U.S. patent application number 11/172050 was filed with the patent office on 2005-12-08 for dosage forms for treatment of benign prostatic hyperplasia.
This patent application is currently assigned to Threshold Pharmaceuticals, Inc.. Invention is credited to Tidmarsh, George.
Application Number | 20050271723 11/172050 |
Document ID | / |
Family ID | 32777423 |
Filed Date | 2005-12-08 |
United States Patent
Application |
20050271723 |
Kind Code |
A1 |
Tidmarsh, George |
December 8, 2005 |
Dosage forms for treatment of benign prostatic hyperplasia
Abstract
A method for treatment or prophylaxis of benign prostatic
hyperplasia by administration of lonidarrine or a lonidamine analog
is provided. Also provided are unit dosage forms of lonidamine or
an analog, useful for such treatment and prophylaxis.
Inventors: |
Tidmarsh, George; (Portola
Valley, CA) |
Correspondence
Address: |
TOWNSEND AND TOWNSEND AND CREW, LLP
TWO EMBARCADERO CENTER
EIGHTH FLOOR
SAN FRANCISCO
CA
94111-3834
US
|
Assignee: |
Threshold Pharmaceuticals,
Inc.
Redwood City
CA
|
Family ID: |
32777423 |
Appl. No.: |
11/172050 |
Filed: |
June 29, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11172050 |
Jun 29, 2005 |
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10759337 |
Jan 16, 2004 |
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60496163 |
Aug 18, 2003 |
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60488265 |
Jul 18, 2003 |
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60472907 |
May 22, 2003 |
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60460012 |
Apr 2, 2003 |
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60458846 |
Mar 28, 2003 |
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60458665 |
Mar 28, 2003 |
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60458663 |
Mar 28, 2003 |
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60442344 |
Jan 23, 2003 |
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60441110 |
Jan 17, 2003 |
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Current U.S.
Class: |
424/468 ;
514/406 |
Current CPC
Class: |
A61K 31/70 20130101;
A61P 13/08 20180101; A61K 31/416 20130101; G01N 2800/342
20130101 |
Class at
Publication: |
424/468 ;
514/406 |
International
Class: |
A61K 031/416; A61K
009/22 |
Claims
What is claimed is:
1. A unit dose formulation of lonidamine that is not a 150 mg
tablet.
2. A unit dose formulation of lonidamine comprising an amount of
lonidamine in the range of 10 mg to 100 mg or in the range of 200
mg to 5000 mg.
3. A unit dose formulation of lonidamine that is not an oral dosage
form.
4. A unit dose formulation of lonidamine that is not in the form of
a pill, tablet, capsule, caplet, troche, elixir, oral suspension,
syrup, wafer, or lozenge.
5. A unit dose formulation of a lonidamine analog.
6. A sustained release formulation of lonidamine or a lonidamine
analog.
7. The sustained release formulation of claim 6 that comprises (a)
a reservoir system; (b) a matrix system; (c) a microencapsulation
system; (d) a coated granule system; (e) a solvent-activated
system; or, (f) a polymeric degradation system.
8. A method for treating or preventing benign prostatic hypertrophy
(BPH) comprising administering the formulation of any of claims 1-7
to a human subject in need of such treatment or prevention.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 10/759,337 (filed Jan. 16, 2004) which claimed
the benefit of U.S. provisional application Nos. 60/496,163 (filed
18 Aug. 2003), 60/488,265 (filed Jul. 18, 2003); 60/472,907 (filed
22 May 2003), 60/460,012 (filed 2 Apr. 2003), 60/458,846 (filed 28
Mar. 2003), 60/458,665 (filed 28 Mar. 2003), 60/458,663 (filed 28
Mar. 2003), 60/442,344 (filed 23 Jan. 2003), and 60/441,110 (filed
17 Jan. 2003). Each of the aforementioned applications is
incorporated herein by reference in its entirety for all
purposes.
FIELD OF THE INVENTION
[0002] The invention relates to treatment and prevention of benign
prostatic hyperplasia, and has application in the field of medicine
and related fields, including chemistry, medicinal chemistry, and
molecular biology.
BACKGROUND OF THE INVENTION
[0003] Benign Prostatic Hyperplasia (BPH), a disease in which
prostate epithelial cells grow abnormally and block urine flow,
afflicts more than 10 million adult males in the United States, and
many millions more throughout the rest of the world. Until
relatively recently, surgical intervention was the only treatment
of the disease, and even today, surgery is the treatment of last
resort, almost inevitably relied upon when other treatments are
not, or cease to be, effective. Prostate surgery and recovery
therefrom is painful, and the surgery itself may not be effective
and poses the risk of serious side effects. For a recent review,
see Barry, 2001 (full citations are provided below).
[0004] Only two classes of drugs are currently available to treat
the symptoms of BPH. One class includes compounds that inhibit
production of the active form of testosterone (dihyrdotestosterone
or DHT). Use of drugs in this class can cause a loss of libido and
loss of muscle mass and tone in males and is associated with an
increased occurrence of high grade prostate cancer. In addition,
this therapy is limited by the very long delay (months) between
first administering the drug and significant reduction in prostate
size in the patient. The second class of currently used drugs for
BPH, alpha adrenergic blockers, acts by relaxing the smooth
muscles, allowing urine to pass through the urethra more freely.
While this class of drugs reduces symptoms more rapidly than the
first, it does not reduce the size of the prostate or prevent it
from growing larger, which can lead to eventual surgical
intervention.
[0005] Thus, there is a significant, unmet need for drugs that can
treat the underlying disease condition of BPH without serious side
effects. The present invention meets that need.
SUMMARY OF THE INVENTION
[0006] The invention provides methods and compositions for treating
BPH in a human subject by administration of lonidamine or a
lonidamine analog to the subject. Pharmaceutical compositions
useful for treatment of BPH, including sustained release
formulations, are also provided. In one embodiment, the formulation
is orally administered and permits once-a-day dosing of a
therapeutically effective dose of the compound.
BRIEF DESCRIPTION OF THE FIGURES
[0007] FIG. 1 shows structures for lonidamine (I, R.dbd.Cl),
tolnidamine (I, R.dbd.CH.sub.3), AF-2364 (II) and AF-2785
(III).
[0008] FIG. 2 shows the expression of HIF-1.alpha. in LNCaP cells
under normoxic and hypoxic conditions and in the presence and
absence of lonidamine. FIG. 2A shows an assay using a nuclear
extract. FIGS. 2B and 2C show an assay using a whole cell
extract.
[0009] FIG. 3 shows the expression of HIF-1.alpha. in PC-3 cells
under normoxic and hypoxic conditions and in the presence and
absence of lonidamine. FIGS. 3A and 3C show an assay using a
nuclear extract. FIG. 3B shows an assay using a whole cell
extract.
[0010] FIG. 4 shows lonidamine-induced apoptosis in LNCaP (FIG. 4A)
and PC-3 (FIG. 4B) cells.
[0011] FIG. 5 shows lonidamine-induced apoptosis in prostate
epithelial cells.
[0012] FIG. 6 shows lonidamine-induced apoptosis in prostate
epithelial cells (FIG. 6A) and prostate stromal cells (FIG.
6B).
[0013] FIG. 7 shows the effect of 0-600 .mu.M lonidamine on
expression of HIF-1.alpha. and other proteins as determined in
whole cell extracts from LNCaP cells cultured under hypoxic
conditions.
[0014] FIG. 8 shows the effect of 0-600 .mu.M lonidamine on
expression of HIF-1.alpha. and other proteins as determined in
nuclear extracts from LNCaP cells cultured under hypoxic
conditions.
DETAILED DESCRIPTION OF THE INVENTION
[0015] 1. Definitions
[0016] The following definitions are provided to assist the reader.
Unless otherwise defined, all terms of art, notations and other
scientific or medical terms or terminology used herein are intended
to have the meanings commonly understood by those of skill in the
chemical and medical arts. In some cases, terms with commonly
understood meanings are defined herein for clarity and/or for ready
reference, and the inclusion of such definitions herein should not
necessarily be construed to represent a substantial difference over
the definition of the term as generally understood in the art.
[0017] As used herein, "treating" a condition or patient refers to
taking steps to obtain beneficial or desired results, including
clinical results. For purposes of this invention, beneficial or
desired clinical results include, but are not limited to,
alleviation or amelioration of one or more symptoms of BPH,
diminishment of extent of disease, delay or slowing of disease
progression, amelioration, palliation or stabilization of the
disease state, and other beneficial results described below.
[0018] As used herein, "reduction" of a symptom or symptoms (and
grammatical equivalents of this phrase) means decreasing of the
severity or frequency of the symptom(s), or elimination of the
symptom(s).
[0019] As used herein, "administering" or "administration of" a
drug to a subject (and grammatical equivalents of this phrase)
includes both direct administration, including self-administration,
and indirect administration, including the act of prescribing a
drug. For example, as used herein, a physician who instructs a
patient to self-administer a drug and/or provides a patient with a
prescription for a drug is administering the drug to the
patient.
[0020] As used herein, a "manifestation" of BPH refers to a
symptom, sign, anatomical state (e.g., prostate size),
physiological state (e.g., PSA level), or report (e.g., AUASI
score) characteristic of a subject with BPH.
[0021] As used herein, a "therapeutically effective amount" of a
drug is an amount of a drug that, when administered to a subject
with BPH, will have the intended therapeutic effect, e.g.,
alleviation, amelioration, palliation or elimination of one or more
manifestations of BPH in the subject. The full therapeutic effect
does not necessarily occur by administration of one dose, and may
occur only after administration of a series of doses. Thus, a
therapeutically effective amount may be administered in one or more
administrations.
[0022] As used herein, a "prophylactically effective amount" of a
drug is an amount of a drug that, when administered to a subject,
will have the intended prophylactic effect, e.g., preventing or
delaying the onset (or reoccurrence) of disease or symptoms, or
reducing the likelihood of the onset (or reoccurrence) of disease
or symptoms. The full prophylactic effect does not necessarily
occur by administration of one dose, and may occur only after
administration of a series of doses. Thus, a prophylactically
effective amount may be administered in one or more
administrations.
[0023] As used herein, "TID" and "QD" have their ordinary meanings
of "three times a day" and "once daily," respectively.
[0024] As used herein, "alkyl" refers to a monovalent alkane
(hydrocarbon) derived radical containing from 1 to 15 carbon atoms.
It may be straight, branched or cyclic and may be unsubstituted or
substituted with substituent groups including but not limited to
hydroxyl, halide, alkoxyl, and nitrile. Alkoxy groups that can be
used include but are not limited to methooxy. Illustrative straight
or branched alkyl groups include methyl, ethyl, propyl, isopropyl,
butyl and t-butyl.
[0025] As used herein, "aryl" refers to moieties that include one
or more monocyclic or fused ring aromatic systems. Such moieties
include any moiety that includes one or more monocyclic or bicyclic
fused ring aromatic systems, including but not limited to phenyl
and naphthyl. Aryl groups may be unsubstituted or substituted with
substituent groups as listed for the particular substituted
aryl.
[0026] As used herein, "heteroaryl" refers to monocyclic aromatic
groups having 5 or 6 ring atoms, or fused ring bicyclic aromatic
groups having 8 to 10 atoms, in which the ring atoms are C, O, S,
SO, SO.sub.2, or N and at least one of the ring atoms is a
heteroatom, i.e., O, S, SO, SO.sub.2, or N. Heteroaryl groups may
be unsubstituted or substituted with substituent groups as listed
for the particular substituted heteroaryl. Examples of monocyclic
aromatic heteroaryl groups include but are not limited to pyridyl.
