U.S. patent application number 09/938846 was filed with the patent office on 2002-09-26 for alpha-difluoromethylornithine (dfmo) suppresses polyamine levels in the human prostate.
Invention is credited to Gerner, Eugene W., Meyskens, Frank L. JR., Simoneau, Anne R..
Application Number | 20020137797 09/938846 |
Document ID | / |
Family ID | 22854168 |
Filed Date | 2002-09-26 |
United States Patent
Application |
20020137797 |
Kind Code |
A1 |
Meyskens, Frank L. JR. ; et
al. |
September 26, 2002 |
Alpha-Difluoromethylornithine (DFMO) suppresses polyamine levels in
the human prostate
Abstract
The present invention concerns a method for treating and/or
inhibiting prostate cancer in a patient comprising administering
.alpha.-difluoromethylornithine (DFMO) in an amount and duration
sufficient to stabilize or reduce the amount of polyamine produced
by the hyperplastic cells, wherein said polyamine is spermine,
spermidine or a combination of spermine and spermidine. Methods of
administration, dosing, and combinational therapies are
described.
Inventors: |
Meyskens, Frank L. JR.;
(Irvine, CA) ; Simoneau, Anne R.; (Long Beach,
CA) ; Gerner, Eugene W.; (Tucson, AZ) |
Correspondence
Address: |
FULBRIGHT & JAWORSKI L.L.P.
A REGISTERED LIMITED LIABILITY PARTNERSHIP
SUITE 2400
600 CONGRESS AVENUE
AUSTIN
TX
78701
US
|
Family ID: |
22854168 |
Appl. No.: |
09/938846 |
Filed: |
August 24, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60227714 |
Aug 24, 2000 |
|
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Current U.S.
Class: |
514/564 |
Current CPC
Class: |
A61P 43/00 20180101;
A61K 33/04 20130101; G11B 7/22 20130101; A61K 31/198 20130101; A61K
31/355 20130101; A61K 31/355 20130101; A61K 2300/00 20130101; A61K
2300/00 20130101; A61P 35/00 20180101; A61K 31/198 20130101; A61K
2300/00 20130101; G01N 33/57434 20130101; A61K 33/04 20130101 |
Class at
Publication: |
514/564 |
International
Class: |
A61K 031/198 |
Goverment Interests
[0002] The government may own rights in the present invention
pursuant to grant number P30CA62203U19 from Public Health Service
and CA81886 from the National Cancer Institute, National Institutes
of Health, Department of Health and Human Services.
Claims
What is claimed is:
1. A method for decreasing spermine and/or spermidine levels in a
human prostate cell comprising administering to said cell
.alpha.-difluoromethylomithine (DFMO) in an amount and duration
sufficient to decrease the spermine and/or spermidine levels in
said cell.
2. The method of claim 1, wherein said prostate cell is a cancer
cell.
3. The method of claim 1, wherein said prostate cell is a
non-cancerous cell.
4. The method of claim 1, wherein said prostate cell is a benign
hyperplastic cell.
5. The method of claim 1, wherein said cell is in a patient.
6. The method of claim 1, wherein said DFMO is substantially free
of the L enantiomer.
7. The method of claim 5, wherein said duration is about 3 to 8
weeks.
8. The method of claim 5, wherein said duration is 2 to 6
months.
9. The method of claim 5, wherein said duration is at least 6
months.
10. The method of claim 5, wherein said duration is at least 1
year
11. The method of claim 5, wherein said duration is at least 10
years.
12. The method of claim 5, wherein said duration is at least 20
years.
13. The method of claim 5, wherein said duration is at least 40
years.
14. The method of claim 5, wherein said amount is about 0.1 to 2.0
gm/m.sup.2/day.
15. The method of claim 5, wherein said amount is at least about
0.5 gm/m.sup.2/day.
16. The method of claim 5, wherein said amount is about 0.5
gm/m.sup.2/day.
17. The method of claim 5, wherein said amount is about 0.1 to 2.0
g/day.
18. The method of claim 5, wherein said amount is about 0.25-1.5
g/day.
19. The method of claim 5, wherein said amount is about 0.5-1.0
g/day
20. The method of claim 5, wherein DFMO is administered orally.
21. The method of claim 1, wherein spermine levels are reduced.
22. The method of claim 21, wherein said spermine levels are
reduced about 20% as compared to the spermidine levels in said cell
prior to said treatment.
23. The method of claim 21, wherein said spermine levels are
reduced about 30% as compared to the spermidine levels in said cell
prior to said treatment.
24. The method of claim 21, wherein said spermine levels are
reduced about 40% as compared to the spermidine levels in said cell
prior to said treatment.
25. The method of claim 21, wherein said spermine levels are
reduced about 50% as compared to the spermidine levels in said cell
prior to said treatment.
26. The method of claim 21, wherein said spermine levels are
reduced about 60% as compared to the spermidine levels in said cell
prior to said treatment.
27. The method of claim 21, wherein said spermine levels are
reduced about 70% as compared to the spermidine levels in said cell
prior to said treatment.
28. The method of claim 1, wherein spermidine levels are
reduced.
29. The method of claim 28, wherein said spermidine levels are
reduced about 30% as compared to the spermidine levels in said cell
prior to said treatment.
30. The method of claim 28, wherein said spermidine levels are
reduced about 45% as compared to the spermidine levels in said cell
prior to said treatment.
31. The method of claim 28, wherein said spermidine levels are
reduced about 55% as compared to the spermidine levels in said cell
prior to said treatment.
32. The method of claim 28, wherein said spermidine levels are
reduced about 65% as compared to the spermidine levels in said cell
prior to said treatment.
33. The method of claim 28, wherein said spermidine levels are
reduced about 75% as compared to the spermidine levels in said cell
prior to said treatment.
34. The method of claim 28, wherein said spermidine levels are
reduced about 85% as compared to the spermidine levels in said cell
prior to said treatment.
35. The method of claim 28, wherein said spermidine levels are
reduced about 99% as compared to the spermidine levels in said cell
prior to said treatment.
36. The method of claim 1, wherein the spermidine/spermine ratio of
said cell also is decreased.
37. The method of claim 36, wherein said spermidine levels are
reduced about 99% as compared to the spermidine levels in said cell
prior to said treatment.
38. The method of claim 1, further comprising a reduction in
putrescine levels of at least 50%.
39. The method of claim 38, further comprising a reduction in
putrescine levels of at least 70%.
40. The method of claim 39, further comprising a reduction in
putrescine levels of at least 90%.
41. A method of treating a human subject afflicted with prostate
cancer comprising administering DFMO to said subject in an amount
and duration sufficient to reduce spermine and/or spermidine levels
in prostate cells of said subject.
42. The method of claim 41, wherein said DFMO is substantially free
of the L enantiomer.
43. The method of claim 5, wherein said duration is about 3 to 8
weeks.
44. The method of claim 5, wherein said duration is 2 to 6
months.
45. The method of claim 5, wherein said duration is at least 6
months.
46. The method of claim 5, wherein said duration is at least 1
year
47. The method of claim 5, wherein said duration is at least 10
years.
48. The method of claim 5, wherein said duration is at least 20
years.
49. The method of claim 5, wherein said duration is at least 40
years.
50. The method of claim 41, wherein said amount is about 0.1 to 2.0
gm/m.sup.2/day.
51. The method of claim 41, wherein said amount is at least about
0.5 gm/m.sup.2/day.
52. The method of claim 41, wherein said amount is about 0.5
gm/m.sup.2/day.
53. The method of claim 5, wherein said amount is about 0.1 to 2.0
g/day.
54. The method of claim 5, wherein said amount is about 0.25-1.5
g/day.
55. The method of claim 5, wherein said amount is about 0.5-1.0
g/day
56. The method of claim 41, wherein DFMO is administered
orally.
57. The method of claim 41, further comprising a second
therapy.
58. The method of claim 57, wherein said second therapy comprises
reducing dihydroxytestosterone.
59. The method of claim 57, wherein said second therapy comprises
dietary antioxidants.
60. The method of claim 59, wherein said dietary antioxidant is
selenium, vitamin E or both.
61. The method of claim 57, wherein said second therapy comprises
retinoids.
62. The method of claim 57, wherein said second therapy comprises
prostatectomy.
63. The method of claim 57, wherein said second therapy comprises
low polyamine diet.
64. The method of claim 57, wherein said second therapy comprises
an inhibitor of polyamine oxidase.
65. The method of claim 57, wherein said second therapy comprises
radiation.
66. The method of claim 57, wherein said second therapy comprises
hormonal therapy.
67. The method of claim 66, wherein said hormonal therapy comprises
using luperon.
68. The method of claim 66, wherein said hormonal therapy comprises
using zoledex.
69. The method of claim 66, wherein said hormonal therapy comprises
using fultamide.
70. The method of claim 66, wherein said hormonal therapy comprises
using casadex.
71. The method of claim 41, wherein spermine levels are
decreased.
72. The method of claim 41, wherein spermidine levels are
decreased.
73. The method of claim 41, wherein the spermidine/spermine ratio
also is decreased.
74. The method of claim 41, further comprising diagnosis.
75. The method of claim 74, wherein said diagnosis comprises
analysis of prostate specific antigen (PSA).
76. The method of claim 74, wherein said diagnosis comprises
prostate biopsy.
77. The method of claim 74, wherein said diagnosis comprises rectal
exam.
78. The method of claim 74, wherein diagnosis comprises analysis of
PSA and rectal exam.
79. A method for inhibiting development of prostate cancer in a
human subject comprising administering DFMO to said subject in an
amount and duration sufficient to reduce spermine and/or spermidine
levels in prostate cells of said subject.
