U.S. patent application number 15/849373 was filed with the patent office on 2018-05-03 for novel treatment of prostate carcinoma.
The applicant listed for this patent is Pellficure Pharmaceuticals, Inc.. Invention is credited to Per Borgstrom.
Application Number | 20180116977 15/849373 |
Document ID | / |
Family ID | 44504259 |
Filed Date | 2018-05-03 |
United States Patent
Application |
20180116977 |
Kind Code |
A1 |
Borgstrom; Per |
May 3, 2018 |
NOVEL TREATMENT OF PROSTATE CARCINOMA
Abstract
Disclosed herein are naphthoquinone analogs, such as plumbagin,
pharmaceutical compositions that include naphthoquinone analogs,
such as plumbagin, and methods of treating diseases and/or
conditions such as cancer with naphthoquinone analogs, such as
plumbagin. Also included are combination therapies wherein a
naphthoquinone analog, such as plumbagin, and a hormone therapy
agent are provided to a subject suffering from a condition such as
cancer.
Inventors: |
Borgstrom; Per; (La Jolla,
CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Pellficure Pharmaceuticals, Inc. |
La Jolla |
CA |
US |
|
|
Family ID: |
44504259 |
Appl. No.: |
15/849373 |
Filed: |
December 20, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15405653 |
Jan 13, 2017 |
9877932 |
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15849373 |
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14829443 |
Aug 18, 2015 |
9655868 |
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15405653 |
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13813394 |
Mar 27, 2013 |
9132105 |
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PCT/US2011/046474 |
Aug 3, 2011 |
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14829443 |
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61370534 |
Aug 4, 2010 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 33/5088 20130101;
A61K 31/6615 20130101; A61K 31/58 20130101; A61K 31/05 20130101;
A61K 31/57 20130101; A61K 31/565 20130101; A61K 31/167 20130101;
A61K 31/58 20130101; A61K 31/05 20130101; A61K 31/122 20130101;
A61K 31/57 20130101; A61K 38/09 20130101; A61P 13/08 20180101; A61K
31/167 20130101; A61K 31/565 20130101; A61P 43/00 20180101; G01N
33/5011 20130101; A61P 5/28 20180101; A61K 31/122 20130101; A61K
31/4166 20130101; A61K 31/277 20130101; A61K 2300/00 20130101; A61K
2300/00 20130101; A61K 2300/00 20130101; A61K 2300/00 20130101;
A61K 45/06 20130101; A61K 2300/00 20130101; A61K 31/277 20130101;
A61K 31/6615 20130101; A61K 31/4166 20130101; A61P 35/00 20180101;
A61K 2300/00 20130101; A61K 2300/00 20130101; A61K 2300/00
20130101; A61K 2300/00 20130101; A61K 31/56 20130101; A61K 2300/00
20130101; A61K 38/09 20130101 |
International
Class: |
A61K 31/122 20060101
A61K031/122; A61K 31/05 20060101 A61K031/05; A61K 38/09 20060101
A61K038/09; A61K 31/6615 20060101 A61K031/6615; A61K 45/06 20060101
A61K045/06; A61K 31/565 20060101 A61K031/565; A61K 31/57 20060101
A61K031/57; A61K 31/4166 20060101 A61K031/4166; A61K 31/56 20060101
A61K031/56; G01N 33/50 20060101 G01N033/50; A61K 31/58 20060101
A61K031/58; A61K 31/277 20060101 A61K031/277; A61K 31/167 20060101
A61K031/167 |
Claims
1. (canceled)
2. A composition comprising: a compound of Formula (I) or a
pharmaceutically acceptable salt of Formula (I): ##STR00019##
wherein: R.sup.1 is hydrogen, R.sup.3 is --OH, and R6 is hydrogen
or --OH; or R.sup.1 is methyl, R.sup.3 is hydrogen, and R6 is
hydrogen; R.sup.2 is hydrogen; R.sup.4 is hydrogen; and R.sup.5 is
hydrogen.
3. The composition of claim 2, wherein the compound of Formula (I)
is juglone.
4. The composition of claim 2, wherein the compound of Formula (I)
is naphthazarin.
5. The composition of claim 2, wherein the compound of Formula (I)
is menadione.
6. The composition of claim 2, wherein said composition is provided
in a product combination, which comprises an androgen deprivation
therapy agent that reduces the production of testosterone.
7. The composition of claim 6, wherein the androgen deprivation
therapy agent is selected from the group consisting of finasteride,
leuprolide, triptorelin, goserelin, abarelix, degarelix, and
abiraterone.
8. The composition of claim 6, wherein the androgen deprivation
therapy agent decreases the subject's serum testosterone level to
about 5-20% of a healthy male subject.
9. The composition of claim 6, wherein said product combination
inhibits the growth of prostate cancer.
10. The composition of claim 6, wherein said product combination
inhibits or delays the onset of castration-resistant prostate
cancer.
11. The composition of claim 6, wherein the compound of Formula (I)
is formulated for oral administration.
12. The composition of claim 6, wherein the compound of Formula (I)
and the androgen deprivation therapy agent are formulated in a
single formulation or a single dosage.
13. The composition of claim 6, wherein the androgen deprivation
therapy agent is formulated for oral administration.
14. The composition of claim 6, wherein the compound of Formula (I)
and the androgen deprivation therapy agent are formulated for oral
administration.
15. The composition of claim 9, wherein said prostate cancer is
androgen dependent prostate cancer.
16. The composition of claim 6, wherein the androgen deprivation
therapy agent decreases the subject's serum testosterone level to
at least about .ltoreq.50 ng/dL.
17. The composition of claim 6, wherein the androgen deprivation
therapy agent decreases the subject's serum testosterone level to
at least about .ltoreq.20 ng/dL.
18. The composition of claim 6, wherein the product combination
results in a decrease in prostate cancer tumor size.
19. A method of inhibiting or delaying the growth of prostate
cancer, comprising administering to a subject having prostate
cancer a therapeutically effective amount of a composition
comprising a compound of Formula (I) or a pharmaceutically
acceptable salt of Formula (I): ##STR00020## wherein: R.sup.1 is
hydrogen, R.sup.3 is --OH, and R6 is hydrogen or --OH; or R.sup.1
is methyl, R.sup.3 is hydrogen, and R6 is hydrogen; R.sup.2 is
hydrogen; R.sup.4 is hydrogen; and R.sup.5 is hydrogen.
20. The method of claim 19, wherein the compound of Formula (I) is
juglone.
21. The method of claim 19, wherein the compound of Formula (I) is
naphthazarin.
22. The method of claim 19, wherein the compound of Formula (I) is
menadione.
23. The method of claim 19, wherein the compound of Formula (I) is
administered to the subject in combination with, subsequent to, or
concomitantly with, an androgen deprivation therapy that reduces
the production of testosterone; and wherein the growth of prostate
cancer is inhibited or delayed.
24. The method of claim 23, wherein the androgen deprivation
therapy is surgical orchiectomy.
25. The method of claim 23, wherein the androgen deprivation
therapy comprises administration of a compound selected from the
group consisting of finasteride, leuprolide, triptorelin,
goserelin, abarelix, degarelix, and abiraterone.
26. The method of claim 23, wherein the androgen deprivation
therapy decreases the subject's serum testosterone level to about
5-20% of a healthy male subject.
27. The method of claim 23, wherein said method inhibits the growth
of prostate cancer.
28. The method of claim 23, wherein said method inhibits or delays
the onset of castration-resistant prostate cancer.
29. The method of claim 23, wherein the compound of Formula (I) is
administered to the subject orally.
30. The method of claim 23, wherein the androgen deprivation
therapy is administered to the subject orally.
31. The method of claim 23, wherein the compound of Formula (I) and
the androgen deprivation therapy are administered to the subject
orally.
32. The method of claim 23, wherein said prostate cancer is
androgen dependent prostate cancer.
33. The method of claim 23, wherein said prostate cancer is
castration-resistant prostate cancer.
34. The method of claim 23, wherein the androgen deprivation
therapy decreases the subject's serum testosterone level to at
least about .ltoreq.50 ng/dL.
35. The method of claim 23, wherein the androgen deprivation
therapy decreases the subject's serum testosterone level to at
least about .ltoreq.20 ng/dL.
36. The method of claim 23, wherein the method results in a
decrease in prostate cancer tumor size.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation application of U.S. Ser.
No. 15/405,653 filed Jan. 13, 2017, which is a continuation
application of U.S. Ser. No. 14/829,443, filed on Aug. 18, 2015,
which issued as U.S. Pat. No. 9,655,868 on May 23, 2017, which is a
divisional application of U.S. Ser. No. 13/813,394, filed on Mar.
27, 2013, which issued as U.S. Pat. No. 9,132,105 on Sep. 15, 2015,
which is a national phase application of International Patent
Application No. PCT/US2011/046474, filed on Aug. 3, 2011, which
designated the United States and was written in English and which
claims the benefit of priority to U.S. Provisional Application No.
61/370,534, filed Aug. 4, 2010. The disclosures of the
aforementioned applications are hereby expressly incorporated by
reference in their entireties.
FIELD OF THE INVENTION
[0002] Aspects of the present application relate to the fields of
chemistry, biochemistry and medicine. More particularly, disclosed
herein are naphthoquinone analogs, such as plumbagin,
pharmaceutical compositions that include naphthoquinone analogs,
such as plumbagin, and methods of treating diseases and/or
conditions with naphthoquinone analogs, such as plumbagin. Also
included are combination therapies, wherein a naphthoquinone
analog, such as plumbagin, and a hormone therapy agent, such as a
hormonal ablation compound, are provided to a subject having a
cancer, such as a prostate cancer.
BACKGROUND OF THE INVENTION
[0003] Prostate cancer develops in the prostate and is typically
slow growing; however, some prostate cancers are aggressive.
Prostate cancer cells are typically androgen/testosterone/DHT
dependent and may metastasize from the prostate to other parts of
the body, particularly the bones and lymph nodes. Treatment options
for prostate cancer that remains within the prostate include
watchful waiting/active surveillance, external beam radiation
therapy, brachytherapy, cryosurgery, HIFU, and surgery. Hormonal
therapy and chemotherapy are often reserved for disease that has
spread beyond the prostate. However, there are exceptions in that
radiation therapy may be used for some advanced tumors, and
hormonal therapy may be used for some early stage tumors.
[0004] After one to three years of hormonal therapy, it is common
that prostate cancer cells resume growth despite the
androgen/testosterone/DHT blockade. Previously referred to as
"hormone-refractory prostate cancer" or "androgen-independent
prostate cancer," the term castration-resistant prostate cancer
(CRPC) is now commonly used. Chemotherapeutic agents and
immunotherapy have been shown to prolong survival after CRPC but
the survival benefit is limited. Despite the efforts of many, the
need for more cancer treatments, in particular prostate cancer
treatments, is manifest.
SUMMARY
[0005] Some embodiments disclosed herein relate to a method of
inhibiting or delaying the growth of prostate cancer by providing a
subject having prostate cancer with a therapeutically effective
amount of a compound of Formula (I), or a pharmaceutically
acceptable salt of Formula (I), while reducing the amount of an
androgen in the subject. In some embodiments, the amount of
androgen can be reduced by providing the subject with an
anti-androgen compound, an estrogen, a luteinizing
hormone-releasing hormone (LHRH) agonist, or a LHRH antagonist. In
some embodiments, the amount of androgen can be reduced by
providing the subject with a steroidal anti-androgen or a
non-steroidal anti-androgen. In some embodiments, the amount of
androgen can be reduced by providing the subject with cyproterone
acetate, abiraterone, finasteride, flutamide, nilutamide,
bicalutamide, ethylstilbestrol (DES), megestrol acetate,
fosfestrol, estamustine phosphate, leuprolide, triptorelin,
goserelin, histrelin, buserelin, abarelix and/or degarelix. In some
embodiments, the method of inhibiting or delaying the growth of
prostate cancer can reduce the subject's serum testosterone level
to between about 20-50 ng/dL. In some embodiments, the method of
inhibiting or delaying the growth of prostate cancer can reduce the
subject's serum testosterone level to less than about 50 ng/dL. In
some embodiments, the method of inhibiting or delaying the growth
of prostate cancer can reduce the subject's serum testosterone
level to less than about 20 ng/dL.
[0006] Some embodiments disclosed herein relate to a method for
identifying a compound that inhibits or delays prostate cancer cell
growth by providing a pseudo-orthotopic chamber mouse model,
wherein the mouse model has prostate cancer; reducing the level of
an androgen in said mouse model; providing the mouse model with a
compound of Formula (I) or a pharmaceutically acceptable salt or a
prodrug thereof; and evaluating whether the compound is effective
in inhibiting the growth of prostate cancer cells.
[0007] Some embodiments disclosed herein relate to a method of
inhibiting or delaying the onset of castration-resistant prostate
cancer (CRPC) by classifying a subject as a member of a population
that is at risk for developing CRPC; providing said subject with a
therapeutically effective amount of a compound of Formula (I), or a
pharmaceutically acceptable salt of Formula (I), while reducing the
amount of an androgen in said subject; and evaluating an inhibition
or delay of prostate cancer cell growth or a marker thereof or the
onset of CRPC.
[0008] Some embodiments disclosed herein relate to a method of
identifying a compound that inhibits or delays prostate cancer cell
growth by contacting prostate cancer cells with a compound of
Formula (I) in the absence of androgen; determining the presence or
absence of an inhibition or delay in prostate cancer cell growth;
and classifying the compound into a population that inhibits or
delays prostate cancer cell growth in the absence of androgen, or
into a population that does not inhibit or delay prostate cancer
cell growth.
