CZ20002716A3 - Novel crystalline form of 6-hydroxy-3-[4-/2-(piperidin-1-yl)ethoxy/phenoxy]-2-(4-methoxyphenyl)benzo[b]thiophene hydrochloride - Google Patents

Abstract

The present invention is directed to a novel crystalline hydrate of 6-hydroxy-3-(4-[2-(piperidin-1-yl)ethoxy]phenoxy)-2-(4-methoxyphenyl)benzo[b]thiophene hydrochloride and uses for same, including inhibition of disease states associated with estrogen deprivation including cardiovascular disease, hyperlipidemia, and osteoporosis; and inhibition of other pathological conditions such as endometriosis, uterine fibrosis, estrogen-dependent cancer (including breast and uterine cancer), prostate cancer, benign prostatic hyperplasia, CNS disorders including Alzheimer's disease, prevention of breast cancer, and up-regulating ChAT.

Description

Technical field

The present invention relates to a novel non-stoichiometric hydrated crystalline form of 6-hydroxy-3- [4- / 2- (piperidin-1-yl) ethoxy / phenoxy] -2- (4-methoxyphenyl) benzo [b] thiophene hydrochloride (FI), a pharmaceutical composition comprising FI; one or more pharmaceutical carriers, diluents or excipients; and to a method of using F-I to inhibit pathological conditions, as well as to use F-I to manufacture medicaments to inhibit these conditions and to up-regulate choline acetyltransferase (ChAT), as well as to manufacture medicaments to up-regulate the enzyme.

BACKGROUND OF THE INVENTION

6-Hydroxy-3- [4- [2- (piperidin-1-yl) ethoxy] phenoxy] -2- (4-methoxyphenyl) benzo [b] thiophene (arzoxifene) hydrochloride was first disclosed in U.S. Pat. No. 5,510,357 and especially described in U.S. Patent No. 5,723,474 (474) and European Patent Application No. 0729956. Arzoxifene is a non-steroidal mixed estrogen antagonist / agonist useful, inter alia, for lowering serum cholesterol and inhibiting hyperlipidemia, osteoporosis, to estrogen dependent cancers including breast and uterine cancers, endometriosis, central nervous system diseases including Alzheimer's disease, aortic smooth muscle cell proliferation, and restenosis.

Arzoxifene is particularly suitable, and it is clinical

evaluated for the treatment of receptor positive metastatic breast cancer; as adjunctive therapy in receptor positive patients following appropriate systemic or local therapy; to reduce relapses of invasive and non-invasive breast cancers; and ductal carcinoma in situ (DCIS). Arzoxifene is also useful in combination with radiotherapy, aromatase inhibitors, luteinizing hormone releasing factor (LHRH) analogs and acetylcholinesterase inhibitors.

Powder X-ray diffraction (XRD), thermogravimetry (TGA), proton magnetic nuclear resonance ( ^Χ Η NMR) and Karl Fischer (KF) analysis of bulk arzoxifene isolated according to US Patent No. 5,723,474 later showed that the material was hydrated , slightly crystalline and contained in its lattice a variable amount of organic volatiles (ethyl acetate).

Weakly crystalline materials are typically less desirable than highly crystalline for processing compositions. In addition, it is generally undesirable to prepare medicaments containing a substantial amount of organic solvents (e.g. ethyl acetate), due to the potential toxicity of the solvent to the recipient thereof and changes in drug strength as a function of the solvent.

Although arzoxifene prepared by the process of US Patent No. 5,723,474 can be used as a medicament, it would be highly desirable and advantageous to find a more crystalline form of arzoxifene that does not contain an organic solvent in its crystal lattice that could be reproducibly and efficiently prepared in an industrial. scale.

* · © © © © © © * * * * * * * * * «« * * * * «« * ««

SUMMARY OF THE INVENTION

The present invention relates to a novel non-stoichiometric hydrated crystalline form of 6-hydroxy-3- [4- / 2- (piperidin-1-yl) ethoxy / phenoxy] -2- (4-methoxyphenyl) benzo [b] thiophene hydrochloride (FI), whose X-ray diffractogram contains the following 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 ° at 2Θ when the record is obtained from a copper radiation source.

The present invention further relates to a pharmaceutical composition comprising F-I; one or more pharmaceutical carriers, diluents or excipients; and optionally estrogen, optionally progestin, optionally aromatase inhibitor, optionally LHRH analogue, and optionally acetylcholinesterase (AChE) inhibitor.

The invention further relates to methods of using FI to inhibit pathological conditions such as uterine fibrosis, endometriosis, aortic smooth muscle cell proliferation, restenosis, breast cancer, uterine cancer, prostate cancer, benign prostate hyperplasia, bone loss, osteoporosis, cardiovascular disease, hyperlipidemia, central nervous system diseases, and Alzheimer's disease; and for the use of FI for the manufacture of a medicament for inhibiting these conditions.

The present invention further relates to a method of using F-I for up-regulating choline acetyltransferase (ChAT) and for using F-I for using F-I for the manufacture of medicaments by up-regulating this enzyme.

• * · · · · · · · · · ·

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a representative S-II differential scanning calorimetry (DSC) / thermogravimetry (TGA) record.

Figure 2 is a representative differential scanning calorimetry (DSC) / thermogravimetry (TGA) F-I plot.

Figure 3 is a representative differential scanning calorimetry (DSC) / thermogravimetry (TGA) F-III plot.

Figure 4 shows the moisture sorption isotherms of 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

The loose arzoxifene prepared as described in U.S. Patent No. 5,723,474 (Example 41, crystallization from ethanol / ethyl acetate, filtration and vacuum drying to constant weight at room temperature) was characterized by an X-ray diffraction pattern and was found to be weakly crystalline.

NMR confirmed that the bulk material contained 6% ethyl acetate.

The crystallization process of US Patent No. 5,723,474 is subsequently modified by adding ethanol to a suspension of crude arzoxifene in ethyl acetate under reflux. After cooling and vacuum filtration, the solid formed by this modified process is highly crystalline

a mixed solvate of arzoxifene ethyl acetate and water (hereinafter S-II), in which the starting material for F-I was later found.

F-I can be prepared by removing ethyl acetate from the crystal lattice of S-II by vacuum drying / heating S-II at elevated temperatures. The time and temperature required to heat S-II to prepare F-I will vary from case to case, but is of the order of 5 days at about 100 ° C. High temperatures are required in this procedure to induce conversion of S-II to F-I, since by suspending S-II in water at room temperature or storing the sample for three weeks at 98% relative humidity, conversion to F-I is not achieved. In addition, even by drying S-II in a conventional high temperature dryer, desolvatization of the materials is not achieved, suggesting that vacuum is also required to displace ethyl acetate from the S-II crystal lattice.

Preferably, F-I is readily prepared and isolated at room temperature by crystallizing arzoxifene (or any polymorph / solvate thereof) from tetrahydrofuran. This crystallization is preferably carried out by initially dissolving arzoxifene in wet tetrahydrofuran (with 1 to 10% by volume water, preferably 2.5 to 7.5% by volume, and most preferably 4.5 to 5.5% by volume), followed by removing said water by means of atmospheric pressure distillation. An example of such distillation is described below in Example 2. When F-I is prepared by this improved crystallization process, a total level of related substances (TRS) of <0.5% can be expected.

A suitable arzoxifene starting material for this crystallization includes S-II, F-III, arzoxifene prepared by the process disclosed in US Patent No. 5,723,474, or any of the following: · But not limited to a mixture thereof. It is not important which form of arzoxifene is started because crystallization from tetrahydrofuran according to the methods described herein results in F-I crystals. For industrial scale synthesis of F-I, it may be advantageous to induce crystallization by inoculation of F-I.

F-III, another non-stoichiometric arzoxifene hydrate, is readily prepared and isolated at room temperature to crystallize arzoxifene (or any polymorph / solvate thereof) from a mixture of isopropyl alcohol (IPA) and water. The volume ratio of water to IPA is generally between about 1: 1 and 9: 1. More preferably, the ratio is between 2.5 and 5.6: 1. Most preferably, the ratio is between 3: 5.6: 1.

1. The ratio of IPA to water is not essential for inducing crystallization, but it affects the yield. For industrial scale synthesis of F-III, it may be advantageous to induce crystallization by F-III. A suitable arzoxifene starting material for the above crystallization comprises S-II,

F-I, arzoxifene prepared as described in U.S. Pat.