80. A method for inhibiting prostate cancer metastasis in a human
subject with primary prostate cancer comprising administering DFMO
to said subject in an amount and duration sufficient to reduce
spermine and/or spermidine levels in prostate cells of said
subject.
81. A method for inhibiting prostate cancer progression in a human
subject having Stage 1 or Stage 2 prostate cancer comprising
administering DFMO to said subject in an amount and duration
sufficient to reduce spermine and/or spermidine levels in prostate
cells of said subject.
82. A method of rendering a human unresectable prostate cancer
tumor resectable comprising administering DFMO to said subject in
an amount and duration sufficient to reduce spermine and/or
spermidine levels in prostate cells of said subject.
83. A method of inhibiting growth of a prostate cancer tumor in a
human subject comprising administering DFMO to said subject in an
amount and duration sufficient to reduce spermine and/or spermidine
levels in prostate cells of said subject.
84. A method of treating benign prostate hyperplasia in a human
subject afflicted with benign prostate hyperplasia comprising
administering DFMO to said subject in an amount and duration
sufficient to stabilize or reduce the amount of polyamine produced
by the hyperplastic cells, wherein said polyamine is spermine,
spermidine or a combination of spermine and spermidine.
85. The method of claim 84, wherein the polyamine is spermine.
86. The method of claim 84, wherein the polyamine is
spermidine.
87. The method of claim 84, wherein the levels of prostate specific
antigen (PSA) produced by the hyperplastic cells also are
stabilized or reduced upon treatment with DFMO.
88. A method for treating benign prostatic hyperplasia in a human
subject afflicted with benign prostatic hyperplasia comprising
administering, for a sufficient duration, a therapeutically
effective amount of DFMO, as measured by a reduction or
stabilization of polyamine levels produced by the hyperplastic
cells, together with a therapeutic effective amount of a second
therapeutic agent selected from an .alpha.-1 adrenergic receptor
blocker, a 5-.alpha.-reductase enzyme blocker, and a combination of
an .alpha.-1 adrenergic receptor blocker, and a 5-.alpha.-reductase
enzyme blocker.
89. The method of claim 88, wherein the polyamine is spermine.
90. The method of claim 88, wherein the polyamine is
spermidine.
91. The method of claim 88, wherein the second agent is an
.alpha.-1 adrenergic receptor blocker.
92. The method of claim 91, wherein the .alpha.-1 adrenergic
receptor blocker is terazosin, doxazosin tamsulosin, prazicin or
alfuzosin.
93. The method of claim 88, wherein the second agent is a
5-.alpha.-reductase enzyme blocker.
94. The method of claim 93, wherein the 5-.alpha.-reductase enzyme
blocker is finasteride.
95. The method of claim 88, wherein the second agent is a
hormone.
96. A method for treating benign prostatic hyperplasia in a human
subject afflicted with benign prostatic hyperplasia comprising
administering, over a sufficient duration, a therapeutically
effective amount of DFMO, as measured by a reduction or
stabilization of polyamine levels produced by the hyperplastic
cells, together with a therapeutically effective amount of saw
palmetto extract.
97. The method of claim 96, wherein polyamine is spermine.
98. The method of claim 96, wherein polyamine is spermidine.
99. The method of claim 96, wherein both spermine and spermidine
levels are decreased or stabilized.
Description
[0001] This application claims priority to U.S. Provisional
Application Ser. No. 60/227,714 filed Aug. 24, 2000, herein
incorporated by reference.
BACKGROUND OF THE INVENTION
[0003] I. Field of the Invention
[0004] The present invention relates generally to the field of
cancer therapy. More particularly, it concerns the use of DFMO
(.alpha.-difluoromethylornithine), an irreversible enzyme inhibitor
of the biosynthetic pathway of polyamines, in treating prostate
cancer.
[0005] II. Description of Related Art
[0006] Because rapidly-proliferating cell-growth disease states are
widespread throughout the world and affect a significant proportion
of the population, they have been the subject of intensive research
efforts. Unfortunately, despite such efforts and despite some
successes, the overall control of these diseases has not been
satisfactory. Recently, however, promising therapeutic methods for
treatment of such disease states have been developed employing
irreversible inhibitors of enzymes involved in the biosynthesis of
the polyamines necessary for cell growth. Particularly useful
enzyme inhibitors are those which produce in vivo irreversible
inhibition of ornithine decarboxylase (ODC), the enzyme which
catalyzes the decarboxylation of ornithine to putrescine.
[0007] .alpha.-Difluoromethylornithine (DFMO) is an irreversible
inhibitor of ornithine decarboxylase (ODC) and causes depletion in
the intracellular concentrations of putrescine and its derivative,
spermidine (Pegg, 1988). Levels of spermine, which is derived from
spermidine, are not as markedly affected by the enzyme inhibition.
DFMO was initially synthesized for therapeutic anticancer usage,
but it was found not to be an active cytotoxic agent in
chemotherapy trials against human cancer (McCann and Pegg, 1992),
except perhaps demonstrating moderate activity in the treatment of
malignant brain tumors (Levin et al., 1987). In general, the
compound was nontoxic, with the significant exception of hearing
loss, which was reversible after the drug treatment was
discontinued (Meyskens et al., 1986). The onset of the hearing loss
could be associated with total cumulative dose (Croghan et al.,
1991).
[0008] In experimental animal models, DFMO has been found to be a
potent inhibitor of carcinogenesis that is especially active in
preventing carcinogen-induced epithelial cancers of many organs,
including those of the colon (Weeks et al, 1982; Thompson et al,
1985; Nowels et al., 1986; Nigro et al, 1987). DFMO acts late in
the tumor-promotion phase in animals, but the precise mechanism by
which it inhibits the development of polyps and cancers is unknown.
Effects on cell transformation, invasion, and angiogenesis by
omithine decarboxylase and polyamines have been reported (Auvinen,
1997); for example, overexpression of ODC enhances cellular
transformation and invasion (Kubota et al, 1997).
[0009] Molecular mechanisms to explain this phenomenon include
activation of mitogen-activated protein (MAP) kinase activity,
secretion of matrix metalloproteinases (Wallon et al, 1994; Kubota
et al., 1997) and pathways with oncongenes (c-myc and ras)
(Meyskens et al, 1999; Clifford et al, 1995). A comprehensive
review of the potential cellular interactions of polyamines has
been recently presented (Auvinen, 1997).
[0010] Prostate cancer is the most commonly diagnosed malignancy in
United States males, and is the second leading cause of male cancer
deaths in the U.S. (Feuer et al, 1999). As the incidence of
prostate cancer increases with the age, the number of men who will
be diagnosed with prostate cancer will increase as medical care and
overall health improve. As such, the prevention of prostate cancer
is of national medical concern. Current strategies for prostate
cancer prevention have focused on changing the hormonal milieu in
the prostate (Proscar), or adding antioxidants, (selenium, and
vitamin E) or retinoids to the diet. Another approach is to
suppress the polyamine levels in the prostate, an avenue suggested
by studies indicating that ornithine decarboxylase (ODC), the first
enzyme in the polyamine pathway, is over-expressed in human
prostate cancer tissue (Mohan et al, 1999).
[0011] ODC activity and polyamine content are higher in prostatic
tissue when compared to other mammalian tissues (Dunzendorfer,
1978). In 1979, Danzin et al. treated rats with DFMO for two weeks
and found maximum inhibition of prostatic ODC activity within 4-6
hours to 10% of baseline activity and sustained suppression to 50%
of baseline activity throughout the study. The prostate was more
sensitive to DFMO than the testis, thymus, spleen or liver, and the
prostate and thymus showed a reduction in organ weight. Danzin et
al. (1979) also looked at the effect of DFMO on prostatic regrowth
with external testosterone after castration. After castration, the
prostate decreased in size as did polyamine content. With androgen
supplementation, prostatic atrophy was readily reversed, and
polyamine content increased. DFMO markedly slowed prostatic
(ventral lobe) weight gain during androgen treatment.
[0012] Heston et al. (1982) measured the suppressive effects of
DFMO on prostate cancer cell lines in vitro and in vivo (after the
inoculation of tumor cell into the flasks of rats to produce gross
tumors). In vitro ODC activity was highest in the two androgen
independent prostate cancer cell lines, followed by the ventral
lobe, a slow growing cancer cell line and the dorsal lobe. In vivo
prostate tumor bearing animals treated with DFMO intraperitoneally
for 18 days developed tumors with half the wet weight of untreated
controls, with no systemic toxicity. In another study, total
polyamine suppression (treatment consisting of a polyamine-free
diet, DFMO, a second inhibitor of polyamine oxidase, and intestinal
tract decontamination) was evaluated on the growth of the MAT-LyLu
prostate cancer cell line transplanted into the flanks of rats
(Movlinoux et al., 1991). In human subjects, reports of polyamine
deprivation leading to decreased PSA titers in patients with
prostate cancer was commented on by Kergozien (1996).
[0013] In 1999, Messing et al. published their results of two weeks
of oral DFMO on human prostate polyamine levels. In their study men
were randomized to receive either two weeks of DFMO 0.5gm/m.sup.2
(n=15) or placebo (n=10) prior to radical prostatectomy or
cystoprostatectomy. At the time of surgery, tissue for the
polyamine analysis was taken after surgical removal of the prostate
using ex vivo core biopsies. When palpable, suspected prostate
cancer nodules were sampled. The number of prostate cores taken
ranged from two to six per subject, but the majority of the cores
did not show cancer despite the researchers efforts to sample
cancerous areas. ODC activity and polyamine levels were measured in
the cores. In addition, each subject's plasma testosterone, serum
PSA, and prostatic acid phosphatase levels were measured, as well
as ODC activity from volar arm skin punch biopsies. Fourteen men
completed two weeks of DFMO with no toxicity. Mean putrescine
levels were statistically lower in the DFMO treated group (1.43
versus 1.95, p=0.03). Significantly, there were no differences
found in ODC activity, spermidine and spermine levels measured in
their study (Messing et al., 1999).
