EA010240B1 - 1-alpha-fluoro-25-hydroxy-16,23e-diene-26,27-bishomo-20-epicholecalciferol and use for treatment thereof - Google Patents

Abstract

The use of 1-alpha-fluoro-25-hydroxy-16,23E-diene-26,27-bishomo-20-epicholecalciferol, 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.

Description

The present invention relates to the use of 1a-fluoro-25-hydroxy-16,23E-diene-26,27-bishomo20-epicholecalciferol (compound A) for the manufacture of a medicament for the prevention and / or treatment of benign prostatic hyperplasia (BPH) and associated symptoms. The invention further relates to a method for the prevention and / or treatment of benign prostatic hyperplasia (BPH) and associated symptoms by administering 1α-fluoro-25-hydroxy-16,23E-diene-26,27-bishomo-20-epicholecalciferol in an amount effective for prevention and / or treating said disease, alone or in combination with other active agents.

BPH is a common disease in the elderly, which affects about 50% of men aged 60 and 90% at the age of 85. BPH is a specific histopathological formation, which is characterized by hyperplasia of stromal and epithelial cells.

For more than a hundred years, it is believed that two etiological factors in the pathogenesis of BPH are the aging of the body and the presence of functional testes. However, with the accumulation of information about the prostate gland, this concept seems insufficient, since it does not take into account all aspects of the pathogenesis of BPH. An additional etiological factor plays a significant role in the regulation of prostate growth. For example, evidence has been obtained that the growth of the prostate gland is directly controlled by special growth factors that are produced by the cells of the gland itself, acting locally on neighboring cells by the paracrine mechanism or on the producing cells of the gland by the autocrine mechanism. Therefore, much attention is currently being paid to developing a treatment strategy based on the inhibition of intraprostatic growth factors.

BPH is a common cause of chronic symptoms of urinary tract insufficiency, which affect the filling phase (irritation symptoms) and the emptying phase (obstructive syndrome) of the urination cycle. These symptoms affect the social, psychological, domestic and professional, physical and sexual aspects of the patient’s life and have a strong negative effect on the quality of life. In addition, BPH can cause more acute urological complications, especially acute urinary retention (AIK), more frequent complications of BPH and less frequent recurrent urinary tract infections, dilatation (expansion) of the upper urinary tract, the formation of stones in the bladder and recurrent hematuria.

BPH treatment is associated with extremely high social costs, which in the United States alone amounted to $ 4 billion in 1993 and are projected to be $ 26 billion in 2003.

The current treatment regimen for BPH includes oral administration of 5a-reductase inhibitors (finasteride and dutasteride, recently approved ΕΌΑ) and α 1 receptor antagonists (terazosin, doxazosin, tamsulosin, as well as silodosin, A1O-8507b, KBx-2258, etc.) . Each of these therapeutic approaches has both advantages and disadvantages associated with various mechanisms of action. While antagonists of the α 1 receptor reduce the symptoms related to the symptoms of urinary tract insufficiency (LCH) with high efficiency, these drugs are ineffective in reducing prostate volume and, therefore, are not suitable for the prevention of surgical intervention. In contrast, 5a-reductase inhibitors, such as finasteride and dutasteride, reduce the formation of dihydrotestosterone (ENT), reduce the size of the prostate gland, and prevent surgery. In addition, the latest results of clinical trials (over 7 years) for the prevention of prostate cancer, in which more than 18,000 healthy elderly men took part, have shown that finasteride can prevent or slow down the development of prostate cancer (see Tjoshrzop Ι.Μ. et al., Νο \ ν д ап 1 па ап 6 оит оит оит оит о о Меб Меб Mebush, 349, 215-224 (2003)). However, as expected (see KazzaYap U.8., Lapse !, 361, 60-62 (2003)), finasteride has antiandrogenic side effects with regard to sexual function, such as decreased potency, desire and gynecomatology, which significantly reduces the possibility of its use as an agent that prevents the development of a tumor. In addition, treatment with finasteride is associated with an increased detection of high-grade prostate cancer, possibly because with the low-androgenic state induced by finasteride, the selection of the most aggressive, androgen-insensitive malignant cells occurs (see Zsatbsho R.T., no. 349, 297-299 (2003)).

Thus, there is an urgent need for a new class of drugs for the treatment of BPH that can prevent acute urinary retention and at the same time eliminate the associated surgical interventions, i.e., reduce androgen-induced growth of the prostate gland and do not directly affect androgen receptors ( AK), and therefore do not have anti-androgenic prostatic and extraprostatic side effects, for example, side effects on the sexual aspects of a patient’s life but. Due to the blocking of signals from local growth factors of the prostate gland, such drugs can be used not only for the treatment of BPH, but also for the prevention of prostate cancer, possibly without selection of AK-insensitive malignant clones.

- 1 010240

As indicated in the text of the application, the applicants found that not having a hypercalcemic effect, a well-tolerated analogue of vitamin B ₃ , 1a-fluoro-25-hydroxy-16,23E-diene-26,27-bishomo-20epicholecalciferol (compound A), is the first an example of such a drug, since this compound can affect BPH in an independent of the androgen receptor way, affecting many pathways controlling the cell growth of BPH, including proliferation of the prostate mediated by growth factors.

1,25-Dihydroxyvitamin ϋ ₃ [1,25 (OH) 2O ₃ ], an activated form of vitamin B ₃ , is a secosteroid hormone that not only plays a central role in bone formation and calcium metabolism, but also takes part in the regulation of immune response, differentiation and apoptosis of many types of cells, including malignant cells.

However, the problem of the therapeutic use of calcitriol is its natural ability to induce hypercalcemia and hyperphosphatemia. In this regard, analogues of calcitriol have been developed that preserve biological activity, but are devoid of hypercalcemic side effects.

No. 8 5939408 and EP 808833 disclose a number of analogues of 1.25 (ONDV3, including 1a-fluoro-25-hydroxy16,23E-diene-26,27-bishomo-20-epicholecalciferol (compound A). In FR 5939408 and EP 808833 are described, that these compounds induce differentiation and inhibit the proliferation of various skin cell lines and cancer cells and are used to treat hyperproliferative skin diseases such as psoriasis, neoplastic diseases such as leukemia, breast cancer and sebaceous diseases such as acne, seborrheic dermatitis and osteoporosis.

Recently, in a number of works of the applicant it has been unexpectedly found that, unlike other analogues of 1.25 (ONDV3, compound A

significantly reduces the growth of human prostate cells ίη νίΐτο due to the induction of apoptosis and inhibits the growth of the prostate gland ίη νίνο without affecting testosterone and dihydrotestosterone levels. In addition, such inhibition of prostate growth is achieved with the introduction of doses that do not cause hypercalcemia. Thus, compound A is an effective pharmacological agent for the treatment of benign prostatic hyperplasia.

As described in the examples, compound A reduces the size of the prostate gland. In addition, like finasteride, compound A reduces the proliferative activity of testosterone ίη νίΐτο and ίη νίνο. However, it is significant that, unlike finasteride, compound A does not inhibit the activity of 5-reductase type 1 and type 2 and can inhibit cell growth in BPH induced not only by testosterone, but also by digitrotestosterone. These antiandrogenic properties of Compound A are independent of the interaction with the AP receptor, since it has been found that Compound A does not bind to AP or does not exhibit AR agonist or antagonist activity. In addition, compound A does not affect the secretion of sex hormone. In addition, as established in our studies, compound A does not have a significant effect on the level of RPA and, therefore, when treating with compound A, there is no danger of masking this important sign of possible prostate cancer.

Another significant advantage of compound A is that, when tested ίη νίνο, this drug, in contrast to finasteride, is able to inhibit the growth of basal cells and testosterone-stimulated growth of bladder cells, and, therefore, such a drug can be used for prevention and / or treating human bladder dysfunction. In studies of ίη νίνο on rat bladder with outlet obstruction, as a model of bladder dysfunction, the therapeutic effect of compound A was established. This property of compound A is of undoubted interest, since bladder dysfunction is a frequent and painful complication of BPH. Thus, compound A is able to reduce the size of the prostate gland and reduce the intensity of bladder dysfunction, i.e. cause an improvement in the function of the bladder and reduce the intensity of the symptoms of BPH associated with the bladder, with the simultaneous direct effect of compound A on the prostate gland and bladder. It is believed that this action is independent of an improvement in bladder symptoms, which is supposed to be simply the result of a reduction in the size of the prostate gland. Bladder symptoms include over-activated bladder, and indicators of improvement in bladder function include a decrease in non-empty bladder contractions and residual

- 2 010240 urine.

Thus, the present invention relates to the use of 1a-fluoro-25-hydroxy-16,23Udien-26,27-bishomo-20-epicholecalciferol for the manufacture of a medicament for the prevention and / or treatment of benign prostatic hyperplasia. Pharmaceutically acceptable esters and salts of compound A are also within the scope of the invention.

The invention also relates to the use of 1a-fluoro-25-hydroxy-16,23E-diene-26,27-bishomo-20epicholecalciferol or a pharmaceutically acceptable salt or ester thereof, for the manufacture of a medicament for the prevention and / or treatment of benign prostatic hyperplasia glands.

The invention also relates to a method for the prevention and / or treatment of benign prostatic hyperplasia in patients who require such prophylaxis or treatment, comprising administering a therapeutically effective amount of 1a-fluoro-25-hydroxy-16,23E-diene-26,27 bisgomo-20- epicholecalciferol or a pharmaceutically acceptable salt or ester thereof.

The invention also relates to the use of 1a-fluoro-25-hydroxy-16,23E-diene-26,27-bishomo-20epicholecalciferol or a pharmaceutically acceptable salt or ester thereof, for the prevention and / or treatment of benign prostatic hyperplasia.

The invention also includes 1a-fluoro-25-hydroxy-16,23E-diene-26,27-bishomo-20-epicholecalciferol or a pharmaceutically acceptable salt or ester thereof for use in the prevention and / or treatment of benign prostatic hyperplasia.

The invention also relates to the use of 1a-fluoro-25-hydroxy-16,23E-diene-26,27-bishomo-20epicholecalciferol or a pharmaceutically acceptable salt or ester thereof for the prevention and / or treatment of benign prostatic hyperplasia in the absence of antiandrogenic prostatic and extraprostatic side effects. The invention also relates to a method for the prevention and / or treatment of benign prostatic hyperplasia in the absence of antiandrogenic prostatic and extraprostatic side effects in patients who require such prophylaxis or treatment, said method comprising administering a therapeutically effective amount of 1a-fluoro-25-hydroxy-16 , 23E-diene-26,27-bishomo-20-epicholecalciferol or a pharmaceutically acceptable salt or ester thereof.