[0009] Some embodiments disclosed herein relate to a method of
making a prostate cancer therapeutic by contacting prostate cancer
cells with a compound of Formula (I) in the absence of androgen;
determining the presence or absence of an inhibition or delay in
prostate cancer cell growth; selecting a compound of Formula (I)
that inhibits prostate cancer cell growth in the absence of
androgen; and formulating the compound that inhibits or delays
prostate cancer cell growth in the absence of androgen for
administration to a subject suffering from prostate cancer.
[0010] Some embodiments disclosed herein relate to a combination of
a compound of Formula (I) or a pharmaceutically acceptable salt of
Formula (I) and a hormone therapy agent for inhibiting or delaying
prostate cancer cell growth or the onset of castration-resistant
prostate cancer (CRPC). In some embodiments, the hormone therapy
agent can be cyproterone acetate, abiraterone, finasteride,
flutamide, nilutamide, bicalutamide, ethylstilbestrol (DES),
megestrol acetate, fosfestrol, estamustine phosphate, leuprolide,
triptorelin, goserelin, histrelin, buserelin, abarelix or degarelix
or any combination of one or more of said compounds.
[0011] Some embodiments disclosed herein relate to a combination of
a compound of Formula (I) or a pharmaceutically acceptable salt of
Formula (I) and a hormone therapy agent for use in decreasing
prostate tumor size. In some embodiments, the hormone therapy agent
can be cyproterone acetate, abiraterone, finasteride, flutamide,
nilutamide, bicalutamide, ethylstilbestrol (DES), megestrol
acetate, fosfestrol, estamustine phosphate, leuprolide,
triptorelin, goserelin, histrelin, buserelin, abarelix or degarelix
or any combination of one or more of said compounds.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] FIG. 1 shows the effect of naphthoquinone analogs on
PTEN-P2/GFP cell proliferation.
[0013] FIG. 2 shows the effect of naphthoquinone analogs on
PTEN-P2/GFP cell proliferation.
[0014] FIG. 3 shows the dose response of plumbagin in PTEN-P2/GFP
cells.
[0015] FIG. 4 compares the growth of tumors without treatment, with
castration alone, with plumbagin alone, and the combination of
castration and plumbagin.
[0016] FIG. 5 shows the effect of plumbagin at 0.1 mg/kg, 0.3 mg/kg
and 1 mg/kg, given in combination with castration.
[0017] FIG. 6 illustrates the effect of adding plumbagin after
surgical castration.
[0018] FIG. 7 illustrates increasing apoptosis (AP) and mitosis
(MI) after daily administration of plumbagin ip (2 mg/kg).
[0019] FIG. 8 illustrates the effect of plumbagin in human LNCaP
cells in the absence of dihydrotestosterone.
DETAILED DESCRIPTION
I. Definitions
[0020] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as is commonly understood by one
of ordinary skill in the art. All patents, applications, published
applications and other publications referenced herein are
incorporated by reference in their entirety unless stated
otherwise. In the event that there are a plurality of definitions
for a term herein, those in this section prevail unless stated
otherwise.
[0021] As used herein, any "R" group(s) such as, without
limitation, R, R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5,
R.sup.6, R.sup.7, R.sup.8, R.sup.9, R.sup.10, R.sup.11, R.sup.12,
R.sup.13, and R.sup.14 represent substituents that can be attached
to the indicated atom. An R group may be substituted or
unsubstituted.
[0022] As used herein, "C.sub.a to C.sub.b" in which "a" and "b"
are integers refer to the number of carbon atoms in an alkyl,
alkenyl or alkynyl group, or the number of carbon atoms in the ring
of a cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl or
heteroalicyclyl group. That is, the alkyl, alkenyl, alkynyl, ring
of the cycloalkyl, ring of the cycloalkenyl, ring of the
cycloalkynyl, ring of the aryl, ring of the heteroaryl or ring of
the heteroalicyclyl can contain from "a" to "b", inclusive, carbon
atoms. Thus, for example, a "C.sub.1 to C.sub.4 alkyl" group refers
to all alkyl groups having from 1 to 4 carbons, that is,
CH.sub.3--, CH.sub.3CH.sub.2--, CH.sub.3CH.sub.2CH.sub.2--,
(CH.sub.3).sub.2CH--, CH.sub.3CH.sub.2CH.sub.2CH.sub.2--,
CH.sub.3CH.sub.2CH(CH.sub.3)-- and (CH.sub.3).sub.3C--. If no "a"
and "b" are designated with regard to an alkyl or alkenyl group,
the broadest range described in these definitions is to be
assumed.
[0023] As used herein, "alkyl" refers to a straight or branched
hydrocarbon chain that comprises a fully saturated (no double or
triple bonds) hydrocarbon group. The alkyl group may have 1 to 20
carbon atoms (whenever it appears herein, a numerical range such as
"1 to 20" refers to each integer in the given range; e.g., "1 to 20
carbon atoms" means that the alkyl group may consist of 1 carbon
atom, 2 carbon atoms, 3 carbon atoms, etc., up to and including 20
carbon atoms, although the present definition also covers the
occurrence of the term "alkyl" where no numerical range is
designated). The alkyl group may also be a medium size alkyl having
1 to 10 carbon atoms. The alkyl group could also be a lower alkyl
having 1 to 6 carbon atoms. The alkyl group of the compounds may be
designated as "C.sub.1-C.sub.4 alkyl" or similar designations. By
way of example only, "C.sub.1-C.sub.4 alkyl" indicates that there
are one to four carbon atoms in the alkyl chain, i.e., the alkyl
chain is selected from methyl, ethyl, propyl, iso-propyl, n-butyl,
isobutyl, sec-butyl, and t-butyl. Typical alkyl groups include, but
are in no way limited to, methyl, ethyl, propyl, isopropyl, butyl,
isobutyl, tertiary butyl, pentyl and hexyl. The alkyl group may be
substituted or unsubstituted.
[0024] As used herein, "alkenyl" refers to an alkyl group that
contains in the straight or branched hydrocarbon chain one or more
double bonds. An alkenyl group may be unsubstituted or
substituted.
[0025] The term "halogen" as used herein, means any one of the
radio-stable atoms of column 7 of the Periodic Table of the
Elements, such as, fluorine, chlorine, bromine and iodine.
[0026] Whenever a group is described as being "optionally
substituted" that group may be unsubstituted or substituted with
one or more of the indicated substituents. Likewise, when a group
is described as being "unsubstituted or substituted" if
substituted, the substituent may be selected from one or more the
indicated substituents. If no substituents are indicated, it is
meant that the indicated "optionally substituted" or "substituted"
group may be substituted with one or more group(s) individually and
independently selected from alkyl, alkenyl, alkynyl, cycloalkyl,
cycloalkenyl, cycloalkynyl, aryl, heteroaryl, heteroalicyclyl,
aralkyl, heteroaralkyl, (heteroalicyclyl)alkyl, hydroxy, protected
hydroxyl, alkoxy, aryloxy, acyl, mercapto, alkylthio, arylthio,
cyano, halogen, thiocarbonyl, O-carbamyl, N-carbamyl,
O-thiocarbamyl, N-thiocarbamyl, C-amido, N-amido, S-sulfonamido,
N-sulfonamido, C-carboxy, protected C-carboxy, O-carboxy,
isocyanato, thiocyanato, isothiocyanato, nitro, silyl, sulfenyl,
sulfinyl, sulfonyl, haloalkyl, haloalkoxy, trihalomethanesulfonyl,
trihalomethanesulfonamido, amino, mono-substituted amino group and
di-substituted amino group, and protected derivatives thereof.
[0027] The term "naphthoquinone analog" refers to a compound of
Formula (I) wherein R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5,
and R.sup.6 are as defined herein.
[0028] The term "pharmaceutically acceptable salt" refers to a salt
of a compound that does not cause significant irritation to an
organism to which it is administered and does not abrogate the
biological activity and properties of the compound. In some
embodiments, the salt is an acid addition salt of the compound.
Pharmaceutical salts can be obtained by reacting a compound with
inorganic acids such as hydrohalic acid (e.g., hydrochloric acid or
hydrobromic acid), sulfuric acid, nitric acid and phosphoric acid.
Pharmaceutical salts can also be obtained by reacting a compound
with an organic acid such as aliphatic or aromatic carboxylic or
sulfonic acids, for example formic, acetic, succinic, lactic,
malic, tartaric, citric, ascorbic, nicotinic, methanesulfonic,
ethanesulfonic, p-toluensulfonic, salicylic or naphthalenesulfonic
acid. Pharmaceutical salts can also be obtained by reacting a
compound with a base to form a salt such as an ammonium salt, an
alkali metal salt, such as a sodium or a potassium salt, an
alkaline earth metal salt, such as a calcium or a magnesium salt, a
salt of organic bases such as dicyclohexylamine,
N-methyl-D-glucamine, tris(hydroxymethyl)methylamine,
C.sub.1-C.sub.7 alkylamine, cyclohexylamine, triethanolamine,
ethylenediamine, and salts with amino acids such as arginine and
lysine.
[0029] It is understood that, in any compound described herein
having one or more chiral centers, if an absolute stereochemistry
is not expressly indicated, then each center may independently be
of R-configuration or S-configuration or a mixture thereof. Thus,
the compounds provided herein may be disatereomerically pure,
disatereomerically enriched, or may be stereoisomeric mixtures. In
addition it is understood that, in any compound described herein
having one or more double bond(s) generating geometrical isomers
that can be defined as E or Z, each double bond may independently
be E or Z a mixture thereof. Likewise, it is understood that, in
any compound described, all tautomeric forms are also intended to
be included.
[0030] The term "pharmaceutical composition" refers to a mixture of
a compound disclosed herein with other chemical components, such as
diluents or carriers. The pharmaceutical composition facilitates
administration of the compound to an organism. Pharmaceutical
compositions can also be obtained by reacting compounds with
inorganic or organic acids such as hydrochloric acid, hydrobromic
acid, sulfuric acid, nitric acid, phosphoric acid, methanesulfonic
acid, ethanesulfonic acid, p-toluenesulfonic acid and salicylic
acid. Pharmaceutical compositions will generally be tailored to the
specific intended route of administration.
[0031] The term "physiologically acceptable" defines a carrier,
diluent or excipient that does not abrogate the biological activity
and properties of the compound.
[0032] As used herein, a "carrier" refers to a compound that
facilitates the incorporation of a compound into cells or tissues.
For example, without limitation, dimethyl sulfoxide (DMSO) is a
commonly utilized carrier that facilitates the uptake of many
organic compounds into cells or tissues of a subject.
[0033] As used herein, a "diluent" refers to an ingredient in a
pharmaceutical composition that lacks pharmacological activity but
may be pharmaceutically necessary or desirable. For example, a
diluent may be used to increase the bulk of a potent drug whose
mass is too small for manufacture and/or administration. It may
also be a liquid for the dissolution of a drug to be administered
by injection, ingestion or inhalation. A common form of diluent in
the art is a buffered aqueous solution such as, without limitation,
phosphate buffered saline that mimics the composition of human
blood.
[0034] As used herein, an "excipient" refers to an inert substance
that is added to a pharmaceutical composition to provide, without
limitation, bulk, consistency, stability, binding ability,
lubrication, disintegrating ability etc., to the composition. A
"diluent" is a type of excipient.
[0035] As used herein, a "subject" refers to an animal that is the
object of treatment, observation or experiment. "Animal" includes
cold- and warm-blooded vertebrates and invertebrates such as fish,
shellfish, reptiles and, in particular, mammals. "Mammal" includes,
without limitation, mice, rats, rabbits, guinea pigs, dogs, cats,
sheep, goats, cows, horses, primates, such as monkeys, chimpanzees,
and apes, and, in particular, humans. In some embodiments, the
subject is human.
[0036] As used herein, the terms "treating," "treatment,"
"therapeutic," or "therapy" do not necessarily mean total cure or
abolition of the disease or condition. Any alleviation of any
undesired signs or symptoms of a disease or condition, to any
extent can be considered treatment and/or therapy. Furthermore,
treatment may include acts that may worsen the patient's overall
feeling of well-being or appearance.
[0037] The term "therapeutically effective amount" is used to
indicate an amount of an active compound, or pharmaceutical agent,
that elicits the biological or medicinal response indicated. For
example, a therapeutically effective amount of compound can be the
amount needed to prevent, alleviate or ameliorate symptoms of
disease or prolong the survival of the subject being treated. This
response may occur in a tissue, system, animal or human and
includes alleviation of the signs or symptoms of the disease being
treated. Determination of a therapeutically effective amount is
well within the capability of those skilled in the art, in view of
the disclosure provided herein. The therapeutically effective
amount of the compounds disclosed herein required as a dose will
depend on the route of administration, the type of animal,
including human, being treated, and the physical characteristics of
the specific animal under consideration. The dose can be tailored
to achieve a desired effect, but will depend on such factors as
weight, diet, concurrent medication and other factors which those
skilled in the medical arts will recognize.
[0038] As used herein, the term "hormone therapy agent" refers to
anti-androgens (including steroidal anti-androgens and
non-steroidal anti-androgens), estrogens, luteinizing
hormone-releasing hormone (LHRH) agonists, and LHRH antagonists, as
well as, hormonal ablation therapy. Exemplary hormone therapy
agents include, but are not limited to, cyproterone acetate,
abiraterone, finasteride, flutamide, nilutamide, bicalutamide,
ethyl stilbestrol (DES), megestrol acetate, fosfestrol, estamustine
phosphate, leuprolide, triptorelin, goserelin, histrelin,
buserelin, abarelix and degarelix.
[0039] As used in this specification, whether in a transitional
phrase or in the body of the claim, the terms "comprise(s)" and
"comprising" are to be interpreted as having an open-ended meaning.