[0014] In light of these results, it remains unclear that DFMO is
useful in treating patients with prostate cancer, especially with
of the lack of any effect on spermine levels.
SUMMARY OF THE INVENTION
[0015] Thus, the present invention contemplates a method of
decreasing spermine and/or spermidine levels in a human prostate
cell comprising administering to said cell DFMO in an amount and
duration sufficient to decrease the levels in said cell. The
prostate has high levels of polyamines when compared to other
organs. It also differs from other organs in the amount and in the
percent reduction of spermine upon exposure to DFMO using the
methods of the present invention.
[0016] In a preferred embodiment, the human prostate cell is a
cancer cell, a non-cancerous cell or a benign hyperplastic cell.
The cell may be in a patient during the administering of DFMO, and
the DFMO employed may be substantially free of the L-enantiomer, or
a be enriched in D-enantiomer relative to a racemic {fraction
(50/50)} mixture of D-and L-isomers.
[0017] In a preferred embodiment, the DFMO will be administered for
about 3, 4, 5, 6, 8, 10 or 12 weeks. The duration of DFMO
administration may also be longer and continue for 2, 3, 4, 5, 6,
8, 10 or more than 12 months. The duration of DFMO administration
may be 2, 3, 4, 5, 10, 15, 20, 25, 30, 40 or 50 years, or may be
for the life of the patient. In a preferred embodiment, the amount
of DFMO administered is about 0.1 to 2.0 gm/m.sup.2/day, or more
preferred, about 0.5 gm/m.sup.2/day. The amount of DFMO may be
about 0.1 to 2 g/day, or more preferred 0.25-1.5 g/day, or even
more preferred 0.5-1.0 g/day. The DFMO may be administered via any
common route. In a preferred embodiment, the DFMO is administered
orally.
[0018] In preferred embodiments the spermine levels are reduced.
This reduction is preferentially about 10%, 20%, 30%, 40%, 50%,
60%, 70% or 80% as compared to the spermidine levels in said cell
prior to said treatment. In a preferred embodiment, the spermidine
levels are reduced. This reduction is preferentially about 30%,
45%, 55%, 65%, 75%, 85%, 95% or 99% as compared to the spermidine
levels in said cell prior to said treatment. In another preferred
embodiment, the spermidine/spermine ratio of said cell also is
decreased. In yet another embodiment, the putrecine level is reduce
about 50%, 60%, 70%, 80%, 90%, 95% or 99%.
[0019] The present invention also concerns a method of treating a
human subject afflicted with prostate cancer which comprises
administering DFMO to the subject in an amount and duration
sufficient to reduce spermine levels, the spermidine levels, or the
spermidine/spermine ratio in prostate cells of said subject.
[0020] Yet another embodiment comprises a second therapy, which can
include singly or in combination: reducing dihydroxytestosterone,
administering dietary antioxidants such as selenium, vitamin E or
both, administering retinoids, prostatectomy, a low polyamine diet,
inhibiting polyamine oxidase, radiation or a hormonal therapy such
as luperon, zoledex, fultamide or casadex.
[0021] In a further embodiment, the effectiveness of the DFMO
therapy according to the present invention can be determined in the
treatment of prostate cancer by diagnostic methods including, but
is not limited to, analysis of prostate specific antigen (PSA), a
prostate biopsy, a rectal exam, or analysis of PSA and rectal
exam.
[0022] Other embodiments include methods for inhibiting development
of prostate cancer in a subject at risk, inhibiting prostate cancer
metastasis in a subject with primary prostate cancer, inhibiting
prostate cancer progression in subjects having Stage 1 or Stage 2
prostate cancer, rendering a unresectable prostate cancer tumor
resectable, and inhibiting growth of a prostate cancer tumor. These
methods comprise administering DFMO to a human subject in an amount
and duration sufficient to reduce spermine and/or spermidine levels
in prostate cells of said subject.
[0023] Also within the scope of the invention is a method of
treating benign prostate hyperplasia in a human subject afflicted
with benign prostate hyperplasia comprising administering DFMO to
said subject in an amount and duration sufficient to stabilize or
reduce the amount of polyamine produced by the hyperplastic cells,
wherein said polyamine is spermine, spermidine or a combination of
spermine and spermidine. The levels of prostate specific antigen
(PSA) produced by the hyperplastic cells could also be stabilized
or reduced upon treatment with DFMO.
[0024] A further embodiment of the invention includes a method for
treating benign prostatic hyperplasia in a human subject afflicted
with benign prostatic hyperplasia comprising administering, for a
sufficient duration, a therapeutically effective amount of DFMO, as
measured by a reduction or stabilization of polyamine (spermine or
spermidine) levels produced by the hyperplastic cells, together
with a therapeutic effective amount of a second therapeutic agent
selected from: an a-1 adrenergic receptor blocker such as
terazosin, doxazosin, prazosin, indoramin, tamsulosin, prazicin and
alfuzosin; a 5-.alpha.-reductase enzyme blocker such as
finasteride; an azasteroid derivative; a combination of an
.alpha.-1 adrenergic receptor blocker and a 5-.alpha.-reductase
enzyme blocker; a potassium channel opener such as minoxidil; or a
retinoic acid derivative. In a preferred embodiment, the second
therapeutic agent is saw palmetto extract.
BRIEF DESCRIPTION OF THE DRAWINGS
[0025] The following drawings form part of the present
specification and are included to further demonstrate certain
aspects of the present invention. The invention may be better
understood by reference to one or more of these drawings in
combination with the detailed description of specific embodiments
presented herein.
[0026] FIG. 1 Polyamine content in prostatic tissue obtained by
core biopsy before (solid bar) and after (open bar) 28 days of
DFMO. FIG. 1A. putrescine content; FIG. 1B. spermidine content; and
FIG. 1C. spermine content.
[0027] FIG. 2 Spermidine/spermine ratio in prostatic tissue
obtained by core biopsy before (solid bar) and after (open bar) 28
days of DFMO.
DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
[0028] I. The Present Invention
[0029] This invention describes the use of DFMO
(.alpha.-difluoromethylomi- thine) as a method for reducing the
polyamine levels in the human prostate. DFMO is able to
significantly decrease the levels of putrescine, spermidine (Spd)
and spermine (Spm) in the prostate of male subjects given 28 days
of oral DFMO. Thus, DFMO may be utilized as in therapeutic
applications in the treatment of prostate cancer. Suprisingly, the
amount and percent reduction of spermine levels in the prostate
with the administration of DFMO are greater than in other tissues
in patients receiving DFMO therapy, provided that the DFMO is
administered in a sufficient amount and for a sufficient time
duration to cause such a decrease.
[0030] II. DFMO (alpha-difluoromethylornithine)
[0031] Numerous highly proliferative types of cancer are associated
with increased levels of the polyamines putrescine, spermidine, and
spermine in blood and urine. Studies have shown that this is
related to increased polyamine synthesis by the rate-limiting
enzyme, ornithine decarboxylase (ODC). Of particular interest in
the instant application is prostate cancer.
[0032] The pathway for polyamine synthesis begins with L-ornithine.
This natural amino acid, although not normally incorporated into
proteins, is part of the urea cycle which metabolizes arginine to
ornithine and urea. Ornithine is converted by ornithine
decarboxylase (ODC) to putrescine and CO.sub.2, the rate-limiting
step in the production of polyamines. With the addition of
propylamine donated from S-adenosylmethionine, putrescine is
converted to spermidine. Spermidine is then converted to spermine
by spermine synthetase, again in association with the
decarboxylation of S-adenosylmethionine. Putrescine, spermidine and
spermine represent the three major polyamines in mammalian tissues.
Polyamines are found in animal tissues and microorganisms and are
known to play an important role in cell growth and proliferation.
Although the exact mechanism of the role of the polyamines in cell
growth and proliferation is not known, it appears that the
polyamines may facilitate macromolecular processes such as DNA,
RNA, or protein synthesis. Polyamine levels are known to be high in
the testes, ventral prostate, and thymus, in psoriatic skin
lesions, and in other cells undergoing rapid growth processes.
[0033] It also is well known that the rapid proliferation of tumor
tissue is marked by an abnormal elevation of polyamine levels.
Hence, the polyamines also may play an important role in the
maintenance of tumor growth. Thus, ODC inhibitors, such as DFMO,
may exert their therapeutic effect by blocking the formation of the
polyamines and thereby slowing, interrupting, or arresting the
proliferation and metastases of the tumor tissue.
[0034] DFMO (alpha-difluoromethylornithine, Eflornithine,
Ornidyl.RTM.) is a structural analog of the amino acid L-omithine
and has a chemical structure: 1
[0035] The DFMO can be employed in the methods of the invention as
a racemic ({fraction (50/50)}) mixture of D- and L-enantiomers, or
preferably as a mixture of D- and L-isomers where the D-isomer is
enriched relative to the L-isomer, for example, 70%, 80%, 90% or
more by weight of the D-isomer relative to the L-isomer. Most
preferably, the DFMO employed is substantially free of the
L-enantiomer.