The invention also relates to the use of 1a-fluoro-25-hydroxy-16,23E-diene-26,27-bishomo-20epicholecalciferol or a pharmaceutically acceptable salt or ester thereof for the prevention and / or treatment of benign prostatic hyperplasia along with the simultaneous prophylaxis and / or treatment for bladder dysfunction. The invention also relates to a method for the prevention and / or treatment of benign prostatic hyperplasia along with the simultaneous prophylaxis and / or treatment of bladder dysfunction in patients who require such prophylaxis or treatment, said method comprising administering a therapeutically effective amount of 1a-fluoro-25- hydroxy-16,23E-diene-26,27-bishomo-20-epicholecalciferol or a pharmaceutically acceptable salt or ester thereof.

1a-Fluoro-25-hydroxy-16,23E-diene-26,27-bis-homo-20-epicholecalciferol is a known compound which is prepared as described in I8 5939408, incorporated by reference in the application description.

Esters include pharmaceutically acceptable labile esters that hydrolyze in the body to release Compound A.

Salts of Compound A include adducts and complexes which are formed upon interaction with alkali and alkaline earth metal ions and their salts, such as sodium, potassium and calcium ions and their salts, such as calcium chloride, calcium malonate and the like.

Compound A or a salt or ester thereof can be used in monotherapy or can be administered in combination with known BPH active agents, for example, an alpha adrenergic receptor blocking agent, such as an α 1 receptor antagonist (e.g. terazosin, doxazosin or tamsulosin, or also silodosin, AY-8507b or KBx-2258) or a 5α-reductase inhibitor (e.g. finasteride or dutasteride). The term “BPH active agent” includes agents that are active or relatively known to be active in the treatment or prophylaxis of BPH, such as the compounds described above. The components of the combination are mixed with compound A or its salts or esters in various ratios and administered separately, sequentially or simultaneously in separate or combined pharmaceutical formulations. Appropriate doses of known therapeutic agents are readily determined by one skilled in the art. The combination of compound A with two or more (for example, three) BPH active compounds can be, for example, a combination of an a1 receptor antagonist and a 5a reductase inhibitor. When administered in combination with compound A (or a salt or ester thereof), the components of the combination can be used at lower doses compared to using such a component alone, even at a subtherapeutic dose.

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Thus, the invention also includes the use of the above, wherein the drug is administered separately, sequentially or simultaneously as separate or combined pharmaceutical formulations comprising a second BPH active agent.

Thus, the invention includes the use of 1a-ator-25-hydroxy-16,23E-diene-26,27-bishomo-20-epicholecalciferol or a pharmaceutically acceptable salt or ester thereof in combination with a second BPH active agent to produce a drug intended for the prevention and / or treatment of benign prostatic hyperplasia.

The invention also relates to a method for the prevention and / or treatment of benign prostatic hyperplasia in a patient who needs such prophylaxis or treatment, the method comprising administering a therapeutically effective amount of 1a-fluoro-25-hydroxy-16,23E-diene26,27-bis-homo- 20-epicholecalciferol or a pharmaceutically acceptable salt or ester thereof, separately, sequentially or simultaneously in separate or combined pharmaceutical formulations comprising a second BPH active agent.

These combinations are usually prepared for use in the form of pharmaceutical formulations. The pharmaceutical compositions thus obtained are also an object of the present invention.

Dosages and the sequence of administration of the active ingredients in the pharmaceutical compositions of the invention can be varied so that the amount of the active ingredient is effective to provide the desired therapeutic response, taking into account the particular patient, composition and route of administration, the indicated amount of the active ingredient being non-toxic to the patient. The dose of compound A can be from 0.1 to 300 μg per day, for example, 50-150 μg per day, for example, 75 or 150 μg per day. Unit doses preferably contain 50-150 μg, for example 75 or 150 μg, and are preferably administered once a day.

A particularly preferred dose of Compound A is that maximum dose that is tolerated by the patient and does not cause hypercalcemia or other undesirable side effects, such as hypercalciuria. Preferably, compound A is administered at a concentration of from about 0.001 μg to about 100 μg per kg body weight, from about 0.001 to about 10 μg / kg, or from about 0.001 to about 100 μg / kg body weight. The ranges of the above values are also part of the present invention.

As noted above, compound A can be administered as a pharmaceutically acceptable salt or ester, however, preferably, compound A is used as such, i.e., not as an ester or salt.

The indicated doses can be taken in the form of an appropriate pharmaceutical composition by single administration, multiple administration or in the form of controlled release preparations to achieve the most effective results, preferably orally once or twice a day (preferably once a day), for example, for a month. In some situations, an alternative dose may be sufficient to achieve the desired therapeutic response.

The choice of exact doses and composition and the most favorable treatment regimen depends on the pharmacological properties of the composition, nature and severity of the condition to be treated, and the physical and mental state of the recipient.

Typical delivery methods include the oral, parenteral (including subcutaneous, intramuscular and intravenous), rectal, buccal (including sublingual), pulmonary, transdermal and intranasal methods, the most preferred oral method. The introduction is continuous and intermittent (for example, in the form of a jet injection).

The invention also relates to a pharmaceutical composition comprising 1a-fluoro-25-hydroxy16,23E-diene-26,27-bishomo-20-epicholecalciferol or a pharmaceutically acceptable salt or ester thereof, and a pharmaceutically acceptable carrier for the prevention and / or treatment of benign prostatic hyperplasia.

The invention also relates to a packaged composition, which comprises a pharmaceutical composition comprising 1a-fluoro-25-hydroxy-16,23E-diene-26,27-bishomo-20-epicholecalciferol or a pharmaceutically acceptable salt or ester thereof, and a pharmaceutically acceptable carrier, and contains instructions for using the composition for the treatment of benign prostatic hyperplasia.

As noted above, such compositions can be prepared for parenteral (subcutaneous, intramuscular or intravenous) administration, especially in the form of liquid solutions or suspensions; for oral or buccal administration, especially in the form of tablets or capsules; for pulmonary or intranasal administration, especially in the form of powders, nasal drops or aerosols; and for rectal or transdermal administration.

Compositions can usually be administered in unit dosage form and can be prepared by any method known in the art of pharmaceuticals, for example, as described in Vetschop's Rhattaceis1 8c1eise8, 17 ^4b ., Musk Ryschidid Sotraiu, Eyoi, RA (1985). Formulations for parenteral administration may contain as

- 4 010240 excipients, sterile water or saline, alkylene glycols such as propylene glycol, polyalkylene glycols such as polyethylene glycol, vegetable oils, hydrogenated naphthalenes, etc. Formulations for nasal administration are 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 sprays. Typical buccal excipients include sugars, calcium stearate, magnesium stearate, pregelatinized starch, and the like.

Compositions for oral administration may include one or more physiologically acceptable carriers and / or excipients and may be in solid or liquid state. Tablets and capsules are prepared using binding agents, for example, syrup, gum arabic, gelatin, sorbitol, tragacanth or polyvinylpyrrolidone; 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, carboxymethyl cellulose or edible fats; emulsifiers such as lecithin or gum arabic; vegetable oils such as almond oil, coconut oil, cod liver oil or peanut butter; preservatives such as bottled hydroxyanisole (BHA) and bottled hydroxytoluene (BHT). Liquid compositions can be encapsulated, for example, in gelatin to obtain standard forms.

Preferred oral solid forms include tablets, two-chamber hard capsules, and soft gelatine capsules (WEUs). Of particular interest are WEU capsules, since they have a distinct advantage over the other two forms (see Zeadeg N., Zoya deiayi sarki1ek: κοίπΐίοη ί т у ей 1 1 1,,,,,,, Р аг се, та 1 се се.. К к к к 9 9 (1985)). Such advantages of WEU capsules include: a) the uniformity of the dosage form is optimized in WEU capsules, since the drug is dissolved and dispersed in a liquid, which is then accurately metered into capsules, b) drugs in the form of WEU capsules have good biocompatibility, since the drug dissolves, solubilizes or disperses in an aqueous or oily medium and, therefore, upon release in the body, these solutions dissolve or emulsify to form dispersions of the lobe GOVERNMENTAL means with a large surface area, c) preventing degradation of drugs that are sensitive to oxidation during long storage because the dry shell of soft gelatin provides a barrier to oxygen diffusion.

The composition of the dry shell usually includes about 40-60% gelatin, about 2030% plasticizer (such as glycerin, sorbitol or propylene glycol) and about 30-40% water. In addition, the composition may also include other components, such as preservatives, colorants, opacifiers and flavorings. The liquid contents of the capsule include a solid drug, dissolved, solubilized or dispersed (mixed with a suspending agent such as beeswax, hydrogenated castor oil or polyethylene glycol 4000) or a liquid drug in a carrier or combination of carriers, such as mineral oil, vegetable oils, triglycerides, glycols, polyols and surfactants.

Typical formulations use soft gelatin capsules of 2 sizes, white, opaque, oval in shape, containing a liquid composition comprising, as active ingredient, compound A dissolved in migliol 812 (triglyceride containing C ₈ -C ₁₂ fatty acids from fractionated coconut oil) mixed with bottled hydroxyanisole (BHA) and bottled hydroxytoluene (BHT) as preservatives. Soft gelatin capsules may contain from 0.01 to 25 mg, for example 75 or 150 μg of compound A. Soft gelatin capsules should be stored at a temperature of 2-8 ° C without light.

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

The compositions are preferably obtained in a nitrogen atmosphere under yellow light.

The present invention is illustrated by the following examples and figures.

In FIG. 1 shows inhibition of BPH cell proliferation by calcitriol and compound A (Comp. A).

FIG. 1A. Incubation in the presence of calcitriol (circles) or compound A (squares) at increasing concentrations from 10 ^-18 to 10 ^-7 M for 48 h causes a significant dose-dependent inhibition of BPH cell growth (* P <0.01 compared to the control). Analysis using the ABNT program shows that two secosteroids exhibit the same maximum inhibition (1 _max = 43 ± 1%), but are characterized by different inhibitory activity (compound A: -1ode 1C ₅₀ -15.8 ± 0.5; calcitriol: -1ode 1C ₅₀ = 10.2 ± 0.6; P <0.005). The results are presented as percent inhibition (mean ± ZEM) compared to the relative controls in three different triple experiments

- 5 010240 nom repeat.

