U.S. patent application number 11/977502 was filed with the patent office on 2008-08-28 for method for treating benign prostatic hyperplasia.
This patent application is currently assigned to BioXell S.p.A.. Invention is credited to Luciano Adorini, Enrico Colli.
Application Number | 20080207769 11/977502 |
Document ID | / |
Family ID | 34315450 |
Filed Date | 2008-08-28 |
United States Patent
Application |
20080207769 |
Kind Code |
A1 |
Adorini; Luciano ; et
al. |
August 28, 2008 |
Method for treating benign prostatic hyperplasia
Abstract
The use of
1-alpha-fluoro-25-hydroxy-16,23E-diene-26,27-bishomo-20-epi-cholecalcifer-
ol, or a pharmaceutically acceptable salt or ester thereof, for the
manufacture of a medicament for the prevention and/or treatment of
benign prostatic hyperplasia (BPH) and associated symptoms.
Inventors: |
Adorini; Luciano; (Milan,
IT) ; Colli; Enrico; (Milan, IT) |
Correspondence
Address: |
EDWARDS ANGELL PALMER & DODGE LLP
P.O. BOX 55874
BOSTON
MA
02205
US
|
Assignee: |
BioXell S.p.A.
Milan
IT
|
Family ID: |
34315450 |
Appl. No.: |
11/977502 |
Filed: |
October 24, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10903211 |
Jul 29, 2004 |
7332482 |
|
|
11977502 |
|
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Current U.S.
Class: |
514/729 |
Current CPC
Class: |
A61P 13/08 20180101;
A61P 13/06 20180101; A61K 31/593 20130101 |
Class at
Publication: |
514/729 |
International
Class: |
A61K 31/047 20060101
A61K031/047; A61P 13/08 20060101 A61P013/08 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 24, 2003 |
GB |
0322395.5 |
Nov 3, 2003 |
GB |
0325598.1 |
Claims
1-6. (canceled)
7. A packaged formulation including a pharmaceutical composition
comprising
1-alpha-fluoro-25-hydroxy-16,23E-diene-26,27-bishomo-20-epi-cholecalcifer-
ol, or a pharmaceutically acceptable salt or ester thereof, and a
pharmaceutically acceptable carrier packaged with instructions for
use in the prevention and/or treatment of benign prostatic
hyperplasia.
8-9. (canceled)
10. A package formulation according to claim 7 wherein the
composition comprising
1-alpha-fluoro-25-hydroxy-16,23E-diene-26,27-bishomo-20-epi-cholecalcifer-
ol, or pharmaceutically acceptable salt or ester thereof, is
provided in unit dose form.
11-12. (canceled)
13. A package formulation according to claim 10 wherein the unit
dose of
1-alpha-fluoro-25-hydroxy-16,23E-diene-26,27-bishomo-20-epi-cholecalcifer-
ol is 50 to 150 .mu.g.
14-17. (canceled)
18. A pharmaceutical formulation comprising
1-alpha-fluoro-25-hydroxy-16,23E-diene-26,27-bishomo-20-epi-cholecalcifer-
ol dissolved in fractionated coco oil together with one or more
preservatives.
19. A pharmaceutical formulation according to claim 18 wherein the
fractionated coco oil is Miglyol 812.
20. A pharmaceutical formulation according to claim 18 wherein the
preservatives are selected from butylated hydroxytoluene, butylated
hydroxyanisole and mixtures thereof.
21. A packaged formulation according to claim 7 where the
composition is presented as a capsule.
22. A formulation according to claim 18 which is presented as a
capsule.
23-26. (canceled)
27. A packaged formulation including a pharmaceutical composition
comprising
1-alpha-fluoro-25-hydroxy-16,23E-diene-26,27-bishomo-20-epi-cholecalcifer-
ol, or a pharmaceutically acceptable salt or ester thereof, and a
pharmaceutically acceptable carrier packaged with instructions for
use in the prevention and/or treatment of benign prostatic
hyperplasia together with concurrent prevention and/or treatment of
bladder dysfunction.
28. A package formulation according to claim 27 wherein the
composition comprising
1-alpha-fluoro-25-hydroxy-16,23E-diene-26,27-bishomo-20-epi-cholecalcifer-
ol, or pharmaceutically acceptable salt or ester thereof, is
provided in unit dose form.
29. A package formulation according to claim 28 wherein the unit
dose of
1-alpha-fluoro-25-hydroxy-16,23E-diene-26,27-bishomo-20-epi-cholecalcifer-
ol is 50 to 150 .mu.g.
30. A packaged formulation according to claim 27 where the
composition is presented as a capsule.
Description
RELATED APPLICATIONS
[0001] This application claims priority to GB 0322395.5, filed Sep.
24, 2003, and to GB 0325598.1, filed Nov. 3, 2003, both of which
applications are incorporated herein in their entireties by this
reference.
FIELD OF THE INVENTION
[0002] The present invention is concerned with the use of
1-alpha-fluoro-25-hydroxy-16,23E-diene-26,27-bishomo-20-epi-cholecalcifer-
ol (Compound A) for the manufacture of a medicament for the
prevention and/or treatment of benign prostatic hyperplasia (BPH)
and associated symptoms. It is further concerned with a method for
preventing and/or treating benign prostatic hyperplasia and
associated symptoms by administering
1-alpha-fluoro-25-hydroxy-16,23E-diene-26,27-bishomo-20-epi-cholecalcifer-
ol in an amount effective to prevent and/or to treat such disease
alone or in combination with further active agents.
BACKGROUND OF THE INVENTION
[0003] BPH is a common disorder in elderly men, occurring in
approximately 50% of men aged 60 years and in 90% of those aged 85
years. BPH is a specific histopathological entity characterized by
stromal and epithelial cell hyperplasia.
[0004] For over a century, the two known etiologic factors for the
pathogenesis of BPH have been aging and the presence of functional
testes. However, as the science of prostate biology advances, this
concept becomes inadequate as it does not cover all aspects of BPH
pathogenesis. Additional etiologic factors play a significant role
in regulating prostatic growth. In particular, evidence has emerged
that prostatic growth is under the immediate control of specific
growth factors produced by prostatic cells, acting locally on
adjacent cells in a paracrine mechanism or to the same cells in an
autocrine mechanism. Therefore much effort is currently being put
into identifying therapeutic strategies aimed at inhibiting
intraprostatic growth factors.
[0005] BPH is a common cause of chronic lower urinary tract
symptoms which may affect both the filling (irritative symptoms)
and voiding (obstructive symptoms) phases of the micturition cycle.
These symptoms affect the social, psychological, domestic,
occupational, physical and sexual lives of the patients leading to
a profound, negative impact on their quality of life. In addition
to this, BPH can cause more acute urological complications,
particularly acute urinary retention (AUR), often considered the
most serious complication of BPH and less frequently recurrent
urinary tract infections, upper urinary tract dilatation, bladder
stone formation and recurrent hematuria.
[0006] BPH management is associated with extremely high social
costs, estimated to be 4 billion dollars in 1993 and projected to
be 26 billion dollars in 2003 in the USA alone.
[0007] The current medical treatment for BPH consists of orally
administered 5 alpha reductase inhibitors (finasteride and
dutasteride, recently approved by the FDA) and alpha 1 receptor
antagonists (terazosin, doxazosin, tamsulosin as well as silodosin,
AIO-8507L, RBx-2258 etc). Each of these therapeutic options is
associated with both advantages and disadvantages relating to their
different mechanisms of action. Although alpha 1 receptor
antagonists are very effective in reducing symptoms related to
lower urinary tract symptoms (LUTS), they are ineffective in
reducing the prostate volume and therefore in preventing
BPH-related surgery. Conversely, 5 alpha reductase inhibitors like
finasteride and dutasteride, by decreasing dihydrotestosterone
(DHT) formation, reduce prostate size and the need for surgery.
[0008] In addition, recent results from the seven-year Prostate
Cancer Prevention Trial, involving more than 18.000 healthy aged
man, demonstrated that finasteride can prevent or delay the
appearance of prostate cancer (see Thompson I M, et al. New England
Journal of Medicine (2003) 349 p 215-224). However, as expected
(see Kassabian V S, Lancet (2003) 361 p 60-62), finasteride was not
free from anti-androgenic adverse effects on sexual function, such
as decreased sexual potency, sexual desire and gynecomastia, that
substantially lessen its attractiveness as a cancer-preventing
agent. In addition, finasteride treatment was associated with an
increased detection of high-grade prostate cancer, probably because
the finasteride-induced low androgen state selected the most
aggressive, androgen-insensitive malignant growing cells (see
Scardino P T, New England Journal of Medicine (2003) 349 p
297-299).
[0009] Thus there is an unmet need for a new class of drugs for
medical therapy of BPH, which should be able to prevent acute
urinary retention, together with its related need for surgery, by
decreasing androgen-induced prostate growth but without directly
interfering with androgen receptors (AR), and therefore without
anti-androgenic prostatic and extra-prostatic adverse effects, for
example, sexual side effects. Such medicaments, by disrupting
intra-prostatic growth factor signalling, might be useful not only
for treating BPH but also for preventing prostate cancer, possibly
without selecting AR-insensitive, malignant clones.
SUMMARY OF THE INVENTION
[0010] As described herein, the Inventors have determined that the
non-hypercalcemic, well-tolerated, vitamin D.sub.3 analogue
1-alpha-fluoro-25-hydroxy-16,23E-diene-26,27-bishomo-20-epi-cholecalcifer-
ol (Compound A), is a primary example of such a medicament, as it
can act against BPH in an androgen-receptor independent manner by
targeting multiple pathways controlling BPH cell growth, including
growth factor-mediated prostate proliferation.
[0011] 1,25-dihydroxyvitamin D.sub.3 [1,25(OH).sub.2D.sub.3], the
activated form of vitamin D.sub.3, is a secosteroid hormone that
not only plays a central role in bone and calcium metabolism, but
is also involved in the regulation of the immune response and the
differentiation and apoptosis of many cell types, including
malignant cells.
[0012] However, a problem with the therapeutic use of calcitriol is
its natural ability to induce hypercalcemia and hyperphosphatemia.
Hence, analogues of calcitriol retaining biological activity but
devoid of hypercalcemic side effects, have been developed.
[0013] U.S. Pat. No. 5,939,408 and EP808833 disclose a number of
1,25(OH).sub.2D.sub.3 analogues including the compound
1-alpha-fluoro-25-hydroxy-16,23E-diene-26,27-bishomo-20-epi-cholecalcifer-
ol (Compound A). U.S. Pat. No. 5,939,408 and EP808833 disclose that
the compounds induce differentiation and inhibition of
proliferation in various skin and cancer cell lines and are useful
for the treatment of hyperproliferative skin diseases such as
psoriasis, neoplastic diseases such a leukemia, breast cancer and
sebaceous gland diseases such as acne and seborrheic dermatitis and
osteoporosis.
