U.S. patent application number 10/383748 was filed with the patent office on 2004-03-18 for method and pharmaceutical compositions forthe treatment of cancer.
This patent application is currently assigned to Ramot At Tel Aviv University Ltd.. Invention is credited to Margalit, Rimona, Peer, Dan.
Application Number | 20040054014 10/383748 |
Document ID | / |
Family ID | 25488062 |
Filed Date | 2004-03-18 |
United States Patent
Application |
20040054014 |
Kind Code |
A1 |
Margalit, Rimona ; et
al. |
March 18, 2004 |
Method and pharmaceutical compositions forthe treatment of
cancer
Abstract
A method of treating a subject having cancer, particularly a
multidrug resistance cancer, which comprises administering to the
subject at least one chemotherapeutic agent and at least one
3-aryloxy-3-phenylpropylamine and pharmaceutical compositions and
kits for implementing the method.
Inventors: |
Margalit, Rimona; (Givataim,
IL) ; Peer, Dan; (Kiryat Ono, IL) |
Correspondence
Address: |
G.E. EHRLICH (1995) LTD.
c/o ANTHONY CASTORINA
SUITE 207
2001 JEFFERSON DAVIS HIGHWAY
ARLINGTON
VA
22202
US
|
Assignee: |
Ramot At Tel Aviv University
Ltd.
|
Family ID: |
25488062 |
Appl. No.: |
10/383748 |
Filed: |
March 10, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10383748 |
Mar 10, 2003 |
|
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PCT/IL02/00750 |
Sep 10, 2002 |
|
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Current U.S.
Class: |
514/651 |
Current CPC
Class: |
A61P 43/00 20180101;
A61K 31/70 20130101; A61P 35/00 20180101; A61P 35/02 20180101; A61K
31/40 20130101; A61K 31/135 20130101; A61K 31/40 20130101; A61K
2300/00 20130101; A61K 31/70 20130101; A61K 2300/00 20130101 |
Class at
Publication: |
514/651 |
International
Class: |
A61K 031/137 |
Claims
What is claimed is:
1. A method of treating a subject suspected as having, or having a
multidrug resistance cancer, the method comprising administering to
the subject a chemotherapeutically effective amount of at least one
chemotherapeutic agent and a chemosensitizing effective amount of
at least one 3-aryloxy-3-phenylpropylamine.
2. The method of claim 1, wherein said multidrug resistant cancer
is inherent.
3. The method of claim 1, wherein said multidrug resistant cancer
is acquired.
4. The method of claim 1, wherein said administering said at least
one chemotherapeutic agent and said at least one
3-aryloxy-3-phenylpropylamin- e is performed substantially at the
same time.
5. The method of claim 1, wherein said chemosensitizing effective
amount ranges between about 0.1 mg/M.sup.2 and about 10
mg/M.sup.2.
6. The method of claim 1, wherein said cancer is selected from the
group consisting of leukemia, lymphoma, carcinoma and sarcoma.
7. The method of claim 1, wherein said at least one
3-aryloxy-3-phenylpropylamine is administered orally.
8. The method of claim 1, wherein said at least one
3-aryloxy-3-phenylpropylamine is of the formula: 5wherein each R'
is independently hydrogen or methyl; R is naphthyl or 6R" and R'"
are halo, trifluoromethyl, C.sub.1-C.sub.4 alkyl, C.sub.1-C.sub.3
alkoxy or C.sub.3-C.sub.4 alkenyl; and n and m are 0, 1 or 2; and
acid addition salts thereof formed with pharmaceutically acceptable
acids.
9. The method of claim 1, wherein said at least one
3-aryloxy-3-phenylpropylamine is selected from the group consisting
of 3-(p-isopropoxyphenxoy)-3-phenylpropylamine methanesulfonate,
N,N-dimethyl 3-(3',4'-dimethoxyphenoxy)-3-phenylpropylamine
p-hydroxybenzoate, N,N-dimethyl
3-(alpha-naphthoxy)-3-phenylpropylamine bromide, N,N-dimethyl
3-(beta-naphthoxy)-3-phenyl-1-methylpropylamine iodide,
3-(2'-methyl-4',5'-dichlorophenoxy)-3-phenylpropylamine nitrate,
3-(p-t-butylphenoxy)-3-phenylpropylamine glutarate, N-methyl
3-(2'-chloro-p-tolyloxy)-3-phenyl-1-methylpropylamine lactate,
3-(2',4'-dichlorophenoxy)-3-phenyl-2-methylpropylamine citrate,
N,N-dimethyl 3-(m-anisyloxy)-3-phenyl-1-methylpropylamine maleate,
N-methyl 3-(p-tolyloxy)-3-phenylpropylamine sulfate, N,N-di methyl
3-(2',4'-di fluorophenoxy)-3-phenylpropylamine 2,4-dinitrobenzoate,
3-(o-ethylphenoxy)-3-phenylpropylamine dihydrogen phosphate,
N-methyl-(2'-chloro-4'-isopropylphenoxy)-3-phenyl-2-methylpropylamine
maleate, N,N-dimethyl
3-(2'-alkyl-4'-fluorophenoxy)-3-phenylpropylamine succinate,
N,N-dimethyl 3-(o-isopropoxyphenoxy)-3-phenyl-propylamine
phenylacetate, N,N-dimethyl
3-(o-)bromophenoxy)-3-phenyl-propylamine beta-phenylpropionate,
N-methyl 3-(p-iodophenoxy)-3-phenyl-propylamine propiolate,
N-methyl 3-(3-n-propylphenoxy)-3-phenyl-propylamine decanoate, and
N-methyl 3-(p-trifluoromethylphenoxy)-3-phenylpropylamine.
10. The method of claim 1, wherein said at least one
3-aryloxy-3-phenylpropylamine is N-methyl
3-(p-trifluoromethylphenoxy)-3-- phenylpropylamine or a
pharmaceutically acceptable salt thereof.
11. The method of claim 1, wherein said at least one
chemotherapeutic agent is selected from the group consisting of an
alkylating agent, an antimetabolite, a natural product, a
miscellaneous agent, a hormone and an antagonist.
12. A method of treating a subject suspected as having, or having a
multidrug resistance cancer, the method comprising administering to
the subject, substantially at the same time, a chemotherapeutically
effective amount of at least one chemotherapeutic agent and a
chemosensitizing effective amount of at least one
3-aryloxy-3-phenylpropylamine.
13. The method of claim 12, wherein said multidrug resistant cancer
is inherent.
14. The method of claim 12, wherein said multidrug resistant cancer
is acquired.
15. The method of claim 12, wherein said chemosensitizing effective
amount ranges between about 0.1 mg/M.sup.2 and about 10
mg/M.sup.2.
16. The method of claim 12, wherein said cancer is selected from
the group consisting of leukemia, lymphoma, carcinoma and
sarcoma.
17. The method of claim 12, wherein said at least one
3-aryloxy-3-phenylpropylamine is administered orally.
18. The method of claim 12, wherein said at least one
3-aryloxy-3-phenylpropylamine is of the formula: 7wherein each R'
is independently hydrogen or methyl; R is naphthyl or 8R" and R'"
are halo, trifluoromethyl, C.sub.1-C.sub.4 alkyl, C.sub.1-C.sub.3
alkoxy or C.sub.3-C.sub.4 alkenyl; and n and m are 0, 1 or 2; and
acid addition salts thereof formed with pharmaceutically acceptable
acids.
19. The method of claim 12, wherein said at least one
3-aryloxy-3-phenylpropylamine is selected from the group consisting
of 3-(p-isopropoxyphenxoy)-3-phenylpropylamine methanesulfonate,
N,N-dimethyl 3-(3',4'-dimethoxyphenoxy)-3-phenylpropylamine
p-hydroxybenzoate, N,N-dimethyl
3-(alpha-naphthoxy)-3-phenylpropylamine bromide, N,N-dimethyl
3-(beta-naphthoxy)-3-phenyl-1-methylpropylamine iodide,
3-(2'-methyl-4',5'-dichlorophenoxy)-3-phenylpropylamine nitrate,
3-(p-t-butylphenoxy)-3-phenylpropylamine glutarate, N-methyl
3-(2'-chloro-p-tolyloxy)-3-phenyl-1-methylpropylamine lactate,
3-(2',4'-dichlorophenoxy)-3-phenyl-2-methylpropylamine citrate,
N,N-dimethyl 3-(m-anisyloxy)-3-phenyl-1-methylpropylamine maleate,
N-methyl 3-(p-tolyloxy)-3-phenylpropylamine sulfate, N,N-dimethyl
3-(2',4'-difluorophenoxy)-3-phenylpropylamine 2,4-dinitrobenzoate,
3-(o-ethylphenoxy)-3-phenylpropylamine dihydrogen phosphate,
N-methyl-(2'-chloro-4'-isopropylphenoxy)-3-phenyl-2-methylpropylamine
maleate, N,N-dimethyl
3-(2'-alkyl-4'-fluorophenoxy)-3-phenylpropylamine succinate,
N,N-dimethyl 3-(o-isopropoxyphenoxy)-3-phenyl-propylamine
phenylacetate, N,N-dimethyl
3-(o-)bromophenoxy)-3-phenyl-propylamine beta-phenylpropionate,
N-methyl 3-(p-iodophenoxy)-3-phenyl-propylamine propiolate,
N-methyl 3-(3-n-propylphenoxy)-3-phenyl-propylamine decanoate, and
N-methyl 3-(p-trifluoromethylphenoxy)-3-phenylpropylamine.
20. The method of claim 12, wherein said at least one
3-aryloxy-3-phenylpropylamine is N-methyl
3-(p-trifluoromethylphenoxy)-3-- phenylpropylamine or a
pharmaceutically acceptable salt thereof.
21. The method of claim 12, wherein said at least one
chemotherapeutic agent is selected from the group consisting of an
alkylating agent, an antimetabolite, a natural product, a
miscellaneous agent, a hormone and an antagonist.
22. A method of treating a subject suspected as having, or having a
multidrug resistance cancer, the method comprising administering to
the subject a chemotherapeutically effective amount of at least one
chemotherapeutic agent and a chemosensitizing effective amount of
at least one 3-aryloxy-3-phenylpropylamine, said chemosensitizing
effective amount ranges between about 0.1 mg/M.sup.2 and about 10
mg/M.sup.2.
23. The method of claim 22, wherein said multidrug resistant cancer
is inherent.
24. The method of claim 22, wherein said multidrug resistant cancer
is acquired.
25. The method of claim 22, wherein said administering said at
least one chemotherapeutic agent and said at least one
3-aryloxy-3-phenylpropylamin- e is performed substantially at the
same.
26. The method of claim 22, wherein said cancer is selected from
the group consisting of leukemia, lymphoma, carcinoma and
sarcoma.
27. The method of claim 22, wherein said at least one
3-aryloxy-3-phenylpropylamine is administered orally.
28. The method of claim 22, wherein said at least one
3-aryloxy-3-phenylpropylamine is of the formula: 9wherein each R'
is independently hydrogen or methyl; R is naphthyl or 10R" and R'"
are halo, trifluoromethyl, C.sub.1-C.sub.4 alkyl, C.sub.1-C.sub.3
alkoxy or C.sub.3-C.sub.4 alkenyl; and n and m are 0, 1 or 2; and
acid addition salts thereof formed with pharmaceutically acceptable
acids.
29. The method of claim 22, wherein said at least one
3-aryloxy-3-phenylpropylamine is selected from the group consisting
of 3-(p-isopropoxyphenxoy)-3-phenylpropylamine methanesulfonate,
N,N-dimethyl 3-(3',4'-dimethoxyphenoxy)-3-phenylpropylamine
p-hydroxybenzoate, N,N-dimethyl
3-(alpha-naphthoxy)-3-phenylpropylamine bromide, N,N-dimethyl
3-(beta-naphthoxy)-3-phenyl-1-methylpropylamine iodide,
3-(2'-methyl-4',5'-dichlorophenoxy)-3-phenylpropylamine nitrate,
3-(p-t-butylphenoxy)-3-phenylpropylamine glutarate, N-methyl
3-(2'-chloro-p-tolyloxy)-3-phenyl-1-methylpropylamine lactate,
3-(2',4'-dichlorophenoxy)-3-phenyl-2-methylpropylamine citrate,
N,N-dimethyl 3-(m-anisyloxy)-3-phenyl-1-methylpropylamine maleate,
N-methyl 3-(p-tolyloxy)-3-phenylpropylamine sulfate, N,N-dimethyl
3-(2',4'-difluorophenoxy)-3-phenylpropylamine 2,4-dinitrobenzoate,
3-(o-ethylphenoxy)-3-phenylpropylamine dihydrogen phosphate,
N-methyl-(2'-chloro-4'-isopropylphenoxy)-3-phenyl-2-methylpropylamine
maleate, N,N-dimethyl
3-(2'-alkyl-4'-fluorophenoxy)-3-phenylpropylamine succinate,
N,N-dimethyl 3-(o-isopropoxyphenoxy)-3-phenyl-propylamine
phenylacetate, N,N-dimethyl
3-(o-)bromophenoxy)-3-phenyl-propylamine beta-phenylpropionate,
N-methyl 3-(p-iodophenoxy)-3-phenyl-propylamine propiolate,
N-methyl 3-(3-n-propylphenoxy)-3-phenyl-propylamine decanoate, and
N-methyl 3-(p-trifluoromethylphenoxy)-3-phenylpropylamine.
30. The method of claim 22, wherein said at least one
3-aryloxy-3-phenylpropylamine is N-methyl
3-(p-trifluoromethylphenoxy)-3-- phenylpropylamine or a
pharmaceutically acceptable salt thereof.
31. The method of claim 22, wherein said at least one
chemotherapeutic agent is selected from the group consisting of an
alkylating agent, an antimetabolite, a natural product, a
miscellaneous agent, a hormone and an antagonist.
32. A method of selecting a chemotherapeutic agent for which
3-aryloxy-3-phenylpropylamine is a chemosensitizer comprising:
assaying cytotoxicity of a candidate chemotherapeutic agent in the
presence and in the absence of a 3-aryloxy-3-phenylpropylamine; and
selecting a candidate chemotherapeutic agent as a chemotherapeutic
agent for which 3-aryloxy-3-phenylpropylamine is a chemosensitizer
when the cytotoxicity of the candidate agent is greater in the
presence of 3-aryloxy-3-phenylpropylamine than in the absence of
3-aryloxy-3-phenylpropylamine.
33. The method of claim 32, wherein said assaying is performed with
multidrug resistant cells.
34. The method of claim 32, wherein said assaying is performed
using a 3-aryloxy-3-phenylpropylamine at a dose that ranges between
about 1 .mu.M and about 10 .mu.M.
35. The method of claim 32, wherein when said assaying is performed
in the presence of a 3-aryloxy-3-phenylpropylamine, said
3-aryloxy-3-phenylpropy- lamine and said candidate chemotherapeutic
agent are administered substantially at the same time.
36. The method of claim 32, wherein said
3-aryloxy-3-phenylpropylamine is of the formula: 11wherein each R'
is independently hydrogen or methyl; R is naphthyl or 12R" and R'"
are halo, trifluoromethyl, C.sub.1-C.sub.4 alkyl, C.sub.1-C.sub.3
alkoxy or C.sub.3-C.sub.4 alkenyl; and n and m are 0, 1 or 2; and
acid addition salts thereof formed with pharmaceutically acceptable
acids.
37. The method of claim 32, wherein said
3-aryloxy-3-phenylpropylamine is selected from the group consisting
of 3-(p-isopropoxyphenxoy)-3-phenylpro- pylamine methanesulfonate,
N,N-dimethyl 3-(3',4'-dimethoxyphenoxy)-3-pheny- lpropylamine
p-hydroxybenzoate, N,N-dimethyl 3-(alpha-naphthoxy)-3-phenylp-
ropylamine bromide, N,N-dimethyl
3-(beta-naphthoxy)-3-phenyl-1-methylpropy- lamine iodide,
3-(2'-methyl-4',5'-dichlorophenoxy)-3-phenylpropylamine nitrate,
3-(p-t-butylphenoxy)-3-phenylpropylamine glutarate, N-methyl
3-(2'-chloro-p-tolyloxy)-3-phenyl-1-methylpropylamine lactate,
3-(2',4'-dichlorophenoxy)-3-phenyl-2-methylpropylamine citrate,
N,N-dimethyl 3-(m-anisyloxy)-3-phenyl-1-methylpropylamine maleate,
N-methyl 3-(p-tolyloxy)-3-phenylpropylamine sulfate, N,N-dimethyl
3-(2',4'-di fluorophenoxy)-3-phenylpropylamine 2,4-dinitrobenzoate,
3-(o-ethylphenoxy)-3-phenylpropylamine dihydrogen phosphate,
N-methyl-(2'-chloro-4'-isopropylphenoxy)-3-phenyl-2-methylpropylamine
maleate, N,N-dimethyl
3-(2'-alkyl-4'-fluorophenoxy)-3-phenylpropylamine succinate,
N,N-dimethyl 3-(o-isopropoxyphenoxy)-3-phenyl-propylamine
phenylacetate, N,N-dimethyl
3-(o-)bromophenoxy)-3-phenyl-propylamine beta-phenylpropionate,
N-methyl 3-(p-iodophenoxy)-3-phenyl-propylamine propiolate,
N-methyl 3-(3-n-propylphenoxy)-3-phenyl-propylamine decanoate, and
N-methyl 3-(p-trifluoromethylphenoxy)-3-phenylpropylamine.