That is, the terms are to be interpreted synonymously with the
phrases "having at least" or "including at least." When used in the
context of a process, the term "comprising" means that the process
includes at least the recited steps, but may include additional
steps. When used in the context of a compound, composition or
device, the term "comprising" means that the compound, composition
or device includes at least the recited features or components, but
may also include additional features or components. The section
below describes some of the compounds that can be used to treat
cancer, or inhibit or delay the growth of cancer cells, especially
prostate cancer cells alone or in combination with one or more
androgen deprivation therapies (e.g., castration, hormonal
castration, hormonal ablation, or hormone therapy).
II. Compounds of Formula (I)
[0040] Some embodiments disclosed herein relate to a compound of
Formula (I), a pharmaceutically acceptable salt thereof, and
methods of using these compounds with and without a hormone therapy
agent, as described herein, to inhibit, delay, treat, or prevent
prostate cancer cell growth or prostate cancer in a subject in need
thereof. Formula (I):
##STR00001##
wherein: R.sup.1 can be selected from hydrogen, halogen, an
optionally substituted C.sub.1-18 alkyl, an optionally substituted
C.sub.2-18 alkenyl, --OR.sup.7 and --SR.sup.8; R.sup.2 can be
selected from hydrogen, halogen, an optionally substituted
C.sub.1-6 alkyl, an optionally substituted C.sub.2-6 alkenyl,
--OR.sup.9 and --SR.sup.10; R.sup.3 can be selected from hydrogen,
an optionally substituted C.sub.1-6 alkyl, and --OR.sup.11; R.sup.4
can be selected from hydrogen, an optionally substituted C.sub.1-6
alkyl, and --OR.sup.12; R.sup.5 can be selected from hydrogen, an
optionally substituted C.sub.1-6 alkyl, and --OR.sup.14; R.sup.6
can be selected from hydrogen, an optionally substituted C.sub.1-6
alkyl, and --OR.sup.14; and R.sup.7, R.sup.8, R.sup.9, R.sup.10,
R.sup.11, R.sup.12, R.sup.13, and R.sup.14 can be independently
selected from hydrogen and an optionally substituted C.sub.1-6
alkyl.
[0041] In some embodiments, R.sup.1 can be hydrogen. In some
embodiments, R.sup.1 can be halogen. In some embodiments, R.sup.1
can be chloro. In some embodiments, R.sup.1 can be an optionally
substituted C.sub.1-18 alkyl. Examples of optionally substituted
C.sub.1-18-alkyls include, but are not limited to, optionally
substituted variants of the following: methyl, ethyl, n-propyl,
isopropyl, n-butyl, isobutyl, tert-butyl, pentyl, hexyl, heptyl,
octyl, nonanyl, decanyl, undecanyl, dodecanyl, tridecanyl,
tetradecanyl, pentadecanyl, hexadecanyl, heptadecanyl, octadecanyl,
and phytanyl. Optionally substituted C.sub.1-18-alkyls can be
branched or straight-chained. In some embodiments, R.sup.1 can be
an optionally substituted C.sub.1-6 alkyl. In some embodiments,
R.sup.1 can be methyl. In some embodiments, R.sup.1 can be t-butyl.
In some embodiments, R.sup.1 can be an optionally substituted
C.sub.2-18 alkenyl. Examples of optionally substituted
C.sub.2-18-alkenyls include, but are not limited to, optionally
substituted variants of the following: ethenyl, propenyl, butenyl,
pentenyl, hexenyl, heptenyl, octenyl, nonenyl, decenyl, undecenyl,
dodecenyl, tridecenyl, tetradecenyl, pentadecenyl, hexadecenyl,
heptadecenyl, octadecenyl, and phytenyl. Optionally substituted
C.sub.2-18-alkenyls can be branched or straight-chained, and can
include one or more double bonds. In some embodiments, R.sup.1 can
be an optionally substituted C.sub.2-6 alkenyl. In some
embodiments, R.sup.1 can be --OR.sup.7, wherein R.sup.7 is
hydrogen. In some embodiments, R.sup.1 can be --OR.sup.7, wherein
R.sup.7 is an optionally substituted C.sub.1-6 alkyl. In some
embodiments, R.sup.1 can be --OR.sup.7, wherein R.sup.7 is methyl.
In some embodiments, R.sup.1 can be --SR.sup.8, wherein R.sup.8 is
hydrogen. In some embodiments, R.sup.1 can be --SR.sup.8, wherein
R.sup.8 is an optionally substituted C.sub.1-6 alkyl. In some
embodiments, R.sup.1 can be --SR.sup.8, wherein R.sup.8 is
C.sub.1-6 alkyl optionally substituted with hydroxy. In some
embodiments, R.sup.1 can be --SR.sup.8, wherein R.sup.8 is
--CH.sub.2CH.sub.2OH.
[0042] In some embodiments, R.sup.2 can be hydrogen. In some
embodiments, R.sup.2 can be halogen. In some embodiments, R.sup.2
can be chloro. In some embodiments, R.sup.2 can be an optionally
substituted C.sub.1-6 alkyl. Examples of optionally substituted
C.sub.1-6-alkyls include optionally substituted variants of the
following: methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl,
tert-butyl, pentyl (branched and straight-chained), and hexyl
(branched and straight-chained). In some embodiments. R.sup.2 can
be methyl. In some embodiments, R.sup.2 can be an optionally
substituted C.sub.2-6 alkenyl. Examples of optionally substituted
C.sub.2-6-alkenyls include optionally substituted variants of the
following: ethenyl, propenyl, butenyl, pentenyl (branched and
straight-chained), and hexenyl (branched and straight-chained). In
some embodiments, R.sup.2 can be
--CH.sub.2--CH.dbd.C(CH.sub.3).sub.2. In some embodiments, R.sup.2
can be --OR.sup.9, wherein R.sup.9 is hydrogen. In some
embodiments, R.sup.2 can be --OR.sup.9, wherein R.sup.9 is an
optionally substituted C.sub.1-6 alkyl. In some embodiments,
R.sup.2 can be --OR.sup.9, wherein R.sup.9 is methyl. In some
embodiments, R.sup.2 can be --SR.sup.10, wherein R.sup.10 is
hydrogen. In some embodiments, R.sup.2 can be --SR.sup.10, wherein
R.sup.10 is an optionally substituted C.sub.1-6 alkyl. In some
embodiments, R.sup.2 can be --SR.sup.1', wherein R.sup.10 is
C.sub.1-6 alkyl optionally substituted with hydroxy. In some
embodiments, R.sup.2 can be --SR.sup.0, wherein R.sup.10 is
--CH.sub.2CH.sub.2OH.
[0043] In some embodiments, R.sup.3 can be hydrogen. In some
embodiments. R.sup.3 can be an optionally substituted C.sub.1-6
alkyl. In some embodiments, R.sup.3 can be --OR.sup.11, wherein
R.sup.11 is hydrogen. In some embodiments, R.sup.3 can be
--OR.sup.11, wherein R.sup.11 is an optionally substituted
C.sub.1-6 alkyl.
[0044] In some embodiments, R.sup.4 can be hydrogen. In some
embodiments, R.sup.4 can be an optionally substituted C.sub.1-6
alkyl. In some embodiments, R.sup.4 can be t-butyl. In some
embodiments, R.sup.4 can be --OR.sup.12, wherein R.sup.12 is
hydrogen. In some embodiments, R.sup.4 can be --OR.sup.2, wherein
R.sup.12 is an optionally substituted C.sub.1-6 alkyl.
[0045] In some embodiments, R.sup.5 can be hydrogen. In some
embodiments, R.sup.5 can be an optionally substituted C.sub.1-6
alkyl. In some embodiments, R.sup.5 can be --OR.sup.13, wherein
R.sup.13 is hydrogen. In some embodiments, R.sup.5 can be
--OR.sup.13, wherein R.sup.13 is an optionally substituted
C.sub.1-6 alkyl.
[0046] In some embodiments, R.sup.6 can be hydrogen. In some
embodiments, R.sup.6 can be an optionally substituted C.sub.1-6
alkyl. In some embodiments, R.sup.6 can be --OR.sup.14, wherein
R.sup.14 is hydrogen. In some embodiments, R.sup.6 can be
--OR.sup.14, wherein R.sup.13 is an optionally substituted
C.sub.1-6 alkyl.
[0047] In some embodiments, R.sup.7, R.sup.8, R.sup.9, R.sup.10,
R.sup.11, R.sup.12, R.sup.13, and R.sup.14 can be independently
selected from hydrogen. In some embodiments, R.sup.7, R.sup.8,
R.sup.9, R.sup.10, R.sup.11, R.sup.12, R.sup.13, and R.sup.14 can
be independently selected from C.sub.1-6 alkyl. In some
embodiments, R.sup.7, R.sup.8, R.sup.9, R.sup.10, R.sup.11,
R.sup.12, R.sup.13, and R.sup.14 can be independently selected from
C.sub.1-6 alkyl, wherein the C.sub.1-6 alkyl can be optionally
substituted with a group selected from halogen, hydroxy, and
C.sub.1-4 alkyl.
[0048] In some embodiments. R.sup.1 can be selected from hydrogen,
halogen, an optionally substituted C.sub.1-6 alkyl, --OR.sup.7 and
--SR.sup.8; R.sup.2 can be selected from hydrogen, halogen, an
optionally substituted C.sub.1-6 alkyl, --OR.sup.9 and --SR.sup.10;
R.sup.3 can be selected from hydrogen and --OR.sup.11; R.sup.4 can
be selected from hydrogen and an optionally substituted C.sub.1-6
alkyl; R.sup.5 can be hydrogen; R.sup.6 can be selected from
hydrogen and --OR.sup.14; and R.sup.7, R.sup.8, R.sup.9, R.sup.10,
R.sup.11, R.sup.12, R.sup.13, and R.sup.14 can be independently
selected from hydrogen and an optionally substituted C.sub.1-6
alkyl.
[0049] In some embodiments, R.sup.1, R.sup.2, R.sup.3, R.sup.4,
R.sup.5 and R.sup.6 can each be hydrogen. In some embodiments,
R.sup.1 can be methyl; R.sup.3 can be --OH; and R.sup.2, R.sup.4,
R.sup.5 and R.sup.6 can each be hydrogen. In some embodiments,
R.sup.3 and R.sup.6 can each be --OH; and R.sup.1, R.sup.2, R.sup.4
and R.sup.5 can each be hydrogen. In some embodiments, R.sup.3 can
be --OH; and R.sup.1, R.sup.2, R.sup.4, R.sup.5 and R.sup.6 can
each be hydrogen. In some embodiments, R.sup.1 and R.sup.2 can each
be --SCH.sub.2CH.sub.2OH; and R.sup.3, R.sup.4, R.sup.5 and R.sup.6
can each be hydrogen. In some embodiments, R.sup.1 and R.sup.2 can
each be --OCH.sub.3; and R.sup.3, R.sup.4, R.sup.5 and R.sup.6 can
each be hydrogen. In some embodiments, R.sup.1 can be --OCH.sub.3;
and R.sup.2, R.sup.3, R.sup.4, R.sup.5 and R.sup.6 can each be
hydrogen. In some embodiments, R.sup.1 can be methyl; and R.sup.2,
R.sup.3, R.sup.4, R.sup.5 and R.sup.6 can each be hydrogen. In some
embodiments, R.sup.1 and R.sup.2 can each be chloro; and R.sup.3,
R.sup.4, R.sup.5 and R.sup.6 can each be hydrogen. In some
embodiments, R.sup.1 can be --OH; and R.sup.2, R.sup.3, R.sup.4,
R.sup.5 and R.sup.6 can each be hydrogen. In some embodiments,
R.sup.1 can be phytenyl; R.sup.2 can be methyl; and R.sup.3,
R.sup.4, R.sup.5 and R.sup.6 can each be hydrogen. In some
embodiments, R.sup.1 and R.sup.4 can each be t-butyl; and R.sup.2,
R.sup.3, R.sup.5 and R.sup.6 can each be hydrogen. In some
embodiments. R.sup.1 can be --OH; R.sup.2 can be
--CH.sub.2--CH.dbd.C(CH.sub.3).sub.2; and R.sup.3, R.sup.4, R.sup.5
and R.sup.6 can each be hydrogen.
[0050] In some embodiments, at least one of R.sup.1, R.sup.2,
R.sup.3, R.sup.4, R.sup.5 and R.sup.6 cannot be hydrogen. In some
embodiments, when R.sup.1 is methyl; and R.sup.2, R.sup.4, R.sup.5
and R.sup.6 are each hydrogen; then R.sup.3 cannot be --OH. In some
embodiments, when R.sup.1, R.sup.2, R.sup.4 and R.sup.5 are each
hydrogen; then at least one of R.sup.3 and R.sup.6 cannot be --OH.
In some embodiments, when R.sup.1, R.sup.2, R.sup.4, R.sup.5 and
R.sup.6 are each hydrogen; then R.sup.3 cannot be --OH. In some
embodiments, when R.sup.3, R.sup.4, R.sup.5 and R.sup.6 are each
hydrogen; then at least one of R.sup.1 and R.sup.2 cannot be
--SCH.sub.2CH.sub.2OH. In some embodiments, when R.sup.3, R.sup.4,
R.sup.5 and R.sup.6 are each hydrogen; then at least one of R.sup.1
and R.sup.2 cannot be --OCH.sub.3. In some embodiments, when
R.sup.2, R.sup.3, R.sup.4, R.sup.5 and R.sup.6 are each hydrogen;
then R.sup.1 cannot be --OCH.sub.3. In some embodiments, when
R.sup.2, R.sup.3, R.sup.4, R.sup.5 and R.sup.6 are each hydrogen;
then R.sup.1 cannot be methyl. In some embodiments, when R.sup.3,
R.sup.4, R.sup.5 and R.sup.6 are each hydrogen; then at least one
of R.sup.1 and R.sup.2 cannot be chloro. In some embodiments, when
R.sup.2, R.sup.3, R.sup.4, R.sup.5 and R.sup.6 are each hydrogen;
then R.sup.1 cannot be --OH. In some embodiments, when R.sup.2 is
methyl; and R.sup.3, R.sup.4, R.sup.5 and R.sup.6 are each
hydrogen; then R.sup.1 cannot be phytenyl. In some embodiments,
when R.sup.2, R.sup.3, R.sup.5 and R.sup.6 are each hydrogen; then
at least one of R.sup.1 and R.sup.4 cannot be t-butyl. In some
embodiments, when R.sup.2 is --CH.sub.2--CH.dbd.C(CH.sub.3).sub.2;
and R.sup.3, R.sup.4, R.sup.5 and R.sup.6 are each hydrogen; then
R.sup.1 cannot be --OH.
