HRP20000503A2 - A NOVEL CRYSTALLINE FORM OF 6-HYDROXY-3-(4-/2-(PIPERIDIN-1-YL)ETHOXY/PHENOXY)-2-(4-METHOXYPHENYL)BENZO/b/THIOPHENE HYDROCHLORIDE - Google Patents

Description

Background of the invention

6-Hydroxy-3-(4-[2-(piperidin-1-yl)ethoxy]phenoxy)-2-(4-methoxyphenyl)benzo[b]thiophene hydrochloride (arzoxifene) was first described in U.S. Pat. patent no. 5, 510, 357, and was subsequently described in detail in U.S. Pat. patent 5, 723, 474 ('474), as well as in European application 0729956. Arzoxifene is an asteroid mixed estrogen antagonist/agonist, which is used, among other things, to lower cholesterol in the serum, and to prevent hyperlipidemia, osteoporosis, cancers affected by estrogen (including breast cancer and uterine cancer), endometriosis, central nervous system disorders, including Alzheimer's disease, aortic smooth muscle cell proliferation and restenosis.

More precisely, the effect of arzoxifene on receptor-positive breast cancer that has metastasized has been clinically tested, and it is used for its treatment, for the adjunctive treatment of receptor-positive patients with appropriate systemic or local therapy; to reduce the likelihood of recurrence of invasive and non-invasive breast cancer, to reduce the likelihood of invasive breast cancer and ductal carcinoma in situ (DCIS). Arzoxifene is also used in combination with radiotherapy, aromatase inhibitors, LHRH analogues, and acetyl choline esterase (AChE) inhibitors.

X-ray powder structural analysis (XRD), thermogravimetry (TGA), 1H nuclear magnetic resonance, (1H NMR) and Karl Fischer (KF) analysis of crude arzoxyphene, isolated according to the procedure described in '474, indicate that the substance is hydrated, poorly crystalline, and that it contains different amounts of volatile organic substances (such as ethyl acetate) in its lattice.

It is characteristic of weakly crystalline substances that they are much less desirable in the process of manufacturing pharmaceutical preparations compared to crystalline substances. Also, in general, it is not desirable to produce such pharmaceutical preparations that contain significant amounts of organic solvent (for example ethyl acetate), considering the possible toxic effect of the solvent itself on patients, as well as the change in the content of the pharmaceutical preparation depending on the solvent.

Although arzoxifene prepared according to the process described in '474 can be used as a pharmaceutical preparation, it would be highly desirable and convenient to obtain a more highly crystalline form of arzoxifene that would not contain an organic solvent in its crystal lattice, and which could be efficiently and reproducibly produced commercially .

The essence of invention

This invention relates to new non-stoichiometric crystalline hydrated forms of 6-hydroxy-3-(4-[2-(piperidin-1-yl)ethoxy]phenoxy-2-(4-methoxyphenyl)benzo[b]thiophene hydrochloride (F-I) whose the stacking pattern obtained by X-ray diffraction includes the following reflections: 7.9 ± 0.2; 10.7 ± 0.2; 14.9 ± 0.2; 15.9 ± 0.2; 18.3 ± 0.2 and 20 ,6 ± 0.2° in 2θ, obtained at a temperature of 25 ± 2 °C, and relative humidity (RH) of 35 ± 10 %, with a copper lamp as a source of radiation.

Furthermore, this invention relates to pharmaceutical preparations that include F-I, one or more pharmaceutical carriers, diluents, or excipients, and if necessary estrogen, i.e. progestin, i.e. aromatase inhibitor, i.e. LHRH analog, i.e. acetyl choline esterase (AChE) inhibitor.

In addition, this invention relates to ways of using F-I for the purpose of preventing pathological conditions such as: uterine fibrosis, endometriosis, aortic smooth muscle cell proliferation, restenosis, breast cancer, uterine cancer, prostate cancer, benign prostatic hyperplasia, bone loss, osteoporosis, cardiovascular diseases, hyperlipidemia, disorders of the central nervous system, Alzheimer's disease, as well as the use of F-I in the production of drugs that prevent the aforementioned.

Furthermore, this invention relates to ways of using F-I for the purpose of regulating acetyltransferase (ChAT), as well as using F-I in the production of drugs that regulate the aforementioned.

Short description of the pictures

Figure 1 shows a representative S-II differential scanning calorimetry (DSC)/TGA graph.

Figure 2 shows a representative graph of F-I differential scanning calorimetry (DSC)/TGA.

Figure 3 shows a representative F-III differential scanning calorimetry (DSC)/TGA graph.

Figure 4 shows moisture sorption isotherms for F-I and F-III.

Figure 5 shows the desolvation of S-II as a function of drying time and temperature.

Detailed description of the invention

Crude arzoxifene prepared according to the procedure described in '474 (Example 41, crystallization from a mixture of ethanol and ethyl acetate, filtering and drying the precipitate in vacuo to constant weight at room temperature) was characterized by X-ray powder structural analysis, where it was determined that it was weakly crystalline. 1H NMR confirmed that the raw material contained about 6% ethyl acetate.

The crystallization procedure described in '474 was substantially modified in such a way that ethanol is added to a suspension of crude arzoxifene which is heated with the recovery of ethyl acetate. After cooling and vacuum filtering, the solid obtained in this modified procedure is, by its characteristics, a highly crystalline mixture of ethyl acetate and water that solvates arzoxifene (designated as S-II), which was later discovered to be the starting substance for the preparation of F-I.

F-I can be prepared by removing ethyl acetate from the crystal lattice of S-II' by vacuum drying/melting S-II at elevated temperatures. The time and temperature required to melt S-II for the purpose of preparing F-I varies from batch to batch, but most often it is around 5 days at a temperature of 100 °C. High temperatures are necessary for the conversion of S-II to F-I, according to this procedure, given that from a crude mixture of compound S-II in water at room temperature, as well as a sample stored at a relative humidity of 98% during for three weeks no F-I was created. Furthermore, drying S-II in a convex oven at high temperatures did not result in desolvation of the substance, so it can be concluded that the use of vacuum is also necessary to extract ethyl acetate from the S-II' grid.

Preferably, F-I is easily prepared and isolated at room temperature by crystallization of arzoxyphene (or any of its polymorphs/solvates) from tetrahydrofuran. It is convenient for this crystallization to be carried out by first dissolving arzoxifene in moist tetrahydrofuran (1-10% water by volume, preferably 2.5-7.5%, and most preferably 4.5-5.5%), followed by removal of water by distillation at atmospheric pressure. An example of such crystallization is exhaustively described in Example 2. If compound F-I is prepared according to this improved procedure, the amount of total related substances (TRS) can be expected to be <0.5%.

Suitable arzoxifene starting materials for the above crystallization include (but are not limited to) S-II, F-III, and arzoxifene prepared according to the process described in '474, or any mixture thereof. It does not matter which form of arzoxifene one starts with, since crystallization from isopropyl alcohol and water results in the formation of F-I crystals, in accordance with the described procedure. In a commercial stepwise synthesis, it is convenient to carry out F-I crystallization with the addition of crystallization centers.

F-III is easily prepared and isolated at room temperature by crystallization of arzoxifene from a mixture of isopropyl alcohol (IPA) and water (or any of its polymorphs/solvates). The ratio of water to isopropyl alcohol (v:v) is generally from 1:1 to 9:1. More preferably, the ratio is between 2.5 and 5.6:1. Most preferably, the ratio is between 3 and 5. 6 : 1. The ratio of isopropanol to water does not critically affect the crystallization of compound F-III, but it does affect the yield. In a commercial stepwise synthesis, it is convenient to carry out the crystallization of F-III with the addition of crystallization centers. Suitable arzoxifene starting materials for the above crystallization include (but are not limited to) S-II, F-I, and arzoxifene prepared according to the process described in '474, or any mixture thereof.

Characterization and differentiation of S-II, F-I and F-III

DSC/TGA and XRD methods were used in the characterization of S-II, F-I and F-III. TGA is often used to differentiate between different solid forms of a substance because the temperature(s) at which a physical change in a substance takes place is usually a characteristic of polymorphic substances or solvates. DSC is a technique that is often used to image polymorphic compounds and the formation of solvates. Finally, XRD is a technique that detects the arrangement in a crystalline substance.