723,474, or any mixture thereof, but is not limited thereto.

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

DSC / TGA and XRD methods were used to characterize S-II, F-I and F-III. TGA is often very suitable for distinguishing between different solid forms of materials because the temperature (s) at which a physical change occurs in the material is typically characteristic of a polymorph or solvate. DSC is a technique that is often used to serially test compounds for polymorphism or solvate formation. Finally, XRD is a technique for detecting long-ordered formations in crystalline material.

• ·

Arzoxifene prepared by the process disclosed in US Patent No. 5,723,474 gives X-ray diffractograms with low signal to noise ratios and an elevated base characteristic of weakly crystalline materials. Therefore, comparisons of F-I and F-III are made to the material (S-II) produced by the above-discussed modified process of crystallizing arzoxifene (by adding ethanol to a suspension of arzoxifene in ethyl acetate at reflux). Representative DSC / TGA records for S-II, F-I and F-III are shown in Figures 1, 2 and 3. DSC record for

S-II shows a broad isotherm beginning at about 62 ° C, corresponding to the loss of ethyl acetate and water from the crystal lattice. The endotherm starting at about 152 ° C is melting. TGA weight loss of about 2.5% occurs at the same time as the first reversal, while the remaining 0.5% weight loss occurs until the onset of melting, suggesting that some solvent molecules are more tightly bound in the lattice.

The DSC record for F-I shows a broad endotherm starting at about 75 ° C, followed by a second endotherm starting at about 155 ° C, corresponding to melting. The TGA record for F-I shows a gradual weight loss of 0.3% followed by a steep loss of 1.5%, which together represent dehydration of the grid. The onset of the first DSC reversal and the corresponding TGA weight loss vary slightly due to differences in heating rates. Initial weight loss represents weakly bonded hydration molecules of water, while the second weight loss corresponds to about 0.5 moles of water present in the grid at very low relative humidity (below 5%

- see moisture sorption data).

The DSC record for F-III shows a broad, low-temperature endotherm at about 30 ° C, followed by a second broad and relatively weak endotherm beginning at about 70 ° C

and a final tip beginning at about 146 ° C corresponding to melting. A sharp 1.5% (about 0.5 mole) weight loss on TGA is consistent with the first endotherm corresponding to a loss of weakly bound water molecules, while an additional about 1.6% weight loss above 60 ° C is a loss of heavily bound water molecules, it is those that are present at very low relative humidity. The weight loss observed above 170 ° C corresponds to the decomposition of F-III.

The X-ray diffractograms F-I and F-III show sharp peaks and flat bases characteristic of highly crystalline materials. The location of the angular peak at 2Θ and the corresponding I / Ιθ data for representative FI, F-III and S-II samples are shown in Table 1. Although many of the intense reflections are generally at similar diffraction angles, each form gives a different powder diffraction pattern, allowing clear distinction between

S-II, F-I, and F-III.

It is well known in the crystallography art that, for any given polymorph, the relative intensities of the diffraction peaks may vary due to the preferential orientation due to factors such as crystal morphology. Where the effects of preferred orientation are present, the peak intensities are altered, but the locations of the characteristic peaks are unchanged. See, for example, The United States Pharmacopeia No. 23, National Formulary No. 18, 1843-1844 (1995). Therefore, F-I can be identified on the basis of peak intensities as well as their location by the presence of peaks at 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 ° at 2 °; if the record is obtained from a copper radiation source.

• · ·

Table 1

S-1 : ι F-I F-III 20 ° I / I _o (¾) 20 ° I / I _o (¾) 20 ° i / i _o (¾) 4.67 . 1.3 4.92 2.6 4.63 20.8 5.03 6 7.69 34.6 7.82 100 ALIGN! 6.83 5.8 7.91 100 ALIGN! 9.29 16.9 7.17 16.1 9.89 2.5 10.16 22.7 7.73 100 ALIGN! 10.22 2 10.35 5.4 9.03 b ³ -. 10.74 7.4 13.77 10.7 9.31 1.7 14.86 9.1 13.97 15.2 9.66 - 15.45 2.3 15.06 6.9 10.27 1.6 15.92 15.9 15.71 22.3 10.47 2, 2 16.67 1.7 15.87 7, 4 10.91 6.3 16.98 3.1 16.35 34.5 13.63 2.1 18.28 17.8 16.77 L 12.3 14.09 4.6 18.56 7 17.28 10 15.10 4.1 20.58 13.1 17.62 47.9 15.52 10.5 20.85 8.8 18.09 43, 9 16.45 g., 1 .. 21.64 3.9 20.43 42 16.67 7.6 22, 19 4.8 20.80 33.6 17.21 4.9 22.65 2.9 21.31 42, 7 17.53 2.4 23, 28 3.4 21.71 13 18.33 28.2 23.97 11.8 21.85 14.5 18.69 11.1 24.31 6.3 22.13 12.8 19.37 3.5 25.52 3.9 22.26 16.3 20.29 8.6 26.20 3.4 23.51 13, 2 20.64 r 17.2 26.47 3.1 23.69 15, 9 21.02 12.7 28.84 6.4 23.91 25.6 21.68 5.1 30.13 3.5 24.31 38.7 22.01 8.3 31.12 2.9 25.22 8 22.29 8 25.67 8.9 23.17 7.8 27.05 18, 9 23, 39 9.1 27.89 13, 3 24.30 13.6 28.24 8.6 25.76 3.4 28.71 21.3 28.10 b-8 ...... 29.89 8, 9 28.73 10.9 30.24 18.7 29.42 3.2 30.88 5.8 30,00 3.7 31.44 7, 6 30.89 2.1 33, 06 4,5 31.34 2.4 34.36 6 31.70 1.1

• · · · · · · · · · · · ·

S-II F-I F-III 2Θ (°) i / i _o (¾) 20 ° i / i _o (¾) 2θ (o) I / I _o (¾) 32.81 1 32.91 0.8 33.48 2 26.05 4 26.63 5.5 27.01 3.1 27, 49 2.8

Further characteristics of F-I and F-III

F-I and F-III hygroscopic studies were performed. The moisture sorption isotherms of F-I and F-III are shown in FIG.

4. After initial exposure of the samples to approximately 5% relative humidity, F-I and F-II immediately increased weight by 1.5% and 1.7% humidity, equivalent to approximately 0.5 moles of water. Both forms exhibit continuous moisture uptake over the entire humidity range, which is probably due to the incorporation of water molecules into the grid.

The difference in moisture absorption of these two forms is likely to reflect the amount of water that can be incorporated into the two grids (i.e., the amount of space available in the water grid that water molecules can fill).

The lack of hysteresis in the sorption-desorption isotherms F-I and F-III suggests that crystalline forms rapidly reach equilibrium at any given humidity.

The moisture sorption profiles F-I and F-III reveal that these forms are inherently non-stoichiometric hydrates. At room relative humidity (about 50% relative humidity), the FI contains about 1.7% water, corresponding to 0.5 mol water, while F-III absorbs about 3.0% water, corresponding to about 0.85 mol water . The bulk forms F-I and F-III achieve a rapidly equilibrium state with the atmosphere such that the water content of analytical techniques reflects the relative humidity at the time of data collection. The case-by-case differences in DSC data are likely to be due to samples being hydrated to a different extent due to different environmental conditions in which they were stored.

X-ray diffractograms were obtained for samples F-I and F-III stored at different relative humidity (0,

22, 50 and 80%). At relative humidity, there is a gradual shift of the initial (zero relative humidity) F-III peaks to about 13.8, 17.6, 18.0, 20.5 and 24.0 ° at 2Θ, as well as a slight shift of less intense peaks at increased relative humidity. These changes observed in F-III in X-ray diffractograms indicate that there is a change in cell unit dimensions, it can be assumed that due to the penetration of weakly bound water molecules with increasing relative humidity. The continuous shift of the peaks with moisture correlates well with moisture sorption data, which show a gradual weight gain over this relative humidity range, providing evidence of the formation of variable hydrates.