[0014] It has now surprisingly been found in several studies
conducted by the Inventors that, unlike certain other
1,25(OH).sub.2D.sub.3 analogues, the 1,25(OH).sub.2D.sub.3 analogue
Compound A:
##STR00001##
significantly reduces the growth of human BPH cells in vitro via
induction of their apoptosis and reduces prostatic growth in vivo,
with no effects on testosterone and dihydrotestosterone levels.
Furthermore, this inhibition of prostate growth is achieved at
non-hypercalcemic doses. Thus, Compound A is an effective
pharmacologic agent for the treatment of benign prostatic
hyperplasia.
[0015] As described in the Examples herein, Compound A reduces
prostate size. Furthermore, as observed with finasteride, Compound
A counteracts against the in vitro and in vivo proliferative
activity of testosterone. Significantly however, and unlike
finasteride, Compound A does not inhibit type-1 or type-2 5
alpha-reductase activity and can counteract not only testosterone
but even dihydrotestosterone induced BPH cell growth. These
anti-androgenic properties of Compound A are independent from
interaction with the AR, as shown by the failure of Compound A both
to bind to the AR, or to act as an AR agonist or antagonist.
Furthermore, Compound A does not affect sex hormone secretion.
Furthermore, in our studies, Compound A has no significant effect
on PSA levels thus there appears to be no danger of treatment with
Compound A masking this important indicator of possible prostate
cancer.
[0016] A further significant advantage of Compound A is that in
vitro studies have revealed that this drug, unlike finasteride, is
capable of inhibiting the basal and testosterone-stimulated growth
of bladder cells and is expected to be useful in preventing and/or
treating of bladder dysfunction in humans. In vivo studies in a
validated rat bladder outlet obstruction model of bladder
dysfunction have also demonstrated the beneficial effect of
Compound A. This is significant because bladder dysfunction is a
common and troublesome sequela of BPH. Thus Compound A is capable
of reducing prostate size and ameliorating bladder dysfunction,
i.e., improving bladder function and bladder related symptoms of
BPH at the same time through direct effect of Compound A both on
the prostate and on the bladder. This effect is expected to go
beyond the improvement in bladder symptoms that would be expected
merely as a result of prostate size reduction. Bladder symptoms
include overactive bladder and indicators of improved bladder
function include reduction in non-voiding contractions and residual
urine.
[0017] Thus, the present invention provides the use of
1-alpha-fluoro-25-hydroxy-16,23E-diene-26,27-bishomo-20-epi-cholecalcifer-
ol in the manufacture of a medicament for the prevention and/or
treatment of benign prostatic hyperplasia. Also considered within
the scope of the invention are pharmaceutically acceptable esters
and salts of compound A.
[0018] The invention thus provides the use of
1-alpha-fluoro-25-hydroxy-16,23E-diene-26,27-bishomo-20-epi-cholecalcifer-
ol, or a pharmaceutically acceptable salt or ester thereof, in the
manufacture of a medicament for the prevention and/or treatment of
benign prostatic hyperplasia
[0019] The invention also provides a method for preventing and/or
treating benign prostatic hyperplasia, in patients in need of such
prevention or treatment comprising administering a therapeutically
effective amount of
1-alpha-fluoro-25-hydroxy-16,23E-diene-26,27-bishomo-20-epi-cholecalcifer-
ol or a pharmaceutically acceptable salt or ester thereof.
[0020] The invention also provides the use of
1-alpha-fluoro-25-hydroxy-16,23E-diene-26,27-bishomo-20-epi-cholecalcifer-
ol, or a pharmaceutically acceptable salt or ester thereof, for the
prevention and/or treatment of benign prostatic hyperplasia.
[0021] The invention also provides
1-alpha-fluoro-25-hydroxy-16,23E-diene-26,27-bishomo-20-epi-cholecalcifer-
ol, or a pharmaceutically acceptable salt or ester thereof, for use
in the prevention and/or treatment of benign prostatic
hyperplasia.
[0022] The invention also provides the use of
1-alpha-fluoro-25-hydroxy-16,23E-diene-26,27-bishomo-20-epi-cholecalcifer-
ol, or a pharmaceutically acceptable salt or ester thereof, for the
prevention and/or treatment of benign prostatic hyperplasia without
anti-androgenic prostatic and extra-prostatic adverse effects. It
also provides a method for preventing and/or treating benign
prostatic hyperplasia without anti-androgenic prostatic and
extra-prostatic adverse effects, in patients in need of such
prevention or treatment, comprising administering a therapeutically
effective amount of
1-alpha-fluoro-25-hydroxy-16,23E-diene-26,27-bishomo-20-epi-cholecalcifer-
ol or a pharmaceutically acceptable salt or ester thereof.
[0023] The invention also provides the use of
1-alpha-fluoro-25-hydroxy-16,23E-diene-26,27-bishomo-20-epi-cholecalcifer-
ol, or a pharmaceutically acceptable salt or ester thereof, for the
prevention and/or treatment of benign prostatic hyperplasia
together with concurrent prevention and/or treatment of bladder
dysfunction. It also provides a method for preventing and/or
treating benign prostatic hyperplasia with concurrent prevention
and/or treatment of bladder dysfunction, in patients in need of
such prevention or treatment, comprising administering a
therapeutically effective amount of
1-alpha-fluoro-25-hydroxy-16,23E-diene-26,27-bishomo-20-epi-cholecalcifer-
ol or a pharmaceutically acceptable salt or ester thereof.
BRIEF DESCRIPTION OF THE DRAWINGS
[0024] FIG. 1 shows inhibition of BPH cell proliferation by
calcitriol and Compound A ("Cmpd A").
[0025] FIG. 2 shows the effect of Compound A, cyproterone acetate
and finasteride on androgen-stimulated BPH cell growth.
[0026] FIG. 3 shows the lack of agonistic or antagonistic
properties of Compound A on human AR.
[0027] FIG. 4 shows inhibition of rat ventral prostate growth by
Compound A or finasteride.
[0028] FIG. 5 shows the effect of Compound A and finasteride on
clusterin gene expression in the rat ventral prostate.
[0029] FIG. 6 shows the morphological effects of Compound A on
ventral prostate of castrated and T-supplemented rats.
[0030] FIG. 7 shows the morphological effects of Compound A and
finasteride on the prostate of intact, adult rats.
[0031] FIG. 8 shows results of a chronic toxicity study in
dogs.
[0032] FIG. 9 shows results of a chronic toxicity study in
dogs.
[0033] FIG. 10 shows the effect of Compound A on
testosterone-stimulated bladder cell growth. "hB"=human bladder
[0034] FIG. 11 shows the effect of Compound A and other comparator
compounds on stimulated and basal bladder cell growth. "T 10
nM"=testosterone; F 1 nM"=finasteride.
[0035] FIG. 12 shows the effect of a vitamin D compound on bladder
weight.
[0036] FIG. 13 shows the effect of a vitamin D compound on
spontaneous non-voiding contraction frequency.
[0037] FIG. 14 shows the effect of a vitamin D compound on
spontaneous non-voiding contraction amplitude.
[0038] FIG. 15 shows the effect of a vitamin D compound on
micturition pressure.
[0039] FIG. 16 shows the effect of a vitamin D compound on residual
urine.
[0040] FIG. 17 shows the effect of a vitamin D compound on the
contractile response of bladder strips to EFS (Electrical Field
Stimulation).
DETAILED DESCRIPTION OF THE INVENTION
[0041]
1-Alpha-fluoro-25-hydroxy-16,23E-diene-26,27-bishomo-20-epi-choleca-
lciferol is a known compound and its preparation is described in
U.S. Pat. No. 5,939,408, the description of which is incorporated
herein by reference.
Esters include pharmaceutically acceptable labile esters that may
be hydrolysed in the body to release Compound A.
[0042] Salts of Compound A include adducts and complexes that may
be formed with alkali and alkaline earth metal ions and metal ion
salts such as sodium, potassium and calcium ions and salts thereof
such as calcium chloride, calcium malonate and the like.
[0043] Compound A, or salt or ester thereof, can be used as a
monotherapy or it can be administered in combination with known
BPH-active agents, for example an alpha-, adrenergic receptor
blocking agent such as an alpha 1 receptor antagonist (for example
terazosin, doxazosin or tamsulosin or else silodosin, AIO-8507L or
RBx-2258) or a 5 alpha-reductase inhibitor (for example finasteride
or dutasteride). The expression "BPH-active agent" includes those
agents capable of or known to have activity in treating or
preventing BPH such as the aforementioned example substances. The
combination partner can be admixed with the compound A or its salts
or esters in various ratios and can be administered separately,
sequentially or simultaneously in separate or combined
pharmaceutical formulations. Appropriate doses of known therapeutic
agents will be readily appreciated by those skilled in the art.
Combination of A with two or more, e.g., 3 BPH-active compounds may
be envisaged, e.g., combination with an alpha 1 receptor antagonist
and a 5 alpha-reductase inhibitor. When administered in combination
with Compound A (or salt or ester) the combination partner(s) may
be used at lower doses compared to that used when the combination
partner is administered alone, perhaps even a dose which is
sub-therapeutic when administered alone.
[0044] Thus the invention also provides the use as defined above
wherein the medicament is administered separately, sequentially or
simultaneously in separate or combined pharmaceutical formulations
with a second BPH-active agent.
[0045] Thus the invention also provides the use of
1-alpha-fluoro-25-hydroxy-16,23E-diene-26,27-bishomo-20-epi-cholecalcifer-
ol, or a pharmaceutically acceptable salt or ester thereof, in
combination with a second BPH-active agent in the manufacture of a
medicament for the prevention and/or treatment of benign prostatic
hyperplasia.
[0046] The invention also provides a method for preventing and/or
treating benign prostatic hyperplasia, in patients in need of such
prevention or treatment comprising administering a therapeutically
effective amount of
1-alpha-fluoro-25-hydroxy-16,23E-diene-26,27-bishomo-20-epi-cholecalcifer-
ol or a pharmaceutically acceptable salt or ester thereof
separately, sequentially or simultaneously in separate or combined
pharmaceutical formulations with a second BPH-active agent.
[0047] The combinations referred to above may conveniently be
presented for use in the form of a pharmaceutical formulation. The
pharmaceutical formulations thus produced also represent a further
aspect of the invention.