38. The method of claim 32, wherein said
3-aryloxy-3-phenylpropylamine is N-methyl
3-(p-trifluoromethylphenoxy)-3-phenylpropylamine or a
pharmaceutically acceptable salt thereof.
39. The method of claim 32, wherein said chemotherapeutic agent is
selected from the group consisting of an alkylating agent, an
antimetabolite, a natural product, a miscellaneous agent, a hormone
and an antagonist.
40. A pharmaceutical composition comprising as a
chemotherapeutically active ingredient at least one
chemotherapeutic agent and as a chemosensitization active
ingredient at least one 3-aryloxy-3-phenylpropy- lamine.
41. The pharmaceutical composition of claim 40, packaged in a
packaging material and identified in print in or on said packaging
material, for use in the treatment of a multidrug resistance
cancer.
42. The pharmaceutical composition of claim 41, wherein said
multidrug resistant cancer is inherent.
43. The pharmaceutical composition of claim 41, wherein said
multidrug resistant cancer is acquired.
44. The pharmaceutical composition of claim 40, wherein said at
least one 3-aryloxy-3-phenylpropylamine is of the formula:
13wherein each R' is independently hydrogen or methyl; R is
naphthyl or 14R" and R'" are halo, trifluoromethyl, C.sub.1-C.sub.4
alkyl, C.sub.1-C.sub.3 alkoxy or C.sub.3-C.sub.4 alkenyl; and n and
m are 0, 1 or 2; and acid addition salts thereof formed with
pharmaceutically acceptable acids.
45. The pharmaceutical composition of claim 40, wherein said at
least one 3-aryloxy-3-phenylpropylamine is selected from the group
consisting of 3-(p-isopropoxyphenxoy)-3-phenylpropylamine
methanesulfonate, N,N-dimethyl
3-(3',4'-dimethoxyphenoxy)-3-phenylpropylamine p-hydroxybenzoate,
N,N-dimethyl 3-(alpha-naphthoxy)-3-phenylpropylamine bromide,
N,N-dimethyl 3-(beta-naphthoxy)-3-phenyl-1-methylpropylamine
iodide, 3-(2'-methyl-4',5'-dichlorophenoxy)-3-phenylpropylamine
nitrate, 3-(p-t-butylphenoxy)-3-phenylpropylamine glutarate,
N-methyl 3-(2'-chloro-p-tolyloxy)-3-phenyl-1-methylpropylamine
lactate, 3-(2',4'-dichlorophenoxy)-3-phenyl-2-methylpropylamine
citrate, N,N-dimethyl 3-(m-anisyloxy)-3-phenyl-1-methylpropylamine
maleate, N-methyl 3-(p-tolyloxy)-3-phenylpropylamine sulfate,
N,N-dimethyl 3-(2',4'-difluorophenoxy)-3-phenylpropylamine
2,4-dinitrobenzoate, 3-(o-ethylphenoxy)-3-phenylpropylamine
dihydrogen phosphate,
N-methyl-(2'-chloro-4'-isopropylphenoxy)-3-phenyl-2-methylpropylamine
maleate, N,N-dimethyl
3-(2'-alkyl-4'-fluorophenoxy)-3-phenylpropylamine succinate,
N,N-dimethyl 3-(o-isopropoxyphenoxy)-3-phenyl-propylamine
phenylacetate, N,N-dimethyl
3-(o-)bromophenoxy)-3-phenyl-propylamine beta-phenylpropionate,
N-methyl 3-(p-iodophenoxy)-3-phenyl-propylamine propiolate,
N-methyl 3-(3-n-propylphenoxy)-3-phenyl-propylamine decanoate, and
N-methyl 3-(p-trifluoromethylphenoxy)-3-phenylpropylamine.
46. The pharmaceutical composition of claim 40, wherein said at
least one 3-aryloxy-3-phenylpropylamine is N-methyl
3-(p-trifluoromethylphenoxy)-3-- phenylpropylamine or a
pharmaceutically acceptable salt thereof.
47. The pharmaceutical composition of claim 40, wherein said at
least one chemotherapeutic agent is selected from the group
consisting of an alkylating agent, an antimetabolite, a natural
product, a miscellaneous agent, a hormone and an antagonist.
48. A pharmaceutical kit comprising as a chemotherapeutically
active ingredient at least one chemotherapeutic agent and as a
chemosensitization active ingredient at least one
3-aryloxy-3-phenylpropy- lamine, wherein said at least one
chemotherapeutic agent and said at least one
3-aryloxy-3-phenylpropylamine are individually packaged within the
pharmaceutical kit.
49. The pharmaceutical kit of claim 48, identified in print for use
in the treatment of a multidrug resistance cancer.
50. The pharmaceutical kit of claim 49, wherein said multidrug
resistant cancer is inherent.
51. The pharmaceutical kit of claim 49, wherein said multidrug
resistant cancer is acquired.
52. The pharmaceutical kit of claim 48, wherein said at least one
3-aryloxy-3-phenylpropylamine is of the formula: 15wherein each R'
is independently hydrogen or methyl; R is naphthyl or 16R" and R'"
are halo, trifluoromethyl, C.sub.1-C.sub.4 alkyl, C.sub.1-C.sub.3
alkoxy or C.sub.3-C.sub.4 alkenyl; and n and m are 0, 1 or 2; and
acid addition salts thereof formed with pharmaceutically acceptable
acids.
53. The pharmaceutical kit of claim 48, wherein said at least one
3-aryloxy-3-phenylpropylamine is selected from the group consisting
of 3-(p-isopropoxyphenxoy)-3-phenylpropylamine methanesulfonate,
N,N-dimethyl 3-(3',4'-dimethoxyphenoxy)-3-phenylpropylamine
p-hydroxybenzoate, N,N-dimethyl
3-(alpha-naphthoxy)-3-phenylpropylamine bromide, N,N-dimethyl
3-(beta-naphthoxy)-3-phenyl-1-methylpropylamine iodide,
3-(2'-methyl-4',5'-dichlorophenoxy)-3-phenylpropylamine nitrate,
3-(p-t-butylphenoxy)-3-phenylpropylamine glutarate, N-methyl
3-(2'-chloro-p-tolyloxy)-3-phenyl-1-methylpropylamine lactate,
3-(2',4'-dichlorophenoxy)-3-phenyl-2-methylpropylamine citrate,
N,N-dimethyl 3-(m-anisyloxy)-3-phenyl-1-methylpropylamine maleate,
N-methyl 3-(p-tolyloxy)-3-phenylpropylamine sulfate, N,N-dimethyl
3-(2',4'-difluorophenoxy)-3-phenylpropylamine 2,4-dinitrobenzoate,
3-(o-ethylphenoxy)-3-phenylpropylamine dihydrogen phosphate,
N-methyl-(2'-chloro-4'-isopropylphenoxy)-3-phenyl-2-methylpropylamine
maleate, N,N-dimethyl
3-(2'-alkyl-4'-fluorophenoxy)-3-phenylpropylamine succinate,
N,N-dimethyl 3-(o-isopropoxyphenoxy)-3-phenyl-propylamine
phenylacetate, N,N-dimethyl
3-(o-)bromophenoxy)-3-phenyl-propylamine beta-phenylpropionate,
N-methyl 3-(p-iodophenoxy)-3-phenyl-propylamine propiolate,
N-methyl 3-(3-n-propylphenoxy)-3-phenyl-propylamine decanoate, and
N-methyl 3-(p-trifluoromethylphenoxy)-3-phenylpropylamine.
54. The pharmaceutical kit of claim 48, wherein said at least one
3-aryloxy-3-phenylpropylamine is N-methyl
3-(p-trifluoromethylphenoxy)-3-- phenylpropylamine or a
pharmaceutically acceptable salt thereof.
55. The pharmaceutical kit of claim 48, wherein said at least one
chemotherapeutic agent is selected from the group consisting of an
alkylating agent, an antimetabolite, a natural product, a
miscellaneous agent, a hormone and an antagonist.
56. A pharmaceutical composition comprising as an active ingredient
at least one 3-aryloxy-3-phenylpropylamine, the pharmaceutical
composition being packaged and indicated for use in
chemosensitization, in combination with a chemotherapeutic agent
and/or in a medical condition for which chemosensitization is
beneficial.
57. The pharmaceutical composition of claim 56, wherein said at
least one 3-aryloxy-3-phenylpropylamine is of the formula:
17wherein each R' is independently hydrogen or methyl; R is
naphthyl or 18R" and R'" are halo, trifluoromethyl, C.sub.1-C.sub.4
alkyl, C.sub.1-C.sub.3 alkoxy or C.sub.3-C.sub.4 alkenyl; and n and
m are 0, 1 or 2; and acid addition salts thereof formed with
pharmaceutically acceptable acids.
58. The pharmaceutical composition of claim 56, wherein said at
least one 3-aryloxy-3-phenylpropylamine is selected from the group
consisting of: 3-(p-isopropoxyphenxoy)-3-phenylpropylamine
methanesulfonate, N,N-dimethyl
3-(3',4'-dimethoxyphenoxy)-3-phenylpropylamine p-hydroxybenzoate,
N,N-dimethyl 3-(alpha-naphthoxy)-3-phenylpropylamine bromide,
N,N-dimethyl 3-(beta-naphthoxy)-3-phenyl-1-methylpropylamine
iodide, 3-(2'-methyl-4',5'-dichlorophenoxy)-3-phenylpropylamine
nitrate, 3-(p-t-butylphenoxy)-3-phenylpropylamine glutarate,
N-methyl 3-(2'-chloro-p-tolyloxy)-3-phenyl-1-methylpropylamine
lactate, 3-(2',4'-dichlorophenoxy)-3-phenyl-2-methylpropylamine
citrate, N,N-dimethyl 3-(m-anisyloxy)-3-phenyl-1-methylpropylamine
maleate, N-methyl 3-(p-tolyloxy)-3-phenylpropylamine sulfate,
N,N-dimethyl 3-(2',4'-difluorophenoxy)-3-phenylpropylamine
2,4-dinitrobenzoate, 3-(o-ethylphenoxy)-3-phenylpropylamine
dihydrogen phosphate,
N-methyl-(2'-chloro-4'-isopropylphenoxy)-3-phenyl-2-methylpropylamine
maleate, N,N-dimethyl
3-(2'-alkyl-4'-fluorophenoxy)-3-phenylpropylamine succinate,
N,N-dimethyl 3-(o-isopropoxyphenoxy)-3-phenyl-propylamine
phenylacetate, N,N-dimethyl
3-(o-)bromophenoxy)-3-phenyl-propylamine beta-phenylpropionate,
N-methyl 3-(p-iodophenoxy)-3-phenyl-propylamine propiolate,
N-methyl 3-(3-n-propylphenoxy)-3-phenyl-propylamine decanoate, and
N-methyl 3-(p-trifluoromethylphenoxy)-3-phenylpropylamine.
59. The pharmaceutical composition of claim 56, wherein said at
least one 3-aryloxy-3-phenylpropylamine is N-methyl
3-(p-trifluoromethylphenoxy)-3-- phenylpropylamine or a
pharmaceutically acceptable salt thereof.
60. The pharmaceutical composition of claim 56, wherein said at
least one chemotherapeutic agent is selected from the group
consisting of an alkylating agent, an antimetabolite, a natural
product, a miscellaneous agent, a hormone and an antagonist.
Description
[0001] This application is a continuation-in-part of
PCT/IL02/00750, filed Sep. 10, 2002, which claims the benefit of
priority from U.S. patent application Ser. No. 09/948,621, filed
Sep. 10, 2001.
FIELD AND BACKGROUND OF THE INVENTION
[0002] The present invention relates generally to the field of
oncology, and to methods and pharmaceutical compositions for
enhancing the activity of a cancer chemotherapeutic agent. More
particularly, the present invention concerns the use of a
3-aryloxy-3-phenylpropylamine such as fluoxetine [(N-methyl
3-(p-trifluoromethylphenoxy)-3-phenylpropylamine] as a
chemosensitizer for enhancing the cytotoxicity of a
chemotherapeutic agent, especially in drug-resistant tumors and
more particularly in the case of inherent and/or acquired Multidrug
Resistance (MDR). Methods and compositions are provided for the
treatment of cancers such as, but not limited to, leukemia,
lymphoma, carcinoma and sarcoma (including glioma) using a
3-aryloxy-3-phenylpropylamine, fluoxetine in particular, as a
chemosensitizer.
[0003] Many of the most prevalent forms of human cancer resist
effective chemotherapeutic intervention. Some tumor populations,
especially adrenal, colon, jejunal, kidney and liver carcinomas,
appear to have drug-resistant cells at the outset of treatment
(Barrows, L. R., "Antineoplastic and Immunoactive Drugs", Chapter
75, pp 1236-1262, in: Remington: The Science and Practice of
Pharmacy, Mack Publishing Co. Easton, Pa., 1995). In other cases, a
resistance-conferring genetic change occurs during treatment; the
resistant daughter cells then proliferate in the environment of the
drug. Whatever the cause, resistance often terminates the
usefulness of an antineoplastic drug.
[0004] Clinical studies suggest that a common form of multidrug
resistance in human cancers results from the expression of the MDR1
gene that encodes P-glycoprotein. This glycoprotein functions as a
plasma membrane, energy-dependent, multidrug efflux pump that
reduces the intracellular concentration of cytotoxic drugs. This
mechanism of resistance may account for de novo resistance in
common tumors, such as colon cancer and renal cancer, and for
acquired resistance, as observed in common hematologic tumors such
as acute nonlymphocytic leukemia and malignant lymphomas. Although
this type of drug resistance may be common, it is by no means the
only mechanism by which cells become drug resistant. MDR is
effected via an extrusion mechanism (Tan B, Piwnica-Worms D, Ratner
L., Multidrug resistance transporters and modulation. Curr. Opin.
Oncol, 2000 September;12(5):450-8). The influx of chemotherapeutic
drugs into cells is mainly by passive diffusion across the cell
membrane, driven by the drug's electrochemical-potential gradient.
In multidrug resistance cells there are energy-dependant extrusion
channels that actively pump the drug out of the cells, reducing its
intracellular concentration below lethal threshold. The first pump
identified was named Pgp (for P-glycoprotein), the second was named
MRP (for Multidrug Resistant associate Protein) and several more
have been identified in recent years (Tan et al. 2000, ibid.). All
of them are naturally occurring proteins, and their physiological
roles are assumed to involve detoxification of cells. In multidrug
resistance cells they are present, for reasons yet unknown, in a
significantly higher number of copies than in other non-multidrug
resistance cells. Hereinafter, these proteins acting as extrusion
pumps or channels in multidrug resistance cells are referred to,
interchangeably, as "MDR pumps", "MDR extrusion pumps", "extrusion
pumps", "MDR channels", "MDR extrusion channels" and "extrusion
channels".
[0005] Chemical modification of cancer treatment involves the use
of agents or maneuvers that are not cytotoxic in themselves, but
modify the host or tumor so as to enhance anticancer therapy. Such
agents are called chemosensitizers. Pilot studies using
chemosensitizers indicate that these agents may reverse resistance
in a subset of patients. These same preliminary studies also
indicate that drug resistance is multifactorial, because not all
drug-resistant patients have P-glycoprotein-positive tumor cells
and only a few patients appear to benefit from the use of current
chemosensitizers.
[0006] Chemosensitization research has centered on agents that
reverse or modulate multidrug resistance in solid tumors by
modulating the activity of the MDR extrusion pumps.
Chemosensitizers known to modulate the function of MDR extrusion
pumps, e.g., P-glycoprotein, include: calcium channel blockers
(Verapamil, indicated for the treatment of hypertension),
calmodulin inhibitors (trifluoperazine), indole alkaloids
(reserpine), quinolines (quinine), lysosomotropic agents
(chloroquine), steroids, (progesterone), triparanol analogs
(tamoxifen), detergents (cremophor EL), and cyclic peptide
antibiotics (cyclosporines, indicated to prevent host vs. graft
disease) (DeVita, V. T., et al., in Cancer, Principles &
Practice of Oncology, 4th ed., J. B. Lippincott Co., Philadelphia,
Pa., pp 2661-2664, 1993; Sonneveld P, Wiemer E. Inhibitors of
multidrug resistance., Curr Opin Oncol 1997
November;9(6):543-8).