In FIG. 1B presents the effect of compound A at increasing concentrations (from 10 ^-18 to 10 ^-7 M) on the proliferation of BPH cells stimulated by T (10 nM, squares), BOR (10 ng / ml, circles) or IE8 (1-3) 1CP -1 (10 ng / ml, triangles). Compound A also causes significant inhibition (* P <0.01 compared to cells treated with T or CP) of BPH cell growth in the presence of all stimulating agents with the same value of I _max = 66.6 ± 7.3%. However, compound A is more effective in inhibiting the action of T (-1Ode 1C ₅₀ = 16.4 ± 0.6) compared with two other growth factors (Oe8 (1-3) UR-1: -1One 1C ₅₀ = 12.7 ± 0.6; and KOR: -1Od 1C ₅₀ = 14.2 ± 0.6; P <0.0001). The results are presented in% deviation (mean ± 8EM) from maximum stimulation in three different experiments with triplicate.

In FIG. Figure 2 shows the effect of compound A, cyproterone acetate and finasteride on the growth of BPH cells stimulated by androgen. BPH cells were incubated for 48 h in the presence of compound A (1 nM) or antiandrogens (finasteride, P, 1 nM, cyproterone acetate, Sur, 100 nM) in the presence of T (10 nM, Fig. 2 A) or ΌΗΤ (10 nM) , Fig. 2B). The figure also shows the results obtained using unstimulated BPH cells (Fig. 2A). The results are presented in percent deviation (mean ± 8EM) compared with the relative controls in three different experiments with a four-fold repetition. Compound A and cyproterone acetate significantly block cell growth induced by T and ΌΗΤ, while finasteride was only active against T. stimulated cells. * P <0.01 compared to control, ° P <0.01 compared to androgen-stimulated cells.

In FIG. 3 shows the absence of agonistic or antagonistic properties of compound A with respect to the human AK receptor. PC3 cells deficient in AK were stably transfected with human AK and placed in 24-well plates with a density of 2x10 ⁴ cells per well. After 24 hours, cells were transfected with AK-responsive plasmid p8PP and after 48 hours the cells were incubated in the presence of increasing concentrations of β (squares) or compound A (circles) (Fig. 3A), or at a fixed concentration of β (3 nM) in the presence of bicalutamide (squares) ) or compound A (circles) (Fig. 3B) for 18 hours. Results (average values from three experiments) are presented as percent bioluminescence per μg of total protein. When evaluating agonistic activity, the enzyme activity in the presence of 100 nM ΌΗΤ was taken as 100% of luciferase activity (Fig. 3A), while when assessing antagonistic activity, the enzyme activity at 3 nM приним was taken as 3 nM ΌΗΤ (Fig. 3B).

In FIG. Figure 4 shows the inhibition of growth of the rat ventral prostate by Compound A or finasteride.

FIG. 4A. Castrated rats injected with T enanthate (30 mg / kg / week) were orally administered for 5 days per week (two consecutive weeks) with vehicle or compound A in increasing doses (10, 30, 100 and 300 μg / kg) or finasteride ( P, 10 and 40 mg / kg). The mass of the ventral prostate was represented as% deviation (mean ± 8EM) from the gland mass of intact rats treated with vehicle ( ^L P <0.05, * P <0.01 compared to control rats, ° P <0.01 compared to rats given T).

FIG. 4B. Carrier (control) or compound A in increasing doses (10, 30, 100 and 300 mcg / kg) or finasteride (P, 10 and 40 mg / kg). The mass of the ventral prostate was represented as% deviation (mean ± 8EM) from the gland mass of the control intact rats receiving the vehicle (* P <0.01 compared to control rats).

In FIG. Figure 5 shows the effect of compound A and finasteride on the expression of the clusterin gene in the rat ventral prostate.

FIG. 5A. Northern blotting (hybridization with a radioactive probe) of clusterin mRNA expression in the ventral prostate of intact rats receiving vehicle (track 1) or orchiectomized rats (track 2). Tracks 3–6 show the expression of the clusterin gene in orchiectomized rats receiving T-enanthate (30 mg / kg) for two weeks and receiving an oral carrier (track 3), compound A (300 μg / kg, track 4, and 100 μg / kg, track 5) or finasteride (40 mg / kg, track 6). 10 μg of total RNA was added to each track. The corresponding expression of ΟΛΡΩΗ (gel stained with ethidium bromide) is given below on the blot. The blot is presented for two separate experiments.

FIG. 5B. Northern blotting (hybridization with a radioactive probe) of clusterin mRNA expression in the ventral prostate of adult intact rats given orally for 1 month (5 times a week, 27 injections) vehicle (track 1), compound A in increasing doses (10, 30 μg / kg, tracks 2 and 3) or finasteride (40 mg / kg, track 4). 10 μg of total RNA was added to each track. Corresponding expression of CAPA (ethidium bromide stained gel) is shown below on the blot. The blot is presented for two separate experiments.

In FIG. 6 shows the morphological changes in the action of compound A on the ventral prostate gland of castrated rats and rats treated with T (Fig. 5A, B, C and E). Characteristic

- 6 010240 ferments obtained using transverse sections of whole prostate glands immunostained with monoclonal antibodies to rat clusterin and secondarily stained with hematoxylin. In rats castrated four days earlier and treated with a vehicle, a clusterin label is found in the cytoplasm of atrophic cuboid epithelial cells (Fig. A, 10x). After a two-week intake of T (Fig. B, 10x), a clusterin label was practically not detected. In contrast, cluster-positive cells are still present in rats treated with T and various doses of Compound A (100 μg / kg, Fig. C, 300 μg / kg, Fig. E, black arrows). In FIG. Ό and E show slices corresponding to slices in FIG. C and E in order to detect DNA fragmentation, which was determined by the method of single-stranded end labeling of dUTP (4ITP) (ΤυΝΕΕ method), mediated by terminal deoxynucleotidyl transferase (TBT). In a two-week treatment (9 administrations) with compound A (100 μg / kg, Fig. Ό, 300 μg / kg, Fig. E), massive apoptosis was induced in most epithelial and stromal cells. It should be noted (black arrows) that all clusterin-positive cells undergo apoptosis, and in a significant part of the cells that do not contain clusterin, fragmentation of the nucleus is also observed.

In FIG. 7 shows morphological changes in the action of compound A and finasterol on the prostate gland of intact adult rats.

FIG. 7A, B, Ό, Ε, E and H - characteristic images were obtained using cross sections of whole prostate glands immunostained with monoclonal antibodies to rat clusterin and secondarily stained with hematoxylin. In FIG. 7A (10x) primary antibodies are absent. In FIG. 7B (10x) shows that in untreated adult rats there are only a few, individual epithelial cells in some glands contain a label (black arrows). In contrast, in the prostate glands of rats treated with increasing concentrations of Compound A, cuboidal epithelial cells with signs of atrophy contain clusterin, which appears in a dose-dependent manner (see black arrows, 10 μg / kg, Fig. 7Ό; 30 μg / kg, Fig. 7E; 100 μg / kg, Fig. 7E, 10x). Similar results were obtained using finasteride (40 mg / kg, Fig. 7H, 10x).

In FIG. 7C, C, and I show serial sections corresponding to the sections in FIG. 7B, Ό and E, respectively, in order to detect DNA fragmentation, which was determined by the single-stranded end labeling of dUTP (άυΤΡ) (ΤυΝΕΕ method), mediated by terminal deoxynucleotidyl transferase (TBT). It should be noted (black arrows) that all clusterin-positive cells undergo apoptosis, and even in a significant part of the cells that do not contain clusterin, fragmentation of the nucleus is also detected.

In FIG. Figure 8 shows the results of a chronic toxicity test in dogs. An apparent decrease in prostate mass is observed after 9 months of treatment with Compound A compared to animals receiving placebo.

In FIG. Figure 9 shows the results of a chronic toxicity test in dogs. The reduction in prostate mass after recovery with Compound A treatment compared to placebo-treated animals.

In FIG. Figure 10 shows the effect of compound A on bladder cell growth stimulated by testosterone, ppm = human bladder.

In FIG. 11 shows the effect of compound A when compared with other compounds on the growth of stimulated and basal cells of the bladder. T 10 nM = testosterone, E 1 nM = finasteride.

In FIG. 12 shows the effect of vitamin Ό on the mass of the bladder.

In FIG. 13 shows the effect of vitamin Ό on the frequency of involuntary contraction of an empty (bladder).

In FIG. 14 shows the effect of vitamin Ό on the amplitude of involuntary contraction of an unfilled (bladder).

In FIG. Figure 15 shows the effect of vitamin Ό on urinary pressure.

In FIG. 16 shows the effect of vitamin Ό on residual urine.

In FIG. Figure 17 shows the effect of vitamin Ό on the frequency of the contractile response of bladder strips when stimulated by an electric field.

Examples

Example 1. The effect of compound A on BPH cells ίη νίΐτο.

