CH698144B1 - Analogue of vitamin D3 - Google Patents

Abstract

Use of 1-α-fluoro-25-hydroxy-16,23E-diene-26,27-bishomo-20-epi-cholecalciferol, or a pharmaceutically acceptable salt or ester thereof, in the manufacture a drug for the prevention and / or treatment of benign prostatic hyperplasia (BPH) and its associated symptoms.

Description

       

  The present invention relates to the use of 1- [alpha] -fluoro-25-hydroxy-16,23E-diene-26,27-bishomo-20-epi-cholecalciferol (compound A) for the manufacture of a a medicament for the prevention and / or treatment of benign prostatic hyperplasia (BPH) and its associated symptoms.

  

[0002] BPH is a common disorder in older men, occurring in approximately 50% of men aged 60 and 90% of men aged 85 years. BPH is a specific histopathological entity characterized by hyperplasia of stromal and epithelial cells.

  

[0003] For more than a century, the two known etiological factors of the pathogenesis of BPH have been aging and the presence of functional testes. However, as prostate science progresses, this concept becomes unsatisfactory as it does not cover all aspects of the pathogenesis of BPH. Additional etiological factors play a significant role in regulating prostate growth. In particular, it has become apparent that growth of the prostate is under the immediate control of specific growth factors produced by prostatic cells, acting locally on adjacent cells in a paracrine mechanism or on the same cells in an autocrine mechanism.

   Therefore, a large effort is currently being made to identify therapeutic strategies to inhibit intraprostatic growth factors.

  

[0004] BPH is a common cause of chronic lower urinary tract symptoms that may affect the filling (irritating symptoms) and discharge (obstructive symptoms) phases of the urination cycle. These symptoms affect the social, psychological, domestic, professional, physical and sexual life of patients and lead to a profound and negative impact on their quality of life. In addition to this, BPH may cause more acute urologic complications, particularly acute urinary retention (URA), often considered to be the most serious complication of BPH and less frequently recurrent urinary tract infections. dilation of the upper urinary tract, the formation of stones in the bladder and recurrent hematuria.

  

[0005] The management of BPH is associated with extremely high social costs estimated at $ 4 billion in 1993 and is expected to be $ 26 billion in 2003 in the United States alone.

  

[0006] The current medical treatment of BPH consists in orally administering 5-alpha-reductase inhibitors (finasteride and dutasteride, recently accepted by the FDA) and [alpha] 1 receptor antagonists (the terazosin, doxazosin, tamsulosin as well as silodosin, AIO-8507L, RBx-2258, etc.). Each of these therapeutic options is associated with advantages and disadvantages regarding their different mechanisms of action. While [alpha] 1 receptor antagonists are highly effective in reducing symptoms related to lower urinary tract symptoms (STUI), they are ineffective in reducing prostate volume and therefore in avoiding surgical intervention. BPH.

   Conversely, 5- [alpha] -reductase inhibitors such as finasteride and dutasteride, by decreasing the formation of dihydrotestosterone (DHT), reduce the size of the prostate and the need for surgery. In addition, recent results from the seven-year Prostate Cancer Prevention Trial (PCPT), which involved more than 18,000 healthy older men, have shown that finasteride can prevent or delay the onset of prostate cancer (see Thompson IM, et al., New England Journal of Medicine (2003) 349, pp. 215-224).

   However, as expected (see Kassabian VS, Lancet (2003) 361, p 60-62), finasteride was not free of anti-androgenic adverse effects on sexual function, such as efficacy. diminished sexual desire, sexual desire and gynecomastia, which significantly weakens its attractiveness as a cancer In addition, finasteride treatment has been associated with increased detection of high-grade prostate cancer, possibly because the weakly androgen-induced state of finasteride has selected malignant growth-stage malignant cells. most aggressive androgenic effect (see Scardino PT, New England Journal of Medicine (2003) 349, pp. 297-299).

  

Thus the needs have not been met for a new class of drugs for medical therapy of BPH, which should be able to prevent acute urinary retention, as well as the need for surgery related to it by decreasing androgen-induced growth of the prostate but without directly interfering with the androgen receptors (AR), and therefore without anti-androgenic adverse effects in the prostate and outside the prostate, for example, sexual side effects. Such drugs, by disrupting the signals of intraprostatic growth factors, could be useful not only for treating BPH but also for preventing prostate cancer, possibly without having to select malignant clones insensitive to AR.

  

As described herein, the inventors have determined that the non-hypercalcemic vitamin D3 analogue, well tolerated, 1- [alpha] -fluoro-25-hydroxy-16,23E-diene-26,27-bishomo -20-epi-cholecalciferol (compound A), is a prime example of such a drug, since it can act against BPH in an androgen receptor-independent manner by targeting multiple pathways that control the growth of B cells. BPH, including growth factor-induced prostate proliferation.

  

[0009] 1,25-Dihydroxyvitamin D3 [1,25 (OH) 2D3], the activated form of vitamin D3, is a secosteroid hormone that plays not only a central role in the metabolism of bone and calcium, but which is also involved in the regulation of the immune response and in the differentiation and apoptosis of many types of cells, including malignant cells.

  

[0010] However, a problem with the therapeutic use of calcitriol is its natural ability to induce hypercalcemia and hyperphosphatemia. Therefore, calcitriol analogues maintaining biological activity but free of hypercalcemic side effects have been developed.

  

US 5,939,408 and EP 808,833 disclose a number of 1,25 (OH) 2D3 analogs comprising the compound 1- [alpha] -fluoro-25-hydroxy-16,23E-diene- 26,27-bishomo-20-epi-cholecalciferol (compound A). US 5,939,408 and EP 808,833 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, diseases neo-plastics such as leukemia, breast cancer and sebaceous gland diseases such as acne and seborrheic dermatitis and osteoporosis.

  

It has now been found, surprisingly, in several studies carried out by the inventors that, unlike some other analogs of 1,25 (OH) 2D3, the analogous compound of 1,25 (OH) 2D3, composed of AT:

  <EMI ID = 2.1>

Compound A

  

Significantly reduces the growth of human BPH cells in vitro by inducing their apoptosis and reduces prostate growth in vivo, without effects on testosterone and dihydrotestosterone levels. In addition, this inhibition of prostate growth is achieved at non-hypercalcemic doses. Thus, compound A is an effective pharmacological agent for the treatment of benign prostatic hyperplasia.

  

As described by the examples mentioned herein, compound A reduces the size of the prostate. In addition, as observed with finasteride, compound A prevents the proliferative activity in vitro and in vivo of testosterone. Significantly however, and unlike finasteride, compound A does not inhibit the activity of type 1 or type 2 5- [alpha] reductase and can prevent not only the growth of BPH cells. induced by testosterone but also that induced by dihydrotestosterone. These anti-androgenic properties of compound A are independent of the interaction with AR, as shown by the fact that compound A can not bind to RA, or can act as an agonist or antagonist of RA. RA. In addition, compound A does not affect the secretion of sex hormones.

   Moreover, in our studies, compound A has no significant effect on PSA levels, it appears that there is no danger of treatment with compound A, thus masking this important indicator of possible cancer. of the prostate.

  

Another significant advantage of the compound A is that in vitro studies have revealed that this drug, unlike finasteride, is able to inhibit the basic growth and stimulated by testosterone bladder cells and one s' expects it to be useful in the prevention and / or treatment of bladder dysfunction in humans. In vivo studies in a validated model of bladder neck obstruction in bladder dysfunction rats also demonstrated the beneficial effect of compound A. This result is significant because bladder dysfunction is a common sequel. and boring of BPH.

   Thus compound A is able to reduce the size of the prostate and improve bladder dysfunction i.e. to improve bladder function and bladder-related BPH symptoms at the same time by a direct effect of compound A on both the prostate and the bladder. This effect can be expected to go beyond the improvement in bladder symptoms that would simply be expected as a result of the reduction in prostate size. Symptoms of the bladder include overactive bladder and indicators of improved bladder function include reduction of bladder contractions and post-voiding residue.

  

Thus, the present invention provides the use of 1- [alpha] -fluoro-25-hydroxy-16,23E-diene-26,27-bishomo-20-epi-cholecalciferol for the manufacture of a medicament for the prevention and / or treatment of benign prostatic hyperplasia. Also within the scope of the invention are esters and pharmaceutically acceptable salts of compound A.

  

The invention thus proposes the use of 1- [alpha] -fluoro-25-hydroxy-16,23E-diene-26,27-bishomo-20-epi-cholecalciferol, or a salt or a salt thereof. a pharmaceutically acceptable ester thereof, for the manufacture of a medicament for the prevention and / or treatment of benign prostatic hyperplasia.

  

[0018] 1- [alpha] -Fluoro-25-hydroxy-16,23-diene-26,27-bishomo-20-epi-cholecalciferol is a known compound and its preparation is described in US Pat. No. 5,939,408, whose description is incorporated herein by reference.

  

Esters include pharmaceutically acceptable, labile esters that can be hydrolysed in the body to release compound A.