Arzoxifene prepared according to the procedure described in '474 had XRD patterns with a weak signal-to-noise ratio, as well as an elevated baseline, indicating a poorly crystalline substance. Therefore, F-I and F-III were compared with S-II prepared according to the above-described modified procedure for crystallization of arzoxifene (adding ethanol to a suspension of arzoxifen in boiling ethyl acetate).

Representative DSC/TGA graphs of compounds S-I, F-II and F-III are shown respectively in Figures 1, 2 and 3. The DSC graph of compound S-II shows a broad endotherm starting at around 62 °C, which corresponds to the removal of ethyl of acetate and water from the crystal lattice. The endotherm, which starts at around 152 °C, represents the melting point. TGA mass loss is approximately 2.5% simultaneously with the first transition, while the remaining 0.5% mass loss occurs at the beginning of melting, which indicates the fact that some solvent molecules are more strongly bound in the crystal lattice.

The DSC graph of compound F-I shows a broad endotherm starting at about 75 °C, followed by a second endotherm starting at about 155 °C, which corresponds to the melting point. The TGA graph of compound F-I shows a gradual mass loss of 0.3%, followed by a sharp loss of 1.5%, which together represent dehydration of the crystal lattice. The onset of the first DSC transition and the corresponding TGA mass loss are slightly offset due to the difference in the amount of heat. Weakly bound water of hydration is responsible for the initial mass loss, while the second mass loss is related to about 0.5 moles of water present in the lattice at very low relative humidity (below 5% - see data obtained for moisture sorption).

The DSC graph of compound F-III shows a broad, low-temperature endotherm at about 30 °C, followed by a second relatively weak endotherm starting at about 70 °C, and a final transition starting at about 146 °C, which corresponds to melting point. A sharp 1.5% mass loss (about 0.5 mol) in TGA coincides with the first endotherm corresponding to the loss of loosely bound water molecules, while an additional mass loss of about 1.6% above 60 °C represents the loss of more tightly bound molecules water, for example those that are present even at very low relative humidity. The observed mass loss after 170 °C corresponds to the decomposition of compound F-III.

The XRD patterns of compounds F-I and F-III have characteristic sharp reflexes and a flat base line, which indicates that they are highly crystalline substances. The angular position of the peak in 2θ, and the corresponding I/Io data for representative samples of compounds F-I and F-III, as well as S-II are shown in Table 1. Although many intense reflections are at generally the same diffraction angles, each form has its own pattern in in the form of a powder, on the basis of which it is possible to clearly distinguish compounds S-II, F-I and F-III.

It is well known in crystallography that with any polymorph, the relative intensities of the diffraction peaks can vary depending on the more favorable orientation, which is influenced by the crystal morphology. Where the effect of more favorable orientation is present, the peak intensities differ, but the characteristic peak positions of the polymorphs remain unchanged. See, e.g., United States Pharmacopoeia (USP) #23, National Formulary #18, pages 1843-1844, 1995. Therefore, based on peak intensity as well as peak position, F-I can be identified in the presence of peaks 7.9 ± 0.2; 10.7 ± 0.2; 14.9 ± 0.2; 15.9 ± 0.2; 18.3 ± 0.2 and 20.6 ± 0.2° in 2θ; obtained at a temperature of 25 ± 2 °C, and a relative humidity (RH) of 35 ± 10 %, with a copper lamp as a source of radiation.

Table 1

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Further characterization of compounds F-I and F-III

The hygroscopicity test was performed on compounds F-I and F-III. The moisture sorption isotherms for F-I and F-III are shown in Figure 4. After the initial exposure of the samples to relative humidity in the amount of about 5%, an immediate increase in mass due to the effect of moisture of 1.5% and 1.7% was recorded, respectively, for compounds F-I and F-III, which is equivalent to about 0.5 moles of water. Both forms continuously absorb moisture regardless of the amount of moisture in the space, which causes water molecules to embed in the crystal lattice.

The difference in the amount of moisture absorbed in these two forms probably reflects the amount of water that can be incorporated into the two lattices (eg, the amount of available space in the lattice that can accommodate water molecules). The lack of hysteresis in the sorption-desorption isotherm of compounds F-I and F-III indicates the fact that the crystal forms equilibrate very quickly at any given humidity.

Moisture sorption diagrams of compounds F-I and F-III show that these forms are not essentially stoichiometric hydrates. At room relative humidity (about 50% RH), compound F-I contains about 1.7% water, which corresponds to an amount of 0.5 mol of water, while F-III sorbs 3.0% of water, which corresponds to an amount of 0.85 mol water. Crude forms F-I and F-III equilibrate rapidly with the atmosphere, so the amount of water obtained by analytical techniques reflects the relative humidity at the time of data collection. Differences between individual batches, with regard to DSC data, probably arise due to different hydration of the samples due to the different conditions in which they were stored.

XRD patterns were obtained for samples F-I and F-III stored in rooms with different relative humidity (0, 22, 50 and 80 %). There is a stepwise shift of the initial (0 % RH) F-III peaks to about 13.8; 17.6; 18.0; 20.5 and 24.0° in 2θ, as well as a slight shift of less intense peaks, accompanied by an increase in relative humidity. These observed changes in the XRD pattern of compound F-III indicate that the dimensions of the unit cell are changing, presumably to accommodate loosely bound water molecules due to an increase in relative humidity. The continuous shift of the peaks due to moisture coincides well with the data on moisture sorption, which indicate a gradual increase in mass within the RH region, as well as the fact that different hydrated forms are formed.

A similar experiment was performed on compound F-I to determine whether varying relative humidity would have a similar effect on its crystal lattice (0, 25, 52, 73 and 95% RH). A very small shift was observed in relation to 0% RH reflexes at around 7.7; 18.3; 18.5; 20.5; and 20.8° in 2θ, as the relative humidity increased. Peaks at about 7.7; 20.8 and 24.1 appeared a little wider and were less resolved as the relative humidity increased, suggesting that water sorbed in the amorphous compounds (or plasticized the solid), especially if the relative humidity moved in the range from 73 to 95 %. The peak shift observed in the XRD patterns of compound F-I is less dramatic compared to the peak shift of F-III that was exposed to different relative humidities. This fact indicates that the crystal lattice of compound F-I does not have the same mode of expansion and/or contraction as is the case with the crystal lattice of compound F-III.

F-I and F-III were found to be stable over the entire relative humidity range, despite the ability of compound F-III to sorb almost twice as much water. The two forms were found to have comparable crystal size, morphology, water solubility and release rate.

A drying test was performed to monitor the desolvation of S-II as a function of drying time and temperature (see Figure 5). XRD patterns were recorded at different time intervals during the desolvation experiment. Many of the diffraction peaks obtained from the desolvation study of compound S-II appear at similar angles to those of F-I, thus confirming that the crystal lattices of S-II and F-I are very similar. Disappearance of diffraction peaks at about 6.8; 7.2 and 14° in 2θ even after minimal drying indicates that these reflections can be attributed to crystallographic planes that contain the partial density of the electron cloud of ethyl acetate molecules.

F-I was formed with extended melting of the solvated substance in vacuum and high temperature. Using XRD, it was determined that the F-I obtained in this way had a high degree of crystallinity. Therefore, the substance formed by crystallization from ethanol and ethyl acetate, after which it is dried under vacuum for only a few hours, as described in '474, had very low crystallinity, which is explained by the fact that such a procedure results in obtaining a partially desolvated compound S-II.

F-I and F-III have several advantages over the previously described forms of arzoxifene. Relative to arzoxifene produced by the process described in '474, F-I and F-III are significantly more stable at room temperature, and are therefore more suitable for pharmaceutical development, eg development of dosage forms. It can also be said that F-I and F-III are significantly more crystalline than the form stated in '474. Crystalline substances are generally less hygroscopic and more stable (eg, they are less susceptible to chemical decomposition and maintain a constant content) compared to amorphous substances, and are therefore more desirable in the production of preparations. Furthermore, in contrast to arzoxifene produced according to the process described in '474, which contains ethyl acetate and water in its crystal lattice, F-I and F-III contain only water.