A similar experiment was performed on F-I to determine whether changes in relative humidity would have a similar effect on its lattice (relative humidity 0, 25, 52, 73 and 95%). As the relative humidity increases, a very slight shift of the peaks is observed, which at relative humidity is 0 to about 7.7, 18.3, 18.5, 20.5, 20.8 ° at 2θ. The tips at about 7.7, 20.8, and 24.1 also expand slightly at higher relative humidity and become less distinguishable, suggesting that water is absorbed into the amorphous components (or plasticizes the solid), especially at relative humidity. 73 and 95%. Peak shifts in X-ray diffractograms are less dramatic in F-I than peak shifts observed when F-III has been exposed to different relative humidities. This suggests that the F-I lattice does not undergo the same expansion and / or contraction as the F-III lattice.

F-I and F-III have been found to be stable over the relative humidity range, despite the ability of F-III to absorb nearly twice as much water. These two forms were found to have comparable crystal size, morphology, water solubility, and dissolution rate.

Drying studies were performed to monitor desolvation of S-II as a function of drying time and temperature (see Figure 5). X-ray diffraction patterns were recorded at various points in the desolver experiment. Many diffraction peaks from the S-II design study appear at similar angles in F-I, confirming that the gratings

S-II and F-I are very similar. The disappearance of the diffraction peaks at about 6.8, 7.2 and 14.0 ° at 2 ° already after minimal drying suggests that these reflections may be attributed to crystallographic planes containing a partial electron density of the ethyl acetate molecules.

Strong heating of the solvate material under vacuum at high temperatures gives F-I. The F-I prepared in this manner exhibits a high degree of crystallinity in X-ray diffraction. Therefore, the material formed by crystallization from a solution of ethanol and ethyl acetate followed by drying under vacuum for only a few hours, as disclosed in US Patent No. 5,723,474, exhibits very poor crystallinity since such a process results in partially desolvated S-II.

F-I and F-III have several advantages over the previously described forms of arzoxifene, as previously known in the art.

·· ··· »· ·

With respect to arzoxifene produced by the processes of US Patent No. 5,723,474, F-I and F-XII are more stable at room temperature and are therefore more adaptable to pharmaceutical developments, such as the development of dosage formulations. In addition, FI and F-III are much more crystalline than the forms disclosed in US Patent No. 5,723,474. Crystalline materials are generally less hygroscopic and more stable (e.g., less prone to chemical degradation, maintain steady strength) than amorphous materials and are therefore more desirable for processing preparations. Further, unlike the forms of arzoxifene produced by the methods of US Patent No. 5,723,474, which contain ethyl acetate and water in their lattice, F-I and F-III contain only water.

Ways of characterization

DSC measurements are performed on a TA Instruments 2920 Modulated DSC connected to a Thermal Analyst 3100 and equipped with a cooling system. The samples (3-5 mg) are heated in corrugated aluminum pans from 10 to 240 ° C at a heating rate of 2 ° C / min.

TGA analyzes are performed on a TA Instruments 2050 Thermogravimetric Analyzer connected to a Thermal Analyst 3100. Samples (5-10 mg) are heated in open pans from 25 to 250 ° C at a heating rate of 5 ° C / min.

X-ray diffraction records are obtained on a Siemens D5000 X-ray powder diffractometer equipped with a CuKce source (0.1854056 nm), and a Kevex solid state detector operating at 50 kV and 40 mA. Each sample is taken between 4 ° and 25 ° at 20. The samples are allowed to equilibrate for at least 30 min at the desired temperature and / or relative humidity prior to data collection.

· · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·

Hygroscopic measurements of F-I and F-III are performed using VTI methods as follows. Each sample is dried under vacuum at 60 ° C until weight loss is no longer detected, at which point the sample chamber is placed at 0% relative humidity. Moisture sorption isotherms are obtained at 25 ° C using a vacuum moisture equilibrium VTI under the following conditions: sample size 10-15 mg, adsorption / desorption range 0-95% relative humidity, step interval 5%, interval between samples 10 min.

The following examples further illustrate the processes for preparing the hydrates of this invention. The examples are not intended to limit the scope of the procedures in any way and are not so construed.

DETAILED DESCRIPTION OF THE INVENTION

Preparation 1

S-II

Crude arzoxifene (1.58 g of the material prepared as described in Example 41 of US Patent No. 5,723,474, the contents of which are incorporated herein by reference) is suspended in 28 mL of ethyl acetate and heated to reflux. 18 ml of ethanol are added to induce dissolution. The solution was kept under reflux for 20 minutes and then allowed to cool to room temperature. The precipitate was collected by vacuum filtration and washed with 30 mL of ethyl acetate to give 1.05 g of a powdery white solid.

···············

Example 1

F-I of S-II

The S-II is dried for 118 hours in a vacuum oven under a vacuum of 100 psi at 100 ° C to give F-I.

Example 2

Improved process for preparing F-I from arzoxifene

A 1 liter three-neck round bottom flask equipped with a reflux condenser and overhead stirrer was charged with 25.0 g of arzoxifene, 475 mL of tetrahydrofuran and 25 mL of water. The reaction vessel was then prepared for simple distillation. The reaction mixture was heated to reflux and 250 ml of distillate removed. Remove the heat briefly and add 250 mL of fresh anhydrous tetrahydrofuran to the flask. Atmospheric distillation continues to remove an additional 250 mL of distillate. The heating was removed briefly with 250 ml of fresh tetrahydrofuran and a further 250 ml of distillate removed. An additional 250 mL of tetrahydrofuran was added and the reaction mixture was refluxed. With this addition of tetrahydrofuran a white precipitate is formed. The reaction mixture was allowed to cool slowly with stirring for 3 hours, during which time additional solids precipitated and the suspension reached room temperature. The crystalline suspension was filtered and dried at 50 ° C for 48 hours under vacuum and gently purged with nitrogen. 22.50 g (90.0% yield) are obtained. X-ray diffraction analysis will show that the wet cake and dry solid spectra are substantially identical and substantially identical to the previously prepared F-I. In the differential scanning calorimetry analysis, 1c of the melting point was obtained at 157 ° C, while the thermal gravimetric analysis showed a 1.5% mass loss between room temperature and 100 ° C. HPLC-free purity was 88.1% versus the theoretical purity of 92.9%. HPLC analysis showed a total level of related substances of 0.44%.

Example 3

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

261 ml tetrahydrofuran, 45 ml water, 6.14 g concentrated sulfuric acid and 6-benzyloxy-3- [4- / 2- (piperidin-1-yl) ethoxy / phenoxy] -2- (4-methoxyphenyl) benzo [b] The thiophene (S-oxide) (with an HPLC purity of 99% and an HPLC determined total level of related substances of 0.35%) was mixed and stirred until a homogeneous mixture was formed. 10% Pd / C (5.6 g suspended in 22 ml water was added and rinsed with 5 ml water). The resulting suspension was evacuated and covered with 60 psi of hydrogen. The reaction temperature is set at 30 ° C. After 2 hours 5.6 g of 10% Pd / C are added together with 30 ml of water. The Hydrogenaee was allowed to continue for another 22 hours at 40 psi and 30 ° C. An additional 4.40 g of 10% Pd / C in 30 mL of water was added and the hydrogenation was continued at 40 psi and 30 ° C for an additional 2.5 hours. The catalyst was removed by filtration and the pH of the filtrate was adjusted to 7.24 with 50% sodium hydroxide. To the two-phase solution stirred for 30 minutes, 8.66 g of sodium chloride dissolved in 18 ml of water was added. The phases are separated and the aqueous phase is back extracted with 50 ml of tetrahydrofuran. The organic phases are combined and evaporated by atmospheric distillation to a volume of 50 ml. 180 ml of methanol were added to the concentrate at 24 ° C over a period of 1 hour. The resulting crystalline suspension was stirred at 24 ° C for 30 minutes, cooled to 0 ° C and stirred for 1 hour. The solids were collected by filtration and washed successively with 39 mL water and 39 mL methanol, then dried overnight at 50 ° C under vacuum. 15.52 g (67.8% yield) were obtained.

A 10g portion of the above product was recrystallized from tetrahydrofuran and water as described in Example 2.

Explanatory notes

The term "effective amount" as used herein means an amount of F-I that is capable of inhibiting the conditions or their deleterious effects described herein. When F-I is co-administered with an estrogen, a progestin, an aromatase inhibitor, an LHRH analogue, or an AChE inhibitor, the term "effective amount" also means an amount of such a compound capable of producing the effect intended.