  

The salts of compound A include adducts and complexes which may be formed with alkali and alkaline earth metal ions such as sodium, potassium and calcium ions and salts thereof such as calcium chloride, calcium malonate and the like.

  

Compound A, or a salt or an ester thereof, may be used alone or may be administered in combination with known HBP-active agents, for example, an agent [alpha] -adrenergic receptor blocking such as an [alpha] 1 receptor antagonist (eg, terazosin, doxazosin, or tamsulosin or silodosin, AIO-8507L or RBX-2258) or an inhibitor of 5- [alpha] reductase (eg finasteride or dutasteride). The term "BPH active agent" includes those agents capable or known to have activity for the treatment or prevention of BPH such as the above-mentioned exemplary substances.

   The combined agent in combination can be mixed with 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. The combination of A with two or more HBP-active compounds, for example, 3 may be considered for example, a combination with an [alpha] 1 receptor antagonist and a 5- [alpha] inhibitor. ] reductase.

   When administered in combination with the compound A (or a salt or an ester) the combined agent (s) may be used at doses lower than those used when the combined agent in combination is administered alone, perhaps even a dose that is below the therapeutic level when administered alone.

  

Thus the invention also provides the use as defined above wherein the drug is in separate pharmaceutical formulations or combined with a second active agent against BPH.

  

Thus, the invention also proposes the use of 1- [alpha] -fluoro-25-hydroxy-16,23E-diene-26,27-bishomo-20-epi-cholecalciferol, or a salt or a salt thereof. a pharmaceutically acceptable ester thereof, in combination with a second BPA active agent for the manufacture of a medicament for the prevention and / or treatment of benign prostatic hyperplasia .

  

The above-named combinations can conveniently be presented for use as a pharmaceutical formulation. The pharmaceutical formulations thus produced also represent one aspect of the invention.

  

The dosage levels and the response time of the administration of the active substances in the pharmaceutical formulations of the invention can be changed in order to obtain an amount of the active substance which is effective to obtain the desired therapeutic response for a particular patient, a composition, and a mode of administration, without being toxic to the patient. An exemplary dose range of compound A ranges from about 0.1 to about 300 [micrograms] per day, for example, about 50 to about 150 [micrograms] per day, for example, about 75 or about 150 micrograms per day. A unit dose formulation preferably contains from 50 to 150 [micro] g for example, 75 or 150 [micro] g and is preferably administered once a day.

  

Specifically, a preferred dose of compound A is the maximum dose that can tolerate a patient and to which he will not develop hypercalcemia or other undesirable side effects such as hypercalciuria. Compound A is preferably administered at a concentration of about 0.001 [micro] g to about 100 [micro] g per kilogram of body weight, about 0.001 to about 10 [micro] g / kg or about 0.001 [micro] g to about 100 [micro] g / kg body weight. The intermediate ranges below the values mentioned are also intended to be part of the invention.

  

As noted above, compound A may be administered in the form of a pharmaceutically acceptable salt or ester thereof, however, compound A is preferably used as it is; it is not used in the form of an ester or a salt thereof.

  

This assay can be provided in a conventional pharmaceutical formulation by single administration, by multiple applications, or by controlled release, as needed to obtain the most effective results, preferably once or twice daily (particularly a daily dose) for example, orally. In certain situations, periodic dosing may be appropriate to achieve the desired therapeutic response.

  

The choice of the exact dose and formulation and the most appropriate mode of delivery will be influenced by, inter alia, the pharmacological properties of the formulation, the nature and severity of the condition being treated, and the physical state and mental acuity of the recipient.

  

Representative delivery modes include the oral, parenteral (including subcutaneous, intramuscular and intravenous), rectal, oral (including sublingual), pulmonary, transdermal, and intranasal routes, most preferably by oral route. Administration may be continuous or intermittent (for example, by rapid injection).

  

The invention also provides a pharmaceutical composition comprising 1- [alpha] -fluoro-25-hydroxy-16,23E-diene-26,27-bishomo-20-epi-cholecalciferol, or a pharmaceutically acceptable salt or ester and a pharmaceutically acceptable carrier for the prevention and / or treatment of benign prostatic hyperplasia.

  

The invention also provides a packaged formulation which contains a pharmaceutical composition comprising 1- [alpha] -fluoro-25-hydroxy-16,23-diene-26,27-bishomo-20-epi-cholecalciferol, or a salt or a pharmaceutically acceptable ester thereof, and a pharmaceutically acceptable carrier packaged with a method of use for the treatment of benign prostatic hyperplasia.

  

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 oral 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.

  

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 the Remington article, Pharmaceutical Sciences, 17. <e> edition, 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, vegetable oils, naphthalenes hydrogenated and similar products.

   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 as nasal drops or metered dose inhaler. For oral administration typical excipients include sugars, calcium stearate, magnesium stearate, pregelatinized starch, and the like.

  

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 sulphate.

   The liquid compositions may contain conventional additives such as suspending agents, for example, sorbitol syrup, methylcellulose, sugar syrup, gelatin, carboxymethylcellulose, or edible fats; emulsifiers 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). The liquid compositions may be encapsulated in, for example, gelatin to provide a unit dosage form.

  

[0036] Preferred solid oral dosage forms include tablets, hard shell capsules and soft and elastic gelatin capsules (SEG). 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 to using SEG capsules are: a) the uniformity of the dose content is optimized in the SEG capsules because the drug is dissolved or dispersed in a liquid that can be metered into the capsules precisely; b) drugs formulated as SEG capsules show good bioavailability because the drug is dissolved, solubilized or dispersed in a water-miscible or oily liquid and so once released into the body the solutions dissolve or are emulsified to produce drug dispersions having a high active area; and c) the degradation of drugs that are sensitive to oxidation during long-term storage is avoided because the dry shell of the soft gelatin provides a barrier against the diffusion of oxygen.

  

The dry shell formulation typically comprises a gelatin concentration of about 40% to 60%, a plasticizer concentration (such as glycerin, sorbitol or propylene glycol) of about 20% to 30% and a water concentration of about 30 to 40%. Other materials such as preservatives, dyes, opacifiers and flavors may also be present. The liquid filling material comprises a solid drug which has been dissolved, solubilized or dispersed (with suspending agents such as beeswax, hydrogenated castor oil or polyethylene glycol 4000) or a liquid medicine in vehicles or combinations of carriers such as mineral oil, vegetable oils, triglycerides, glycols, polyols and surfactants.

  

In an exemplary formulation, the soft gelatin capsules are size 2 gelatin capsules, white, opaque and oval containing a liquid filler comprising the active substance, compound A, dissolved in Miglyol 812 (triglyceride of C8-C12 fatty acids from fractionated coconut oil) 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, for example 75 or 150 [micro] g of compound A. The soft gelatin capsules should be stored at a temperature of 2 to 8 [deg. ] C and in the shelter of light.

  

Formulations containing compound A, or a pharmaceutically acceptable salt or ester thereof, optionally in combination with a second active agent against BPH, may be prepared by mixing the ingredients.

  

The formulations are preferably prepared under nitrogen and in an orange light.

  

The present invention is now described by means of the following nonlimiting examples, with reference to the figures, in which:

  

FIG. 1 shows inhibition of proliferation of BPH cells by calcitriol and compound A ("Cmpd A"). Panel A shows that incubation for 48 h with increasing concentrations (10 <-> <18> to 10 <-7> M) calcitriol (circles) or compound A (squares) resulted in significant and dose-dependent growth inhibition of BPH cells (* P <0.01 relative to the control). The ALLFIT analysis indicates that both secosteroids share the same maximal inhibition (Imax = 43 + - 1%), but show a marked difference in activity (-logIC50 for compound A = 15.8 + - 0.3; logIC50 for calcitriol = 10.2 + -0.6, P <0.005). The results are expressed in% inhibition (mean + - SEM) relative to their relative controls in 3 different experiments carried out in triplicate.

   Panel B shows the effect of increasing concentrations (10 <-> <18> to 10 (-7) M) of compound A on proliferation of T-stimulated BPH cells (10 nM, squares), KGF (10 ng / ml, circles) or Des (1-3) IGF-I (10 ng / ml). ml, triangles). Compound A induces significant inhibition (* P <0.01 compared to T- or GF-treated cells) of BPH cell growth also in the presence of all stimuli tested with a similar Imax = 66.6 + - 7.3%. However, compound A was more effective in inhibiting the effect of T (-logIC50 = 16.4 + - 0.6), than the other two GFs, = 12.7 + - 0.6, and -logIC50 of KG F = 14.2 + - 0.6, P <0.0001). The results are expressed in% of the variation (mean + - SEM) with respect to the maximum stimulation in 3 different experiments performed in triplicate.

  

FIG. 2 shows the effect of compound A, cyproterone acetate and finasteride on the growth of androgen stimulated BPH cells. BPH cells were incubated for 48 h with compound A (1 nM) or antiandrogens (finasteride, F, 1 nM, cyproterone acetate, Cyp, 100 nM) in the presence of T (10 nM). nM, panel A) or DHT (10 nM, panel B). The results obtained for unstimulated HBP cells are also shown (panel A). The results are expressed in% of the variation (mean + - SEM) relative to their relative controls in three different experiments performed in four copies.