[0007] A review of studies where chemosensitizing agents were used
concluded the following: (i) cardiovascular side effects associated
with continuous, high-dose intravenous Verapamil therapy are
significant and dose-limiting; (ii) dose-limiting toxicities of the
chemosensitizers, trifluoperazine and tamoxifen, was attributed to
the inherent toxicity of the chemosensitizer and not due to
enhanced chemotherapy toxicity; (iii) studies using high doses of
Cyclosporin A as a chemosensitizer found hyperbilirubinemia as a
side effect; and (iv) further research is clearly needed to develop
less toxic and more efficacious chemosensitizers to be used
clinically (DeVita et al., 1993, ibid.).
[0008] For example, while Verapamil is effective in hypertension
treatment at the 2-4 .mu.M range, for MDR reversal it requires the
dose range of 10-15 .mu.M, while at 6 .mu.M it is already in the
toxic domain.
[0009] Tumors that are considered drug-sensitive at diagnosis but
acquire an MDR phenotype at relapse, pose an especially difficult
clinical problem. At diagnosis, only a minority of tumor cells may
express proteins such as P-glycoprotein, which act as extrusion
pumps and treatment with chemotherapy provides a selection
advantage for the few cells that are, for example, P-glycoprotein
positive early in the course of disease. Another possibility is
that natural-product-derived chemotherapy actually induces the
expression of MDR1, leading to P-glycoprotein-positive tumors or
other MDR pump-positive tumors at relapse. Using chemosensitizers
early in the course of disease may prevent the emergence of MDR by
eliminating the few cells that are MDR-pump positive at the
beginning. In vitro studies have shown that selection of
drug-resistant cells by combining Verapamil and Doxorubicin does
prevent the emergence of P-glycoprotein, but that an alternative
drug resistance mechanism develops, which is secondary to altered
topoisomerase II function (Dalton, W. S., Proc. Am. Assoc. Cancer
Res. 31:520, 1990).
[0010] More efficacious and less toxic chemosensitizers are
urgently needed to improve the outcome of chemotherapy. Clinical
utility of a chemosensitizer depends upon its ability to enhance
the cytotoxicity of a chemotherapeutic drug and also on its low
toxicity in vivo. The present inventors have addressed these
problems and provide herein a new class of chemosensitizers that
permit new approaches in cancer treatment.
[0011] 3-Aryloxy-3-phenylpropylamines and their use to treat
depression are described in, for example, U.S. Pat. Nos. 4,018,895
and 6,258,853. Fluoxetine [(N-methyl
3-(p-trifluoromethylphenoxy)-3-phenylpropylamine], known better by
its commercial name Prozac, is a well-known approved drug,
indicated for psychiatric treatments (Cookson J, Duffett R.,
Fluoxetine: therapeutic and undesirable effects. Hosp Med 1998
August;59(8):622-6). It is known to be an SSRI (Selective Serotonin
Reuptake Inhibition) agent, and this activity is considered to be
related to its mechanism of action in its capacity as a psychiatric
drug (Cookson et al., 1998, ibid.).
[0012] WO 94/18961, WO 92/11035, and U.S. Pat. Nos. 5,798,339 and
5,859,065, which are incorporated by reference as if fully set
forth herein, disclose methods of treating cancer using histamine
antagonists followed by chemotherapy. Specifically, the methods
described in these documents are directed at increasing the
cytotoxicity and inhibiting the adverse side effects of a
chemotherapeutic agent used in chemotherapy, and are effected by
administering the chemotherapeutic agent following the
administration of a histamine antagonist. WO 94/18961 teaches in
this respect that histamine antagonists inhibit normal cell
proliferation, while promoting malignant cell proliferation and
further teaches DPPE analogs as preferred compounds that act as
histamine antagonists that affect cell proliferation as described.
WO 94/18961 recites fluoxetine amongst other psychiatric agents
which can also act as histamine antagonists, but further states
that this group of compounds, at their effective histamine
antagonizing concentration, cause adverse side effects, such as
cardiac arrhythmia.
[0013] In the experiments described in WO 94/18961, doses
equivalent to 20-40 mg/M.sup.2 of fluoxetine were employed. These
experiments show that at this dose range, fluoxetine promotes the
proliferation of fibrosarcoma cells and inhibits the proliferation
of concavalin A-stimulated normal lymphocytes. It will be
appreciated in this regard that the known, acceptable safety limit
of fluoxetine is 80 mg/M.sup.2, while the safe, substantially side
effect-free, daily dose range of fluoxetine is below 10-15
mg/M.sup.2.
[0014] According to WO 94/18961 and WO 92/11035, the histamine
antagonists are administered prior to the administration of the
chemotherapeutic agent. WO 92/11035 clearly indicates that the
antagonist compound is administered about 15 to about 90 minutes,
preferably, about 30 to about 60 minutes, prior to the
administration of the chemotherapeutic agent, in order to permit
the antagonist to inhibit the binding of intracellular histamine to
its receptor in normal cells and thereby, in effect, inhibit the
proliferation of normal cells and hence provide chemoprotection to
such cells.
[0015] Although WO 94/18961 teaches the use of fluoxetine in a
method of treating cancer, as a compound which is administered in
combination with a chemotherapeutic drug, WO 94/18961 does not
teach the use of fluoxetine as a chemosensitizer used for enhancing
the cytotoxic effect of a chemotherapeutic agent in the treatment
of multidrug resistance cancer cells.
[0016] Rather, WO 94/18961 teaches that fluoxetine acts as a
compound that inhibits proliferation of normal cells while
promoting the proliferation of cancer cells, when administered
prior to the chemotherapeutic drug, at a dose which causes adverse
side effects. Both WO 94/18961 and WO 92/11035 do not address the
issue of multidrug resistance cancer cells and fail to indicate the
use of the methods disclosed therein for treating multidrug
resistance cancer. In fact, the methods taught in these
publications, employ, as is described in the Examples section
thereof, cancer cells that are known to be susceptible to
chemotherapeutic treatment, such as S-10 and fibrosarcoma cells.
Furthermore, U.S. Pat. Nos. 5,798,339 and 5,859,065, which
correspond to these international publications, specifically recite
methods of treating cancer cells which are susceptible to
chemotherapy treatment. Based on the mechanism of action of
histamine antagonists disclosed in these publications, one would be
reluctant from administering fluoxetine at its histamine
antagonizing dose to a cancer patient not only because of its
associated side effects, but also because it is said and shown to
enhance the proliferation of cancer cells at these
concentrations.
[0017] Hence, 3-Aryloxy-3-phenylpropylamines in general and
fluoxetine in particular have not hitherto been indicated as
chemosensitizers for the treatment of multidrug resistance
cancer.
SUMMARY OF THE INVENTION
[0018] While reducing the present invention to practice it was
unexpectedly found that fluoxetine, a member of the
3-aryloxy-3-phenylpropylamines family of compounds, induces a
significant enhancement of the cytotoxic effect of conventional
chemotherapeutic drugs, acting via totally different cytotoxic
mechanisms, at a dose range well below fluoxetine's toxicity limits
and further well below fluoxetine's side effect-free limit. Such an
enhancement of the cytotoxic effect of chemotherapeutic drugs is
particularly advantageous in the treatment of multidrug resistance
cancer cells.
[0019] Hence, the present invention provides methods,
pharmaceutical compositions and kits for chemosensitization using a
3-aryloxy-3-phenylpropylamine as a chemosensitizing agent.
[0020] As used herein, the term "chemosensitization" means an
increase or an enhancement of the measured cytotoxicity of a
chemotherapeutic agent on multidrug resistance cells in the
presence of a chemosensitizing agent, as is compared to the level
of cytotoxicity exerted by the chemotherapeutic agent in the
absence of the chemosensitizing agent.
[0021] As shown herein, 3-aryloxy-3-phenylpropylamines act as
chemosensitizing agents, rendering inherent and acquired multidrug
resistant cancer cells more sensitive to chemotherapy.
[0022] Hence, in one aspect, the present invention provides a
method of treating a subject suspected of having, or having, a
multidrug resistance (MDR) cancer. The method comprises
administering to the subject a chemotherapeutically effective
amount of a chemotherapeutic agent and a chemosensitizing effective
amount of a 3-aryloxy-3-phenylpropylamine. The cancer may be
leukemia, lymphoma, carcinoma or sarcoma.
[0023] In a preferred embodiment, the chemotherapeutic agent and
the 3-aryloxy-3-phenylpropylamine are administered substantially at
the same time.
[0024] In another preferred embodiment, the chemosensitizing dose
of the 3-aryloxy-3-phenylpropylamine is within its safety range,
and moreover, it is within its side effect-free range, so as to
avoid adverse side effects. Preferably the range used is between
about 0.1 mg/M.sup.2 and about 10 mg/M.sup.2.
[0025] As used herein, the term about indicates .+-.20%.
[0026] The phrases "side effect-free range" and "side effect-free
limit" indicate a dose range and a maximal dose, respectively, that
are within the safety limit and are further below the minimal dose
that substantially induces adverse side effects. In other words,
these phrases indicate safe, substantially free of side effects,
range and limit, respectively. As is demonstrated hereinabove,
fluoxetine, for example, induces side effects even at
concentrations that are within its safety limit. As the safety
limit of fluoxetine is a daily dose of 80 mg/M.sup.2, fluoxetine
induces side effects such as cardiac arrhythmia at a dose range of
20-40 mg/M.sup.2. Hence, the side effect-free range of fluoxetine
includes daily doses that are below its side effect-free limit,
namely, below 15 mg/M.sup.2, preferably, below 10 mg/M.sup.2.
[0027] In another aspect of the invention,
3-aryloxy-3-phenylpropylamines may be used as topical
chemosensitizers. For example, Table 2 below indicates that
5-fluorouracil is used topically in the treatment of premalignant
skin lesions. The inventors of the present invention envision the
use of 3-aryloxy-3-phenylpropylamines to enhance the cytotoxicity
of chemotherapeutic agents formulated applicable for topical
administration.
[0028] A method for selecting a chemotherapeutic agent for which
3-aryloxy-3-phenylpropylamine acts as a chemosensitizer is a
further aspect of the present invention. The method comprises (i)
assaying cytotoxicity of a candidate chemotherapeutic agent in the
presence and in the absence and optionally at different
concentrations of a 3-aryloxy-3-phenylpropylamine; and (ii)
selecting a candidate chemotherapeutic agent as a chemotherapeutic
agent for which 3-aryloxy-3-phenylpropylamine is a chemosensitizer
when the cytotoxicity of the candidate agent is greater in the
presence of 3-aryloxy-3-phenylpropylamine than in the absence of
3-aryloxy-3-phenylpropylamine. A presently preferred in vitro assay
is the MTT cytotoxicity assay which is described in the examples
section. An exemplary in vivo assay is described in, for example,
U.S. Pat. No. 5,776,925, which is incorporated herein by
reference.
[0029] Preferably, the method according to this aspect of the
present invention is performed with inherent or acquired multidrug
resistant cells, with a dose of 3-aryloxy-3-phenylpropylamine that
ranges between about 1 .mu.M and about 15 .mu.M, preferably, about
1 .mu.M and about 12 .mu.M and/or while administering the
3-aryloxy-3-phenylpropylamine and the candidate chemotherapeutic
agent substantially at the same time.
[0030] According to further aspects of the present invention, there
are provided pharmaceutical compositions and pharmaceutical
kits.
[0031] In one embodiment, the pharmaceutical composition of the
invention comprises a 3-aryloxy-3-phenylpropylamine as a
chemosensitizing agent and a chemotherapeutic agent.
[0032] The pharmaceutical composition is preferably packaged in a
packaging material and is identified in print in or on the
packaging material for use in the treatment of inherent and
acquired multidrug resistance cancer.
[0033] In another embodiment, the pharmaceutical composition of the
invention comprises a 3-aryloxy-3-phenylpropylamine and the
pharmaceutical composition is packaged in a packaging material and
is identified in print in or on the packaging material for use in
chemosensitization.
[0034] The pharmaceutical kit of the present invention comprises a
3-aryloxy-3-phenylpropylamine as a chemosensitizing agent and a
chemotherapeutic agent, which are individually packaged in the
kit.
[0035] A 3-aryloxy-3-phenylpropylamine used as a chemosensitizer in
accordance with the teachings of the present invention is
preferably of the formula: 1
[0036] wherein each R' is independently hydrogen or methyl;
[0037] R is naphthyl or 2
[0038] R" and R'" are halo, trifluoromethyl, C.sub.1-C.sub.4 alkyl,
C.sub.1-C.sub.3 alkoxy or C.sub.3-C.sub.4 alkenyl; and
[0039] n and m are 0, 1 or 2; and acid addition salts thereof
formed with pharmaceutically acceptable acids.
[0040] In the above formula when R is naphthyl, it can be either
alpha-naphthyl or beta-naphthyl. R" and R'" when they are halo,
C.sub.1-C.sub.4 alkyl, C.sub.1-C.sub.3 alkyloxy or C.sub.3-C.sub.4
alkenyl represent, illustratively, the following atoms or groups:
fluoro, chloro, bromo, iodo, methyl, ethyl, isopropyl, n-propyl,
n-butyl, isobutyl, sec-butyl, t-butyl, methoxy, ethoxy, n-propoxy,
isopropoxy, allyl, methallyl, crotyl and the like. R thus can
represent o, m and p-trifluoromethylphenyl, o, m and
p-chlorophenyl, o, m and p-bromophenyl, o, m and p-fluorophenyl, o,
m and p-tolyl, xylyl including all position isomers, o, m and
p-anisyl, o, m and p-allylphenyl, o, m and p-methylallylphenyl, o,
m and p-phenetolyl(ethoxyphenyl), 2,4-dichlorophenyl,
3,5-difluorophenyl, 2-methoxy-4chlorophenyl,
2-methyl-4-chlorophenyl, 2-ethyl-4-bromophenyl,
2,4,6-trimethylphenyl, 2-fluoro-4-trifluoromethylphenyl,
2,4,6-trichlorophenyl, 2,4,5-trichlorophenyl and the like.
[0041] Also included within the scope of the present invention are
the pharmaceutically acceptable salts of the amine bases
represented by the above formula formed with non-toxic acids. These
acid addition salts include salts derived from inorganic acids such
as: hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid,
hydrobromic acid, hydriodic acid, nitrous acid, phosphorous acid
and the like, as well as salts of non-toxic organic acids including
aliphatic mono and dicarboxylates, phenyl-substituted alkanoates,
hydroxy alkanoates and alkanedioates, aromatic acids, aliphatic and
aromatic sulfonic acids etc. Such pharmaceutically-acceptable salts
thus include: sulfate, pyrosulfate, bisulfate, sulfite, bisulfite,
nitrate, phosphate, monohydrogenphosphate, dihydrogenphosphate,
metaphosphate, pyrophosphate, chloride, bromide, iodide,
fluorodide, acetate, propionate, decanoate, caprylate, acrylate,
formate, isobutyrate, caprate, heptanoate, propiolate, oxalate,
malonate, succinate, suberate, sebacate, fumarate, maleate,
butyne-1,4-dioate, hexyne-1,6-dioate, benzoate, chlorobenzoate,
methylbenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate,
phthalate, terephthalate, benzenesulfonates, toluenesulfonate,
chlorobenzenesulfonate, xylenesulfonate, phenylacetate,
phenylpropionate, phenylbutyrate, citrate, lactate,
beta-hydroxybutyrate, glycollate, malate, tartrate,
methanesulfonate, propanesulfonates, naphthalene-1-sulfonate,
naphthalene-2-sulfonate, mandelate and the like salts.
[0042] Compounds illustrative of the scope of this invention
include the following:
[0043] 3-(p-isopropoxyphenxoy)-3-phenylpropylamine
methanesulfonate;
[0044] N,N-dimethyl 3-(3',4'-dimethoxyphenoxy)-3-phenylpropylamine
p-hydroxybenzoate;
[0045] N,N-dimethyl 3-(alpha-naphthoxy)-3-phenylpropylamine
bromide;
[0046] N,N-dimethyl 3-(beta-naphthoxy)-3-phenyl-1-methylpropylamine
iodide;
[0047] 3-(2'-methyl-4',5'-dichlorophenoxy)-3-phenylpropylamine
nitrate;
[0048] 3-(p-t-butylphenoxy)-3-phenylpropylamine glutarate;
[0049] N-methyl
3-(2'-chloro-p-tolyloxy)-3-phenyl-1-methylpropylamine lactate;
[0050] 3-(2',4'-dichlorophenoxy)-3-phenyl-2-methylpropylamine
citrate;
[0051] N,N-dimethyl 3-(m-anisyloxy)-3-phenyl-1-methylpropylamine
maleate;
[0052] N-methyl 3-(p-tolyloxy)-3-phenylpropylamine sulfate;
[0053] N,N-dimethyl 3-(2',4'-difluorophenoxy)-3-phenylpropylamine
2,4-dinitrobenzoate;
[0054] 3-(o-ethylphenoxy)-3-phenylpropylamine dihydrogen
phosphate;
[0055]
N-methyl-(2'-chloro-4'-isopropylphenoxy)-3-phenyl-2-methylpropylami-
ne maleate;
[0056] N,N-dimethyl
3-(2'-alkyl-4'-fluorophenoxy)-3-phenylpropylamine succinate;
[0057] N,N-dimethyl 3-(o-isopropoxyphenoxy)-3-phenyl-propylamine
phenylacetate;
[0058] N,N-dimethyl 3-(o-)bromophenoxy)-3-phenyl-propyl amine
beta-phenylpropionate;
[0059] N-methyl 3-(p-iodophenoxy)-3-phenyl-propylamine
propiolate;
[0060] N-methyl 3-(3-n-propylphenoxy)-3-phenyl-propylamine
decanoate; and preferably,
[0061] N-methyl
3-(p-trifluoromethylphenoxy)-3-phenylpropylamine.