[0051] Examples of compounds of Formula (I) include, but are not
limited to the following:
##STR00002##
[0052] In some embodiments, the compound of Formula (I) can be a
dimer, such that one of R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5
or R.sup.6 has the structure of Formula (I). For example, in some
embodiments, the compound of Formula (I) can be Lawsone dimer:
##STR00003##
[0053] The section below describes some of the conventional
therapies that can be used to inhibit or delay prostate cancer cell
growth and/or treat or prevent prostate cancer. It should be
understood that the inventive therapies described herein can be
performed with and without any of the conventional therapies for
prostate cancer including anyone or more of the therapies described
in the following section.
III. Prostate Cancer
[0054] There were an estimated 192,280 new cases of prostate cancer
diagnosed in the U.S. in 2009 and an estimated 27,360 deaths. About
90% of patients with advanced disease will develop bone metastases,
associated with severe pain, loss of mobility, and spinal cord
compression. Other affected organs may include the liver, lungs and
brain. Advanced prostate cancer is resistant to hormone therapy,
radiation and conventional chemotherapy. Although the 5-year
survival rate is close to 100% for local disease, it drops to 30%
for advanced cancer.
[0055] There have been some advances in the treatment of prostate
cancer recently, including new surgical approaches and improvements
in radiotherapy. For example:
[0056] 1) In 1986, surgeons developed a technique (using da Vinci
Prostatectomy) that allowed the removal of the prostate while
minimizing nerve damage, thereby decreasing adverse side
effects.
[0057] 2) In addition, clinical researchers improved a
long-established radiotherapy technique known as brachytherapy,
which involves the implantation of a small amount of radioactive
material (seeds) into the prostate. This radiation therapy method
is an effective treatment for early-stage prostate cancer.
[0058] 3) There have also been advances in hormonal therapy for
prostate cancer including the development of gonadotropin-releasing
hormone (GnRH) agonists, which inhibit the ability of the pituitary
gland to stimulate the testes to make testosterone.
[0059] 4) Advances have also been made in chemotherapy for prostate
cancer. In 2004, results from two large NCI-sponsored clinical
trials showed that use of the drug docetaxel could prolong the
survival of men who had advanced prostate cancer which no longer
responded to hormonal therapy.
[0060] Unfortunately, should the prostate-specific antigen (PSA)
level remain above zero after radical prostatectomy is performed,
with conventional therapy or with advanced therapy using da Vinci
Prostatectomy, this indicates that the prostate cancer has spread
outside the capsule, i.e., disseminated disease, and to date, there
is no curable treatment for this.
[0061] Thus, all current hormonal, as well as, chemotherapy
treatment regimens for such disseminated androgen dependent
prostate cancers are palliative. Subsequently, even if there have
been advances in the treatment of prostate cancer, finding new
strategies for treatment of disseminated disease remains a crucial
challenge. The section below provides more details on the use of
compounds of Formula (I) to inhibit or delay the growth of cancer
cells, in particular prostate cancer cells.
IV. Compounds of Formula (I) as Anticancer Agents
[0062] Compounds of Formula (I) have significant anti-cancer
properties. For example, plumbagin
(5-hydroxy-2-methylnaphthalene-1,4-dione) is a naturally occurring
naphthoquinone that can be found in various medicinal herbal
species, including Plumbago zeylanica, Statice limonium, and
Limonium carolinianum. Plumbagin has demonstrated anticancer effect
toward fibrosarcomas (ED.sub.50 0.75 mg/kg body weight) and P388
lymphocytic leukemia (ED.sub.50 4 mg/kg body weight), induced
regression of hepatoma, and has inhibited growth and invasion of
hormone-refractory prostate cancer. Aziz et al., Cancer Res. 2008,
68(21):9024-322. Furthermore, plumbagin has shown to be a promising
chemopreventive/anticarcinogenic agent against intestinal
neoplasia.
[0063] Without wishing to be bound by theory, it is contemplated
that the primary mechanism of cytotoxic action of plumbagin and
other quinoid compounds is due to redox-cycling and electrophilic
arylation. Plumbagin can be reduced by electron transfer from
flavoprotein to a semiquinone radical, which can, in turn, reduce
oxygen to superoxide. The resulting superoxide can consequently be
converted into hydrogen peroxide, hydroxyl radicals, and/or
peroxynitrite, all of which are highly reactive oxygen species
(ROS) with potent cytotoxic and tumoricidial effects.
[0064] While still not wishing to be bound by theory, an additional
antitumor mechanism of plumbagin and related quinones can involve
direct arylation of intracellular thiols leading to depletion of
glutathione (GSH). Depletion of GSH may ultimately result in
alkylation of cellular macromolecules and in their inactivation.
Moreover, it has been shown that low dose concentrations of
plumbagin (5 umol/L) can inhibit expression of multiple molecular
targets, including protein kinase Cq (PKCq), phosphatidylinositol
3-kinase (PI3K), AKT, activation of transcription factors activator
protein-1 (AP-1), nuclear factor-.kappa.B (NF-.kappa.B), and signal
transducer and activator of transcription 3 (Stat3) in prostate
carcinoma cells. Such activities may contribute to the tumoricidial
effects of plumbagin.
[0065] Studies using plumbagin in pre-clinical models have revealed
that treatment with plumbagin can result in slower growth of
androgen independent prostate cancer, and that the mechanism behind
the slower growth may be due to apoptosis of prostate tumor
cells.
[0066] It is contemplated that several compounds of Formula (I)
have anti-cancer activity and that this anti-cancer activity,
especially with respect to prostate cancer, can be significantly
improved (e.g., synergy can be obtained) when the compounds are
provided in conjunction with a blockade of
testosterone/androgen/DHT (e.g., castration, a hormone treatment
therapy, such as hormonal ablation). For example, it is believed
that the administration of menadione (vitamin K3) to a subject in
need thereof will effectively inhibit the growth of prostate cancer
cells and thereby reduce the incidence of fatal prostate cancer.
The combination of menadione with an antioxidant, such as ascorbic
acid, alpha lipoic acid, n-acetyl cysteine (NAC), lycopene,
tocopherol, tocotrienol, or others may also be beneficial. The
combination of menadione and mitomycin C can also be beneficial in
treating subjects with advanced solid tumors, advanced lung cancer,
and advanced gastrointestinal cancer. By administering a
combination of menadione and an antioxidant or plurality of
antioxidants, such as vitamin C, to subjects having prostate
cancer, it is contemplated that a reduction in tumor cell numbers
and PSA (prostate cancer specific antigen) will be obtained.
[0067] In a phase I/IIa trial, a combination of menadione and
vitamin C were given to patients with prostate cancer that had
previously failed the standard of care treatment regimen (i.e.,
radical prostatectomy, radiotherapy and/or hormonal ablation). Ten
of the patients in the trial had received hormonal ablation therapy
prior to the trial but these patients were not exposed to hormonal
ablation therapy at the time of receiving the combination of
vitamin C and menadione. See Tareen et al., Int. J. Med. Sci, 2008,
5:62. In this study, treatment was tested in patients with late
stage disease (aggressive, recurrent). It is likely that the
patients that had previously received hormone therapy had become
hormone-resistant at the time of the trial (which is probably why
disease was progressing in these patients).
[0068] It is contemplated herein that a significantly improved
inhibition of prostate cancer cell growth can be obtained when
castration, hormonal castration, hormonal ablation, or hormone
therapy are provided during the time a patient receives the
combination of antioxidant (e.g., ascorbic add) with a compound of
Formula (I), such as, menadione. Provided herein is an improved
method for treating a subject suffering from prostate cancer with a
compound of Formula (I) and androgen ablation therapy to subjects
with PSA values above zero after radical prostatectomy, i.e., when
they have androgen-dependent disseminated disease. Today there is
no cure for this and patients currently receive only palliative
treatment, including hormone therapy alone. The data provided
herein demonstrates that the combination of plumbagin at the time
of hormone therapy is better than hormone-therapy alone.
[0069] 2,3-Bis[(2-hydroxyethyl)thio]-1,4-naphthoquinone (NSC 95397)
can be a potent inhibitor of the dual-specificity phosphatase
Cdc25, which is involved in cell cycle regulation. NSC 95397 can
inhibit the activity of mitogen-activated protein kinase
phosphatases MKP-1 and MKP-3. This compound has been studied in
combination with chemotherapy drugs such as doxorubicin, etoposide,
oxaliplatin, and docetaxel. NSC 95397 has been studied in
neuroendocrine tumor cells, human pancreatic carcinoma cells, and
bronchial carcinoma cells. Furthermore, this compound has been used
in prostate cancer cells so as to examine the role of the Cdc25
phosphatase in regulation of the mitogen activated protein kinase
(MAP-kinase) pathway. See Nemoto et al., Prostate, 2004, 58:95.
Nevertheless, the effect of NSC 95397 on the growth or survival of
prostate cancer cells was not reported by Nemoto. It is
contemplated that NSC 95397 can be used to inhibit prostate cancer
cell growth and that a significantly improved inhibition of
prostate cancer cell growth can be obtained when castration,
hormonal castration, hormonal ablation, or hormone therapy are
provided before, during, and/or after the time a patient receives
the NSC 95397.
[0070] Juglone is believed to be a peptidyl-prolyl cis/trans
isomerase (PIN-I) inhibitor. Juglone has been studied in
combination with etoposide in human cancer cells and beta-lapachone
can improve the effect of radiation in laryngeal epidermoid
carcinoma cells. It is contemplated that the compounds of Formula
(I) are highly oxidative and induce oxidative stress in cells.
Accordingly, it is contemplated that juglone can be used to inhibit
prostate cancer cell growth and that a significantly improved
inhibition of prostate cancer cell growth can be obtained when
castration, hormonal castration, hormonal ablation, or hormone
therapy are provided before, during, and/or after the time a
patient receives the juglone.
[0071] Naphthazarin may be a microtubule depolymerzing agent and
2,3-Dimethoxy-1,4-naphthoquinone (DMNQ) may inhibit DNA
topoisomerase-I. It is contemplated that naphthazarin and/or
2,3-dimethoxy-1,4-naphthoquinone (DMNQ) can be used to inhibit
prostate cancer cell growth and that a significantly improved
inhibition of prostate cancer cell growth can be obtained when
castration, hormonal castration, hormonal ablation, or hormone
therapy are provided before, during, and/or after the time a
patient receives naphthazarin and/or
23-dimethoxy-1,4-naphthoquinone (DMNQ).
[0072] As mentioned above, although treating a subject that has
cancer (e.g., prostate cancer) with one or more compounds of
Formula (I) alone or in a combination of compounds of Formula (I)
can inhibit the growth of cancerous cells, a significantly improved
inhibition of cancer cell growth (e.g., prostate cancer cell
growth) can be obtained by providing one or more of the compounds
of Formula (I), separately or in a mixture or combination, in
conjunction with a therapy that reduces the androgen levels of the
patient (e.g., castration, hormonal castration, hormonal ablation,
or hormone therapy). That is, some embodiments include methods of
inhibiting cancer cell growth (e.g., prostate cancer cell growth)
or treating or preventing a cancer (e.g., prostate cancer), wherein
a subject having a cancer (e.g., prostate cancer) is provided one
or more compounds of Formula (I) (e.g., plumbagin) while reducing
the amount of androgens in the subject (e.g., providing castration,
hormonal castration, hormonal ablation, or hormone therapy).
Optionally, the inhibition of cancer (e.g., prostate cancer) or a
marker thereof (e.g., PSA) is evaluated after the treatment (e.g.,
after the combination of plumbagin and hormone therapy is
provided). Stated differently, some embodiments of the invention
include a combination of one or more of the compounds of Formula
(1), formulated for administration separately or together, and an
androgen deprivation therapy (e.g., castration, hormonal
castration, hormonal ablation, or hormone therapy) for use in
inhibiting or delaying the growth of prostate cancer cells or
treating or preventing prostate cancer. The section below describes
some of the approaches that can be used to deplete the levels of
androgen in the subject so as to provide the treatments and
treatment protocols described above.
V. Hormone Therapy
[0073] Hormone therapy for treating prostate cancer, or inhibiting
or delaying prostate cancer cell growth, can also be called
androgen deprivation therapy (ADT), chemical castration, or
androgen ablation therapy. Androgens can fuel the growth of
prostatic cells, including both healthy prostatic cells and
cancerous prostatic cells. In some embodiments, a subject suffering
from prostate cancer is provided with a hormone therapy agent that
reduces the subject's androgen levels. In some embodiments, the
androgen that is decreased in the subject is testosterone,
dihydrotestosterone (DHT), androsterone, androstenediol,
androstenedione, dehydroepiandrosterone (DHEA), and
dehydroepiandrosterone sulfate (DHEA-S). In some embodiments, a
subject's serum testosterone level is decreased with one or more
anti-androgen agents or androgen ablation agents. Preferably, the
androgen deprivation therapy is provided during a period in which
one or more compounds of Formula (1) are provided.
