean plasma concentration of SAHA capable of inhibiting a histone deacetylase in vivo in a subject over a period of at least two hours following administration by administering to said subject an effective amount of a pharmaceutical composition comprising SAHA or a pharmaceutically acceptable salt or hydrate thereof, and a pharmaceutically acceptable carrier or diluent.
[0086] Furthermore, the present invention also provides a method of inducing differentiation of tumor cells in a subject having a tumor comprising producing a mean plasma concentration of at least 10 nM of SAHA in vivo in a subject over a period of at least two hours following administration by administering to said subject an effective amount of a pharmaceutical composition comprising SAHA or a pharmaceutically acceptable salt or hydrate thereof, and a pharmaceutically acceptable carrier or diluent.
[0087] Furthermore, the present invention provides a method of inhibiting the activity of a histone deacetylase in a subject, comprising producing a mean plasma concentration of SAHA capable of inhibiting a histone deacetylase in vivo in a subject over a period of at least two hours following administration by administering to said subject an effective amount of a pharmaceutical composition comprising SAHA or a pharmaceutically acceptable salt or hydrate thereof, and a pharmaceutically acceptable carrier or diluent.
[0088] Furthermore, the present invention provides a method of inhibiting the activity of a histone deacetylase in a subject, comprising producing a mean plasma concentration of at least 10 nM of SAHA in vivo in a subject over a period of at least two hours following administration by administering to said subject an effective amount of a pharmaceutical composition comprising SAHA or a pharmaceutically acceptable salt or hydrate thereof, and a pharmaceutically acceptable carrier or diluent.
[0089] In another preferred embodiment, the composition provides a mean plasma concentration of the HDAC inhibitor (e.g., SAHA) capable of inhibiting histone deacetylase over a period of at least 2 hours following administration, which is preferably at a concentration of at least about 10 nM. In another embodiment, the composition provides a mean plasma concentration of the HDAC inhibitor of at least about 10 nM over a period of at least 8 hours following administration. In yet another preferred embodiment, the composition provides a mean plasma concentration of the HDAC inhibitor of at least about 10 nM over a period of at least 10 hours following administration. In yet another preferred embodiment, the composition provides a mean plasma concentration of the HDAC inhibitor of at least about 10 nM over a period of at least 12 hours following administration. Non-limiting examples of mean plasma concentrations are about 10 nM, 25 nM, 40 nM, 45 nM, 50 nM, 100 nM, 1 μM, 2 μM, 2.5 μM, 5 μM 10 μM, 25, μM, 50 μM, 100 μM and the like. It should be apparent to a person skilled in the art that these doses are in no way limiting the scope of this invention, and that any mean plasma concentration which is capable of inhibiting a histone deacetylase is suitable.
[0090] In one preferred embodiment, the composition provides a mean plasma concentration of the HDAC inhibitor (e.g., SAHA) capable of selectively inducing terminal differentiation, cell growth arrest and/or apoptosis of neoplastic cells, or inducing differentiation of tumor cells in a tumor, wherein the concentration is maintained over a period of at least 2 hours following administration, which is preferably at a concentration of at least about 2.5 μM. In another embodiment, the composition provides a mean plasma concentration of the HDAC inhibitor of at least about 2.5 μM over a period of at least 8 hours following administration. In yet another preferred embodiment, the composition provides a mean plasma concentration of the HDAC inhibitor of at least about 2.5 μM over a period of at least 10 hours following administration. In yet another preferred embodiment, the composition provides a mean plasma concentration of the HDAC inhibitor of at least about 2.5 μM over a period of at least 12 hours following administration. Non-limiting examples of mean plasma concentrations are about 10 nM, 25 nM, 40 nM, 45 nM, 50 nM, 100 nM, 1 μM, 2 μM, 2.5 μM, 5 μM 10 μM, 25, μM, 50 μM, 100 μM and the like. It should be apparent to a person skilled in the art that these doses are in no way limiting the scope of this invention, and that any mean plasma concentration which is capable of inducing terminal differentiation, cell growth arrest and/or apoptosis of neoplastic cells is suitable.