   Compound A and cyproterone acetate significantly blocked T- and DHT-induced growth, while finasteride was only effective against T. * P <0.01 with respect to the control; [Deg.] P <0.01 compared to androgen treated cells.

  

FIG. 3 shows that compound A lacks agonist or antagonistic properties on human AR. The AR-deficient PC3 cell line stably transfected with human AR was transferred to a 24-well plate with a density of 2 x 10 <4> cells / well. After 24 h, the cells were transfected with the AR adapted plasmid pLSPP and, 48 h later, the 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. The results (the average of three transfection experiments) are expressed as a percentage of bioluminescence by [micro] g of all the proteins.

   In order to evaluate the agonist activity, a 100% activity against luciferase was fixed in the presence of 100 nM DHT (panel A), whereas in order to examine the antagonist activity an activity 100% against luciferase was fixed with DHT at 3 nM (panel B).

  

FIG. 4 shows the inhibition of the growth of the ventral prostate in rats by compound A or finasteride. Panel A: Castrated rats, injected with T-enanthate (30 mg / kg / week), were orally treated for 5 days / week for two consecutive weeks with a vehicle or with increasing doses of Compound A (10, 30, 100 and 300 [micro] g / kg) or finasteride (F, 10 and 40 mg / kg). The weight of the ventral prostate is expressed in% of the variation (mean + - SEM) of the weight of the intact, vehicle-treated rats (^ P <0.05, * P <0.01 compared with control rats, [deg.] P <0.01 relative to the rats to which T was added).

   Panel B: intact adult rats were orally treated for one month (5 times / week for a total of 27 administrations) with vehicle (control) or increasing concentrations of compound A (10, 30, 100 and 300 [micro] g / kg) or finasteride (F, 10 and 40 mg / kg). The weight of the ventral prostate is expressed in% of the variation (average + SEM) of the weight of the control rats treated with the vehicle (* P <0.01 relative to the control rats).

  

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 intact (line 1) or orchidectomized (line 2) rats treated with a vehicle. Lanes 3 to 6 represent clusterin gene expression in orchidectomized rats supplemented with T-enanthate (30 mg / kg) for two weeks and treated orally with a vehicle (line 3) compound A (300 [micro] g / kg, line 4 and 100 [micro] g / kg, line 5) or finasteride (40 mg / kg, line 6). Each line was loaded with 10 [micro] g of total RNA. The corresponding expression of GAPDH and the ethidium bromide staining of the gel are shown below the stain.

   The task is representative of two separate experiments. Panel B: Northern analysis of clusterin mRNA expression in the ventral prostate of intact adult rats treated orally for 1 month (5 times / week, 27 administrations) with a vehicle (line 1), increasing concentrations of compound A (10, 30 [micro] g / kg, line 2 and 3) or finasteride (40 mg / kg, line 4). Each line was loaded with 10 μg of total RNA. The corresponding expression of GAPDH and the ethidium bromide staining of the gel are shown below the stain. The task is representative of two separate experiments.

  

FIG. Figure 6 shows the morphological effects of compound A on the ventral prostate of castrated rats and to which were added T. Panels A, B, C and E. Representative fields obtained from cross sections of whole prostate glands to which were applied immunostaining with a monoclonal antibody against rat clusterine and counterstaining with hematoxylin. In rats treated with castrated vehicles 4 days earlier, clusterin labeling was detectable in the cytoplasm of atrophic cuboidal epithelial cells (panel A, 10x). After two weeks of T supplements (panel B, 10x), almost all clusterin labeling has disappeared.

   Conversely, clusterin-positive cells were still present in T-treated rats and different doses of compound A (100 [micro] g / kg, panel C, 300 [micro] g / kg, panel E, black arrows). Panels D and F show sections consecutive to those used in panels C and E, to highlight DNA fragmentation as assessed by the TUNEL final labeling test by terminal deoxynucleotidyl transferase (TdT).

  

Two weeks of treatment (9 administrations) with compound A (100 [micro] g / kg, panel D; 300 [micro] g / kg, panel F) induced massive apoptosis of the majority of epithelial cells and stromal. It should be noted (black arrows), that all clusterin-positive cells have undergone apoptosis, while a substantial portion of clusterless cells also show nuclear fragmentation.

  

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 monoclonal antibody to rat clusterine and counterstaining with hematoxylin. In panel A (10x) the primary antibody was omitted. Panel B (10x) shows that in untreated adult rats, only a few scarce epithelial cells were labeled in some glands (black arrows).

   Conversely, in the prostate glands of rats treated with increasing concentrations of compound A, cubic epithelial cells characterizing atrophy were stained with clusterin as a function of dose (see black arrows, [micro] g / kg, panel D; [micro] g / kg, panel E, 100 [micro] g / kg, panel F, 10x). Similar results were obtained with finasteride (40 mg / kg, panel H, 10x). Panels C, G and I show periodic consecutive slices, to those shown in panels B, D and F, respectively, to highlight DNA fragmentation as assessed by TUNEL labeling assay by terminal deoxynucleotidyl transferase (TdT).

   It should be noted (black arrows), that all clusterin-positive cells have undergone apoptosis, while a substantial portion of clusterless cells also show nuclear fragmentation.

  

FIG. 8 shows the results of a chronic toxicity study in dogs. An apparent reduction in prostate weight is shown after 9 months of treatment with compound A versus placebo.

  

FIG. 9 shows the results of a chronic toxicity study in dogs. A reduction in the weight of the prostate after healing using a treatment with compound A compared to a placebo.

  

FIG. 10 shows the effect of compound A on testosterone stimulated cell growth of the bladder. "hB" = human bladder.

  

FIG. 11 shows the effect of compound A and other comparator compounds on stimulated and basic bladder cell growth. "T 10 nM" = testosterone; "F 1nM" = finasteride.

  

FIG. 12 shows the effect of a vitamin D compound on the weight of the bladder.

  

FIG. 13 shows the effect of a vitamin D compound on the frequency of spontaneous bladder contractions.

  

FIG. 14 shows the effect of a vitamin D compound on the amplitude of spontaneous bladder contractions.

  

FIG. Shows the effect of a vitamin D compound on the pressure of urination.

  

FIG. 16 shows the effect of a vitamin D compound on the post-void residue.

  

FIG. 17 shows the effect of a vitamin D compound on the contractile response of bladder bands to EFS (electrical stimulation).

Examples

Example 1 Effects of Compound A on In Vitro BPH Cells Materials and Methods Materials

  

The minimum essential medium (MEM), DMEM-F12 mixture 1: 1, Ham F12 medium, phosphate buffered saline (PBS), fraction V bovine serum albumin (BSA), glutamine , geneticin, collagenase type IV, vitamin D3, testosterone (T), dihydrotestosterone (DHT), cyproterone acetate, reduced form of [beta] -nicotinamide adenine dinucleotide 3-phosphate (NADPH) dithiothreitol (DTT), phenylmethylsulfonyl fluoride (PMSF) and a kit for measuring serum calcium were purchased from Sigma (St. Louis, MO). The protein measurement kit was obtained from Bio-Rad Laboratories, Inc. (Hercules, CA). Fetal bovine serum (FBS) was purchased from Unipath (Bedford, UK).

   Rat-specific anti-clusterin monoclonal antibody (mouse monoclonal IgG) was purchased from UPSTATE Biotechnology (Lake Placid, NY). The Apop Tag Kit for In Situ Terminal Marking (ISEL) was obtained from Oncor (MD, USA). CHO 1827 and CHO 1829 cells 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 kindly offered by Merck Sharp and Dohme Research Laboratories (Rahway, NJ) Bicalutamide was kindly donated by AstraZeneca (AstraZeneca, Milan, Italy) .The 1- [alpha] -fluoro-25-hydroxy-16,23E-diene-26,27-bishomo analogue -20-epi-cholecalciferol (compound A) was provided by Bioxell (Bioxell, Milan, Italy).

   The keratinocyte growth factor (KGF) was obtained from Pepro Tech EC (London, England) and the insulin-like growth factor type 1, human [Des (1-3) IGF-I] was purchased from GroPep Limitated (Adelaide, Australia). The POD in situ cell death detection kit for TUNEL terminal deoxynucleotidyl transferase (TdT) labeling was obtained from Roche Diagnostics Corporation (Indianapolis, IN). Plastic articles for cell cultures were purchased from Falcon (Oxnard, CA). Disposable filtration units for the preparation of growth media were purchased from PBI International (Milan, Italy).

   Lipofectamine 2000 and Opti-MEM I medium for luciferase transfection were obtained from Invitrogen, Life Technologies (San Giuliano Milanese, Milan, Italy). Thin layer chromatography silica plates were obtained from Merck (Darmstad, Germany). Testosterone enanthate (T-enanthate) was obtained from Geymonat (Anagni, Italy). The Total Testosterone Detection Kit Coat-A-Count <(R)> was purchased from Medical System (Genova Struppa, Italy). Rat luteinizing hormone (rLH) assay systems [ <125> I] were obtained from Amersham Pharmacia Biotech (Piscataway, NJ).