The term inhibit or inhibit includes their generally accepted meaning, ie, preventing, preventing, reducing, alleviating, calming, slowing, arresting or reversing, the progression or severity of the pathological conditions or their consequences described herein.

The terms prevention, prophylaxis, prophylactic and avoidance are used interchangeably herein and mean reducing the likelihood that the F-I recipient will experience or develop any of the pathological conditions or their consequences described herein.

The terms estrogen deprivated and estrogen deprivation mean conditions, whether or not. naturally occurring or clinically induced, in which the woman is unable to produce endogenous estrogenic hormones in an amount sufficient to maintain estrogen-dependent functions such as menstruation, bone homeostasis, neuronal function, cardiovascular condition, etc. Such estrogen deprivation situations are due to menopause and surgical or chemically induced ovariectomy, including but not limited to functional equivalents thereof, e.g., aromatase inhibitor, GnRH agonist or antagonist, ICI 182780 and the like. Diseases associated with estrogen deprivation include, but are not limited to, bone loss, osteoporosis, cardiovascular disease, and hyperlipidemia.

The term estrogen, as used herein, includes steroid compounds with estrogenic activity, such as 17β-estradiol, estrone, conjugated estrogen (Premarin ^® ), equine estrogen 17E-ethinylestradiol, and the like. A preferred estrogen-based compound is Premarin, ^<R> and norethylnodrel.

The term progestin, as used herein, includes compounds with progestin activity such as, for example, progesterone, norethylnodrel, nongestrel, megestrol acetate, norethindrone, and the like. The preferred progestin-based agent is norethindrone.

The term aromastase inhibitor as used herein includes compounds capable of inhibiting aromatase, for example, commercially available inhibitors such as aminoglutemide (CYTANDREN ^<R> ), anastrazole (ARIMIDEX ^<R> ), letrozole (FEMARA ^<R> ), formestane (LENATRON ^{<R) >} ), exemestane (AROMASIN ^(R5 ) and the like).

The term LHRH analogue refers to an analogue of the factor releasing the · · · · · · · · · · · · · · · · · · · · · · · · · · · · Luteinizing hormone that inhibits estrogen production in premenopausal women, including, for example, goserelin (ZOLADEX ^® ), leuprolide (LUPRON ^® ) and the like.

The term AChE inhibitor includes acetylcholinesterase inhibitor compounds such as physostigmine salicylate, tacrine hydrochloride, donepezil hydrochloride and the like.

The term up-regulating ChAT means increasing the enzymatic activity of ChAT, i.e., promoting the conversion of choline to acetylcholine. This support will include increasing the efficiency and / or rate of reaction of ChAT and choline and / or increasing the amount of ChAT present at the site of action. This increase in the amount of enzyme present may be due to gene regulation or at another stage of synthesis during enzyme formation and / or a decrease in enzyme deactivation and metabolism.

Selected testing procedures

General procedure for preparation of rats

75-day-old Sprague Dawley rats (200-225 g) are obtained from Charles River Laboratories (Portage, MI), unless otherwise stated, and the animals are either ovariectomized (OVX) at Charles River Laboratories. or undergo Sham surgery and leave after one week. After delivery, the animals are housed in hanging metal cages for one week, in groups of three or four per cage, with access to food and water ad libitum (dietary calcium content approximately 0.5%). The room temperature is maintained at 22.2 ° C ± 1.7 ° C with a minimum relative humidity of 40%. The lighting period is 12 h light and 12 h dark.

· · 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 · · * ·

5 · ··· * * ·· · · · · ·

Dosing regimen and tissue harvesting

After a one-week acclimatization period (i.e., two weeks after ovariectomy), daily dosing of F-I is started. 17E-Ethinylestradiol or F-I are administered orally, unless otherwise indicated, as a suspension in 1% carboxymethylcellulose or dissolved in 20% cyclodextrin. Animals are dosed daily for 4 days. At the end of the dosing regimen, the animals are weighed and anesthetized with ketamine / xylazine (2: 1, v / v) and blood samples taken by cardiac puncture. The animals are then sacrificed by asphyxiation in CO, a midline incision is taken to remove the uterus and freshly determined for weight. 17α-Ethinylestradiol is obtained from Sigma Chemical Co., St. Petersburg. St. Louis, MO.

Cardiovascular disease / hyperlipidemia

Blood samples were allowed to clot for two hours at room temperature, followed by centrifugation for 10 minutes at 3000 rpm to give serum. Serum cholesterol is determined by a high-performance cholesterol assay from Boehringer Manheim Diagnostics. Briefly, cholesterol is oxidized to cholest-4-en-3-one and hydrogen peroxide. The hydrogen peroxide then reacts in the presence of peroxidase with phenol and 4-aminophenazone to form a β-quinone imine dye, which is determined spectrophotometrically at 500 nm. The cholesterol concentration is then calculated from the standard curve. The whole test is automated using the Biomek Automated Workstation.

9 9 9 9 9 9 9 9 9 9 9 9 9 9 9

9 * · 9 9 9 »9 9 999 ·»

Uterine eosinophil peroxidase assay

The uteri obtained as described above are maintained at 4 ° C until enzymatic analysis. The uteri are then homogenized in 50 volumes of 50 mM Tris buffer (pH = 8.0) containing 0.0005% Triton X-100. After adding 0.01% hydrogen peroxide and 10 mM O-phenylenediamine (final concentration) to Tris buffer, the absorbance was monitored at 450 nm for 1 minute. The presence of eosinophils in the uterus is an indicator of the estrogenic activity of the compound. The maximum speed of the 15 second interval is determined above the initial, linear portion of the reaction curve.

Test procedure for inhibition of bone loss (Osteoporosis)

In accordance with the above general procedures, rats are treated daily for 35 days (six rats per treatment group) and sacrificed by carbon dioxide asphyxiation on day 36. A 35-day time period is sufficient to maximize the reduction in bone density measured as described herein. At the time of sacrifice, the uterus is removed, the external tissues are removed, and the liquid content is drained, then its fresh weight is determined to confirm the estrogen deficiency associated with ovariectomy. The uterine weight is usually reduced by about 75% in response to ovariectomy. The uterus is then placed in 10% neutral pH buffered formalin to allow subsequent histological analysis.

Right femurs are excised and digitized with generated X-rays, and distal metaphyses are analyzed with an image analysis program (NIH image). The proximal tibia in these animals is scanned by quantitative tomography. They are administered in accordance with the above procedures

- ώΖ,

orally tested animals orally F-I or ethinyl estradiol (EE) in 20% hydroxypropyl-S-cyclodextrin. F-I is also suitable in combination with estrogen or progestin.

MCF-7 proliferation assay

MCF-7 breast adenocarcinoma cells (ATCC HTB 22) are maintained in phenol red-free MEM (minimal essential medium); Louis, MO) supplemented with 10% (v / v) fetal bovine serum (FBS), 2 mM

L-glutamine, 1 mM sodium pyruvate, 10 mM HEPES (acid

N- (2-hydroxyethyl) piperazine-N '- (2-ethanesulfonic), non-essential amino acids and bovine insulin at a concentration of 1 gg / ml (maintenance medium). 10 days prior to the experiment, MCF-7 cells were transferred to bovine serum-stripped 10% activated carbon-coated maintenance medium (DCC-FBS assay medium), instead of 10% FBS to deplete internal steroid stocks. Cells

MCF-7 is removed from the maintenance flask using cell separation medium (Ca ² '/ Mg ² * HBSS-free (phenol red-free) supplemented with 10 mM HEPES and 2 mM EDTA). Cells are washed twice with assay medium and adjusted to 80,000 cells / ml.

Approximately 100 ml (8000 cells are added to flat-bottom microculture wells (Costar 3596) and incubated at 37 ° C in 5% CO ₂ in a humidified incubator for 48 hours to allow cellular adherence and equilibrium after transfer. A dilution series of drug or DMSO is prepared as a diluent control in assay medium and 50 mL is transferred to microculture triplicate and then 50 mL of assay medium is transferred to a final volume of 200 mL. CO is revived with 2 microcultures with tritiated thymidine (37 kBq / well for 4 hours).

hours followed by thawing and harvesting of microcultures, using a semi-automatic Skatron cell collector. Samples are counted by liquid scintillation counting using a Wallac counter

BetaPlace S.