Examples of bicyclic fused ring heteroaryl groups include but are
not limited to indazolyl, pyrrolopyrymidinyl, indolizinyl,
pyrazolopyridinyl, triazolopyridinyl, pyrazolopyrimidinyl,
triazolopyrimidinyl, pyrrolotriazinyl, pyrazolotriazinyl,
triazolotriazinyl, pyrazolotetrazinyl, hexaaza-indenly, and
heptaaza-indenyl. Unless indicated otherwise, the arrangement of
the hetero atoms within the ring may be any arrangement allowed by
the bonding characteristics of the constituent ring atoms.
[0027] As used herein, the terms "heterocycloalkyl" and
"heterocyclyl" refer to a monocyclic or fused ring multicyclic
cycloalkyl group at least a portion of which is not aromatic and in
which one or more of the carbon atoms in the ring system is
replaced by a heteroatom selected from O, S, SO, SO.sub.2, or N.
Examples of heterocyclyl groups include but are not limited to
piperidinyl, morpholinyl, pyrrolidinyl, tetrahydrofuranyl,
tetrahydroimidazo[4,5-c]pyridinyl, imidazolinyl, piperazinyl,
pyrrolidine-2-onyl, and piperidin-2-onyl.
[0028] As used herein, "cycloalkyl" refer to a monocyclic or fused
ring multicyclic group at least a portion of which is not aromatic
and in which the ring atoms are carbon.
[0029] As used herein "heterocycloalkenyl" refers to a monocyclic
or fused ring multicyclic group in which one or more of the carbon
ring atoms is replaced by a hetero atom, the ring system is at
least partially not aromatic, and the ring system includes at least
one carbon-carbon double bond.
[0030] 2. Benign Prostatic Hyperplasia and the Effects of
Lonidamine and Lonidamine Analogs
[0031] The present invention provides compositions and methods
useful in the treatment of benign prostatic hyperplasia (BPH). In
particular, the invention relates to the use of lonidamine (LND)
for the treatment or prevention of BPH. Additionally, the invention
relates to the use of lonidamine analogs for the treatment or
prevention of BPH. To aid in understanding the invention, a brief
discussion of BPH (also referred to as benign prostatic
hypertrophy) and the properties of lonidamine and its bioactive
analogs is provided below.
[0032] BPH involves overgrowth (hyperplasia) of cells in the
prostate, resulting in enlargement of the prostate and leading to
lower urinary tract symptoms and disease. The prostate gland
contains secretory epithelial cells in a stroma of connective
tissue and smooth muscle (see Barry, 2003, for a more detailed
description of prostate anatomy), and BPH involves hyperplasia of
the epithelial component. The secretory epithelial component in the
normal prostate is remarkable in that the level of zinc in this
tissue is very high compared to other normal tissues. A consequence
of the high zinc levels is that, through a mechanism involving zinc
inhibition of the enzyme m-aconitase, the generation of energy via
the tricarboxylic acid (TCA) cycle and oxidative phosphorylation is
substantially reduced in the secretory epithelium, making this
tissue far more dependent than other organs and tissues upon
glycolysis as an energy source. The zinc inhibition of m-aconitase,
a key enzyme in the TCA cycle, results in at least a substantial
reduction in, and perhaps a near complete blockade of, the TCA
cycle in prostate epithelial cells. Another physiological result of
the zinc-based inhibition of m-aconitase is the diversion of
citrate from the TCA cycle, enabling the prostate to secret large
quantities of citrate, used by the sperm as an energy source, into
the seminal fluid. See, generally, Costello, 1999; Costello et al.,
2000; Costello and Franklin, 2000.
[0033] As other normal cells in the body do not accumulate zinc to
a level inhibitory to the metabolism of citrate, prostate
epithelial cells are uniquely dependent on glycolysis (anaerobic
metabolism). The present invention relates in part to the discovery
of these cells' susceptibility to the drug lonidamine, which allows
lonidamine to be administered, as described herein, to treat or
prevent BPH in humans. Lonidamine is the generic name for
1-(2,4-dichlorobenzyl)-1H-indazole-3-carboxylic acid, and has also
been referred to in the medical literature as
1-[(2,4-dichlorophenyl)methyl]-1H-indazole-3-carboxylic acid,
AF1890, diclondazolic acid (DICA), and Doridamina.TM.. See FIG. 1.
Lonidamine was first identified as an antispermatogenic agent, and
subsequently used in the treatment of breast, cervical, lung and
prostate cancers, in a few countries in Europe. See Silvestrini,
1981; Gatto et al., 2002. The mechanisms of action of lonidamine in
spermatogenesis and cancer may not be completely understood.
However, it has been suggested that lonidamine's anticancer
properties result at least in part from a lonidamine-mediated
disruption of the mitochondrial membrane, resulting in reduced
activity of mitochondrially-bound hexokinase and interference with
ATP production by the glycolytic pathway and oxidative
phosphorylation. See, Floridi et al., 1981, Fanciulli et al., 1996,
and references numbered 15-22 therein; and Gatto, 2002. Also see
Kaplan, 2000. Without intending to be bound by a specific mechanism
for the effects of lonidamine in benign prostatic hyperplasia, it
is believed that lonidamine inhibits glycolysis and/or impairs the
already diminished mitochondrial function in prostate epithelial
cells, starving these cells, relative to the normal cells in the
body, of energy. Without intending to be bound by a specific
mechanism, it is believed that, due to this energy deprivation,
enough of the hyperplastic, epithelial cells are destroyed or
otherwise reduced in size to reduce the size of the prostate and
thereby relieve the condition and its clinical consequences.
[0034] Accordingly, administration of lonidamine to a human subject
diagnosed with, or exhibiting symptoms of, BPH provides benefits
such as reduction of severity or frequency of one or more symptoms,
reduction in prostate size or rate of enlargement, improvement in
perceived quality of life, and reversion of other manifestations of
BPH toward a more normal state. Further, administration of
lonidamine to a human subject in need of prophylaxis for BPH
provides benefits such as a reduction in likelihood that BPH will
appear, reappear or progress in the subject. Still further,
administration of a lonidamine analog to a human subject is
similarly effective for treatment and prophylaxis of BPH. In
another embodiment, administration of lonidamine or its analogs to
a human subject as described herein can be efficacious in the
treatment of acute urinary retention. These and other aspects of
the invention are discussed in greater detail below. Section 3,
below, describes certain lonidamine analogs useful for treatment
and prophylaxis of BPH. Section 4 relates to synthesis and forms of
lonidamine and lonidamine analogs. Section 5 describes patient
populations for whom administration of lonidamine and lonidamine
analogs provides benefit. Section 6 describes methods of
administration of lonidamine (e.g., dose, route, schedule and
duration of administration). Section 7 describes combination
therapies in which lonidamine or an analog is administered in
combination with another drug or therapy. Section 8 describes
exemplary dosage forms. Section 9 provides examples of the use and
effects of lonidamine. The description below is organized into
sections for convenience only, and disclosure found in any
organizational section is applicable to any aspect of the
invention.
[0035] 3. Lonidamine Analogs
[0036] As noted above, in addition to lonidamine, a variety of
compounds related to lonidamine are useful for the treatment and
prevention of BPH. Useful compounds are generally structurally
similar to, are bioisosteres of, or are pharmacophores of
lonidamine, as described below, and have biological activity(s)
similar to those of lonidamine, as also discussed below. Such
compounds can be referred as "bioactive lonidamine analogs,"
"lonidamine analogs," or, in some cases, simply, "analogs."
[0037] Structural characteristics of lonidamine analogs. Based, in
part, on the structure of lonidamine and related compounds known to
have pharmaceutical activities similar to that of lonidamine,
certain lonidamine analogs, including novel analogs provided by the
present invention, suitable for use in treatment or prophalaxis of
BPH are described by the formula, 1
[0038] where R.sub.1, R.sub.2, X, Y, n and 2
[0039] are defined below:
[0040] R.sub.1 represents --COOH or a derivative or bioisostere of
the --COOH group. R.sub.1 is usually selected from an acid group of
formula --COOH; an amide of formula --CONR.sub.3R.sub.4, where
R.sub.3 and R.sub.4 may be independently alkyl or hydrogen, with
hydrogen preferred; a hydrazide of formula --CONHNR.sub.6R.sub.7,
where R.sub.6 and R.sub.7 are usually --H or --CH.sub.3; a
substituted ester of formula --COOR.sub.5, with R.sub.5 being a
residue easily hydrolyzed in the subject after administration and
generally a straight chain or branched chain alkyl group
substituted with one or more hydroxyl groups, more usually a
straight chain or branched chain methyl, ethyl, or propyl group
substituted with one or more hydroxyl groups, more usually still an
ethyl group substituted with one hydroxyl group or a straight chain
or branched chain propyl group substituted with two hydroxyl
groups, and most usually --CH.sub.2CH.sub.2OH,
--CH.sub.2CH(OH)CH.sub.2OH, or --CH.sub.2(CH.sub.2OH).sub.2.
R.sub.1 may also be the carboxylate anion of formula --COO.sup.-,
in which case the lonidamine or lonidamine analog will be
associated with a counter ion, Z.sup.+, where Z.sup.+ is a
pharmaceutically acceptable cation.
[0041] R.sub.2 represents a substituted or unsubstituted aryl or
heteroaryl group. Usually, R.sub.2 is a substituted aryl group;
more usually, a substituted phenyl group; more usually still, a
phenyl group substituted by one, two, or three substituents
independently selected from halo and alkyl substituents,
particularly --Cl, --Br, --I, CF.sub.3 and --CH.sub.3 substituents.
When R.sub.2 is a substituted phenyl group, R.sub.2 is usually
--Cl, --Br, --I, CF.sub.3 or --CH.sub.3, monosubstituted phenyl,
substituted at the 2, 3, or 4 position; dichloro, dibromo,
dimethyl, or chloro and methyl disubstituted phenyl, substituted at
the 2 and 3 or 2 and 4 positions; or 2, 4, 5 trichlophenyl. When
R.sub.2 is a substituted phenyl group, R.sub.2 is more usually
2,4-dichlorophenyl or 4-chloro-2-methylphenyl.
[0042] X represents a straight chain or branched chain, saturated
or unsaturated hydrocarbon linkage group. When X is a saturated
hydrocarbon linkage group, X is usually a straight chain linkage
group and usually X has the formula --(CH.sub.2).sub.p--, with p
equal to 1, 2, or 3. When X is a saturated hydrocarbon linkage
group, X is most usually a methylene group, --(CH.sub.2)--. When X
is an unsaturated hydrocarbon linkage group, X is usually a
straight chain linkage group, most usually --(CH.dbd.CH)--.
[0043] Y represents a moiety of formula --CHR.sub.7--, where
R.sub.7 is hydrogen or a straight chain or branched chain alkyl
group, more usually R.sub.7 is hydrogen or or a straight chain
alkyl group, more usually still R.sub.7 is hydrogen, methyl, ethyl,
or n-propyl, more usually still R.sub.7 is hydrogen or methyl, and
most usually R.sub.7 is hydrogen (i.e., Y is most usually
--CH.sub.2--).
[0044] n is zero or, most usually, one. 3
[0045] is a core ring system that may generally be an aryl,
heteroaryl, cycloalkyl or heterocyclyl ring system. The Ar core
ring system usually includes 2 fused rings. The fused rings may
generally be 4-, 5-, 6-, 7-, or 8-membered rings, more usually 5-
or 6-membered rings. The core ring system is most usually fused 5-
and 6-membered rings. The fused ring atoms may generally be any
atom, usually carbon or hetero atoms, more usually carbon and
nitrogen group atoms, and more usually still carbon and nitrogen.