Characterization procedures

DSC measurements were performed on a TA device 2920 for modulated DSC connected to a thermal analyzer (Thermal Analyst 3100), which was additionally equipped with a cooling system. Samples (3-5 mg) were heated in corrugated aluminum crucibles starting from 10 °C and up to 240 °C, with a heating rate of 2 °C/min.

TGA analysis was performed on a TA device for thermogravimetric analysis (2050 Thermogravimetric Analyzer) connected to a device for thermal analysis 3100. Samples (5-10 mg) were heated in corrugated aluminum crucibles starting from 25 °C and up to 250 °C, with a heating rate of 5 °C/min.

XRD patterns were obtained on a Siemens D5000 X-ray powder diffractometer, equipped with a CuKα source (λ = 1.54056 Ĺ) and a Kevex detector for solids, operating at a voltage of 50 kV and a current of 40 mA. Each sample is recorded between 40 and 350 in 2θ. The samples were equilibrated for at least 30 minutes at the desired temperature and/or relative humidity before measurements were taken.

Hygroscopicity measurements were made on compounds F-i and F-III using the VTI procedure described below. Each sample was dried in a vacuum at a temperature of 60 °C to a constant mass, at which time the relative humidity in the sample chamber was 0%. Moisture sorption isotherms were obtained at 25 °C using a VTI vacuum device for moisture determination, under the following conditions: sample mass is 10-15 mg, absorption/desorption in the range 0-95% relative humidity, increase interval is 5%, sample collection interval 10 minutes.

The following examples describe further procedures for preparing the hydrates of this invention. These examples are not intended to limit the scope of these procedures in any respect, and should not be construed as such.

Preparations

Preparation 1

S-II

Crude arzoxifene (1.58 g of product prepared according to the procedure of Example 41 in U.S. Patent 5,723,474, the conclusions of which are incorporated herein by reference) is suspended in 28 ml of ethyl acetate and heated to reflux. Ethanol (18 ml) was added to the solution. The solution is heated at reflux for 20 minutes, after which it is cooled to room temperature. The precipitate is isolated by vacuum filtration and washed with 30 ml of ethyl acetate, which results in the formation of 1.05 g of a powdery, white solid.

Examples

Example 1

F-I from S-II

S-II was dried in a vacuum oven (-25 in. Hg) at 100 °C for 118 hours to give compound F-I.

Example 2

Improved procedure for the preparation of compound F-I from arzoxifene

A reflux condenser and a dropping funnel containing 25.0 g of arzoxifene, 475 ml of THF and 25 ml of water, and a distillation apparatus were mounted on a liter round, three-necked flask. The reaction mixture is heated to boiling, during which 250 ml of distillate is predistilled. The apparatus is quickly cooled, and an additional 250 ml of THF is added to the flask, whereby 250 ml of the distillate is re-distilled at atmospheric pressure. The apparatus is quickly cooled, and an additional 250 ml of THF is added to the flask, whereby 250 ml of the distillate is re-distilled at atmospheric pressure. An additional 250 ml of THF is added to the flask, while the reaction mixture is continuously boiling. After adding this amount of THF, a white precipitate forms. The reaction mixture is slowly cooled over 3 hours, during which an additional amount of precipitate is formed. The raw mixture is cooled to room temperature. The crystalline mixture is filtered and dried under vacuum at 50 °C for 48 hours with a gentle nitrogen purge. The yield of the reaction is 22.50 g (90.0 %). XRD analysis showed that the spectra of the wet precipitate and the dry solid are essentially the same, and that they are essentially the same compared to the spectrum obtained for F-I (which was previously prepared). DSC analysis determined the melting point at 157 °C, while TGA analysis determined a mass loss of 1.5% by comparing the results obtained at room temperature and at 100 °C. The HPLC purity calculated on a pure basis was 88.1%, compared to the theoretical content of 92.9%. The total amount of related substances obtained by HPLC analysis was 0.44%.

Example 3

F-I derived from [6-benzyloxy-3-[4-[2-(piperidin-1-yl)ethoxy]phenoxy]-2-(4-methoxyphenyl)]benzo[b]thiophene-(S-oxide)

THF (261 mL), water (45 mL), concentrated sulfuric acid (6.14 g) and [6-benzyloxy-3-[4-[2-(piperidin-1-yl)ethoxy]phenoxy]-2 -(4-methoxyphenyl)]benzo[b]thiophene-(S-oxide) (HPLC: content 99%, HPLC: total amount of related substances 0.35%), and mix until a homogeneous mixture is obtained. Add 10% Pd/C (5.6 g mixed with 22 ml of water) and wash with 5 ml of water. The resulting crude mixture is evacuated and filled with hydrogen at a pressure of 60 psi. The reaction temperature is set to 30 °C. After 2 hours, the next amount of 10% Pd/C (5.6 g) is added along with 30 ml of water. Hydration at a temperature of 30 °C and a pressure of 60 psi is carried out for the next 22 hours. An additional amount of 4.40 g of 10% Pd/C with 30 ml of water is added, and hydration is carried out at 60 psi and a temperature of 30°C for an additional 2.5 hours. The catalyst is removed by filtration, and the pH of the filtrate is adjusted to 7.24 using a 50% sodium hydroxide solution. Sodium chloride (8.66 g) dissolved in water (18 ml) was added, and the two-layer solution was stirred for 30 minutes. The layers are separated and the aqueous layer is re-extracted with 50 ml of THF. The organic layers are combined and concentrated by distillation at atmospheric pressure to a volume of 50 ml. At a temperature of 24 °C, methanol (180 ml) was added to the concentrate over 1 hour. The resulting crystalline crude mixture was stirred for 30 minutes at a temperature of 24 °C, then cooled to 0 °C and stirred for 1 hour. The solid is isolated by filtration, and washed with 39 ml of water and 39 ml of methanol, after which it is dried under vacuum overnight at a temperature of 50 °C. The yield is 15.52 g (67.8 %).

Part of the product described above (10 g) is recrystallized from THF and water according to the procedure described in Example 2.

Terms used

The term "effective amount" herein refers to the amount of compound F-I required to prevent the conditions (described herein) or their adverse effects. When F-I is co-administered with an estrogen, progestin, aromatase inhibitor, LHRH analog, or AChE inhibitor, the term “effective amount” also refers to the amount of such substance capable of producing the intended effect.

The terms "inhibiting (preventing)" and "inhibiting (preventing)" include their general meanings, e.g. prevention, prohibition, containment, reduction, amelioration, slowing down, halting, i.e. prevention of disease progression, i.e. the severity of pathological conditions, i.e. their health consequences (described here).

The terms "prevention", "prevention of what", "prophylaxis", "prophylactic" and "prevent" are used interchangeably herein and refer to reducing the likelihood that a patient receiving Compound F-I will become ill. , that is, the development of any of the aforementioned pathological conditions, or the appearance of their consequences.

The terms "estrogen deficiency" and "estrogen deficiency" refer to a condition that can be naturally caused or clinically induced, in which a woman cannot produce a sufficient amount of endogenous estrogen hormones that are necessary to maintain functions that depend on estrogen, such as menstruation. , homeostasis of bone mass, nervous function, state of the cardiovascular system, etc. Such estrogen-deficient conditions increase (but are not limited to) menopause, as well as surgical or chemical ovariectomies, including its functional equivalents, e.g. drugs containing an aromatase inhibitor, GnRH agonists or antagonists, ICI 182780, etc. Diseases associated with estrogen deficiency include (but are not limited to) bone loss, osteoporosis, cardiovascular disease, and hyperlipidemia.

The term "estrogen" as used herein includes steroids that have estrogenic activity, such as, for example, 17β-ethynyl estradiol, estrone, conjugated estrogen (Premarin®), equine estrogen 17β-ethinyl estradiol, etc. The most suitable estrogen-based compounds are Premarin® and Noretilnodrel.