DMBA induced breast tumor inhibition

Estrogen-dependent breast tumors are induced in female Sprague-Dawley rats, purchased from Harlan Industries, Indianapolis, Indiana. At approximately 55 days of age, rats receive 20 mg 7,12-dimethylbenz [a] anthracene (DMBA) in a single dose. About 6 weeks after DMBA administration, the mammary glands are palpated at weekly intervals for tumor discovery. Whenever 1 or more tumors occur, the longest and shortest radius of each tumor is measured using a metric caliper, measurements are recorded and these animals are selected for experimentation. The intention is to evenly distribute different tumor sizes in the treatment and control groups so that tumors of average size are evenly distributed among the test groups. The control and test groups contain 5 to 9 animals per experiment.

F-I is administered by intraperitoneal injection in 2% gum arabic or orally. Orally administered compounds are either dissolved or suspended in 0.2 ml corn oil. Each therapy, including gum arabic and corn oil therapy, is administered to each test animal once daily. After initial tumor measurement and selection of test animals, tumors are measured as described above each week. Treatment and measurement of the animals is continued for 3 to 5 weeks after which the final tumor areas are determined. The change in mean tumor area is determined for each compound and control therapy.

· · ♦ ······················································ · ··· ♦ ·

Uterine fibrosis testing procedures

Test 1: 3 to 20 women with uterine fibrosis are given F-I. The amount of compound administered is from 0.1 to 1000 mg per day and the administration period is 3 months. The effects on uterine fibrosis in women are monitored during the administration period and up to 3 months after discontinuation.

Test 2: The same procedure as that used in Test 1 except that the administration period is 6 months.

Test 3: The same procedure as that used in Test 1 except that the administration period is 1 year.

Test 4: Prolonged estrogen stimulation is used to induce leiomyoma in sexually mature guinea pigs.

Animals are dosed 3-5 times a week with estradiol injections for 2 to 4 months or until tumors develop. Therapy containing F-I or vehicle is administered daily for 3 to 16 weeks, after which the animals are sacrificed, the uteri are collected and analyzed for tumor regression.

Test 5: Human leiomyoma tissue is implanted in the peritoneal cavity and / or uterine myometry of sexually adult castrated female nude mice. Exogenous estrogen is delivered to induce the growth of the explanted tissue. In some cases, harvested tumor cells are cultured in vitro prior to implantation. Therapies containing F-I or vehicle are administered daily by gavage for 6 to 16 weeks and implants are removed to measure growth or regression. At the time of sacrifice, the uteri are collected to assess the state of the organs.

• ·

Test 6: Tissue from human uterine fibroid tumors is harvested and maintained in vitro as primary untransformed cultures. Surgically collected samples are passed through a sterile mesh or screen, or optionally detached from surrounding tissue to form a single cell suspension. Cells are maintained in medium containing 10% serum and antibiotic. The growth rate in the presence and absence of estrogen is determined. Cells are evaluated for their ability to produce the complement component C3 and their response to growth factors and growth hormone. In vitro cultures are evaluated for their proliferative response to progestin, GnRH, F-I and vehicle treatment. Steroid hormone receptor levels are evaluated weekly to determine if significant cellular characteristics are maintained in vitro. Tissue from 5 to 25 patients is used.

Test 7: The ability of F-I to inhibit the proliferation of LT-derived leiomyoma cells is essentially measured as described by Fuchs-Young et al., Inhibition of Estrogen - Stimulated Grouwth of Uterine Leiomyomas by Selective Estrogen Receptor Modulators, Mol. Car., 17 (3). 151-159 (1996), which is incorporated herein by reference in its entirety.

Testing of endometriosis

Test 1: 12-30 adult CD female rats were used as test animals. It is divided into 3 groups of equal numbers. The estration cycle of all animals is monitored. The day before estration, surgery is performed on each female. The females in each group are removed from the left corner of the uterus, cut into small squares, and the squares are easily sewn to various sites adjacent to the mesenteric bloodstream. In addition, females in Group 2 have ovaries removed. Starting from the day after surgery, animals in Groups 1 and 2 receive intraperitoneal injections of water for 14 days, while animals in Group 3 receive intraperitoneal injections of 1.0 mg F-I per kg body weight for the same period. After 14 days of treatment, each female is sacrificed and endometrial explants, adrenal glands, remaining uterus and, if possible, ovaries are collected and prepared for histological examination. The adrenal and ovarian weight is determined.

Test 2: 12-30 adult female CD rats are used as test animals. It is divided into 2 equal groups. The estration cycle of all animals is monitored. The day before estration, surgery is performed on each female. The females in each group are removed from the left corner of the uterus, cut into small squares, and the squares are easily sewn at various sites adjacent to the mesenteric bloodstream. From approximately 50 days after surgery, animals in Group 1 receive intraperitoneal injections of water for 21 days, while animals in Group 2 receive intraperitoneal injections of 1.0 mg F-I per kg body weight for the same time. After 21 days of therapy, each female is sacrificed and endometrial explants, and adrenal glands are collected and weighed. The explants are measured as an indication of growth. The estration cycle is monitored.

Test 3: Autologous grafts of endometrial tissue are used to induce endometriosis in rats and / or rabbits. Reproductive adult female animals undergo bilateral oopphorectomy and estrogen is delivered exogenously to provide a specific and constant level of the hormone. Autologous endometrial tissue is implanted on the peritoneum of 5 to 150 animals and estrogen is delivered to induce the growth of the explanted tissue. Therapy that takes

consists of a compound of the invention administered daily by gastric gavage for 3 to 16 weeks and the implants are removed and evaluated for growth or regression. At sacrifice, the intact horn of the uterus is taken to evaluate the condition of the endometrium.

Test 4: Tissue from human lesions of the endometrium is implanted on the peritoneum of sexually mature, castrated female nude mice. Exogenous estrogen is supplied to induce growth of the explanted tissue. In some cases, harvested endometrial cells are cultured in vitro prior to implantation. Therapy consisting of F-I is administered daily by gastric tube for 3 to 16 weeks and implants are removed and evaluated for growth or regression. At sacrifice, the uteri are removed to evaluate the condition of the intact endometrium.

Test 5: Tissue from human endometrial lesions is harvested and maintained in vitro as primary untransformed cultures. Surgical specimens are passed through a sterile mesh or screen or optionally torn from surrounding tissue to form a single cell suspension. Cells are maintained in medium containing 10% serum and antibiotic. The growth rate in the presence and absence of estrogen is determined. Cells are evaluated for their ability to produce the complement component C3 and their response to growth factors and growth hormone. In vitro cultures are evaluated for their proliferative response to progestins, GnRH, F-I and vehicle. Steroid hormone receptor levels are evaluated weekly to determine if significant cellular characteristics are maintained in vitro. Tissue from 5 to 25 patients is used.

CNS diseases, including Alzheimer's disease

Estrogens, such as 17S-estradiol, regulate transcription of genes by binding to estrogen receptors (ERs), which are located in the cytoplasm of certain cell populations. Ligand activation of ER is a prerequisite for the nuclear transport of a complex binding to a palindromic consensus sequence of 13 bp (estrogen-responsive element, ERE) that triggers a transcriptional apparatus that culminates in the activation of the respective target genes. Various estrogen-regulated genes have been identified. These include cytoskeletal proteins, enzymes of neurotransmitter biosynthesis and metabolism, as well as other hormones and neuropeptides. EREs have been identified in many estrogen-responsive genes, including vitellogenin, c-fos, prolactin, and luteinizing hormone.

ERE-like sequences in p75 ^{ns: c} 'and trkA have been identified as important in the central nervous system, both of which serve as neurotrophin signaling molecules: nerve growth factor (NGF), brain-derived nerve growth factor (BDNGF) and neurotrophin-3. .

BDNF, like NGF, has been shown to promote the survival of cholinergic neurons in culture. It is believed that if the interactions between neurotrophins and estrogens are important for the development and survival of basal forebrain neurons (which degenerate in Alzheimer's disease), then a clinical condition in which estrogen deficiency exists (such as after menopause) may contribute to the loss of these neurons.