Materials and methods

Materials

Minimum Essential Medium (MEM), ΌΜΕΜ-Ρ12, 1: 1, mixture, Ham E12 medium, saline with phosphate buffer (PB8), bovine serum albumin (BSA), fraction V, glutamine, geneticin, type IV collagenase, vitamin Ό ₃ , testosterone (T), dihydrotestosterone (INT), cyproterone acetate, reduced form of β-nicotinamide adenine dinucleotide-3′-phosphate (NADP), dithiothreit (DTT), phenylmethyl sulfonifluoride (PM8E) and a calcemia measurement kit (81 MO). A kit for determining protein was obtained at the company Kab-BaLaogaFpek, 1 ps. (Negi1e§, CA). Fetal calf serum (EB8) was obtained from υηίραΐΐι (VeLogb, IK). Monoclonal antibodies of rat anti-clusterin (monoclonal mouse 1dC) specific for the beta chain were obtained on

- 7 010240 to the company υΡδΤΆΤΕ Vyuyesypoduodu (Lake ΡΙαοίά. ΝΥ). The Aror Tad Κ11 kit for end labeling ίη κίΐη (method 18EB) was obtained from Opsog (MO, USA). CHO 1827 and CHO 1829 cells were obtained at 8egopo 1p1egpayopa (Geneva, Switzerland). 1pk1ade1 p1ik obtained at the company Raskagb (81. Bosch. MO). Finasteride (purified preparation) (17c- (No. ter-butyl) carbamoyl-4-aza-5a-androst-1-en-3-one) was kindly provided by the company Megsk 8yagr & Sing ypYogaUpek (Rainau. Νυ). Bicalutamide is kindly provided by Ak1ta2epes (Ak1ga2epesa. Milan. Italy). An analogue of 1a-fluoro-25-hydroxy-16.23E-diene-26.27 bisgomo-20-epicholecalciferol (compound A) was obtained from Vuehe 11 (Vuehe 11, Milan. Italy). Keratinocyte growth factor (ΚΟΕ) was obtained at the company Rergo Theseus EC (London. England). and human insulin-like growth factor-1 | Oek (1-3) UN-1 | obtained at Ogorer Ytjeb (Adelaide. Australia). Set ΡΟΌ for determining cell death w κίΐη by the method of single-stranded end labeling άυΤΡ (method ΤυΝΕΕ). mediated by terminal deoxynucleotidyl transferase (ΤάΤ). received at the company Rosie P1adpokys8 Sogrogayop (1pY1aparoY8. ΙΝ). Plastic dishes for cell cultures obtained at the company Ea1sop (Ohpagy. CA). Disposable filter modules for obtaining a nutrient medium are obtained from ΡΒΙ 1n1egpa1yupa (Milan. Italy). Lipofectamine 2000 and Ory-Met I medium for transfection of luciferase were obtained at 1 Puygodep. L1e йesyηo1od ^ e8 (8ap OshNapo Myapeke. Milan. Italy). Plates for thin layer chromatography (TLC) were obtained from Megsk (Pagschkp. Germany). Testosterone enanthate (T enanthate) was obtained at Oeutopa1 firm (Apad. Italy). A set of Coa1-ACoip1 for determining total testosterone was obtained at MeFsa1 8uk1et (Oepoua 81girra. Italy). A system for [ ¹²⁵ S] analysis of rat luteinizing hormone (hHH) was obtained at the company Ategyat Hya ^ tas ^ a Vu1esy (V ^ 8ca1a ^ ay. N1).

BPH cells

Human BPH cells were obtained. cultivated and used. as described earlier in the article Szheksyu S. et al. S1pysa1 apy Epiopododu Me1aLo11kt. 85.2576-2583 (2000). Cells were obtained from prostate tissue. taken from five patients. who underwent suprapubic adenomectomy of BPH (surgery to remove prostate adenoma). after discussion and approval by the local ethics committee. Patients did not receive any pharmacological treatment for three months before surgery.

Cells of the SNO-1827 and SNO-1829 lines. transfected with 5a reductase

CHO-1827 and CHO-1829 cells. transfected with 5a-reductase type 1 (5aR-1) or type 2 (5aR-2). respectively. (see 81 eegk ^ .. His1od. 58. 17-24 (2001)) were cultivated in the medium of Hem G12. containing 5% HS8.

Cell line PC3. transfected AR

Human PC3 adenocarcinoma cells. stably transfected with plasmid p5HyAP-A. containing the human androgen receptor (yAR). as previously described (see Vopassogk1. et al., Episode 1. 141. 3172-3182 (2000)). cultivated in vials with an area of 75 cm ² on Hem G-12 medium. containing 50 μg / ml geneticin. 10% HS8. penicillin (100 units / ml) and streptomycin (100 mg / ml).

BPH fabrics

Prostate tissue for binding analysis was obtained from patients. who underwent suprapubic adenomectomy of BPH (surgery to remove prostate adenoma). Patients did not receive any pharmacological treatment for three months before surgery. After surgery, tissues were immediately immersed in liquid nitrogen and stored at -80 ° C until analysis.

Rat tissue

The ventral prostate glands of rats were rapidly excised. weighed and quickly frozen in dry ice. Immunohistochemical experiments were performed on adjacent cryostatic sections with a thickness of 14 μm in order to directly compare tissue morphology. clusterin expression and localization of apoptosis using the ΤυΝΕΕ method. For extraction of total DNA and Western blotting (protein hybridization with a labeled probe), ventral prostate glands were used. derived from 4-6 animals.

BPH Cell Proliferation Assay

For analysis of cell proliferation, 4 × 10 ⁴ BPH cells were plated in 12-well plates in an appropriate culture medium. kept in the environment. not containing red dye and serum and containing 0.1% BSA. for 24 hours and then treated with a stimulating agent for 48 hours. Cells in the medium. phenol red and serum free and containing 0.1% BSA. used as a control. Then the cells were trypsinized and each experimental point was obtained after measurement on a hemocytometer. calculating the average value from the data. obtained in at least six different sections of each well. as previously described (see Szheksuy S. et al. 1OiGiP1O! C11hci1 apy Epiospodiu Me1aOo11ct. 85. 2576-2583 (2000). The experiments were carried out using increasing concentrations (from 10 ^-18 to 10 ^-7 M) of calcitriol or compound And in the presence or absence of a fixed concentration of T (10 nM). ΚΟΕ or Pe8 (1-3) 1OG-1 (10 ng / ml). Cell growth analysis was also performed using a fixed concentration of androgens (10 nM) at

- 8 010240 in the absence or absence of compound A (1 nM, 10 nM) or the anti-androgens of finasteride (G, 1 nM) and cyproterone acetate (Sur, 100 nM). Cell growth analysis was also performed using a fixed concentration of T (10 nM) or CE (10 ng / ml) in the presence or absence of compound A (10 nM). In this experiment, each experimental point was determined from three different experiments in triplicate or quadruple repetition. The results are presented in percent deviation (mean ± 8EM) compared with the maximum stimulation of T or CE.

End tagging ίη kyi (18EB method)

Method 18EB was performed on BPH cells using an Aror Tad reagent kit including peroxidase to determine apoptosis ίη δίΐιι. according to the manufacturer’s methodology. Cells were incubated in the presence of T (10 nM), KSE (10 ng / ml) or Os8 (1-3)! CE-1 (10 ng / ml) in the presence or absence of 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 areas of each section when analyzing five different sections. The results were presented as mean ± 8EM from the results of three different experiments.

5a-reductase inhibition assay

Inhibition analysis of 5a-reductase was carried out on CHO-1827 cells transfected with 5aB-1, or on CHO-1829 cells transfected with 5aB-2, as described previously (see Siagpa A. et al., 1it1 furniture 1, 1 Kit1ku, 43, 37188 -3735 (2000)). Compound A was added at a concentration of 10 ^-9 to 10 ^-5 M, finasteride was used as a control inhibitor in each experiment.

Binding analysis

The analysis of the binding of cytosolic fractions to BPH fragments was carried out as described previously (see Szheksuy S. et al., Episode 1, 144, 3046-3057 (2003), (at a final protein concentration of 1.8 mg / ml). Cytosol fractions were incubated in the presence of increasing concentration (0.125, 0.25, 0.5, 1 nM) [ ³ N] -B1881 (specific activity 83.5 Ci / mmol) in the absence or presence of [ ³ N] -B1881 (1 nM) increasing concentrations unlabeled B1881 (10 ^-1 -10 ^-6 M), INT (10 ^-10 -10 ^-6 M), T (10 ^-10 -10 ^-6 M), bicalutamide (10 ^-10 -10 ^-4 M) and compounds a (10 ^-10 -10 ^-4 M). to prevent V1881 progesterone receptor binding in each 1 μM triamcinolone acetonide was added to the test tube. The separation of the bound and unbound ligand was carried out as described previously (see S. Hexuy et al., Episode 1, 144, 3046-3057 (2003)). The protein content was determined by the Bradford method using BSA as standard.

Analysis of luciferase activity

PC3 cells stably transfected with human AB were placed in 24-well plates at a density of 2x10 ⁴ in Hem's E12 medium containing 10% EC8. After 24 hours, the cells were transfected (in an amount of 750 ng per well) with plasmid p8PP containing the wild-type sequence MMTU-LTP linked to the firefly luciferase gene (see M. RakhkhadN et al., Apauysa Vusyet1kyu, 204, 315-323 (1992 )) when using lipofectamine 2000 (1 mg / ml) according to the manufacturer's method. After 48 hours, cells were incubated in the presence of ΌΗΤ (10 ^-12 -10 ^-6 M) or bicalutamide (10 ^-9 -10 ^-5 M) in the presence of 3 nM ΌΗΤ and an equimolar concentration of compound A for 18 hours. Steroids and an analogue of compound A dissolved in ethanol. Transfected cells incubated in the presence of ethanol were used only as a positive control.

The analysis of luciferase activity was carried out on a Bertold luminometer according to the manufacturer's method (Lysiegake Akkau 8uk1et, firm Rgoteda, Milan, Italy). Cells were lysed directly in the plate by adding 200 μl of lysis buffer solution. Luciferase activity was determined in 20 μl of cell lysate 10 s after adding 100 μl of luciferin. Total protein was determined in 20 μl of cell lysate. At least three independent double-repeat analyzes were performed.

results

Upon incubation of BPH cells in the presence of increasing concentrations of calcitriol or compound A, inhibition of cell growth is observed (Fig. 1A). Both compounds inhibit cell proliferation in a dose-dependent manner. The analysis according to the ATE1T program (see EE Leap A. et al., Atepsap 1oit1 οί RPuk1o1odu, 235, E97-E102 (1978)) showed that despite the insignificant statistical difference in the values of the maximum inhibition of cell growth by calcitriol and compound A (compound A (compound A) in the figures) (I _max = 43 ± 1%), these compounds differ in relative activity, i.e. for compound A, the relative activity is several logarithmic units higher compared to calcitriol (compound A: -1ode 1C ₅₀ = 15.8 + 0.3; calcitriol: -1ode 1C ₅₀ = 10.2 ± 0.6, P <0.005) .

BPH cell proliferation increases significantly (p <0.01) in the presence of testosterone (T) (156 ± 8%) and growth factors (CE), such as (Iek (1-3) 1CE-1 (194 ± 6%) or KSE (183 ± 5%). When cell growth is stimulated in the presence of T or CE for 48 hours (Fig. 1B), the inhibitory effect of compound A becomes more pronounced (I _max = 66.6 ± 7.3%). see E. E. Leap et al., Atepsap 1oit1 οί Ryukyuodu, 235, E97-E102 (1978)) of the inhibition schedules shows that Compound A is more effective against BPH cells stimulated by T (-1Od 1C ₅₀ k = 16.4 ± 0.6) than the other two and growth factors (-1ode 1C ₅₀ k-12.7 ± 0.6 in the case of IEC (1-3) 1CE-1 and -1ode 1C ₅₀ k = 14.2 ± 0.6 in the case of KSE, respectively, P < 0.0001).