[0048] Dosage levels and time course of administration of the
active ingredients in the pharmaceutical formulations of the
invention may be varied so as to obtain an amount of the active
ingredient which is effective to achieve the desired therapeutic
response for a particular patient, composition, and mode of
administration, without being toxic to the patient. An exemplary
dose range of Compound A is from 0.1 to 300 .mu.g per day, for
example 50-150 .mu.g per day, e.g., 75 or 150 .mu.g per day. A unit
dose formulation preferably contains 50-150 .mu.g, e.g., 75 or 150
.mu.g and is preferably administered once per day.
[0049] Specifically, a preferred dose of Compound A is the maximum
that a patient can tolerate and not develop hypercalcemia or other
undesirable side effects such as hypercalcuria. Preferably Compound
A is administered at a concentration of about 0.001 .mu.g to about
100 .mu.g per kilogram of body weight, about 0.001-about 10
.mu.g/kg or about 0.001 .mu.g-about 100 .mu.g/kg of body weight.
Ranges intermediate to the above-recited values are also intended
to be part of the invention.
[0050] As noted above, Compound A may be administered as a
pharmaceutically acceptable salt or ester thereof however
preferably Compound A is employed as is, i.e., it is not employed
as an ester or a salt thereof.
[0051] This dosage may be delivered in a conventional
pharmaceutical formulation by a single administration, by multiple
applications, or via controlled release, as needed to achieve the
most effective results, preferably once or twice daily (especially
once daily), e.g., by mouth. In certain situations, alternate day
dosing may prove adequate to achieve the desired therapeutic
response.
[0052] The selection of the exact dose and formulation and the most
appropriate delivery regimen will be influenced by, inter alia, the
pharmacological properties of the formulation, the nature and
severity of the condition being treated, and the physical condition
and mental acuity of the recipient.
[0053] Representative delivery regimens include oral, parenteral
(including subcutaneous, intramuscular and intravenous), rectal,
buccal (including sublingual), pulmonary, transdermal, and
intranasal, most preferably oral. Administration may be continuous
or intermittent (e.g., by bolus injection).
[0054] The invention also provides a pharmaceutical composition
comprising
1-alpha-fluoro-25-hydroxy-16,23E-diene-26,27-bishomo-20-epi-cholecalcifer-
ol, or a pharmaceutically acceptable salt or ester thereof, and a
pharmaceutically acceptable carrier for the prevention and/or
treatment of benign prostatic hyperplasia.
[0055] The invention also provides a packaged formulation which
includes a pharmaceutical composition comprising
1-alpha-fluoro-25-hydroxy-16,23E-diene-26,27-bishomo-20-epi-cholecalcifer-
ol, or a pharmaceutically acceptable salt or ester thereof, and a
pharmaceutically-acceptable carrier packaged with instructions for
use in the treatment of benign prostatic hyperplasia.
[0056] As mentioned above, such compositions may be prepared for
parenteral (subcutaneous, intramuscular or intravenous)
administration, particularly in the form of liquid solutions or
suspensions; for oral or buccal administration, particularly in the
form of tablets or capsules; for pulmonary or intranasal
administration, particularly in the form of powders, nasal drops or
aerosols; and for rectal or transdermal administration.
[0057] The compositions may conveniently be administered in unit
dosage form and may be prepared by any of the methods well-known in
the pharmaceutical art, for example as described in Remington's
Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton,
Pa., (1985). Formulations for parenteral administration may contain
as excipients sterile water or saline, alkylene glycols such as
propylene glycol, polyalkylene glycols such as polyethylene glycol,
oils of vegetable origin, hydrogenated naphthalenes and the like.
Formulations for nasal administration may be solid and may contain
excipients, for example, lactose or dextran, or may be aqueous or
oily solutions for use in the form of nasal drops or metered spray.
For buccal administration typical excipients include sugars,
calcium stearate, magnesium stearate, pregelatinated starch, and
the like.
[0058] Orally administrable compositions may comprise one or more
physiologically compatible carriers and/or excipients and may be in
solid or liquid form. Tablets and capsules may be prepared with
binding agents, for example, syrup, acacia, gelatin, sorbitol,
tragacanth, or poly-vinylpyrollidone; fillers, such as lactose,
sucrose, corn starch, calcium phosphate, sorbitol, or glycine;
lubricants, such as magnesium stearate, talc, polyethylene glycol,
or silica; and surfactants, such as sodium lauryl sulfate. Liquid
compositions may contain conventional additives such as suspending
agents, for example sorbitol syrup, methyl cellulose, sugar syrup,
gelatin, carboxymethylcellulose, or edible fats; emulsifying agents
such as lecithin, or acacia; vegetable oils such as almond oil,
coconut oil, cod liver oil, or peanut oil; preservatives such as
butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT).
Liquid compositions may be encapsulated in, for example, gelatin to
provide a unit dosage form.
[0059] Preferred solid oral dosage forms include tablets, two-piece
hard shell capsules and soft elastic gelatin (SEG) capsules. SEG
capsules are of particular interest because they provide distinct
advantages over the other two forms (see Seager, H., "Soft gelatin
capsules: a solution to many tableting problems"; Pharmaceutical
Technology, 9, (1985)). Some of the advantages of using SEG
capsules are: a) dose-content uniformity is optimized in SEG
capsules because the drug is dissolved or dispersed in a liquid
that can be dosed into the capsules accurately; b) drugs formulated
as SEG capsules show good bioavailability because the drug is
dissolved, solubilized or dispersed in an aqueous-miscible or oily
liquid and therefore when released in the body the solutions
dissolve or are emulsified to produce drug dispersions of high
surface area; and c) degradation of drugs that are sensitive to
oxidation during long-term storage is prevented because the dry
shell of soft gelatin provides a barrier against the diffusion of
oxygen.
[0060] The dry shell formulation typically comprises of about 40%
to 60% concentration of gelatin, about a 20% to 30% concentration
of plasticizer (such as glycerin, sorbitol or propylene glycol) and
about a 30 to 40% concentration of water. Other materials such as
preservatives, dyes, opacifiers and flavours also may be present.
The liquid fill material comprises a solid drug that has been
dissolved, solubilized or dispersed (with suspending agents such as
beeswax, hydrogenated castor oil or polyethylene glycol 4000) or a
liquid drug in vehicles or combinations of vehicles such as mineral
oil, vegetable oils, triglycerides, glycols, polyols and
surface-active agents. In an example formulation, the soft gelatin
capsules are size 2, white, opaque, oval gelatin capsules
containing a liquid fill consisting of the active ingredient,
Compound A, dissolved in Miglyol 812 (triglyceride of fractionated
C.sub.8-C.sub.12 coconut oil fatty acids) with butylated
hydroxytoluene (BHT), and butylated hydroxyanisole (BHA), as
preservatives. The soft gelatin capsules can be formulated to
contain between 0.01 and 25 mg, e.g., 75 or 150 .mu.g of Compound
A. Soft gelatin capsules should be stored at 2-8.degree. C. and
protected from light.
[0061] Formulations containing Compound A, or a pharmaceutically
acceptable salt or ester thereof, optionally in combination with a
second BPH-active agent, may be prepared by mixing the
ingredients.
Formulations are preferably prepared under nitrogen in amber
light.
EXAMPLES
[0062] The present invention will now be described with reference
to the following non-limiting examples, with reference to the
figures, in which:
[0063] FIG. 1 shows inhibition of BPH cell proliferation by
calcitriol and Compound A ("Cmpd A"). Panel A Incubation for 48 h
with increasing concentrations (10.sup.-18-10.sup.-7 M) of
calcitriol (circles) or Compound A (squares) resulted in a
significant and dose-dependent inhibition of BPH cell growth
(*P<0.01 vs control). ALLFIT analysis indicates that the two
secosteroids share the same maximal inhibition
(I.sub.max=43.+-.1%), but show a marked difference in the rank of
potency (-logIC.sub.50 Compound A=15.8.+-.0.3; -logIC.sub.50
calcitriol=10.2.+-.0.6, P<0.005). Results are expressed as %
inhibition (mean .+-.SEM) over their relative controls in 3
different experiments performed in triplicate. Panel B Effect of
increasing concentrations (10.sup.-18-10.sup.-7 M) of Compound A on
BPH cell proliferation stimulated by T (10 mM, squares), KGF (10
ng/ml, circles) or Des(1-3)IGF-I (10 ng/ml, triangles). Compound A
induced a significant inhibition (*P<0.01 vs T- or GF-treated
cells) of BPH cell growth also in presence of all the stimuli
tested with similar I.sub.max=66.6.+-.7.3%. However, Compound A was
more potent in inhibiting the effect of T (-log
IC.sub.50=16.4.+-.0.6), than of the other two GFs, (-log IC.sub.50
Des(1-3)IGF-I=12.7.+-.0.6, and -log IC.sub.50 KGF=14.2.+-.0.6,
P<0.0001). Results are expressed as % variation (mean .+-.SEM)
over the maximal stimulation in 3 different experiments performed
in triplicate.
[0064] FIG. 2 shows the effect of Compound A, cyproterone acetate
and finasteride on androgen-stimulated BPH cell growth. BPH cells
were incubated for 48 h with Compound A (1 nM) or anti-androgens
(finasteride, F, 1 nM; cyproterone acetate, Cyp, 100 nM) in the
presence of T (10 nM, Panel A) or DHT (10 nM, Panel B). Results
obtained in unstimulated BPH cells are also shown (Panel A).
Results are expressed as % variation (mean .+-.SEM) over their
relative controls in three different experiments performed in
quadruplicate. Compound A and cyproterone acetate significantly
blocked both T- and DHT-induced growth, while finasteride was
effective only against T. *P<0.01 vs control; .degree. P<0.01
vs androgen-treated cells.
[0065] FIG. 3 shows the lack of agonistic or antagonistic
properties of Compound A on human AR. The AR deficient PC3 cell
line stably transfected with the human AR was plated in 24 well
plates at a density of 2.times.10.sup.4 cells/well. After 24 h, the
cells were transfected with the AR-responsive plasmid pLSPP and, 48
h later, cells were incubated with increasing concentrations of DHT
(squares) or Compound A (circles) (panel A), or with a fixed
concentration of DHT (3 nM) in the presence of bicalutamide
(squares) or Compound A (circles) (panel B) for 18 h. Results (the
mean of three transfection experiments) are expressed as percentage
of bioluminescence per .mu.g of total proteins. To evaluate
agonistic activity 100% luciferase activity was set in the presence
of DHT 100 nM (panel A), whereas to test antagonistic activity 100%
luciferase activity was set with DHT 3 nM (panel B).