[0074] In some embodiments, a subject suffering from prostate
cancer is classified as a subject in need of a therapy for prostate
cancer and said subject is provided a hormone therapy agent that
reduces the subject's androgen levels while said subject is
receiving one or more compounds of Formula (1), such as plumbagin,
or a compound presented in Table 1. Optionally, the inhibition in
prostate cancer cell growth or an inhibition in prostate cancer
advancement is evaluated. Optionally, the delaying prostate cancer
cell growth or delaying prostate cancer advancement is evaluated. A
subject can be identified as one in need of a therapy for prostate
cancer using conventional clinical pathology including, biopsy, CT
scan, MR!, digital examination, Gleason score, or PSA level.
Patients today also get PET scans, which are very important since
they evaluate the activity of the tumor cells (glucose metabolism).
Similarly, the inhibition or delay of cancer cell growth in said
subject after receiving the treatment can be evaluated using
conventional clinical pathology including, biopsy, CT scan, MRI,
digital examination, Gleason score, or PSA level.
[0075] In some embodiments, the hormone therapy agent that can be
used with anyone or more of the methods or treatments described
herein is selected from the group consisting of an antiandrogen
(including steroidal anti androgens and nonsteroidal
antiandrogens), an estrogen, a luteinizing hormone-releasing
hormone (LHRH) agonist, and a LHRH antagonist. Steroidal anti
androgen agents include, but are not limited to, cyproterone
acetate and finasteride. Nonsteroidal antiandrogens include, but
are not limited to, flutamide, nilutamide and bicalutamide.
Estrogen agents include, but are not limited to, ethylstilbestrol
(DES), megestrol acetate, fosfestrol, and estamustine phosphate.
LHRH agonist agents include, but are not limited to, leuprolide,
triptorelin, goserelin, histrelin and buserelin. LHRH antagonist
agents include, but are not limited to, abarelix and degarelix.
Desirably, one or more of the compounds selected from the group
consisting of cyproterone acetate, finasteride, flutamide,
abiraterone, nilutamide, bicalutamide, ethylstilbestrol (DES),
megestrol acetate, fosfestrol, estamustine phosphate, leuprolide,
triptorelin, goserelin, histrelin, buserelin, abarelix and
degarelix are used in the methods and treatments (compositions)
described herein, wherein one or more of the compounds of Formula
(I) (e.g., a compound of Table 1) are provided before, during,
and/or after providing said cyproterone acetate, finasteride,
flutamide, abiraterone, nilutamide, bicalutamide, ethyl stilbestrol
(DES), megestrol acetate, fosfestrol, estamustine phosphate,
leuprolide, triptorelin, goserelin, histrelin, buserelin, abarelix
or degarelix.
[0076] As mentioned above, prostate cancer can be treated by
hormone therapy agents, however, hormone therapy agents alone can
result in the development of castration-resistant prostate cancer
(CRPC). For example, hormonal therapy can initially deliver a
response in a subject suffering from prostate cancer, however, the
return of hormone-refractory tumors invariably prevents long-term
patient survival. More effective strategies are needed to extend
life expectancy and improve the quality of life for patients with
advanced prostate cancer. Accordingly, some aspects of the present
invention concern methods for ameliorating or inhibiting or
reducing or delaying the onset of castration-resistant prostate
cancer (CRPC) or treatments (e.g., compositions used for the
purpose of ameliorating or inhibiting or reducing or delaying the
onset of CRPC), whereby one or more of the compounds of Formula (I)
(e.g., a compound from Table 1) are provided before, during and/or
after providing cyproterone acetate, finasteride, abiraterone,
flutamide, nilutamide, bicalutamide, ethyl stilbestrol (DES),
megestrol acetate, fosfestrol, estamustine phosphate, leuprolide,
triptorelin, goserelin, histrelin, buserelin, abarelix or
degarelix. Optionally, the inhibition in prostate cancer cell
growth, an inhibition in prostate cancer advancement, or delaying
the onset of CRPC is evaluated. Optionally, a patient with prostate
cancer is classified as a subject in need of an agent that
ameliorates, reduces, delays, or inhibits the onset of CRPC prior
to receiving one or more of the combination therapies described
herein. A subject can be identified as one in need of a therapy for
prostate cancer using conventional clinical pathology including,
biopsy, CT scan. MRI, digital examination, Gleason score, or PSA
level.
[0077] Patients today also get PET scans, which are very important
since they evaluate the activity of the tumor cells (glucose
metabolism).
[0078] Similarly, the inhibition or delay of cancer cell growth in
said subject after receiving the treatment can be evaluated using
conventional clinical pathology including, biopsy, CT scan, MRI,
digital examination, Gleason score, or PSA level. The section below
describes the combination therapies in greater detail.
VI. Combination Therapies
[0079] In some embodiments, the compounds disclosed herein, such as
a compound of Formula (I) (e.g., a compound of Table 1), or a
pharmaceutically acceptable salt thereof, or a pharmaceutical
composition that includes a compound described herein, can be used
in combination with one or more additional agent(s). Some
embodiments disclosed herein relate to a method of ameliorating or
treating a neoplastic disease that can include administering to a
subject suffering from a neoplastic disease a therapeutically
effective amount of one or more compounds described herein (e.g., a
compound of Formula (I), or a pharmaceutically acceptable salt
thereof), in combination with one or more hormone therapy agents
(referred to as "combination therapy"). Examples of additional
agents that can be used in combination with a compound of Formula
(I), or a pharmaceutically acceptable salt thereof, or a
pharmaceutical composition that includes a compound of Formula (I),
or a pharmaceutically acceptable salt thereof, include, but are not
limited to, agents that can decrease the subject's serum androgen
levels (e.g., cyproterone acetate, abiraterone, finasteride,
flutamide, nilutamide, bicalutamide, ethyl stilbestrol (DES),
megestrol acetate, fosfestrol, estamustine phosphate, leuprolide,
triptorelin, goserelin, histrelin, buserelin, abarelix or
degarelix).
[0080] In some embodiments, the neoplastic disease can be cancer.
In some embodiments, the neoplastic disease can be a tumor such as
a solid tumor. In an embodiment, the neoplastic disease can be
prostate cancer and in some embodiments the prostate cancer can be
CRPC. In some embodiments, the prostate cancer is androgen
dependent. Therefore, in some embodiments, a compound of Formula
(I), or a pharmaceutically acceptable salt thereof, or a
pharmaceutical composition that includes a compound of Formula (I),
or a pharmaceutically acceptable salt thereof, is used in
combination with one or more hormone therapy agents for the purpose
of treating a subject that has prostate cancer, for inhibiting the
growth of prostate cancer cells, for delaying prostate cancer, for
decreasing the size of a prostate tumor, or for inhibiting the
onset or development of CRPC.
[0081] In some embodiments, a compound of Formula (I), or a
pharmaceutically acceptable salt thereof, or a pharmaceutical
composition that includes a compound of Formula (I) (e.g., one or
more of the compounds of Table 1), or a pharmaceutically acceptable
salt thereof, is used in combination with surgical orchiectomy
and/or one or more of the hormone therapy agents cyproterone
acetate, finasteride, abiraterone, flutamide, nilutamide,
bicalutamide, ethylstilbestrol (DES), megestrol acetate,
fosfestrol, estamustine phosphate, leuprolide, triptorelin,
goserelin, histrelin, buserelin, abarelix or degarelix such that a
"combination therapy" is provided.
[0082] Normal serum testosterone ranges between 1000-300 ng/dL. In
some embodiments, a subject is provided a combination therapy, as
described herein, whereby a reduction in the treated subject's
serum testosterone level to at least about .ltoreq.80, .ltoreq.70,
.ltoreq.60, .ltoreq.50, .ltoreq.40, .ltoreq.30, .ltoreq.20, or
.ltoreq.10 ng/dL is obtained. In some embodiments, a subject is
provided a combination therapy that reduces the subject's serum
testosterone level to at least about .ltoreq.50 ng/dL. In some
embodiments, a subject is treated with a combination therapy that
results in a reduction in the subject's serum testosterone level to
at least about .ltoreq.20 ng/dL. In some embodiments, a subject is
treated with a combination therapy, as described herein, that
reduces the subject's serum testosterone level to at least about or
any number in between the range of 120-70, 100-60, 80-40, 70-30,
50-20, 40-10, 30-10, or 20-10 ng/dL. In some embodiments, a subject
is treated with a combination therapy that produces a reduction in
the subject's serum testosterone level to about .ltoreq.95%,
.ltoreq.90%, .ltoreq.80%, .ltoreq.70%, .ltoreq.60%, or .ltoreq.50%
that of a healthy male. In some embodiments, a subject is treated
with a combination therapy that results in a reduction in the
subject's serum testosterone level to the range of at least about
or any number in between the range of about 5-20%, 10-30%, 20-40%,
30-50%, 40-60%, or 50-70% that of a healthy male.
[0083] Intermittent hormonal therapy (IHT) is an alternative to
continuous hormonal therapy, which may delay progression of
hormone-refractory disease (i.e., CRPC). For example, intermittent
therapy can be used for a period of 6 months on, followed by a
period of 6 months off. In some embodiments, one or more hormonal
therapy agents is provided for one month on, followed by one month
off. In some embodiments, one or more hormonal therapy agents is
provided for three months on, followed by three months off.
Accordingly, one or more of the compounds of Formula (I), e.g., a
compound of Table 1, can be provided before, during and/or after
IHT, as described above, so as to reduce or inhibit or delay the
onset of CRPC.
[0084] A non-limiting list of example combination of compounds of
Formula (I), or a pharmaceutically acceptable salt thereof, or a
pharmaceutical composition that includes a compound described
herein, with one or more hormonal therapy agents are provided in
Tables 1 and 2. Table 1 provides a shorthand name for each compound
of Formula (I) and a shorthand name for each hormonal therapy
agent. Each numbered X compound in Table 2 has a corresponding
compound structure provided in Table 1. Likewise, each numbered Y
therapy in Table 2 has a corresponding therapy provided in Table 1.
Therefore, each "X:Y" entry in Table 2 provides an example of a
combination of a compound of Formula (I) and a hormonal therapy
agent that can be used to treat a subject suffering from prostate
cancer. For example, the combination designated as "F02:AT04" in
Table 2 provides a combination of
##STR00004##
(plumbagin), and flutamide that can be used to treat a subject
suffering from prostate cancer. Each of the combinations provided
in Table 2 can be used with one, two, three or more additional
agents described herein.
TABLE-US-00001 TABLE 1 Additional Compound of Formula (I) Therapy
##STR00005## cyproterone acetate (AT01) ##STR00006## finasteride
(AT02) ##STR00007## bicalutamide (AT03) ##STR00008## flutamide
(AT04) ##STR00009## nilutamide (AT05) ##STR00010## bicalutamide
(AT06) ##STR00011## ethylstilbestrol (DES) (AT07) ##STR00012##
megestrol acetate (AT08) ##STR00013## fosfestrol (AT09)
##STR00014## estamustine phosphate (AT10) ##STR00015## leuprolide
(AT11) ##STR00016## triptorelin (AT12) ##STR00017## goserelin
(AT13) ##STR00018## histrelin (AT14) -- buserelin (AT15) --
abarelix (AT16) -- degarelix (AT17) -- surgical orchiectomy
(AT18)
TABLE-US-00002 TABLE 2 X:Y X:Y X:Y X:Y X:Y X:Y X:Y F01:AT02
F02:AT02 F03:AT02 F04:AT02 F05:AT02 F06:AT02 F07:AT02 F01:AT03
F02:AT03 F03:AT03 F04:AT03 F05:AT03 F06:AT03 F07:AT03 F01:AT04
F02:AT04 F03:AT04 F04:AT04 F05:AT04 F06:AT04 F07:AT04 F01:AT05
F02:AT05 F03:AT05 F04:AT05 F05:AT05 F06:AT05 F07:AT05 F01:AT06
F02:AT06 F03:AT06 F04:AT06 F05:AT06 F06:AT06 F07:AT06 F01:AT07
F02:AT07 F03:AT07 F04:AT07 F05:AT07 F06:AT07 F07:AT07 F01:AT08
F02:AT08 F03:AT08 F04:AT08 F05:AT08 F06:AT08 F07:AT08 F01:AT09
F02:AT09 F03:AT09 F04:AT09 F05:AT09 F06:AT09 F07:AT09 F01:AT10
F02:AT10 F03:AT10 F04:AT10 F05:AT10 F06:AT10 F07:AT10 F01:AT11
F02:AT11 F03:AT11 F04:AT11 F05:AT11 F06:AT11 F07:AT11 F01:AT12
F02:AT12 F03:AT12 F04:AT12 F05:AT12 F06:AT12 F07:AT12 F01:AT13
F02:AT13 F03:AT13 F04:AT13 F05:AT13 F06:AT13 F07:AT13 F01:AT14
F02:AT14 F03:AT14 F04:AT14 F05:AT14 F06:AT14 F07:AT14 F01:AT15
F02:AT15 F03:AT15 F04:AT15 F05:AT15 F06:AT15 F07:AT15 F01:AT16
F02:AT16 F03:AT16 F04:AT16 F05:AT16 F06:AT16 F07:AT16 F01:AT17
F02:AT17 F03:AT17 F04:AT17 F05:AT17 F06:AT17 F07:AT17 F01:AT18
F02:AT18 F03:AT18 F04:AT18 F05:AT18 F06:AT18 F07:AT18 F08:AT02
F09:AT02 F10:AT02 F11:AT02 F12:AT02 F13:AT02 F14:AT02 F08:AT03
F09:AT03 F10:AT03 F11:AT03 F12:AT03 F13:AT03 F14:AT03 F08:AT04
F09:AT04 F10:AT04 F11:AT04 F12:AT04 F13:AT04 F14:AT04 F08:AT05
F09:AT05 F10:AT05 F11:AT05 F12:AT05 F13:AT05 F14:AT05 F08:AT06
F09:AT06 F10:AT06 F11:AT06 F12:AT06 F13:AT06 F14:AT06 F08:AT07
F09:AT07 F10:AT07 F11:AT07 F12:AT07 F13:AT07 F14:AT07 F08:AT08
F09:AT08 F10:AT08 F11:AT08 F12:AT08 F13:AT08 F14:AT08 F08:AT09
F09:AT09 F10:AT09 F11:AT09 F12:AT09 F13:AT09 F14:AT09 F08:AT10
F09:AT10 F10:AT10 F11:AT10 F12:AT10 F13:AT10 F14:AT10 F08:AT11
F09:AT11 F10:AT11 F11:AT11 F12:AT11 F13:AT11 F14:AT11 F08:AT12
F09:AT12 F10:AT12 F11:AT12 F12:AT12 F13:AT12 F14:AT12 F08:AT13
F09:AT13 F10:AT13 F11:AT13 F12:AT13 F13:AT13 F14:AT13 F08:AT14
F09:AT14 F10:AT14 F11:AT14 F12:AT14 F13:AT14 F14:AT14 F08:AT15
F09:AT15 F10:AT15 F11:AT15 F12:AT15 F13:AT15 F14:AT15 F08:AT16
F09:AT16 F10:AT16 F11:AT16 F12:AT16 F13:AT16 F14:AT16 F08:AT17
F09:AT17 F10:AT17 F11:AT17 F12:AT17 F13:AT17 F14:AT17 F08:AT18
F09:AT18 F10:AT18 F11:AT18 F12:AT18 F13:AT18 F14:AT18
[0085] The order of administration of a compound of Formula (I), or
a pharmaceutically acceptable salt thereof, with one or more
additional hormone therapy agent(s) can vary. In some embodiments,
a compound of Formula (I), or a pharmaceutically acceptable salt
thereof, can be administered prior to all additional hormone
therapy agents. In other embodiments, a compound of Formula (I), or
a pharmaceutically acceptable salt thereof, can be administered
prior to at least one additional hormone therapy agent. In still
other embodiments, a compound of Formula (I), or a pharmaceutically
acceptable salt thereof, can be administered concomitantly with one
or more additional hormone therapy agents. In yet still other
embodiments, a compound of Formula (I), or a pharmaceutically
acceptable salt thereof, can be administered subsequent to the
administration of at least one additional hormone therapy agent. In
some embodiments, a compound of Formula (I), or a pharmaceutically
acceptable salt thereof, can be administered subsequent to the
administration of all additional hormone therapy agents.