The term "progestin" as used herein includes compounds having progesterone activity, such as eg progesterone, norethylnodrel, nongestrel, megestrol acetate, norethindrone, etc. Norethindrone is the most preferred progestin-based compound.

The term "aromatase inhibitor" includes herein compounds capable of inhibiting aromatase, e.g. commercially available inhibitors such as aminoglutamide (CYTANDREN®), anastrazole (ARIMIDEX®), letrozole (FEMARA®), formestane (LENATRON®), exemestane (AROMASIN®) and similar.

The term "LHRH analog," as used herein, refers to a luteinizing hormone-releasing hormone analog that inhibits estrogen production in premenopausal women, including, for example, goserlin (ZOLADEX®), leuprolide (LUPRON®), and the like.

The term "AChE inhibitor" as used herein includes compounds that inhibit acetyl choline esterase, eg, physostigmine salicylate, tacrine hydrochloride, doneprezil hydrochloride, and the like.

The term "ChAT regulation" refers to an increase in the enzymatic activity of the ChAT compound, eg, it supports the conversion of choline to acetylcholine. This assistance includes increasing the efficiency and/or speed of the reaction of ChAT and choline and/or increasing the amount of the ChAT compound present at the site of action. This increase in the enzyme present may be caused by gene regulation or some other synthetic level of enzyme formation and/or a decrease in enzyme deactivation and metabolism.

Selected control procedures

General Rat Preparation Procedure: 75-day-old (unless otherwise specified) female Sprague Dawley rats (weight 200 to 225 g) were obtained from Charles River Laboratories (Portage, MI). The animals are subjected to bilateral ovariectomy (OVX), or they are exposed to Sham's surgery at the aforementioned institution "Charles River Laboratories", and after one week they are shipped on request. After arrival, they are placed in metal hanging cages in groups of 3 or 4 animals per cage, and have free access to food (calcium content is about 0.5%) and water for one week. The room temperature is maintained in the range of 22.2 °C ± 1.7 °C with a minimum humidity of 40%. The photoperiod in the room was divided into 12 hours of light and 12 hours of darkness.

Tissue sampling after the dosed diet: After one week in which the adaptation procedure to the environment was carried out (that is, two weeks after OVX), the daily dosing of compound F-I begins. 17α-ethynyl estradiol or F-I is administered orally (unless otherwise decided) in the form of a 1% carboxymethylcellulose suspension or in the form of a 20% cyclodextrin solution. The animals receive a daily dose for 4 days. After the dosed diet, the animals are weighed and anesthetized with a mixture of ketamine and xylazine (2:1, v:v), and a blood sample is taken by cardiac puncture. After that, the animals are sacrificed by suffocation with the help of CO2, the uterus is removed by means of an incision along the medial line, and the wet weight of the uterus is determined. 17α-ethynyl estradiol was purchased from Sigma Chemical Co., St. Louis, MO.

Cardiovascular diseases/hyperlipidemia

Blood samples are allowed to clot at room temperature for 2 hours. The serum is obtained after centrifugation for 10 minutes at a speed of 3000 revolutions/min. Cholesterol in serum is determined using the procedure for determining the content of the company Boehringer Mannheim Diagnostics. Briefly, cholesterol oxidizes to cholest-4-en-3-one and hydrogen peroxide. Hydrogen peroxide further reacts with phenol and 4-aminophenazone in the presence of peroxidase, which results in the formation of a p-quinone imine color, which is read spectrophotometrically at 500 nm. After that, the cholesterol concentration is determined with the help of a standard curve. The entire process of content determination is automated (a Biomek automated workstation is used).

Uterine eosinophil peroxidase (EPO) content

The above-mentioned uteri are stored at a temperature of 4 °C until the time of enzymatic analysis. Then the uteri are homogenized in 50 volumes of 50 mM Tris buffer (pH about 8.0) containing 0.005% Triton X-100. After addition of 0.1% hydrogen peroxide solution and 10 mM O-phenylenediamine (final concentration) to Tris buffer, the increase in absorbance is monitored for one minute at 450 nm. The presence of eosinophils in the uterus indicates the estrogenic effect of the compound. The maximum speed in the interval of 15 seconds is determined on the initial, linear part of the reaction curve.

Control procedure to prevent loss of bone mass (osteoporosis)

Following the general preparation procedure described in the previous part of the text, rats are treated daily for 35 days (6 rats in one experimental group) and sacrificed on the 36th day by suffocation using carbon dioxide. A period of time of 35 days is sufficient for the maximum decrease in bone density (measured and described here). At the time of liquidation, the uteri are removed, separated from the nonessential, extraneous tissue, and the liquid content is discarded before the wet mass is determined to confirm the estrogen deficiency associated with total ovariectomy. Uterine mass is routinely reduced by about 75% as a result of ovariectomy. After that, the uteri are placed in 10% neutral formalin buffer in order to perform the entire histological analysis.

The right femurs are removed, and digitized X-rays are produced that are analyzed using an analysis program (NIH image) on the distal metaphysis. The proximal aspect of the tubules of these animals is also recorded by quantitative computed tomography. According to the above procedure, F-I or ethinyl estradiol (EE2) is administered orally to test animals via a 20% solution of hydroxypropyl β-cyclodextrin. F-I is also useful in combination with estrogen and progestin.

MCF-7 proliferation content

MCF-7 breast adenocarcinoma cells (ATCC HTB 22) are maintained in MEM (minimum essential medium, without phenol red, Sigma, St. Louis, MO) supplemented with 10% fetal bovine serum (FBS) (V/V) , L-glutamine (2 mM), sodium pyruvate (1 mM), HEPES {(N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid], 10 mM}, non-essential amino acids and bovine insulin (1 ug/ml) (maintenance medium).Ten days before assay, MCF-7 cells were plated in assay medium containing 10% dextran on charcoal, and pure fetal bovine serum (DCC-FBS ) instead of 10% FBS to deplete internal stores of steroids. MCF-7 cells were removed from the maintenance dishes using cell dissociation medium (Ca2+/Mg2+ without HBSS (without phenol red) supplemented with 10 mM HEPES and 2 mM EDTA).Cells are washed twice with assay medium and adjusted to 80,000 cells/ml.About 100 ml (8,000 cells) are added to flat bottom containers (Costar 3596), and in select at 37 °C in a humidified incubator with 5% CO2, for 48 hours, so that after the transfer, cell growth and equilibration occur. Prepare a series of solutions containing the medicinal substance or DMSO as a control diluent in the test medium, and transfer 50 ml to triplicate microcultures, after which 50 ml of the test medium is added to the final volume of 200 ml.

After a further 48 hours of incubation at 37 °C in a humidified incubator with 5% CO2, the microcultures were pulsed with tritium-enriched thymidine (1 uCi per container) for 4 hours. The growth of the cultures is stopped by freezing at -70 °C for 24 hours, followed by immersion and "harvesting" (collection) of the microcultures using the so-called "Skatron semi-automatic cell harvester". Samples are counted by liquid scintillation using a Wallac BetaPlace β counter.

Inhibition of DMBA-induced breast cancer

Estrogen-dependent mammary tumors were produced in female Sprague-Dawley rats (ordered from Harlan Industries, Indianapolis, Indiana). Around the 55th day of life, rats are given a single dose of 20 mg of 7, 12-dimethylbenz[a]anthracene (DMBA) orally. After about 6 weeks after DMBA administration, the mammary glands are examined by palpation at weekly intervals in order to detect the formation of tumors. Each time one or more tumors appear, the shortest and longest diameters of each tumor are measured using a caliper, the measurements are recorded, and the animals are selected for the experiment. An attempt is made to uniformly distribute the different tumor sizes into the treated or controlled groups, so that the average tumor size is equally distributed among the tested groups. Control groups and tested groups (for each experiment) contain 5 to 9 animals.

F-I is given either intraperitoneally in the form of injections with 2% acacia, or orally. The orally administered compound was either dissolved or suspended in 0.2 ml of corn oil. Each treatment, including control treatments with acacia and corn oil, was administered once daily to each test animal. After the initial measurement of the tumors, and the selection of the animals on which the experiment is performed, the tumors are measured once a week according to the previously described method. Treatments and measurements on animals are carried out over 3 to 5 weeks. Within this time period, the final surface area of the tumor is determined. For each compound and control treatment, the change in mean tumor area is determined.