[0036] III. Anti-tumor Properties and Toxicity of DFMO
[0037] DFMO is relatively non-toxic to the host while producing
inhibition of putrescine synthesis in tumors. Oral DFMO in humans
is associated with ototoxicity (hearing loss) which limits
recommended doses for chemoprevention to a single 0.5 gm/m.sup.2
dose given daily. The other dose limiting toxic effect of DFMO is
thrombocytopenia (abnormally few platelets in the blood), which
occurs in about fifty percent of patients, leukopenia (abnormally
few leukocytes), or anemia. Another side effect of DFMO is nausea
and vomiting, which occurs in up to ninety percent of the patients.
All these toxic effects are relatively harmless and reversible and
cease upon withdrawal of the drug.
[0038] The effect of an ODC inhibitor for the control of the growth
rate of rapidly proliferating tumor tissue has been assessed in
standard animal tumor models. For example, the anti-tumor effect of
DFMO has been demonstrated in the following animal tumor models:
L1210 leukemia in mice, EMT6 tumor in Balb/C mice,
7,12-dimethylbenzanthracene-induced (DMBA-induced) mammary tumor in
rats, and DFMO Morris 7288C or 5123 hepatoma in Buffalo rats. In
addition, the anti-tumor effect of DFMO in combination with various
cytotoxic agents has been demonstrated as follows: (a) in
combination with vindesine or adriamycin in L1210 leukemia in mice,
in Morris 7288C hepatoma in Buffalo rats, and in EMT6 tumor in
mice, (b) in combination with cytosine arabinoside in L1210
leukemia in mice, (c) in combination with methotrexate in L1210
leukemia in mice, (d) in combination with cyclophosphamide in EMT6
tumor in mice and in DMBA-induced tumor in mice, (e) in combination
with BCNU in mouse glioma 26 brain tumor, and (f) in combination
with MGBG in L1210 leukemia in mice, in Morris 7288C hepatoma in
Buffalo rats, in P388 lymphocytic leukemia in mice, and in S-180
sarcoma in mice.
[0039] Although DFMO can effectively block tumor putrescine
biosynthesis, the resultant antitumor effect is cytostasis, not
cytotoxicity. For example, DFMO reduces the growth rate of an MCA
sarcoma, but does not produce tumor regression. This finding is
consistent with reports of other investigators who showed that DFMO
is a cytostatic agent. However, studies indicate that a significant
role may exist for DFMO agents, permitting the future development
of combination chemotherapeutic regimens which incorporate
DFMO.
[0040] The initial promise of DFMO as a therapeutic ODC inhibitor
for use in the treatment of various neoplasias has dimmed somewhat
because, although DFMO does, in fact, irreversibly inhibit ODC
activity, cells treated in vivo with DFMO significantly increase
their uptake of exogenous putrescine as described in U.S. Pat. No.
4,925,835. The intercellular transport mechanisms of the cell do an
"end run" around the DFMO-impaired ODC activity by importing
putrescine from the extra-cellular milieu. Therefore, DFMO's effect
in vivo is far poorer than in vitro. So, while DFMO treatment
effectively inhibits intracellular putrescine neogenesis, it also
results in increased uptake of extracellular putrescine, thereby
offsetting its ODC inhibitory effect.
[0041] This problem is compounded by the fact that putrescine is
present in many common foods, such as orange juice, which contains
approximately 400 ppm putrescine. This makes it virtually
impossible to provide a patient a nutritionally sufficient diet
which is free of putrescine. Therefore, DFMO-treated cells are
capable of importing sufficient amounts of extracellular putrescine
to support cell division.
[0042] Strategies to make DFMO more acceptable to human patients
are described in U.S. Pat. No. 4,859,452 (incorporated by
reference). Formulations of DFMO are described which include
essential amino acids in combination with either arginine or
ornithine to help reduce DFMO-induced toxicities.
[0043] Topical application of this drug to lesions that are
superficial allow a chemopreventive cancer therapy that has minimal
or no significant systemic uptake in human beings. One object of
this invention is to provide a salve that can be used as a chronic
topical chemopreventive agent against warts and superficial
anogenital HPV lesions that are often precancerous. The salve also
may be applied directly to cancerous lesions on the anogenital
areas. It is known in the art that a topically applied, five
percent solution of DFMO blocks the synthesis of DNA in mouse
epidermis. In vitro putrescine levels decreased to twenty-five
percent of control, while spermine and spermidine levels were not
affected U.S. Pat. No. 4,859,452 Further, application of a ten
percent DFMO cream in ten patients suffering from psoriasis was
shown to reduce cutaneous spermine levels by sixty-six percent,
with a marginal improvement in psoriatic lesions.
[0044] IV. D-DFMO
[0045] Toxicity of DFMO can be greatly reduced by using the
D-enantiomer of DFMO, or mixtures of D- and L-isomers which are
enriched for the D-isomer content such that the D-isomer comprises
at least 60%, and preferably more than 90% by weight of the
isomeric mixture. D-DFMO, while still an inhibitor of ODC, has
lower toxicity, including ototoxicity, in animal models. In a study
on guinea pigs, the enantiomers of DFMO did not show significant
toxicity. The D-form of DFMO was found to have no significant
effects on either the compound action potential or cochlear
microphonic. An evaluation of auditory function found that the L-
form of DFMO produced significant disruption of normal cochlear
potentials.
[0046] The use of D-DFMO or enriched D-isomer mixtures can overcome
many of the problems associated with the use of racemic (50/50)
D,L-DFMO. D-DFMO or enriched D-DFMO isomer mixtures may be
administered at a dosage higher than a racemic mixture, due to
lower anticipated toxicity associated with the D enantiomer. In
three separate studies using concentrations from 0.6 .mu.M to 80
.mu.M D-, L-, and D,L-DFMO, the effective concentration level of
each which inhibits 50% of the ODC activity (K,) was determined.
Both enantiomers, as well as the racemic mixture, were inhibitory.
The Ki of D-DFMO was four fold lower than the L-form and 3 fold
lower than the mixture. (U.S. Patent Application entitled
"D-enantiomer of DFMO and methods of use therefor," filed Jul. 1,
2000).
[0047] V. DFMO and the Human Prostate
[0048] DFMO and its use in the treatment of benign prostatic
hypertrophy are described in two patents, U.S. Pat. Nos. 4,413,141
and 4,330,559. U.S. Pat. No. 4,413,141 describes DFMO as being an
inhibitor of ODC, both in vitro and in vivo. Administration of DFMO
reportedly causes a decrease in putrescine and spermidine
concentrations in cells in which these polyamines are normally
actively produced. Additionally, DFMO has been shown to be capable
of slowing neoplastic cell proliferation when tested in standard
tumor models. U.S. Pat. No. 4,330,559 describes the use of DFMO and
DFMO derivatives for the treatment of benign prostatic hypertrophy.
Benign prostatic hypertrophy, like many disease states
characterized by rapid cell proliferation, is accompanied by
abnormal elevation of polyamine concentrations. The treatment
described within this reference can be administered to a patient
either orally, or parenterally. However, as pointed out above, a
more recent study by Messing et al. (1999) failed to detect any
reduction in spermidine or spermine levels in cancerous prostates
after treatment with DFMO over a two-week period at 0.5 gm/m.sup.2
given orally.
[0049] Recently the role of polyamines in prostate cancer has been
revisited. Mohan et al. (1999) measured ODC activity in benign and
malignant tissues from the same patient and found the cancerous
portion to have levels almost three times that of benign tissue
(1142+100 vs. 427+51). In addition they evaluated the ODC activity
of prostatic fluid obtained by massage and found higher levels in
men with prostate cancer compared to men with benign hypertrophy
(3847+162 vs. 2742+67).
[0050] Chemoprevention for prostate cancer is being studied using
both hormonal and antioxidant agents. The first large scale
chemopreventive study for prostate cancer began in 1993 by the
Southwest Oncology Group (SWOG), and 18,000 men were randomized to
finasteride (Proscar) or placebo for seven years. The hypothesis
being tested was that a reduction in the levels of
dihydrotestosterone (DHT) in the prostate by blocking the
conversion of testosterone to DHT by 5 alpha reductatase would
reduce prostate cancer. Results are expected to be available in
2004. A smaller study looked at surrogate end markers at time zero
and after one year of Proscar treatment or no treatment (Cote et
al., 1998). The study group included men with an elevated PSA and
negative prostate needle biopsies. The study demonstrated an
increase in cancer diagnosed after a one year follow up biopsy in
the men on Proscar (8 of 27 men vs. 1 of 25 men with no treatment
(p=0.025)). A small Phase II study of oral fenretinide in 22 men at
high risk for prostate cancer was undertaken by Pienta et al.
(1997). This trial closed early when 8 men with negative prestudy
biopsies developed malignancy while on study. With these two small
studies unable to demonstrate protective benefits from the agent of
interest, there should be a role in prostate cancer
chemoprevention, for different agents, such as DFMO.
[0051] VI. Diagnosis of Prostate Cancer
[0052] The most commonly utilized current tests for prostate cancer
are digital rectal examination (DRE) and analysis of serum prostate
specific antigen (PSA). While PSA is specific to prostate tissue,
it is produced by normal and benign as well as malignant prostatic
epithelium, resulting in a high false-positive rate for prostate
cancer detection (Partin & Oesterling, 1994).
[0053] Other markers that have been used for prostate cancer
detection include prostatic acid phosphatase (PAP) and prostate
secreted protein (PSP). PAP is secreted by prostate cells under
hormonal control (Brawn et al., 1996). It has less specificity and
sensitivity than does PSA. As a result, it is used much less now,
although PAP may still have some applications for monitoring
metastatic patients that have failed primary treatments. In
general, PSP is a more sensitive biomarker than PAP, but is not as
sensitive as PSA (Huang et al., 1993). Like PSA, PSP levels are
frequently elevated in patients with BPH as well as those with
prostate cancer.