The following experiment was performed on ovariectomized rats (prepared as described above) to determine the similarities and / or differences between F-I treatment

and estrogen for gene expression in various regions of the brain. Six week old rats were dosed daily with subcutaneous injections of 0.03 mg / kg estradiol benzoate, FI or vehicle (control). After five weeks of therapy, the animals are sacrificed and their brains are removed and hippocampuses are harvested by micropreparation. The hippocampuses are rapidly frozen in liquid nitrogen and stored at -70 ° C. Total RNA is prepared from stored tissue of the respective treatment and control groups and reverse transcribed using a 3'-oligonucleotide primer selected for specific mRNA (poly-A +) populations. Polymerase chain reactions (PCR) were performed in a cocktail consisting of: random 5-oligonucleotides (10 base pairs in length, total 150), reaction buffer, Taq polymerase and ³² PdTCP.

After 40 rounds of amplification, the reaction products were sized on a 6% TBE-urea gel, dried and applied to the X-ray film. The resulting mRNA display sets are compared between groups.

Use of F-I in conjunction with estrogen

Peri- and postmenopasal women often undergo hormone replacement therapy (HRT) to combat the negative consequences associated with a decrease in circulating endogenous estrogen, for example to treat hot flashes. On the other hand, HRT is associated with an increased risk of some cancers, including uterine and breast cancers. F-I can be used in conjunction with hormone replacement therapy to inhibit these risks.

Use of F-I in conjunction with an aromatase inhibitor

By definition, the ovaries of postmenopausal women are not functional. The only source of female estrogen is the conversion of adrenal androgens into estrogens by the enzyme aromatase found in peripheral tissues (including fat, muscle and breast tumors themselves). Therefore, drugs that inhibit aromatase (aromatase inhibitors) cause estrogen depletion in postmenopausal women. Estrogen deprivation mediated by aromatase inhibition is an important treatment option in patients with metastatic breast cancer. During therapy with an aromatase inhibitor, the lack of circulating estrogen may cause unintended side effects, for example on serum lipid levels. F-I can be used to inhibit these negative effects

Use of F-I in conjunction with LHRH analogue

Continuous exposure to the LHRH analogue (luteinizing hormone releasing factor) inhibits estrogen production in premenopausal women by desensitizing the pituitary gland, which no longer stimulates the ovary to produce estrogen. The clinical effect is pharmacological oofrectomy, which is reversible upon disappearance of the LHRH analogue. During LHRH analog therapy, circulating estrogen deficiency may cause unintended side effects, for example on serum lipid levels. F-I can be used to inhibit these negative effects.

Increased levels of acetylcholine

Patients with Alzheimer's disease are known to have significantly lower levels of cholinergic neurons in

hippocampus than their counterparts who do not suffer from Alzheimer's disease. The progressive loss of these cholinergic neurons appears to mirror the progressive loss of memory and cognitive functions in these patients. One reason for the loss of these neurons is believed to be a decrease or decrease in neurotransmitter function, acetylcholine.

The level of acetylcholine in a neuron is basically determined by the balance between its biosynthesis and biodegradation. The cholinacetyltransferase (ChAT) enzyme is primarily responsible for its synthesis and acetylcholinesterase (AChE) is degraded.

The following experiment was performed to determine the effect of F-I on ChAT levels. Following the general procedure for the preparation of the rats mentioned above, 40 rats are administered daily by subcutaneous injection or gavage at a dose of 3 mg / kg F-I per day in a vehicle containing 10% cyclodextrin, 0.3 or 0.03 mg estradiol benzoate or vehicle as a control. Animals are treated for 3 or 10 days. There are 20 animals in each dosing regimen. After appropriate time intervals, the animals are sacrificed and their brains removed. Individual brain parts are homogenized and analyzed. Homogenized hippocampus and frontal cortex samples are processed and ChAT activity determined by radiolabeled acetylcholine biosynthesis analysis. This procedure can be found in Schoepp et al., J. Neural Transmiss. 78: 183-193 (1989), which is incorporated herein by reference.

As expected, they are. in ovariectomized animals, reduced ChAT levels by more than 50% (p <0.001) compared to surrogate controls.

In other embodiments of the invention, F-I is used in combination with an AChE inhibitor. The use of an AChE inhibitor increases acetylcholine levels by blocking its degradation through inhibition of AChE.

Benign prostatic hyperplasia (BPH)

For background information on the association between estrogen action and treatment of BPH and prostate cancer, see PCT Patent Application No. WO98 / 07274, dated October 15, 1998.

In the experiments described below, the ability of F-I to bind to estrogen receptors in several human prostate cancer cell lines was evaluated.

LNCaP, DU-45 and PC-3 human prostate cancer cell line lysates are prepared in TEG medium containing 50 nM Tris HCl at pH = 7.4, 1.5 mM ethylenediaminetetraacetic acid (EDTA), 0.4 M potassium chloride, 10 % glycerol,

0.5 mM 2-ME and 10 mM sodium molybdate, further containing 0.1 mg / ml protease inhibitors 0.1 mg / ml pepstatin, 2 mg / ml leupeptin, 5 mg / ml aprotinin and 0.1 mM phenylmethylsulfonyl fluoride (PMSF) ) (TEGP).

Cell lysates are centrifuged and the pellets are resuspended in cold TEGP (1 ml TEGP per 100 mg pellets) and exposed to ultrasonic waves (dosed cycles 70%, output 1.8) for 30 seconds on a Branson Model 450 Sonifier. The lysates were pelleted by centrifugation at 10,000 x-g for 15 minutes at 4 ° C, after which the supernatant was collected and used either immediately or stored at -70 ° C.

• · · · · · · · · · · · · · · · · · · ·

Competitive binding test

The binding buffer is TEG in which 0.4 M potassium chloride is replaced with 50 mM sodium chloride and to which 1 mg / ml ovalbumin (TEGO) was added. F-I is diluted to 20 mM in TEGO from which 3-fold serial dilutions are prepared. The tests are performed on round bottom polypropylene microplates in triplicated microwells. 35 ml of tritium-labeled 17E-estradiol (0.5 mM, specific activity 2.224 TBq / mmol, DuPont-New England Nuclear, Boston, MA) and 35 ml of cold competing test compound (0.1 nM to 5 mM) are added to each well. ) or TEGO and after incubation for 5 minutes at 4 ° C with shaking of 70 ml MCF-7 cell line lysate.

The plates were incubated for 24 hours at 4 ° C, after which time 70 ml of dextran coated charcoal (DCC) was added to each well, then shaken vigorously for 8 min at 4 ° C. The wells are then centrifuged at 1500 x g at 4 ° C for 10 min. The supernatant is removed from each well into a flexible polystyrene microplate for scintillation counting on a Wallac Micobeta Model 1450. Radioactivity is expressed as counts per minute (DPM) after correcting the counting efficiency (35-40%) and background. Further checks are total count and total count with DCC to determine the lower limit of counts obtainable from DCC. The results of these competitive binding assays are expressed as the mean percent binding (% binding) + / standard deviation using the formula:

DPM. - DPM test. ceXlc compounds. number + · DCC% binding = ------------------------------------------- x 100

DPM - DPM netest.sXonceniny o®Xlc. + DCC count • · ·· ·· · ··

Prevention of breast cancer

The present invention also relates to administration of F-I to a recipient at risk of developing breast cancer de novo. The term de novo as used herein means, in the first phase, the lack of transformation or metamorphosis of normal breast cells into cancerous or malignant degenerate cells. Such transformation may occur at the stage of the same cells or daughter cells due to the evolutionary process, or may occur in a unique key case. This de novo process is in contrast to metastasis, colonization or spread of already transformed or malignant degenerate cells from the primary tumor site to a new location.

A person who has no particular risk of developing breast cancer is one who may develop de novo breast cancer, for whom there is no evidence or suspicion of the possibility of developing a disease beyond normal risk and for whom the disease has never been diagnosed. The greatest risk factor contributing to the development of breast cancer is a personal history of the disease, or an earlier history of a personal history of the disease, even though it is in remission and there is no evidence of its presence. Another risk factor is the family history of the disease.

Induction of breast tumors in rats by the administration of carcinogen N-nitroso-N-methylurea is a well accepted animal model for breast cancer studies and has been found to be useful in analyzing the effects of chemoprotective agents.

day-old female Sprague-Dawley rats were given intravenous (study 1) or intraperitoneal dose (study 2) 50 mg N-nitroso-N- 35 in two separate studies.