BPH cells

  

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 suspubian adenomectomy for BPH, after informed consent and approval by the local ethics committee. Patients received no pharmacological treatment in the 3 months prior to surgery.

CHO-1827 and CHO-1829 cell lines transfected with 5- [alpha] reductase

  

CHO-1827 and CHO-1829 cells, transfected with 5- [alpha] -reductase type 1 (5 [alpha] R-1) or type 2 (5 [alpha] R-2), respectively (see Steers, W. Urology (2001) 58, pp. 17-24), were maintained in Ham F12 medium supplemented with 5% FCS.

PC3 cell lines transfected by AR

  

PC3 human prostate adenocarcinoma cells, stably transfected with plasmid p5HbhAR-A containing the human androgen receptor (hAR) as previously described (see Bonaccorsi L, et al., Endocrinology (2000) 141). , pp. 3172-3182), were grown in 75 cm culture flasks <2> in Ham F12 medium containing 50 [micro] g / ml geneticin, 10% FCS, penicillin (100 U / ml) and streptomycin (100 mg / ml).

HBP fabric

  

Prostate tissues for a fixation test were obtained from patients who underwent a suspubian adenomectomy for BPH. No pharmacological treatment was performed in the 3 months prior to surgery. After the surgery, the tissues were immediately placed in liquid nitrogen and stored at -80 [deg.] C until treatment.

Rat tissues

  

Glands of the rat ventral prostate were quickly excised, weighed and quickly frozen in dry ice. Immunohistochemistry experiments were performed in contiguous cryostatic sections of 14 [micro] m thickness for direct comparison of tissue morphology, clusterin expression, and apoptosis localization by assay. TUNEL. For total RNA extraction and Western blot analysis, rat ventral prostates from 4 to 6 animals were pooled.

Analysis of the proliferation of BPH cells

  

For all cell proliferation assays, 4 x 10 <4> BPH cells were inoculated into 12-well plates in their growth medium, starved in red medium and serum-free containing 0.1% BSA for 24 h, and then treated with specific stimuli for 48 hours. h. The cells in a phenol-based, serum-free red medium containing 0.1% BSA were used as controls. Then the cells were trypsinized, and each experimental point was derived from a hemocytometer count, 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: 2576-2583).

   The experiments were carried out using increasing concentrations (10 <-> <18> to 10 <-> <7> M) Calcitriol or Compound A with or without a fixed concentration of T (10 nM), KG F or Des (1-3) IGF-I (10 ng / ml). Growth assays were also performed using a fixed concentration of androgens (10 nM) with or without compound A (1 nM, 10 nM) or anti-androgenic finasteride (F, 1 nM) and cyproterone (Cyp, 100 nM). Growth assays were also performed using a fixed concentration of T (10 nM) or GF (10 ng / mL) with or without compound A (10 nM). In the same experiment, each experimental point was repeated in three or four copies and the experiments were carried out 3 times.

   The results are expressed in% of the variation (mean + - SEM) with respect to the maximal stimulation induced by T or GF.

In situ Terminal Marking (ISEL)

  

The ISEL was carried out on BPH cells using Apoptop apoptosis in situ detection kit peroxidase according to the manufacturer's instructions. The 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 fields separated by slide in five different slides. The results are expressed as an average + - SEM of three separate experiments.

Inhibition test of 5- [alpha] reductase

  

The 5- [alpha] -reductase inhibition assay was performed using CHO 1827 cells, transfected with [alpha] R-1, or CHO 1829 cells, transfected with [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 of 10 <-9> to 10 <-5> M, using finasteride as a control inhibitor in each experiment.

Link test

  

[0069] Fixation assays on cytosol fractions of HBP fragments were carried out as previously described (see Crescioli C et al., Endocrinology (2003) 144, pp. 3046-3057), (final protein concentration: 1.8 mg / ml). Incubations of cytosolic fractions were performed with increasing concentrations (0.125, 0.25, 0.5, 1 nM) of [ <3> H] -R1881 (specific activity: 83.5 Ci / mmol) in the absence or presence ([ <3> H] -R1881: 1 nM) increasing concentrations of cold R1881 (10 <-> <10> to 10 <-6> M), of DHT (10 <-10> to 10 <-6> M), of T (10) <-10> to 10 <-6> M), of bicalutamide (10 <-10> to 10 <-4> M), and compound A (10 <-10> to 10 <-4> M). To prevent the attachment of R1881 to the progesterone receptor, 1 [micro] M triamcinolone acetonide was added to each tube.

   Separation of bound and unfixed ligand was performed as previously described (see Crescioli C et al., Endocrinology (2003) 144, pp. 3046-3057). The protein content was determined by the known Bradford method, using BSA as a standard.

Luciferase test

  

PC3 cells stably transfected with human AR were transferred to 24-well plates at a density of 2 x 10 <4> in a Ham F12 setting plus 10% FCS. After 24 hours, the cells were transfected with 750 ng / well of plasmid pLSPP containing the wild type sequence configuration of the MMTV-LTR attached to the firefly luciferase gene (see Pazzagli M. et al., Analytical Biochemistry (1992)). 204, pp. 315-323), using lipofectamine 2000 (1 mg / ml) according to the manufacturer's instructions. After 48 h, the cells were incubated with DHT (10 <-12> to 10 <-6> M) or bicalutamide (10 <-9> to 10 <-5> M), in the presence of 3 nM DHT, and with an equimolar concentration of compound A for 18 h. Steroids and compound analog A were dissolved in ethanol.

   Transfected cells incubated with ethanol only served only as positive controls.

  

The assay of luciferase was performed using 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 [micro] l of a lysis buffer solution. Luciferase activity was measured on [micro] l of cell lysate for 10 s after the addition of 100 [micro] 1 of luciferin. Measurement of the total proteins was carried out on 20 [micro] l of cell lysate. At least three independent analyzes have been made in two copies.

Results

  

Incubation of BPH cells with increasing concentrations of calcitriol or Compound A inhibited cell growth (Panel A, Panel 1). Both compounds inhibit cell proliferation as a function of dose. ALLFIT analysis (see De Lean A, et al., American Journal of Physiology (1978) 235, pp. E97-E102) indicated that although maximal inhibition of calcitriol and compound A ("Cmpd A" on the figures) was not statistically significantly different (Imax = 43 + - 1%), their relative activity was, compound A being several log units more efficient than calcitriol, (-logIC50 of compound A = 15.8 + - 0.3 compared to -logIC50 of calcitriol = 10.2 + - 0.6, P <0.005).

  

The proliferation of BPH cells was substantially 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 hours with T or GF (Figure 1, panel B), the inhibitory effect of compound A was even more pronounced (Imax = 66.6 + -7.3%). Mathematical modeling (see De Lean A, et al., American Journal of Physiology (1978) 235, pp. E97-E102) of inhibition curves indicated that Compound A was more potent in T-stimulated BPH cells. (-logIC50s = 16.4 + - 0.6) than with the two other growth factors (-logIC50s = 12.7 + - 0.6, and -logIC50 = 14.2 + - 0.6 for Des ( 1-3) IGF-I and KGF, respectively; <0.0001).

  

Compound A (1 nM) antagonized proliferation of HBP cells stimulated not only by T but also by DHT to a similar extent to AR antagonist cyproterone acetate (Cyp, 100 nM; 2panel A and B). Reciprocally, finasteride inhibitor of 5- [alpha] reductase (F, 1 nM) antagonized only T-induced cell growth (Figure 2, panel A). In addition, compound A reduced growth even in cells not stimulated by androgen (Fig 2, panel A).

  

To evaluate the potential anti-androgenic properties of compound A, in addition to inhibiting the growth of BPH cells, we studied its interaction with AR. First, we eliminated the possibility that compound A binds to RA by performing competitive studies in human homogenates of BPH, using synthetic androgen [ <3> H] -R1881 as a labeled ligand. LIGAND analysis (see Munson PJ et al., Analytical Biochemistry (1980) 107, pp. 220-239,) indicated that unlabeled R1881, DHT, T, and AR antagonist bicalutamide completely displaced fixation. from [ <3> H] -R1881 (Table I). Conversely, Compound A did not compete with the fixation of [ <3> H] -R1881 at any concentration examined (Table I).

   These results were confirmed and prolonged using a luciferase reporter gene assay. In PC3 cells expressing full-length AR coupled with a luciferase reporter gene, DHT stimulated a dose-dependent increase in luciferase activity (EC50 = 2 + - 1.3 nM, panel A), whereas that bicalutamide inhibited DHT-stimulated activity (IC50 = 194 + -80 nM, panel B). In this system, the increasing concentration of compound A neither stimulated nor inhibited the increase in AR-induced luciferase activity (Figure 3). Finally, in order to verify whether compound A interacts with the formation of DHT, the active metabolite of T, we performed experiments in CHO cells transfected with 5- [alpha] -reductase type 1 and type 2 .