Uterine Fibrosis Testing Procedures

Test 1: F-I is given to women (between 3 and 20) who have uterine fibrosis. The amount of compound given is from 0.1 to 1000 mg per day, and the duration of administration is 3 months. Women are observed during administration of the compound, and up to 3 months after discontinuation of administration, in order to observe the effect on uterine fibrosis.

Test 2: The same procedure as for Test 1 is used, with the exception that the administration time was 6 months.

Test 3: The same procedure as for Test 1 was used, with the exception that the administration time was 1 year.

Test 4: Prolonged estrogen stimulation is used to induce leiomyoma in sexually mature guinea pigs. The animals are dosed with estradiol 3-5 times a week in the form of injections for 2-4 months, or until tumor growth occurs. Treatment consists of daily administration of compound F-I or vehicle for 3-16 weeks, after which the animals are sacrificed and the uteri collected and analyzed to determine whether tumor regression has occurred.

Test 5: Human leiomyoma tissue is implanted into the peritoneal cavity and/or uterine myometrium of sexually mature, castrated, female nude mice. Exogenous estrogen is administered to stimulate the growth of the explanted tissue. In some cases, the extracted tumor cells are cultured in vitro before implantation. Treatment in which F-I or vehicle is administered includes daily gastric lavage for 3-16 weeks, and the implants are removed and their growth or regression is measured. At the time of sacrifice, the uteri are removed to assess the condition of the organs.

Test 6: Tissue from fibroid tumors of human uterus is sampled and maintained in vitro, in the form of primary unaltered culture. Surgical specimens are pushed through a sterile sieve, or alternatively, separated from the surrounding tissue in order to prepare a suspension of the same type of cells. Cells are grown in a medium containing 10% serum and an antibiotic. Growth rate is determined in the presence and absence of estrogen. The cells are tested to determine their ability to produce the complement compound C3, as well as their response to growth factors and growth hormones. In vitro, cultures are tested for their proliferative response following treatment with progestin, GnRH, F-I and vehicle. Steroid hormone receptor levels are assayed daily to determine whether important cell characteristics are maintained in vitro. Tissue from 5-25 patients is used.

Test 7: The ability of compound F-I to inhibit proliferation of estrogen-stimulated leiomyoma-derived ELT cell lines is measured according to the protocol described in Fuchs-Young et al., "Growth inhibition of estrogen-stimulated uterine leiomyomas by selective estrogen receptor modulators", Mol. Car., 17(3) : 151-159 (1996), whose conclusions are included here in the literature review.

Endometriosis testing procedures

Test 1: From 12 to 30 adult CD female rats that are related to each other are used as experimental animals. They are divided into three equal groups. The mating cycle of all animals is observed. On the day of peak fertility, a surgical procedure is performed on each female. For females in each group, the left horn of the uterus is removed, cut into small squares, which are then loosely sutured together in different places near the mesenteric blood flow. Females from the 2nd group have their ovaries removed. Starting from the day after surgery, animals in groups 1 and 2 are given intraperitoneal injections of water for 14 days, while animals in group 3 are given intraperitoneal injections of compound F-I at a concentration of 1.0 mg per kilogram of body weight for the same period of time. period. After 14 days of treatment, all animals are sacrificed, and endometrial explants, adrenal glands, the remaining part of the uterus and ovaries (where possible) are removed and then prepared for histological examination. Ovaries and adrenal glands are weighed.

Test 2: From 12 to 30 adult CD female rats that are related to each other are used as experimental animals. They are divided into two equal groups. The mating cycle of all animals is observed. On the day of peak fertility, a surgical procedure is performed on each female. For females in each group, the left horn of the uterus is removed, cut into small squares, which are then loosely sutured together in different places near the mesenteric blood flow. After approximately 50 days after surgery, animals in the 1st group were given intraperitoneal injections of water for 21 days, while animals in the 2nd group were given intraperitoneal injections of compound F-I at a concentration of 1.0 mg per kilogram of body weight for the same period of time. After the 21st day of treatment, all animals are sacrificed, endometrial explants and adrenal glands are removed and weighed. Measured explants indicate growth. Fertility cycle is observed.

Test 3: Autographs of endometrial tissues are used to induce endometriosis in rats and/or rabbits. A bilateral oophorectomy is performed on females of reproductive age, and estrogen is given exogenously to ensure a specific constant hormone level. Autologous endometrial tissue is implanted into the peritoneal cavity in 5-150 animals, with estrogen administered to stimulate growth of the explanted tissue. The treatment containing the compound described in this invention is based on daily gastric lavage (by which the compound is introduced) for 3-16 weeks, after which the implants are removed and their growth or regression is measured. At the time of sacrifice, the intact uterine horn is removed to assess the condition of the endometrium.

Test 4: Human endometrial tissue, created as a result of injury, is implanted into the peritoneal cavity of sexually mature, castrated, female nude mice. Exogenous estrogen is administered to stimulate the growth of the explanted tissue. In some cases, the extracted tumor cells are cultured in vitro before implantation. Treatment in which F-I or vehicle is administered includes daily gastric lavage for 3-16 weeks, and the implants are removed and their growth or regression is measured. At the time of sacrifice, the uteri are removed to assess the condition of the organs.

Test 5: Human endometrial tissue, formed as a result of injury, is sampled and maintained in vitro, in the form of a primary unchanged culture. Surgical specimens are pushed through a sterile sieve, or alternatively, separated from the surrounding tissue in order to prepare a suspension of the same type of cells. Cells are grown in a medium containing 10% serum and an antibiotic. Growth rate is determined in the presence and absence of estrogen. The cells are tested to determine their ability to produce the complement compound C3, as well as their response to growth factors and growth hormones. In vitro, the cultures are tested for their proliferative response, followed by treatment with progestin, GnRH, F-I and vehicle. Steroid hormone receptor levels are assayed daily to determine whether important cell characteristics are maintained in vitro. Tissue from 5-25 patients is used.

Central nervous system disorders including Alzheimer's disease

Estrogens, such as 17β-estradiol, regulate gene transcription by binding to estrogen receptors (ER) located in the cytoplasm of certain cell populations. Activation of the estrogen receptor ligand is a necessary prerequisite for the nuclear transfer of the complex, where binding to the palindromic DNA sequence, which contains 13 base pairs (estrogen-responsive element or ERE), begins with the assembly of the transcription apparatus, which culminates in the activation of target genes. A variety of estrogen-regulated genes have been identified. This includes cytoskeletal proteins, neurotransmitters of biosynthetic and metabolic enzymes and receptors, as well as other hormones and neuropeptides. ERE has been identified in many estrogen-responsive genes, including vitellogenin, c-fos, prolactin, and luteinizing hormone.

ERE-like sequences were identified in p75ngr and trkA (which was significant for the central nervous system), both of which serve as neurotrophin signaling molecules: neural growth factor (NGF), brain-derived neural growth factor (BDNGF), and neurotrophin-3 .

BDNF as well as NGF have been shown to enhance the survival of cholinergic neurons in culture. It has been found that if the interaction between neurotrophins and estrogen is important for the development and survival of the basal forebrain nerves (where degenerative changes occur in Alzheimer's disease), then clinical conditions in which there is a lack of estrogen (such as after menopause) may contribute to the disappearance of these nerves. .

The following experiment is performed on ovariectomized rats (according to the previously described procedure) in order to determine the similarities and/or differences between compound F-I and estrogen that affect gene expression in different areas of the brain. 6-week-old rats are dosed daily with estradiol benzoate, F-I or vehicle (control) via subcutaneous injections (0.03 mg/kg). After the five-week treatment, the animals are sacrificed, their brains are removed, and the ammonites are connected by microdissection. Amon's horns are quickly frozen at -70 °C in liquid nitrogen. Total RNA is prepared from combined tissues obtained by appropriate treatment, as well as from control groups, and reverse transcribed with the help of a 3' oligonucleotide primer selected for a special mRNA (pli-A+) population. Polymerization chain reactions (PCR) are performed in a mixture consisting of: randomly selected 5' oligonucleotides (the length of which is 10 base pairs, 150 in total), reaction buffer, Taq polymerase and 32PdTCP.