[0066] FIG. 4 shows inhibition of rat ventral prostate growth by
Compound A or finasteride. Panel A: Castrated rats, injected with T
enanthate (30 mg/Kg/week), were orally treated for 5 day/week for
two consecutive weeks with vehicle or with increasing doses of
Compound A (10, 30, 100 and 300 .mu.g/Kg) or finasteride (F, 10 and
40 mg/Kg). Ventral prostate weight is expressed as % variation
(mean .+-.SEM) of the weight of intact, vehicle-treated, rats (
P<0.05, *P<0.01 vs control rats, .degree. P<0.01 vs
T-supplemented rats). Panel B: Intact adult rats were orally
treated for over one month (5 times/week for a total of 27
administration) with vehicle (control) or increasing concentrations
of Compound A (10, 30, 100 and 300 .mu.g/Kg) or finasteride (F, 10
and 40 mg/Kg). Ventral prostate weight is expressed as % variation
(mean .+-.SEM) of the weight of control, vehicle-treated rats
(*P<0.01 vs control rats).
[0067] FIG. 5 shows the effect of Compound A and finasteride on
clusterin gene expression in the rat ventral prostate. Panel A.
Northern analysis of clusterin mRNA expression in the ventral
prostate of vehicle-treated intact (lane 1) or orchidectomized
(lane 2) rats. Lanes 3-6 show clusterin gene expression in
orchidectomized rats supplemented for two weeks with T enanthate
(30 mg/Kg) and orally treated with vehicle (lane 3), Compound A
(300 .mu.g/Kg, lane 4 and 100 .mu.g/Kg, lane 5) or finasteride (40
mg/Kg, lane 6). Every lane was loaded with 10 .mu.g of total RNA.
The corresponding GAPDH expression and the ethidium bromide
staining of the gel are shown below the blot. The blot is
representative of two separate experiments. Panel B. Northern
analysis of clusterin mRNA expression in the ventral prostate of
adult intact rats orally treated for over 1 month (5 times/week, 27
administrations) with vehicle (lane 1), increasing concentrations
of Compound A (10, 30, .mu.g/Kg, lane 2 and 3) or finasteride (40
mg/Kg, lane 4). Every lane was loaded with 10 .mu.g of total RNA.
The corresponding GAPDH expression and the ethidium bromide
staining of the gel are shown below the blot. The blot is
representative of two separate experiments.
[0068] FIG. 6 shows the morphological effects of Compound A on
ventral prostate of castrated and T-supplemented rats. Panels A, B,
C and E. Representative fields obtained from cross-sections of
whole prostate glands immunostained with a monoclonal antibody
against rat clusterin and counterstained with haematoxylin. In
vehicle-treated rats castrated 4-days earlier, clusterin labelling
is detectable in the cytoplasm of the atrophic cuboidal epithelial
cells (Panel A, 10.times.). After two-week T supplementation (Panel
B, 10.times.), almost all the clusterin labelling disappeared.
Conversely, clusterin positive cells were still present in rats
treated with T and different doses of Compound A (100 .mu.g/Kg,
Panel C; 300 .mu.g/Kg, Panel E, black arrows). Panels D and F shows
sections consecutive to those in Panel C and E, to highlight DNA
fragmentation as assessed by terminal deoxynucleotidyl transferase
(TdT)-mediated dUTP nick end-labelling (TUNEL). Two-week treatment
(9 administrations) with Compound A (100 .mu.g/Kg, Panel D; 300
.mu.g/Kg, Panel F) induced massive apoptosis in the majority of
epithelial and stromal cells. Note (black arrows), that all the
clusterin-positive cells were undergoing apoptosis, while also a
consistent portion of clusterin unlabeled cells shows nuclear
fragmentation.
[0069] FIG. 7 shows the morphological effects of Compound A and
finasteride on the prostate of intact, adult rats. Panels A, B, D,
E, F and H. Representative fields obtained from cross-sections of
whole prostate glands immunostained with a monoclonal antibody
against rat clusterin and counterstained with haematoxylin. In
Panel A (10.times.) the primary antibody was omitted. Panel B
(10.times.) shows that in untreated adult rats only few, scanty
epithelial cells were labelled in some glands (black arrows).
Conversely, in prostate glands from rats treated with increasing
concentrations of Compound A cuboidal epithelial cells showing the
hallmark of atrophy were dose-dependently stained for clusterin
(see black arrows, 10 .mu.g/Kg, Panel D; 30 .mu.g/Kg, Panel E, 100
.mu.g/Kg, Panel F, 10.times.). Similar results were obtained with
finasteride (40 mg/Kg, Panel H, 10.times.). Panels C, G and I shows
serial, consecutive slices to those depicted in Panel B, D and F,
respectively, to highlight DNA fragmentation as assessed by
terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick
end-labelling (TUNEL). Note (black arrows), that all the
clusterin-positive cells were undergoing apoptosis, while also a
consistent portion of clusterin unlabeled cells shows nuclear
fragmentation.
[0070] FIG. 8 shows results of a chronic toxicity study in dogs. A
clear reduction of prostate weight is shown after 9 months of
treatment with Compound A relative to placebo.
[0071] FIG. 9 shows results of a chronic toxicity study in dogs. A
reduction of prostate weight after recovery from treatment with
Compound A relative to placebo.
[0072] FIG. 10 shows the effect of Compound A on
testosterone-stimulated bladder cell growth. "hB"=human bladder
[0073] FIG. 11 shows the effect of Compound A and other comparator
compounds on stimulated and basal bladder cell growth. "T 10
nM"=testosterone; F 1 nM"=finasteride.
[0074] FIG. 12 shows the effect of a vitamin D compound on bladder
weight.
[0075] FIG. 13 shows the effect of a vitamin D compound on
spontaneous non-voiding contraction frequency.
[0076] FIG. 14 shows the effect of a vitamin D compound on
spontaneous non-voiding contraction amplitude.
[0077] FIG. 15 shows the effect of a vitamin D compound on
micturition pressure.
[0078] FIG. 16 shows the effect of a vitamin D compound on residual
urine.
[0079] FIG. 17 shows the effect of a vitamin D compound on the
contractile response of bladder strips to EFS (Electrical Field
Stimulation).
Example 1
Effects of Compound A on BPH Cells In Vitro
Materials and Methods
Materials
[0080] Minimum Essential Medium (MEM), DMEM-F12 1:1 mixture, Ham's
F12 medium, phosphate buffered saline (PBS), bovine serum albumin
(BSA) fraction V, glutamine, geneticine, collagenase type IV,
vitamin D.sub.3, testosterone (T), dihydrotestosterone (DHT),
cyproterone acetate, .beta.-nicotinamide adenine dinucleotide
3'-phosphate reduced form (NADPH), dithithreitol (DTT),
phenylmethylsulfonyl fluoride (PMSF) and a kit for measuring
calcemia were purchased from Sigma (St. Louis, Mo.). The protein
measurement kit was from Bio-Rad Laboratories, Inc. (Hercules,
Calif.). Fetal bovine serum (FBS) was purchased from Unipath
(Bedford, UK). Monoclonal anti-rat clusterin antibody (mouse
monoclonal IgG) specific for beta-chain was from UPSTATE
Biotechnology (Lake Placid, N.Y.). Apop Tag kit for in situ end
labelling (ISEL) was from Oncor (MD, USA). CHO 1827 and CHO 1829
were provided by Serono International (Geneva, Switzerland).
Instagel plus was purchased from Packard (St Louis, Mo.).
Finasteride (pure substance)
(17.beta.-(N,t-butyl)carbamoyl-4-aza-5.alpha.-androst-1-en-3-one)
was a kind gift from Merck Sharp & Dohme Reaserch Laboratories
(Rahway, N.J.). Bicalutamide was a kind gift from AstraZeneca
(AstraZeneca, Milan, Italy). Analogue
1-.alpha.-fluoro-25-hydroxy-16,23E-diene-26,27-bishomo-20-epi-cholecalcif-
erol (Compound A) was provided by Bioxell (Bioxell, Milan, Italy).
Keratinocyte growth factor (KGF) was from Pepro Tech EC (London,
England) and insulin-like growth factor-I, human, [Des(1-3)IGF-I]
was purchased from GroPep Limited (Adelaide, Australia). In Situ
Cell Death Detection Kit POD for terminal deoxynucleotidyl
transferase (TdT) mediated dUTP nick end-labelling (TUNEL) were
from Roche Diagnostics Corporation (Indianapolis, Ind.). Plastic
ware for cell cultures was purchased from Falcon (Oxnard, Calif.).
Disposable filtration units for growth media preparation were
purchased from PBI International (Milan, Italy). Lipofectamine 2000
and Opti-MEM I Medium for luciferase transfection were from
Invitrogen, Life Technologies (San Giuliano Milanese, Milan,
Italy). Thin-layer chromatography (TLC) silica plates were obtained
from Merck (Darmstad, Germany). Testosterone enanthate (T
enanthate) was from Geymonat (Anagni, Italy). Coat-A-Count.RTM.
Total Testosterone detection kit was purchased from Medical System
(Genova Struppa, Italy). Rat luteinizing hormone (rLH) [.sup.125I]
assay systems were from Amersham Pharmacia Biotech (Piscataway,
N.J.).
BPH Cells
[0081] Human BPH cells, prepared, maintained and used as previously
described in Crescioli C, et al. Journal of Clinical and
Endocrinology Metabolism (2000) 85 p 2576-2583, were obtained from
prostate tissues derived from 5 patients, who underwent suprapubic
adenomectomy for BPH, after informed consent and approval by the
Local Ethical Committee. Patients did not receive any
pharmacological treatment in the 3 months preceding surgery.
[0082] 5.alpha. reductase-transfected CHO-1827 and CHO-1829 cell
lines CHO-1827 and CHO-1829 cells, transfected with 5.alpha.
reductase type I (5.alpha.R-1) or type 2 (5.alpha.R-2),
respectively (see Steers W. Urology (2001) 58 p 17-24), were
maintained in Ham's F12 medium supplemented with 5% FCS.
AR-Transfected PC3 Cell Line
[0083] Human prostate adenocarcinoma PC3 cells, stably transfected
with the plasmid p5HbhAR-A containing human androgen receptor (hAR)
as previously described (see Bonaccorsi L, et al. Endocrinology
(2000) 141 p 3172-3182), were grown in 75 cm.sup.2 culture flasks
in Ham's F-12 medium containing 50 .mu.g/ml geneticine, 10% FCS,
penicillin (100 U/ml) and streptomycin (100 mg/ml).
BPH Tissue
[0084] Prostatic tissues for binding assay were obtained from
patients who underwent suprapubic adenomectomy for BPH. No
pharmacological treatment was performed in the 3 months preceding
surgery. After surgery, the tissues were immediately placed in
liquid nitrogen and stored at -80.degree. C. until processing.