- 9 010240

Compound A (1 nM) exhibits an antagonistic effect not only with respect to proliferation of BPH cells stimulated by T and ΌΤΗ, but also to some extent with respect to cells stimulated with cyproterone acetate (Sur, 100 nM, Fig. 2A and 2B), which is an antagonist AK In contrast, the 5a-reductase inhibitor finasteride (E, 1 nM) exerts an antagonistic effect only against T-induced cell growth (Fig. 2A). In addition, compound A reduces the growth of even those cells that have not been stimulated by anti-androgen (Fig. 2A).

To assess the potential antiandrogenic properties of compound A, in addition to inhibiting the growth of BPH cells, we studied its binding to the AK receptor. First, we ruled out the possibility of binding of compound A to AK due to the study of competitive binding in human BPH homogenates using synthetic androgen [ ³ Η] K1881 as a labeled ligand. An analysis of the data on the program ΕΙΟΑΝΏ (see Mipcop R.1. Et al., Apauysa1 VusyeshschNu, 107, 220239 (1980)) showed that unlabeled K1881, ΌΗΤ, T and the antagonist AK bicalutamide completely suppress the binding of [ ³ H] -K1881 ( table I). Conversely, compound A does not compete for binding (AK receptor) to [ ³ H] -K1881 at any concentration studied (Table I). These results were confirmed and supplemented by analysis of the luciferase reporter gene. In PC3 cells expressing the full AK gene associated with the reported luciferase gene, ΌΗΤ stimulated a dose-dependent increase in luciferase activity (EC ₅₀ = 2 ± 1.3 nM, Fig. 3A), while bicalutamide inhibited активность-stimulated activity (1C ₅₀ = 194 ± 80 nM, Fig. 3B). In this system, an increase in the concentration of compound A does not stimulate or inhibit the increase in luciferase activity mediated by AK (Fig. 3). Finally, in order to identify the relationship between Compound A and the biosynthesis of ΌΗΤ, the active metabolite T, experiments were performed using CHO cells transfected with 5a-reductases of type 1 type 2. The results were compared with data obtained with finasteride (E). While E inhibits the conversion of T to ΌΗΤ with the expected values of 1C ₅₀ (for 5a-reductase type 1 1C ₅₀ = 659 ± 100 nM, for 5a-reductase type 2 1C ₅₀ = 53.7 ± 11 nM, n = 3) Compound A does not compete with any of the isoenzymes up to the micromolar level (data not shown).

Ligand AK Affinity Constants (Ka nmol / L) K1818 0.16 ± 0.06 ONT 0.07 + 0.03 T 1.89 ± 0.94 Bicalutamide 159 ± 82 Compound A > 100,000

Table I. The affinity constants of androgen agonists (K1881, ΌΗΤ, Τ), antagonist (bikatuamida) and compound A in human BPH homogenates according to competition binding to the radioactive labeled ligand [ ³ H] -K1881.

According to the 18Eb method (n = 3, Table II), compound A suppresses BPH cells, at least in part, due to the activation of programmed cell death. The percentage of apoptotic nuclei increases significantly (270%) after incubation in the presence of compound A (10 nM) for 48 h (P <0.01 compared to the control). In contrast, when processing T cells (10 nM) or growth factors (OE, 10 ng / ml), the number of apoptotic BPH cells decreases significantly (P <0.01) compared to untreated cells (^ e8 (1-3) IOE-I = -42%, CFU = -54%, T = -27%). However, even in the presence of OE or T, compound A causes a significant (more than 250%) and significant (P <0.01) increase in the number of KEB-positive BPH cells. Apoptotic Index (%)

The control Compound A The control 18.55 ± 0.8 68.44 ± 1.26 ^a ϋβ ₃ (1-3) ΙΟΡ-Ι 10.69 ± 0.6 ^a 45.85 ± 0.66 ^a ' ^b ' ⁱⁿ KOR 8.5 ± 0.42 ^a 44.46 ± 0.57 ^a ' ^{b, c} T 13.56 ± 0.72 ^a 49.0611.87 ^ ’“

Table II. The effect of compound A (10 nM), growth factors (OE, 10 ng / ml), or T (10 nM) on DNA fragmentation in BPH cells. Apoptotic index (%) means the number of stained nuclei according to ^ Eb in BPH cells in each of at least five separate sections on the slice. The results are presented as mean ± 8EM in three separate experiments. Compound A is capable of inducing apoptosis in untreated BPH cells, as well as in BPH cells simultaneously incubated in the presence of growth factors (OE) or T (a: P <0.01 compared to the control, b: P <0.01 compared to cells treated with compound A, B: P <0.01 compared with cells treated with OE or T).

Example 2. Antiproliferative properties of compound A in models of prostate growth ίη νίνο.

Animal Testing Procedures

Male rats, 8gadie EaMeu (28 days old), were acquired in Cialis erythrocytes Kex Ь Lebola eryte8 (Ca

- 10 010240

Besso, Italy). All described animal tests were carried out in accordance with accepted standards for the treatment of animals. Castration was performed through the scrotum under anesthesia with ketamine / xylazine. Three days after castration, rats (5-8 animals in the group) were treated or not treated with T enanthate (30 mg / kg) with two separate subcutaneous injections once a week. For 5 days of the first week and 4 days of the second week, the rats were orally administered with vehicle (migliol 812), compound A (10, 30, 100 and 300 mg / kg) or finasteride (10 and 40 mg / kg), a total of 9 injections, and after 1 day the animals were killed.

In another embodiment, an intact adult male Zrtadie Ea \\ '1eu (weighing 250 g) was administered orally with a carrier (migliol 812), compound A (10, 30, 100 and 300 mg / kg) or finasteride (10 and 40 mg / kg) 5 days a week for 5 consecutive weeks and two additional days for the sixth week, a total of 27 injections, unless otherwise indicated. After the completion of each experiment, blood was collected from animals for analysis on the content of calcium and hormones.

Northern hybridization analysis

Total RNA was extracted using the RIARak kit! Firm Mo1eci1ag §u81esh (8ap O1edo, CA). Blotting, labeling, hybridization conditions and probes (full-length cDNA of clusterin 1.5 ko long and full-size cDNA 1,2 1.2 ko long) were obtained by the described methods (Be! Ϊ́πζζί et al., Vusyeshyu1 1oyta1, 257, 293-296 (1989 ) and MapieSh et al., VusyeshShtu apb Ce11 Vyuodu, 72, 515-521 (1994)). Autoradiograms were read by densitometric scanning on a LKV and Itaxap XB densitometer.

Immunohistochemistry

All cryostatic sections obtained from control or treated rats were processed in parallel, as described previously (AChapsoNe et al., Proposal et al., 167, 197-204 (2000)). In each experiment, three alternative sections of the prostate glands of three different rats were analyzed. In the negative controls obtained in the absence of specific antibodies in the reaction medium, no specific staining was observed. Secondary staining was performed with hematoxylin, coverslips were treated with Eiky! (company O. Kshb1et Oshn & Co, Germany). High-resolution digital color photographs were taken with a CCD camera through a microscope.

Ίη Shi DNA fragmentation analysis (TiIEB method)

DNA fragmentation in cryostatized sections of the prostate revealed by single-stranded end-labeling with dUTP (BITP) (ΤϋΝΕΕ) mediated by terminal deoxynucleotidyl transferase (TBT) was performed using the ίη Kyi cell death detection kit (manufacturer ΡΟΌ, VosNe). Apoptotic nuclei positive according to the method of TIIE were recorded in the form of digital color images with a large increase when taking a CCD camera through a microscope.

Secondary staining was performed with eosin, coverslips were treated with an EikSn kit (O. K1pb1eg Oshbn & Co, Germany).

Calcium determination

Calcium levels were determined using a commercial colorimetric assay kit (company SCa) according to the manufacturer's method.

Determination of testosterone and tHN

The hormones T and bH were determined using commercial kits for radioimmunoassay according to the manufacturer's methods. To measure the level of T in serum (blood), the samples were first added to 4 volumes of diethyl ether, stirred by carefully turning the tubes over for 15 minutes and then centrifuged at 2000 rpm for 5 minutes. The aqueous phase was frozen with dry ice, the organic phase was separated and evaporated to dryness in a nitrogen atmosphere. The dried residue was transferred to a buffer solution for analysis (1 vol./ 1 vol., Samples taken from intact rats; 4 vol. / 1 vol., Samples taken from castrated rats).

Statistical analysis

Statistical analysis was performed using one-way analysis of variance ΑΝΟνΑ and, where appropriate, paired or unpaired student criteria. Binding data (with the receptor) was analyzed using the LUAIO program (Mipcop et al., Apaubsa1 Wusieshtu, 107, 139-220 (1980)).

The ABBNT computer program (Ee Leap A. et al., Ashepsap 1oopt1 o! Ryu8yuodu, 235, E97-E102 (1978)) was used to analyze sigmoidal dose-response curves to estimate half-inhibition values from the maximum value (1С ₅₀ ) and stimulation values at half the maximum value (EC ₅₀ ), as well as the maximum inhibitory (I _max ) and stimulating (E _max ) actions. Data were presented as mean ± 8EM.

results

To analyze the antiproliferative properties of compound A in prostate growth models, castrated and intact rats were treated with increasing concentrations of compound A (10-300 μg / kg) or finasteride (10, 40 mg / kg). As shown in FIG. 4A, castration significantly reduces the mass of the ventral prostate, while a two-week treatment with test enanthate testosterone ron (T) (30 mg / kg) not only completely restores, but even stimulates its growth. Compound A in any doses tested completely suppresses T-stimulated proliferation (hypertrophy) of the prostate, reducing the mass of the ventral prostate to a lower level than untreated rats. Similar results were obtained using finasteride (10, 40 mg / kg). When treating intact adult rats with compound A for one month, a significant decrease in the mass of the ventral prostate gland was observed, and the maximum decrease (30%) was observed at the highest dose used (300 μg / mg). At this dose, the inhibitory effect of compound A on prostate growth is comparable to that of finasteride at a dose of 10 or 40 mg / kg (Fig. 4B). In all experiments, when various doses of compound A were orally administered, very low hypercalcemia was observed only when the highest dose (300 μg / mg) was administered (Table III). No other significant side effects were observed.