[0086] In some embodiments, a subject suffering from prostate
cancer is treated by surgical orchiectomy (i.e., removal of the
testes). In some embodiments, a compound of Formula (I), or a
pharmaceutically acceptable salt thereof, can be administered after
surgical orchiectomy. In some embodiments, a compound of Formula
(I), or a pharmaceutically acceptable salt thereof, can be
administered before and after surgical orchiectomy.
Determining and Evaluating Anti-Cancer Activity
[0087] Animal Models
[0088] Animal models are pivotal to further our understanding of
the mechanisms of (progressive) growth of cancer. Currently used
rodent tumor models, including transgenic tumor models, (using
genetically modified mice susceptible to develop cancer), as well
as implantation of human tumors under the skin in immunodeficient
mice, do not sufficiently represent clinical cancer, especially
with regard to metastasis and drug sensitivity. Preclinical tumor
model systems employed to evaluate potential new treatment
strategies should aim to represent the process and patterns of
metastasis of their clinical counterparts as closely as
possible.
[0089] A syngeneic pseudo-orthotopic in vivo model was developed to
study the early steps of prostate cancer. Chambers are surgically
placed into the dorsal skinfold of male mice. Briefly, male mice
(25-30 g body weight) are anesthetized and placed on a heating pad.
Two symmetrical titanium frames are implanted into the dorsal
skinfold. A circular layer is excised from one of the skin layers.
The underlying muscle and subcutaneous tissues are covered with a
glass coverslip incorporated in one of the frames. After a recovery
period of 2-3 days, stroma tissue and tumor cells are carefully
placed in the chamber.
[0090] Tumor-derived cell lines can be grown directly in the
chamber, corresponding to the traditional subcutaneous model.
However, it was found that various minced tissues implanted in the
chambers survive and revascularize, and that tumor-derived cell
lines adapt to these various stroma after co-implantation, which
points to this approach as an orthotopic model as well as a model
for initial steps in metastasis.
[0091] For example, mouse prostate tissue can be grafted in the
chamber. The graft develops its own vasculature and serve as
orthotopic stroma for the tumor. A small number of prostate cancer
cells (e.g., TRAMP-C2 cells derived from a TRAMP mouse) can be
implanted on top of the prostate stroma. The tumor microenvironment
can be important for the progression of different types of cancer,
and orthotopic implantation of cancer cells can recapitulate human
disease much more closely than subcutaneous implantation. Tumors
can grow faster and develop better vasculature when the cancer
cells are implanted into the relevant organ. Co-implanting mouse
prostate cancer cells with prostate stroma can provide the tumor
cells with an environment that better reflects the clinical disease
compared to purely subcutaneous models. Re-vascularized stromal
tissue and implanted tumors can remain viable for long periods of
time using this method, for example, up to 90 days.
[0092] Phosphate and Tensin Homolog (PTEN) Deficient Model
[0093] Mouse cells derived from the PTEN (phosphatase and tensin
homolog deleted in chromosome 10) deficient model of prostate
cancer can be used to study prostate cancer. The tumor suppressor
PTEN is one of the most frequently mutated genes in human prostate
cancer. Loss of PTEN can result in constitutively high PI3-kinase
and Akt activities, which may lead to increased migration,
invasiveness, cell proliferation and survival. Loss of PTEN can
play a major role in the pathogenesis of human prostate cancer.
Alteration of at least one PTEN allele is observed in approximately
60% of primary tumors. Loss of PTEN can be associated with higher
Gleason scores and poor prognosis, cancer progression toward
hormone-independence, resistance to chemotherapy or to
radiotherapy, and bone metastasis. PTEN-deficient mice have an
increased incidence of cancer, similarly to the human genetic
predisposition to cancer known as Cowden syndrome, which is caused
by germline mutation in the PTEN gene. In these respects, the
PTEN-deficient model appears to mimic human development quite
closely. Thus, heterozygous disruption of the PTEN gene can result
in spontaneous development of tumors in several tissues and
prostatic intraepithelial neoplasia (PIN) lesions in the prostate.
Prostate-specific homozygous loss of PTEN can be sufficient to
induce prostate tumors, which can progress into metastatic disease.
Heterozygous loss of PTEN, on the other hand, can cause PIN with a
late latency.
[0094] Germline homozygous deletion of PTEN may result in embryonic
lethality due to PTEN ablation. This can be overcome through the
conditional inactivation of the gene using the Cre-LoxP system. A
transgenic mouse can be generated that displays expression of the
Cre recombinase specifically in the epithelial cells of the
prostate through the use of the prostate-specific probasin promoter
(PB-Cre4 mice). By crossing these animals with mice that have
floxed PTEN alleles, it can be possible to generate both
heterozygous and homozygous mice in which PTEN is deleted
specifically in the prostate epithelium. Progression of prostate
cancer in this model is very similar to the progression of prostate
cancer as observed in humans. For example, in this model epithelial
hyperplasia was observed, followed by dysplasia, PIN, invasive
adenocarcinoma, and finally metastases to the lymph nodes and to
the lung. Similar to human cancer, the PTEN-null mice first regress
following androgen ablation, and then become
androgen-independent.
[0095] Epithelial cell lines can be derived from a prostate tumor
dissected from a homozygous PTEN.sup.L/L/PBCre+ mouse. At least two
clonal cell lines (PTEN-P2 and PTEN-P8) are heterozygous
PTEN.sup.L/+. The remaining allele can be silenced by forced
expression of the Cre recombinase in vitro (PTEN-CaP2 and PTEN-CaP8
cells). Loss of the second allele can increase
anchorage-independent growth and confer tumorigenesis in vivo.
Spontaneous androgen-independence can occur in vivo, even though
the PTEN-CaP2 and PTEN-CaP8 cells express the androgen
receptor.
[0096] The implementation of PTEN prostate cells in the animal
models disclosed herein can be highly relevant to human prostate
cancer, and can allow detailed observation of the growth and/or
regression of prostate tumors in response to different treatment
regimens. Implantation in syngeneic mice respects many aspects of
normal tumor growth. For example, two pairs of mouse prostate
cancer cells (PTEN-P2/8 and PTEN-CaP2/8) can facilitate examination
of metastasis in a mouse model of prostate cancer that is relevant
to human cancer.
[0097] IntraVital Microscopy (NM)
[0098] IntraVital Microscopy (IVM) can be used to visualize tumors
in animals and analyze various aspects of cancer physiology such as
tumor vascularization, cell migration and metastasis. An advantage
of IVM includes the real-time analysis of dynamic processes with
single-cell resolution. IntraVital microscopy offers the
possibility to follow tumor growth in a non-invasive,
non-destructive manner. The application of IVM can be limited to
animal models that bear visually accessible tumors. Therefore, the
dorsal skinfold chamber model described above can be compatible
with IVM. Using IVM can permit a number of parameters to be
measured in living animals and as a function of time, including
tumor growth, angiogenesis, infiltration by immune cells, tumor
cell migration, cell cycle entry, mitosis (cell-division) and
apoptosis (programmed cell death), all in the context of the host
and in real time.
VIII. Pharmaceutical Compositions
[0099] Some embodiments described herein relate to a pharmaceutical
composition, that can include a therapeutically effective amount of
a one or more compounds described herein (e.g., a compound of
Formula (I), (e.g., a compound in Table 1) or a pharmaceutically
acceptable salt thereof, and/or a hormone therapy agent) and a
pharmaceutically acceptable carrier, diluent, excipient or
combination thereof. In some embodiments, the pharmaceutical
composition can include a single diastereomer of a compound of
Formula (I), or a pharmaceutically acceptable salt thereof, (for
example, a single diastereomer is present in the pharmaceutical
composition at a concentration of greater than 99% compared to the
total concentration of the other diastereomers). In other
embodiments, the pharmaceutical composition can include a mixture
of diastereomers of a compound of Formula (I), or a
pharmaceutically acceptable salt thereof. For example, the
pharmaceutical composition can include a concentration of one
diastereomer of >about 50%, .gtoreq.60%, .gtoreq.70%,
.gtoreq.80%, .gtoreq.90%, .gtoreq.95%, or .gtoreq.98%, as compared
to the total concentration of the other diastereomers. In some
embodiments, the pharmaceutical composition includes a racemic
mixture of diastereomers of a compound of Formula (I), or a
pharmaceutically acceptable salt thereof.
[0100] Some embodiments described herein relates to a
pharmaceutical composition, that can include a therapeutically
effective amount a compound of Formula (I), an additional hormone
therapy agent, and a pharmaceutically acceptable carrier, diluent,
excipient or combination thereof. Some embodiments described herein
relates to a pharmaceutical composition, that can include a
therapeutically effective amount a compound of Formula (1), and a
pharmaceutically acceptable carrier, diluent, excipient or
combination thereof. Some embodiments relate to a pharmaceutical
composition that can include a therapeutically effective amount of
a hormone therapy agent and a pharmaceutically acceptable carrier,
diluent, excipient or combination thereof.
[0101] The pharmaceutical compositions described herein can be
administered to a human patient per se, or in pharmaceutical
compositions where they are mixed with other active ingredients, as
in combination therapy, or carriers, diluents, excipients or
combinations thereof. Proper formulation is dependent upon the
route of administration chosen. Techniques for formulation and
administration of the compounds described herein are known to those
skilled in the art.
[0102] The pharmaceutical compositions disclosed herein may be
manufactured in a manner that is itself known, e.g., by means of
conventional mixing, dissolving, granulating, dragee-making,
levigating, emulsifying, encapsulating, entrapping or tableting
processes. Additionally, the active ingredients are contained in an
amount effective to achieve its intended purpose. Many of the
compounds used in the pharmaceutical combinations disclosed herein
may be provided as salts with pharmaceutically compatible
counterions.
[0103] Multiple techniques of administering a compound and/or agent
exist in the art including, but not limited to, oral, rectal,
topical, aerosol, injection and parenteral delivery, including
intramuscular, subcutaneous, intravenous, intramedullary
injections, intrathecal, direct intraventricular, intraperitoneal,
intranasal and intraocular injections.
[0104] One may also administer the compound and/or agent in a local
rather than systemic manner, for example, via injection of the
compound directly into the infected area, often in a depot or
sustained release formulation. Furthermore, one may administer the
compound and/or agent in a targeted drug delivery system, for
example, in a liposome coated with a tissue-specific antibody. The
liposomes will be targeted to and taken up selectively by the
organ.
[0105] The compositions may, if desired, be presented in a pack or
dispenser device which may contain one or more unit dosage forms
containing the active ingredient. The pack may for example comprise
metal or plastic foil, such as a blister pack. The pack or
dispenser device may be accompanied by instructions for
administration. The pack or dispenser may also be accompanied with
a notice associated with the container in 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 drug for human or veterinary
administration. Such notice, for example, may be the labeling
approved by the U.S. Food and Drug Administration for prescription
drugs, or the approved product insert. Compositions that can
include a compound and/or agent described herein formulated in a
compatible pharmaceutical carrier may also be prepared, placed in
an appropriate container, and labeled for treatment of an indicated
condition.
IX. Dosing
[0106] As will be readily apparent to one skilled in the art, the
useful in vivo dosage to be administered and the particular mode of
administration will vary depending upon the age, weight, the
severity of the affliction, and mammalian species treated, the
particular compounds employed, and the specific use for which these
compounds are employed. The determination of effective dosage
levels, that is the dosage levels necessary to achieve the desired
result, can be accomplished by one skilled in the art using routine
methods, for example, human clinical trials and in vitro
studies.