Rat Tissues
[0085] Rat ventral prostate glands were rapidly excised out,
weighed and quickly frozen in dry ice. Immunohistochemistry
experiments were performed in 14 .mu.m-thick contiguous cryostatic
sections for direct comparison of tissue morphology, clusterin
expression and apoptosis localization by TUNEL. For total RNA
extraction and Western blot analysis, rat ventral prostates from 4
to 6 animals were pooled.
BPH Cell Proliferation Assay
[0086] For all cell proliferation assay, 4.times.10.sup.4 BPH cells
were seeded onto 12-well plates in their growth medium, starved in
red- and serum-free medium containing 0.1% BSA for 24 h, and then
treated with specific stimuli for 48 h. Cells in phenol red- and
serum-free medium containing 0.1% BSA were used as controls.
Thereafter, cells were trypsinized, and each experimental point was
derived from hemocytometer counting, averaging at least six
different fields for each well, as previously reported (see
Crescioli C, et al. Journal of Clinical and Endocrinology
Metabolism (2000) 85 p 2576-2583). Experiments were performed using
increasing concentrations (10.sup.-18-10.sup.-7M) of calcitriol or
Compound A with or without a fixed concentration of T (10 nM), KGF
or Des(1-3)IGF-I (10 ng/ml). Growth assays were also carried out
using a fixed concentration of androgens (10 nM) with or without
Compound A (1 nM, 10 nM) or the anti-androgens finasteride (F, 1
nM) and cyproterone acetate (Cyp, 100 nM). Growth assays were also
performed using a fixed concentration of T (10 nM) or GFs (10
ng/ml) with or without Compound A (10 nM). In the same experiment,
each experimental point was repeated in triplicate or quadruplicate
and experiments were performed 3 times. Results are expressed as %
variation (mean .+-.SEM) over the maximal T or GF-induced
stimulation.
In Situ End Labeling (ISEL)
[0087] ISEL was performed onto BPH cells using Apop Tag in situ
apoptosis detection kit peroxydase following the manufacturer's
instruction. Cells were incubated with T (10 nM), KGF (10 ng/ml) or
Des(1-3)IGF-I (10 ng/ml) with or without Compound A (10 nM). The
percentage of apoptotic cells (the number of stained cells divided
by the total number of cells) was calculated in at least five
separate fields per slide in five different slides. Results are
expressed as mean .+-.SEM from three separate experiments.
5.alpha. Reductase Inhibition Test
[0088] 5.alpha. reductase inhibition assay was performed using CHO
1827 cells, transfected with 5.alpha.R-1, or CHO 1829 cells,
transfected with 5.alpha.R-2, as described (see Guarna A, et al.
Journal of Medicinal Chemistry (2000) 43 3718-3735.). Compound A
was added in a concentration range from 10.sup.-9 to 10.sup.-5 M,
using finasteride as a control inhibitor in each experiment.
Binding Assay
[0089] Binding assay on cytosol fractions of BPH fragments were
carried out as previously reported (see Crescioli C et al.
Endocrinology (2003) 144 p 3046-3057), (final protein
concentration: 1.8 mg/ml). Incubations of cytosolic fractions were
carried out with increasing concentration (0.125, 0.25, 0.5, 1 nM)
of [.sup.3H]-R1881 (specific activity: 83.5 Ci/mmol) in the absence
or in the presence ([.sup.3H]-R1881:1 nM) of increasing
concentrations of cold R1881 (10.sup.-10-10.sup.-6 M), DHT
(10.sup.-10-10.sup.-6 M), T (10.sup.-10-10.sup.-6 M), bicalutamide
(10.sup.10-10.sup.-4 M), and Compound A (10.sup.-10-10.sup.-4 M).
To prevent R1881 binding to progesterone receptor, 1 .mu.M
triamcinolone acetonide was added to each tube. Separation of bound
and unbound ligand was performed as previously described (see
Crescioli C et al. Endocrinology (2003) 144 p 3046-3057). Protein
content was determined by the known method of Bradford, using BSA
as a standard.
Luciferase Assay
[0090] PC3 cells stably transfected with human AR were plated in
24-well plates at a density of 2.times.10.sup.4 in Ham's F12 plus
10% FCS. After 24 hours, the cells were transfected with 750
ng/well of pLSPP plasmid containing the wilde-type sequence
configuration of the MMTV-LTR linked to the firefly luciferase gene
(see Pazzagli M. et al. Analytical Biochemistry (1992) 204 p
315-323.), using Lipofectamine 2000 (1 mg/ml) according to the
manufacturer's instructions. After 48 h, the cells were incubated
with DHT (10.sup.-12-10.sup.-6 M) or bicalutamide
(10.sup.-9-10.sup.-5-M), in the presence of 3 nM of DHT, and with
equimolar concentration of Compound A for 18 h. Steroids and
Compound A analogue were dissolved in ethanol. Transfected cells
incubated with ethanol only served as positive controls.
[0091] The Luciferase assay was performed with a Berthold
luminometer according to the manufacturer's instructions
(Luciferase Assay System, Promega, Milan, Italy). The cells were
lysed directly in the plate with 200 .mu.l of lysis buffer.
Luciferase activity was measured on 20 .mu.l of cell lysate for 10
s after addition of 100 .mu.l of luciferine. Total protein
measurement was performed on 20 .mu.l of cell lysate. At least
three independent assays were done in duplicate.
Results
[0092] Incubation of BPH cells with increasing concentrations of
calcitriol or Compound A inhibits cell growth (FIG. 1 panel A).
Both compounds inhibited dose-dependently cell proliferation.
ALLFIT (see De Lean A, et al. American Journal of Physiology (1978)
235 p E97-E102) analysis indicated that, although maximal
inhibition of calcitriol and Compound A ("Cmpd A" in the Figures)
was not statistically significantly different (I.sub.max=43.+-.1%),
their relative potency was, Compound A being several log units more
effective than calcitriol (-logIC.sub.50 Compound A=15.8.+-.0.3 vs
-logIC.sub.50 calcitriol=10.2.+-.0.6, P<0.005).
[0093] BPH cell proliferation was significantly increased
(P<0.01) by testosterone (T) (156.+-.8%), and growth factors
(GF), such as Des(1-3)IGF-I (194.+-.6%) or KGF (183.+-.5%). When
cell growth was stimulated for 48 h with T or GFs (FIG. 1, panel B)
the inhibitory effect of Compound A was even more pronounced
(I.sub.max=66.6.+-.7.3%). Mathematical modelling (see De Lean A, et
al. American Journal of Physiology (1978) 235 p E97-E102) of
inhibition curves indicated that Compound A was more potent in BPH
cells stimulated with T (-log IC.sub.50s=16.4.+-.0.6) than with the
other two growth factors (-log IC.sub.50s=12.7.+-.0.6, and -log
IC.sub.50=14.2.+-.0.6 for Des(1-3)IGF-I and KGF, respectively;
P<0.0001).
[0094] Compound A (1 nM) antagonized not only T- but also
DHT-stimulated BPH cell proliferation to an extent similar to the
AR antagonist cyproterone acetate (Cyp, 100 nM; FIG. 2 panel A and
B). Conversely, the 5.alpha.-reductase inhibitor finasteride (F, 1
nM) antagonized only T-induced cell growth (FIG. 2 panel A). In
addition, Compound A reduced growth even in androgen-unstimulated
cells (FIG. 2, panel A).
[0095] To evaluate potential anti-androgenic properties of Compound
A, in addition to BPH cell growth inhibition, we investigated its
interaction with the AR. First, we ruled out the possibility that
Compound A binds to the AR by performing competition studies in
human BPH homogenates, using the synthetic androgen [3H]-R1881 as
labelled ligand. LIGAND analysis (see Munson P J et al. Analytical
Biochemistry (1980) 107 p 220-239.) of the data indicated that
unlabeled R1881, DHT, T, and the AR antagonist bicalutamide
completely displaced [.sup.3H]-R1881 binding (Table I). Conversely,
Compound A did not compete for [.sup.3H]-R1881 binding at any
concentration tested (Table I). These results were confirmed and
extended using a luciferase reporter gene assay. In PC3 cells
expressing the full length AR coupled to a luciferase report gene,
DHT stimulated a dose-dependent increase in luciferase activity
(EC.sub.50=2.+-.1.3 nM, panel A), while bicalutamide inhibited
DHT-stimulated activity (IC.sub.50=194.+-.80 nM, panel B). In this
system, increasing concentration of Compound A neither stimulated
nor inhibited AR-mediated luciferase activity increase (FIG. 3).
Finally to verify whether or not Compound A interacts with the
formation of DHT, the active metabolite of T, we performed
experiments in CHO cells transfected with type 1 and type 2
5.alpha. reductase. Results were compared to those obtained with
finasteride (F). While F inhibited T conversion into DHT with the
expected IC.sub.50s, (IC.sub.50 for 5.alpha. reductase type
1=659.+-.100 nM and IC.sub.50 for 5.alpha. reductase type 2,
=53.7.+-.11 nM, n=3), Compound A did not interfere with either
isoenzyme up to the micromolar range (data not shown).
TABLE-US-00001 TABLE I Affinity constants of androgen agonists
(R1881, DHT, T), antagonist (bicalutamide) and Compound A in human
BPH homogenates as detected by [.sup.3H]R1881 binding. Affinity
constants AR Ligand (K.sub.d nmol/L) R1881 0.16 .+-. 0.06 DHT 0.07
.+-. 0.03 T 1.89 .+-. 0.94 Bicalutamide 159 .+-. 82 Compound A
>100000
[0096] The effect of Compound A in BPH cells was, at least in part,
due to activation of programmed cell death as detected by ISEL
(n=3, Table II). The percentage of apoptotic nuclei dramatically
increased (270%) after a 48 h exposure to 10 nM Compound A
(P<0.01 vs control). Conversely, treatment with T (10 .mu.M), or
GFs (10 ng/ml) significantly (P<0.01) reduced the number of
apoptotic BPH cells as compared to untreated cells
(Des(1-3)IGF-I=-42%; KGF=-54%; T=-27%). However, even in the
presence of GFs or T, Compound A induced a sustained (more than
250%) and significant (P<0.01) increase in the number of
ISEL-positive BPH cells.
Apototic index (%)
TABLE-US-00002 TABLE II Effect of Compound A (10 nM), GFs (10
ng/ml) or T (10 nM) on DNA fragmentation in BPH cells. Control
Compound A Control 18.55 .+-. 0.8 68.44 .+-. 1.26 .sup.a
Des(1-3)IGF-I 10.69 .+-. 0.6 .sup.a 45.85 .+-. 0.66 .sup.a,b,c KGF
8.5 .+-. 0.42 .sup.a 44.46 .+-. 0.57 .sup.a,b,c T 13.56 .+-. 0.72
.sup.a 49.06 .+-. 1.87 .sup.a,b,c Apoptotic index (%) represents
the number of stained nuclei, as detected by ISEL, over BPH cells
in each of at least 5 separate fields per slide. Results are
expressed as mean .+-. SEM in three separate experiments. Compound
A is able to induce apoptosis in untreated BPH cells as well as in
BPH cells simultaneously incubated with GFs or T (.sup.aP < 0.01
vs control; .sup.bP < 0.01 vs Compound A-treated cells; .sup.cP
< 0.01 vs GF- or T-treated cells).