   The results were compared with those obtained with finasteride (F). While F inhibited the conversion of T to DHT with the expected IC50s, (IC50 for 5- [alpha] -reductase type 1 = 659 + - 100nM and IC50 for 5- [alpha] -reductase type 2 = 53.7 + - 11nM, n = 3). Compound A did not interfere with either isoenzyme up to the micromolar range (data not shown).
 <tb> AR Ligand <sep> Constants of affinity
(Kd nmol / L)


   <Tb> R1881 <sep> 0.16 + - 0.06


   <Tb> DHT <sep> 0.07 + - 0.03


   <Tb> T <sep> 1.89 + - 0.94


   <Tb> Bicalutamide <sep> 159 + - 82


   <tb> Compound A <sep >> 100,000

Table I androgenic agonist affinity constants (R1881, DHT, T), antagonist (bicalutamide) and compound A in human homogenates of BPH as detected by [3H] R1881 binding

  

The effect of compound A in BPH cells was, at least in part, due to activation of programmed cell death as detected by the ISEL test (n = 3, Table II). The percentage of apoptotic nuclei increased significantly (270%) after 48 h exposure to 10 nM compound A (P <0.01 compared to control). Conversely, treatment with T (10 nM), or GFs (10 ng / ml) significantly reduced (P <0.01) the number of apoptotic cells of BPH compared with 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 (over 250%) and significant (P <0.01) of the number of ISEL positive HBP cells.

Apoptotic index (%)

  

[0077]
 <Tb> <September> Control <sep> Compound A


   <Tb> control <sep> 18.55 + - 0.8 <sep> 68.44 + - 1.26 <a>


   <tb> Des (1-3) IGF-I <sep> 10.69 + - 0.6 <a> <sep> 45.85 + - 0.66 <a,> <b, c>


   <Tb> KGF <sep> 8.5 + - 0.42 <a> <sep> 44.46 + - 0.57 <a,> <b, c>


   <Tb> T <sep> 13.56 + - 0.72 <a> <sep> 49.06 + - 1.87 <> <a> <,> <b, c>

Table II: Effect of compound A (10 nM), GFs (10 ng / ml) or T (10 nM) on DNA fragmentation in BPH cells.

  

The apoptotic index (%) represents the number of colored nuclei, as detected by the ISEL test, on all the BPH cells in each of at least 5 fields separated by slide. The results are expressed as mean + - SEM in three separate experiments. Compound A can induce apoptosis in untreated BPH cells as well as in BPH cells incubated simultaneously with GFs or T (a: P <0.01 compared to control; b: P <0.01 relative to cells treated with compound A; c: P <0.01 compared to cells treated with GF or T).

Example 2: Antiproliferative Properties of Compound A in In Vivo Prostate Growth Models Protocols Involving Animals

  

[0079] Male Sprague Dawley rats (28 days old) were purchased from Charles River Laboratories (Calco, Lecco, Italy). All the described animal experiments have been carried out in accordance with accepted standards for animal health. Castration was performed scrotally under anesthesia with a ketamine / xylazine mixture. Three days after castration, the rats (5-8 animals per group) were either treated with T-enanthate (30 mg / kg) or not with two separate subcutaneous injections per week. The rats were orally treated for 5 days the first week, and 4 days the second week with a vehicle (miglyol 812), compound A (10, 30, 100 and 300 [micro] g / kg) or finasteride (10 and 40 mg / kg) for a total of 9 administrations, and sacrificed a day later.

  

Alternatively, intact adult male rats, Sprague Dawley (weight 250 g) were treated orally with a vehicle (miglyol 812), compound A (10, 30, 100 and 300 [micro] g / kg) or finasteride (10 and 40 mg / kg) 5 days / week for 5 consecutive weeks and for two additional days on 6 <e> week, for a total of 27 administrations, unless otherwise indicated. Blood for measurements of calcium and hormone content was obtained at the end of each experimental protocol.

Northern analysis of hybridization

  

The total RNA was extracted using RNAFast obtained from Molecular System (San Diego, CA). Fingerprinting, labeling, hybridization and probes (full-length cDNA clusters of 1.5 Kb rat and full-length cDNA of 1.2 Kb GAPDH) were performed according to published procedures (Bettuzzi et al. Biochemical Journal, (1989), 257, pp. 293-296 and Marinelli et al., Biochemistry and Cell Biology (1994), 72, pp. 515-521). Quantification of autoradiograms was obtained by densitometric scanning using an LKB Ultrascan XL densitometer.

immunohistochemistry

  

All the cryostatic sections obtained from the controls and the treated rats were transformed in parallel as described above (Astancolle et al., Journal of Endocrinology, (2000), 167, p 197-204). For each experimental condition, 3 alternating sections of 3 different rat prostates were examined. Negative controls, performed excluding the specific antibody of the reaction, showed no specific staining. Counterstaining was performed with hematoxylin, and coverslips were mounted with Eukitt (O. Kindler GmbH and Co. Germany). High-power color digital images were acquired by a CCD camera through the microscope.

In situ DNA fragmentation analysis (TUNEL)

  

The fragmentation of DNA in the cryostatic sections of the prostate, evaluated by the TUNEL labeling assay by the terminal deoxynucleotidyl transferase (TdT), was performed using the detection kit for cell death in situ (POD, Roche) as recommended by the manufacturer. TUNEL positive apoptotic nuclei have been documented by high magnification color digital images acquired by a CCD camera through the microscope. Counterstaining was done with eosin, and coverslips were mounted with Eukitt (0 Kindler Gmbh and Co., Germany).

Calcium measurements

  

Serum calcium levels were measured using a commercially available colorimetric assay (Sigma) according to manufacturers' instructions.

Measurement of testosterone and rLH

  

Serum levels of T and rLH hormones were determined using commercially available radioimmunoassay kits, according to manufacturers' instructions. To measure the serum T level in rats, the 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 collected and evaporated to dryness under a stream of nitrogen. The dry extract was reconstituted in the buffer solution of the assay as follows: (1 vol: 1 vol) in intact rats, and (4 vol: 1 vol) in castrated rats.

  

[0086] Statistical Analysis

  

The statistical analysis was performed by one-way ANOVA and by paired or unpaired Student t-tests, if appropriate. The binding data was analyzed using the LIGAND computer program (Munson et al., Analytical Biochemistry (1980), 107, pp. 220-139).

  

The ALLFIT computer program (De Lean et al., American Journal of Physiology (1978), 235, pp. E97-E102), was used for the analysis of sigmoidal dose-response curves to obtain estimates. values corresponding to half of the maximum of the inhibition (IC50) and values corresponding to half of the maximum of the stimulation (EC50) as well as the effects of the maximal inhibition (Imax) and of the maximum stimulation (Emax) . The data were expressed in (mean + - SEM).

Results

  

In order to examine the anti-proliferative properties of compound A on models of prostate growth in vivo, castrated and intact rats were orally treated with increasing concentrations of compound A (10 to 300 μg / kg). ) or finasteride (F) (10, 40 mg / kg). As shown in fig. 4, panel A, castration significantly reduced the weight of the ventral prostate, while a two-week treatment with testosterone enanthate (T) (30 mg / kg) not only completely restored, but also stimulated its growth. Compound A, at any dose tested, completely blunted the T-stimulated prostate overgrowth, reducing the weight of the ventral prostate to a lower value than untreated rats. Similar results were obtained with finasteride (10, 40 mg / kg).

   A one-month treatment on intact adult rats with Compound A significantly decreased the weight of the ventral prostate, with a maximal reduction (30%) at the highest dose tested (300 [micro] 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 of finasteride (Figure 4, panel B). In all experimental protocols, oral administration of different doses of compound A caused very modest hypercalcemia only at the highest dose tested (300 [micro] g / kg) (Table III). No other perceptible side effects were observed.
 <Tb> <September> Serum Calcium


   <Tb> Witness <sep> 10.2 + - 0.16


   <tb> Compound A 10 [micro] g / kg <sep> 10.16 + - 0.24


   <tb> Compound A 30 [micro] g / kg <sep> 9.87 + - 0.15


   <tb> Compound A 100 [micro] g / kg <sep> 10.55 + - 0.18


   <tb> Compound A 300 [micro] g / kg <sep> 10.85 + - 0.1

Table III Calcium (mg / dl) in castrated rats in which T was replaced after different doses (10, 30, 100, 300 [micro] g / kg) of compound A. Compound A never varied Serum calcium levels in castrated rats in which T was replaced by T-enanthate (30 mg / kg / week) compared to controls. Similar results were obtained in intact rats (not shown). The results represent the mean + SEM of the rats / group.