The number of carbon atoms in the core ring system is usually 7.
The core ring system usually contains 2 hetero atoms, where the
preferred hetero atom is nitrogen. Generally, one or more of the
fused rings may be aromatic. When the core ring system is fused 5-
and 6-membered rings, the core ring system is usually aromatic over
both fused rings. The fused 5- and 6-membered ring system is most
usually an indazole.
[0046] More particularly, lonidamine analogs for use according to
the methods of the invention, and certain of the novel analogs
provided by the invention, include analogs of the formula 4
[0047] where R.sub.1, R.sub.2, X, Y, and n are generally as above
or, in a preferred version,
[0048] R.sub.2 is --Cl, --Br, --I, or --CH.sub.3, monosubstituted
phenyl, substituted at the 2, 3, or 4 position; dichloro, dibromo,
dimethyl, or chloro and methyl disubstituted phenyl, substituted at
the 2 and 3 or 2 and 4 positions; or 2, 4, 5 trichlophenyl;
[0049] Y is --(CH.sub.2)--; and
[0050] n is zero, and R.sub.1 is --COOH, --CONH.sub.2,
--CONHNH.sub.2, --CONHN(CH.sub.3).sub.2, --CH.sub.2CH.sub.2OH,
--CH.sub.2CH(OH)CH.sub.2OH- , or CH.sub.2(CH.sub.2OH).sub.2; or
[0051] n is one, R.sub.1 is --COOH, and X is --CH.dbd.CH--.
[0052] In one embodiment, the lonidamine analog is a
1,3-substituted-indazole, such as a 1-halobenzyl-1H-indazole. In
another embodiment, the lonidamine analog is a 3-substituted
1-benzyl-1H-indazole. In another embodiment, the lonidamine analog
is a 1-substituted-indazole-3-carboxylic acid, such as a
1-halobenzyl-1H-indazole-3-carboxylic acid.
[0053] Bioisosteres. In addition, lonidamine analogs that may be
used in the treatment methods of the invention include bioisosteres
and pharmacophores of lonidamine and analogs described herein.
Bioisosterism is a well-known tool for predicting the biological
activity of compounds, based upon the premise that compounds with
similar size, shape, and electron density can have similar
biological activity. To form a bioisostere of a given molecule, one
replaces one or more atoms or groups with known bioisosteric
replacements for that atom or group. Known bioisosteric
replacements include, for example, the interchangeability of --F,
--OH, --NH.sub.2, --Cl, and --CH.sub.3; the interchangeability of
--Br and -i-C.sub.3H.sub.7; the interchangeability of --I and
-t-C.sub.4H.sub.9; the interchangeability of --O--, --S--, --NH--,
--CH.sub.2, and --Se--; the interchangeability of --N.dbd.,
--CH.dbd., and --P.dbd. (in cyclic or noncyclic moieties); the
interchangeability of phenyl and pyridyl groups; the
interchangeability of --C.dbd.C-- and --S-- (for example, benzene
and thiophene); the interchangeability of an aromatic nitrogen
(R.sub.1--N(R.sub.3)--R.sub.2) for an unsaturated carbon
(R.sub.1--C(.dbd.R.sub.3)--R.sub.2); and the interchangeability of
--CO--, --SO--, and --SO.sub.2--. These examples are not limiting
on the range of bioisosteric equivalents and one of skill in the
art will be able to identify other bioisosteric replacements known
in the art. See, e.g., Patani and LaVoie, 1996; and Burger,
1991.
[0054] Pharmacophores. In addition to the lonidamine analogs
described herein, lonidamine analogs that may be used in the
methods of the invention can generally be any pharmacophore of
lonidamine and the lonidamine analogs described above. Often a
reasonable quantitative prediction of the binding ability of a
known molecule can be made based on the spatial arrangement of a
small number of atoms or functional groups in the molecule. Such an
arrangement is called a pharmacophore, and once the pharmacophore
or pharmacophores in a molecule have been identified, this
information can be used to identify other molecules containing the
same or similar pharmacophores. Such methods are well known to
persons of ordinary skill in the art of medicinal chemistry, and as
the structural information described in this application identifies
the pharmacophore of lonidamine and the lonidamine analogs relevant
to treatment of BPH, those of skill in the art can identify other
LND analogs that comprise the pharmacophore and so are useful in
treating BPH. An example of programs available to perform
pharmacophore--related searches is the program 3D Pharmacophore
search from the Chemical Computing Group (see
http://www.chemcomp.com/ fdept/prodinfo.htm).
[0055] A lonidamine analog of particular interest is tolnidamine
(1-(4-chloro-2-methylbenzyl)-1H-indazole-3-carboxylic acid, AF
1923); see Ansari et al., 1998; Corsi et al., 1976. Tolnidamine
(TND) differs from lonidamine by the presence of a methyl
substituent, rather than a chlorine substituent, in position 2 of
the benzyl group. Other analogs of lonidamine with biological
activity have been described in the following publications: U.S.
Pat. No. 3,895,026 entitled "Substituted
1-Benzyl-1H-indazole-3-Carboxylic Acids and Derivatives Thereof;"
Corsi et al., 1976, "1-Halobenzyl-1H-Indazole-3-Carboxylic Acids. A
New Class of Antispermatogenic Agents," Journal of Medicinal
Chemistry 19:778-83; Silvestrini, 1981, "Basic and Applied Research
n the Study of Indazole Carboxylic Acids," Chemotherapy 27:9-20;
Lobl et al., 1981, "Effects of Lonidamine (AF 1890) and its
analogues on follicle-stimulating hormone, luteinizing hormone,
testosterone and rat androgen binding protein concentrations in the
rat and rhesus monkey," Chemotherapy 27:61-76; U.S. Pat. No.
6,001,865 entitled "3-Substituted 1-Benzyl-1H-Indazole Derivatives
As Antifertility Agents"; and Cheng et al., 2001, "Two new male
contraceptives exert their effects by depleting germ cells
prematurely from the testis," Biol Reprod. 65:449-61, which
describe AF-2364 and AF-2785 and other compounds (see FIG. 1).
[0056] Functional characteristics of lonidamine analogs. Lonidamine
analogs suited for use in the invention are those that interfere
with cellular energy metabolism of prostate epithelial cells when
administered to a human, non-human primate, or other mammal. As is
usual in the pharmaceutical arts, not every structural analog of a
compound (e.g., lonidamine) is pharmacologically active. Active
forms can be identified by routine screening of analogs for the
activity of the parent compound. A variety of assays and tests can
be used to assess pharmacological activity of lonidamine analogs,
including in vitro assays, such as those described below and
elsewhere herein, in vivo assays of prostate function (including
citrate production and ATP production) in humans, non-human
primates and other mammals, in vivo assays of prostate size in
humans, non-human primates and other mammals, and/or clinical
studies.
[0057] Apoptosis assay in cell lines. As shown in Example 3,
lonidamine induces apoptosis in cell lines derived from human
prostate cells. The induction of apoptosis is significantly greater
in LNCaP cells (ATCC NO. CLR-1740), a prostate-derived cell line
that is citrate-producing, than in PC3 cells (ATCC NO. CLR-1435), a
prostate-derived cell line that is citrate-oxidizing, consistent
with the susceptibility of the citrate-producing prostate cells to
metabolic inhibitors such as lonidamine. In some embodiments of the
invention in which a lonidamine analog is used for treatment or
prevention of BPH or its manifestations, an analog with similar
apoptosis-inducing activity is selected. Thus, in some embodiments
of the invention, a lonidamine analog that induces apoptosis
(enhances caspase 3 activity) in citrate-producing prostate cells,
such as LNCaP cells, is administered to treat BPH. In some
embodiments of the invention, a lonidamine analog that induces
apoptosis in LNCaP cells to a significantly greater degree than in
PC3 cells is administered to treat BPH. In some embodiments of the
invention, the induction of apoptosis by the lonidamine analog is
at least about 2-fold greater in LNCaP cells than in PC3 cells (and
sometimes at least about 3-fold greater, at least about 4-fold
greater, or at least about 10-fold greater) when assayed at the
concentration of analog at which the difference in the level of
apoptosis in the two cell lines is greatest (provided that the
concentration of analog used in the assay is not greater than 1
mM).
[0058] Apoptosis assay in primary cell cultures. As shown in
Example 3, lonidamine induces apoptosis in primary cultures of
human prostate epithelial cells. The induction of apoptosis is
significantly greater in primary cultures of prostate epithelial
cells than in primary cultures of human prostate stromal cells,
consistent with the susceptibility of citrate-producing prostate
cells to metabolic inhibitors such as lonidamine. In some
embodiments of the invention in which a lonidamine analog is
administered for treatment or prevention of BPH or its
manifestations, an analog with apoptosis-inducing activity similar
to that of lonidamine is selected. Thus, in some embodiments of the
invention, a lonidamine analog that induces apoptosis in prostate
epithelial cells is administered to treat BPH. In some embodiments
of the invention, a lonidamine analog that induces apoptosis in
primary cultures of prostate epithelial cells to a significantly
greater degree than in primary cultures of human prostate stromal
cells is used. In some embodiments of the invention, the lonidamine
analog does not significantly induce apoptosis in stromal cells. In
some embodiments of the invention, induction of apoptosis by the
lonidamine analog is at least 2-fold greater in epithelial cells
than in stromal cells (and sometimes at least 4-fold greater,
sometimes at 10-fold greater, and sometimes at least 20-fold
greater) when assayed at the concentration of analog at which the
difference in the level of apoptosis in the two cell lines is
greatest (provided that the concentration of analog used in the
assay is not greater than 1 mM).
[0059] HIF-1.alpha. expression assays. As shown in Example 2,
lonidamine reduced HIF-1.alpha. expression/accumulation (measured
in the nuclear fraction) in cells cultured under conditions of
hypoxia by almost 2-fold at 200 micromolar and by more than 5 fold
(i.e., more than 10-fold) at higher lonidamine concentrations.
Thus, in some embodiments of the invention, an energolytic agent
reduces HIF-1.alpha. expression (prevents HIF-1.alpha.
accumulation) in LNCaP cells cultured under hypoxic conditions by
at least about 2-fold, at least about 5-fold or at least about
10-fold compared to culture in the absence of lonidamine.
[0060] In the figures corresponding to Example 2, the effect of
lonidamine on HIF-1.alpha. expression in prostate cells appears
more pronounced in LNCaP cells than in PC3 cells cultured under
hypoxic conditions (oxygen level <0.1%). Some lonidamine analogs
useful for treatment of BPH according to the present invention may
have a similar effect.
[0061] The results of these experiments do not definitively
establish the mechanism or specificity of inhibition of
HIF-1.alpha. by lonidamine. Lonidamine's effect on HIF-1.alpha.
levels may be due entirely or in part to a general inhibition of
protein synthesis, described as an activity of lonidamine by
Floridi et al., 1985. Lonidamine's effect on HIF-1.alpha. levels
could also be due entirely or in part to lonidamine's effect on
oxygen utilization by mitochondria. Hagen et al., 2003, reported
that HIF-1.alpha. is constitutively synthesized but degraded in the
presence of oxygen. It is possible that, under hypoxic conditions,
inhibition of mitochondrial respiration by lonidamine reduces
oxygen consumption by mitochondria. This in turn could lead to
enhanced activity of the oxygen-dependent enzyme, prolyl hydrolase,
which plays a role in the HIF-1.alpha. degradation pathway.