[0107] The dosage may range broadly, depending upon the desired
effects and the therapeutic indication. Alternatively dosages may
be based and calculated upon the surface area of the patient, as
understood by those of skill in the art. Although the exact dosage
will be determined on a drug-by-drug basis, in most cases, some
generalizations regarding the dosage can be made. The daily dosage
regimen for an adult human patient may be, for example, an oral
dose of between 0.01 mg and 3000 mg of each active ingredient,
preferably between 1 mg and 700 mg, e.g. 5 to 200 mg. The dosage
may be a single one or a series of two or more given in the course
of one or more days, as is needed by the subject. In some
embodiments, an active ingredient will be administered for a period
of continuous therapy, for example for a week or more, or for
months or years. In some embodiments, an active ingredient can be
administered one time per day.
[0108] Multiple doses can be administered to a subject. For
example, an active ingredient can be administered once per month,
twice per month, three times per month, every other week (qow),
once per week (qw), twice per week (biw), three times per week
(tiw), four times per week, five times per week, six times per
week, every other day (qod), daily (qd), twice a day (qid), or
three times a day (tid), over a period of time ranging from about
one day to about one week, from about two weeks to about four
weeks, from about one month to about two months, from about two
months to about four months, from about four months to about six
months, from about six months to about eight months, from about
eight months to about 1 year, from about 1 year to about 2 years,
or from about 2 years to about 4 years, or more.
[0109] In some embodiments, a compound of Formula (I) or a
pharmaceutically acceptable salt thereof, and a hormone therapy
agent can be cyclically administered to a patient. Cycling therapy
involves the administration of a first active ingredient for a
period of time, followed by the administration of a second active
ingredient for a period of time and repeating this sequential
administration. Cycling therapy can reduce the development of
resistance to one or more therapies, avoid or reduce the side
effects of one or more therapies, and/or improve the efficacy of
treatment. In some embodiments, a compound of Formula (I) or a
pharmaceutically acceptable salt thereof, and a hormone therapy
agent are administered in a cycle of less than about 3 weeks, about
once every two weeks, about once every 10 days, or about once every
week. The number of cycles can be from about 1 to about 12 cycles,
or from about 2 to about 10 cycles, or from about 2 to about 8
cycles.
[0110] In some embodiments, the active ingredient can be a compound
of Formula (I), or a pharmaceutically acceptable salt thereof. In
some embodiments, the active ingredient can be a hormone therapy
agent. In some embodiments, both an active ingredient of compound
of Formula (I), or a pharmaceutically acceptable salt thereof, and
an active ingredient of a hormone therapy agent are administered to
a subject.
[0111] The daily dosage regimen for an adult human patient may be
the same or different for two active ingredients provided in
combination. For example, a compound of Formula (I) can be provided
at a dose of between 0.01 mg and 3000 mg, while a hormone therapy
agent can be provided at a dose of between 1 mg and 700 mg. The
dosage or each active ingredient can be, independently, a single
one or a series of two or more given in the course of one or more
days, as is needed by the subject. In some embodiments, the active
ingredients will be administered for a period of continuous
therapy, for example for a week or more, or for months or years. In
some embodiments, a compound of Formula (I), or a pharmaceutically
acceptable salt thereof, can be administered one time per day. In
some embodiments, the hormone therapy agent can be administered
once a week.
[0112] In instances where human dosages for active ingredients have
been established for at least some condition, those same dosages
may be used, or dosages that are between about 0.1% and 500%, more
preferably between about 25% and 250% of the established human
dosage. Where no human dosage is established, as will be the case
for newly-discovered pharmaceutical compositions, a suitable human
dosage can be inferred from ED.sub.50 or ID.sub.50 values, or other
appropriate values derived from in vitro or in vivo studies, as
qualified by toxicity studies and efficacy studies in animals.
[0113] In cases of administration of a pharmaceutically acceptable
salt, dosages may be calculated as the free base. As will be
understood by those of skill in the art, in certain situations it
may be necessary to administer the active ingredients disclosed
herein in amounts that exceed, or even far exceed, the
above-stated, preferred dosage range in order to effectively and
aggressively treat particularly aggressive diseases or
infections.
[0114] Dosage amount and interval may be adjusted individually to
provide plasma levels of the active moiety which are sufficient to
maintain the modulating effects, or minimal effective concentration
(MEC). The MEC will vary for each active ingredient but can be
estimated from in vitro data. Dosages necessary to achieve the MEC
will depend on individual characteristics and route of
administration. However, HPLC assays or bioassays can be used to
determine plasma concentrations. Dosage intervals can also be
determined using MEC value. Compositions should be administered
using a regimen which maintains plasma levels above the MEC for
10-90% of the time, preferably between 30-90% and most preferably
between 50-90%. In cases of local administration or selective
uptake, the effective local concentration of the drug may not be
related to plasma concentration.
[0115] It should be noted that the attending physician would know
how to and when to terminate, interrupt, or adjust administration
due to toxicity or organ dysfunctions. Conversely, the attending
physician would also know to adjust treatment to higher levels if
the clinical response were not adequate (precluding toxicity). The
magnitude of an administrated dose in the management of the
disorder of interest will vary with the severity of the condition
to be treated and to the route of administration. The severity of
the condition may, for example, be evaluated, in part, by standard
prognostic evaluation methods. Further, the dose and perhaps dose
frequency, will also vary according to the age, body weight, and
response of the individual patient. A program comparable to that
discussed above may be used in veterinary medicine.
[0116] Active ingredients disclosed herein can be evaluated for
efficacy and toxicity using known methods. For example, the
toxicology of a particular active ingredient, or of a subset of the
active ingredients, sharing certain chemical moieties, may be
established by determining in vitro toxicity towards a cell line,
such as a mammalian, and preferably human, cell line. The results
of such studies are often predictive of toxicity in animals, such
as mammals, or more specifically, humans. Alternatively, the
toxicity of particular compounds in an animal model, such as mice,
rats, rabbits, or monkeys, may be determined using known methods.
The efficacy of a particular active ingredient may be established
using several recognized methods, such as in vitro methods, animal
models, or human clinical trials. When selecting a model to
determine efficacy, the skilled artisan can be guided by the state
of the art to choose an appropriate model, dose, route of
administration and/or regime.
EXAMPLES
[0117] Additional embodiments are disclosed in further detail in
the following examples, which are not in any way intended to limit
the scope of the claims.
Example 1
[0118] Compounds of Formula (I) can be prepared by methods known in
the art. Additionally, many compounds of Formula (I) are naturally
occurring organic compounds that can be isolated from plants.
Furthermore, many compounds of Formula (I) are commercially
available.
[0119] Plumbagin is soluble in alcohol, acetone, chloroform,
benzene, and acetic acid. Plumbagin has been used in preparation
with Ethanol (in vitro) and in preparation with DMSO (in vitro) or
DMSO with PEG 30% (in vivo).
Example 2
[0120] Cell culture: PTEN-P2/GFP are cells that stably express
histone H2B-GFP fusion protein. Kanda et al. (Kanda T, Sullivan K
F, Wahl G M. Histone-GFP fusion protein enables sensitive analysis
of chromosome dynamics in living mammalian cells. Curr Biol 1998
Mar. 26; 8(7):377-85) developed a highly sensitive method for
observing chromosome dynamics in living cells. They fused the human
Histone H2B gene to the gene encoding the GFP, which was
transfected into human HeLa cells to generate a stable line
constitutively expressing H2B-GFP. The H2B-GFP fusion protein was
incorporated into chromatin without affecting cell cycle
progression. We have generated cDNA encoding a Histone H2B-GFP
fusion protein under the 5'LTR in the LXRN retroviral cassette, and
have introduced it into a number of humans, as well as, murine
cancer cell lines by retroviral transduction.
[0121] Cells are grown in DMEM medium containing 10% FBS, 2 mM
L-glutamine, 100 U/ml penicillin/100 .mu.g/ml streptomycin,
insulin-selenium-transferrin (5 .mu.g/ml insulin), and DHT
10.sup.-8M final. Androgen withdrawal is achieved by keeping the
cells in phenol red-free DMEM medium containing 10%
charcoal-treated FBS and the same supplements as in the normal
medium except for DHT. Cells are maintained in a humidified
incubator at 37.degree. C. and 5% CO.sub.2. G418 (100 .mu.g/ml) is
added to maintain stable expression of H2B-GFP.
[0122] Cell counting: Cells in 12-well plates are washed once with
PBS, detached using Trypsin, and transferred to a suspension vial
in a final volume of 10 ml PBS. Cells are counted using a
COULTER.TM. Multisizer II instrument (Beckman Coulter Inc.,
Hialeah, Fla.) gated for the appropriate cell size and corrected
for particulate debris.
[0123] Animal model and surgical techniques: Animal experiments
have been approved as appropriate. All surgical procedures are
performed in a sterile laminar flow hood. Dorsal skinfold chambers
and surgical instruments are autoclaved before use. Saline used to
keep tissue moist during surgical preparation is mixed with
gentamicin (50 .mu.l/ml).
[0124] Male Nude mice (25-35 g body weight) are anesthetized (7.3
mg ketamine hydrochloride and 2.3 mg xylazine/100 g body weight,
i.p.) and placed on a heating pad. Two symmetrical titanium frames
are implanted into a dorsal skinfold, so as to sandwich the
extended double layer of skin. A 15 mm full thickness circular
layer is excised. The underlying muscle (M. cutaneous max.) and
subcutaneous tissues are covered with a glass coverslip
incorporated in one of the frames. After a recovery period of 2-3
days, prostate tissue and cancer cell spheroids are carefully
placed in the chamber. Small circular Band Aids are applied on the
backside of the chamber after surgery to prevent scratching. Before
surgery, Buprenorphine (0.1 mg/kg) will be given IP. After surgery
Meloxicam will be given in the drinking water for 4 days Meloxicam
(5.0 mg/ml), is added at 35 .mu.l per 100 ml of water to be
medicated.
[0125] Preparation of stroma: A male donor mouse is euthanized and
the anterior prostate tissue is excised, put in a Petri dish with
antibiotics (gentamicin 50 .mu.l/ml), and minced with fine scissors
into small pieces (<1 mm.sup.2) for implantation.
[0126] Preparation of tumor spheroids: Liquid overlay plates are
generated using 1% Agarose melted in DMEM that is added to
round-bottom 96-well plates (50 ul/well). Cancer cells grown as
pre-confluent monolayers are trypsinized, diluted to a final volume
of 250,000 tumor cells/ml. Viability is determined using Trypan
blue. The cells are plated at 100 ul/well into the agarose-coated
plates. After 48 hrs the cells form spheroids, which are picked and
washed in serum-free medium before implantation into the mouse
chambers. Viability is determined using Trypan blue. The size of
the implanted spheroid can be determined precisely to minimize
variations between animals.
[0127] Surgical Castration: Mice are anesthetized with 7.3 mg
ketamine hydrochloride and 2.3 mg xylazine/100 g body weight, i.p.
A lateral incision across the scrotum is made and the testes are
individually ligated and excised. The wound was cauterized. The
incision was then sutured and sealed with Nexaband.RTM.
acrylic.
[0128] Intravital microscopy: Fluorescence microscopy is performed
using a Mikron Instrument Microscope equipped with epi-illuminator
and video-triggered stroboscopic illumination from a xenon arc
(MV-7600, EG&G). A silicon intensified target camera (SIT68,
Dage-MTI) is attached to the microscope. A Hamamatsu image
processor (Argus 20) with firmware version 2.50 (Hamamatsu Photonic
System) is used for image enhancement and for the capture of images
to a computer. A Zeiss Plan Neoflour 1.25.times./0.035 objective is
used to obtain an over-view of the chamber and to determine tumor
size. A Zeiss A-Plan 10.times./0.25 objective is used to capture
images for calculation of vascular parameters. A Zeiss Achroplan
20.times./0.5 W objective is used to capture images for calculation
of mitotic and apoptotic indices. Our system permits evaluation of
the following parameters.
[0129] Tumor area (A.sub.T) is defined as number of pixels with
photo density above 75 (256 gray levels), i.e.,
A.sub.T=.SIGMA.A.sub.k, for 75<k<255.
[0130] Number of Tumor cells: When tumors are heterogeneous,
changes in A.sub.T do not directly reflect tumor growth. An
estimate of the number of tumor cells (N.sub.TC) can be obtained by
fitting to a quadratic function of an intensity index, e.g.
N.sub.TC=-3.296.times.10.sup.-12+190.6.times.I.sub.T+7.7310.sup.-2.times.-
(I.sub.T).sup.2, where the index of intensity is given by
I.sub.T=.SIGMA.A.sub.k*k, for 75<k<255.
[0131] Mitotic and Apoptotic Indices: At each time point, two
peripheral and two central .times.20 fields of the tumor are
captured with a FITC filter and an integrated frame grabber. Only
mitotic figures in metaphase-telophase (MI) are included in the
mitotic indices to exclude the potential artifact of nuclear
membrane distortion. Apoptotic/Pyknotic nuclei are defined as
H.sub.2B-GFP labeled nuclei with a cross sectional area <30
.mu.m.sup.2. Nuclear karyorrhexis (NK), easily distinguishable by
the vesicular nuclear condensation and brightness of H2BGFP, is
included within this apoptotic indices.
[0132] Image Analysis of Vascular Parameters: For each spheroid,
video recordings are used to calculate length, area and vascular
density of the neovasculature being induced by the implanted tumor
spheroids. Vascular parameters are analyzed from the video
recording using Image-Pro Plus. Photomicrographs obtained with the
.times.10 objective, are "flattened" to reduce the intensity
variations in the background pixels. An Area of Interest (AOI) is
selected to eliminate distorted areas, and thresholding is used to
segment the picture into objects and background. This panel is used
to calculate the vascular area (Av). The picture is skeletonized to
calculate the vascular length (L.sub.V). The average tumor vessel
diameter D.sub.V is calculated as A.sub.V/L.sub.V, and the vascular
density (.sub.V) is calculated as L.sub.V per tumor area. Finally,
we calculate the growth rate of the total area of tumor
vasculature.