After 40 amplification cycles, the reaction products are separated on a 6% TBE-urea gel, dried and exposed to X-ray film. The resulting mRNA samples are compared within the treated groups.

Use of F-I together with estrogen

Peri- and postmenopausal women often receive hormone replacement therapy (HRT) to counteract the negative effects associated with reduced circulating endogenous estrogen, eg to affect hot flashes. But HRT is associated with an increased risk of certain cancers, including breast and uterine cancer. F-I can be administered together with HRT to prevent these risks.

Use of F-I together with an aromatase inhibitor

By definition, the ovaries of postmenopausal women are not functioning. The only source of estrogen is through the conversion of adrenal androgens into estrogens by the enzyme aromatase, which is found in peripheral tissues (including adipose tissue, muscle, as well as the breast tumor itself). Therefore, drugs that inhibit aromatase (aromatase inhibitors) deplete circulating estrogen in postmenopausal women. Estrogen deficiency due to inhibition of aromatase represents an important option in the treatment of patients with metastasized breast cancer. During aromatase inhibitor therapy, the lack of circulating estrogen can cause negative, unintended side effects, eg affecting serum lipid levels. F-I can be used to prevent these negative effects.

Use of F-I together with an LHRH analogue

Continuous exposure to LHRH analogues prevents estrogen production in premenopausal women due to desensitization of the pituitary gland, which then no longer stimulates the ovaries to produce estrogen. The clinical effect is "medical oophrectomy". It is reversible after cessation of LHRH analogue action. During LHRH analogue therapy, the lack of circulating estrogen can cause negative, unintended side effects, eg affecting serum lipid levels. F-I can be used to prevent these negative effects.

Increased levels of acetyl choline

It is known that patients suffering from Alzheimer's disease have a noticeably lower level of cholinergic nerves in the horn than people who do not suffer from this disease. In these patients, the progressive disappearance of these cholinergic nerves seems to be reflected in the progressive loss of memory as well as cognitive functions. It is believed that one of the reasons for the disappearance of these nerves is the loss or reduced function of the neurotransmitter - acetylcholine.

The level of acetylcholine in the nerve is determined based on where the balance is between its biosynthesis and biodegradation. The enzyme choline acetyltransferase (ChAT) is primarily responsible for its synthesis, and the enzyme acetylcholinesterase (AChE) for its degradation.

In order to determine the effect of compound F-I on ChAT levels, the following experiment was performed: Following the general procedure for preparing rats (described in the previous section of the text), 40 rats received a daily dose of compound F-I via subcutaneous injections or orally in the amount of 3 mg/kg in a vehicle containing cyclodextrin, estradiol benzoate in the amount of 0.03 to 0.3 mg/kg per day, or received a control vehicle. Animals are treated for 3 to 10 days. There are 20 animals in each group with a special diet. At appropriate time intervals, the animals are sacrificed and their brains are removed. Certain parts of the brain are homogenized and examined. Homogeneous mixtures obtained from Adon's horns and frontal cortex are processed, and ChAT activity is determined using a radiolabeled acetylcholine biosynthesis test. This procedure can be found in the article by Schoepp et al., J. Neural Transmiss., 78 183-193, 1989, the conclusions of which are incorporated herein by reference.

As expected, ChAT levels were decreased in OVX animals (>50%, p<0.001) compared to sham controls.

In other compositions of the present invention, F-I is used together with an AChE inhibitor. Using an AChE inhibitor increases the level of acetylcholine by blocking its degradation via AChE.

Benign prostatic hyperplasia (BPH)

For more information on the relationship between estrogen action and the treatment of BPH and prostate cancer, see PCT application no. WO 98/07274, date of international publication: 15.10.1998.

In the experiments described below, the ability of compound F-I to bind to estrogen receptors in several human prostate cancer cells was examined.

Lysates of LNCaP, Du-45, and PC-3 human prostate cancer cell lines were prepared in TEG medium including 50 nM Tris HCl, pH 7.4, 1.5 mM ethylenediamine tetraacetic acid, (EDTA) 0.4 M KCl, 10 % solution of glycerol, 0.5 mM 2-ME and 10 mM sodium molybdate, as well as protease inhibitors - pepstatin (1 mg/ml), leupeptin (2 mg/ml), aprotinin (5 mg/ml) and phenylmethylsulfonyl fluoride (PMSF, 0.1 mM) (TEGP).

Cell lysates are centrifuged, and the pellet is resuspended in cold TEGP (1 ml TEGP/100 mg pellet), and placed in an ultrasonic bath (Branson Model 450) for sonication for 30 seconds (cycling efficiency 70%, product 1.8). The lysate is precipitated after centrifugation for 15 minutes at a speed of 10,000 G at a temperature of 4 °C. Immediately after centrifugation, the supernatant is separated and used immediately or stored at -70 °C.

Competitive binding assay: The binding buffer is TEG in which 0.4 M KCl has been replaced by 50 mM NaCl, and 1 mg/ml ovalbumin (TEGO) has been added to it. F-I is diluted to 20 nM with TEGO. Triplicate series of solutions are made from this starting solution. The test is carried out in rounded polypropylene microvessels in triple microtanks. 35 ml of tritiated 17β-estradiol (0.5 nM, specific activity of 60.1 Ci/nmol, DuPont-New England Nuclear, Boston, MA) and 35 ml of chilled competition compound (0.1 nM - 5 mM) or TEGO, followed by 70 ml of MCF-7 cell line lysate with an incubation process at 4 °C for 5 minutes, with shaking.

The vessels are incubated for 24 hours at a temperature of °C, after which 70 ml of dextran-coated charcoal (DCC) is added to each vessel. Then the dishes are centrifuged for 10 minutes at 1500 G, at a temperature of 4 °C. The supernatant is separated from each container and transferred to a flexible microvessel for scintillation counting using a Wallac Micobeta Model 1450 meter. Radioactivity is expressed as disintegrations per minute (DPM) after correction for measurement efficiency (35-40%) and background. Additionally, total activity and total activity + DCC are checked to determine the lowest limit for DCC extraction activity. The result of the competitive binding test is expressed as the mean value of binding percentage (% binding) + / - standard deviation, and the expression formula is:

DPM tested compound - DPM total activity + DCC

% binding = ------------------------------------------------------------- ------ x 100

DPM untested compound - DPM total activity + DCC

Breast cancer prevention

This invention also relates to the administration of F-I to patients at risk of developing "de novo" breast cancer. The term "de novo" here means the absence of transformation or metamorphosis of normal breast cells into cancerous or malignant cells in the first stage. But such a transformation can happen gradually in the same or daughter cells in the evolutionary process, or it can happen in a single, decisive case. This de novo process is in contrast to metastasis, colonization or spread of already transformed or malignant cells from primary tumor sites to new areas.

A person who does not have any particular risk of developing breast cancer can get de novo breast cancer, where there is no reason to suspect the development of a potential disease with a higher than normal risk. Such people have never been diagnosed with cancer. The greatest risk that contributes to the development of breast cancer is a personal history of the disease or a previous occurrence of the disease, even if it is a withdrawal without evidence of its presence. The next risk in the occurrence of the disease is family history.

Inducing breast tumors in rats by administering the carcinogen N-nitroso-N-methylurea is a well-accepted procedure for testing breast cancer, and is considered suitable for analyzing the effects of chemopreventive substances.

In two separate studies, 55-day-old female Sprague-Dawley rats were given intravenously (Study 1) or intraperitoneally (Study 2) a dose of 50 mg of N-nitroso-N-methylurea per kilogram of body weight one week prior to administration of a diet different amounts of compound F-I, (Z)-2-[4-(1,2-diphenyl-1-butenyl)phenoxy]-N,N-dimethylethanamine base (tamoxifen base), or a control sample are mixed.

In Test 1, dietary doses of 60 mg/kg dietary ration and 20 mg/kg dietary ration were roughly divided into comparable doses of 3 and 1 mg/kg body weight of experimental animals.