[0062] Hexokinase activity. As discussed above, and without
intending to be bound to any specific mechanism, the effects of
lonidamine on the prostate may be mediated, at least in part, by
its effects on mitochondria and mitochondrial hexokinase activity
in secretory epithelial cells. Accordingly, some lonidamine analogs
useful in the present invention have hexokinase inhibitory activity
as great or greater than that of lonidamine. Assays for hexokinase
activity are known in the art. See Fanciulli et al., 1996, and
Floridi et al., 1981.
[0063] Antispermatogenic activity. Likewise, it is believed that
the antispermatogenic activity of lonidamine results, at least in
part, from energolytic effects in germ cells. Some lonidamine
analogs useful in the present invention have antispermatogenic
activity as great, or greater, than that of lonidamine. Assays for
antispermatogenic activity are known in the art. See, e.g., Grima
et al., 2001; Lohiya et al., 1991.
[0064] In addition to in vitro assays, energolytic agents can be
evaluated in vivo for use in the methods of the invention. For
example and without limitation, suitable assays include
measurements of prostate function and activity.
[0065] In vivo measurements of prostate function. The effect of a
compound on prostate function, and, in particular, on respiration,
can be assessed by monitoring prostate tissue metabolism following
administration of the compound. Some lonidamine analogs useful in
the present invention will detectably reduce ATP, citrate, and/or
lactate production by the prostate in animals (including humans,
non-human primates and other mammals). ATP, citrate, and/or lactate
levels can be monitored directly and/or indirectly in vivo using
techniques of magnetic resonance spectroscopy (MRS) or other
methods. See, for example, Narayan and Kurhanewicz, 1992;
Kurhanewicz et al., 1991; Thomas et al., 1990, for MRS assays that
can be applied for this purpose.
[0066] In vivo measurements of prostate size. The effect of a
compound on prostate size can be assessed following administration
of the compound using standard methods (for example,
ultrasonography or digital rectal examination, for humans, and
ultrasonography and/or comparison of organ weight in animals).
Assays can be conducted in humans or, more usually, in healthy
non-human animals or in monkey, dog, rat, or other animal models of
BPH (see, Jeyaraj et al., 2000; Lee et al., 1998; Mariotti et al.,
1982), Some lonidamine analogs useful in the present invention will
detectably reduce prostate size in such assays and animal
models.
[0067] Clinical trials. Clinical trials, such as that described for
lonidamine in the Example, infra, can be used to assess the
therapeutic effects of lonidamine analogs.
[0068] The activity of a lonidamine analog of interest in any of
the aforementioned assays can be compared with that of lonidamine
to provide guidance concerning dosage schedules for the compound,
and other information. Generally, lonidamine analogs with greater
biological activity per mg than lonidamine are of special
interest.
[0069] 4. Synthesis and Forms of Lonidamine and Lonidamine
Analogs
[0070] Lonidamine and lonidamine analogs and derivatives can be
prepared using by well known synthetic methods. Synthesis of
lonidamine is described in U.S. Pat. No. 3,895,026 and Germany
Patent No. 2,310,031. Synthesis of exemplary lonidamine analogs,
including tolnidamine (TND), is described in the art (see, e.g.,
Corsi et al., 1976, "1-Halobenzyl-1H-Indazole-3-Carboxylic Acids. A
New Class of Antispermatogenic Agents", Journal of Medicinal
Chemistry 19:778-83; Cheng et al., 2001, "Two new male
contraceptives exert their effects by depleting germ cells
prematurely from the testis" Biol Reprod. 65:449-61; Silvestrini,
1981, "Basic and Applied Research in the Study of Indazole
Carboxylic Acids" Chemotherapy 27:9-20; Lobl et al., 1981, "Effects
of Lonidamine (AF 1890) and its analogues on follicle-stimulating
hormone, luteinizing hormone, testosterone and rat androgen binding
protein concentrations in the rat and rhesus monkey" Chemotherapy
27:61-76; U.S. Pat. Nos. 3,895,026 and 6,001,865). It will be
appreciated, of course, that lonidamine analogs useful in the
practice of the invention are not limited to those for which
specific structures are provided in this disclosure or the cited
references, and that the compounds described above are provided for
illustration and not to limit the present invention. It also will
be clear that lonidamine analogs useful in the methods of the
present invention are not limited to those now described herein or
elsewhere in the pharmaceutical and patent literature; the
ordinarily skilled practitioner guided by the present disclosure
can synthesize novel analogs suitable for use according to the
present invention using routine methods of medicinal chemistry.
[0071] In certain embodiments, lonidamine or a lonidamine analog is
provided in the form of a pharmaceutically acceptable salt.
Pharmaceutically acceptable salts include addition salts with
acids, as well as the salts with bases. Salts with bases are, for
example, alkali metal or alkaline earth metal salts, such as
sodium, potassium, calcium or magnesium salts, or ammonium salts,
such as those with ammonia or suitable organic amines, e.g.
diethylamine, di-(2-hydroxyethyl)-amine or
tri-(2-hydroxyethyl)-amine. Suitable acids for the formation of
acid addition salts are, for example, mineral acids, such as
hydrochloric, hydrobromic, sulphuric or phosphoric acid, or organic
acids, such as organic sulphonic acids, for example,
benzenesulphonic, 4-toluenesulphonic or methanesulphonic acid, and
organic carboxylic acids, such as acetic, lactic, palmitic,
stearic, malic, maleic, fumaric, tartaric, ascorbic or citric
acid.
[0072] Administration of ester, amide and prodrug derivatives of
lonidamine and analogs is also contemplated in the practice of the
present invention (see, e.g., U.S. Pat. No. 6,146,658, for general
information regarding preparation of such derivatives from a
compound of interest) as is administration of polymorphic forms,
enantiomeric forms, tautomeric forms, solvates, hydrates, and the
like.
[0073] 5. Patients For Whom Administration of Lonidamine Provides
Benefit
[0074] The present invention provides that administration of
lonidamine to men afflicted with, or susceptible to, BPH can be
therapeutically effective. Accordingly, in one aspect of the
invention, lonidamine or a lonidamine analog is administered to a
subject in need of treatment for BPH. In one embodiment, the
subject in need of treatment is a human male who does not have
cancer. As used herein, "a subject in need of treatment for BPH" is
a man diagnosed with BPH. BPH is diagnosed using art-known methods
and criteria. The most common test is the digital rectal
examination in which a physician determines whether the prostate is
of a normal size and firmness. Other diagnostic assays include a
urine flow rate test, determination of post void residual urine
volume (e.g., by palpitation of the abdomen, drainage of residual
urine, x-ray urogramography, or ultrasonography), moderate or
severe symptom scores on the American Urologic Association Symptom
Index (AUASI; Barry et al., 1992) or International Prostate Symptom
Score (IPSS; Barry et al., 2001), and other tests known in the
art.
[0075] Desired clinical results of treatment for BPH include, but
are not limited to, alleviation or amelioration of one or more
symptoms of BPH (see below), a reduction in prostate size (see
below), a reduction in AUASI or IPSS scores compared to base line
measurements prior to commencement of therapy (for example, by 3
points or more, such as by 5 points or more), AUASI or IPSS scores
less than 8, a reduction in serum PSA by at least about 20%, such
as by at least about 40%, a serum PSA less than 4, such as less
than 2, improvement in urodynamic parameters, and other desired
results that will be recognized by a treating physician as
indicative of a reduction in severity of BPH in a subject. An
assessment of the response to treatment can be made at any time
following the first administration of the drug. For example, an
assessment is made about 30 days, about 60 days, or about 90 days
after beginning treatment. Alternatively, assessment can be made
about 6, 12, 18, 24 or more months after beginning treatment.
Alternatively, an assessment can be made less than about 30 days,
about 30 days, about 60 days, or about 90 days after a course of
treatment ends.
[0076] In a related aspect, lonidamine or a lonidamine analog is
administered to a human subject exhibiting a symptom associated
with BPH to reduce the frequency or severity of the symptom. As
used herein, "a symptom associated with BPH" refers to any one or
more of the following symptoms: (1) urinary urgency; (2) terminal
dribbling of urine; (3) frequent urination; (4) nocturia; (5) a
weak/slow stream of urine; (6) a sense of incomplete emptying; (7)
intermittency; (8) straining; (9) dysuria; (10) hematuria; (11)
acute urinary retention; (12) urinary tract infection; (13)
incontinence. Administration of lonidamine or a lonidamine analog
according to the methods of the invention typically results in a
reduction in severity, or elimination, of one or more of these
symptoms; usually results in either a reduction in severity of, or
elimination of, all of these symptoms; and often results in
elimination of all of these symptoms.
[0077] In another related aspect, lonidamine or a lonidamine analog
is administered to reduce prostate size in a human subject in need
of such reduction. As used herein, "a subject in need of reduction
of prostate size" is a man having an enlarged prostate gland as
determined by (1) imaging (e.g., ultrasonography, magnetic
resonance imaging) or (2) one or more signs or symptoms resulting
directly or indirectly from compression of the urethra by the
prostate (e.g., including the symptoms of BPH discussed herein). A
reduction in serum PSA (prostate specific antigen) is also a useful
proxy for reduction of prostate volume. Although varying among
individuals, enlarged prostates often exceed 30 grams, 40 grams, or
50 grams in size. The degree of reduction of prostate size will
vary from subject to subject due to a number of factors, including
the degree of enlargement at the time of onset of therapy, but will
typically be a reduction of at least about 10% volume, more often
at least about 25%, sometimes at least about 40%, sometimes at
least about 50%, and sometimes an even greater than 50% reduction
in prostate size is observed. This reduction can be determined by
imaging or other methods. Serum PSA can also in some instances
serve as a useful proxy for prostate volume.
[0078] In a related aspect, lonidamine or an analog is administered
to a subject with a serum PSA level greater than 2 ng/ml. PSA is
secreted only by the epithelial cells of the prostate. For men with
BPH, higher PSA levels suggest a relatively higher ratio of
epithelial cell proliferation to stromal cell proliferation than in
men with lower PSA levels. The present invention provides a number
of diagnostic methods suitable for use in determining patients who
should respond favorably to treatment with lonidamine or an analog.
Thus, lonidamine treatment can provide a therapeutic benefit to
subjects with PSA levels greater than 2 ng/ml. Accordingly,
subjects predicted to benefit significantly from treatment in
accordance with the invention can be selected in a population of
men with BPH by identifying subjects with a serum PSA value greater
than 2 ng/ml. In one embodiment of the invention, the subject has a
PSA level greater than about 4 ng/ml. Because higher PSA levels are
also, and perhaps more closely, associated with prostate cancer
than with BPH, in one embodiment, the subject selected for therapy
with lonidamine or an analog has a PSA level less than about 10
ng/ml.
[0079] In one aspect of the invention, lonidamine or a lonidamine
analog is administered to a subject who would benefit from
prophylaxis of BPH. In one example, "a subject who would benefit
from prophylaxis of BPH" is a man previously treated for BPH by
surgery, transurethral microwave thermotherapy, transurethral
needle ablation, transurethral electrovaporization, laser therapy,
balloon dilatation, prostatic urethral stent, drug therapy, or
other therapy and not currently diagnosed with or exhibiting
symptoms of BPH. In another example, a subject who would benefit
from prophylaxis of BPH is a man at increased risk for developing
BPH due to age (e.g., men older than 40, older than 50, older than
60, or older than 70 years of age). In another example, a subject
who would benefit from prophylaxis of BPH is a man who is
asymptomatic, or has symptoms sufficiently mild so that no clear
diagnosis of BPH can be made, but who has an elevated serum PSA
level (e.g., PSA>2 ng/ml or, in some cases, >4 ng/ml).