  

In order to better understand the molecular mechanisms responsible for the reduction of the weight of the prostate induced by the compound A, the expression of the clusterin and protein gene and the morphological characteristics of the apoptosis were evaluated by the TUNEL test for labeling with terminal deoxynucleotidyl transferase (TdT). Clusterin is an ubiquitous gene strictly related to cell cycle arrest and atrophy, the expression of which is reduced by androgens. Fig. 5, panel A, shows the prostatic expression of clusterin mRNA, as detected by Northern analysis, in orchidectomized rats to which T was added or not. Castration significantly increased clusterin mRNA abundance, whereas this effect was completely reversed by a two-week T administration.

   Simultaneous treatment with different concentrations of compound A (300 and 100 [micro] g / kg) or F (40 mg / kg) partially blunted the decrease in T-induced clusterin gene expression in rats. intact (Fig. 5, panel B), one-month administration of different concentrations of compound A (30 and 100 [micro] g / kg) induced a sustained increase in clusterin gene expression in the prostate comparable to, or even superior to, that induced by 40 mg / kg F.

  

[0091] The local expression of clusterin in the prostate of orchidectomized rats is shown in FIG. 6. Castration induced marked and widespread atrophy in the prostate gland, and almost all cuboidal epithelial cells adjoining the lumen of the gland were clusterin positive (panel A). Replacement of T (panel B) reversed the morphological features of atrophy and uniformly reduced clusterin staining. Such an effect was avoided by the simultaneous administration of compound A (panels C and E). Panels D, and F show TUNEL test results in sections adjacent to those shown in panels C and E.

   Treatment with compound A (100 [micro] g / kg, panel D, and 300 [micro] g / kg, panel F) induced obvious nuclear fragmentation in epithelial and stromal cells, and apoptosis was detectable at both in positive cells and in clusterin negative cells. The first two panels of fig. Figure 7 shows the morphology of the prostate gland of intact rats treated for clusterin detection, with (panel A) or without (panel B) omission of the primary antibody. It should be noted that clusterin labeling is almost absent in the prostate of untreated adult rats (panel B), since it is a nuclear fragmentation (TUNEL, panel C). Conversely, treatment with different doses of compound A induced the expression of clusterin (panels D to F) and apoptosis (C, G and I).

   Panel F compares the effect of finasteride (40 mg / kg) on clusterin positivity in the prostate gland.

  

In order to eliminate the possibility that compound A reduces prostate growth in vivo by interfering with pituitary or testicular function, serum levels of rat luteinizing hormone (rLH) and T were measured in castrated and intact rats. As expected (Table IV, panel A), castration significantly reduced T while increasing serum levels of rLH. Administration of T-enanthate (30 mg / kg) (two weeks) completely reversed the effect of orchiectomy. Oral treatment with compound A (100 and 300 [micro] g / kg) of castrated rats in which T was replaced did not significantly affect serum levels of rLH or T. Similar results were obtained in intact rats (Table IV, panel B).

   In fact, chronic administration (1 month) of compound A (10, 30, 100 [micro] g / kg) or F (40 mg / kg) to intact rats did not alter serum levels of rLH and T.

Panel A

  

[0093]
 <Tb> <September> rLH <September> T


   <tb> control (intact rats) <sep> 2.36 + - 0.46 <sep> 11.5 + - 2.44


   <Tb> neutered <sep> 20.64 + - 6 * <sep> 0.9 + - 0.32 *


   <tb> castrated + T replaced <sep> 2.08 + - 0.36 <sep> 21.25 + - 4.12


   <tb> castrated + T replaced + compound A 100 [micro] g / kg <sep> 1.8 + - 0.2 <sep> 11.13 + - 1.02


   <tb> castrated + T replaced + compound A 300 [micro] g / kg <sep> 3.15 + - 0.65 <sep> 15.73 + - 2.75

Panel B

  

[0094]
 <Tb> <September> rLH <September> T


   <tb> control (intact rats) <sep> 2 + - 0.16 <sep> 11.98 + - 2.87


   <Tb> Finasteride <sep> 2.2 + - 0.4 <sep> 18.11 + - 3.23


   <tb> Compound A 10 [micro] g / kg <sep> 2.22 + - 0.25 <sep> 19.13 + - 3.83


   <tb> Compound A 30 [micro] g / kg <sep> 2.32 + - 0.36 <sep> 9.39 + - 2


   <tb> Compound A 100 [micro] g / kg <sep> 1.96 + - 0.13 <sep> 11 + - 2.14

Table IV Serum levels of rLH (ng / ml) and T (nM) in castrated rats in which T (panel A) or intact (panel B) were replaced after treatment with different doses of compound A.

Panel A.

  

[0095] Castration significantly reduced the serum T (* P <0.01 compared to control) while it increased the serum level of rLH (* P <0.05 compared to control). After treatment with T-enanthate (30 mg / kg / week) serum levels of rLH and T were restored. Compound A at all doses tested did not significantly affect serum levels of rLH or T.

Panel B.

  

The chronic administration (1 month) of F (40 mg / kg) or of compound A (10, 30 and 100 [micro] g / kg) did not vary the serum level of rLH nor the rate serum T in intact rats.

  

This study demonstrates that Compound A reduces the size of the prostate in intact rats to a degree similar to finasteride. In addition, like finasteride, compound A suppresses the proliferative activity in vitro and in vivo of testosterone. However, unlike finasteride, compound A does not inhibit the activity of type 1 or type 2 5- [alpha] reductase and can inhibit the growth of BPH cells induced not only by T but also by DHT. These anti-androgenic properties of compound A are independent of the interaction with AR, as shown by the fact that compound A can not bind to RA, and can not act as an AR agonist or antagonist in PC3 cells transfected with AR.

   In addition, compound A did not affect the secretion of sex hormones because, in rats, plasma levels of gonadotropin and T were unchanged by daily administration of compound A for up to one month. Therefore, compound A acts downstream of the interaction between ligand and AR receptor. Without wishing to be bound by the theory this action most likely occurs through the interruption of the relay between testosterone and growth factor.

  

Very low concentrations of Compound A were able to completely prevent not only the proliferation of T-stimulated BPH cells, but also the proliferation induced by the two most important intraprostatic growth factors: IGF-I and KGF. In addition, even in the presence of T or GFs, compound A induced apoptosis in BPH cells. Compound A-induced cell death program also appeared in the prostate both intact and orchidectomized rats in which T was replaced and was characterized by a diffuse appearance of DNA fragmentation with increased concomitant expression of clusterin gene and protein.

   Clusterin is a protein closely regulated in the prostate by androgens (Bettuzzi et al., Biochemical Journal (1989), 257, pp. 293-296). Although clusterin function is still not well understood, it is markedly increased under conditions of atrophy of the gland (Bettuzzi et al., Oncogene (2002), 21, 4328-4334, and Bettuzzi et al. Journal of Endocrinology (1992), 132, pp. 361-367), and apoptosis (Leskov et al., Journal of Biological Chemistry (2003), 278, pp. 11590-11600). Thus, the induction of clusterin by treatment with compound A is consistent with the ability of this compound to inhibit proliferation and induce apoptosis in prostate cells.

  

[0099] 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.

  

A nine-month toxicity study was conducted in four groups of Beagle male dogs, which were treated daily with oral gavage with 0.5 [micro] g, 1.5 [micro] g and 5 [micro] g / kg of body weight / day of compound A (in the vehicle Miglyol 812) or with the vehicle alone. This treatment was followed by a two-month cure period for the highest dose group, [micro] g, after which the prostate weight was measured. In addition to the entirely favorable toxicity data, a lower prostate weight was observed at the end of treatment with compound A (see Fig. 8) and after healing (see Fig. 9).

   The results after healing were analyzed statistically using a one-tailed Student t test and found to be substantially 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: Soft gelatin capsule as an oral dosage form

  

A capsule intended for oral administration is formulated under nitrogen and in an orange light from 0.01 to 25.0 mg of compound A in 150 mg of fractionated coconut oil (for example, Miglyol 812). ), with 0.015 mg of butylated hydroxytoluene (BHT) and 0.015 mg of butylated hydroxyanisole (BHA), filled into a soft gelatin capsule.

  

The capsule is prepared using the following method:
1. BHT and BHA are suspended in fractionated coconut oil (e.g. Miglyol 812) and heated to about 50 ° C with stirring until dissolved.
2. Compound A is dissolved in the solution of Step 1 at 50 [deg.] C.
3. The solution in Step 2 is cooled to room temperature.
4. The solution from Step 3 is poured into soft gelatin capsules.

  

All manufacturing steps are performed under a nitrogen atmosphere and protected from natural light.

Example 4A: Soft gelatin capsule as an oral dosage form

  

A capsule intended for oral administration is formulated under nitrogen and in an orange light: 150 [micro] g of compound A in 150 mg of fractionated coconut oil (Miglyol 812), with 0.015 mg of hydroxytoluene Butyl (BHT) and 0.015 mg of butylated hydroxyanisole (BHA), filled into a soft gelatin capsule.

Example 4B: Soft gelatin capsule as an oral dosage form

  

A capsule intended for oral administration is formulated under nitrogen and in an orange light: 75 [micro] g of compound A in 150 mg of fractionated coconut oil (Miglyol 812), with 0.015 mg of hydroxytoluene Butyl (BHT) and 0.015 mg of butylated hydroxyanisole (BHA), filled into a soft gelatin capsule.