[0054] Another serum marker associated with prostate disease is
prostate specific membrane antigen (PSMA) (Horoszewicz et al.,
1987; Carter et al., 1996; Murphy et al., 1996). PSMA is a Type II
cell membrane protein and has been identified as Folic Acid
Hydrolase (FAH) (Carter et al., 1996). Antibodies against PSMA
react with both normal prostate tissue and prostate cancer tissue
(Horoszewicz et al., 1987). Murphy et al. (1995) used ELISA to
detect serum PSMA in advanced prostate cancer. As a serum test,
PSMA levels are a relatively poor indicator of prostate cancer.
However, PSMA may have utility in certain circumstances. PSMA is
expressed in metastatic prostate tumor capillary beds (Silver et
al., 1997) and is reported to be more abundant in the blood of
metastatic cancer patients (Murphy et al., 1996). PSMA messenger
RNA (mRNA) is down-regulated 8-10 fold in the LNCaP prostate cancer
cell line after exposure to 5-.alpha.-dihydroxytestosterone (DHT)
(Israeli et al., 1994).
[0055] As indicated earlier, DFMO acts late in the tumor promotion
stage of epithelial carcinogenesis. A mechanism for the inhibition
of colon carcinogenesis by DFMO includes suppression of the matrix
metalloproteinase matrilysin (Wallon et al., 1994), a secreted
protease which is involved in tumor invasion. This mechanism is
operational in prostate cancer, since DFMO also suppresses
interleukin-1.beta.(IL-1.beta- .)-induced matrilysin expression in
LNCaP human prostate cancer derived cells.
[0056] A relatively new potential biomarker for prostate cancer is
human kallekrein 2 (HK2) (Piironen et al., 1996). HK2 is a member
of the kallekrein family that is secreted by the prostate gland. In
theory, serum concentrations of HK2 may be of utility in prostate
cancer detection or diagnosis, but the usefulness of this marker is
still being evaluated.
[0057] VII. Methods of Treating Cancer
[0058] In a particular aspect, the present invention provides
methods for the treatment of prostate cancer. Treatment methods
will involve treating an individual with an effective amount of a
therapeutic composition containing DFMO. An effective amount is
described, generally, as that amount sufficient to detectably and
repeatedly to ameliorate, reduce, minimize or limit the extent of a
disease or its symptoms. More rigorous definitions may apply,
including elimination, eradication or cure of disease.
[0059] DFMO may be administered at a dose of about 0.05 to about
20.0 gm/m.sup.2/day. Preferred doses of DFMO to be administered are
from about 0.1 to about 15.0 gm/m.sup.2/day, or from about 0.1 to
12 gm/m.sup.2/day, or from about 0.1 to 10 gm/m.sup.2/day, or from
about 0.1 to 8 gm/m.sup.2/day, or from about 0.1 to 6
gm/m.sup.2/day, or from about 0.1 to 4 gm/m.sup.2/day, or from
about 0.1 to 2 gm/m.sup.2/day, or from about 0.1 to 1
gm/m.sup.2/day, or from about 0.1 to 0.5 gm/m.sup.2/day or about
0.5 gm/m.sup.2/day. DFMO may also be administered at a dose of
about 0.1 to 2.0 g/day, or from about 0.25 to 1.5 g/day, or more
preferred from about 0.5 to 1.0 g/day.
[0060] To kill cells, inhibit cell growth, inhibit metastasis,
decrease tumor size and otherwise reverse or reduce the malignant
phenotype of tumor cells, using the methods and compositions of the
present invention, one would generally contact a "target" cell with
the therapeutic composition. This may be combined with compositions
comprising other agents effective in the treatment of cancer. These
compositions would be provided in a combined amount effective to
kill or inhibit proliferation of the cell. This process may involve
contacting the cells with DFMO and the agent(s) or factor(s) at the
same time. This may be achieved by contacting the cell with a
single composition or pharmacological formulation that includes
both agents, or by contacting the cell with two distinct
compositions or formulations, at the same time, wherein one
composition includes DFMO and the other includes the second
agent.
[0061] Administration of the therapeutic DFMO composition of the
present invention to a patient will follow general protocols for
the administration of chemotherapeutics, taking into account the
toxicity, if any, of DFMO. It is expected that the treatment cycles
would be repeated as necessary. It also is contemplated that
various standard therapies, as well as surgical intervention, may
be applied in combination with the described therapy.
[0062] Where clinical application of a DFMO therapy is
contemplated, it will be necessary to prepare the complex as a
pharmaceutical composition appropriate for the intended
application. Generally this will entail preparing a pharmaceutical
composition that is essentially free of pyrogens, as well as any
other impurities that could be harmful to humans or animals. One
also will generally desire to employ appropriate salts and buffers
to render the complex stable and allow for complex uptake by target
cells.
[0063] Aqueous compositions of the present invention comprise an
effective amount of the compound, dissolved or dispersed in a
pharmaceutically acceptable carrier or aqueous medium. Such
compositions can also be referred to as inocula. The phrases
"pharmaceutically or pharmacologically acceptable" refer to
molecular entities and compositions that do not produce an adverse,
allergic or other untoward reaction when administered to an animal,
or a human, as appropriate. 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 pharmaceutical 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 also
can be incorporated into the compositions.
[0064] The compositions of the present invention may include
classic pharmaceutical preparations. Dispersions also can be
prepared in glycerol, liquid polyethylene glycols, and mixtures
thereof and in oils. Under ordinary conditions of storage and use,
these preparations contain a preservative to prevent the growth of
microorganisms.
[0065] Depending on the particular cancer to be, administration of
therapeutic compositions according to the present invention will be
via any common route so long as the target tissue is available via
that route. This includes oral, nasal, buccal, rectal, vaginal or
topical. Topical administration would be particularly advantageous
for treatment of skin cancers. Alternatively, administration will
be by orthotopic, intradermal, subcutaneous, intramuscular,
intraperitoneal or intravenous injection. Such compositions would
normally be administered as pharmaceutically acceptable
compositions that include physiologically acceptable carriers,
buffers or other excipients.
[0066] In certain embodiments, ex vivo therapies also are
contemplated. Ex vivo therapies involve the removal, from a
patient, of target cells. The cells are treated outside the
patient's body and then returned.
[0067] The treatments may include various "unit doses." Unit dose
is defined as containing a predetermined-quantity of the
therapeutic composition calculated to produce the desired responses
in association with its administration, i.e., the appropriate route
and treatment regimen. The quantity to be administered, and the
particular route and formulation, are within the skill of those in
the clinical arts. Also of import is the subject to be treated, in
particular, the state of the subject and the protection desired. A
unit dose need not be administered as a single injection but may
comprise continuous infusion over a set period of time.
[0068] One of the preferred embodiments of the present invention
involves the use of therapeutic compositions of DFMO with specific
target cancer cells. Of particular interest are prostate cancer
cells.
[0069] According to the present invention, one may treat the cancer
by directly injection a tumor with the DFMO or analog composition.
Alternatively, the tumor may be infused or perfused with the
composition using any suitable delivery vehicle. Local or regional
administration, with respect to the tumor, also is contemplated.
Finally, systemic administration may be performed. Continuous
administration also may be applied where appropriate, for example,
where a tumor is excised and the tumor bed is treated to eliminate
residual, microscopic disease. Delivery via syringe or
catherization is preferred. Such continuous perfusion may take
place for a period from about 1-2 hours, to about 2-6 hours, to
about 6-12 hours, to about 12-24 hours, to about 1-2 days, to about
1-2 wk or longer following the initiation of treatment. Generally,
the dose of the therapeutic composition via continuous perfusion
will be equivalent to that given by a single or multiple
injections, adjusted over a period of time during which the
perfusion occurs. For tumors of >4 cm, the volume to be
administered will be about 4-10 ml (preferably 10 ml), while for
tumors of <4 cm, a volume of about 1-3 ml will be used
(preferably 3 ml). Multiple injections delivered as single dose
comprise about 0.1 to about 0.5 ml volumes. The viral particles may
advantageously be contacted by administering multiple injections to
the tumor, spaced at approximately 1 cm intervals.
[0070] In certain embodiments, the tumor being treated may not, at
least initially, be resectable. Treatments with therapeutic DFMO
compositions may increase the resectability of the tumor due to
shrinkage at the margins or by elimination of certain particularly
invasive portions. Following treatments, resection may be possible.
Additional treatments subsequent to resection will serve to
eliminate microscopic residual disease at the tumor site.
[0071] Other factors that cause DNA damage and have been used
extensively include what are commonly known as y-rays, X-rays,
and/or the directed delivery of radioisotopes to tumor cells. Other
forms of DNA damaging factors are also contemplated such as
microwaves and UV-irradiation. It is most likely that all of these
factors effect a broad range of damage on DNA, on the precursors of
DNA, on the replication and repair of DNA, and on the assembly and
maintenance of chromosomes. Dosage ranges for X-rays range from
daily doses of 50 to 200 roentgens for prolonged periods of time (3
to 4 wk), to single doses of 2000 to 6000 roentgens. Dosage ranges
for radioisotopes vary widely, and depend on the half-life of the
isotope, the strength and type of radiation emitted, and the uptake
by the neoplastic cells.