[0062] The present invention successfully addresses the
shortcomings of the presently known configurations by identifying
new chemosensitizers which efficiently act at concentrations well
below their toxicity and which are of particular efficacy in
chemosensitizing multi drug resistant (MDR) cancer cells.
BRIEF DESCRIPTION OF THE DRAWINGS
[0063] The invention is herein described, by way of example only,
with reference to the accompanying drawings. With specific
reference now to the drawings in detail, it is stressed that the
particulars shown are by way of example and for purposes of
illustrative discussion of the preferred embodiments of the present
invention only, and are presented in the cause of providing what is
believed to be the most useful and readily understood description
of the principles and conceptual aspects of the invention. In this
regard, no attempt is made to show structural details of the
invention in more detail than is necessary for a fundamental
understanding of the invention, the description taken with the
drawings making apparent to those skilled in the art how the
several forms of the invention may be embodied in practice.
[0064] In the drawings:
[0065] FIG. 1 is a bar graph demonstrating the increase in death of
C6 cells and, separately, PANC-1 cells (% from untreated control)
as a function of treatment media, at 24 hours post administration.
CS--15 .mu.M fluoxetine alone, CT--0.1 .mu.g/ml Doxorubicin alone,
CT+CS--combination of the two. Each bar is an average of 32-64
repeats, and the error bars represent the standard deviations. The
star (*) indicates statistical significance P<0.001 (two-tails
student t-test) compared to the treatment with the chemotherapeutic
drug alone.
[0066] FIG. 2 is a bar graph demonstrating the increase in death of
C6 cells and, separately, PANC-1 cells (% from untreated control)
as a function of treatment media, at 24 hours post administration.
CS--15 .mu.M fluoxetine alone, CT--30 .mu.g/ml Mitomycin C alone,
CT+CS--combination of the two. Each bar is an average of 32-64
repeats, and the error bars represent the standard deviations. The
star (*) indicates statistical significance P<0.001 (two-tails
student t-test) compared to the treatment with the chemotherapeutic
drug alone.
[0067] FIG. 3 is a dose response curve for the effects of
fluoxetine on the survival of PANC-1 cells treated with 0.1
.mu.g/ml Doxorubicin or, separately, 0.3 .mu.g/ml Vinblastine, 48
hours post administration. The points are experimental, each an
average of 32-64 repeats (sd levels which are similar to those in
FIGS. 1 and 2, are not shown in order to reduce symbol crowding).
The solid curves are non-theoretical, drawn to emphasize the trends
in the data.
[0068] FIG. 4 is a bar graph demonstrating LD.sub.50 doses of
fluoxetine effect in potentiating tumor treatment by the
chemotherapeutic drugs Doxorubicin and, separately, Vinblastine,
for five different cell lines. Data was taken from analysis of dose
response curves similar to those shown in FIG. 3, for each of the
cell lines, obtained under the same drug species, drug dose and
treatment period listed in the legend to FIG. 3.
[0069] FIGS. 5a-b are bar graphs demonstrating the effect of
various concentrations (1-20 .mu.M) of fluxetine on the survival of
P388/WT (denoted WT) non-resistant cell line (FIG. 5a) and P388/ADR
(denoted ADR) DOX-resistant cell line. Each bar is an average of 64
wells and the standard deviation (sd) is expressed by the error
bar.
[0070] FIGS. 6a-b are bar graphs demonstrating the effect of
various concentrations (0.5, 1 and 5 .mu.g/ml) of doxorubicin
(denoted as DOX) with and without 5 .mu.M fluoxetine on the cells
death of non-resistant cell line P388/WT (FIG. 6a) and the superior
chemosensitizing effect of fluoxetine, as compared with Verapamil
and Cyclosporin A (denoted as CsA), on MDR-acquired cells
(P388/ADR) treated with various concentrations (1, 5 and 10
.mu.g/ml) of DOX (FIG. 6b). Each bar is an average of 64 wells and
the standard deviation (sd) is expressed by the error bar.
[0071] FIG. 7 is a scheme illustrating the MDR mechanism. The
chemotherapeutic drug, denoted CT, usually gains entry into the
cell by self-diffusion, this influx driven by the electrochemical
gradient of the drug across the cell membrane. The intracellular
drug concentration is reduced below lethal threshold, by
ATP-dependant extrusion through the MDR pumps embedded in the cell
membrane.
[0072] FIG. 8 demonstrates the efflux of intracellular doxorubicin
(DOX) from C6 cells, under unidirectional flux conditions. The
efflux is expressed as f(t), the cumulative quantity of DOX that
diffused out of the cells at time t, normalized to the total
intracellular DOX concentration at time=0. The points are the
experimental data, open squares--for cells loaded with 0.1 .mu.g/ml
DOX and open circles for cells loaded with 0.1 .mu.g/ml DOX
together with 15 .mu.M fluoxetine. The solid curves are
non-theoretical, drawn to emphasize the trends in the data.
[0073] FIG. 9 demonstrates the efflux of intracellular doxorubicin
(DOX) from P388/ADR cells, under unidirectional flux conditions.
The efflux is expressed as the cumulative quantity of DOX that
diffused out of the cells at time=t, normalized to the total
intracellular DOX concentration at time=0. The points are the
experimental data, each an average of ten determinations with the
sd represented by the error bars, filled squares--for cells loaded
with 0.1 .mu.g/ml DOX, open squares--for cells loaded with 0.1
.mu.g/ml DOX together with 5 .mu.M Verapamil, open triangles--for
cells loaded with 0.1 .mu.g/ml DOX together with 5 .mu.M
Cyclosporin A (CsA) and open circles for cells loaded with 0.1
.mu.g/ml DOX together with 5 .mu.M fluoxetine. The solid curves are
non-theoretical, drawn to emphasize the trends in the data.
[0074] FIG. 10 is a bar graph demonstrating the
chemosensitizer-induced intracellular accumulation of Rhodamine-123
in C-26 cells, expressed in Rhodamine-123 intracellular
fluorescence. Each bar is an average of three independent
experiments and the standard deviation (sd) is expressed by the
error bar.
[0075] FIG. 11 is a bar graph demonstrating the
chemosensitizer-induced intracellular accumulation of Rhodamine-123
in P388/ADR cells, expressed in Rhodamine-123 intracellular
fluorescence. Each bar is an average of three independent
experiments and the standard deviation (sd) is expressed by the
error bar.
[0076] FIGS. 12a-c present confocal microscopy images demonstrating
the intracellular accumulation of Rhodamine-123 in monolayers of
C-26 cells incubated with 5 .mu.M Rhodamine-123 alone (FIG. 12a), 5
.mu.M Rhodamine-123 and 15 .mu.M Verapamil (FIG. 12b) and 5 .mu.M
Rhodamine-123 and 15 .mu.M fluoxetine (FIG. 12c).
[0077] FIGS. 13a-b present confocal microscopy images demonstrating
the intracellular accumulation of Doxorubicin in monolayers of
P388/ADR cells incubated with 5 .mu.g/ml Doxorubicin alone (FIG.
13a) and 5 .mu.g/ml Doxorubicin and 5 .mu.M fluoxetine (FIG.
13b).
[0078] FIG. 14 presents comparative plots demonstrating an increase
in a solid tumor volume with time, in each of the tested groups in
in vivo studies of a solid tumor model. Each point in the plots
represents an experimental measurement and is an average of 5
animals. The error bars represent the SEM and the curves are
non-theoretical, indicating the trends in the data. The results
presented in the left-hand side plots were obtained with Mitomycin
C (MMC) as the chemotherapeutic drug and the results presented in
the right-hand side plots were obtained with Doxorubicin (DOX) as
the chemotherapeutic drug.
[0079] FIG. 15 presents the survival data in each of the tested
groups in the solid tumor model of FIG. 14.
[0080] FIGS. 16a-b are bar graphs demonstrating the increase in
lung weight (FIG. 16a) and the number of tumor metastasis (FIG.
16b) in different mice groups injected with B16F10 cells. Each bar
is an average of all the animals in the group and the sd is
represented by the error bars.
[0081] FIG. 17 presents the survival data obtained in each of the
tested groups in the lung metathesis model of FIGS. 16a-b.
[0082] FIGS. 18a-b present comparative plots demonstrating the
change in body weight with time, in each of the tested groups in
the acquired MDR model: open squares--saline; open
circles--doxorubicin; open triangles--fluoxetine; and filled
circles--a combination of doxorubicin and fluoxetine. FIG. 18a
presents the data obtained in mice transplanted with non-resistant
P388/WT and FIG. 18b presents the data obtained in mice
transplanted with MDR-acquired P388/ADR. Each point in the plots
represents an experimental measurement and is an average of 5
animals. The error bars represent the standard deviation and the
curves are non-theoretical, indicating the trends in the data.
[0083] FIGS. 19a-b presents the survival data in each of the tested
groups described in FIG. 18. FIG. 19a presents the data obtained in
mice transplanted with non-resistant P388/WT and FIG. 19b presents
the data obtained in mice transplanted with MDR-acquired P388/ADR.
Each line in the plots connects the symbols representing the daily
survival state of the group, the symbols themselves were omitted in
order to avoid cluttered drawings.
[0084] FIG. 20 presents plots demonstrating the effect of
fluoxetine on the pharmacokinetics of doxorubicin (DOX) in mice
bearing B16F10.9 lung tumors. Each point in the plots represents an
experimental measurement and is an average of 10 animals. The error
bars represent the standard deviations and the curves are
non-theoretical, indicating the trends in the data.
[0085] FIG. 21 presents bar graphs demonstrating the effect of
fluoxetine on the biodistribution of doxorubicin (DOX) in mice
bearing B16F10.9 lung tumors. Each bar is an average of 5 animals
and the error bars represent the standard deviations.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0086] The present invention is of methods and pharmaceutical
compositions which can be used in chemosensitization. Specifically,
the present invention can be used to render cancer cells and, in
particular inherent and/or acquired multidrug resistant cancer
cells, more sensitive to chemotherapeutic agents, hence increase
the cytotoxic effect of such agents on cells.
[0087] The principles and operation of the methods and
pharmaceutical compositions according to the present invention may
be better understood with reference to the drawings and
accompanying descriptions.
[0088] Before explaining at least one embodiment of the invention
in detail, it is to be understood that the invention is not limited
in its application to the details set forth in the following
description or exemplified by the Examples. The invention is
capable of other embodiments or of being practiced or carried out
in various ways. Also, it is to be understood that the phraseology
and terminology employed herein is for the purpose of description
and should not be regarded as limiting.
[0089] The present invention results from the discovery that
3-aryloxy-3-phenylpropylamines act as efficient chemosensitizers on
multidrug resistance cells at non-toxic concentrations.
Chemosensitization using a 3-aryloxy-3-phenylpropylamine refers to
an enhancement of cytotoxicity on the part of a chemotherapeutic
agent when that agent is administered to multidrug resistance cells
in conjunction with administering a
3-aryloxy-3-phenylpropylamine.
[0090] Hence, the invention relates to a novel treatment for
effecting tumor (both solid and non-solid) chemotherapy, based on
the combination of at least one chemotherapeutic drug that are used
in, for example, standard therapy protocols in the clinic such as,
but not limited to, Doxorubicin, Vinblastine and Mitomycin C; and
at least one 3-aryloxy-3-phenylpropylamine, preferably fluoxetine
(also known as Prozac), a drug approved and widely used for
psychiatric situations such as depression.
[0091] It is shown herein that a combined treatment of at least one
chemotherapeutic drug and at least one
3-aryloxy-3-phenylpropylamine leads to significant increases in
efficacy of the cytotoxic drugs, up to 5-fold for a single dose,
and at doses well below safety limits and side effect-free limits
known for 3-aryloxy-3-phenylpropylamines. Moreover, the novel
treatment is especially effective in tumors that are resistant to
the chemotherapeutic drugs, i.e., inherent and acquired multidrug
resistance tumors.
[0092] It will be appreciated in this respect that other multidrug
resistance reversal agents, such as Verapamil and Cyclosporin A,
are not used in the clinic due to their toxicity at the required
dose levels.
[0093] In addition, 3-aryloxy-3-phenylpropylamines, such as
fluoxetine, can be administered orally, which is easier on the
patient, and its presently indicated effect of mood improvement
will also be beneficial to the cancer patient.
[0094] According to the present invention the chemotherapeutic
agent may be, for example, one of the following: an alkylating
agent such as a nitrogen mustard, an ethylenimine and a
methylmelamine, an alkyl sulfonate, a nitrosourea, and a triazene;
an antimetabolite such as a folic acid analog, a pyrimidine analog,
and a purine analog; a natural product such as a vinca alkaloid, an
epipodophyllotoxin, an antibiotic, an enzyme, a taxane, and a
biological response modifier; miscellaneous agents such as a
platinum coordination complex, an anthracenedione, an
anthracycline, a substituted urea, a methyl hydrazine derivative,
or an adrenocortical suppressant; or a hormone or an antagonist
such as an adrenocorticosteroid, a progestin, an estrogen, an
antiestrogen, an androgen, an antiandrogen, or a
gonadotropin-releasing hormone analog. Specific examples of
alkylating agents, antimetabolites, natural products, miscellaneous
agents, hormones and antagonists, and the types of cancer for which
these classes of chemotherapeutic agents are indicated are provided
in Table 2. Preferably, the chemotherapeutic agent is a nitrogen
mustard, an epipodophyllotoxin, an antibiotic, or a platinum
coordination complex. A more preferred chemotherapeutic agent is
Bleomycin, Vinblastine, Doxorubicin, Paclitaxel, etoposide, 4-OH
cyclophosphamide, or cisplatinum.
[0095] Presently preferred chemotherapeutic agents are Doxorubicin,
Mitomycin C and/or Vinblastine, which are the chemotherapeutic
drugs employed in the in vitro and/or the in vivo experiments
described in the Examples section that follows, yet the use of
other chemotherapeutic drugs in context of the present invention is
also applicable.
[0096] As is well known in the art, Vinblastine, Mitomycin C and
Doxorubicin are cytotoxic drugs toward which many tumors exhibit
drug resistance and therefore serve as representative examples for
the chemosensitization effect of 3-aryloxy-3-phenylpropylamine.
[0097] Furthermore, as is delineated in Table 1 below, these drugs
differ from one other by their chemical structure, mechanism of
action and the location of their cellular targets. For example,
Vinblastine acts in the cytosol, via depolymerization of
microtubules, Mitomycin C acts in the nucleus via DNA alkylation
and Doxorubicin acts in both the cytosol and the nucleus, as well
as in the cell membrane, having different effects in each of these
locations.
1TABLE 1 Intracellular location of drug Cytotoxic drug target
Mechanisms of action Vinblastine The cytosol Depolymerization of
Microtubules Mitomycin C The nucleus DNA alkylation Doxorubicin The
nucleus DNA intercalation, blocking synthesis of DNA and RNA The
cytosol DNA strands scission by affecting topoisomerase II The cell
Altering membrane fluidity and ion membrane transport Generation of
semiquinone free radicals and oxygen radicals
[0098] Hence, as these drugs act via different pathways, the
present inventors envision that 3-aryloxy-3-phenylpropylamines may
be used as chemosensitizers for enhancing the cytotoxicity of a
variety of chemotherapeutic agents having different mechanisms of
action.
[0099] A listing of currently available chemotherapeutic agents
according to class, and including diseases for which the agents are
presently indicated, is provided as Table 2 below. Each of these
exemplary chemotherapeutic agents can be used in the context of the
present invention.