Example 2
Anti-Proliferative Properties of Compound A in In Vivo Models of
Prostate Growth
Animal Protocols
[0097] Male Sprague Dawley rats (28 days old) were purchased from
Charles River Laboratories (Calco, Lecco, Italy). All animal
experimentation described was conducted in accord with accepted
standards of animal care. Castration was performed via the scrotal
route under ketamine/xylazine anaesthesia. Three days after
castration, rats (5-8 animals per group) were treated or not with T
enanthate (30 mg/Kg) in two separate weekly sc injections. Rats
were orally treated for 5 days the first week, and 4 days the
second week with vehicle (Miyglyol 812), Compound A (10, 30, 100
and 300 .mu.g/Kg) or finasteride (10 and 40 mg/Kg) for a total of 9
administrations, and sacrificed one day later.
[0098] Alternatively, intact, adult male Sprague Dawley rats
(weight 250 g) were dosed orally with vehicle (Miglyol 812),
Compound A (10, 30, 100 and 300 .mu.g/Kg) or finasteride (10 and 40
mg/Kg) 5 days/week for 5 consecutive weeks and for two additional
days the 6.sup.th week, for a total of 27 administrations, unless
otherwise specified. Blood for calcium and hormone measurements was
obtained at the end of each experimental protocol.
Northern Hybridisation Analysis
[0099] Total RNA was extracted using RNAFast from Molecular System
(San Diego, Calif.). Blotting, labelling, hybridization conditions
and probes (rat clusterin 1.5 Kb full-length cDNA and GAPDH 1.2 Kb
full-length cDNA) were performed according to the reported
procedures (Bettuzzi et al, Biochemical Journal, (1989), 257, p
293-296 and Marinelli et al, Biochemistry and Cell Biology, (1994),
72, p 515-521). Quantitation of the autoradiograms was obtained by
densitometric scanning using an LKB Ultrascan XL densitometer.
Immunohistochemistry
[0100] All the cryostatic sections obtained from controls and
treated rats were processed in parallel as previously described
(Astancolle et al, Journal of Endocrinology, (2000), 167, p
197-204). For every experimental condition, 3 alternate sections
from 3 different rat prostates were examined. Negative controls,
made by excluding the specific antibody from the reaction, showed
no specific staining. Counterstaining was performed with
haematoxylin, and cover slips were mounted with Eukitt (O. Kindler
GmbH & Co, Germany). Digital high-magnification colour images
were acquired by a CCD camera through the microscope.
In Situ DNA Fragmentation Analysis (TUNEL)
[0101] DNA fragmentation in prostate cryostatic sections, assessed
by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick
end-labelling (TUNEL), was performed using the In Situ Cell Death
Detection Kit (POD, Roche) as recommended by the manufacturer.
TUNEL positive apoptotic nuclei were documented by digital
high-magnification colour images acquired by a CCD camera through
the microscope. Counterstaining was performed with eosin, and cover
slips were mounted with Eukitt (O Kindler GmbH & Co,
Germany).
Calcium Measurements
[0102] Serum calcium levels were measured with a commercially
available colorimetric assay (Sigma), according to the
manufacturers' instructions.
Testosterone and rLH Measurement
[0103] Serum levels of T and rLH hormones were determined by
commercially available radioimmunoassay kits, according to the
manufacturers' instructions. To measure serum T in rats, samples
were first added to 4 volumes of diethyl ether, mixed by gentle
inversion for 15 min and then centrifuged for 5 min at 2000 rpm.
The aqueous phase was frozen in dry ice and the organic phase was
recovered and evaporated to dryness under a nitrogen stream. The
dried extract was reconstituted in the assay buffer as follows: (1
vol: 1 vol) in intact rat, and (4 vol: 1 vol) in castrated
rats.
Statistical Analysis
[0104] Statistical analysis was performed by one-way ANOVA and
paired or unpaired Student's t tests, when appropriate. Binding
data were analysed using the computerized program LIGAND (Munson et
al, Analytical Biochemistry, (1980), 107, p 220-139). The computer
program ALLFIT (DeLean et al, American Journal of Physiology,
(1978), 235, p E97-E102) was used for the analysis of sigmoid
dose-response curves to obtain estimates of half-maximal inhibition
values (IC.sub.50) and half-maximal stimulatory values (EC.sub.50)
as well as maximal inhibitory (I.sub.max) and stimulatory
(E.sub.max) effects. Data were expressed as (mean .+-.SEM).
Results
[0105] To test the anti-proliferative properties of Compound A in
in vivo models of prostate growth, castrated and intact rats were
orally treated with increasing concentrations of Compound A (10-300
.mu.g/Kg) or finasteride (F) (10, 40 mg/Kg). As shown in FIG. 4,
panel A, castration dramatically reduced ventral prostate weight,
while a two-week treatment with testosterone (T) enanthate (30
mg/Kg) not only completely restored, but further stimulated its
growth. Compound A, at any dose tested, completely blunted
T-stimulated prostate over-growth, reducing ventral prostate weight
below that of untreated rats. Similar results were obtained with
finasteride (10, 40 mg/Kg). A one-month treatment of intact adult
rats with Compound A significantly decreased ventral prostate
weight, with a maximal reduction (30%) at the highest dose tested
(300 .mu.g/Kg). At this dose, the inhibitory effect of Compound A
on prostate growth was comparable to that induced by 10 or 40 mg/Kg
finasteride (FIG. 4, panel B). In all the experimental protocols,
oral administration of different doses of Compound A caused a very
modest hypercalcemia only at the highest dose tested (300 .mu.g/Kg)
(Table III). No other discernible side effects were observed.
TABLE-US-00003 TABLE III Calcemia (mg/dl) in T-replaced castrated
rats after different doses (10, 30, 100, 300 .mu.g/Kg) of Compound
A. calcemia Control 10.2 .+-. 0.16 Compound A 10 .mu.g/Kg 10.16
.+-. 0.24 Compound A 30 .mu.g/Kg 9.87 .+-. 0.15 Compound A 100
.mu.g/Kg 10.55 .+-. 0.18 Compound A 300 .mu.g/Kg 10.85 .+-. 0.1
Compound A never changed calcium serum levels in castrated rats
replaced with T enanthate (30 mg/Kg/week) as compared to controls.
Similar results were obtained in intact rats (not shown). Results
represent the mean .+-. SEM of rats/group.
[0106] To better understand the molecular mechanisms underlying
Compound A-induced prostate weight reduction, the expression of
clusterin gene and protein and the morphological hallmarks of
apoptosis were evaluated by terminal deoxynucleotidyl transferase
(TdT) mediated dUTP nick end-labelling (TUNEL). Clusterin is an
ubiquitous product gene, strictly related to cell cycle arrest and
atrophy, the expression of which is down regulated by androgens.
FIG. 5 panel A, shows the prostatic expression of clusterin mRNA,
as detected by Northern analysis, in orchidectomized rats
supplemented or not supplemented with T. Castration dramatically
up-regulated clusterin mRNA abundance, while this effect was
completely reverted by a two-week administration of T. The
simultaneous treatment with different concentrations of Compound A
(300 and 100 .mu.g/Kg) or F (40 mg/Kg) partially blunted the
T-induced down-regulation of clusterin gene expression. In intact
rats (FIG. 5 panel B), a one-month administration of different
concentrations of Compound A (30 and 100 .mu.g/Kg) induced a
sustained increase in clusterin gene expression in the prostate,
comparable, or even higher, than that induced by 40 mg/Kg F.
[0107] The local expression of clusterin in the prostate of
orchidectomized rats is shown in FIG. 6. Castration induced a
marked and widespread atrophy in the prostate gland, and nearly all
the cuboidal epithelial cells facing the gland lumen were clusterin
positive (panel A). T-replacement (panel B) reverted the
morphological hallmarks of atrophy and consistently reduced
clusterin staining. Such an effect was prevented by the
simultaneous administration of Compound A (panels C and E). Panels
D and F show TUNEL results in sections adjacent to those shown in
panels C and E. Compound A treatment (100 .mu.g/Kg, panel D and 300
.mu.g/Kg, panel F) induced an evident nuclear fragmentation in
epithelial and stromal cells, and apoptosis was detectable in both
clusterin positive and negative cells. The first two panels of FIG.
7 show the morphology of the prostate gland of an intact rats
processed for clusterin detection, with (panel A) or without (panel
B) the omission of the primary antibody. Note that clusterin
labelling is almost absent in the prostate of untreated adult rats
(panel B), as it is nuclear fragmentation (TUNEL, panel C).
Conversely, treatment with different doses of Compound A induced
clusterin expression (panels D-F) and apoptosis (C, G and I). Panel
F shows, for comparison, the effect of finasteride (40 mg/Kg) on
clusterin positivity in the prostate gland.
[0108] To rule out the possibility that Compound A reduced in vivo
prostate growth by interfering with pituitary or testis function,
rat luteinizing hormone (rLH) and T serum levels were measured in
castrated and intact rats. As expected (Table IV, panel A),
castration significantly reduced T while it increased rLH serum
levels. T enanthate (30 mg/Kg) administration (two weeks)
completely reverted the effect of orchidectomy. Oral treatment with
Compound A (100 and 300 .mu.g/Kg) of T-replaced castrated rats did
not significantly affect rLH or T serum levels. Similar results
were obtained in intact rats (Table IV, panel B). In fact, chronic
administration (1 month) of Compound A (10, 30, 100 .mu.g/Kg) or F
(40 mg/Kg) to intact rats did not modify rLH and T serum
levels.
TABLE-US-00004 TABLE IV rLH (ng/ml) and T (nM) serum levels in
T-replaced castrated (panel A) or intact (panel B) rats after
treatment with different doses of Compound A. rLH T Panel A control
(intact rats) 2.36 .+-. 0.46 11.5 .+-. 2.44 Castrated 20.64 .+-. 6*
0.9 .+-. 0.32* castrated + T-replaced 2.08 .+-. 0.36 21.25 .+-.
4.12 castrated + T-replaced + 1.8 .+-. 0.2 11.13 .+-. 1.02 Compound
A 100 .mu.g/Kg castrated + T-replaced + 3.15 .+-. 0.65 15.73 .+-.