[0072] VIII. Combinational Therapy with DFMO
[0073] The present invention contemplates that DFMO may be used in
combination with other therapies. There is an increasing body of
experimental and epidemiological data suggesting that aspirin, and
some other NSAIDs, exert a chemopreventive action on colorectal
cancers and maybe also on stomach, esophagus (Thun et al., 1993)
and even bladder (Earnest et al., 1992) cancers. Aspirin,
ibuprofen, piroxicam (Reddy et al., 1990;
[0074] Singh et al., 1994), indomethacin (Narisawa, 1981), and
sulindac (Piazza et al., 1997; Rao et al., 1995), effectively
inhibit colon carcinogenesis in the azoxymethane AOM-treated rat
model and flurbiprofen has demonstrated anti-tumor effects in the
APC(Min)+mouse model (Wechter et al., 1997). NSAIDs also inhibit
the development of tumors harboring an activated Ki-ras (Singh and
Reddy, 1995). Studies have been conducted in which DFMO was
combined with aspirin to evaluate its chemopreventive effect in to
AOM-treated rats. The combination of aspirin and DFMO administered
after AOM was found to be synergistic (Li et al., 1999). The
results demonstrated that the aspirin and DFMO combination could
prevent colon cancer when administered after AOM (Li et al.,
1999).
[0075] The combination of DFMO with the chemotherapeutic agent
piroxicam has been shown to have a synergistic chemopreventive
effect in the AOM-treated rat model of colon carcinogenesis (Reddy
et al., 1990), although DFMO exerted a greater suppressive effect
than piroxicam on Ki-ras mutation and tumorigenesis when each agent
was administered separately (Singh et al., 1993; Reddy et al.,
1990; Kulkarni et al., 1992). In one study, administration of DFMO
or piroxicam to AOM-treated rats reduced the number of tumors
harboring Ki-ras mutations from 90% to 36% and 25% respectively
(Singh et al., 1994). The Apc mutant Min mouse model was used to
test piroxicam and DFMO to determine that combined treatment was
much more effective than either agent alone and resulted in a
significant number of mice totally free of any intestinal adenomas
(Jacoby et al., 2000). Both DFMO and piroxicam also reduced the
amount of biochemically active p21 ras in existing tumors. (Singh
et al., 1993). Despite the success of the drugs in model systems,
phase I trials conducted with this combination resulted in a range
of adverse side effects (Carbone et al., 1998).
[0076] Because DFMO is an effective inhibitor of ODC, some
researchers are attempting to use DFMO as part of a conjunctive
treatment in combination with interferon. U.S. Pat. No. 4,499,072,
describe improving the polyamine-depletion effects of ODC
inhibitors (including DFMO) by using interferon in combination with
the ODC inhibitor. Additionally, it describes the use of both an
ODC inhibitor and interferon in conjunction with a known cytotoxic
agent such as methotrexate. U.S. Pat. No. 5,002,879, describe a
similar conjunctive therapy in which an ODC inhibitor, preferably
DFMO, is used in combination with lymphokine-activated killer (LAK)
cells and interleukin-2.
[0077] Cancer therapies also include a variety of combination
therapies of DFMO with both chemical and radiation based
treatments. Combination chemotherapies include, for example,
cisplatin (CDDP), carboplatin, procarbazine, mechlorethamine,
cyclophosphamide, camptothecin, ifosfamide, melphalan,
chlorambucil, bisulfan, nitrosurea, dactinomycin, daunorubicin,
doxorubicin, bleomycin, plicomycin, mitomycin, etoposide (VP16),
tamoxifen, taxol, transplatinum, 5-fluorouracil, vincristin,
vinblastin and methotrexate or any analog or derivative variant
thereof For precancerous conditions such as benign prostatic
hyperplasia, a second therapeutic agent selected from cc-1
adrenergic receptor blocker such as terazosin, doxazosin, prazosin,
bunazosin, indoramin, tamsulosin, prazicin or alfuzosin; a
5-.alpha.-reductase enzyme blocker such as finasteride or an
azasteroid derivative; a combination of an .alpha.-1 adrenergic
receptor blocker, and a 5-.alpha.-reductase enzyme blocker, a
potassium channel opener such as minoxidil, and a retinoic acid
derivative. In a preferred embodiment, the second therapeutic agent
is saw palmetto extract.
[0078] Various combinations may be employed, for instance where
DFMO composition is "A" and the radio-, chemotherapeutic or other
therapeutic agent is "B":
[0079] A/B/A B/A/B B/B/A A/A/B A/B/B B/A/A A/B/B/B B/A/B/B B/B/B/A
B/B/A/B A/A/B/B A/B/A/B A/B/B/A B/B/A/A B/A/B/A B/A/A/B A/A/A/B
B/A/A/A A/B/A/A A/A/B/A
[0080] The terms "contacted" and "exposed," when applied to a cell,
are used herein to describe the process by which a therapeutic
composition and a chemotherapeutic or radiotherapeutic agent are
delivered to a target cell or are placed in direct juxtaposition
with the target cell. To achieve cell killing or stasis, both
agents are delivered to a cell in a combined amount effective to
kill the cell or prevent it from dividing.
[0081] The DFMO therapy may precede or follow the other agent
treatment by intervals ranging from minutes to weeks. In
embodiments where the other agent and DFMO are applied separately
to the cell, one would generally ensure that a significant period
of time did not expire between the time of each delivery, such that
the agent and DFMO would still be able to exert an advantageously
combined effect on the cell. In such instances, it is contemplated
that one would contact the cell with both modalities within about
12-24 h of each other and, more preferably, within about 6-12 h of
each other, with a delay time of only about 12 h being most
preferred. In some situations, it may be desirable to extend the
time period for treatment significantly, however, where several
days (2, 3, 4, 5, 6 or 7) to several weeks (1, 2, 3, 4, 5, 6, 7 or
8) lapse between the respective administrations.
[0082] IX. Pharmaceutical Compositions
[0083] Aqueous compositions of the present invention comprise an
effective amount of DFMO, dissolved or dispersed in a
pharmaceutically acceptable carrier or aqueous medium. The phrases
"pharmaceutically or pharmacologically acceptable" refer to
molecular entities and compositions that do not produce an adverse,
allergic or other untoward reaction when administered to an animal,
or a human, as appropriate.
[0084] 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
pharmaceutical 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. For human administration,
preparations should meet sterility, pyrogenicity, general safety
and purity standards as required by FDA Office of Biologics
standards.
[0085] The biological material should be extensively dialyzed to
remove undesired small molecular weight molecules and/or
lyophilized for more ready formulation into a desired vehicle,
where appropriate. The active compounds will then generally be
formulated for parenteral administration, e.g., formulated for
injection via the intravenous, intramuscular, sub-cutaneous,
intralesional, or even intraperitoneal routes. The preparation of
an aqueous composition that contains an headpin agent as an active
component or ingredient will be known to those of skill in the art
in light of the present disclosure. Typically, such compositions
can be prepared as injectables, either as liquid solutions or
suspensions; solid forms suitable for using to prepare solutions or
suspensions upon the addition of a liquid prior to injection can
also be prepared; and the preparations can also be emulsified.
[0086] The pharmaceutical forms suitable for injectable use include
sterile aqueous solutions or dispersions; formulations including
sesame oil, peanut oil or aqueous propylene glycol; 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 syringability 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.
[0087] Solutions of the active compounds as free base or
pharmacologically acceptable salts can be prepared in water
suitably mixed with a surfactant, such as hydroxypropylcellulose.
Dispersions can also be prepared in glycerol, liquid polyethylene
glycols, and mixtures thereof and in oils. Under ordinary
conditions of storage and use, these preparations contain a
preservative to prevent the growth of microorganisms.
[0088] DFMO of the present invention can be formulated into a
composition in a neutral or salt form. Pharmaceutically acceptable
salts, include the acid addition salts (formed with the free amino
groups of the protein) and which are formed with inorganic acids
such as, for example, hydrochloric or phosphoric acids, or such
organic acids as acetic, oxalic, tartaric, mandelic, and the like.
Salts formed with the free carboxyl groups can also be derived from
inorganic bases such as, for example, sodium, potassium, ammonium,
calcium, or ferric hydroxides, and such organic bases as
isopropylamine, trimethylamine, histidine, procaine and the
like.
[0089] The carrier can also 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 or sodium chloride. Prolonged absorption of the
injectable compositions can be brought about by the use in the
compositions of agents delaying absorption, for example, aluminum
monostearate and gelatin.
[0090] 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 The
preparation of more, or highly, concentrated solutions for direct
injection is also contemplated, where the use of DMSO as solvent is
envisioned to result in extremely rapid penetration, delivering
high concentrations of the active agents to a small area.
[0091] Upon formulation, solutions will be administered in a manner
compatible with the dosage formulation and in such amount as is
therapeutically effective. The formulations are easily administered
in a variety of dosage forms, such as the type of injectable
solutions described above, but drug release capsules and the like
can also be employed.
[0092] For parenteral administration in an aqueous solution, for
example, the solution 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. For example, one dosage
could be dissolved in 1 ml of isotonic NaCl solution and either
added to 1000 ml of hypodermoclysis fluid or injected at the
proposed site of infusion, (see for example, "Remington's
Pharmaceutical Sciences" 15th Edition, pages 1035-1038 and
1570-1580). Some variation in dosage will necessarily occur
depending on the condition of the subject being treated. The person
responsible for administration will, in any event, determine the
appropriate dose for the individual subject.
[0093] In addition to the compounds formulated for parenteral
administration, such as intravenous or intramuscular injection,
other pharmaceutically acceptable forms include, e.g., tablets or
other solids for oral administration; liposomal formulations; time
release capsules; and any other form currently used, including
cremes.