Example 3
Effect of Naphthoguinone Analogs on PTEN-P2/GFP Cell
Proliferation
[0133] PTEN-P2/GFP prostate cancer cells were plated at a density
of 8000 cells/well in 96-well plates (triplicates) in growing
medium containing 10% Fetal Bovine Serum and DHT. The next day,
increasing concentrations of a naphthoquinone analog (diluted from
10 mM DMSO stock solutions) were incubated for 24 hrs. Cell
viability was determined by the formazan-based cytotoxicity assay
"CellTiter96Aquaeous nonradioactive proliferation assay" (Promega).
The results are shown in Tables 3 and 4, and FIGS. 1 and 2.
TABLE-US-00003 TABLE 3 conc (.mu.M) % viability .sigma. % viability
.sigma. % viability .sigma. % viability .sigma. 1,4- 2-methoxy-1,4-
Phylloquinone naphthoquinone naphthoquinone NSC 95397 (K1) 0 100.0
3.6 100.0 2.2 100.0 3.7 100.0 2.7 1 100.0 3.3 93.1 3.9 103.7 5.6
107.9 6.1 2 97.0 0.7 88.4 1.3 100.6 4.4 108.1 5.2 3 93.9 2.6 86.9
1.9 86.6 5.1 106.4 7.1 4 90.4 1.8 85.0 3.2 68.4 6.3 106.8 8.1 5
90.1 0.9 82.5 1.0 53.8 3.5 108.0 6.8 7 88.7 1.3 67.2 2.2 47.1 4.3
110.8 7.5 10 83.0 2.5 43.5 8.2 36.3 1.7 110.1 9.2 25 47.3 3.0 0.3
0.4 15.1 7.5 109.1 7.4 50 0.1 0.5 -0.4 0.2 4.8 1.0 109.8 8.2
2,6-di-tert- butyl-1,4- Lawsone naphtoquinone Lapachol dimer
Juglone 0 100.0 1.7 100.0 3.2 100.0 2.1 100.0 3.0 1 103.9 3.0 106.4
6.2 98.3 2.4 90.7 2.7 2 104.5 4.7 105.3 6.1 98.6 2.3 85.1 3.1 3
104.7 6.3 102.4 7.0 97.4 3.6 80.6 3.6 4 103.0 7.2 102.8 5.6 95.1
2.9 80.5 1.2 5 104.0 6.3 99.8 4.8 96.8 3.1 58.2 6.0 7 102.8 2.7
99.7 6.5 96.1 3.7 2.3 2.3 10 102.2 6.2 99.6 5.4 98.6 1.7 1.1 0.5 25
102.9 3.6 96.2 5.6 99.4 1.8 2.5 0.2 50 92.9 6.0 86.1 4.4 97.9 1.8
5.3 1.9 Naphthazarin Menadione DMNQ Lawsone 0 100.0 3.6 100.0 2.5
100.0 3.3 100.0 3.4 1 73.4 5.6 95.0 3.1 95.0 3.9 90.1 1.3 2 36.4
2.6 93.1 1.8 91.0 1.5 85.1 1.9 3 8.9 4.6 90.2 3.7 88.5 2.1 81.3 3.5
4 1.5 0.7 93.5 1.7 85.9 3.4 82.5 3.5 5 1.7 0.6 89.4 3.7 85.3 7.9
80.4 3.5 7 2.2 0.6 94.0 2.0 58.3 4.2 80.3 2.9 10 2.4 0.9 77.5 7.0
2.4 1.8 83.1 1.9 25 5.4 0.7 2.4 0.7 0.9 0.6 87.0 2.4 50 9.1 0.5 2.9
0.7 0.8 0.5 96.0 1.5 Dichlon Plumbagin conc (.mu.M) % viability
.sigma. % viability .sigma. 0 100.0 3.3 100.0 0.8 1 96.5 2.0 94.6
2.2 2 95.6 1.7 90.9 3.3 3 92.3 3.6 88.9 2.1 4 92.2 2.3 73.0 0.7 5
91.8 1.7 32.9 6.0 7 99.3 4.2 0.4 0.4 10 87.1 1.1 0.6 0.2 25 89.2
3.0 -- -- 50 4.9 7.3 -- --
TABLE-US-00004 TABLE 4 Compound IC50 (.mu.M) Naphtazarin 1.65
Plumbagin 4.55 Juglone 5.3 NSC 95397 6.2 DMNQ 7.35 2-methoxy-1,4-
8.95 naphtoquinone Menadione 14.5 1,4-naphtoquinone 24.1 Dichlon
37.75 Phylloquinone (K1) >50 2,6-di-tert-butyl-1,4- >50
naphtoquinone Lapachol >50 Lawson >50 Lawsone dimer
>50
Example 4
Dose Response Plumbagin in PTEN-P2/GFP Cells
[0134] PTEN-P2/GFP mouse cancer cells were placed in androgen
withdrawal medium in the presence or absence of DHT
(dihydrotestosterone) at a final concentration of 10-.sup.8 M.
Plumbagin was added at the indicated concentrations for 24 hours.
The absence of DHT simulates surgical or chemical castration. Cells
were trypsinized and counted using a Cell Coulter counter
Multisizer II, which excludes debris. Results represent cell
numbers as percent of control (in which the number of cells in the
absence of drug is 100%). FIG. 3 is a graph that shows the mean of
two separate experiments, each run in duplicates. The results are
shown in Table 5 and FIG. 3. The results indicate that in vitro,
the combination treatment of plumbagin with simulated surgical or
chemical castration was more efficient than treatment with
plumbagin alone.
[0135] Androgen withdrawal medium: DMEM high-glucose phenol-red
free, with the following additives: 10% charcoal-treated Fetal
Bovine Serum, 25 ug/ml bovine pituitary extract, 5 ug/ml insulin, 6
ng/ml EGF recombinant.
TABLE-US-00005 TABLE 5 .mu.M plumbagin % control % control Average
noDHT 0 100.01 100.00 100.00 1 73.70 95.96 84.83 2 42.90 37.14
40.02 4 10.12 1.57 5.84 8 0.45 0.22 0.33 with DHT 0 100.00 100.00
100.00 1 106.93 61.29 84.11 2 94.18 19.16 56.67 4 22.62 4.89 13.76
8 0.85 0.40 0.62
Example 5
In Vivo Effect of Plumbagin Combined with Castration in the
Pseudo-Orthotopic Chamber Model for Prostate Cancer
[0136] Platinum chambers were placed in the dorsal skinfold of nude
mice by surgery. Two days later, minced prostate tIssUe from BalbC
mice (syngeneic) was grafted into the chambers and allowed to
vascularize for 7 to 10 days. Small tumor cells spheroids were
implanted into each chamber. Tumor cells PTEN-P2 stably transfected
with H2B-GFP fusion protein (PTEN-P2/GFP) were used in these
experiments. When tumor vascularization was established (about 5-7
days), the animals were surgically castrated to inhibit androgen
production. Surgical castration induces androgen deprivation, and
is known in the art to effectively mimic clinical hormone therapy.
The mice were treated with plumbagin soon after castration.
Plumbagin administration schedule was 1 mg/kg (DMSO and PEG30%) via
intraperitoneal injection, once/day. The results unexpectedly
indicate that the combination treatment of plumbagin with
castration was more efficient in vivo than either treatment alone.
Therefore, this experiment provides an important indication that
castration (whether surgical or chemical) in combination with
plumbagin can provide a significant improvement over therapies that
were previously known in the art.
[0137] Furthermore, the results demonstrate that treatment with
castration only, or treatment with plumbagin only, did not lead to
a marked decrease in tumor size. However, the combination treatment
of castration with plumbagin unexpectedly resulted in significant
decreases in tumor size. As such, the combination therapies
described herein provide significant improvements in treating
prostate cancer over therapies that were previously known in the
art.
[0138] FIG. 4 compares the growth of tumors without treatment,
castration alone, plumbagin alone, and the combination of
castration and plumbagin.
[0139] FIG. 5 shows the effect of plumbagin at 0.1 mg/kg, 0.3 mg/kg
and 1 mg/kg, given in combination with castration. In FIGS. 4 and
5, day 0 is the first day of plumbagin treatment.
Example 6
In Vivo Effect of Plumbagin Combined with Castration in the
Pseudo-Orthotopic Chamber Model for Prostate Cancer
[0140] Platinum chambers were placed in the dorsal skinfold of nude
mice by surgery. Two days later, minced prostate tissue from BalbC
mice (syngeneic) was grafted into the chambers and allowed to
vascularize for 7 to 10 days. Small tumor cells spheroids were
implanted into each chamber. Tumor cells PTEN-P2 stably transfected
with H2B-GFP fusion protein (PTEN-P2/GFP) were used in these
experiments. The animals were surgically castrated about three
weeks after implantation to inhibit androgen production. Surgical
castration induces androgen deprivation, and is known in the art to
effectively mimic clinical hormone therapy. Two weeks after
castration, the mice were treated daily with plumbagin at 2 mg/kg
ip.
[0141] FIG. 6 illustrates the effect of adding plumbagin after
surgical castration.
[0142] FIG. 7 illustrates increasing apoptosis CAP) and mitosis
(MI) after daily administration of plumbagin ip (2 mg/kg). This
figure illustrates that underlying the rapid tumor regression,
there was an increase; rA apoptosis, but also that mitosis
increased, which was interpreted as cell cycle arrest.
[0143] The results unexpectedly indicate that the combination
treatment of plumbagin with castration was more efficient in vivo
than castration alone. Therefore, this experiment provides an
important indication that castration (whether surgical or chemical)
in combination with plumbagin can provide a significant improvement
over therapies that were previously known in the art.
[0144] Furthermore, the results demonstrate that treatment with
castration only, did not lead to a marked decrease in tumor size.
However, the combination treatment of castration with plumbagin
unexpectedly resulted in significant decreases in tumor size. As
such, the combination therapies described herein provide
significant improvements in treating prostate cancer over therapies
that were previously known in the art.
Without wishing to be bound by theory, the observations indicate
that
[0145] tumor regression is likely caused by a combination of
decreased vascularization due to androgen withdrawal, together with
tumor cell growth arrest or with tumor cells apoptosis due mostly
to plumbagin treatment. Thus, the efficacy of the combination was
much better in vivo than can be observed in vitro because the
separate effects of each treatment on distinct biological
compartments (vasculature stroma possibly inflammatory cells) are
not represented in the culture of cell lines.
Example 7
Dose Response Plumbagin in Human LNCaP Cells
[0146] LNCaP hormone-sensitive human prostate cancer cells were
placed in androgen withdrawal medium in the absence of DHT
(dihydrotestosterone). The absence of DHT simulates surgical or
chemical castration. The androgen withdrawal medium was phenol-red
free DMEM high-glucose containing 10% charcoal-treated Fetal Bovine
Serum.
[0147] Plumbagin was added at the indicated concentrations in Table
6 for 24 hours. Cells were trypsinized and counted using a Cell
Coulter counter Multisizer II, which excludes debris. The results
in Table 6 represent cell numbers as percent of control (in which
the number of cells in the absence of drug is 100%). FIG. 8
illustrates the effect of plumbagin in human LNCaP cells. The
results indicate that in vitro, the combination treatment of
plumbagin with simulated surgical or chemical castration is more
efficient than treatment castration alone.
TABLE-US-00006 TABLE 6 Plumbagin cell number/20 cone. (.mu.M) trial
1 trial 2 trial 3 trial 4 trial 5 trial 6 trial 7 mean % control 0
7245 7376 7551 7603 7327 8047 7562 7530 100 0.5 6422 5989 6453 6475
-- -- -- 6335 84.13 1 6997 7139 6769 6490 -- -- -- 6849 90.95 2
5324 5282 4522 4821 -- -- -- 4987 66.23 4 3005 3082 3327 3300 -- --
-- 3179 42.21 6 1396 1500 1323 1352 -- -- -- 1393 18.50 8 330 283
284 287 -- -- -- 296 3.93
Example 8
In Vivo Effect of Plumbagin Combined with Chemical Castration
[0148] Platinum chambers are placed in the dorsal skinfold of nude
mice by surgery. Two days later, minced prostate tissue from BalbC
mice (syngeneic) are then grafted into the chambers and allowed to
vascularize for 7 to 10 days. Small tumor cells spheroids are then
implanted into each chamber. Tumor cells PTEN-P2 stably transfected
with H2B-GFP fusion protein (PTEN-P2/GFP) are then used in these
experiments. When tumor vascularization is established (about 5-7
days), the animals are treated with an antiandrogen compound (e.g.,
cyproterone acetate) to induce androgen deprivation. The mice are
then treated with plumbagin or an' analog thereof (e.g., a compound
from Table 1). Plumbagin or analog thereof is administered
according to the schedule: Img/kg (DMSO and PEG30%) via
intraperitoneal injection, once/day. Control mice that are not
treated with cyproterone acetate are analyzed in parallel. Also,
mice treated with cyproterone acetate, but not treated with
plumbagin are analyzed in parallel. The results will show that the
combination of plumbagin (or analog thereof) with the anti androgen
compound (e.g., cyproterone acetate) will inhibit prostate cancer
cell growth more efficiently than treatment with plumbagin (or
analog thereof) or antiandrogen compound (e.g., cyproterone
acetate) alone.
[0149] It will be understood by those of skill in the art that
numerous and various modifications can be made without departing
from the spirit of the present disclosure. Therefore, it should be
clearly understood that the forms disclosed herein are illustrative
only and are not intended to limit the scope of the present
disclosure.
* * * * *