[0080] Thus, in some cases, the subject to whom lonidamine is
administered in accordance with the methods of the invention is a
man who has previously been treated for BPH, while in other cases
the subject is a man who has not previously been treated for BPH.
Similarly, it will be clear that any references in this section to
administration of lonidamine apply equally to administration of a
biologically active lonidamine analog.
[0081] In one embodiment of the invention, the subject in need of
treatment or prophylaxis for BPH either is not also under treatment
for cancer or does not have cancer. In a related embodiment, the
subject in need of treatment or prophylaxis for BPH has not been
diagnosed as having cancer. In one embodiment, the subject in need
of treatment or prophylaxis for BPH does not have cancer. In one
embodiment, the subject in need of treatment has a cancer other
than prostate cancer but does not have prostate cancer. As used
herein, "cancer" has its ordinary medical meaning and refers to a
malignancy (including head, neck, prostate and breast cancers,
leukemias and lymphomas), generally characterized by clonality,
autonomy, anaplasia, and metastasis (see Mendelsohn, 1991).
[0082] In one embodiment, the invention provides a method of
treating BPH in a patient by administering lonidamine to the
patient. In a related embodiment, the invention provides a method
for treating BPH comprising (a) administering lonidamine to a
patient diagnosed with BPH and (b) determining whether one or more
manifestations of BPH are reduced in the patient. In one
embodiment, the invention provides a method for treating BPH by (a)
diagnosing BPH in a patient, (b) administering lonidamine to the
patient and (c) determining whether one or more manifestations of
BPH are reduced in said patient. In one embodiment, the invention
provides a method of treating BPH in a patient by administering a
lonidamine analog to the patient. In a related embodiment, the
invention provides a method for treating BPH comprising (a)
administering a lonidamine analog to a patient diagnosed with BPH
and (b) determining whether one or more manifestations of BPH are
reduced in the patient. In one embodiment, the invention provides a
method for treating BPH by (a) diagnosing BPH in a patient, (b)
administering a lonidamine analog to the patient and (c)
determining whether one or more manifestations of BPH are reduced
in said patient. In the foregoing embodiments, optionally the
subject is not diagnosed with or under treatment for cancer;
optionally has a PSA less than or equal to 2 ng/ml, optionally has
a PSA greater than 2 ng/ml and less than 10 ng/ml.
[0083] In another aspect, the invention provides a method entailing
(a) advertising the use of lonidamine, or a lonidamine analog, for
treatment of BPH, and (b) selling lonidamine or a lonidamine analog
to individuals for use for treatment of BPH. In one embodiment, the
advertising makes reference to a trademark that identifies an
lonidamine product and the lonidamine sold in step (b) is
identified by the same trademark. It will be appreciated that the
individuals to whom lonidamine is sold include corporate persons
(corporations) and the like and "selling BPH to individuals for use
for treatment of BPH" includes selling to, for example, a medical
facility for distribution to patients for treatment of BPH.
[0084] In another embodiment, the invention provides a method of
treating acute urinary retention in a human by administering
lonidamine or a lonidamine analog to the human. Because acute
urinary retention can be a symptom of BPH, this embodiment of the
invention is applicable to any subject who suffers from acute
urinary retention but has not been diagnosed as having BPH when
lonidamine or a lonidamine analog is first administered.
[0085] 6. Dose, Route, Schedule and Duration of Administration
[0086] A variety of routes and dosage schedules are appropriate for
administration of lonidamine and lonidamine analogs according to
the invention.
[0087] A preferred mode of delivery of lonidamine and lonidamine
analogs to a patient is oral delivery. Preferred dosage forms for
oral administration are pills, tablets, capsules, caplets, and the
like, especially as formulated for sustained release. Other
suitable forms for oral administration include troches, elixirs,
suspensions, syrups, wafers, lozenges, and the like. Other modes of
administration are also contemplated, including parenteral,
inhalation spray, transdermal, rectal, intraprostatic injection
(e.g., of lonidamine-containing microparticles) and other routes.
Lonidamine and lonidamine analogs may be formulated in suitable
dosage unit formulations containing conventional non-toxic
pharmaceutically acceptable carriers, adjuvants and vehicles
appropriate for each route of administration. In one embodiment,
the dosage form is the 150 mg unit dosage form marketed in Italy
under the trade name DORIDAMINA.
[0088] The dose, schedule and duration of administration of
lonidamine and lonidamine analogs will depend on a variety of
factors, including the age, weight and health of the subject, the
severity of BPH symptoms, if any, the subject's medical history,
co-treatments, therapeutic goal (e.g., therapy or prophalaxis), the
mode of administration of the drug, the formulation used, patient
response to the drug, and the like. For illustration rather than
limitation, three general categories of dosing for administration
of lonidamine and lonidamine analogs can be described: high dosing,
low dosing, and intermediate dosing. For reference, the standard
lonidamine dose used for the treatment of the specific types of
cancer for which lonidamine has been approved in a few countries in
Europe is 150 mg po TID for about thirty days.
[0089] Low dosing. Low dosing is contemplated for the treatment and
prophylaxis of BPH. Exemplary low doses of lonidamine or a
lonidamine analog include, without limitation, doses in the range
of 1-300 mg per day (total daily dosage), more often in the range
of 5-300 mg/day, or sometimes in the range of 5-70 mg/day. Other
exemplary low dose ranges include 1-25 mg/day, 20-45 mg/day, 40-65
mg/day, 40-70 mg/day, 50-100 mg/day, 50-200 mg/day, and 50-300
mg/day. In one embodiment, the low dose is 150 mg administered
orally once per day; the Doridamina unit dose form can be used in
this embodiment. In another embodiment, the low dose is 75 mg
administered orally twice daily; the Doridamina unit dose form can
be used in this embodiment by splitting it into two equal
parts.
[0090] As noted, the daily dosages recommended herein may be
divided for, for example, two-, three- or four-times per day
administration. In one embodiment, the drug is formulated for
administration once-per day. In one embodiment, the drug is
formulated for administration less frequently than once per day. In
another embodiment, a modified-release form of the drug is
used.
[0091] Administration of low doses of lonidamine can be daily,
every other day, five days on, two days off, and other schedules
determined by the administering physician.
[0092] An advantage of low dose schedules of the invention is that
this dose may be continued to be administered for weeks to months
while limiting or eliminating the unwanted, albeit usually mild,
side effects reported for higher doses of lonidamine (principally
myalgia and testicular pain).
[0093] A low dose schedule can be used for therapy or prophalaxis.
In one embodiment, a low dose form is used for a maintenance dose
after a higher initial, priming or loading dose.
[0094] High dosing. In another embodiment, BPH is treated in
accordance with the methods of the invention by administering to a
BPH patient a higher dose of lonidamine or a lonidamine analog
(usually for a shorter period of time than for low doses).
Exemplary high doses include, without limitation, total daily doses
greater than 0.5 g, such as doses in the range 0.5-5 g/day, 0.5-3
g/day, 0.5-1 g/day and 1-3 g/day, or higher doses. The daily
dosages may be divided, for example, for two-, three- or four-time
per day administration. In one embodiment, the drug is formulated
for administration once-per day, or less frequently than once per
day. In one embodiment, a modified-release form of the drug is
used. Alternatively, a high dose can be administered on a one-time,
once-a-week, once every two weeks, or once-a-month basis (e.g.,
0.5-5 g/administration) or by other schedules to be determined by
the administering physician.
[0095] A high dose schedule can be used for therapy or prophalaxis.
In one embodiment, a high dose is administered in combination with,
or following, a surgical or other non-drug treatment for BPH.
[0096] Intermediate dosing. In another embodiment, BPH is treated
in accordance with the methods of the invention by administering
lonidamine or a lonidamine analog to a BPH patient at a dose
intermediate between a high dose and a low dose. Exemplary
intermediate doses include, without limitation, doses greater than
300 and less than 500 mg/day, such as doses in the range>300-400
or 400<500 (e.g., 450 mg/day). The daily dosages may be divided,
for example, for two-, three- or four-times per day administration.
In an embodiment, the drug is formulated for administration
once-per day or less frequently than once per day. In one
embodiment, a modified-release form of the drug is used.
Alternatively, this intermediate dose can be administered on a
one-time, once-a-week, once every two weeks, or once-a-month basis
(e.g., 300-500 mg/administration) or by other schedules to be
determined by the administering physician. In one embodiment, the
daily dosage is 150 mg of lonidamine or a lonidamine analog taken
three times a day.
[0097] An intermediate dose schedule may be used for therapy or
prophylaxis. In one embodiment, an intermediate dose is
administered in combination with, or following, a surgical or other
non-drug treatment for BPH.
[0098] It will be appreciated that these dosing schedules are for
illustration and not limitation, and that a dosing schedule may
change during a course of therapy based on, for example, a
patient's response to the therapy or the use of a lonidamine analog
that has an activity/dose profile significantly different from that
of lonidamine.
[0099] Duration. In therapeutic and prophylactic applications,
lonidamine or the lonidamine analog can be administered a single
time or many times over periods as long as several months or years.
In one embodiment of the invention, lonidamine or an analog is
administered to a symptomatic (e.g., experiencing difficulty in
urination) BPH patient only until the symptoms abate or disappear,
and then treatment is stopped unless and until symptoms reappear.
When symptoms reappear, administration of lonidamine or an analog
is resumed. In another embodiment, treatment continues after
symptoms disappear or are reduced to an acceptable target level, at
least for a period of time, such as a week, two weeks, a month or
several months. In another embodiment, the drug is administered to
an asymptomatic subject to prevent the development or reoccurence
of symptoms (i.e., prophylactically administered).
[0100] 7. Treatment Combinations
[0101] Lonidamine and lonidamine analogs can be administered to a
BPH patient in combination with other agents or procedures intended
to treat BPH, ameliorate symptoms of BPH, potentiate the effects of
the lonidamine or lonidamine analog, or provide other therapeutic
benefit. Administration of an agent "in combination with" includes
parallel administration (administration of both the agents to the
patient over a period of time, such as administration of lonidamine
and tamsulosin on alternate days for one month), co-administration
(in which the agents are administered at approximately the same
time, e.g., within about a few minutes to a few hours of one
another), and co-formulation (in which the agents are combined or
compounded into a single dosage form suitable for oral or
parenteral administration). Exemplary agents for administration in
combination with lonidamine or lonidamine analogs include, but are
not limited to, zinc, alpha-blockers, 5-alpha-reductase inhibitors,
and plant extracts. Other agents for administration in combination
with lonidamine or lonidamine analogs include other metabolic
inhibitors, including but not limited to other hexokinase
inhibitors and other inhibitors of glycolysis, including but not
limited to 2-deoxy-D-glucose and an inhibitor, direct or indirect,
of HIF-1.alpha..
[0102] Zinc: As discussed above, high concentrations of zinc in the
secretory epithelial cells of the prostate inhibit m-aconitase,
increasing the dependence of that tissue on glycolysis for energy
production. In accordance with the methods of the present
invention, it may in some patients be beneficial to co-administer
zinc (e.g., zinc chloride, zinc gluconate, zinc sulfate, zinc
acetate, zinc aspatate, zinc citrate, zinc glycerate, zinc oxide,
zinc picolinate, etc.) with a drug composition of the invention, to
maximize the efficacy of the treatment. For example and not
limitation, 15-300 mg/day zinc can be administered for this
purpose, typically 30-50 mg/day are administered.