Example 5: Reduction of Prostate Weight in Human Clinical Trials

  

A randomized, 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-cholecalciferol) in patients with BPH.

  

[0107] The primary inclusion criterion was that male patients were diagnosed with BPH and had a prostate volume> 40 ml as determined by transrectal ultrasonography (USTR).

  

[0108] Statistical methods: the primary efficacy analyzes were performed on a population treated according to the protocol (Per-Protocol: PP) and, as a support, the same analyzes had to be made on the population to be treated. Patients who could be assessed by the protocol-based analysis were all randomized patients who adhered to the protocol criteria who followed the entire study without major protocol violations and who presented valid assessments of prostate volume. Valid patients for the population to be treated (TTIs) were all randomized patients who received at least one dose of the test drug and for whom the volume of the prostate at baseline and after 12 weeks was available.

  

[0109] All randomized patients who took at least one dose of the study drug were evaluated for safety analysis.

  

The comparability of the treatment group was evaluated at baseline for all patients with descriptive significance. The data were processed by the chi-square test for the nominal variables, and by the ANOVA model for the continuous variables.

  

The descriptive statistics were calculated by means of usual methods: the mean, the standard deviation, the minimum and maximum values on continuous variables, and the absolute and relative frequencies for the nominal variables. Descriptive statistics were made for each treatment and for each visit / week.

  

Centers having less than 4 patients were pooled.

  

The primary efficacy variable was the percentage change in prostate volume as measured by the centralized MRI axial scan. An ANOVA model was used for analysis, with treatment and center as fixed effects.

  

The study participants received a capsule of 150 [micro] g (according to Example 4A with the drug omitted in the case of placebo) once daily in the morning. The duration of treatment was 12 weeks. The number of patients involved was as follows:
 <Tb> <sep> Compound A <September> Placebo <September> Total


   <tb> Randomized patients <September> 57 <September> 62 <September> 119


   <tb> Patients who have completed the treatment <September> 56
(98.3%) <September> 60
(96.8%) <September> 116
(97.5%)


   <tb> Patients who interrupted or did not follow treatment <September> 0 <September> 2
(3.2%) <September> 2
(1.7%)


   <tb> Insufficient therapeutic effect <September> 1
(1.8%) <September> 0 <September> 1
(0.8%)

Percentage change in prostate volume measured by axial scanning

  

The percentage change in prostate volume in the PP population was -1.89 + - 5.2 in the group of compound A versus 4.99 + - 5.99 in the placebo group with a significant p value <0.0001 in favor of compound A. The evaluation of the difference between treatments (compound A minus placebo) was -7.24 with a 95% confidence limit of -9.54 and -4, 94. The effect of the center was also significant (p = 0.0176). The same analysis performed on the ITT population confirmed the results (p = 0001), that is, compound A was more effective than placebo in reducing prostate volume.

  

In patients with a basal volume of the prostate> = 80 ml the difference between the treatment groups (p = <0.0001) was clearer compared to patients with a basal volume of the prostate <60 ml (p = 0.0320) particularly in the PP population.

  

In patients aged between 61 and 70 years the difference between the treatments, still in favor of the compound A, was more obvious than with the treatments of the older patients (age> 70 years). In fact, in the ITT population, the difference between treatments in patients> 70 years old had a significance of p = 0.0540 compared to p = <0, 0001 for other classes of patients.

answering Machines

  

The proportion of responders observed with compound A was 27.5%, in the PP population, with an unchanged proportion of 65% and only 7.5% of patients were not responders. In the placebo group the responder class was nil, in fact, the patients were equitably divided into a class of patients with no change and a class of responders (50% in each class). The chi-square test comparing proportions confirmed the observed results for the primary efficacy variable, p = <0.0001, that is, compound A was more effective than placebo in reducing prostate volume.

  

In the ITT population, the results were confirmed, the proportion of responders in the group of compound A being 28.8%, with a value of p determined by the chi-square test. <0.0001 between treatments.

  

The mean reduction in prostate volume relative to baseline volume in responder patients was -6.88 ± 2.5 in the PP population, while the mean difference in patients with no change is -0.35 + - 2.3 in the group of compound A versus 0.40 + - 2.0 in the placebo group. For non-responder patients the mean difference was 3.93 + - 0.8 in the group of compound A versus 7.48 + - 4.9 in the placebo group, confirming the better efficacy of compound A to control and reduce the volume of the prostate.

Percentage change in prostate volume measured by paraxial scan and centralized transient

  

The support analyzes with a paraxial and transient acquisition confirmed the results obtained with an axial scan. In particular, in the PP population, for paraxial acquisition the percentage change was -1.30 + - 6.9 in the group of compound A versus 2.57 + - 6.8 in the placebo group p = 0.0172; while for the transient scan the percentage change was -0.22 + - 9.6 for the group of compound A versus 6.18 + - 10.9 for the placebo group, p = 0.0028. Also, in the ITT population there was a significant difference between treatments in favor of compound A, regarding the reduction of prostate volume.

Total serum PSA and hormone levels

  

The mean PSA change in the compound A group was 0.23 + -1.3 compared to 0.43 + - 1.7 in the placebo group, for the PP population. No significant difference was observed between treatments (p = 0.2722).

  

Also, for testosterone, in the PP population, there was no significant difference between the groups (p = 0.2150), with an average change of 0.07 + - 1.5 in the group of compound A versus 0.22 + - 1.4 in the placebo group.

  

There was no difference between the treatment groups for dihydrotestosterone (p = 0.7257 - PP population), with a mean change observed in the PP population of -30.77 + - 227.71 in the group of compound A versus -166.76 + - 490.26 in the placebo group.

  

There was no difference in LH hormone (p = 0.9320 - PP population), with an average change in compound A of -0.02 + -1.7 compared to -0.00 + - 1.8 in the placebo group for the PP population.

  

The observed mean changes in PSA and hormone levels were around zero, except for DHT, compound A did not alter hormone levels.

  

The same results were confirmed in the ITT population.

safety

  

The number of patients with at least one adverse event was 31 (17 in the group of compound A, 14 in the placebo group); no patient withdrew because of adverse events, and only one patient in the placebo group experienced a serious adverse event: acute cholecystitis, resolved by hospitalization.

  

The number of patients with adverse events related to treatment with compound A was 3 (5.26%), while the number of patients with treatment-related adverse events was 6 (9.68%). in the placebo group. The events related to compound A were: dizziness, headache, decreased libido and hot flashes, while placebo-related events were: increased phosphate in the urine, headache, syncope, decreased libido (3 patients), hypercalciuria, erectile dysfunction and hot flushes.

  

Calciuria values monitored over the entire duration of the study in the compound A group did not differ significantly from the placebo group.

Conclusion

  

In this short study constituting proof of the concept of the effect of compound A on the size of the prostate in patients with BPH, the drug proved effective. Analysis of the primary variable in the study, ie prostate size assessment, showed a significant difference between compound A and placebo, thereby confirming that the test drug can stop progression of the prostate. disease. The safety profile was good, there was no different incidence of adverse events between compound A and placebo, and no serious adverse events were reported in the compound A group.

   The drug tested was free of any anti-androgenic effects, had no effect on PSA levels, and had no significant effect on calcium homeostasis.

Example 6 The Activity of Compound A on the Growth and Function of Bladder Cells

  

Compound A was effective in inhibiting the basic growth of bladder cells and that stimulated by testosterone. This activity, which has never been reported before, is dose dependent with 1.6 + - 7 x 10 1- [alpha] -fluoro-25-hydroxy-16,23-diene-26,27-bishomo-20-epi-cholecalciferol ("compound A" / "Cmpd A" in the figures) ( on stimulated cells) (see Fig. 10 and Fig. 11).

  

This effect was significantly greater than that of anti-androgenic finasteride widely used in the treatment of urogenital diseases (Fig. 11).

Example 7: The Effect of Compound A on Bladder Dysfunction in a Model of Bladder Neck Obstruction

Experimental part

1. Materials

  

[0134] 1.1. Animals:
Female Sprague-Dawley rats, weighing 200 to 250 g
1.2. grouping

  

[0135] Group A:
BOO rats treated with compound A for 2 weeks, starting one day after creation of obstruction (n = 12)
Group B: BOO rats, treated with a vehicle for 2 weeks, starting one day after the creation of the obstruction (n = 12)
Group C: Sham-operated rats treated with Compound A for 2 weeks, starting one day after surgery (n = 12)
1.3. Studies:
a) Cystometry (approximately 18 hours after the last administration of the drug / vehicle, 12 hours after removal of the obstruction ligation) in a conscious state.
b) Measurements of the weight of the bladder
c) In vitro studies

2. Methods

  

2.1. Obstruction of the bladder neck (BOO):
The bladder and ureterovesical junction were exposed through a lower mid-length abdominal incision. A 0.9 mm metal shank was placed next to the proximal urethra and a ligation with 3/0 silk thread was securely attached around the urethra and stem, which was subsequently removed. The fictitious surgical procedure was therefore performed without placing the ligature. After 13 days, the ligation was removed and a catheter was inserted into the bladder dome and implanted subcutaneously.
2.2. cystometry
The morning after insertion of the catheter, a cystometric examination was performed without any anesthesia or stress in a metabolic cage.