2.75 Compound A 300 .mu.g/Kg Panel B control (intact rats) 2 .+-.
0.16 11.98 .+-. 2.87 Finasteride 2.2 .+-. 0.4 18.11 .+-. 3.23
Compound A 10 .mu.g/Kg 2.22 .+-. 0.25 19.13 .+-. 3.83 Compound A 30
.mu.g/Kg 2.32 .+-. 0.36 9.39 .+-. 2 Compound A 100 .mu.g/Kg 1.96
.+-. 0.13 11 .+-. 2.14 Panel A. Castration significantly reduced
serum T (*P < 0.01 vs control) while it increased serum rLH (*P
< 0.05 vs control). After treatment with T enanthate (30
mg/Kg/week) rLH and T serum levels were restored. Compound A at all
the doses tested did not significantly affect either rLH or T serum
levels. Panel B chronic administration (1 month) of F (40 mg/Kg) or
Compound A (10, 30 and 100 .mu.g/Kg) did change neither rLH nor T
serum levels in intact rats.
[0109] This study demonstrates that Compound A reduces prostate
size in intact rats to an extent similar to finasteride. In
addition, as finasteride, Compound A abolishes the in vitro and in
vivo proliferative activity of testosterone. However, at variance
with finasteride, Compound A does not inhibit type-1 or type-2
5.alpha.-reductase activity and can counteract not only T but even
DHT induced BPH cell growth. These anti-androgenic properties of
Compound A are independent from interaction with the AR, as shown
by the failure of Compound A to bind to the AR, and to act as AR
agonist or antagonist in AR-transfected PC3 cells. Furthermore,
Compound A does not affect sex hormone secretion because, in the
rat, gonadotropin and T plasma levels were unchanged by daily
administration of Compound A for up to one month. Hence, Compound A
acts downstream the AR receptor-ligand interaction. Without wishing
to be bound by theory this action most probably occurs via the
disruption of testosterone-growth factor cross talk.
[0110] Very low concentrations of Compound A were able to
completely antagonize not only T-stimulated BPH cell proliferation,
but also proliferation induced by the two most important
intra-prostatic growth factors: IGF-I and KGF. In addition, even in
the presence of T or GFs, Compound A induced apoptosis in BPH
cells. The Compound A-induced death program was evident also in the
prostate of both intact and T-supplemented orchidectomized rats and
was characterized by the diffuse appearance of DNA fragmentation
with a concomitant increase in clusterin gene and protein
expression. Clusterin is a protein tightly regulated in the
prostate by androgens (Bettuzzi et al, Biochemical Journal, (1989),
257, p 293-296). Although clusterin function is still not well
understood, it is markedly up-regulated in conditions of gland
atrophy (Bettuzzi et al, Oncogene, (2002), 21, p 4328-4334 and
Bettuzzi et al, Journal of Endocrinology, (1992), 132, p 361-367)
and apoptosis (Leskov et al, Journal of Biological Chemistry,
(2003), 278, p 11590-11600). Thus, clusterin induction by Compound
A treatment is consistent with the capacity of this compound to
inhibit proliferation and induce apoptosis in prostate cells.
[0111] In conclusion, this study indicates that Compound A is
effective in reducing prostate cell growth in different
experimental models.
Example 3
Reduction of Prostate Weight in Healthy Dogs Treated with Compound
A
[0112] A 9-month toxicity study was carried out in four groups of
male beagle dogs, which were treated by daily oral gavage with 0.5
.mu.g, 1.5 .mu.g and 5 .mu.g/kg body weight/day of Compound A (in
vehicle Miglyol 812) or with vehicle alone. This treatment was
followed by a 2-month recovery period for the group receiving the
highest dose, 5 .mu.g, after which prostate weights was measured.
In addition to entirely favourable toxicity data, a lower prostate
weight was observed at the end of treatment with Compound A (see
FIG. 8) and after recovery (see FIG. 9). The results after recovery
were analysed statistically via a one-tailed Student's t test and
were found to be significantly different between Compound A and
vehicle (P<0.05). These results further demonstrate the ability
of Compound A to reduce prostate size in vivo.
Example 4
Oral Dosage Form Soft Gelatin Capsule
[0113] A capsule for oral administration is formulated under
nitrogen in amber light from 0.01 to 25.0 mg of Compound A in 150
mg of fractionated coconut oil (e.g., Miglyol 812), with 0.015 mg
butylated hydroxytoluene (BHT) and 0.015 mg butylated
hydroxyanisole (BHA), filled in a soft gelatin capsule.
The capsule is prepared by the following process: 1. BHT and BHA
are suspended in fractionated coconut oil (e.g., Miglyol 812) and
warmed to around 50.degree. C. with stirring, until dissolved. 2.
Compound A is dissolved in the solution from step 1. at 50.degree.
C. 3. The solution from step 2. is cooled to room temperature. 4.
The solution from step 3. is filled into soft gelatin capsules. All
manufacturing steps are performed under a nitrogen atmosphere and
protected from natural light.
Example 4A
Oral Dosage Form Soft Gelatin Capsule
[0114] A capsule for oral administration is formulated under
nitrogen in amber light: 150 .mu.g of Compound A in 150 mg of
fractionated coconut oil (Miglyol 812), with 0.015 mg butylated
hydroxytoluene (BHT) and 0.015 mg butylated hydroxyanisole (BHA),
filled in a soft gelatin capsule.
Example 4B
Oral Dosage Form Soft Gelatin Capsule
[0115] A capsule for oral administration is formulated under
nitrogen in amber light: 75 .mu.g of Compound A in 150 mg of
fractionated coconut oil (Miglyol 812), with 0.015 mg butylated
hydroxytoluene (BHT) and 0.015 mg butylated hydroxyanisole (BHA),
filled in a soft gelatin capsule.
Example 5
Reduction in Prostate Weight in Human Clinical Trials
[0116] A randomised double blind placebo controlled parallel group
study was performed to determine the effect of Compound A
(1-alpha-fluoro-25-hydroxy-16,23E-diene-26,27-bishomo-20-epi-cholecalcife-
rol) in patients with BPH.
[0117] The principal inclusion criterion was that male patients be
diagnosed with BPH and have prostate volume >40 ml as determined
by transrectal ultrasound (TRUS). Statistical methods: primary
efficacy analyses were planned on Per-Protocol (PP) Population and,
as support, the same analyses were to be done on Intent-to-treat
population. Patients evaluatable for the Per-Protocol analysis were
all randomized patients compliant to protocol criteria who
completed the whole course of study without major protocol
violations and have valid assessments of prostate volume. Patients
valid for intent-to-treat (ITT) population were all randomized
patients who received at least one dose of trial medication and for
whom the prostate volume at baseline and at 12 weeks visit were
available.
[0118] All patients randomized who took at least one dose of study
drug were evaluated for safety analysis.
[0119] The treatment group comparability was assessed at baseline
for all patients with descriptive meaning. The data were processed
by the Chi-Square test for the categorical variables, and by the
ANOVA model for the continuous variables.
[0120] Descriptive statistics were calculated by means of usual
methods: mean, standard deviation, minimum and maximum values on
continuous variables, and absolute and relative frequencies for
categorical ones. Descriptive statistics were done per treatment
and per visit/weeks.
Centers with less than 4 patients were pooled.
[0121] The primary efficacy variable was the percentage change of
prostate volume, measured by centralized MRI axial scanning. An
ANOVA model was used for the analysis, with treatment and center as
fixed effects.
[0122] Study participants received 150 .mu.g capsule (as per
Example 4A with drug omitted in the case of placebo) once daily in
the morning. The treatment period was 12 weeks. The number of
patients involved was as follows:
TABLE-US-00005 Compound A Placebo Total Randomised 57 62 119
patients Complete patients 56 (98.3%) 60 (96.8%) 116 (97.5%)
Discontinued or 0 2 (3.2%) 2 (1.7%) lost to follow-up
Unsatisfactory 1 (1.8%) 0 1 (0.8%) therapeutic effect
Percentage Change in Prostate Volume Measured by Axial Scanning
[0123] The percentage change in prostate volume in the PP
population was -1.89.+-.5.2 in the Compound A group vs 4.99.+-.5.99
in placebo group with a significant p vale of <0.0001 in favour
of Compound A. The estimate of difference between treatments
(Compound A minus placebo) was -7.24 with 95% confidence limit of
-9.54 and -4.94. The centre effect was also significant (p=0.0176).
The same analysis performed on the ITT population confirmed the
results (p=0001), that is that Compound A was more effective than
placebo in reducing the prostate volume.
In patients with a baseline prostate volume >=80 ml the
difference between treatment groups (p=<0.0001) was clearer in
comparison with patients with a baseline prostate volume <60 ml
(p=0.0320) especially in the PP population.
[0124] In patients with age between 61-70 years the difference
between treatments, always in favour of Compound A, was more
evident than with respect to older patients (age>70 years). In
fact in the ITT population, the difference between treatments in
patients with age >70 had a significance of p=0.0540 vs
p=<0.0001 for the other classes of patients.
Responders
[0125] The proportion of responders observed with Compound A was
27.5%, in the PP population, with an unchanged proportion of 65%
and only a 7.5% of patients not responder. In placebo group the
class of responders was null, in fact patients were equitably
separated in unchanged and not responder (50% in each class). The
Chi-Square test comparing proportions confirmed the results
observed in the primary efficacy variable, p=<0.0001, that is
Compound A was more effective than placebo in reduction of prostate
volume.
[0126] In the ITT population the results were confirmed, the
proportion of responders in the Compound A group being 28.8%, with
a chi-square p-value of <0.0001 between treatments.
[0127] The mean reduction of prostate volume vs baseline in
responder patients was -6.88.+-.2.5, in the PP population, while
the mean difference in unchanged patients is -0.35.+-.2.3 in the
Compound A group vs 0.40.+-.2.0 in Placebo group. For non responder
patients the mean difference was 3.93.+-.0.8 in the Compound A
group vs 7.48.+-.4.9 in Placebo group that confirms the greater
efficacy of Compound A in controlling and reducing prostate
volume.
Percentage Change of Prostate Volume Measured by Centralized
Paraxial and Transitional Scanning
[0128] The supportive analyses on paraxial and transitional
acquisition confirmed the results obtained on axial scanning. In
particular, in the PP population, for paraxial acquisition the
percentage change was -1.30.+-.6.9 in the Compound A group vs
2.57.+-.6.8 in placebo group, p=0.0172; while for transitional
scanning the percentage change was -0.22.+-.9.6 for Compound A
group vs 6.18.+-.10.9 for Placebo group, p=0.0028. Also in the ITT
population there was a significant difference between treatments in
favour of Compound A, in reduction of prostate volume.