[0103] Alpha-Adrenergic-Blockers: Alpha-blockers alleviate some
symptoms of BPH, without curing the underlying disease. These
agents work by relaxing the muscles at the neck of the bladder and
in the prostate, reducing the pressure on the urethra. Exemplary
alpha-blockers include doxazosin (Cardura), terazosin (Hytrin),
tamsulosin (Flomax), alfuzosin (Xatral), and prazosin (Hypovase).
In one embodiment of the invention, an alpha blocker is
administered in combination with lonidamine or a lonidamine analog
to treat BPH. In another embodiment, the alpha-blocker is
administered at a lower dosage (amount) or less frequently (e.g.,
alternate days rather than daily) than the "standard" dosage (the
dosage that would be indicated for the subject in the absence of
lonidamine administration) in combination with lonidamine or a
lonidamine analog.
[0104] 5-Alpha-Reductase Inhibitors: 5-alpha-reductase inhibitors
inhibit the conversion of testosterone to dihydrotestosterone 2
(DHT), an androgen that contributes to prostate enlargement. An
exemplary 5-alpha-reductase inhibitor is finasteride (Proscar). In
one embodiment of the invention, a 5-alpha-reductase inhibitor is
administered in combination with lonidamine to treat BPH. In
another embodiment, the 5-alpha-reductase inhibitor is administered
at a lower dosage (amount) or less frequently (e.g., alternate days
rather than daily) than the "standard" dosage (the dosage that
would be indicated for the subject in the absence of lonidamine
administration) in combination with lonidamine (or an lonidamine
analog).
[0105] Glycolytic and Mitochondrial Function Inhibitors: Glycolytic
inhibitors, such as 2-deoxy-D-glucose and compounds that inhibit
glucose transport, mitochondrial function inhibitors, mitochondrial
poisons, and hexokinase inhibitors such as 3-bromopyruvate and its
analogs can also be used in combination with lonidamine or a
lonidamine analog to treat BPH. Such inhibitors are known in the
art, and include those described in PCT patent publications WO
01/82926 published 8 Nov. 2001; U.S. Pat. Nos. 6,670,330;
6,218,435; 5,824,665; 5,652,273; and 5,643,883; U.S. patent
application publication Nos. 20030072814; 20020077300; and
20020035071; and U.S. patent application Ser. No. 10/10/754,239.
Such inhibitors can be administered in combination with lonidamine
or lonidamine analogs for therapeutic benefit in the treatment of
BPH.
[0106] Plants: Saw Palmetto (Serenoa repens) or an extract thereof,
or Pygeum Africanum or an extract thereof can be administered in
combination with lonidamine or lonidamine analogs for therapeutic
benefit in the treatment of BPH.
[0107] Procedures. In addition, lonidamine or a lonidamine analog
may be administered in combination with, or prior to, procedures
for treatment of BPH including surgery (transurethral resection of
the prostate; transurethral incision of the prostate; or open
prostatectomy), laser therapy, transurethral microwave
thermotherapy, balloon dilatation, placement of a prostatic
urethral stent, transurethral needle ablation, transurethral
electrovaporization of the prostate, or other non-drug
therapies.
[0108] 8. Dosage Forms
[0109] Unit Dosage Forms. The compounds used in the methods of the
present invention are formulated in compositions suitable for
therapeutic administration. In one embodiment, the methods of the
invention are practiced with lonidamine in the unit dosage form
marketed as Doridamina (by ACRAF) in Italy. New dosage forms of
lonidamine are also provided. For example, the present invention
provides a unit dosage pharmaceutical formulation of lonidamine
that is suitable for oral administration (including tablets,
capsules, caplets, and pills) and contains, in various embodiments,
an amount of lonidamine in a range bounded by a lower limit of (in
mg) 1, 5, 10, and 50 and an upper limit of 10, 20, 40, 50, 70 and
100 (where the higher limit is in mg and greater than the lower
limit) and is especially convenient for certain low dose schedules.
In an other embodiment, the unit dosage form contains an amount of
drug in a range bounded by a lower limit of (in mg) 200, 300, 500
or 1000 and an upper limit of 500, 1000, 3000 or 5000 (where the
higher limit is greater than the lower limit) and is especially
convenient for certain high dose schedules. In yet other
embodiments, the formulation contains between 100 and 200 mg of
compound (e.g., 150 mg), between 200 and 5000 mg, between 200 and
1000 mg, or between 500 and 1000 mg of the compound. Lonidamine
analogs can be similarly formulated.
[0110] In addition to lonidamine and/or lonidamine analogs, solid
unit dosage forms of the invention generally include a
pharmaceutically acceptable carrier. As used herein,
"pharmaceutically acceptable carrier" refers to a solid or liquid
filler, diluent, or encapsulating substance, including for example
excipients, fillers, binders, and other components commonly used in
pharmaceutical preparations, including, but not limited to, those
described below. Methods for formulation of drugs generally are
well known in the art, and the descriptions herein are illustrative
and not limiting. See, e.g., Ansel et al., 1999; Marshall,
1979.
[0111] Hydrophilic binders suitable for use in the formulations of
the invention include copolyvidone (cross-linked
polyvinylpyrrolidone), polyvinylpyrrolidone, polyethylene glycol,
sucrose, dextrose, corn syrup, polysaccharides (including acacia,
guar, and alginates), gelatin, and cellulose derivatives (including
HPMC, HPC, and sodium carboxymethylcellulose).
[0112] Water-soluble diluents suitable for use in the formulations
of the invention include sugars (lactose, sucrose, and dextrose),
polysaccharides (dextrates and maltodextrin), polyols (mannitol,
xylitol, and sorbitol), and cyclodextrins. Non-water-soluble
diluents suitable for use in the formulations of the invention
include calcium phosphate, calcium sulfate, starches, modified
starches, and microcrystalline cellulose.
[0113] Surfactants suitable for use in the formulations of the
invention include ionic and non-ionic surfactants or wetting agents
such as ethoxylated castor oil, polyglycolyzed glycerides,
acetylated monoglycerides, sorbitan fatty acid esters, poloxamers,
polyoxyethylene sorbitan fatty acid esters, polyoxyethylene
derivatives, monoglycerides or ethoxylated derivatives thereof,
sodium lauryl sulfate, lecithins, alcohols, and phospholipids.
[0114] Disintegrants suitable for use in the formulations of the
invention include starches, clays, celluloses, alginates, gums,
cross-linked polymers (PVP, sodium carboxymethyl-cellulose), sodium
starch glycolate, low-substituted hydroxypropyl cellulose, and soy
polysaccharides. Preferred disintegrants include a modified
cellulose gum such as cross-linked sodium
carboxymethylcellulose.
[0115] Lubricants and glidants suitable for use in the formulations
of the invention include talc, magnesium stearate, calcium
stearate, stearic acid, colloidal silicon dioxide, magnesium
carbonate, magnesium oxide, calcium silicate, microcrystalline
cellulose, starches, mineral oil, waxes, glyceryl behenate,
polyethylene glycol, sodium benzoate, sodium acetate, sodium
chloride, sodium lauryl sulfate, sodium stearyl fumarate, and
hydrogenated vegetable oils. Preferred lubricants include magnesium
stearate and talc and combinations thereof.
[0116] The preferred range of total mass for the tablet or capsule
may be from about 40 mg to 2 g, from about 100 mg to 1000 mg, and
from about 300 mg to 750 mg.
[0117] Sustained Release Forms. In addition, the present invention
provides unit dosage forms that are sustained release formulations
of lonidamine or a lonidamine analog to allow once a day (or less)
oral dosing, a frequency sometimes preferred by patients over
multiple day dosing. Such sustained release formulations (including
tablets, capsules, caplets and pills) of the invention usually
contain between 1 mg and 3 g of the active compound, with various
alternative embodiments including those described above for
conventional oral unit doses, such as an amount of drug in a range
bounded by a lower limit of (in mg) 1, 5, 10, and 50 and an upper
limit of 10, 20, 40, 50, 70 and 100 (where the higher limit is
greater than the lower limit) and are especially convenient for
certain low dose or intermediate dose schedules. In another
embodiment, the unit dosage form contains an amount of drug in a
range bounded by a lower limit of (in mg) 200, 300, 500, 750 or
1000 and an upper limit of 500, 1000, 2000, 3000 or 5000 (where the
higher limit is greater than the lower limit).
[0118] In one embodiment, lonidamine or a lonidamine analog in the
sustained release formulations (also called "modified" or
"controlled" release forms) is released over a period of time
greater than 6 hours, e.g., greater than 12 hours, after
administration. In one embodiment, the sustained release
formulation allows once a day dosing to achieve a pharmacodynamic
profile therapeutically equivalent to dosing 150 mg of lonidamine
three times a day.
[0119] Examples of sustained-release formulations for other drugs
that can be modified in accordance with the teachings herein to be
useful in the present invention are well known in the art, and are,
for example, described in U.S. Pat. Nos. 5,968,551; 5,266,331;
4,970,075; 5,549,912; 5,478,577; 5,472,712; 5,356,467; 5,286,493;
6,294,195; 6,143,353; 6,143,322; 6,129,933; 6,103,261; 6,077,533;
5,958,459; and 5,672,360. Sustained-release formulations are also
discussed in the scientific literature, e.g., in ORAL SUSTAINED
RELEASE FORMULATIONS: DESIGN AND EVALUATION, edited by A. Yacobi
and E. Halperin-Walega, Pergamon Press, 1988, which describes a
variety of types of sustained-release dosage forms and drug release
mechanisms, for example single unit (e.g., matrix tablets, coated
tablets, capsules), multiple unit (e.g., granules, beads,
micro-capsules), inert, insoluble matrix, hydrophilic gel matrix
(e.g., bioadhesive, erodible, non-erodible), and ion-exchange resin
sustained-release dosage forms.
[0120] In one embodiment, the present invention provides a method
of treating BPH, by administering once daily to a patient in need
of such treatment a sustained release tablet dosage form comprising
a daily therapeutic dose of lonidamine from about 1 mg to 2 g in a
hydrophilic matrix. The matrix can be, for example and without
limitation, selected from the group consisting of
hydroxypropylmethyl cellulose (by weight percent of about 20-40%),
lactose (5-15%), microcrystalline cellulose (4-6%), and silicon
dioxide (1-5%) having an average particle size ranging from 1-10
microns, often ranging from 2-5 microns, and most often ranging
from about 2-3 microns.
[0121] Illustrative preferred sustained release formulations of the
invention include formulations A and B in the table below.
1 Formulation A B (weight percentage) Lonidamine (milled) 53.8 53.8
HPMC (Methocel K15M, CR) 8 30 Methyl cellulose (Methocel, K100L,
CR) 18 0 Anydrous lactose 12.2 8.2 Microcrystalline cellulose
(Avicel PH101) 5 5 Silicon dioxide (1-10 micron; Syloid 244) 3 3
Total Table Weight (in grams) 1 1
[0122] The sustained release formulations of the invention may be
in the form of a compressed tablet containing an intimate mixture
of lonidamine and a partially neutralized pH-dependent binder that
controls the rate of drug dissolution in aqueous media across the
range of pH in the stomach (typically .about.2) and intestine
(typically .about.5.5).