2TABLE 2 Chemotherapeutic Agents Useful in Neoplastic Disease.sup.1
Class Type of Agent Name Disease.sup.2 Alkylating Nitrogen
Mechlorethamine Hodgkin's disease, non-Hodgkin's Agents Mustards
(HN.sub.2) lymphomas Cyclophosphamide Acute and chronic lymphocytic
Ifosfamide leukemias, Hodgkin's disease, non-Hodgkin's lymphomas,
multiple myeloma, neuroblastoma, breast, ovary, lung, Wilms' tumor,
cervix, testis, soft-tissue sarcomas lphalan Multiple myeloma,
breast, ovary lorambucil Chronic lymphocytic leukemia, primary
macroglobulinemia, Hodgkin's disease, non- Hodgkin 's lymphomas
Estramustine Prostate Ethylenimines Hexamethyl- Ovary and melamine
Methylmelamines Thiotepa Bladder, breast, ovary Alkyl Busulfan
Chronic granulocytic leukemia Sulfonates Nitrosoureas Carmustine
Hodgkin's disease, non-Hodgkin's lymphomas, primary brain tumors,
multiple myeloma, malignant melanoma Lomustine Hodgkin's disease,
non-Hodgkin's lymphomas, primary brain tumors, small-cell lung
Semustine Primary brain tumors, stomach, colon Streptozocin
Malignant pancreatic insulinoma, malignant carcinoid Triazenes
Dacarbazine Malignant melanoma, Hodgkin's Procarbazine disease,
soft-tissue sarcomas Aziridine Antimetabolites Folic Acid
Methotrexate lymphocytic leukemia, Analogs Trimetrexate
choriocarcinoma, mycosis fungoides, breast, head and neck, lung,
osteogenic sarcoma Pyrimidine Fluorouracil Breast, colon, stomach,
pancreas, Analogs Floxuridine ovary, head and neck, urinary
bladder, premalignant skin lesions (topical) Cytarabine Acute
granulocytic and acute Purine Analogs Azacitidine lymphocytic
leukemias and Related Mercaptopurine lymphocytic, acute Inhibitors
granulocytic, and chronic granulocytic leukemias Thioguanine Acute
granulocytic, acute lymphocytic, and chronic granulocytic leukemias
Pentostatin Hairy cell leukemia, mycosis fungoides, chronic
lymphocytic leukemia Fludarabine Chronic lymphocytic leukemia,
Hodgkin's and non-Hodgkin's lymphomas, mycosis fungoides Natural
Vinca Alkaloids Vinblastine (VLB) Hodgkin's disease, non-Hodgkin's
Products lymphomas, breast, testis Vincristine Acute lymphocytic
leukemia, neuroblastoma, Wilms' tumor, rhabdomyosarcoma, Hodgkin's
disease, non-Hodgkin's lymphomas, small-cell lung Vindesine
Vinca-resistant acute lymphocytic leukemia, chronic myolocytic
leukemia, melanoma, lymphomas, breast Epipodophyl- Etoposide
Testis, small-cell lung and other Lotoxins Teniposide lung, breast,
Hodgkin's disease, non-Hodgkin's lymphomas, acute granulocytic
leukemia, Kaposi's sarcoma Antibiotics Dactinomycin
Choriocarcinoma, Wilms' tumor, rhabdomyosarcoma, testis, Kaposi's
sarcoma Daunorubicin Acute granulocytic and acute lymphocytic
leukemias Doxorubicin Soft-tissue, osteogenic, and 4'- other
sarcomas; Hodgkin's Deoxydoxorubicin disease, non-Hodgkin's
lymphomas, acute leukemias, breast, genitourinary, thyroid, lung,
stomach, neuroblastoma Bleomycin Testis, head and neck, skin,
esophagus, lung, and genitourinary tract; Hodgkin's disease, non-
Hodgkin's lymphomas Plicamycin Testis, malignant hypercalcemia
Mitomycin Stomach, cervix, colon, breast, pancreas, bladder, head
and neck Enzymes Asparaginase Acute lymphocytic leukemia Taxanes
Docetaxel Breast, ovarian Paclitaxel Biological Interferon Alfa
Hairy cell leukemia, Kaposi's Response sarcoma, melanoma,
carcinoid, Modifiers cell, ovary, bladder, non-Hodgkin's lymphomas,
mycosis fungoides, multiple myeloma, chronic granulocytic leukemia
Tumor Necrosis Investigational Factor Tumor- Investigational
Infiltrating Lymphocytes Miscellaneous Platinum Cisplatin Testis,
ovary, bladder, head and Agents Coordination Carboplatin neck,
lung, thyroid, cervix, Complexes endometrium, neuroblastoma,
osteogenic sarcoma Anthracenedione Mitoxantrone Acute granulocytic
leukemia, breast Substituted Hydroxyurea Chronic granulocytic
leukemia, Urea polycythemia vera, essential thrombocytosis,
malignant melanoma Methyl Procarbazine Hodgkin's disease Hydrazine
Derivative Adrenocortical Mitotane Adrenal cortex Suppressant
Aminoglutethimide Breast Hormones and Acute and chronic lymphocytic
Antagonists costeroids leukemias, non-Hodgkin's lymphomas,
Hodgkin's disease, breast Progestins Hydroxy- Endometrium, breast
progesterone caproate Medroxy- progesterone acetate Megestrol
acetate Estrogens Diethyistil- Breast, prostate bestrol Ethinyl
estradiol Antiestrogen Tamoxifen Androgens tosterone propionate
Fluoxymesterone Antiandrogen Flutamide Prostate Gonadotropin-
Leuprolide Prostate, Estrogen-receptor- Releasing Goserelin
positive breast hormone analog .sup.1Adapted from Calabresi, P.,
and B. A. Chabner, "Chemotherapy of Neoplastic Diseases" Section
XII, pp 1202-1263 in: Goodman and Gilman's The Pharmacological
Basis of Therapeutics, Eighth ed., 1990 Pergamin Press, Inc.; and
Barrows, L. R., "Antineoplastic and Immunoactive Drugs", Chapter
75, pp 1236-1262, in: Remington: The Science and Practice of
Pharmacy, Mack Publishing Co. Easton, PA, 1995.; both references
are incorporated by reference herein, #in particular for treatment
protocols. .sup.2Neoplasms are carcinomas unless otherwise
indicated.
[0100] 3-aryloxy-3-phenylpropylamine compounds, methods for making
same and methods for using them are described in U.S. Pat. Nos.
4,018,895, 4,314,081, 5,166,437 and 6,258,853, which are
incorporated by reference herein, and further below.
[0101] 3-Aryloxy-3-phenylpropylamines used as chemosensitizers may
be administered before, together with or after administration of
the chemotherapeutic agent. Preferably, the
3-aryloxy-3-phenylpropylamine and the chemotherapeutic agent are
administered substantially at the same time.
[0102] The administration of a chemosensitizer and a
chemotherapeutic agent substantially at the same time is a highly
important and advantageous feature in the treatment of multidrug
resistance (MDR) cells.
[0103] As is discussed in detail hereinabove, MDR is effected via
an extrusion mechanism, which involves energy-dependant extrusion
channels or pumps that actively pump the drug out of the cells,
thereby reducing its intracellular concentration below lethal
threshold. As is further discussed hereinabove, chemosensitizers in
this respect are agents that reverse or modulate multidrug
resistance in MDR cells by modulating the activity of the MDR
extrusion pumps. It is therefore advantageous that the
chemosensitizing agent and the chemotherapeutic agent would be
administered substantially at the same time, in order to allow
their combined action by their dual presence in the treated
cell.
[0104] Hence, the phrase "substantially at the same time", as used
herein, means that the 3-aryloxy-3-phenylpropylamine and the
chemotherapeutic agent are administered in such time intervals that
would allow their dual presence in effective concentrations in the
treated cells. The 3-aryloxy-3-phenylpropylamine and the
chemotherapeutic agent can be administered by different or
identical routes of administration.
[0105] The 3-aryloxy-3-phenylpropylamine may be administered as a
single dose, or it may be administered as two or more doses
separated by a time interval. Where the
3-aryloxy-3-phenylpropylamine is administered as two or more doses,
the time interval between the 3-aryloxy-3-phenylpropylamin- e
administrations may be from about one minute to about 12 hours,
preferably from about 5 minutes to about 5 hours, more preferably
about 4 to 5 hours. The dosing protocol may be repeated; from one
to three times, for example. Administration may be intravenous,
intraperitoneal, parenteral, intramuscular, subcutaneous, oral, or
topical, with oral and intravenous administration being preferred,
and intravenous administration being presently most preferred.
[0106] The 3-aryloxy-3-phenylpropylamine used in the method of the
invention administered in a chemosensitizing effective amount.
[0107] As used herein the phrase "chemosensitizing effective
amount" means that daily dose which results in an enhanced toxicity
by a chemotherapeutic agent, without adverse side effects. The
specific daily dose will vary depending on the particular
3-aryloxy-3-phenylpropylamine used, the dosing regimen to be
followed, and the particular chemotherapeutic agent with which it
is administered. Such a daily dose can be determined without undue
experimentation by methods known in the art or as described
herein.
[0108] As is exemplified in the Examples section that follows, a
presently preferred chemosensitizing effective amount, according to
the present invention, ranges between 0.05 mg/M.sup.2 and 20
mg/M.sup.2, preferably between 0.1 mg/M.sup.2 and 10 mg/M.sup.2,
more preferably between 0.1 mg/M.sup.2 and 7 mg/M.sup.2, more
preferably between 0.1 mg/M.sup.2 and 5 mg/M.sup.2 and most
preferably between 0.4 mg/M.sup.2 and 4 mg/M.sup.2.
[0109] The chemosensitizing effect of fluoxetine as a
representative example of a 3-aryloxy-3-phenylpropylamine, was
tested in vitro by administering fluoxetine, in combination with
different chemotherapeutic drugs, at doses that range between 2
.mu.M and 15 .mu.M, administered every two days. These in vitro
doses correspond to human daily doses that range between about 0.45
mg/M.sup.2 and about 3.5 mg/M.sup.2, respectively.
[0110] The conversion of these in vitro doses to in vivo human
daily doses is performed by first converting the .mu.M fluoxetine
concentrations to units of mg/ml, and thereafter estimating the
corresponding human daily dose in mg/Kg body weight, taking into
account that an average human weight is about 70 Kg, an average
human height is about 1.75 m and that the human blood volume is 5
liters, and further taking into account that the in vitro treatment
in the experiments conducted included a single dose every two days.
The obtained results are then converted into mg/M.sup.2 surface
area units, assuming that an individual weighting 70 Kg and of a
height of 1.75 m has 1.85 M.sup.2 skin surface.
[0111] In the in vivo studies, which are also described in detail
in the Examples section that follows, the daily doses of fluoxetine
were about 0.04 mg/kg body, which correspond to 2.8 mg per 70 Kg
body or to 1.5 mg/M.sup.2, according to the conversion index
described hereinabove.
[0112] The chemosensitizing effective amount of fluoxetine as a
representative 3-aryloxy-3-phenylpropylamine, as is demonstrated by
the in vitro and in vivo studies described herein, is therefore
well below fluoxetine doses that are used in its classical,
psychiatric indications, and are well below both its safety limit
and its side effect-free limit. As is further shown in these in
vitro and in vivo studies, at this low dose, fluoxetine has no
anticancer effect and no adverse side effects.
[0113] The fact that fluoxetine exerts chemosensitizing effect in
the treatment of multidrug resistance cancer at such low doses is
novel and highly advantageous.
[0114] As is discussed in detail hereinabove, a method that
involves administration of fluoxetine for treating cancer is
described in WO 94/19861.
[0115] The fluoxetine is used in this method as a compound that
inhibits proliferation of normal cells, while promoting the
proliferation of cancer cells, via histamine receptor antagonism
mechanism. The fluxetine dose required to achieve these effects,
according to the teachings of WO 94/19861, is 20-40 mg/M.sup.2,
which is about 10-20 fold higher than the dose used in context of
the present invention and about 2 fold higher than the side
effect-free limit determined for fluxetine. As is further indicated
in WO 94/18961, at this dose range, fluoxetine promotes the
proliferation of cancer cells, and is further accompanied by
adverse side effects such as cardiac arrhythmia. Also,
administration of fluoxetine according to the teachings of WO
94/18961 should precede the administration of a chemotherapeutic
agent in order to be effective. In sharp contrast, the
administration of fluoxetine and the chemotherapeutic agent,
according to the present invention, should be substantially at the
same time, for reasons set forth hereinabove.
[0116] Hence, the present invention provides a method of treating
cancer that is superior over to the presently known methods, as it
involves the administration of the chemosensitizing agent at low
concentrations, within its safety and side effect-free limits, and
therefore does not results in adverse side effects.
[0117] A 3-aryloxy-3-phenylpropylamine for use as a chemosensitizer
according to the teachings of the present invention may have
structure I: 3
[0118] wherein each R' is independently hydrogen or methyl;
[0119] R is naphthyl or 4
[0120] R" and R'" are halo, trifluoromethyl, C.sub.1-C.sub.4 alkyl,
C.sub.1-C.sub.3 alkoxy or C.sub.3-C.sub.4 alkenyl; and
[0121] n and m are 0, 1 or 2; and acid addition salts thereof
formed with pharmaceutically acceptable acids.
[0122] In the above formula when R is naphthyl, it can be either
alpha-naphthyl or beta-naphthyl. R" and R'" when they are halo,
C.sub.1-C.sub.4 alkyl, C.sub.1-C.sub.3 alkyloxy or C.sub.3-C.sub.4
alkenyl represent, illustratively, the following atoms or groups:
fluoro, chloro, bromo, iodo, methyl, ethyl, isopropyl, n-propyl,
n-butyl, isobutyl, sec-butyl, t-butyl, methoxy, ethoxy, n-propoxy,
isopropoxy, allyl, methallyl, crotyl and the like. R thus can
represent o, m and p-trifluoromethylphenyl, o, m and
p-chlorophenyl, o, m and p-bromophenyl, o, m and p-fluorophenyl, o,
m and p-tolyl, xylyl including all position isomers, o, m and
p-anisyl, o, m and p-allylphenyl, o, m and p-methylallylphenyl, o,
m and p-phenetolyl(ethoxyphenyl), 2,4-dichlorophenyl,
3,5-difluorophenyl, 2-methoxy-4chlorophenyl,
2-methyl-4-chlorophenyl, 2-ethyl-4-bromophenyl,
2,4,6-trimethylphenyl, 2-fluoro-4-trifluoromethylphenyl,
2,4,6-trichlorophenyl, 2,4,5-trichlorophenyl and the like.
[0123] Also included within the scope of this invention are the
pharmaceutically acceptable salts of the amine bases represented by
the above formula formed with non-toxic acids. These acid addition
salts include salts derived from inorganic acids such as:
hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid,
hydrobromic acid, hydriodic acid, nitrous acid, phosphorous acid
and the like, as well as salts of non-toxic organic acids including
aliphatic mono and dicarboxylates, phenyl-substituted alkanoates,
hydroxy alkanoates and alkanedioates, aromatic acids, aliphatic and
aromatic sulfonic acids etc. Such pharmaceutically-acceptable salts
thus include: sulfate, pyrosulfate, bisulfate, sulfite, bisulfite,
nitrate, phosphate, monohydrogenphosphate, dihydrogenphosphate,
metaphosphate, pyrophosphate, chloride, bromide, iodide,
fluorodide, acetate, propionate, decanoate, caprylate, acrylate,
formate, isobutyrate, caprate, heptanoate, propiolate, oxalate,
malonate, succinate, suberate, sebacate, fumarate, maleate,
butyne-1,4-dioate, hexyne-1,6-dioate, benzoate, chlorobenzoate,
methylbenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate,
phthalate, terephthalate, benzenesulfonates, toluenesulfonate,
chlorobenzenesulfonate, xylenesulfonate, phenylacetate,
phenylpropionate, phenylbutyrate, citrate, lactate,
beta-hydroxybutyrate, glycollate, malate, tartrate,
methanesulfonate, propanesulfonates, naphthalene-1-sulfonate,
naphthalene-2-sulfonate, mandelate and the like salts.
[0124] Compounds illustrative of the scope of this invention
include the following:
[0125] 3-(p-isopropoxyphenxoy)-3-phenylpropylamine
methanesulfonate;
[0126] N,N-dimethyl 3-(3',4'-dimethoxyphenoxy)-3-phenylpropylamine
p-hydroxybenzoate;
[0127] N,N-dimethyl 3-(alpha-naphthoxy)-3-phenylpropylamine
bromide;
[0128] N,N-dimethyl 3-(beta-naphthoxy)-3-phenyl-1-methylpropylamine
iodide;
[0129] 3-(2'-methyl-4',5'-dichlorophenoxy)-3-phenylpropylamine
nitrate;
[0130] 3-(p-t-butylphenoxy)-3-phenylpropylamine glutarate;
[0131] N-methyl
3-(2'-chloro-p-tolyloxy)-3-phenyl-1-methylpropylamine lactate;
[0132] 3-(2',4'-dichlorophenoxy)-3-phenyl-2-methylpropylamine
citrate;
[0133] N,N-dimethyl 3-(m-anisyloxy)-3-phenyl-1-methylpropylamine
maleate;
[0134] N-methyl 3-(p-tolyloxy)-3-phenylpropylamine sulfate;
[0135] N,N-dimethyl 3-(2',4'-difluorophenoxy)-3-phenylpropylamine
2,4-dinitrobenzoate;
[0136] 3-(o-ethylphenoxy)-3-phenylpropylamine dihydrogen
phosphate;
[0137]
N-methyl-(2'-chloro-4'-isopropylphenoxy)-3-phenyl-2-methylpropylami-
ne maleate;
[0138] N,N-dimethyl
3-(2'-alkyl-4'-fluorophenoxy)-3-phenylpropylamine succinate;
[0139] N,N-dimethyl 3-(o-isopropoxyphenoxy)-3-phenyl-propylamine
phenylacetate;
[0140] N,N-dimethyl 3-(o-)bromophenoxy)-3-phenyl-propylamine
beta-phenylpropionate;
[0141] N-methyl 3-(p-iodophenoxy)-3-phenyl-propylamine
propiolate;
[0142] N-methyl 3-(3-n-propylphenoxy)-3-phenyl-propylamine
decanoate; and preferably,
[0143] N-methyl
3-(p-trifluoromethylphenoxy)-3-phenylpropylamine.