[0094] One also may use nasal solutions or sprays, aerosols or
inhalants in the present invention. Nasal solutions are usually
aqueous solutions designed to be administered to the nasal passages
in drops or sprays. Nasal solutions are prepared so that they are
similar in many respects to nasal secretions, so that normal
ciliary action is maintained. Thus, the aqueous nasal solutions
usually are isotonic and slightly buffered to maintain a pH of 5.5
to 6.5. In addition, antimicrobial preservatives, similar to those
used in ophthalmic preparations, and appropriate drug stabilizers,
if required, may be included in the formulation. Various commercial
nasal preparations are known and include, for example, antibiotics
and antihistamines and are used for asthma prophylaxis.
[0095] Additional formulations which are suitable for other modes
of administration include vaginal suppositories and pessaries. A
rectal pessary or suppository may also be used. Suppositories are
solid dosage forms of various weights and shapes, usually
medicated, for insertion into the rectum, vagina or the urethra.
After insertion, suppositories soften, melt or dissolve in the
cavity fluids. In general, for suppositories, traditional binders
and carriers may include, for example, polyalkylene glycols or
triglycerides; such suppositories may be formed from mixtures
containing the active ingredient in the range of 0.5% to 10%,
preferably 1%-2%.
[0096] Oral formulations include such normally employed excipients
as, for example, pharmaceutical grades of mannitol, lactose,
starch, magnesium stearate, sodium saccharine, cellulose, magnesium
carbonate and the like. These compositions take the form of
solutions, suspensions, tablets, pills, capsules, sustained release
formulations or powders. In certain defined embodiments, oral
pharmaceutical compositions will comprise an inert diluent or
assimilable edible carrier, or they may be enclosed in hard or soft
shell gelatin capsule, or they may be compressed into tablets, or
they may be incorporated directly with the food of the diet. For
oral therapeutic administration, the active compounds may be
incorporated with excipients and used in the form of ingestible
tablets, buccal tables, troches, capsules, elixirs, suspensions,
syrups, wafers, and the like. Such compositions and preparations
should contain at least 0.1% of active compound. The percentage of
the compositions and preparations may, of course, be varied and may
conveniently be between about 2 to about 75% of the weight of the
unit, or preferably between 25-60%. The amount of active compounds
in such therapeutically useful compositions is such that a suitable
dosage will be obtained.
[0097] The tablets, troches, pills, capsules and the like may also
contain the following: a binder, as gum tragacanth, acacia,
cornstarch, or gelatin; excipients, such as dicalcium phosphate; a
disintegrating agent, such as corn starch, potato starch, alginic
acid and the like; a lubricant, such as magnesium stearate; and a
sweetening agent, such as sucrose, lactose or saccharin may be
added or a flavoring agent, such as peppermint, oil of wintergreen,
or cherry flavoring. When the dosage unit form is a capsule, it may
contain, in addition to materials of the above type, a liquid
carrier. Various other materials may be present as coatings or to
otherwise modify the physical form of the dosage unit. For
instance, tablets, pills, or capsules may be coated with shellac,
sugar or both. A syrup of elixir may contain the active compounds
sucrose as a sweetening agent methyl and propylparabens as
preservatives, a dye and flavoring, such as cherry or orange
flavor.
[0098] In certain embodiments, the use of liposomes and/or
nanoparticles is contemplated for the formulation and
administration of DFMO or an analog thereof The formation and use
of liposomes is generally known to those of skill in the art, and
is also described below.
[0099] Nanocapsules can generally entrap compounds in a stable and
reproducible way. To avoid side effects due to intracellular
polymeric overloading, such ultrafine particles (sized around 0.1
.mu.m) should be designed using polymers able to be degraded in
vivo. Biodegradable polyalkyl-cyanoacrylate nanoparticles that meet
these requirements are contemplated for use in the present
invention, and such particles may be are easily made.
[0100] Liposomes are formed from phospholipids that are dispersed
in an aqueous medium and spontaneously form multilamellar
concentric bilayer vesicles (also termed multilamellar vesicles
(MLVs). MLVs generally have diameters of from 25 nm to 4 .mu.m.
Sonication of MLVs results in the formation of small unilamellar
vesicles (SUVs) with diameters in the range of 200 to 500 A,
containing an aqueous solution in the core.
[0101] The following information may also be utilized in generating
liposomal formulations. Phospholipids can form a variety of
structures other than liposomes when dispersed in water, depending
on the molar ratio of lipid to water. At low ratios the liposome is
the preferred structure. The physical characteristics of liposomes
depend on pH, ionic strength and the presence of divalent cations.
Liposomes can show low permeability to ionic and polar substances,
but at elevated temperatures undergo a phase transition which
markedly alters their permeability. The phase transition involves a
change from a closely packed, ordered structure, known as the gel
state, to a loosely packed, less-ordered structure, known as the
fluid state. This occurs at a characteristic phase-transition
temperature and results in an increase in permeability to ions,
sugars and drugs.
[0102] Liposomes interact with cells via four different mechanisms:
Endocytosis by phagocytic cells of the reticuloendothelial system
such as macrophages and neutrophils; adsorption to the cell
surface, either by nonspecific weak hydrophobic or electrostatic
forces, or by specific interactions with cell-surface components;
fusion with the plasma cell membrane by insertion of the lipid
bilayer of the liposome into the plasma membrane, with simultaneous
release of liposomal contents into the cytoplasm; and by transfer
of liposomal lipids to cellular or subcellular membranes, or vice
versa, without any association of the liposome contents. Varying
the liposome formulation can alter which mechanism is operative,
although more than one may operate at the same time.
X. EXAMPLES
[0103] The following example is 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.
Example 1
Experimental
[0104] This protocol was approved by the investigational review
board of the University of California Irvine and the Long Beach
Veterans Administration Medical Center. Men between the ages of 50
and 85, who were undergoing a trans-rectal prostate needle biopsy
for either an elevated PSA or abnormal rectal exam, signed a
written consent to undergo four additional core needle biopsies of
the peripheral zone at the time of their routine extant biopsy. The
additional four biopsies were immediately frozen liquid nitrogen
and stored in the minus 70-degree freezer. The sextent biopsy was
sent to Pathology for routine analysis. If the patient elected (1)
radical surgery for prostate cancer (2) transurethral resection for
outlet obstruction, or (3) a second biopsy due to the diagnosis of
atypia, he was then asked to continue participation in the trial
and to take oral DFMO 0.5gm/m.sup.2 for 28 days prior to and the
day of the second procedure. Patients were monitored for side
effects using questionnaires and interview techniques. Coagulation
parameters were carefully assessed prior to surgery. In the
operating room just prior to the surgical procedure four
trans-rectal core biopsies of the peripheral zone were taken and
frozen. The pre and post DFMO specimens were sent together to
Arizona on dry ice for histology and polyamine analysis. Radical
prostatectomy specimens were staged according to the TMN updated
1997 staging system (American Joint Committee on Cancer 1997).
[0105] To determine prostate core polyamine contents, cores were
first thawed, washed in phosphate buffered saline (PBS) and
weighted. Samples were then minced in 0.2 N perchloric acid and
sonicated to disrupt cellular material. Samples were centrifuged at
2,000 .times. g for 5 minutes to separate acid soluble and
insoluble fractions. The acid soluble fraction was evaluated for
polyamine contents, using reverse-phase, ion-pared high performance
liquid chromatography (PHLC) methods, as described elsewhere
(Gerner et al. 1994; Meyskens et al 1994; Meyskens et al. 1998).
Briefly, polyamines are separated on a C18 B Ondapak column,
derivatized with o-pthaldehyde after separation and detected by
absorption of the derivatized material at 750 nm.
[0106] Mean, median, and range for polyamine levels are reported in
nmol/mg protein. The limit of detection of our method is 0.01
nmol/mg.
[0107] Routine sextant biopsies and surgical specimens were
processed and reported according to the protocol established by the
department of Pathology at the Long Beach Veterans Administration
Medical Center.
[0108] Histological Changes were assessed by bright field
microscopy methods. Pre- and post DFMO polyamine values were
compared using the Wilcoxon matched-pairs signed rank test. Thus,
for each patient we were able to take into account both the
magnitude and the direction of changes in polyamines due to
DFMO.
Example 2
Results
[0109] Demographics: Forty-nine men signed consents for
participation in the study. Twenty-two had the first set of four
additional cores at time of sextant biopsy. Based on the routine
biopsy results and patient consent ten men were started on DFMO.
Nine men had the second set of biopsies. One man elected to receive
no treatment for his prostate cancer after starting the DFMO. The
detailed demographics of the participants and their routine biopsy
histopathology results are presented in Table 1.
[0110] The mean age of men who completed the trial was 65.6 years
(median 66 years range 56-73 years). Ethnicity of the group was
White (3), Latino (3), Black (1), Asian (1), and mixed White/Asian
(1). Indications for the biopsy were elevated PSA (5), and abnormal
digital rectal exam (3) or both (1). The average PSA before the
first biopsy for the nine patients was 9.4 ng/ml, (median 5.1 ng/ml
range 1.7-47.2) DFMO was given an average of 28.2 days (median 28
days, range 21-35 days). Compliance was 100% as measured by
documentation and interview. Second procedures were radical
retropubic prostatectomy (RRR) (4), repeat biopsy (4), and
transurethral resection of the prostate (TURP) (1)
[0111] Side Effects: Side effects were reported in four men. Two
men had grade 0 side effects involving mild clinical hearing loss
(not confirmed by pure tone audiology), nausea, diarrhea, and
fatigue in one man, and a sudden one-time weakness in another man.
One man had grade 1 vertigo, along with nausea and epigastric pain.
There was no clinically significant change in the platelet counts,
or protime with the DFMO. Bleeding time was checked prior to
surgery and was not altered by DFMO.