Calcemia The control 10.2 ± 0.16 Compound A 10 μg / kg 10.16 ± 0.24 Compound A 30 μg / kg 9.87 ± 0.15 Compound A 100 μg / kg 10.55 ± 0.18 Compound A 300 μg / kg 1О, 85 ± О, 1

Table III. Calcemia (mg / dl) in castrated rats with testosterone replacement therapy after administration of various doses (10, 30, 100, 300 μg / kg) of compound A. Compound A in no case changes the level of potassium in plasma (blood) in castrated rats with testosterone enanthate replacement therapy (30 mg / kg / week) compared with the control. Similar results were obtained on intact rats (data not shown). The results are presented as mean ± ZEM obtained for rats in this group.

In order to gain a better understanding of the molecular mechanisms of the prostate mass reduction process induced by compound A, we studied the expression of the clusterin gene and protein biosynthesis, as well as the analysis of morphological signs of apoptosis using single-stranded end labeling of DUTP (DTP) mediated by terminal deoxynucleotidyl transferase (TbT) (TONEY method). Clusterin is a ubiquitous (ubiquitous) gene product that is directly involved in stopping the cell cycle and atrophy, the expression of which is suppressed by androgens. In FIG. 5A shows the expression of clusterin mRNA in the prostate gland (according to the Northern analysis) in orchiectomized rats that received or did not receive T. Castration significantly increases the relative content of clusterin mRNA, while the opposite effect is caused by a two-week administration of T. Stimulating administration of various concentrations of the compound A (300 and 100 μg / kg) or E (40 mg / kg) partially inhibits the reduction of clusterin gene expression induced by T. administration. In intact rats (Fig. 5B), administration of various concentrations of the compound (30 and 100 mg / kg) for one month to induce stable overexpression of clusterin gene in the prostate gland, which is comparable or even higher than the expression of growth induced when administered 40 mg / kg E.

Local expression of clusterin in the prostate gland of orchiectomized rats is shown in FIG. 6. Castration causes a noticeable and widely developed atrophy in the prostate gland and almost all cuboidal epithelial cells lining the gland of the gland give a positive reaction to clusterin (Fig. 6A). T-replacement therapy (Fig. 6B) reverses the morphological signs of atrophy and significantly reduces the amount of stained clusterin. The indicated effect is excluded with the simultaneous administration of compound A (Fig. 6C and E). In FIG. 6Ό and E show the results of the analysis by the ΤϋΝΕΕ method of slices adjacent to those shown in FIG. 6C and E. When treated with compound A (100 μg / kg, Fig. 6Ό, and 300 μg / kg, Fig. 6E), there is a clear fragmentation of the nuclei in the cells of the epithelium and stroma, and apoptosis can be detected in clusterin-positive and clusterin-negative cells. In FIG. 7A and B (the first two sections) shows the morphology of the prostate gland of intact rats when stained for the presence of clusterin in the absence (Fig. 7A) or in the presence (Fig. 7B) of primary antibodies. It should be noted that in the prostate gland of untreated adult rats there is practically no clusterin label (Fig. 7B), and no signs of nuclear fragmentation are observed (TONEY method, Fig. 7C). On the contrary, treatment with different doses of compound A causes the expression of clusterin (Fig. 7Ό-Ε) and apoptosis (Fig. 7C, C and I). In FIG. 7E shows for comparison the effect of finasteride (40 mg / kg) on the presence of clusterin-positive color in sections of the prostate gland.

In order to exclude the possibility of reducing the growth of the prostate gland ίη νίνο by compound A due to the effect on the function of the pituitary or testes in castrated and intact rats, the levels of luteinizing hormone (tyN) and T in serum (blood) were measured. As expected (table. GUA), castration significantly reduces the level of T and at the same time increases the level of tyN in serum (blood). The introduction of enanthate T (30 mg / kg) for two weeks completely reverses the effect of orchiectomy. With the oral administration of compound A (100 and 300 μg / kg) to castrated rats undergoing T-replacement therapy, a slight effect on serum (blood) tyN and T levels is observed. Similar results were obtained in intact rats (Table IVΒ). Indeed, for

- 12 010240 long-term administration (within 1 month) of compound A (10, 30, 100 μg / kg) or P (40 mg / kg) in intact rats, there was no change in serum (blood) tH and T levels.

Table ΤΥΑ

KY1 T Control (intact rats) 2.36 + 0.46 11.5 + 2.44 Castrated Rats 20.64 ± b ’ 0.9 ± 0.32 * Castrated rats + T-therapy 2.08 ± 0.36 21.25 ± 4.12 Castrated rats + T-therapy + compound A, 100 mcg / kg 1.8 + 0.2 11.13 ± 1.02 Castrated rats + T-therapy + Compound A, 300 mcg / kg 3.15 ± 0.65 15.73 ± 2.75

TUBE table

IBN t Control (intact rats) 2 ± 0.16 11.98 + 2.87 Finasteride 2.2 + 4 18.11 ± 3.23 Compound A, 10 μg / kg 2.22 ± 0.25 19.13 ± 3.83 Compound A, 30 mcg / kg 2.32 ± 0.36 9.39 ± 2 Compound A, 100 μg / kg 1.96 ± 0.13 11 + 2.14

Table IV. The content of tHN (ng / ml) and T (nM) in the serum (blood) of castrated rats during substitution therapy (Table 1UA) or in intact rats (Table 1UB) after treatment with different doses of compound A.

1UA. Castration significantly reduces the level of T in serum (blood) (P <0.01 compared with the control) and at the same time increases the level of TB (P <0.05 compared with the control). After administration of T enanthate (30 mg / kg / week), the levels of Tb and T are restored. Compound A at all doses tested had a negligible effect on tH and T levels.

1UB. Prolonged administration (within 1 month) of P (40 mg / kg) or compound A (10, 30, 100 μg / kg) does not lead to a change in serum (blood) levels of Hb and T in intact rats.

The results obtained indicate that compound A reduces the size of the prostate in intact rats to a greater extent compared with finasteride. In addition, like finasteride, compound A cancels the proliferative activity of testosterone ίη νίΐτο and ίη νίνο. However, unlike finasteride, compound A does not inhibit the activity of 5a-reductase type 1 or type 2 and can prevent the growth of BPH cells induced not only by T, but also by OHT. These anti-androgenic properties of compound A are independent of the interaction with ΑΒ since it was established that compound A does not bind to ΑΒ, but acts on PC3 cells transfected with в as an agonist or antagonist of ΑΒ. In addition, compound A does not affect the secretion of sex hormone, since in tests on rats the levels of gonadotropin and T in plasma (blood) do not change with the daily administration of compound A for up to one month. Therefore, compound A inhibits the interaction of receptor ΑΒ with the ligand. Without going into theoretical aspects, it can be assumed that such an action is carried out due to the interruption of signal transmission from testosterone to growth factor.

At very low concentrations, compound A is able to completely suppress (exert an antagonistic effect) not only T-stimulated proliferation of BPH cells, but also proliferation induced by the two most important intraprostatic growth factors, UR-1 and CSR. In addition, even in the presence of T or CP, compound A induces apoptosis in BPH cells. Programmed cell death induced by compound A is also observed in the prostate gland of both intact and T-treated orchiectomized rats and is characterized by a diffuse pattern of DNA fragmentation with a concomitant increase in the expression of the clusterin gene and protein. Clusterin is a protein that is strictly regulated by androgens in the prostate gland (ΒοΙΙιιζζί et al., Βίοοΐιοιηίοαΐ. Toyota 1, 257, 293-296 (1989)). Although the function of clusterin has not been fully established, the level of this protein increases markedly with atrophy of the prostate gland (ΒοΙΙιιζζί et al., Osodepe, 21, 43284334 (2002) and ΐΐеζиζζ ^ et al., Toyota1 οб Epbospoduoda, 132, 361-367 (1992 )) and apoptosis ^ 5 & ον et al., Loigpa1 οί Vyuododka SNetMgu. 278, 11590-11600 (2003)). Thus, the induction of clusterin during therapy with compound A is consistent with the ability of this compound to inhibit proliferation and induce apoptosis in the prostate gland.

Thus, the results show that compound A is effective in reducing the growth of prostate cells in various experimental models.

Example 3. The reduction in prostate mass in healthy dogs treated with compound A.

Toxicity tests were carried out for 9 months on four groups of dogs, male beagle breeds, who were daily injected with a compound A dose of 0.5 μg, 1.5 μg and 5 μg / kg body weight / day through a gastric tube (in the carrier migliol 812 ) or one medium. After administration of the drug, the group receiving the maximum dose (5 μg) followed by a rehabilitation period of two months, after which the mass of the prostate gland was measured. Upon completion of the tests, along with very favorable toxicity data after completion of the course of taking compound A (see Fig. 8) and after

- 13 010240 bilitation (see Fig. 9) in animals a lower prostate mass was observed. The results after rehabilitation were statistically processed using the one-factor student criterion and established the presence of a significant difference in the action of compound A and the carrier (P <0.05). These results also demonstrate the ability of compound A to reduce prostate mass ίη νίνο.

Example 4. An oral dosage form in the form of a soft gelatin capsule.

Capsules for oral administration were obtained under a nitrogen atmosphere in yellow light, while one soft gelatin capsule was filled with a mixture containing from 0.01 to 25.0 mg of compound A in 150 mg of fractionated coconut oil (for example, migliol 812), 0.015 mg of butylated hydroxytoluene (BHT) and 0.015 mg of butylated hydroxyanisole (BHA). Capsules were prepared according to the following procedure:

1) BHT and BHA were suspended in fractionated coconut oil (for example, migliol 812) and heated with stirring at approximately 50 ° C until completely dissolved,

2) in the solution obtained in stage 1 at 50 ° C, compound A was dissolved,

3) the solution obtained in stage 2 was cooled to room temperature,

4) the solution obtained in stage 3 was filled in soft gelatin capsules.

All stages were carried out in a nitrogen atmosphere and without access to daylight.

Example 4A Oral dosage form in the form of a soft gelatin capsule.

Capsules for oral administration were obtained under a yellow atmosphere in a nitrogen atmosphere, while one soft gelatin capsule was filled with a mixture containing 150 μg of compound A in 150 mg of fractionated coconut oil (for example, migliol 812), 0.015 mg of butylated hydroxytoluene (BHT), and 0.015 mg of bottled hydroxyanisole (BHA).