Serum Total PSA and Hormone Levels
[0129] The PSA mean change in the Compound A group was 0.23.+-.1.3
vs 0.43.+-.1.7 in placebo group, for PP population. No significant
difference was observed between treatments (p=0.2722). Also for
testosterone, in PP population, there was no significant difference
between groups (p=0.2150), with mean change 0.07.+-.1.5 in the
Compound A group vs 0.22.+-.1.4 in Placebo group.
[0130] There was no difference between treatment groups for
dihydrotestosterone (p=0.7257-PP population), with an observed mean
change in PP population of -30.77.+-.227.71 in the Compound A group
vs -166.76.+-.490.26 in the Placebo group.
[0131] There was no difference in LH hormone (p=0.9320-PP
population), with a mean change in Compound A of -0.02.+-.1.7 vs
-0.00.+-.1.8 in Placebo group for PP population.
[0132] The mean changes observed in PSA and in hormone levels was
around zero, except for DHT, Compound A does not modify the hormone
levels.
[0133] The same results were confirmed in the ITT population.
Safety
[0134] The number of patients with at least one adverse event was
31 (17 in the Compound A group, 14 in placebo group); no patient
dropped out due to adverse events, and only one patient in Placebo
group experienced a serious adverse event: an acute colecystitis,
solved with hospitalization.
[0135] The number of patients with adverse events related to
Compound A treatment was 3 (5.26%), while the number of patients
with adverse events related to treatment was 6 (9.68%) in the
Placebo group. The events related to Compound A were: dizziness,
headache, libido decrease and hot flushes, while the events related
to Placebo were: urine phosphate increase, headache, syncope,
libido decrease (3 patients), hypercalciuria, erectile dysfunction
and hot flushes.
The calciuria values monitored over the course of the study in the
Compound A group did not differ significantly from the Placebo
group.
CONCLUSION
[0136] In this short proof of concept study for effect of Compound
A on prostatic size in patients with BPH, the drug proved to be
efficacious. The analysis of the primary variable of the study,
namely the evaluation of the prostatic size, showed significant
difference between Compound A and placebo, thus confirming that the
tested drug is able to arrest the progression of the disease. The
safety profile was good, there was no different incidence of
adverse events between Compound A and placebo, and no severe
adverse event was reported in Compound A group. The tested drug was
devoid of any antiandrogenic effects, had no effect on PSA levels,
and had no significant effect on the calcium homeostasis.
Example 6
The Activity of Compound A on the Growth and Function of Bladder
Cells
[0137] Compound A has been shown to be effective in inhibiting the
basal and testosterone-stimulated growth of bladder cells. This
activity, never reported before, is dose-dependent with a
1.6.+-.7.times.10.sup.-15 for
1-alpha-fluoro-25-hydroxy-16,23e-diene-26,27-bishomo-20-epi-cholecalcifer-
ol ("Compound A"/"Cmpd A" in the figures) (on stimulated cells)
(see FIG. 10 and FIG. 11).
[0138] This effect was significantly greater than that of the
anti-androgen finasteride widely used in the treatment of
uro-genital diseases (FIG. 11).
Example 7
The Effect of Compound a on Bladder Dysfunction in a Bladder Outlet
Obstruction Model
Experimental
1. Materials
1.1. Animals:
[0139] Female Sprague-Dawley rats, weighing 200-250 g
1.2. Grouping
[0140] Group A: BOO rats, treated with Compound A over 2 weeks,
beginning at day 1 after creation of the obstruction (n=12) Group
B: BOO rats, treated with vehicle over 2 weeks, beginning at day 1
after creation of the obstruction (n=12) Group C: Sham operated
rats, treated with Compound A over 2 weeks, beginning at day 1
after surgery (n=12)
1.3. Studies:
[0141] a) Cystometry (.about.18 hours after last administration of
the drug/vehicle, 12 hours after removal of the obstructing
ligature) under conscious conditions. b) Measurements of bladder
weight c) In vitro investigations
2. Methods
2.1. Bladder Outlet Obstruction (BOO):
[0142] The bladder and urethrovesical junction were exposed through
a lower abdominal midline incision. A 0.9 mm metal rod was placed
alongside the proximal urethra and a 3-0 silk ligature was tied
tightly around the urethra and the rod, which was consequently be
removed. Sham surgery was be performed accordingly, without placing
the ligature. After 13 days the ligature was be removed and a
catheter was be inserted into the bladder dome and tunneled
subcutaneously.
2.2. Cystometry
[0143] The following morning after insertion of the catheter, the
cystometric investigation was performed without any anesthesia or
restraint in a metabolic cage. The amount of voided urine was
measured by means of a fluid collector, connected to a force
displacement transducer. The bladder was continuously filled with
saline at room temperature. The catheter was also connected to a
pressure transducer. After a stabilization period of 30-60 minutes,
when reproducible voiding patterns are achieved, the following
parameters were recorded over a period of 30 minutes: Basal bladder
pressure, micturition pressure, threshold pressure, micturition
interval and volume, and non-voiding contractions. The amount of
residual urine was investigated manually 3 times, at the end of the
cystometry. Bladder capacity was calculated based on the measured
values.
2.3. In Vitro Investigations
2.3.1. Preparations.
[0144] After completion of the cystometries, the rats were
sacrificed by carbon monoxide asphyxiation followed by
exsanguination. The abdomen was accessed through a lower midline
incision whereafter the symphysis was opened. The bladder was
carefully dissected free, and immediately placed in chilled Krebs
solution, and strip preparations were dissected.
2.3.2 Recording of Mechanical Activity.
[0145] The bladder and urethra were separated at the level of the
bladder neck, and semicircular strips were prepared from the middle
third of the detrusor (1.times.2.times.5 mm). All preparations were
used immediately after removal.
[0146] The strips were transferred to 5 ml tissue baths containing
Krebs solution. The Krebs solution was maintained at 37.degree. C.
and bubbled continuously with a mixture of 95% O.sub.2 and 5%
CO.sub.2, resulting in a pH of 7.4. The strips were suspended
between two L-shaped hooks by means of silk ligatures. One hook was
connected to a movable unit allowing adjustment of passive tension,
and the other to a Grass FT03C (Grass Instruments Co, MA, USA)
force transducer. Isometric tension was recorded using a Grass
polygraph (7D). After mounting, the strips were stretched to a
passive tension of 4 mN (the same tension for all preparations) and
allowed to equilibrate for 45-60 min before further experiments
were performed.
2.3.3. Electrical Field Stimulation
[0147] Electrical field stimulation (EFS) was accomplished by means
of two platinum electrodes placed on either side of the
preparations, and was performed using a Grass S48 or S88
stimulator, delivering single square wave pulses at selected
frequencies. The train duration was 5 s, the pulse duration 0.8 ms,
and the stimulation interval 2 min. The polarity of the electrodes
was shifted after each pulse by means of a polarity changing
unit.
2.3.4 Procedure
[0148] Each experiment was started by exposing the preparations to
a high K.sup.+ (124 mM) Krebs solution until two reproducible
contractions are obtained. Then the following experiments were
carried out:
a) Electrical stimulation of nerves was performed and
frequency-response relations obtained, in the presence and absence
of atropine. b) Concentration-response curves were constructed for
carbachol and ATP
Results
[0149] The validated bladder outlet obstruction rat model described
above was used to test the ability of Compound A to control and
treat bladder dysfunction. The objective was to evaluate whether a
vitamin D3 analogue
(1-alpha-fluoro-25-hydroxy-16,23e-diene-26,27-bishomo-20-epi-cholecalcife-
rol--compound "A") at the dose of 150 .mu.g/Kg/daily) can prevent
bladder hypertrophy and bladder dysfunction such as bladder
overactivity.
[0150] In this model a ligature was surgically placed around the
outlet of the catheterized bladder, so that when the catheter was
removed, the bladder experienced increased urethral resistance. The
rats underwent continuous cystometry to evaluate bladder function.
In addition the contractile properties of isolated bladder
preparation in response to nerve stimulation and exogenous stimuli
in vitro were investigated under electrical field stimulation
(EFS).
[0151] The following cystometric parameters were investigated (see
FIGS. 12-16):
[0152] micturition pressure (the maximum bladder pressure during
micturition),
[0153] bladder capacity (residual volume after voiding plus the
volume of saline infused to induce the void)
[0154] micturition volume (volume of the expelled urine)
[0155] residual urine (bladder capacity minus micturition volume)
and
[0156] frequency and amplitude of spontaneously occurring changes
intravesical pressure (non-voiding contractions).
[0157] In this model the analogue under evaluation had a beneficial
effect on bladder function. This effect was evident in the normal
bladder and is maintained in bladder outlet obstruction. In
particular significant differences versus vehicle were observed
in:
[0158] spontaneous non-voiding contraction frequency and amplitude
(FIGS. 13 and 14);
[0159] residual urine (absent with Compound A, FIG. 16);
[0160] micturition pressure (FIG. 15);
[0161] In addition a beneficial effect on bladder function has been
confirmed in the in vitro tests:
[0162] K response;
[0163] response to EFS (FIG. 17);
[0164] response to carbachol.
[0165] Finally a slight decrease in bladder weight was observed
with Compound A (FIG. 12).
[0166] These data demonstrate the use of Compound A (in the dose
range from 50 .mu.g to 300 .mu.g--equivalent to approximately 0.725
to 5 .mu.g/kg of body mass in humans) in the prevention and
treatment of bladder dysfunction, eg overactive bladder, such as
is, for example, demonstrated in patients with BPH.
Abbreviations
[0167] T testosterone DHT dihydrotestosterone GF growth factor BPH
benign prostatic hyperplasia PP per-protocol ITT
intent-to-treat
ANOVA Analysis of Variance
[0168] TRUS Transrectal ultrasound
BOO Bladder Outlet Obstruction
[0169] AR Androgen receptors
PSA Prostate Specific Antigen
[0170] Throughout the specification and the claims which follow,
unless the context requires otherwise, the word `comprise`, and
variations such as `comprises` and `comprising`, will be understood
to imply the inclusion of a stated integer or step or group of
integers but not to the exclusion of any other integer or step or
group of integers or steps.
[0171] The application of which this description and claims forms
part may be used as a basis for priority in respect of any
subsequent application. The claims of such subsequent application
may be directed to any feature or combination of features described
herein. They may take the form of product, composition, process, or
use claims and may include, by way of example and without
limitation, the claims that follow.
INCORPORATION BY REFERENCE
[0172] The entire contents of all patents, published patent
applications and other references cited herein are hereby expressly
incorporated herein in their entireties by reference.
EQUIVALENTS
[0173] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, numerous
equivalents to the specific procedures described herein. Such
equivalents were considered to be within the scope of this
invention and are covered by the following claims.
* * * * *