- methyl urea per kg of body weight one week before being fed ad libitum with mixed amounts of FI, (Z) -2- [4- (1,2-diphenyl-1-butenyl) phenoxy] -Ν , Ν-dimethylethanamine base (tamoxifen base) or control substances.

In Study 1, dietary doses of 60 mg / kg diet and 20 mg / kg diet can be converted to roughly comparable doses of 3 and 1 mg / kg body weight of test animals.

In Study 2, diet doses of 20, 6, 2 and 0.6 mg / kg of diet can be converted to roughly comparable doses of 1, 0.3, 0.1 and 0.03 mg / kg of body weight of the test animals.

Rats are monitored for evidence of toxicity, weighed once a week and palpated for tumor formation. Animals are sacrificed after 13 weeks (study 1) or after 18 weeks (study 2), the tumors are confirmed and their weight after autopsy determined.

Preparations

The term pharmaceutical, when used herein as an adjective, means substantially harmless to the mammal receiving it. By the term pharmaceutical formulation is meant that the carrier, the solvent and the excipient and the active ingredient (s) must be compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.

The F-I is preferably formulated prior to administration. The choice of preparation should be decided by the physician involved, taking into account the same factors affecting the determination of the effective amount.

fefe ··· fe · «fe fe fe fe fe ef ef ef ef ef ef ef ef ef ef ef ef ef ef ef ef ef ef

The total amount of active ingredients in such a formulation will be in the range of from 0.1 to 99.9% by weight of the formulation. Preferably there will be no more than two active ingredients in said formulation. Thus, it is preferred to formulate F-I with a second active ingredient selected from estrogen, progestins, aromatase inhibitor, LHRH analogue, and AChE inhibitor. Most preferred formulations are those in which F-I is the only active ingredient.

The pharmaceutical compositions of this invention may be prepared by methods known in the art using well known and readily available ingredients. For example, FI alone or in combination with estrogen, progestin, aromatase inhibitor, LHRH analogue or AChE inhibitor may be formulated with conventional excipients, diluents or carriers and formed into tablets, capsules, suspensions, solutions, injections, aerosols, powders and the like .

The pharmaceutical compositions of this invention for parenteral administration include sterile aqueous or non-aqueous solutions, dispersions, suspensions, or emulsions, as well as sterile powders that are reconstituted immediately into sterile solutions or suspensions prior to use. Examples of suitable sterile aqueous and non-aqueous carriers, diluents, solvents or humectants include water, saline, ethanol, polyols (such as glycerol, propylene glycol, poles (ethylene glycol) and the like) and suitable mixtures thereof, an oil plant (such as olive oil), injectable organic esters such as ethyloleate. Proper fluidity is maintained, for example, by the use of coating materials such as lecithin, by maintaining the correct particle size in the case of dispersions and suspensions, and by the use of surfactants.

• 9 4 «· 9 * * · · V · 9 · · · · · 9 · 9 9 · 99 9 99

Parenteral compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents, and dispersing agents. Prevention of the action of microorganisms is ensured by the addition of antibacterial or antifungal agents such as paraben, chlorobutanol, phenol, sorbic acid and the like. It may also be desirable to add isotonic agents such as sugars, sodium chloride, and the like. Prolonged absorption of injectables may be brought about by the addition of an agent which delays absorption, such as aluminum monostearate and gelatin.

In some cases, in order to prolong the effect of a drug, it is desirable to slow the absorption of the drug following subcutaneous or intranmuscular injection. This can be achieved by using a liquid suspension of crystalline material of low water solubility or by dissolving or suspending the drug in an oil vehicle. In the case of subcutaneous or intramuscular injection of a suspension containing a form of a drug of low water solubility, the rate of absorption of the drug depends on its rate of dissolution.

Injectable depot preparations F-I are prepared by forming microencapsulated drug matrices in biodegradable polymers such as lactic acid, poly (glycolic), lactic-glycolic acid copolymers, poly (orthoesters) and poly (anhydrides) of these materials, which are known in the art. Depending on the ratio of drug to polymer and the characteristics of the particular polymer used, the rate of drug release can be controlled.

Injectable preparations are sterilized, for example, by filtration through bacteria-retaining filters, or by prior sterilization of the components of the mixture prior to mixing, either during manufacture or just prior to administration (as in the example of a two-chamber syringe package).

Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the FI is admixed with at least one inert pharmaceutical carrier such as sodium citrate or dibasic calcium phosphate and / or a) a filler or extender such as starches, sugars including lactose and glucose, mannitol and silicic acid, b) a binder such as carboxymethylcellulose and other cellulose derivatives, alginates, gelatin, poles (vinylpyrrolidone), sucrose and acacia, c) a wetting agent such as glycerol, d) a disintegrating agent such as agar-agar, calcium carbonate, sodium bicarbonate, potato or tapioca starch, alginic acid, silicates and sodium carbonate, e) a humectant such as glycerol, f) a solubility retarding agent such as paraffin, g) a resorption accelerating agent such as quaternary ammonium compounds, h) wetting agents such as cetyl alcohol and glycerin monostearate, i) adsorbent carriers such as kaolin and bentonite clay and j) lubricants such as talc, stearate calcium or magnesium, solid poles (ethylene glycols), sodium lauryl sulfate and mixtures thereof. In the case of capsules, tablets and pills, the dosage forms may also contain buffering agents.

Solid compositions of a similar type may also contain a fill in a soft or hard gelatin capsule using excipients such as lactose, as well as high molecular weight polyesters (ethylene glycols) and the like.

Solid compositions such as tablets, dragees, capsules, pills, and granules can also be prepared with a coating or coating such as an enteric coating or other coatings well known in the pharmaceutical formulation art. The coatings may contain disintegrating agents or agents that release the active ingredient (s) in a portion of the digestive tract, such as an acid-soluble coating to release the active ingredient (s) in the stomach, or a basic soluble coating to release the active ingredient (s). component (s) in the intestinal tract.

The active ingredient (s) may also be microencapsulated in a slow release coating, the microcapsules being part of a pill or capsule formulation.

Liquid dosage forms for oral administration of F-I include solutions, emulsions, suspensions, syrups, and elixirs. In addition to the active components, the liquid preparations may contain inert diluents commonly used in the art such as water or other pharmaceutical solvents, solubilizing and emulsifying agents such as ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol,

1,3-butylene glycol, dimethylformamide, oils (especially cottonseed, peanut, corn, germ, olive, castor and sesame oil), glycerol, tetrahydrofurfuryl alcohol, polyesters (ethylene glycols), sorbitol fatty acid esters and mixtures thereof.

In addition to inert diluents, liquid oral preparations may also contain adjuvants such as wetting agents, emulsifying and suspending agents, and sweetening, flavoring, and perfuming agents.

Liquid suspensions may contain, in addition to the active ingredient (s), suspending agents such as ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite earth, agar-agar and tract, and mixtures thereof.

Formulations for rectal or intravaginal administration are prepared by mixing FI with a suitable non-irritating excipient such as cocoa butter, polyethylene glycol or any suppository wax that is solid at room temperature but liquid at body temperature and therefore melts in the vagina or rectum cavity thereby releasing the active component (s). The compounds are dissolved in the melted wax, formed into the desired shape and allowed to solidify into the final suppository preparation.

F-I may also be administered in the form of liposomes. As is known in the art, liposomes are generally derived from phospholipids and other lipid substances. Liposome formulations are formed by mono- or multilamellar hydrated liquid crystals that are dispersed in an aqueous medium. Any non-toxic, pharmaceutical, and metabolic-capable lipids capable of forming liposomes can be used. The composition present in the liposome form may contain, in addition to F-I, stabilizing agents, excipient, preservatives and the like. Preferred lipids are phospholipids and phosphatidylcholines (lecithins), both natural and synthetic.

Methods for forming liposomes are known in the art as described, for example, in Prescott, eds., Methods in Cell Biology, Vol. XIV, Academic Press, New York, p. 33 et seq. (1976).

The following examples of means are illustrative only

and is in no way intended to limit the scope of the invention.

Means i

Gelatin capsules

Hard gelatin capsules are prepared using the following ingredients Quantity (mg / capsule) F-I 0.001 - 1000 Starch, NF 0 - 650 Starch, fine powder 0 - 650 Liquid silicone, 350.10 ~ ^s m ² .s ^{_: L} 0-15

The above composition may be varied in accordance with the reasonable variations provided.