[0123] Many materials known in the pharmaceutical art as "enteric"
binders and coating agents have the desired pH dissolution
properties suitable for use in the sustained formulations of the
invention. These include phthalic acid derivatives such as the
phthalic acid derivatives of vinyl polymers and copyolymers,
hydroxyalkylcellulose, alkylcelluloses, cellulose acetates,
hydroxyalkylcellulose acetates, cellulose ethers, alkylcellulose
acetates, and esters thereof, and polymers and copoloymers of lower
alkyl acrylic acids and lower alkyl acrylates, and the partial
esters thereof.
[0124] Preferred pH-dependent binder materials are methacrylic acid
copolymers. Such a copolymer is commercially available from Rohm
Pharma as Eudragit.TM. L-100-55 as a powder or L30D-55 as a 30%
dispersion in water. Other pH-dependent binder materials which may
be used alone or in combination include hydroxypropyl cellulose
phthalate, hydroxypropyl methylcellulose phthalate, cellulose
acetate phthalate, polyvinylacetate phthalate, polyvinylpyrrolidone
phthalate, and the like. One or more pH-dependent binders are
present in the sustained release oral dosage forms of the invention
in an amount ranging from about 1 to 20 weight percent, or from 5
to 12 weight percent, or about 10%.
[0125] The pH-independent binders or viscosity enhancing agents
contained in the sustained release formulations of the invention
include substances such as hydroxypropyl methylcellulose,
hydroxypropyl cellulose, methylcellulose, polyvinylpyrrolidone,
neutral poly(meth)acrylate esters, and the like. The pH-independent
binders are in an amount ranging from 1 to 10 weight percent or
from 1 to 3 weight percent, or about 2%.
[0126] The sustained release formulations of the invention also
contain in some embodiments one or more pharmaceutical excipients
intimately mixed with the lonidamine and the pH-dependent binder,
such as pH-independent binders or film-forming agent, starch,
gelatin, sugars, carboxymethylcellulose, and the like, as well as
other useful pharmaceutical diluents such as lactose, mannitol, dry
starch, microcrystalline cellulose, and the like, and surface
active agents such as polyoxyethylene sorbitan esters, sorbitan
esters, and the like; and coloring agents and flavoring agents.
Lubricants such as talc and magnesium stearate and tableting aids
are also present.
[0127] The sustained release formulations of the invention include
any of the commercially available polymers suitable for use in such
formulations, including but not limited to cellulose, ethyl
cellulose, methyl cellulose, carboxymethyl cellulose,
hydroxypropylmethyl cellulose, hydroxypropylmethyl cellulose
phthalate, hydroxypropyl cellulose, microcrystalline cellulose,
sodium carboxymethyl cellulose, cellulose acetate phthalate,
polyvinyl acetate phthalate, polyvinylpyrrolidone, polyethylene
oxide, polyethylene glycol, zein, alginate, hypromellose phthalate,
methacrylic acid copolymer, Crospovidone, silica aerogel,
pregelatinized starch, corn starch, croscarmellose sodium, sodium
starch glycolate, candelilla wax, paraffin wax, carnauba wax,
montan glycol wax, white wax, Eudragit (polymethacrylic acid
esters), Aquacoat (ethyl cellulose, cellulose acetate phthalate),
Carbopol (acrylic acid polyalkeny polyether copolymer), and
Macrogol (polyethylene glycol).
[0128] The sustained release formulations of the invention include
formulations that are diffusion controlled, such as those that
employ:
[0129] (a) a reservoir system in which the drug is encapsulated in
a polymeric membrane, and water diffuses through the membrane to
dissolve the drug, which then diffuses out of device;
[0130] (b) a monolithic (matrix) system in which the drug is
suspended in a polymeric matrix and diffuses out through long
pathways;
[0131] (c) microencapsulation and coated granule systems in which
particles of drug (or particles of drug and polymer) as small as 1
micron are coated in a polymeric membrane, including embodiments in
which particles coated with polymers with different release
characteristics are delivered together in a capsule;
[0132] (d) solvent-activated systems, including (i) osmotically
controlled devices (e.g. OROS) in which an osmotic agent and the
drug are encapsulated in a semi-permeable membrane, water is pulled
into device due to osmotic gradient, and increased pressure drives
drug out of device through a laser drilled hole; (ii) a hydrogel
swelling system in which drug is dispersed in a polymer and/or a
polymer is coated onto a particle of drug, and the polymer swells
on contact with water (swelling is in some embodiments pH or
enyzmatically controlled), allowing diffusion of drug out of the
device; (iii) a microporous membrane system in which drug is
encapsulated in a membrane that has a component that dissolves on
contact with water (in some embodiments, dissolution is pH or
enyzmatically controlled), leaving pores in the membrane through
which the drug diffuses; and (iv) a wax matrix system in which the
drug and an additional soluble component are dispersed in wax, such
that, when water dissolves the component, diffusion of drug from
the system is allowed; and
[0133] (e) polymeric degradation systems, including (i) bulk
degradation, in which drug is dispersed in polymeric matrix, and
degradation occurs throughout the polymeric structure in a random
fashion, allowing drug release; and (ii) surface erosion, in which
drug is dispersed in polymeric matrix and delivered as the surface
of the polymer erodes.
[0134] In one aspect, the invention provides a method for treating
BPH by administering a unit dose oral pharmaceutical composition
that is a sustained-release formulation containing an effective
amount of lonidamine, such as described above, once per day.
9. EXAMPLES
Example 1
Clinical Trial
[0135] A Phase II randomized dose comparison study of lonidamine
administration for the treatment of symptomatic benign prostatic
hyperplasia is conducted. Patients are males 50 to 80 years of age
with BPH confirmed by ultrasonography, a serum PSA>2, and no
evidence of prostate cancer. Lonidamine (150 mg tablet; Doridamina
formulation) is administered 150 mg p.o. TID (intermediate dosage)
or QD (low dosage) 8 weeks. Patients receiving TID dosing take the
compound 5 days on and 2 days off to aid compliance with the
protocol.
[0136] Patients are assessed at baseline, day 30 and day 60 for
prostate volume by ultrasonography, urine flow, AUASI score, PSA,
adverse events, and serum chemistry to determine whether one of the
two doses provides significantly greater benefit than the other
dose and to measure the reduction in prostate size achieved by the
therapy.
Example 2
Lonidamine Reduces Expression of HIF-1.alpha. in Prostate Cells
[0137] This example shows the effects of lonidamine treatment on
HIF-1.alpha. expression in two cell lines derived from metastatic
lesions of human prostate cancers. LNCaP is a citrate-producing
cell (ATTC No. CRL-1740) while PC3 is citrate oxidizing cell (ATTC
No. CRL-1435). See Franklin et al; 1995. Cells may be obtained from
the American Type Culture Collection (ATCC), P.O. Box 1549,
Manassas, Va. 20108 USA.
[0138] As shown in FIGS. 2 and 3, lonidamine treatment reduced the
level of HIF-1.alpha. protein detected in nuclear (NE) and
whole-cell extract (WCE) preparations. The inhibition was
dose-dependent, and observed under normoxic (PC3 cells only) and
hypoxic conditions (LNCaP cells and PC3 cells). The lonidamine
effect was specific to the HIF-1.alpha. subunit under the
conditions tested and, except at 800 .mu.M concentration, had no
detectable inhibition under the conditions tested on the protein
levels of actin, caspase 3, NF-.kappa.B, or I.kappa.B.alpha..
Lonidamine has, however, been reported to inhibit protein synthesis
generally (see Floridi et al., supra), and the results presented
herein should not be construed as definitive evidence that
lonidamine is a specific inhibitor of HIF-1.alpha. or that
lonidamine's therapeutic effect in the treatment of BPH is in whole
or in part due to its inhibitory effect on the accumulation of
HIF-1.alpha. in any cell type.
[0139] Methods: Cells were plated at a density of 5.times.10.sup.5
cells into a dish, and then maintained in 37.degree. C. incubator
(5% CO.sub.2) for 2 days. Prior to the assay, cells were rinsed
twice with pre-warmed (37.degree. C.) RPMI-1640 Medium (ATCC No.
30-2001; 10 mM HEPES; 1 mM sodium pyruvate; 2 mM L-glutamine; 4500
mg glucose/L; 1500 mg sodium bicarbonate/L). Cells were incubated
with 2 ml culture medium in the absence or presence of lonidamine
at different concentrations for 4 hours at 37.degree. C. either
under normoxia or hypoxia (oxygen level<0.1%). At the end of the
incubation, the dish was placed on ice, and the cells were washed
rapidly twice with cold PBS buffer (4.degree. C.). For nuclear
extracts, cells were lysed with buffer A (10 mM Tris, pH7.5; 1.5 mM
MgCl.sub.2; 10 mM KCl and protease inhibitors and buffer C (0.5 M
NaCl; 20 mM Tris pH7.5; 1.5 mM MgCl.sub.2; 20% glycerol and
protease inhibitors), sequentially. The protease inhibitors used in
the experiments were a cocktail of five protease inhibitors (500 mM
AEBSF-HCl, 1 mg/ml Aprotinin, 1 mM E-64, 500 mM EDTA and 1 mM
Leupeptin; Calbiochem NO 539131). For whole cell lysate, cells were
lysed with 150 mM NaCl; 10 mM Tris ph7.5; 10 mM EDTA; 1% Triton
X-100; 0.5% Deoxycholate, and protease inhibitors. The protein
concentration was measured using a Bio-Rad protein assay. Equal
amounts of protein were loaded on a SDS-PAGE gel. After
transferring of the sample to PVDF membrane, the membrane was
blocked with TBST containing 5% non-fat milk overnight at 4.degree.
C. Subsequently, the membrane was incubated with primary antibodies
(HIF-1.alpha., HIF-1.beta., and actin) and alkaline
phosphatase-conjugated secondary antibody, for two hours each
incubation. To detect the expression of caspase 3, NF-.kappa.B, P65
and I.kappa.B.alpha., the membrane was blocked with TBST containing
5% non-fat milk for 1 h at room temperature, and the proteins were
detected by incubation with the corresponding antibodies overnight
at 4.degree. C. and with the alkaline phosphatase-conjugated
secondary antibody for 1 h. The specific protein was detected using
a colorimetric substrate, and the intensity of each protein was
quantified using an NIH image system.
[0140] In separate experiments carried out generally as above, the
effect of 0-600 .mu.M lonidamine on expression of HIF-1.alpha. and
other proteins was determined in LNCaP whole cell extracts (FIG. 7)
or nuclear extracts (FIG. 8) from cells cultured under hypoxic
conditions.
Example 3
Lonidamine Induces Apoptosis in Citrate-Producing Cells
[0141] To determine whether apoptosis occurs in cells treated with
lonidamine, the effect of lonidamine on cells producing citrate
(LNCaP) and cells oxidizing citrate (PC3) was assessed. As shown in
FIG. 4, lonidamine induced activation of caspase 3 in
citrate-producing cells (LNCaP) to a much greater extent than in
citrate-oxidizing cells (PC3). The activation of caspase 3 is a
time-dependent process (FIG. 5).
[0142] The effect of lonidamine was also examined in primary
cultures of prostate epithelial cells (which accumulate citrate) or
prostate stromal cells (which do not accumulate citrate). As shown
in FIG. 6, lonidamine induced apoptosis only in prostate epithelial
cells in a dose-dependent manner. In contrast, induction of
apoptosis was not observed in prostate stromal cells after
treatment with lonidamine.