Example 4B. Oral dosage form in the form of a soft gelatin capsule.

Capsules for oral administration were obtained under nitrogen in yellow light, while one soft gelatin capsule was filled with a mixture containing 75 μg of compound A in 150 mg of fractionated coconut oil (for example, migliol 812), 0.015 mg of butylated hydroxytoluene (BHT) and 0.015 mg of bottled hydroxyanisole (BHA).

Example 5. The decrease in the mass of the prostate gland in the human body during clinical trials.

Determination of the effect of compound A (1a-fluoro-25-hydroxy-16,23E-diene-26,27-bishomo-20-epicholecalciferol) on patients with BPH was carried out using the double control method with random sampling and the parallel group method with placebo.

When selecting patients for testing, the results of diagnostics of adult patients for the presence of BPH and the volume of the prostate gland> 40 ml according to the results of transrectal ultrasound diagnostics (TKI8) were used as the main criterion.

Statistical Methods

An analysis of the primary effectiveness of the treatment was planned to be carried out on patients for protocol studies (PP), and to confirm the results obtained, similar tests were supposed to be carried out on volunteers (1TT). Patients of the PP category were selected by random sampling, and all patients met the protocol criteria, underwent a full course of examination without violating the main protocol and had a reliable estimate of the volume of the prostate gland. Patients of the 1TT category were selected by random sampling, with all patients receiving at least one dose of the study drug, and the prostate volume in the selected patients corresponded to the baseline. Volunteers underwent clinical trials for 12 weeks.

All randomly selected patients who received at least one dose of the test drug were evaluated for safety analysis.

The comparison in the group was performed according to the baseline for all patients in a descriptive way. Data were processed using the chi-square test for categorical variables and analysis of variance ΑΝΟνΑ for continuous variables.

Descriptive statistics were calculated by conventional methods: mean, standard deviation, minimum and maximum values of continuous variables, and absolute values and relative frequencies of categorical variables. Descriptive statistics were used to evaluate each introduction and each visit per week.

Institutions comprising less than 4 patients were combined.

The percentage of the prostate gland volume, which was determined by the method of centralized nuclear magnetic tomography (MK1) during axial scanning, was taken as a variable of primary efficiency. For data analysis, the ΑΝΟνΑ method was used taking into account the results of treatment and the institutions in which the results were recorded.

Patients received capsules containing 150 μg (see Example 4A, with the exception of capsules containing placebo), once a day in the morning. The treatment period was 12 weeks. The number of patients tested is listed below.

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Compound A Placebo Total patients Randomly selected patients 57 62 119 Real number of patients 56 (98.3%) 60 (96.8%) 116 (97.5%) Test interrupted or lost control 0 2 (3.2%) 2 (1.7%) Poor therapeutic effect 1 (1.8%) 0 1 (0.8%)

The change in the volume of the prostate gland (%) determined by the method of axial scanning.

The percentage change in prostate volume in patients with appropriate pathology (PP) was -1.89 + 5.2 in the group of patients receiving compound A, and 4.99 + 5.99 in the group of patients receiving placebo, with a significant value of P < 0.0001 in favor of compound A. The difference between these values (compound A minus placebo) is -7.24 with a confidence interval of 95% from -9.54 to -4.94. The role of the medical institution is also significant (p = 0.0176). A similar analysis of tests on volunteers confirms the results obtained (p = 0.0176), i.e. Compound A more effectively reduces prostate volume compared to placebo.

In patients with a baseline of prostate volume> 80 ml, the difference between the groups of patients receiving the drug (p <0.0001) is more obvious than patients who have a baseline of prostate volume <60 ml (p = 0 , 0320), primarily in patients of the PP category.

In patients aged 61-70 years, the difference, always in favor of Compound A, was more evident compared with older patients (> 70 years). Indeed, in volunteers (ΙΤΤ), the difference between groups in patients older than 70 years is p = 0.0540 compared to p = 0.0001 in other groups of patients.

Drug Sensitive Patients

In the PP category, the percentage of patients sensitive to Compound A was 27.5%, no changes were observed in 65%, and only 7.5% of the patients were insensitive to treatment. In the placebo group, the number of treatment-sensitive patients was zero, in fact, the patients were divided equally into a group where no changes were observed, and a group of treatment-insensitive (50% in each group). Comparison of percentages using the chi-square criterion confirmed the results of the survey, obtained in the form of a primary efficacy variable p <0.0001, which compound A more effectively reduces the volume of the prostate compared with placebo.

The results obtained in category подтверж confirm that the percentage of patients who are sensitive to treatment in the group receiving compound A is 28.8%, and the difference according to the chi-square criterion is p <0.0001.

In patients sensitive to treatment category PP, the average decrease in prostate volume compared with the baseline is -6.88 ± 2.5, and the average deviation in patients without changes is -0.35 ± 2.3 in the group of patients receiving compound A, compared with 0.40 ± 2.0 in the group of patients receiving placebo. In patients insensitive to treatment, the average deviation is 3.93 ± 0.8 in the group of patients receiving compound A, and 7.48 ± 4.9 in the group of patients receiving placebo, which confirms the higher effectiveness of compound A with respect to regulation size and volume reduction of the prostate gland.

Percentage change in prostate volume determined by centered paraxial and intermediate scan

Control analyzes of the paraxial and intermediate images confirmed the results obtained by axial scanning. In the PP contingent, when receiving a paraxial image, the percentage change is -1.30 ± 6.9 in the group of patients receiving compound A, compared with 2.57 ± 6.8 in the group of patients receiving placebo, p = 0.0172. In the intermediate scan, the percentage change is -0.22 ± 9.6 in the group of patients receiving compound A, compared with 6.18 ± 10.9 in the group of patients receiving placebo, p = 0.0028. Volunteers (ΙΤΤ) who underwent treatment also showed a significant difference in reducing prostate volume in favor of compound A.

The total level of P8A and hormone in serum (blood)

In the PP contingent, the average change in the P8A content in the group of patients receiving compound A was 0.23 ± 1.3, and in the group of patients receiving placebo, 0.43 ± 1.7. Moreover, between the groups of patients who underwent treatment, there was no significant difference (p = 0.2722).

The PP contingent also did not show a significant difference between the groups in terms of testosterone content (p = 0.2150). In the group of patients receiving compound A, the average value was 0.07 ± 1.5, and in the group of patients receiving placebo, 0.22 ± 1.4.

In the PP contingent, there was no difference in the dihydrotestosterone content between the treatment groups (p = 0.7257 in the PP contingent). In the group of patients treated with the compound

- 15 010240

And, the observed mean change was -30.77 ± 227.71, and in the group of patients receiving placebo -166.76 ± 490.26.

There was no difference in the content of LH hormone (p = 0.9220 in the contingent PP), in the group of patients receiving compound A, the average change was -0.02 ± 1.7, and in the group of patients receiving placebo -0.00 ± 1.8.

The observed average changes in the content of P8A and the hormone are approximately equal to zero, with the exception of ΌΗΤ, i.e. Compound A does not change the level of the hormone. Similar results were obtained in the group of volunteers (ΙΤΤ).

Security

The number of patients who had at least one adverse reaction was 31 (17 in the group of patients receiving compound A and 14 in the group of patients receiving placebo). For this reason, not a single patient dropped out of the tests, only one patient from the placebo group had a serious symptom in the form of acute cholecystitis (with subsequent hospitalization).

The number of patients with adverse symptoms in connection with treatment with compound A was 3 people. (5.26%), while the number of patients with adverse symptoms due to placebo was 6 people. (9.68%). Symptoms associated with compound A included dizziness, headache, decreased libido and hot flashes, while symptoms associated with placebo included increased urinary phosphate, headache, fainting, decreased libido (3 patients), hypercalciuria, erectile dysfunction and hot flashes. .

Symptoms of calciuria recorded throughout the course of the trials in the group receiving compound A did not differ significantly from those observed in the group receiving placebo.

Conclusion

The results of the short-term test of the concept described above on the effect of compound A on the size of the prostate in patients with BPH indicate the effectiveness of the drug. When analyzing the primary treatment variable, namely, when assessing the size of the prostate gland, a significant difference was established between compound A and placebo, and thus the fact that the test drug is able to block the development of the disease is confirmed. The safety profile is quite satisfactory because there is no difference between side effects in the case of taking compound A and placebo, and in the group of patients treated with compound A, there are no serious side effects. No antiandrogenic properties were detected in the studied medicinal product, it does not affect the level of P8A and does not have a significant effect on calcium homeostasis.

Example 6. The activity of compound A in relation to the growth and function of bladder cells.

Compound A was found to be effective in inhibiting the growth of basal and testosterone-stimulated bladder cells. The indicated activity of 1a-fluoro-25hydroxy-16,23E-diene-26,27-bishomo-20-epicholecalciferol (compound A or compound A in Fig.) In relation to stimulated cells, not previously described, is dose-dependent and is 1.6 ± 7x 10 ¹⁵ (see. Fig. 10 and Fig. 11).

The specified action significantly exceeds the action of the anti-androgen finasteride, which is widely used for the treatment of diseases of the genitourinary tract (Fig. 11).

Example 7. The effect of compound A on bladder dysfunction in the model of obstruction of the outlet of the bladder.

Experiment description

1. Materials.

1.1 Animals.

Female rats 8gradie-Eate1eu, weighing 200-250 g

1.2 Groups.

Group A: SBI rats treated with Compound A for 2 weeks, starting from the first day after obstruction (n = 12).

Group B: SBI rats treated with the vehicle for 2 weeks, starting from the first day after the creation of obstruction (n = 12).

Group B: mock-operated rats treated with compound A for 2 weeks, starting from the first day after the creation of obstruction (n = 12).

1.3 Methods of examination.

a) Cystometry (~ 18 hours after the last injection of the drug / carrier, 12 hours after the removal of the ligature creating the obstruction) in animals that are conscious.

b) Measurement of the mass of the bladder.

c) Research w uygo.

2. Methods.

- 16 010240

2.1 Bladder obstruction (SBI).

The bladder and urethro-bladder junction were removed through a midline incision in the lower part of the peritoneum. A 0.9 mm thick metal rod was placed along the ureter and 3-0 silk ligature was wrapped around the urethra and the rod, then the rod was removed. False surgery was performed in a similar manner, but without ligature. After 13 days, the ligature was removed, a catheter was inserted into the bladder dome and subcutaneous catheterization was performed.