[0091] In another preferred embodiment, the composition provides a mean plasma concentration of the HDAC inhibitor (e.g., SAHA) effective to induce differentiation of tumor cells in a subject having a tumor, wherein the amount is maintained for a period of at least 2 hours following administration to the subject. In another preferred embodiment, the composition provides a mean plasma concentration of the HDAC inhibitor effective to induce differentiation of tumor cells in a subject having a tumor, wherein the amount is maintained for a period of at least 8 hours following administration to the subject. In another preferred embodiment, the composition provides a mean plasma concentration of the HDAC inhibitor effective to induce differentiation of tumor cells in a subject having a tumor, wherein the amount is maintained for a period of at least about 10 hours following administration to the subject. Non-limiting examples of mean plasma concentrations are about 10 nM, 25 nM, 40 nM, 45 nM, 50 nM, 100 nM, 1 μM, 2 μM, 2.5 μM, 5 μM 10 μM, 25, μM, 50 μM, 100 μM and the like. It should be apparent to a person skilled in the art that these doses are in no way limiting the scope of this invention, and that any mean plasma concentration which is capable of inducing differentiation of tumor cells in a tumor is suitable.
[0092] The methods of the present invention are suitable for practice in vitro and in vivo. If the methods are practiced in vitro, contacting may be effected by incubating the cells with the compound. The concentration of the compound in contact with the cells should be from about I about nM to about 25 mM, for example, from about 10 nM to about 1 mM, from about 40 nM to about 0.5 mM. Non-limiting examples of specific doses are 10 nM, 25 nM, 40 nM, 45 nM, 50 nM, 100 nM, 1 μM, 2 μM, 2.5 μM, 5 μM, 10 μM, 25 μM, 50 μM, 100 μM and the like. The concentration depends upon the individual compound and the state of the neoplastic cells.
[0093] Although the methods of the present invention can be practiced in vitro, it is contemplated that the preferred embodiment for the methods of selectively inducing terminal differentiation, cell growth arrest and/or apoptosis of neoplastic cells will comprise contacting the cells in vivo, i.e., by administering the compounds to a subject harboring neoplastic cells or tumor cells in need of treatment.
[0094] The methods of the present invention may also comprise initially administering to the subject an antitumor agent so as to render the neoplastic cells in the subject resistant to an antitumor agent and subsequently administering an effective amount of any of the compositions of the present invention, effective to selectively induce terminal differentiation, cell growth arrest and/or apoptosis of such cells.
[0095] The antitumor agent may be one of numerous chemotherapy agents such as an alkylating agent, an antimetabolite, a hormonal agent, an antibiotic, colchicine, a vinca alkaloid, L-asparaginase, procarbazine, hydroxyurea, mitotane, nitrosoureas or an imidazole carboxamide. Suitable agents are those agents that promote depolarization of tubulin. Preferably the antitumor agent is colchicine or a vinca alkaloid; especially preferred are vinblastine and vincristine. In embodiments where the antitumor agent is vincristine, the cells preferably are treated so that they are resistant to vincristine at a concentration of about 5 mg/ml. The treating of the cells to render them resistant to an antitumor agent may be effected by contacting the cells with the agent for a period of at least 3 to 5 days. The contacting of the resulting cells with any of the compounds above is performed as described previously. In addition to the above chemotherapy agents, the compounds may also be administered together with radiation therapy.
[0096] The present invention also provides a method of treating a patient having a tumor characterized by proliferation of neoplastic cells which comprises administering to the patient an effective amount of any of the compositions of the present invention above, effective to selectively induce terminal differentiation of such neoplastic cells and thereby inhibit their proliferation.
[0097] The method of the present invention is intended for the treatment of human patients with tumors. However, it is also likely that the method would be effective in the treatment of tumors in other mammals. The term tumor is intended to include any cancer caused by the proliferation of neoplastic cells, such as lung cancer, acute lymphoid myeloma, Hodgkins lymphoma, non-Hodgkins lymphoma, bladder melanoma, renal carcinoma, breast carcinoma, prostate carcinoma, ovarian carcinoma or colorectal carcinoma.
[0098] The administration of the pharmaceutical compositions can be carried out in unit dosages which may be administered orally once a day, twice a day, three times a day and the like. Currently preferred embodiments are once-daily administration, twice-daily administration and three-times daily administration.
Histone Deacetylases and Histone Deacetylase Inhibitors
[0099] Histone deacetylases (HDACs), as that term is used herein, are enzymes that catalyze the removal of acetyl groups from lysine residues in the amino terminal tails of the nucleosomal core histones. As such, HDACs together with histone acetyl transferases (HATs) regulate the acetylation status o