[0144] The 3-aryloxy-3-phenylpropylamines of this invention in the
form of their free bases are high boiling oils, but white
crystalline solids in the form of their acid addition salts. The
compounds can be prepared in several ways. A particularly useful
procedure for preparing compounds represented by the above formula
(in which both R' groups attached to the nitrogen are methyl)
involves the reduction of beta-dimethylaminopropioph- enone
produced by a Mannich reaction to yield N,N-dimethyl
3-phenyl-3-hydroxypropylamine. Replacement of the hydroxyl group
with a halogen, such as chlorine, yields the corresponding
N,N-dimethyl 3-phenyl-3-chloropropylamine. Reaction of this chloro
compound with a suitably substituted phenol, as for example
o-methoxyphenol (guiacol), produces a compound of this invention in
which both R' groups are methyl. Treatment of the N,N-dimethyl
compound with cyanogenbromide serves to replace one N-methyl group
with a cyano group. Hydrolysis of the resulting compound with base
yields a compound of this invention in which only one R' group on
the nitrogen is methyl. For example, treatment of N,N-dimethyl
3-(o-anisyloxy)-3-phenylpropylamine with cyanogen bromide followed
by alkaline hydrolysis of the N-cyano compound yields directly
N-methyl 3-(o-anisyloxy)-3-phenylpropylamine [N-methyl 3-(o-methoxy
phenoxy)-3-phenylpropylamine].
[0145] An alternate preparation of the compounds of this invention
in which only one of the R' groups attached to the nitrogen is
methyl is carried as follows:
[0146] 3-Chloropropylbenzene is reacted with a positive
halogenating agent such N-bromosuccinimide to yield the
corresponding 3-chloro-1-bromopropylbenzene. Selective replacement
of the bromo atom with the sodium salt of a phenol, as for example,
the sodium salt of o-methoxyphenol (guiacol) yields a
3-chloro-1-(1-methoxyphenoxy)-propylbe- nzene [also named as
3-chloro-1-(o-anisyloxy)propylbenzene]. Reaction of the 3-chloro
derivative thus produced with methylamine yields the desired
N-methyl 3-(o-anisyloxy)-3-phenylpropylamine.
[0147] 3-Aryloxy-3-phenylpropylamine compounds in which both R'
groups attached to the nitrogen in the above formula are hydrogen
can be prepared from an intermediate produced in the previous
preparation of the N-methyl compounds such as, for illustrative
purposes, 3-chloro-1-(o-anisyloxy)-propylbenzene prepared by the
reaction of 3-chloro-1-bromobenzene and sodium guiacol. This chloro
compound is reacted with sodium azide to give the corresponding
3-azido-1-(o-anisyloxy)-propylbenzene. Reduction of the azide group
with a metallo-organic reducing agent such as sodium borohydride
yields the desired primary amine. Alternatively, the chloro
compound can be reacted directly with a large excess of ammonia in
a high pressure reactor to give the primary amine.
[0148] 3-Aryloxy-3-phenylpropylamine compounds in which the R'
group on the carbon atom alpha to the nitrogen is methyl can be
prepared by reacting phenyl 2'-propenyl ketone with dimethylamine
[See J. Am. Chem. Soc., 75, 4460 (1953)]. The resulting
3-dimethylaminobutyrophenone is reduced to yield the N,N-dimethyl
3-hydroxy-1-methyl-3-phenylpropylamine. Replacement of the hydroxyl
with chlorine followed by reaction of the chloro-compound with the
sodium salt of a suitably substituted phenol yields the
N,N-dimethyl derivatives of this invention bearing an alpha methyl
group on the propylamine backbone of the molecule. Production of
the corresponding N-methyl derivative can be accomplished by the
aforementioned reaction sequence utilizing cyanogen bromide. The
N-methyl derivative can in turn be converted to the corresponding
primary amine (in which both R' groups on the nitrogen are
hydrogen) by oxidation in neutral permanganate according to the
procedure of Booher and Pohland, Ser. No. 317,969, filed Dec. 26,
1972. Compounds in which the R' group attached to the beta-carbon
atom is methyl are prepared by a Mannich reaction involving
propiophenone, formaldehyde and dimethylamine. The resulting
ketone, an alpha-methyl-beta-dimethylaminopropiophenone, is
subjected to the same reduction procedure as before to yield a
hydroxy compound. Replacement of the hydroxyl with chlorine
followed by reaction of the chloro compound with the sodium salt of
a phenol yields a dimethyl amine compound of this invention.
Conversion of the dimethylamine to the corresponding monomethyl and
primary amines is carried out as before.
[0149] Those 3-aryloxy-3-phenylpropylamine compounds in which the
R' group attached to either the alpha or beta-carbon is methyl have
two asymmetric carbon atoms, the carbon carrying the R' methyl and
the gamma.-carbon carrying the phenoxy and phenyl groups. Thus,
such compounds exist in four diastereomeric forms occurring as two
racemic pairs, the less soluble pair being designated alpha-dl form
and the more soluble the beta-dl form. Each racemate can be
resolved into its individual d and l isomers by methods well known
in the art, particularly, by forming salts with optically active
acids and separating the salts by crystallization.
[0150] A 3-aryloxy-3-phenylpropylamine may be coupled to a
site-directing molecule to form a conjugate for targeted in vivo
delivery. "Site-directing" means having specificity for targeted
sites. "Specificity for targeted sites" means that upon contacting
the 3-aryloxy-3-phenylpropylamine-site-directing-conjugate with the
targeted site, for example, under physiological conditions of ionic
strength, temperature, pH and the like, specific binding will
occur. The interaction may occur due to specific electrostatic,
hydrophobic, entropic or other interaction of certain residues of
the conjugate with specific residues of the target to form a stable
complex under conditions effective to promote the interaction.
Exemplary site-directing molecules contemplated in the present
invention include but are not limited to: oligonucleotides,
polyamides including peptides having affinity for a biological
receptor and proteins such as antibodies; steroids and steroid
derivatives; hormones such as estradiol, or histamine; hormone
mimics such as morphine; and further macrocycles such as sapphyrins
and rubyrins.
[0151] As used herein, a "site-directing molecule" may be an
oligonucleotide, an antibody, a hormone, a peptide having affinity
for a biological receptor, a sapphyrin molecule, and the like. A
preferred site-directing molecule is a hormone, such as estradiol,
estrogen, progesterone, and the like. A site-directing molecule may
have binding specificity for localization to a treatment site and a
biological receptor may be localized to a treatment site. A
3-aryloxy-3-phenylpropyl- amine oligonucleotide-conjugate, where
the oligonucleotide is complementary to an oncogenic messenger RNA,
for example, would further localize chemotherapeutic activity to a
particularly desired site. Antisense technology is discussed in
U.S. Pat. Nos. 5,194,428, 5,110,802 and 5,216,141, all of which are
incorporated by reference herein.
[0152] A couple may be described as a linker, i.e., the covalent
product formed by reaction of a reactive group designed to attach
covalently another molecule at a distance from the
3-aryloxy-3-phenylpropylamine macrocycle. Exemplary linkers or
couples are amides, amine, thiol, thioether, ether, or phosphate
covalent bonds. In most preferred embodiments, site-directing
molecules are covalently bonded to the
3-aryloxy-3-phenylpropylamine via a carbon-nitrogen, carbon-sulfur,
or a carbon-oxygen bond.
[0153] Generally, water soluble 3-aryloxy-3-phenylpropylamines
retaining lipophilicity are preferred for the applications
described herein. "Water soluble" means soluble in aqueous fluids
to about 1 mM or better. "Retaining lipophilicity" means having
greater affinity for lipid rich tissues or materials than
surrounding nonlipid rich tissues. "Lipid rich" means having a
greater amount of triglyceride, cholesterol, fatty acids or the
like.
[0154] Representative examples of useful steroids include any of
the steroid hormones of the following five categories: progestins
(e.g. progesterone), glucocorticoids (e.g., cortisol),
mineralocorticoids (e.g., aldosterone), androgens (e.g.,
testosterone) and estrogens (e.g., estradiol).
[0155] Representative examples of useful amino acids of peptides or
polypeptides include amino acids with simple aliphatic side chains
(e.g., glycine, alanine, valine, leucine, and isoleucine), amino
acids with aromatic side chains (e.g., phenylalanine, tryptophan,
tyrosine, and histidine), amino acids with oxygen and
sulfur-containing side chains (e.g., serine, threonine, methionine,
and cysteine), amino acids with side chains containing carboxylic
acid or amide groups (e.g., aspartic acid, glutamic acid,
asparagine, and glutamine), and amino acids with side chains
containing strongly basic groups (e.g., lysine and arginine), and
proline. Representative examples of useful peptides include any of
both naturally occurring and synthetic di-, tri-, tetra-,
pentapeptides or longer peptides derived from any of the above
described amino acids (e.g., endorphin, enkephalin, epidermal
growth factor, poly-L-lysine, or a hormone). Representative
examples of useful polypeptides include both naturally occurring
and synthetic polypeptides (e.g., insulin, ribonuclease, and
endorphins) derived from the above described amino acids and
peptides.
[0156] The term "a peptide having affinity for a biological
receptor" means that upon contacting the peptide with the
biological receptor, for example, under appropriate conditions of
ionic strength, temperature, pH and the like, specific binding will
occur. The interaction may occur due to specific electrostatic,
hydrophobic, entropic or other interaction of certain amino acid or
glycolytic residues of the peptide with specific amino acid or
glycolytic residues of the receptor to form a stable complex under
the conditions effective to promote the interaction. The
interaction may alter the three-dimensional conformation and the
function or activity of either or both the peptide and the receptor
involved in the interaction. A peptide having affinity for a
biological receptor may include an endorphin, an enkephalin, a
growth factor, e.g. epidermal growth factor, poly-L-lysine, a
hormone, a peptide region of a protein and the like. A hormone may
be estradiol, for example.
[0157] For use as a chemosensitizer, 3-aryloxy-3-phenylpropylamines
are provided as pharmaceutical preparations. A pharmaceutical
preparation of a 3-aryloxy-3-phenylpropylamine may be administered
alone or in combination with pharmaceutically acceptable carriers,
in either single or multiple doses. Suitable pharmaceutical
carriers include inert solid diluents or fillers, sterile aqueous
solution and various organic solvents. The pharmaceutical
compositions formed by combining a 3-aryloxy-3-phenylpropylamine of
the present invention and the pharmaceutically acceptable carriers
are then easily administered in a variety of dosage forms such as
injectable solutions.
[0158] For parenteral administration, solutions of the
3-aryloxy-3-phenylpropylamine in sesame or peanut oil, aqueous
propylene glycol, or in sterile aqueous solution may be employed.
Such aqueous solutions should be suitably buffered if necessary and
the liquid diluent first rendered isotonic with sufficient saline
or glucose. These particular aqueous solutions are especially
suitable for intravenous, intramuscular, subcutaneous and
intraperitoneal administration. In this connection, sterile aqueous
media which can be employed will be known to those of skill in the
art in light of the present disclosure.
[0159] The pharmaceutical forms suitable for injectable use include
sterile aqueous solutions or dispersions and sterile powders for
the extemporaneous preparation of sterile injectable solutions or
dispersions. In all cases the form must be sterile and must be
fluid to the extent that easy use with a syringe exists. It must be
stable under the conditions of manufacture and storage and must be
preserved against the contaminating action of microorganisms, such
as bacteria and fungi. The carrier can be a solvent or dispersion
medium containing, for example, water, ethanol, polyol (for
example, glycerol, propylene glycol, and liquid polyethylene
glycol, and the like), suitable mixtures thereof, and vegetable
oils. The proper fluidity can be maintained, for example, by the
use of a coating, such as lecithin, by the maintenance of the
required particle size in the case of dispersion and by the use of
surfactants. The prevention of the action of microorganisms can be
brought about by various antibacterial and antifungal agents, for
example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal,
and the like. In many cases, it will be preferable to include
isotonic agents, for example, sugars such as mannitol or dextrose
or sodium chloride. A more preferable isotonic agent is a mannitol
solution of about 2-8% concentration, and, most preferably, of
about 5% concentration. 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.
[0160] Sterile injectable solutions are prepared by incorporating
the active compounds in the required amount in the appropriate
solvent with various of the other ingredients enumerated above, as
required, followed by filtered sterilization. Generally,
dispersions are prepared by incorporating the various sterilized
active ingredients into a sterile vehicle which contains the basic
dispersion medium and the required other ingredients from those
enumerated above. 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 a powder of the active ingredient plus any additional desired
ingredient from a previously sterile-filtered solution thereof.
[0161] As used herein, "pharmaceutically acceptable carrier"
includes any and all solvents, dispersion media, coatings,
antibacterial and antifungal agents, isotonic and absorption
delaying agents and the like. The use of such media and agents for
pharmaceutically active substances is well known in the art. Except
insofar as any conventional media or agent is incompatible with the
active ingredient, its use in the therapeutic compositions is
contemplated. Supplementary active ingredients can also be
incorporated into the compositions.
[0162] 3-Aryloxy-3-phenylpropylamines may be co-formulated with a
chemotherapeutic agent. Methods of co-formulating more than a
single active ingredient are well known in the art. Such
co-formulation ensures co-administration of the chemotherapeutic
agent and the 3-aryloxy-3-phenylpropylamine.
[0163] Hence pharmaceutical compositions that comprise a
3-aryloxy-3-phenylpropylamine, as a chemosensitizing agent, and a
chemotherapeutic agent, are provided in accordance with the present
invention. Such pharmaceutical compositions can be formulated as
described hereinabove.
[0164] The pharmaceutical compositions of the present invention may
be presented in a pack or dispenser device, such as a FDA approved
kit, which may contain one or more unit dosage forms containing the
active ingredients. The package may, for example, comprise metal or
plastic foil, such as a blister package. The package or dispenser
device may be accompanied by instructions for administration and
indication. The package or dispenser may also be accompanied by a
notice associated with the container in a form prescribed by a
governmental agency regulating the manufacture, use or sale of
pharmaceuticals, which notice is reflective of approval by the
agency of the form of the compositions or human or veterinary
administration. Such notice, for example, may be of labeling
approved by the U.S. Food and Drug Administration for prescription
drugs or of an approved product insert.
[0165] The pharmaceutical compositions of the present invention can
therefore be packaged in a packaging material and identified in
print in or on the packaging material for use in the treatment of a
multi drug resistance cancer.
[0166] The following examples are included to demonstrate preferred
embodiments of the invention. It should be appreciated by those of
skill in the art that the techniques disclosed in the examples
which follow represent techniques discovered by the inventor to
function well in the practice of the invention, and thus can be
considered to constitute preferred modes for its practice. However,
those of skill in the art should, in light of the present
disclosure, appreciate that many changes can be made in the
specific embodiments which are disclosed and still obtain a like or
similar result without departing from the spirit and scope of the
invention.
[0167] Hence, additional objects, advantages, and novel features of
the present invention will become apparent to one ordinarily
skilled in the art upon examination of the following examples,
which are not intended to be limiting. Additionally, each of the
various embodiments and aspects of the present invention as
delineated hereinabove and as claimed in the claims section below
finds experimental support in the following examples.
EXAMPLES
[0168] Reference is now made to the following examples, which
together with the above descriptions, illustrate the invention in a
non limiting fashion.
In Vitro Studies Materials and Methods
[0169] Chemotherapies (CT):
[0170] Mitomycin C (MMC), Vinblastine (VIN) and Doxorubicin
(DOX).
[0171] Chemosensitizers (CS):
[0172] Fluoxetine, Verapamil and Cyclosporin A.
[0173] Cell Lines:
[0174] MCF-7 (human breast carcinoma), HT1080 (human fibrosarcoma),
U2OS (human osteosarcoma), PANC-1 (human pancreatic
adenocarcinoma), C6 (rat glioblastoma), C26 (murine colon
adenocarcinoma), B16F10 (murine melanoma), P388/WT (murine
leukemia) and its MDR-acquired subline P388/ADR (obtained by
selective growth of P388/WT in the presence of Doxorubicin,
according to the procedure described in Johnson et al., Cancer
Treat. Rep., 62: 1535-1547, 1978.
[0175] Cell Culture Growth and Maintenance Media:
[0176] Dulbecco's modified Eagle's medium (DMEM) supplemented with
10% fetal calf serum (FCS), Penicillin (10,000 units/ml),
Streptomycin (10 mg/ml) and L-Glutamine (200 mM).