[0143] Methods:
[0144] Immunoblotting: Immunoblotting was carried out as described
in Example 2. To detect the expression of caspase 3, the membrane
was blocked with TBST containing 5% non-fat milk for 1 h at room
temperature, and caspase 3 protein was detected by incubation with
caspase 3 antibody overnight at 4.degree. C. and with the alkaline
phosphatase-conjugated secondary antibody for 1 h. The specific
protein was detected using calorimetric substrate, and the
intensity of each protein was quantified using an NIH image
system.
[0145] Primary Cell Cultures: Primary cultures of human prostate
epithelial cells (Cambrex No. CC-2555) and human prostate stromal
cells (Cambrex No. CC-2508) were obtained from Cambrex Bio Science
Rockland, Inc. (191 Thomaston Street, Rockland, Me. 04841).
[0146] Apoptosis Assay: Cells were plated at a density of
2.times.10.sup.4 cells per well in a 96 well plate, and then
maintained in a 37.degree. C. incubator (5% CO.sub.2) for 16 h.
Lonidamine was added into each well at different concentrations,
and then incubated for 6 h at 37.degree. C. To assess the caspase 3
activity, the homogeneous buffer and caspase 3 substrate (Promega
No G7791; Promega Corporation, 2800 Woods Hollow Road, Madison Wis.
USA 53711) were added into each well in the presence or absence of
caspase 3 inhibitor (Promega No G5961). The fluorescence intensity
of cleaved substrate was determined using a fluorescence plate
reader at excitation 485 nm and emission 530 nm.
10. REFERENCES CITED
[0147] Ansari et al., 1998, "Long-term sequelae of tolnidamine on
male reproduction and general body metabolism in rabbits"
Contraception 57:271-79.
[0148] Ansel et al., 1999, Pharmaceutical Dosage Forms and Drug
Delivery Systems 7th ed. Lippincott Williams & Wilkins,
Philadelphia: pp. 1-562.
[0149] Barry et al., 1992, "Symptom index for benign prostatic
hyperplasia" J Urol. 148:1549-57.
[0150] Barry et al., 2001, "Measuring the symptoms and health
impact of benign prostatic hyperplasia and its treatments" BENIGN
PROSTATIC HYPERPLASIA (5TH INTERNATIONAL CONSULTATION ON BPH).
Health Publication, Ltd.
[0151] Barry et al., 2003, "Benign Prostatic Hyperplasic" in
SCIENTIFIC AMERICAN MEDICINE, Dale and Federman Eds., WebMD
Inc.
[0152] Burger, 1991, "Isosterism and Bioisosterism in Drug Design"
A. Prog. Drug Res. 37:287-371.
[0153] Cheng et al., 2001, "Two new male contraceptives exert their
effects by depleting germ cells prematurely from the testis" Biol
Reprod. 65:449-61.
[0154] Corsi et al., 1976, "1-Halobenzyl-1H-Indazole-3-Carboxylic
Acids. A New Class of Antispermatogenic Agents", Journal of
Medicinal Chemistry 19:778-83.
[0155] Costello & Franklin, 2000, "The intermediary metabolism
of the prostate: a key to understanding the pathogenesis and
progression of prostate malignancy" Oncology 59:269-82
[0156] Costello et al., 1999, "Citrate in the diagnosis of prostate
cancer" Prostate 38:237-45.
[0157] Costello et al., 2000, "Zinc causes a shift toward citrate
at equilbrium of the m-aconitase reaction of prostate mitochondria"
J. Inorganic Biochemistry 78:161-65.
[0158] Fanciulli et al., 1996, "Effect of the antitumor drug
lonidamine on glucose metabolism of adriamycin-sensitive and
-resistant human breast cancer-cells" Oncology Research
3:111-120
[0159] Floridi et al., 1981, "Effect of lonidamine on the energy
metabolism of Ehrlich ascites tumor cells" Cancer Res.
41:4661-6.
[0160] Franklin et al; 1995, "Regulation of citrate metabolism by
androgen in the LNCaP human prostate carcinoma cell line."
Endocrine 3:603-607
[0161] Gatto et al., 2002, "Recent studies on lonidamine, the lead
compound of the antispermatogenic indazol-carboxylic acids"
Contraception 65:277-78.
[0162] Grima et al., 2001; 2001, "Reversible inhibition of
spermatogenesis in rats using a new male contraceptive,
1-(2,4-dichlorobenzyl)-indazole-3- -carbohydrazide" Biol Reprod.
64:1500-8.
[0163] Heywood et al., "Toxicological studies on
1-substituted-indazole-3-- carboxylic acids." 1981, Chemotherapy
27:91-97.
[0164] Jeyaraj et al., 2000, "Effects of long-term administration
of androgens and estrogen on rhesus monkey prostate: possible
induction of benign prostatic hyperplasia" J Androl. 21:833-41.
[0165] Kaplan, 2000 "Correspondence re: M. Fanciulli et al., Energy
metabolism of human LoVo colon carcinoma cells: correlation to drug
resistance and influence of lonidamine." Clin Cancer Res.
6:4166-7.
[0166] Kurhanewicz et al., 1991, ".sup.31P Spectroscopy of the
human prostate gland in vivo using a transrectal probe" Magnetic
Resonance in Medicine 22:404-13.
[0167] Kurhanewicz et al., 2000, Radiol Clin North Am
38:115-38.
[0168] Lee et al., 1998, "Chronology and urodynamic
characterization of micturition in neurohormonally induced
experimental prostate growth in the rat" Neurourol Urodyn.
17:55-69.
[0169] Lobl et al., 1981, "Effects of Lonidamine (AF 1890) and its
analogues on follicle-stimulating hormone, luteinizing hormone,
testosterone and rat androgen binding protein concentrations in the
rat and rhesus monkey" Chemotherapy 27:61-76.
[0170] Lohiya et al., 1991, "Antispermatogenic effects of
tolnidamine in langur (Presbytis entellus)" Contraception
43:485-96.
[0171] Mariotti et al., 1982, "Collagen and cellular proliferation
in spontaneous canine benign prostatic hypertrophy" J Urol.
127:795-7.
[0172] Marshall, 1979. "Solid Oral Dosage Forms," MODERN
PHARMACEUTICS, Vol. 7, (Banker and Rhodes, editors), pp.
359-427.
[0173] Mendelsohn, 1991, "Principles of Neoplasia" in HARRISON'S
PRINCIPLES OF INTERNAL MEDICINE, Wilson ed., McGraw-Hill, New York,
p. 1576.
[0174] Narayan and Kurhanewicz, 1992, "Magnetic resonance
spectroscopy in prostate disease: diagnostic possibilities and
future developments." Prostate Suppl. 4:43-50.
[0175] Patani and LaVoie, 1996, "Bioisosterism: A Rational Approach
in Drug Design" Chem. Rev. 96:3147-76.
[0176] Silvestrini et al., 1984, Prog. Med. Chem. 21, G. P. Ellis
and G. B. West, Eds. (Elsevier Science Publishers, Amsterdam), p.
111-35.
[0177] Silvestrini, 1981, "Basic and Applied Research n the Study
of Indazole Carboxylic Acids" Chemotherapy 27:9-20.
[0178] Thomas et al., 1990, ".sup.1H MR spectroscopy of normal and
malignant human prostates in vivo." Journal of Magnetic Resonance
87:610-19.
[0179] Yacobi and Halperin-Walega, 1988, in Oral Sustained Release
Formulations: Design and Evaluation, Pergamon Press
[0180] U.S. 2003/0072814 "Topical Pharmaceutical Composition for
the Treatment of Warts".
[0181] U.S. 2002/0077300 "Screening Method for Cancer Therapeutics
and Stable Antitumor Drug".
[0182] U.S. 2002/0035071 "Mimicking the Metabolic Effects of
Caloric Restriction by Administration of Glucose
Antimetabolites".
[0183] U.S. Pat. No. 3,895,026 "Substituted
1-benzyl-1H-indazole-3-carboxy- lic Acids and derivatives
thereof".
[0184] U.S. Pat. No. 4,970,075 "Controlled Release Bases for
Pharmaceuticals".
[0185] U.S. Pat. No. 5,266,331 "Controlled Release Oxycodone
Compositions".
[0186] U.S. Pat. No. 5,286,493 "Stabilized Controlled Release
Formulations Having Acrylic Polymer Coating".
[0187] U.S. Pat. No. 5,386,467 "Controlled Release Coatings Derived
From Aqueous Dispersions of Zein".
[0188] U.S. Pat. No. 5,472,712 "Controlled Release Formulations
Coated With Aqueous Dispersions of Ethylcellulose".
[0189] U.S. Pat. No. 5,478,577 "Methods of Treating Pain by
Administering 24 Hour Oral Opioid Formulations Exhibiting Rapid
Rate of Initial Rise of Plasma Drug Level".
[0190] U.S. Pat. No. 5,549,912 "Controlled Release Oxycodone
Compositions".
[0191] U.S. Pat. No. 5,643,883 "Glucose-6-Phosphate Uptake
Inhibitors and Novel Uses Thereof".
[0192] U.S. Pat. No. 5,652,273 "Reduction of Hair Growth".
[0193] U.S. Pat. No. 5,672,360 "Method of Treating Pain by
Administering 24 Hour Oral Opioid Formulations".
[0194] U.S. Pat. No. 5,824,665 "Reduction of Hair Growth".
[0195] U.S. Pat. No. 5,958,459 "Opioid Formulations Having Extended
Controlled Released".
[0196] U.S. Pat. No. 5,968,551 "Orally Administrable Opioid
Formulations Having Extended Duration of Effect".
[0197] U.S. Pat. No. 6,001,865"3-substituted 1-benzyl-1H-indazole
derivatives as antifertility agents".
[0198] U.S. Pat. No. 6,077,533 "Powder-Layered Oral Dosage
Forms".
[0199] U.S. Pat. No. 6,103,261 "Opioid Formulations Having Extended
Controlled Release".
[0200] U.S. Pat. No. 6,129,933 "Stabilized Controlled Release
Substrate Having a Coating Derived From an Aqueous Dispersion of
Hydrophobic Polymer".
[0201] U.S. Pat. No. 6,143,322 "Method of Treating Humans With
Opioid Formulations Having Extended Controlled Release".
[0202] U.S. Pat. No. 6,143,353 "Controlled Release Formulations
Coated With Aqueous Dispersions of Acrylic Polymers".
[0203] U.S. Pat. No. 6,146,658 "Prodrugs, their preparation and use
as pharmaceuticals".
[0204] U.S. Pat. No. 6,218,435 "Reduction of Hair Growth".
[0205] U.S. Pat. No. 6,294,195 "Orally Administrable Opioid
Formulations Having Extended Duration of Effect".
[0206] U.S. Pat. No. 6,670,330 "Cancer Chemotherapy With
2-Deoxy-D-Glucose".
[0207] WO 01/82926 "Manipulation of Oxidative Phosphorylation for
Hypersensitizing Tumor Cells to Glycolytic Inhibitors".
[0208] Although the present invention has been described in detail
with reference to specific embodiments, those of skill in the art
will recognize that modifications and improvements are within the
scope and spirit of the invention, as set forth in the claims which
follow. All publications and patent documents (patents, published
patent applications, and unpublished patent applications) cited
herein are incorporated herein by reference as if each such
publication or document was specifically and individually indicated
to be incorporated herein by reference. Citation of publications
and patent documents is not intended as an admission that any such
document is pertinent prior art, nor does it constitute any
admission as to the contents or date of the same. The invention
having now been described by way of written description and
example, those of skill in the art will recognize that the
invention can be practiced in a variety of embodiments and that the
foregoing description and examples are for purposes of illustration
and not limitation of the following claims.
* * * * *
References