[0112] Pathology: (Table 1) Routine pathological diagnosis on the
first biopsy was atypia (2), atypia with inflammation (3), Gleason
sum 5 (1), Gleason sum 6 (2), and Gleason sum 7 (1) (total 9). The
routine pathology specimens at the second procedure demonstrated
BPH (1). BPH and inflammation (1), inflammation (1), atypia (1),
Gleason sum 6 (1), Pt2A Gleason sum 6 (1), pT2b Gleason sum 6 (1),
pT3a Gleason sum 6 (1), and pT4 Gleason sum 8 (1), (total 9).
1TABLE 1 Patient Demographics Patient 1 2 3 4 5 6 7 8 9 Age 56 66
73 69 70 62 67 63 65 Race Black White Latino Asian/White White
White Asian Latino Latino DRE Normal Normal Normal Abnormal
Abnormal Normal Normal Normal Normal PSA 5.8 9.8 2.3 1.7 5.1 2.6
6.3 47.2** 4.1 Biopsy Atypia Gleason 5 Atypia Atypia Atypia Gleason
6 Atypia Gleason 7 Gleason 6 Pathology Inflammation Inflammation
Inflammation Study PSA 4.4 Not done 2.3 1.5 16.6* 11.1* 8.7 23.2
6.8* Days of 28 28 35 30 30 28 25 29 21 DFMO Pre-op PSA 3.7 7.5 2.5
1.4 5 2.3 6.8 35.5 8.2* Procedure Biopsy RRP Biopsy Biopsy TURP RRP
Biopsy RRP RRP Pathology Atypia T3a Gleason 6 Gleason 6 BPH BPH T2b
Inflammation T4 Gleaosn 8 T2a Gleason 6 Inflammation Gleason 6
[0113] Core Histology: No major differences in histology were
observed in core biopsy samples obtained before or after DFMO
treatment. (RB Nagle, personal communication, data not shown).
[0114] PSA: (Table 1) The average PSA before the first biopsy for
the nine patients was 9.4 ng/ml, (median 5.1 ng/ml range 1.7-47.2)
For the four patients with a final benign pathology, we compared
the pre-biopsy PSA or pre-DFMO PSA with the PSA drawn while on
DFMO. The PSA decreased in each patient (patients 1,4,5,7). The PSA
increased in 3 of 5 of the patients with malignant pathology
(patients 3,8,9). Although of these three men, one man's PSA was
very variable (patient 8), and another man's PSA was drawn within 6
weeks of the biopsy and thus the PSA may have been falsely elevated
from the biopsy (patient 9).
[0115] Polyamine Levels: FIG. 1 compares the values of putrescine,
spermidine, spermine, and the spermidine/spermine ratio in the nine
men who had biopsies performed before and after oral DFMO.
Pre-therapy putrescine was detectable prior to DFMO in six men and
non-detectable (<0.01 nmol/mg) in three men. The average
putrescine level was 0.42 nmol/mg (median 0.27, range nd to 0.94).
All men had undetectable levels of putrescine after DFMO. For the
six men with pre-DFMO putrescine level >0.01 the average
decrease from baseline was 97.6%, (p=0.03 1). Spermidine was
measurable in all specimens prior to DFMO. The average pretreatment
level was 1.21 nmol/mg (median 0.81, range 0.49 to 3.82), and
decreased in all specimens after DFMO. The average level of
spermidine after therapy was 0.32 (median 0.21, range nd to 1.18),
and in two specimens the levels were undetectable. The average
percent decrease from baseline was 73.6% (p=0.004). The spermine
levels prior to DFMO were the highest of all the polyamines tested.
Average spermine level prior to DFMO was 29.14 nmol/mg (median
28.85 range 9.88 to 53.66) and after DFMO decreased in all
specimens to an average level of spermine, 14.33 (median 17.40,
range 3.24 to 25.58). The average decrease from baseline was 50.8%
(p=0.004). The spermidine/spermine ratio was calculated for each
specimen. Eight of nine patients had a decrease in this ratio after
DFMO was given. The average decrease from baseline was 50% (median
52% range--25.3% to 97%) (p=0.019). The two patients with
extracapsular prostate cancer had the least decrease from baseline
or an actual increase in spermidine/spermine ratio after treatment
with DFMO (patients 2 and 8).
[0116] Example 3
Discussion
[0117] In this short-term trial, the inventors were able to
demonstrate a significant reduction in the prostate polyamines:
putrescine, spermidine and spermine and the spermidine/spermine
ratio after administration of oral DFMO 0.5gm/m.sup.2 daily for 28
days. Putrescine decreased from baseline by 98% (n=6, p=0.031),
spermidine by 74% (n=9, p=0.004), and spermine by 51% (n=9,
p=0.004). Particularly intriguing was the demonstration of a large
reduction in spermine. This effect of DFMO on spermine levels has
not been observed in other tissues in patients receiving DFMO on
spermine levels has not been observed in other tissues in patients
receiving DFMO therapy (Gerner et al., 1994; Meyskens et al., 1994;
Meyskens et al., 1998).
[0118] A brief review of the inventor's trial design and that of
Messing et al. (1999), discussed supra, points to study differences
that can account for the discordant results between the two
studies. An advantage of the current study was that the inventors
elected to use each male as his own control for polyamine
suppression by using samples from the same male before and after
DFMO. In addition, the samples were not run until both the before
and after samples could be run together to avoid batch differences.
The data shows a wide variation in polyamine levels among the
subjects prior to manipulation, with putrescine demonstrating the
least variability of the polyamines tested. (Put (range
non-detectable (n.d.<0.01)-0.94), Spd (range 0.49-3.82), and Spm
(range 9.96-53.7)). This variability makes it difficult to assess
differences in a small control vs. treatment group, and may be the
answer as to why only putrescine, with the smallest variability,
was significantly changed in the Messing trial. In the inventors'
trial, each man was his own control, and thus the effects of DFMO
were more easily measured. Similar difficulty with the variability
in polyamine levels was addressed by Mitchell et al. (1997). They
reported on polyamine levels in cervical cancer compared to normal
cervical tissue, specifically addressing the potential of using
polyamine levels as intermediate markers. Although there were
differences in the polyamine levels between these types of tissues,
the authors concluded that due to the variability of the polyamine
levels large numbers of subject would be needed to see a
significant result.
[0119] There are also processing issues related to the manner in
which the tissues were managed between these two studies. The
inventors specified peripheral biopsies of the prostate, as the ODC
activities are different in the different prostate lobes of the rat
(they are higher in the ventral lobe as compared to the dorsal
lobe) and it was not known if these same differences would be
observed in the zones of the human prostate (Heston et al., 1982).
In addition, the biopsies taken after 28 days of DFMO just prior to
surgery were carried out just as they had been initially,
transrectally and frozen, to avoid any potential polyamine
degradation or alteration that may occur with cautery or
devasularization of the prostate during surgery. It is unknown what
the impact of ischemia for one to two hours on the prostate is as
it is sytematically devasularized and removed. The unknown effect
of this manipulation is obviated in the study by taking the cores
prior to definitive surgery.
[0120] Another difference between the inventor's study and that of
Messing et al. (1999) is the length of treatment. The subjects
received 4 wks of DFMO (vs 2 wks). Work in the rat prostate
published by Danzin demonstrated that the most significant ODC
inhibition to 10% of the control value by intraperitoneal injection
of DFMO (100 mg/kg every 12 hrs) was seen after 4 to 6 hrs.
Inhibition rose to 50% reduction compared to control by 24 hrs.
Through out the rest of the 8-day study inhibition ranged from 30
to 50% from the control and did not reach a steady state. In the
same paper Danzin measured polyamine levels after two weeks of
treatment and found significant reduction of putrescine, and
spermine levels at low dos DFMO (100 mg/kg) and spermidine levels
at the higher dose (1 gm/kg) (Danzin et al., 1979). The reduction
in putrescine from 202.+-.37 to 4.+-.4 nmol/g after two weeks of
100 mg/kg DFMO makes it difficult to speculate on any further
impact of another 2 wks of treatment on putrescine. But there was
less of a reduction in spermidine (7334.+-.193 to 1188.+-.289) and
no significant reduction in spermine (2968.+-.293 to 2741.+-.346)
at two weeks using 100 mg/kg DFMO. It may be that these later two
polyamines require longer treatment times to see a reduction with
DFMO (Danzin et al. 1979).
[0121] The polyamine levels were evaluated with respect to race,
age, pathology, and days of inhibition with no obvious trends noted
in this small series. The apparent lack of reduction in the
spermidine/spermine ratios with DFMO in the men with
extra-prostatic cancer is of great interest. Of the five men with
prostate cancer, two men were found to have prostate cancer outside
the prostate gland (extracapsular and bladder neck invasion T3 and
T4) and these two men (patients 2 and 8) had little or an actual
increase in the spermidine/spermine ratio with DFMO. Whereas the
specimens with prostate cancer confined to the gland (T2, organ
confined) had a similar reduction in the spermidine/spermine ratio
as that seen with the benign prostate disease (FIG. 2). It is
possible that as the tumor becomes more aggressive, as seen with
extracapsular and bladder neck invasion, alternative polyamine
synthesis or the dysregulation of polyamines occurs. Follow-up of
this observation in a larger cohort would be of great interest. The
polyamine dysregulation could be predictive of a poor prognosis and
that the measured dysregulation of polyamines could be used to
determine which patients need to be treated in a more aggressive
manner or just be observed. A further study would help determine
whether the invasion into other tissues or the dysregulation of
polyamines occurs first. It is also believed that there is a time
before extraprostatic cancer extension when there is measured
dysregulation of polyamines.
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