[0177] Cell Cultures:
[0178] Cells were grown in monolayers in 100.times.20 mm dishes, in
the growth media listed above, at 37.degree. C. in 5% CO.sub.2.
[0179] Cell Survival:
[0180] Cells were grown in monolayers as describe above and seeded
onto 96 multiwell plates at a density of 1.times.10.sup.4 cells/ml,
24 hours prior to an experiment. Twenty four hours later, the media
was replaced by treatment media as is detailed in the Experimental
Results section that follows. The experiments were terminated 24 or
48 hours post media replacement. The quantity of viable cells was
determined by the MTT test, recording the absorbencies in a plate
reader, at two wavelengths: 550 and 650 nm.
[0181] Drug Efflux Measurements:
[0182] Cells were grown in monolayers as described above. Several
days prior to an experiment the cells (at a density in the range of
5.times.10.sup.4-5.times.10.sup.5 cells/ml) were seeded into 24
multiwell culture plates. The experiments were performed when the
cells reached semi-confluency. The efflux experiments were
conducted according to the following protocol: Cells were loaded,
by incubation for 10 hours, with either a non-lethal dose of DOX,
or the same DOX dose combined with fluoxetine, Verapamil or
Cyclosporin A. Control cells received media alone. At the end of
the incubation, the media was removed, the cells washed with
phosphate-buffered saline (PBS), and thereafter incubated with
either efflux media (PBS) alone (for the wells incubated with DOX
only) or efflux media with fluoxetine at the same dose as in the 10
hours incubation. At selected time points, the medium from every
well was collected and replaced with a fresh similar medium. At the
end of the experiment the cells in each well were dissolved by
adding 5% Deoxycholate (DOC) to each well. Samples from the media
collected at each time point, and samples from the final
detergent-dissolved cells, were transferred to a 96 well plate
suitable for a fluorimeter plate reader. Excitation and emission
were at 480 nm and 530 nm, respectively. Calibration curves were
run with each assay using DOX standards dissolved in the
appropriate media (i.e., buffer or buffer/DOC).
[0183] Drug Uptake and Drug Efflux Measurements Using
Rhodamine-123:
[0184] Rhodamine-123 is a well-known substrate of Pgp, the first
MDR extrusion channel identified, and hence serves as a fluorescent
indicator for the presence and activity of a chemosensitizer. As
the accumulation of Rhodamine-123 in Pgp-containing multidrug
resistance cells is increased significantly in the presence of a
chemosensitizer, the effects of a molecule suspected to inhibit MDR
pumps on the intracellular accumulation of Rhodamine-123 has become
a classical test for chemosensitizers.
[0185] The effect of fluoxetine as a chemosensitizer was therefore
measured by fluorescence measurements upon incubating a cell line
with either a chemotherapeutic drug or Rhodamine-123 or with a
combination of fluoxetine and a chemotherapeutic drug or
Rhodamine-123. In the experiments conducted with Rhodamine-123 as a
model, the chemosensitizing effect of fluoxetine was compared to
the effect of the known chemosensitizer Verapamil and, in some
experiments, also to the known chemosensitizer Cyclosporin A.
[0186] The intracellular accumulation of the chemotherapeutic agent
or Rhodamine-123 was measured on C-26 cell line, which is known as
Pgp-containing, inherent MDR cell line, and on the MDR-acquired
cell line P388/ADR, in two different systems: In system I,
suspensions of cells were used and the accumulation was measured by
flow cytometry; In system II, adherent monolayers of cells were
used and the accumulation was measured by confocal microscopy,
according to the following protocols:
[0187] System I: Suspensions of cells in PBS (1.times.10.sup.6
cells/ml) were incubated for 30 minutes, at 37.degree. C., with 5
.mu.M Rhodamine-123 alone and in combination with fluoxetine, at
varying doses. For comparison, experiments were performed also with
Verapamil or Cyclosporin A as a chemosensitizer, at a typical dose
of 15 .mu.M. The cells were thereafter analyzed for intracellular
fluorescence, using a flow cytometer (Becton-Dickinson, USA).
Excitation and emission were at 485 nm and 547 nm,
respectively.
[0188] System II: Cells were grown as monolayers on cover slides,
and were thereafter incubated for one hour at 37.degree. C., with 5
.mu.g/ml Doxorubicin or 5 .mu.M Rhodamine-123, either alone or in
combination with a chemosensitizer as described hereinabove. At the
end of the incubation time, the cells were washed 6 times with PBS,
fixed with a mounting medium (Mounting medium with anti-fading
agents, Biomeda corp., CA, USA) and were analysed by Confocal
microscopy (Zeiss LSM 510). Excitation was at 488 nm and emission
was measured using a band pass filter of 505-550 nm.
Experimental Results
[0189] Testing the Response of Inherent MDR Cell Lines to a Single
Fluoxetine Dose:
[0190] C6 and, in separate experiments, PANC-1 cells were seeded
onto multiwell (96) culture plates, and the experiments were
initiated when the cells reached semi-confluency. The
serum-supplemented cell growth media was replaced by a treatment
media, selected from: (i) the combination of a chemotherapeutic
drug and fluoxetine dissolved in serum supplemented growth media;
(ii) chemotherapeutic drug dissolved in serum supplemented growth
media; (iii) fluoxetine dissolved in serum supplemented growth
media; and (iv) serum supplemented growth media alone (untreated
control). Drug species and dose were the same in (i) and (ii).
Fluoxetine dose was the same in (i) and (iii). The experiment was
terminated 24 hours later, and the number of viable cells was
quantitated using the MTT method.
[0191] Typical results, showing the effects of the various
treatment groups on cell death are shown in FIGS. 1 and 2, for both
cell lines and for the chemotherapeutic drugs Mitomycin C (MMC) and
Doxorubicin (DOX), respectively. The fluoxetine dose used in these
experiments matches the highest safe dose used for accepted
indications of fluoxetine. The data clearly shows that fluoxetine
alone does not affect cell survival at all. At the drug doses
applied (listed in FIGS. 1 and 2), treatment with drug alone was
only mildly effective in causing cell death, at its best no more
than 20%. In contrast, for the four cases studied (2 drugs, 2 cell
lines), the combination treatment caused a significant enhancement
in cell death, which was 3-4 fold, clearly showing the
effectiveness of the combination treatment.
[0192] These experiments were similarly performed with B16F10 and
C-26 cell lines, which are known as drug resistance cells.
Mitomycin C and Doxorubicin were applied, at typical doses of 50
.mu.g/ml and 1.0 .mu.g/ml, respectively, with and without 15 .mu.M
of fluoxetine. The treatment with the drug alone generated cell
death in the range of 10-15%, thus confirming the
inherent-resistant nature of these cell lines. Treatment with a
combination of the drug and fluoxetine increased the cell death to
about of 80-90%, thus demonstrating the increased cell demise
caused by the chemosensitizer.
[0193] Evaluating Fluoxetine Dose Response in Inherent MDR Cell
Lines:
[0194] Studies similar in general to those outlined in the previous
section, were conducted with five cell lines selected for this task
(PANC-1, C6, MCF-7, U20S and HT1080), increasing the length of the
experiment to 48 hours. The studies were performed with DOX and
with Vinblastine (VIN or VLB). The treatment groups were similar to
those listed in the previous sections, with the following
additions: a series of fluoxetine doses were tested, alone and in
combination with the cytotoxic drugs, covering a fluoxetine range
of 0-15 .mu.M.
[0195] As expected from the testing with 15 .mu.M, fluoxetine alone
was not toxic to the cells. For the drug species and respective
doses tested--0.1 .mu.g/ml DOX and 0.3 .mu.g/ml VIN--drugs alone
caused 50% and 10-20% reductions in cell survival, for the
non-resistant and resistant cell lines, respectively. Normalizing,
for each cell line, the survival of cells receiving the
chemotherapeutic drug and fluoxetine, to the survival of the cells
receiving the chemotherapeutic drug alone (i.e., zero fluoxetine)
it was possible to construct fluoxetine dose response curves. A
typical example is shown in FIG. 3, for the PANC-1 cell line, with
both drugs. From such dose response curves, using computer-aided
polynomial curve fitting, it was possible to determine for each
drug and each cell line, an LD.sub.50 for the fluoxetine
potentiation effect. These LD.sub.50 values, for all five cell
lines, each with both drugs, are shown in FIG. 4.
[0196] Comparative Studies on the Response of a Non-Resistant Cell
Line and an MDR-Acquired Cell Line to Fluoxetine:
[0197] Evaluating the response of non-resistant and MDR-acquired
cells to fluoxetine alone: The effect of various concentrations
(1-20 .mu.M) of fluoxetine on the non-resistant P388/WT cell line
and its MDR-acquired subline P388/ADR was tested by incubating the
cells with increasing concentrations of fluoxetine for 4 hours and
thereafter washing and re-incubating the cells with CS-free media
for additional 20 hours. The number of viable cells was quantitated
using the MTT method.
[0198] The obtained data, presented in FIGS. 5a and 5b, clearly
indicate that at the tested concentrations range, 1-20 .mu.M,
fluoxetine has no effect on cells viability in both non-resistant
cells (FIG. 5a) and MDR-Acquired cells (FIG. 5b).
[0199] Testing the Response of MDR-Acquired Cell Lines to a Single
Fluoxetine Dose:
[0200] P388/WT cells were seeded onto multi-well (96) culture
plates, and the experiments were initiated when the cells reached
semi-confluency. The serum-supplemented cell growth media was
replaced by a treatment media, selected from: (i) various
concentrations (0.5, 1 and 5 .mu.g/ml) of a chemotherapeutic drug
(DOX) dissolved in serum supplemented growth media; (ii) a
combination of a chemotherapeutic drug (1, 5 or 10 .mu.g/ml DOX)
and a chemosensitizing agent selected from fluoxetine, Verapamil,
and Cyclosporin A (5 .mu.M) dissolved in serum supplemented growth
media; and (iii) serum supplemented growth media alone (untreated
control). After 4 hours incubation, the treatment media was
replaced with CS- and CT-free media and the experiment was
terminated 24 hours later. The number of viable cells was
quantitated using the MTT method.
[0201] As is shown in FIG. 6a, treatment with 0.5 .mu.g/ml DOX was
sufficient to generate about 50% cell death in the non-resistant
cell line P388/WT, while 100% cell death was observed in these
cells upon increasing the DOX concentration to 5 .mu.g/ml. As
expected for non-resistant cells, a combination treatment of a
chemotherapeutic agent and fluoxetine had no effect on the level of
cells death in this cell line.
[0202] Contrary to the data obtained with the non-resistant cell
line, a well-demonstrated chemosensitizing effect of fluoxetine was
observed with the MDR-acquired cell line P388/ADR. As is shown in
FIG. 6b, treatment with 5 .mu.g/ml DOX, which was enough to kill
all the cells in the non-resistant cell line, generated less then
20% cell death, while doubling the drug concentration increased the
cell death only to about 30%, thus confirming the acquired
resistance of this cell line. While a combined treatment of DOX and
5 .mu.M Verapamil or Cyclosporin A (CsA) generated a modest
increase in cell death, treatment with a combination of DOX and
fluoxetine at the same concentration resulted in significant
increase (about 2-3 fold as compared with Verapamil and CsA) in
cell death, thus demonstrating the enhanced chemosensitizing effect
of fluoxetine on MDR-acquired cells. Several features of these
results are worthy of attention:
[0203] First, in all cases fluoxetine potentiates the cytotoxic
effect of the chemotherapeutic drug.
[0204] Second, the LD.sub.50 range, which spans from 7-10 .mu.M
fluoxetine and 6.5-8 .mu.M fluoxetine, for DOX and VIN,
respectively, is well below the highest safety limit of 15 .mu.M
fluoxetine. This is completely different than the cases of
Verapamil and Cyclosporin, where the dose range for
chemosensitization was well above their safety limit and hence
impractical for clinical applications.
[0205] Third, taking into consideration that in the resistant cell
lines the potentiation has to work on double the number of cells
than in the non-resistant lines, yet the LD.sub.50 range is quite
similar--these data imply that the potentiation effect is more
significant in the MDR lines.
[0206] Fourth, in the non-resistant lines, the effect of fluoxetine
on a given line is not drug-sensitive while in the resistant lines,
fluoxetine is more potent (lower LD.sub.50) with VIN than with
DOX.
[0207] Insights and Results with Respect to the Operating
Mechanisms:
[0208] Without an intention to limit the present invention in any
way, the data presented herein allows speculating some mechanistic
insights with respect to the chemosensitization activity of
3-aryloxy-3-phenylpropylami- ne in general and fluoxetine in
particular.
[0209] The finding that fluoxetine potentiates the cytotoxicity
with different drugs, that have furthermore different killing
mechanisms, rules out a drug-specific effect. Could fluoxetine be
triggering a cell death mechanism that it totally independent of
the presence of the chemotherapeutic drug in the cell? It is
suggest this triggering option is unlikely in view of the finding
that fluoxetine alone is not toxic to the cells--at the same dose
level where it exerts its effect in the presence of a cytotoxic
drug. Since the sites of action for the chemotherapeutic drugs are
intracellular it is reasonable to assume that fluoxetine exerts its
effect(s) inside the cell, also.
[0210] In general, nature has not planned for the introduction of
foreign matter such as drugs, into living biological systems. Hence
nature has made no specific efforts to assist drug entry into
cells. That drugs do gain entry into cells is a fact of life. Drugs
do it by at least two pathways that are not mutually exclusive: (i)
by diffusion across the cell membrane, driven by the drug's
electrochemical-potential gradient; and (ii) by "borrowing a ride"
on natural transport systems designed (by nature) to transport
molecules that are a normal component of a living system. Obviously
both pathways can operate in both directions, namely influx and
efflux. In addition, the interaction of the foreign entity with
biological transport systems can take the form of blockage, where a
foreign matter blocks the passage of other materials through the
transporter.
[0211] The data presented herein reveal that fluoxetine acts on
both MDR and non-multidrug resistance cells, but is more effective
with the former type. This raises at least two possibilities for
fluoxetine's mechanism(s) of action:
[0212] First: Fluoxetine inhibits extrusion channels that pump
chemotherapeutic drugs out of the cells, reducing the intracellular
drug doses below the lethal threshold. The fact that both MDR and
non-multidrug resistance cells have been affected, but to different
extent, fits with the extrusion pumps being natural proteins that
can exist in all cells, but in significantly larger numbers (copies
per cell) in multidrug resistance cells.
[0213] Second: Fluoxetine has two different activities: One is pump
inhibition as above especially (and possibly only) in the multidrug
resistance cells. The other is enhancement of the cellular response
to the chemotherapeutic drug, without any change in the
intracellular drug level. The latter could operate in the
non-multidrug resistance cells alone, or in both types of
cells.
[0214] An experimental method to support or refute the first
mechanism, is the following:
[0215] Cells are loaded with non-lethal doses of a chemotherapeutic
drug alone, or drug and fluoxetine. Upon completion of loading the
extracellular fluid is replaced with buffer alone, and the efflux
of drug into the external media is monitored for several hours. If
fluoxetine inhibits efflux pumps, drug efflux in the systems
receiving the combined treatment should be slower than in those
receiving the drug alone. This expectation was met, as shown by the
following:
[0216] The effect of fluoxetine on DOX efflux from inherent MDR C6
cells and from acquired MDR P388/ADR cells was studied as detailed
under the Methods section above.
[0217] In the experiments conducted with C6 cells, the DOX and
fluoxetine loading doses were 0.1 .mu.g/ml and 15 .mu.M,
respectively, and the obtained data is presented in FIG. 8. The
cumulative quantity of DOX that diffused out of the cells at time=t
was normalized to the total intracellular concentration of DOX at
time=0, and is denoted f(t). The magnitudes of f(t) as function of
time are plotted in FIG. 8, for the cells that received DOX alone
and for the cells that received DOX with fluoxetine.
[0218] The data presented makes it clear that 2 hours suffice for
complete depletion of intracellular DOX from cells that were loaded
with DOX alone. In contrast, DOX efflux was significantly slower in
cells that received both DOX and fluoxetine. At 2 hours, loss of
intracellular DOX (in the combined treatment) was under 40%, and
complete depletion was 450% slower than in the absence of
fluoxetine. The pattern of DOX efflux from the cells loaded with
this drug alone fits dominance of a single pathway. Based on
previous experience, were the efflux seen for the DOX-alone systems
dominated by self diffusion of the drug through the lipid bilayer
membranes, at 2 hours f(t) would range from 10-30%. This clearly
indicates that the single efflux pathway, that provides 100%
depletion at 2 hours, can be assigned to an extrusion pump.
[0219] DOX efflux from cells that received the combined treatment
is at the least bi-phasic, which indicates that DOX diffuses out of
those cells by at least two pathways. The pattern of the fastest
pathway, which dominates efflux at the first 30 minutes and
accounts for .ltoreq.20% of the total depletion, is quite similar
to that of the DOX-alone case. The pattern of the remaining 80%
fits one or more additional, significantly slower, pathways. These
data imply a fluoxetine effect at the transport level, a major part
of which is reduction in the number of active pumps, with possible
minor effects of reduction in the rate constant of DOX efflux
through this pump.