In Trial 2, dietary doses in the amount of 20, 6, 2 and 0.6 mg/kg of dietary ration were roughly divided into comparable doses of 0.3 and 0.1 and 0.03 mg/kg of body weight of experimental animals.

Rats are observed to determine toxicity, and weighed and palpated once a week to determine tumor formation. Animals are sacrificed after 13 weeks (Study 1), or 18 weeks (Study 2), after which the presence of tumors is confirmed and weighed at necropsy.

Preparations

If the term "pharmaceutical" is used here as an adjective, then it means an essentially non-lethal substance administered to mammals. The term "pharmaceutical preparation" means that the carrier, diluent, excipients, and active substance (active substances) must be compatible with the other ingredients of the preparation, so that they are non-lethal by themselves for the patient.

It is convenient to make a preparation containing compound F-I before administration. The choice of the type of preparation depends on the decision of the doctor, who must take into account the same factors that are involved in the process of determining the effective amount.

The total amount of active substances in such preparations accounts for from 0.1 to 99.9% of the mass of the preparation. It is convenient that the preparation does not contain more than two active substances. This means that compound F-I is combined with another active substance selected from the group consisting of estrogen, progestin, aromatase inhibitor, LHRH analog, and AChE inhibitor. The most favorable are those preparations that contain only F-I as an active substance.

The pharmaceutical preparations of this invention are prepared according to known procedures with the use of well-known and available ingredients. For example, F-I, alone or together with estrogen, progestin, aromatase inhibitor, LHRH analog, and AChE inhibitor is mixed with common excipients, diluents or carriers, and preparations in the form of tablets, capsules, suspensions, solutions, injections, aerosols are produced , powder, etc.

The pharmaceutical mixtures of this invention for parenteral administration include sterile aqueous or non-aqueous solutions, dispersions, suspensions, and emulsions, as well as sterile powders that are reconstituted into sterile solutions or suspensions immediately before administration. Examples of suitable sterile aqueous and non-aqueous carriers, diluents, solvents or vehicles include water, physiological saline, ethanol, polyols (such as glycerol, propylene glycol, poly(ethylene glycol), etc.), as well as their respective mixtures, then vegetable oils (such as olive oil) , and organic esters that may be introduced into the body (such as ethyl oleate). The appropriate density must be maintained, for example, by using appropriate coatings (such as lecithin), and by maintaining the appropriate particle size in dispersions, as well as by using surfactants.

Parenteral mixtures may also contain excipients such as preservatives, wetting agents, emulsifiers, and dispersing agents. Prevention of the action of microorganisms is ensured by the inclusion of antibacterial and fungicidal agents, such as paraben, chlorobutanol, phenol sorbic acid, etc. It is also convenient to include isotonizing agents such as sugar, sodium chloride, etc. Prolonged absorption of preparations in the form of injections can be achieved by including substances that delay absorption, such as aluminum monostearate and gelatin.

In some cases, in order to prolong the effect of the drug, it is convenient to slow down the absorption of the drug given subcutaneously or intramuscularly in the form of an injection. This can be achieved by using a liquid suspension of a crystalline substance that dissolves poorly in water, or by dissolving or suspending the medicinal substance in an oily vehicle. In the case of subcutaneous or intramuscular injection of a suspension containing such a form of medicinal substance that is poorly soluble in water, the rate of absorption of the drug depends on its release rate.

Preparations in the form of injections with prolonged effect, containing compound F-I, are produced using microencapsulated matrices of the medicinal substance in biodegradable polymers such as poly(lactic acid), poly(glycolic acid), copolymers of lactic and glycolic acids, poly(orthoesters), and poly(anhydrides) of these substances which are described in professional literature. Depending on the ratio of medicinal substance and polymer, as well as on the properties of the particular polymer used, the rate of drug release can be controlled.

Preparations in the form of injections are sterilized, for example, by filtering through filters that retain bacteria, or by pre-sterilizing the compounds that make up the mixture before mixing them, regardless of whether the procedure is carried out in production, or immediately before administration (as in the example with the syringe packaging with double chamber).

Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. In such solid dosage forms, compound F-I is mixed with at least one inert, pharmaceutical carrier such as, for example, sodium citrate or dicalcium phosphate and/or (a) fillers or extenders such as starches, sugars (including lactose and glucose), mannitol and salicylic acid , (b) binding substances such as carboxymethylcellulose and other cellulose derivatives, alginate, gelatin, poly(vinylpyrrolidine), sucrose and acacia, (c) humectants such as glycerol, (d) disintegrating substances such as agar-agar, calcium carbonate, sodium bicarbonate, potato or tapioca starch, alginic acid, silicate and sodium carbonate, (e) humectants such as glycerol, (f) retarding agents such as paraffin, (g) substances that accelerate absorption such as quaternary ammonium compounds (h) humectants such as cetyl alcohol and glycerin monostearate, (i) absorbers such as kaolin and bentonite and (j) lubricants such as talc, calcium stearate, magnesium stearate solid poly(ethylene glycols ), sodium lauryl sulfate and their mixtures. With capsules, tablets and pills, the dosage form may also contain buffers.

Solid mixtures similar in type may also include filler in soft or hard gelatin capsules with the use of excipients such as lactose, as well as high molecular weight poly(ethylene glycol) and the like.

Solid dosage forms, such as tablets, dragees, capsules, pills, and granules, may also be prepared with a coating or shell, such as enteric coatings or other coatings well known in the pharmaceutical industry. Envelopes may contain opaque substances, or substances that release the active substance (active substances), this means the digestive tract in particular, such as acid-soluble envelopes, where the active substance (active substances) are released in the stomach, or soluble envelopes in a basic environment, during which the active substance (active substances) are released in the intestines.

The active substance(s) can be in the form of microcapsules that are inside envelopes characterized by controlled release, as well as with microcapsules that are part of a pill or capsule.

Liquid dosage forms for oral administration of Compound F-I include solutions, emulsions, suspensions, syrups and elixirs. In addition to the active substance, liquid preparations may contain inert diluents that are common in the pharmaceutical industry, such as water or other pharmaceutical solvents, substances that promote solubility and emulsifiers, such as ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol. , 1, 3-butylene glycol, dimethyl formamide, oils (especially cottonseed oil, corn oil, germ oil, olive oil, castor and sesame oil), glycerol, tetrahydrofurfuryl alcohol, poly(ethylene glycols), sorbitol fatty acid esters, as well as their mixtures.

In addition to inert diluents, liquid oral preparations may contain auxiliary substances such as moisturizers, emulsifiers, suspending substances, sweeteners, aromas and fragrances.

In addition to the active substance, liquid suspensions can also contain suspension substances, such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan ester, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, as well as their mixtures.

Formulations for rectal or intravaginal administration are prepared by mixing compound F-I with suitable non-irritating excipients such as cocoa butter, polyethylene glycol, or any suppository wax that is solid at room temperature but liquid at body temperature, meaning it melts. in the large intestine or vaginal cavity, during which the active substance (active substances) are released. The compounds are dissolved in melted wax, and shaped as desired, after which they are allowed to harden, which results in the formation of the final preparation in the form of suppositories.

F-I can also be administered in the form of liposomes. As known in the art, liposomes are derived from phospholipids or other lipid substances. Liposomal preparations consist of mono- or multilamellar hydrated liquid crystals that are dispersed in an aqueous medium. Any lipid that is non-toxic, that is pharmaceutically acceptable and that can be metabolized in the body and that can form liposomes can be used. The mixtures of this invention that come in the form of liposomes can, in addition to compound F-I, also contain stabilizers, auxiliary substances, preservatives, etc. The most suitable lipids are phospholipids and phosphatidyl choline (lecithin), regardless of whether they are of natural or synthetic origin.

Methods of preparing liposomes are known in the art and are described, for example, in Prescott, Ed., Methods in Cell Biology, Volume XIV, Academic Press, New York, N.Y. (1976), pp. 33 et seq.

The following examples of preparations serve as an illustration and therefore should not limit the scope of this invention.

Preparation 1: Gelatinous capsules

[image]

The ingredients of the preparation described above can be changed according to the necessary modifications.