   The amount of urine evacuated was measured using a liquid 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 to 60 min, when the evacuation models were reproducible, the following parameters were recorded for 30 min: pressure at the base of the bladder, urination pressure, threshold pressure, interval and volume urination, and bladder contractions. The amount of post-voiding residue was manually studied 3 times at the end of the cystometry. The capacity of the bladder was calculated on the basis of the measured values.
2.3. In vitro studies
2.3.1.

   Preparations
Once cystometry was complete, the rats were sacrificed by carbon monoxide asphyxiation followed by exsanguination. The abdomen was examined by a lower incision of intermediate length after which the symphysis was opened. The bladder was carefully dissected freely, and immediately placed in a minced form in Krebs solution, and band preparations were dissected.
2.3.2 Recording the mechanical activity
The bladder and urethra were separated at the bladder neck, and semicircular bands were prepared from the middle third of the detrusor (1 x 2 x 5 mm). All preparations were used immediately after removal.
The strips were transferred to 5 ml tissue baths containing the Krebs solution.

   The Krebs solution was maintained at 37 ° C and continuously bubbled with a mixture of 95% O2 and 5% CO2, resulting in a pH of 7.4. The strips were suspended between two L-shaped hooks with a silk thread ligation. One hook was connected to a mobile unit for adjusting the passive voltage, and the other to a Grass FT03C force transducer (Grass Instruments Co., MA, USA). The isometric tension was recorded using a Grass polygraph (7D). After mounting, the strips were stretched at a passive tension of 4 mN (the same voltage for all preparations) and allowed to return to equilibrium for 45-60 min before the other experiments were performed.
2.3.3.

   Electric stimulation
Electrical stimulation (EFS) was accomplished using two platinum electrodes placed on either side of the preparations, and was performed using a Grass S48 or S88 stimulator, providing simple square pulses at selected frequencies. The duration of the stimulus train was 5 s, the pulse duration 0.8 ms, and the stimulation interval 2 min. The polarity of the electrodes was reversed after each pulse using a polarity change unit.
2.3.4 Process
Each experiment was started by exposing the preparations to a Krebs solution at K <+> high (124 mM) until two reproducible contractions are obtained.

   Then, the following experiments were performed:
a) Electrical stimulation on the nerves was performed and the frequency response was obtained in the presence and absence of atropine.
b) Concentration-response curves were plotted for carbachol and ATP.

Results

  

The validated rat bladder neck obstruction model described above was used to examine the ability of compound A to control and treat bladder dysfunction. The objective was to evaluate whether an analogue of vitamin D3 (1- [alpha] -fluoro-25-hydroxy-16,23e-diene-26,27-bishomo-20-epi-cholecalciferol - compound "A" to the dose of 150 [micro] g / kg / daily) can prevent bladder hypertrophy and bladder dysfunction such as overactive bladder.

  

In this model, a ligation was surgically placed around the neck of the catheterized bladder, so that when the catheter was removed, the bladder experienced increased urethral resistance. Rats underwent continuous cystometry to evaluate bladder function. In addition, the contractile properties of an isolated bladder preparation in response to nerve stimulation and exogenous stimuli in vitro were studied under electrical stimulation (EFS).

  

The following cystometric parameters have been examined (see Figs 12 to 16):
- pressure of urination (the maximum pressure of the bladder during urination),
- the capacity of the bladder (the residual volume after evacuation plus the volume of saline infused to induce evacuation),
- volume of urination (volume of urine expelled)
- the post-voiding residue (capacity of the bladder minus the volume of urination) and
- frequency and amplitude of spontaneous changes in intravesical pressure (vesical contractions).

  

In this model, the evaluated analogue has a beneficial effect on bladder function. This effect was evident in the normal bladder and is maintained in the obstruction of the bladder neck. In particular, significant differences with respect to the vehicle have been observed for:
- the frequency and amplitude of spontaneous bladder contractions (Figures 13 and 14);
the post-voiding residue (absent with compound A, Fig. 16);
- the pressure of urination (Fig. 15).

  

In addition, a beneficial effect on the bladder function has been confirmed in the in vitro tests:
- answer K;
- response to the EFS (Fig. 17);
- response to carbachol.

  

Finally, a slight decrease in the weight of the bladder was observed with compound A (FIG 12).

  

These data demonstrate the use of compound A (in the dose range of 50 to 300 [micro] g - corresponding to approximately 0.725 to 5 [micro] g / kg body weight in humans) for the prevention and treatment of bladder dysfunction, for example, overactive bladder, as, for example, demonstrated in patients with BPH.

Abbreviations

  

[0144]
 <Tb> T: <September> testosterone


   <Tb> DHT: <September> dihydrotestosterone


   <Tb> GF: <sep> growth factor


   <Tb> BPH: <sep> benign prostatic hyperplasia


   <Tb> PP: <sep> according to the protocol


   <Tb> ITT: <sep> to treat


   <Tb> ANOVA: <sep> variance analysis


   <Tb> USTR: <sep> transrectal ultrasonography


   <Tb> BOO: <sep> obstruction of the bladder neck


   <Tb> AR: <sep> androgen receptors


   <Tb> PSA: <sep> specific antigen of the prostate

  

All references including the patents and patent applications cited in this application are incorporated herein by reference in order to cover the widest possible range.

  

[0146] In the entirety of the following description and claims, unless the context indicates a different meaning, the word "includes", and its variations such as "include" and "comprising" will be understood to imply that inclusion of an integer or a step or group of integers but not the exclusion of any other integer or step or group of integers or steps.

  

The priority request may be based on the application of which the present description and claims are part in respect with any subsequent application. The claims of such subsequent application may be directed to any feature or combination of features described herein. They may be in the form of a product, composition, process, or use of the claims and may include, by way of example and without limitation, the following claims.


    

Claims (16)

1. Use of 1- [alpha] -fluoro-25-hydroxy-16,23E-diene-26,27-bishomo-20-epi-cholecalciferol, or a pharmaceutically acceptable salt or ester thereof , for the manufacture of a medicament for the prevention and / or treatment of benign prostatic hyperplasia. 2. Use of 1- [alpha] -fluoro-25-hydroxy-16,23E-diene-26,27-bishomo-20-epi-cholecalciferol for the manufacture of a medicament for the prevention and / or treatment of benign prostatic hyperplasia. 3. Use according to claim 1 or 2 of 1- [alpha] -fluoro-25-hydroxy-16,23E-diene-26,27-bishomo-20-epi-cholecalciferol, or a salt or an ester pharmaceutically acceptable thereof, in combination with a second BPH active agent for the manufacture of a medicament for the prevention and / or treatment of benign prostatic hyperplasia. The use of claim 3 wherein the second HBP active agent is a [alpha] adrenergic receptor blocking agent. 5. Use according to claim 4 wherein the [alpha] -adrenergic receptor blocking agent is selected from terazosin, doxazosin, tamsulosin, silodosin, AIO-8507L and RBx-2258. Use according to claim 3 wherein the second active agent against BPH is a 5- [alpha] reductase inhibitor. The use of claim 6 wherein the 5- [alpha] reductase inhibitor is selected from finasteride and dutasteride. A pharmaceutical formulation comprising 1- [alpha] -fluoro-25-hydroxy-16,23E-diene-26,27-bishomo-20-epi-cholecalciferol, or a pharmaceutically acceptable salt or ester thereof, and a pharmaceutically acceptable carrier for the prevention and / or treatment of benign prostatic hyperplasia. The pharmaceutical formulation of claim 8 further comprising a second active agent against BPH. The pharmaceutical formulation of claim 9 wherein the second BPH active agent is a [alpha] adrenergic receptor blocking agent. The pharmaceutical formulation according to claim 10 wherein the [alpha] -adrenergic receptor blocking agent is selected from terazosin, doxazosin, tamsulosin, silodosin, AIO-8507L and RBx-2258. The pharmaceutical formulation of claim 9 wherein the second HBP active agent is a 5- [alpha] reductase inhibitor. The pharmaceutical formulation of claim 12 wherein the 5- [alpha] reductase inhibitor is selected from finasteride and dutasteride. A pharmaceutical formulation according to any one of claims 8 to 13 which is presented in capsule form. 15. 1- [alpha] -Fluoro-25-hydroxy-16,23E-diene-26,27-bishomo-20-epi-cholecalciferol or a pharmaceutically acceptable salt or ester thereof, as an agent for the prevention and / or the treatment of benign prostatic hyperplasia. 16. 1- [alpha] -fluoro-25-hydroxy-16,23E-diene-26,27-bishomo-20-epi-cholecalciferol as agent for the prevention and / or treatment of benign prostatic hyperplasia.