[0220] In the experiments conducted with P388/ADR cells, the effect
of fluoxetine on drug efflux was compared with the effect of the
known chemosensitizers Verapamil and Cyclosporin A (CsA). The DOX
and chemosensitizer dose loadings were 1 .mu.g/ml and 5 .mu.M,
respectively, and the obtained data is presented in FIG. 9. The
cumulative quantity of DOX that diffused out of the cells at time=t
is presented as its percentage from the total intracellular
concentration of DOX at time=0.
[0221] As is shown in FIG. 9, in these cells one hour suffices for
complete depletion of intracellular DOX from cells that were loaded
with DOX alone. In contrast, and similarly to the results obtained
with C6 cells, DOX efflux was significantly slower in cells that
received both DOX and a chemosensitizer. However, the obtained data
clearly show that in cells treated with fluoxetine as a
chemosensitizer, a 11 fold increase of the time needed for complete
depletion of the intracellular DOX was observed as compared with
cells treated with DOX only, while an increase of only 3 and 5 fold
was observed in cells treated with Verapamil and CsA, respectively,
as chemosensitizers. These data indicate again the superior
chemosensitizing effect of fluoxetine as compared with presently
known chemosensitizers. Support for these data was found in the
drug uptake measurements performed with either a chemotherapeutic
agent or with Rhodamine-123 as an indicator for the effect of a
molecule on MDR extrusion pumps. As is detailed hereinabove in the
methods section, the chemosensitizing effects of fluxetine and
Verapamil on the intracellular level of doxorubicin or
Rhodamine-123 were measured in two different systems.
[0222] As is shown in FIG. 10, in the experiments performed in
suspended C-26 cells, Verapamil, at the standard dose (15 .mu.M),
generated a minor increase of 23% of the intracellular
fluorescence, as compared to the control, chemosensitizing-free
cells, while fluoxetine, at the same dose generated an increase of
140% of the intracellular fluorescence. The experiments further
showed a direct correlation between the fluoxetine dose and the
fluorescence level. By comparing the results obtained with the
known chemosensitizer Verapamil and with fluoxetine, it is clearly
demonstrated that (i) fluoxetine acts as a chemosensitizer by
exerting the same effect as Verapamil on the intracellular
fluorescence level; and (ii) the chemosensitizing potential of
fluoxetine in substantially higher than that of Verapamil.
[0223] The same enhanced chemosensitizing effect of fluoxetine, as
compared with known chemosensitizers, was also observed in the
MDR-acquired cell line. As is shown in FIG. 11, in the experiments
performed in suspended P388/ADR cells incubated with Rhodamine-123,
with or without Verapamil or Cyclosporin A at the standard dose (15
.mu.M), an increase of about 60% and about 110%, respectively, of
the intracellular fluorescence, as compared to the control,
chemosensitizing-free cells, was observed, while fluoxetine, at the
same dose, generated an increase of about 200% of the intracellular
fluorescence.
[0224] Similar results were obtained in the experiments performed
with monolayered cells. FIGS. 12a-c present confocal microscopy
images of C-26 cells incubated with Rhodamine-123 and a
chemosensitizer, which clearly demonstrate that while Verapamil
generated an increase in the intracellular level of Rhodamine-123
(FIG. 12b), as compared with the control, chemosensitizer-free,
cells (FIG. 12a), fluoxetine generated a substantially higher
increase in the intracellular accumulation of Rhodamine-123 (FIG.
12c).
[0225] FIGS. 13a-b present confocal microscopy images of the
MDR-acquired cell line P388-ADR, incubated with doxorubicin, with
or without 5 .mu.M fluoxetine, and clearly show that the
intracellular accumulation of doxorubicin alone is quite poor (FIG.
13a) while adding to the incubation media a rather low dose of
fluoxetine (5 .mu.M) generated a substantial increase in the
intracellular level of DOX.
[0226] Hence, the results obtained by the qualitative and
quantitative measurements of drug uptake provide additional support
for the inhibitory effect of fluoxetine on MDR extrusion pumps,
which was suggested upon the efflux studies described hereinabove.
These results further demonstrate the superior activity of
fluoxetine over known chemosensitizers such as Verapamil and
Cyclosporin A. The chemosensitizing activity of fluoxetine in both
suspended and monolayered cells provides an indication for its in
vivo chemosensitizing activity in both solid and non-solid tumors,
as is further demonstrated hereinbelow.
In Vivo Studies in Inherent MDR Tumors Materials and Methods
[0227] The following in vivo studies were conducted in mice, in two
tumor models: a solid tumor model (also referred to herein as model
1) and a lung metastasis model (also referred to herein as model
2).
[0228] Cells:
[0229] In the solid tumor model, MDR-inherent C-26 cells were
injected into the animal's right-hind footpad.
[0230] In the lung metastasis model, MDR-inherent B16F10 cells were
injected intravenously, into the tail vein.
[0231] Chemosensitizer (CS):
[0232] The chemosensitizer used in both models was fluoxetine.
[0233] In both models the chemosensitizer was administered orally,
via the drinking water. Daily intake was 0.04 mg/kg body weight.
This daily dose is equivalent to a daily dose of 2.8 mg for a human
weighting 70 kg, whereas the approved (safe) range of daily dose of
fluoxetine as an antidepressant is 20-80 mg for a human weighting
70 kg.
[0234] Chemotherapeutic Agents (CT):
[0235] In the solid tumor model, Mitomycin C (MMC) and Doxorubicin
(DOX) were tested separately, each at a dose of 5 mg/kg body.
[0236] In the lung metastasis model, only Doxorubicin, at a dose of
10 mg/kg body, was tested.
[0237] In each model, the animals were divided into 4 groups, 5
animals per group. In the lung metastatic model, a fifth group of
untreated, healthy animals (i.e., animals that were not inoculated
with tumor cells), served as a control group for both models.
[0238] Each of the animal groups was treated with saline,
chemosensitizer, chemotherapeutic agent or a combination of
chemotherapeutic agent and chemosensitizer. The saline and the
chemotherapeutic agent were injected into the tail vein (100
.mu.l). The dosing regimen in the solid tumor model experiments was
3 injections, spaced a week apart and starting at day 5 from tumor
inoculation. The dosing regimen in the lung metastatic model was
also 3 injections, at days 1, 5 and 9 from tumor inoculation.
Experimental Results
[0239] The Solid Tumor Model:
[0240] The effective impact of the combined treatment of fluoxetine
and a chemotherapeutic agent in the solid tumor model is
demonstrated in FIGS. 9 and 10. FIG. 9 clearly demonstrates that
the solid tumor increases fast and exponentially in the groups
treated with saline, a chemosensitizer alone and a chemotherapeutic
agent alone. These results indicate that the tumor retains its drug
resistance nature in vivo. Contrary to that, in the animals
receiving the combination therapy, the appearance of the tumor is
delayed, as compared with the other groups, the tumors are
substantially smaller and the tumor growth is significantly slower.
FIG. 10 indicates the same trend, as it demonstrates that only the
animals receiving the combination therapy were long survivors,
namely, the survival of the animals treated with MMC+CS and DOX+CS
was prolonged 2 and 3 fold, respectively, as compared with animals
treated with saline, CS alone and the respective CT alone.
[0241] The Lung Metastasis Model:
[0242] The results presented in FIGS. 11a and 11b demonstrate the
lung metastatic burden by two measures: the increase in the lung
weight (FIG. 11a) and the number of lung metastasis (FIG. 11b).
These results clearly indicate that, by both measures, animals
treated with saline or a chemosensitizer alone had the highest
metastatic burden. These results further demonstrate that treatment
with Doxorubicin generated only a mild reduction in the metastatic
burden, while the combination treatment of chemosensitizer and a
Doxorubicin generated a substantial reduction thereof.
[0243] This encouraging effect of the combination treatment is
reflected also in the survival results presented in FIG. 12. The
survival data in FIG. 12 show the results obtained in a 75-days
experiment. As is shown in FIG. 12, similar to the pattern of the
solid tumor model (FIG. 10), animals treated with saline, CS alone
or CT alone, died rather early and within short intervals of one
another, while those treated with the combination treatment were
long survivors.
[0244] Of the two tumor models tested, the B16F10 is a more
aggressive tumor. This fact is evident by the shift in the survival
data in FIG. 12, as compared with the data shown in FIG. 10,
towards a shorter time span between the administration of the tumor
cells to the animals and onset of animal demise.
[0245] The obtained results clearly demonstrate the advantageous
features of fluoxetine, as a representative example of a
3-aryloxy-3-phenylpropyla- mine, as a chemosensitizing agent, as is
delineated hereinbelow:
[0246] This chemosensitizing agent changes the course of the tumor
response to chemotherapeutic drugs from poor to excellent, by all
counts: tumor progression, metastatic burden, and survival;
[0247] As the chemosensitizing activity of the CS agent was
demonstrated with two different chemotherapeutic drugs, acting via
different pathways (as is discussed in detail hereinabove), this CS
agent is not drug specific and therefore has the potential to
resolve the drug resistance to additional drugs;
[0248] The chemosensitizer itself at the doses employed has no
detrimental effects with respect to tumor progression;
[0249] The dose range required for chemosensitization is well below
the safe dose in humans; and
[0250] Finally, the chemosensitizer is administerable orally, which
is a patient-friendly route of administration.
In Vivo Studies in Acquired MDR Tumors
[0251] The following in vivo studies were conducted in mice, in two
tumor models: a non-resistant model and an acquired MDR model.
[0252] Cells:
[0253] P388/WT (non-resistant) or P388/ADR (MDR-acquired) murine
leukemia cells were propagated in the peritoneum of BDF.sub.1
female mice by weekly transfer of 0.5 ml of peritoneal fluid
containing 5.times.10.sup.5 cells, to generate an intraperitoneal
ascites model.
[0254] Chemosensitizer (CS):
[0255] The chemosensitizer used in both models was fluoxetine.
[0256] In both models the chemosensitizer was administered orally,
via the drinking water. Daily intake was 0.04 mg/kg body weight,
which is, as described hereinabove, well below the approved (safe)
range of daily dose of fluoxetine as an antidepressant.
[0257] Chemotherapeutic Agents (CT):
[0258] In both models, Doxorubicin (DOX) was tested. Doses of 3
mg/kg body were injected into the lateral tail vain, 1, 5 and 9
days after tumor inoculation.
[0259] Evaluating the Effect of Fluoxetine on Animals Weight and
Survival in Non-Resistant and MDR-Acquired Tumors:
[0260] Comparative experiments were run in parallel with the
sensitive, non-resistant P388/WT model and the corresponding,
MDR-acquired P388/ADR model. In each experiment, 20 animals were
divided into four treatment groups (5 animals per group), each
treated with either saline, a chemosensitizing agent (fluoxetine),
a chemotherapeutic agent (DOX) or a combination of a
chemotherapeutic agent and chemosensitizing agent (DOX and
fluoxetine). A group of untreated healthy animals was served as a
control group. The saline and the chemotherapeutic agent were
injected into the tail vein (100 .mu.l). The fluoxetine was given
in the drinking water, from tumor inoculation and on. Doxorubicin
was injected into the lateral tail vain 1, 5 and 9 days post tumor
inoculation. The effects of the various treatments on tumor
response were monitored through animals' survival and animals' body
weight. The latter was monitored in order to assess the treatment
toxicity.
Experimental Results
[0261] The Effect of Fluoxetine on Animals' Body Weight:
[0262] The changes in animals body weight in the various treatment
groups as compared with the control group, in mice induced with a
non-resistant tumor and an MDR-acquired tumor are presented in
FIGS. 18a and 18b, respectively. The obtained data indicate that
the nature of the tumor did not affect the body weight changes and
further indicate no weight loss in the fluoxetine-only treatment
groups and a minor weight decrease in the groups treated with DOX,
either alone or in combination with fluoxetine. However, as is
clearly shown in FIGS. 18a and 18b, in both tumor models fluoxetine
was found to positively modulate weight loss, thus indicating no
reproducible enhancement of toxicity as a results of the combined,
fluoxetine and DOX, treatment.
[0263] The effect of Fluoxetine on Animal's Survival:
[0264] The survival data of animals implanted with the
non-resistant P388/WT tumor and the MDR-acquired P388/ADR tumor, as
a result of the various treatments described hereinabove are
presented in FIGS. 19a and 19b, respectively.
[0265] As is shown in FIG. 19a, in animals implanted with the
non-resistant cells, the combined treatment of DOX and fluoxetine
had no effect on animal's survival. However, as is shown in FIG.
19b, in sharp contrast, in animals implanted with the MDR-acquired
cells, treatment with a combination of fluoxetine and DOX generated
a significant impact on the animals' survival, resulting in more
than 2-fold elongated life span.
[0266] These results further demonstrate the chemosensitizing
efficacy of fluoxetine, as a representative of a
3-aryloxy-3-phenylpropylamine. As the chemosensitizing activity of
fluoxetine was demonstrated with both MDR-inherent tumor and
MDR-acquired tumor, this CS agent is not tumor specific and
furthermore has the potential to resolve also acquired drug
resistance of various tumors to chemotherapeutic drugs.
The Effect of Fluoxetine on Drug Pharmacokinetics and
Biodistribution Materials and Methods
[0267] Cells:
[0268] B16F10.9 cells were injected intravenously into 12-weeks old
male C57BL/6 mice.
[0269] Chemosensitizer (CS):
[0270] The chemosensitizer used was fluoxetine. The chemosensitizer
was administered orally, via the drinking water. Daily intake was
0.04 mg/kg body weight.
[0271] Chemotherapeutic Agents (CT):
[0272] Doxorubicin (DOX), at a dose of 10 mg/kg body was injected
into the lateral tail vain, 10 days after tumor inoculation.
[0273] Evaluating the Effect of Fluoxetine on Drug Pharmacokinetics
and Biodistribution:
[0274] The animals were divided into two groups, 10 animals per
group, one was treated with DOX only and the other with a
combination of DOX and fluoxetine. Fluoxetine was given in the
drinking water, as described above, from tumor inoculation and on.
DOX was injected at day 10 from tumor inoculation. In an experiment
designated for pharmacokinetics, blood samples were taken up to six
hours post injection of the chemotherapeutic drug and were
processed for assaying their DOX content by fluorescence
measurements. In a parallel experiment, designated for
biodistribution, animals were scarified one hour post injection of
the chemotherapeutic drug, the lungs and other selected organs were
removed and were viewed by a pathologist, weighed and thereafter
processed for assaying their DOX content.
Experimental Results
[0275] The Effect of Fluoxetine on Drug Pharmacokinetics and
Biodistribution:
[0276] The pharmacokinetics of doxorubicin in mice having inherent
MDR lung tumor was evaluated by measuring the drug concentration in
the mice blood as a function of time. The decrease in the DOX
concentration was measured and compared in mice treated with DOX
only and with a combination of DOX and fluoxetine as a
chemosensitizer. The results, presented in FIG. 20, clearly
indicate that fluoxetine does not alter doxorubicin
pharmacokinetics, as the blood clearance of doxorubicin was found
to be the same in both tested groups. These findings further
demonstrate the efficacy of fluoxetine as a chemosensitizer which
acts only within the tumor site and does not affect the
pharmacokinetics of the chemotherapeutic agent.
[0277] The effect of fluoxetine on the biodistribution of
doxorubicin was measured by determining the DOX concentration in
selected organs, one hour post injection, in mice treated with DOX
only and in mice treated with a combination of DOX and fluoxetine.
The results, presented in FIG. 21, clearly show that while
fluoxetine had no effect on the drug concentration in the
tumor-free organs (spleen, kidneys and liver), a major increase
(about 12 fold) in the DOX level was induced by fluoxetine at the
tumor-bearing lungs. Again, these findings provide further support
for the in vivo efficacy of fluoxetine as a chemosensitizer that
acts solely at the tumor site and provides for elevated levels of
the chemotherapeutic drugs thereat.
[0278] It is appreciated that certain features of the invention,
which are, for clarity, described in the context of separate
embodiments, may also be provided in combination in a single
embodiment. Conversely, various features of the invention, which
are, for brevity, described in the context of a single embodiment,
may also be provided separately or in any suitable
subcombination.
[0279] Although the invention has been described in conjunction
with specific embodiments thereof, it is evident that many
alternatives, modifications and variations will be apparent to
those skilled in the art. Accordingly, it is intended to embrace
all such alternatives, modifications and variations that fall
within the spirit and broad scope of the appended claims. All
publications, patents and patent applications mentioned in this
specification are herein incorporated in their entirety by
reference into the specification, to the same extent as if each
individual publication, patent or patent application was
specifically and individually indicated to be incorporated herein
by reference. In addition, citation or identification of any
reference in this application shall not be construed as an
admission that such reference is available as prior art to the
present invention.
* * * * *