2.2 Cystometry.

The next morning after the introduction of the catheter, a cystometric examination was performed without the use of anesthesia or mechanical retention in the chamber to examine the metabolism. The amount of urine output was measured using a fluid collector connected to a mechanical displacement sensor. 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 a reproducible urination regimen was reached, the following parameters were recorded for 30 minutes: basal bladder pressure, urination pressure, threshold pressure, urination interval, volume and unfilled contractions. The amount of residual urine was determined manually three times after completion of cystometry. The bladder capacity was calculated according to the measurement results.

2.3. Research ίη νίΐτο.

2.3.1. Receiving drugs.

After completion of cystometry, rats were sacrificed by carbon monoxide asphyxiation followed by bleeding. The abdominal cavity was opened in the lower section along the section below the midline, and then the symphysis was opened. The bladder was carefully cut off, immediately placed in a chilled Krebs solution and cut into strips.

2.3.2. Registration of mechanical activity.

The bladder and urethra were separated in the neck of the bladder and the middle third of the detrusor was cut into semicircular strips (1x2x5 mm). All preparations were used immediately after extraction. The strips were placed in 5 ml tissue baths containing Krebs solution. The Krebs solution was maintained at a temperature of 37 ° C and a mixture of O ₂ / CO ₂ (95% / 5%) was continuously passed through the solution, maintaining a pH of 7.4. Strips were hung between two b-hooks using silk ligatures. One hook was connected to a movable device, allowing to regulate passive tension, and the other to a Sgaz ET03C mechanical displacement sensor (Stase 1p81gitep18 So, MA, USA). Isometric tension was recorded on a Staz polygraph (7Ό). After mounting the preparations, the strips were pulled with a passive voltage of 4 mN (the same for all preparations) and balanced for 45-60 minutes before further experiments.

2.3.3. Electric field stimulation.

Electric field stimulation (EE8) was performed using two platinum electrodes mounted on opposite ends of the tissue strips, and a Stase 848 or 888 source, which provided the supply of single rectangular vibrational pulses at the selected frequencies. The duration of a series of pulses was 5 s, the pulse duration was 0.8 ms, and the interval between stimulations was 2 min. The polarity of the electrodes was changed after each pulse using a switch.

Testing procedure.

Each experiment was started by keeping the preparations in a Krebs solution with a high concentration of K ⁺ (124 nM) until two reproducible contractions were obtained. Then conducted the following experiments:

a) electrical nerve stimulation was performed and the response frequency response was recorded in the presence or absence of atropine;

b) received graphs of the concentration dependence for carbachol and ATP.

results

To analyze the ability of compound A to regulate and treat bladder dysfunction, a rat artificial bladder outlet obstruction model was used. The purpose of such tests was to determine the ability of a vitamin аналог ₃ analogue (1a-fluoro-25-hydroxy-16,23Udien-26,27-bishomo-20-epicholecalciferol, compound A) at a dose of 150 μg / kg / day to prevent urinary hypertrophy and dysfunction bladder, such as overactive bladder.

In this model, a ligature was placed surgically around the outlet of the bladder with the inserted catheter, then the catheter was removed and the bladder was tested for increased urethral stability. Rats underwent continuous cystometry to assess bladder function. In addition, the contractile properties of isolated bladder preparations were studied in response to nerve stimulation by an electric field (EE8) and to exogenous stimuli ίη νίΐτο.

In this case, the following cystometric parameters were studied (Fig. 12-16):

- 17 010240 pressure during urination (maximum pressure in the bladder during urination), bladder capacity (residual volume after emptying plus the volume of saline that was poured to cause emptying), urination volume (volume of displaced urine), residual urine (volume bladder minus the volume of urination), the frequency and amplitude of spontaneously occurring changes in intravesical pressure (non-empty contractions).

In this model, the studied analogue had a therapeutic effect on the function of the bladder. This action is observed in a normal bladder and is stored on the model of obstruction of the outlet of the bladder. Significant differences when compared with the introduction of the media were primarily observed in the following parameters:

the frequency and amplitude of spontaneous nonempty contractions (Figs. 13 and 14), residual urine (absence with the introduction of compound A, Fig. 16), urination pressure (Fig. 15).

In addition, the therapeutic effect on the function of the bladder was confirmed in the following tests ίη υιΙιό:

response to K ⁺ , response to EEZ (Fig. 17), response to carbachol.

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

The results obtained indicate the possibility of using compound A (at a dose of 50 μg to 300 μg, which is equivalent to 0.725 to 5 mg / kg) for the prevention and treatment of bladder dysfunction, for example, overactive bladder, which is observed in patients with BPH.

The following abbreviations are used in the examples:

T testosterone

ONT dihydrotestosterone

BPH benign prostatic hyperplasia

PP patients with appropriate pathology

ΙΤΤ volunteers

ΑΝΟνΑ analysis of variance

ΤΚϋ8 transrectal ultrasound

SBI obstruction of the outlet of the bladder

AK androgen receptors

P8A specific antigen of the prostate gland

All footnotes mentioned in the description, including patents and patent applications, are incorporated into the text of the application by reference in full.

Unless otherwise indicated, the word is intended to include its variations, such as including and including those used in the description of the application and the claims, mean the inclusion of the stated (claimed) integer or stage or group of integers, and not the exclusion of any other an integer or group of integers or stages.

The application, of which the present description and claims are a part, can be used as a priority in relation to any subsequent application. The claims of such a subsequent application may relate to any distinguishing feature or combination of features described. They may take the form of a product, composition, method, or may use the claims and may include, for example, without limitation, the following claims.

Claims (25)

CLAIM 1. The use of 1a-fluoro-25-hydroxy-16,23E-diene-26,27-bishomo-20-epicholecalciferol 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. The use according to claim 1, where the drug is administered separately, sequentially or simultaneously in the form of separate or combined pharmaceutical formulations with a second agent active against BPH. 3. The use of 1a-fluoro-25-hydroxy-16,23E-diene-26,27-bishomo-20-epicholecalciferol or a pharmaceutically acceptable salt or ester thereof in combination with a second BPH active agent for the manufacture of a medicament, intended for the prevention and / or treatment of benign prostatic hyperplasia. - 18 010240 4. The use according to claim 2 or 3, where the second agent, active against BPH, means an agent that blocks the alpha-adrenergic receptor. 5. The use according to claim 4, where the alpha-adrenergic receptor blocking agent is selected from the range of terazosin, doxazosin, tamsulosin, silodosin, A1O-8507B and BBx-2258. 6. The use according to claim 2 or 3, where the second agent, active against BPH, means a 5α reductase inhibitor. 7. The use according to claim 6, where the 5a-reductase inhibitor is selected from a number of finasteride and dutasteride. 8. The use according to any one of claims 1 to 7, wherein 1a-fluoro-25-hydroxy-16,23E-diene-26,27-bishomo-20epicholecalciferol or a pharmaceutically acceptable salt or ester thereof is obtained in unit dosage form. 9. The use of claim 8, where the standard dose of 1a-fluoro-25-hydroxy-16,23E-diene-26,27-bishomo-20epicholecalciferol contains from 50 to 150 μg of the compound. 10. The use according to any one of claims 1 to 9 for the prevention and / or treatment of benign prostatic hyperplasia in the absence of antiandrogenic prostatic and extraprostatic side effects. 11. The use according to any one of claims 1 to 10 for the prevention and / or treatment of benign prostatic hyperplasia simultaneously with concomitant prophylaxis and / or treatment of bladder dysfunction. 12. The use of a pharmaceutical composition comprising 1a-fluoro-25-hydroxy-16,23E-diene 26,27-bishomo-20-epicholecalciferol or a pharmaceutically acceptable salt or ester thereof and a pharmaceutically acceptable carrier thereof for the prevention and / or treatment of benign prostatic hyperplasia glands. 13. The use of the pharmaceutical composition according to item 12, where 1a-fluoro-25-hydroxy-16,23E-diene-26,27 bisgomo-20-epicholecalciferol or a pharmaceutically acceptable salt or ester thereof is obtained in unit dosage form. 14. The use of the pharmaceutical composition according to item 13, where the standard dose of 1a-fluoro-25-hydroxy16,23E-diene-26,27-bishomo-20-epicholecalciferol contains from 50 to 150 μg of the compound. 15. The use of the composition according to claims 12-14, wherein the pharmaceutical composition further comprises a second agent active against BPH. 16. A method for the prevention and / or treatment of benign prostatic hyperplasia in patients requiring such prophylaxis or treatment, comprising administering a therapeutically effective amount of 1a-fluoro-25-hydroxy-16,23E-diene-26,27-bishomo-20-epicholecalciferol or a pharmaceutically acceptable salt or ester thereof. 17. A method for the prevention and / or treatment of benign prostatic hyperplasia in patients requiring such prophylaxis or treatment, comprising administering a therapeutically effective amount of 1a-fluoro-25-hydroxy-16,23E-diene-26,27-bishomo-20-epicholecalciferol or a pharmaceutically acceptable salt or ester thereof, separately, sequentially or simultaneously, in separate or combined pharmaceutical formulations in admixture with a second BPH active agent. 18. The method of claim 17, wherein the second BPH active agent is an alpha adrenergic receptor blocking agent. 19. The method of claim 18, wherein the alpha adrenergic receptor blocking agent is selected from the range of terazosin, doxazosin, tamsulosin, silodosin, AY-8507B and BBx-2258. 20. The method according to 17, where the second agent active against BPH means a 5α reductase inhibitor. 21. The method according to claim 20, where the 5a-reductase inhibitor is selected from a number of finasteride and dutasteride. 22. The method according to any one of claims 16-21, wherein 1a-fluoro-25-hydroxy-16,23E-diene-26,27-bishomo-20-epicholecalciferol or a pharmaceutically acceptable salt or ester thereof is obtained as a unit dose. 23. The method according to item 22, where the standard dose of 1a-fluoro-25-hydroxy-16,23E-diene-26,27-bishomo-20-epicholecalciferol contains from 50 to 150 μg of the indicated compound. 24. The method according to any one of paragraphs.16-23 for the prevention and / or treatment of benign prostatic hyperplasia in the absence of antiandrogenic prostatic and extraprostatic side effects. 25. The method according to any one of paragraphs.16-23 for the prevention and / or treatment of benign prostatic hyperplasia simultaneously with concomitant prophylaxis and / or treatment of bladder dysfunction.