The tablet preparation is prepared using the following ingredients:

Preparation 2: Tablets

[image]

The ingredients are mixed and compressed into a tablet.

Preparation 3: Tablets containing respectively about 10 and 50 mg of compound F-I can be prepared in the following way:

[image]

The ingredients are mixed and compressed into a tablet.

In an alternative way, as follows, tablets containing from 2.5 - 1000 mg of compound F-I are prepared:

Preparation 4: Tablets

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F-I, starch and cellulose are sieved (pore size 45, U.S.) and mixed well. The polyvinylpyrrolidone solution is mixed with the resulting powder and sieved through a sieve (pore size 14, U.S.). The granules produced in this way are dried at a temperature of 50-60 °C and sieved again through a sieve (pore size 18, U.S.). Sodium carboxymethylcellulose, starch, magnesium stearate and talc, previously sieved through a sieve (pore size 60, U.S.), are added to the granules, and after mixing, the resulting mixture is compressed in a tablet press, resulting in tablets.

Suspensions containing from 0.1 to 1000 mg of medication in a dose of 5 ml are prepared as follows:

Preparation 5: Suspensions

[image]

The medicinal product is sieved through a sieve (pore size 45, U.S.), and mixed with sodium carboxymethylcellulose and syrup to form a smooth paste. After diluting in a little water, benzoic acid solutions, sweetener and color are added while mixing. After that, add the required amount of water to the prescribed volume.

The aerosol is prepared with the following ingredients:

Preparation 6: Aerosol

[image]

F-I is mixed with ethanol, and the mixture is added to a certain amount of propellant 22, cooled to 30 °C, after which it is transferred to the filling apparatus. The required amount is then filled into steel containers and diluted with the remaining amount of propellant. Valves are then mounted on the tanks.

Suppositories are prepared in the following way:

Preparation 7: Suppositories

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F-I is sieved through a sieve (pore size 60, U.S.), and suspended in pre-melted (with minimal heating) saturated glycerides of fatty acids. After that, the mixture is poured into suppository molds with a nominal capacity of 2 g and cooled.

The intravenous preparation is prepared in the following way:

Preparation 8: Intravenous solution

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The solution of the above ingredients is given intravenously to the patient at a rate of about 1 ml per minute.

Preparation 9: Combined capsule I

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Preparation 10: Combined capsule II

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Preparation 11: Combined capsule III

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Dosage

The specific dose of compound F-I to be administered in accordance with the present invention is determined according to the particular circumstances typical of each individual situation. These circumstances include the method of administration, the patient's medical history, the pathological conditions or symptoms being treated, the severity of the condition/symptoms being treated, as well as the age and gender of the patient.

In general, an effective minimum daily dose of compound F-I is 1, 5, 10, 15 or 20 mg. The typical maximum daily dose is 800, 100, 60, 50 or 40 mg. The most typical dose ranges from 15 to 60 mg. The exact dose can be determined, in accordance with the usual practice in medicine, by "titration of the dose", which means that a low dose of the compound is initially administered, which is gradually increased until the moment when the desired therapeutic effect is observed.

Although it may be necessary to titrate the dose to the individual patient, with due attention to the combination therapy discussed in the previous part of the text, typical doses of other active substances are as follows: ethinyl estrogen (0.01-0.03 mg per day) , mestranol (0.05-0.15 mg per day), bound estrogen hormones (e.g. Premarin®, Wyeth-Ayerst; 0.3-2.5 mg), medroxyprogesterone (2.5-10 mg per day), noretininodrel (1.0-10.0 mg per day), nonethindrone (0.5-2.0 mg per day), aminoglutamide (250-1250 mg per day, 250 mg four times per day is preferable), anastrazole (1 -5 mg a day, preferably 1 mg once a day), letrozole (2.5-10 mg a day, preferably 2.5 mg once a day), formestane (250-1250 mg per week, preferably 250 mg twice a week) , exemestane (25-100 mg per day, preferably 25 mg once a day), goserlin (3-15 mg every three months, preferably 3.6-7.2 mg once every three months) and leuprolide (3-15 mg monthly) , more favorable 3.75-7.5 mg once a month).

Method of drug administration

F-I can be administered by various routes, including oral, rectal, transdermal, subcutaneous, intravenous, intramuscular, and intranasal administration. The method of administration of agents based on estrogen and progestin is in accordance with already known methods of administration. F-I, alone or in combination with an estrogen, progestin, or AChE inhibitor, are generally administered by conventional routes.

The pharmaceutical mixtures of this invention can be administered to humans and other mammals (eg, dogs, cats, horses, pigs, etc.), orally, rectally, intravaginally, parenterally, superficially, buccally, sublingually and nasally. The term "parenteral administration" refers herein to routes of administration including intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous, and intra-articular administration by injection or infusion.

Method/length of administration

In most methods of the present invention, F-I is administered continuously 1 to 3 times per day or as often as necessary to deliver an effective amount of compound F-I to the patient. Cyclic therapy can be particularly useful in the treatment of endometriosis or can be used acutely during pain attacks. In case of restenosis, therapy can be limited to short intervals (1-6 months), followed by medical procedures such as angioplasty.

Claims (16)

1. Crystalline compound 6-hydroxy-3-(4-[2-(piperidin-1-yl)ethoxy]phenoxy)-2-(4-methoxyphenyl)benzo[b]thiophene hydrochloride hydrate (F-I) characterized by having X-ray diffraction pattern including the following reflections: 7.9 ± 0.2; 10.7 ± 0.2; 14.9 ± 0.2; 15.9 ± 0.2; 18.3 ± 0.2 and 20.6 ± 0.2° in 2θ; with a copper lamp as a source of radiation. 2. Pharmaceutical preparation, characterized in that it includes the crystalline compound of claim 1; one or more pharmaceutical carriers, diluents, or excipients, and if necessary estrogen, progestin, aromatase inhibitor, LHRH analogue and choline esterase inhibitor (AChE). 3. The preparation from claim 2, characterized in that it includes the crystalline compound from claim 1, one or more pharmaceutical carriers, diluents, or excipients, and estrogen. 4. The preparation from claim 3, characterized in that the estrogen in it comes from the preparation Premarin®. 5. The preparation from claim 2, characterized in that it includes the crystalline compound from claim 1, one or more pharmaceutical carriers, diluents, or excipients, and progestin. 6. The preparation according to claim 5, characterized in that the progestin is selected from the group consisting of norethylnodrel and norethindrone. 7. The preparation from claim 2, characterized in that it includes the crystalline compound from claim 1, one or more pharmaceutical carriers, diluents, or excipients, and an AChE inhibitor. 8. The preparation according to claim 7, characterized in that the AChE inhibitor is selected from the group consisting of physostigmine salicylate, tacrine hydrochloride and donepezil hydrochloride. 9. The preparation from claim 2, characterized in that it includes the crystalline compound from claim 1, one or more pharmaceutical carriers, diluents, i.e. excipients, estrogen, and progestin. 10. The compound of claim 1, characterized in that it is used to prevent pathological conditions selected from the group consisting of: uterine fibrosis, endometriosis, aortic smooth muscle cell proliferation, restenosis, breast cancer, uterine cancer, prostate cancer, benign prostatic hyperplasia, loss bone masses, osteoporosis, cardiovascular diseases, hyperlipidemia, disorders of the central nervous system and Alzheimer's disease. 11. The compound of claim 14, characterized in that it is used to prevent breast cancer. 12. The compound of claim 15, characterized in that it is used in prophylaxis. 13. The compound of claim 14, characterized in that it is used to prevent ovarian cancer. 14. The compound of claim 14, characterized in that it is used to prevent endometrial cancer. 15. The compound of claim 1, characterized in that it is used for the regulation of choline acetyltransferase (ChAT) in mammals. 16. The process for the preparation of the compound from claim 1, characterized in that it includes the crystallization of 6-hydroxy-3-(4-[2-(piperidin-1-yl)ethoxy]phenoxy)-2-(4-methoxyphenyl)benzo[b] thiophene hydrochloride from tetrahydrofuran.