A tablet formulation is prepared using the ingredients listed below.

Means 2

Tablets • Tablets • Tablets • Tablets • Tablets • Tablets

Component

Quantity (mg / tablet)

F-I 2.5 - 1000 Microcrystalline cellulose 200 - 650 Silica, fumed 10 - 650 Stearic acid 5-15

The components are mixed and compressed into tablets.

Means 3

Tablets containing approximately 10 and 50 are prepared as follows: mg F-I may be Component Amount (mg / tablet) Amount (mg / tablet) F-I 11.3 56.5 Lactose, anhydrous 176.8 128.2 Lactose, spray, spec. dry 44.2 32.0

· · ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** **

Component Amount (mg / tablet) Amount (mg / tablet) Povidon 11, 0 13.0 Polysorbate 80 2.5 2.6 Krospovidon (internal) 6.25 6.24 Crosspovidon (external) 6.25 6.5 Magnesium stearate 1.5 1.7 Microcrystalline cellulose (external) 0.0 13.0

The components are mixed and compressed to form tablets.

Tablets, each containing 2.5 mg to 1,000 mg F-I, are optionally prepared as follows:

*

Component Quantity (mg / tablet) F-I 25 - 1000 Starch 45 Microcrystalline cellulose 35 Polyvinylpyrrolidone (as a 10% solution in water) 4 Sodium carboxymethylcellulose 4,5 Magnesium stearate 0.5 Talc 1

F-I, starch and cellulose are sieved through a sieve with a particle size of 0.300 mm and mixed thoroughly. The polyvinylpyrrolidone solution is mixed with the resulting powders and then sieved through a 1.168 mm sieve. The granules so produced are dried at 50 to 60 ° C and sieved through a 0.912 mm sieve. Starch sodium glycolate, magnesium stearate and talc, previously passed through a 0.246 mm sieve, are then added to the granules which are mixed and then compressed on a tabletting machine to obtain tablets.

Suspensions, each containing 0.1 mg to 1000 mg of the drug in

A 5-ml batch is prepared as follows.

Means 5

Suspension

Component Amount (mg / 5 ml) F-I 0.1 - 1000 Sodium carboxymethylcellulose 50 Syrup 1,25 Benzoic acid solution 0,10 ml Flavor according to need Dye according to need Purified water etc. 5 ml

The drug is passed through a 0.300 mm sieve and mixed with sodium carboxymethylcellulose and syrup to form a fine paste. Dilute benzoic acid solution, flavor and color with a small amount of water and add with stirring. A sufficient amount of water is then added to obtain the desired volume.

· · · · · * * * * * * * * * * * * * * * * * * * * *

An aerosol solution is prepared containing the components: following Means 6 Aerosol Component Amount (% by weight) F-I 0.2 Ethanol 25.75 Browned 22 (chlorodifluoromethane) 70,00

The F-I was mixed with ethanol and the mixture was added to a portion of browning 22, cooled to 30 ° C and transferred to a filling machine. The required amount is then added to the stainless steel container and diluted with the remaining calender.

The spray unit is then added to the container.

Suppositories are prepared as follows:

Means 7

Suppositories «· ·« ··· · · ··· · · ··· «······ · · ···

Component

Amount (mg / suppository)

F-I 250 saturated fatty acid glycerides 2 000

The F-I is sieved through a 0.246 mm sieve and suspended in saturated fatty acid glycerides, which are previously thawed using the lowest temperature required. The mixture is then poured into a suppository mold with a nominal capacity of 2 g and allowed to cool.

The intravenous formulation is prepared as follows:

Means 8

Intravenous solution

Component

Amount

F-1 mg

Saline isotonic

000 ml

A solution of the above ingredients is administered to patients intravenously at a rate of about 1 ml per minute.

Means 9

Combined capsule

Component Quantity (mg / capsule) F-I 50 Premarin 1 Avicel pH 101 50 Starch 1500 117.50 Silicone oil 2 Tween 80 0.50 Cab-O-Sil 0.25

Means 10

Combined capsule II

Component Quantity (mg / capsule) F-I 50 Norethylnodrel 5 Avicel pH 101 82.50 Starch 1500 90 Silicone oil 2 Tween 80 0.50 Means 11 Combination tablet Component Quantity (mg / capsule) F-I 50 Premarin 1

Corn starch, NF · NF · · NF škrob NF škrob NF škrob škrob NF škrob NF NF NF NF škrob NF

Component Quantity (mg / capsule) Povidon, K 2 9 -3 0 6 Avicel pH 101 41.50 Avicel pH 102 136.50 Krospovidon XL10 2.50 Magnesium stearate 0.50 Cab-O-Sil 0.50

Dosage

The specific dose of F-I administered according to the present invention is determined by the particular circumstances that characterize each situation. These circumstances include the route of administration, the prior history of the recipient, the pathological condition or symptom being treated, the intensity of the condition or symptom being treated, and the age and sex of the recipient.

The minimum effective daily dose of F-I is generally about 1, 5, 10, 15, or 20 mg. The maximum effective dose is usually about 800, 100, 60, 50 or 40 mg. Most commonly, doses range between 15 and 60 mg. The exact dose can, in accordance with standard medical art practice, be determined by titrating the dose to the recipient, i.e. by initially administering a small dose. Dose of the compound with a gradual dose increase until the desired therapeutic effect is observed.

Although it may be necessary to titrate the dose in the recipient with respect to the therapeutic combinations discussed above, typical doses of non-F-III active ingredients are: ethinyl estrogen (0.01 to 0.03 mg per day), mestranol (0.05 to 0, 15 mg per day), conjugated estrogenic hormones (e.g. Premarin ^CR> , Wyeth-Ayerst; 0.3 to 2.5 mg per day), medroxyprogesterone (2.5 to 10 mg per day), norethylnodrel (1.0 to 10 mg per day) 0 mg per day), nonethindrone (0.5 to 2.0 mg per day), aminoglutemide (250 to 1250 mg per day, preferably 250 mg four times a day), anastrazole (1 to 5 mg per day, preferably 1 mg once) daily), letrozole (2.5 to 10 mg per day, preferably

2.5 mg once a day), formestane (250 to 1250 mg per day, preferably 250 mg twice a day), exemestane (25 to 100 mg per day, preferably 25 mg once a day), goserlin (3 to 15 mg for three months, preferably 3 , 6 to 7.2 mg once every three months), and leuprolide (3 to 15 mg per month, preferably 3.75 to 7 mg).

7.5 mg once a month).

Route of administration

F-I can be administered by a variety of routes including oral, rectal, transdermal, subcutaneous, intravenous, intramuscular and intranasal. The route of administration of each estrogen and progestin-based agent is in accordance with what is known in the art. F-I will be administered alone or in combination with an estrogen, a progestin or an AChE inhibitor in a suitable formulation.

The pharmaceutical compositions of the invention may be administered to humans or other mammals (e.g., dogs,

cats, horses, pigs and the like) orally, rectally, intravaginally, parenterally, topically, buccally or sublingually or nasally. The term parenteral administration means modes of administration that include intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection or infusion.

Method / Length of Administration

In most methods of the invention, F-I is administered continuously 1 to 3 times daily or as often as necessary to deliver an effective amount of F-I to the recipient. Cyclic therapy may be particularly useful in the treatment of endometriosis or may be used acutely during painful attacks of the disease.

In the case of restenosis, treatment may be limited to short (1- to 2-fold)

6-month intervals followed by treatments such as angioplasty.

Claims (11)

PATENT CLAIMS Crystalline hydrate of 6-hydroxy-3- [4- / 2- (piperidin-1-yl) ethoxy / phenoxy] -2- (4-methoxyphenyl) benzo [b] thiophene hydrochloride, or FI, characterized in that The X-ray diffractogram contains the following 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 ° at 20; if it is obtained from a copper radiation source. A pharmaceutical composition comprising the crystalline compound of claim 1; one or more pharmaceutical carriers, diluents or excipients; and optionally estrogen, optionally progestin, optionally aromatase inhibitor, optionally LHRH analog, and optionally acetylcholinesterase or AChE inhibitor. A composition according to claim 2 comprising the crystalline compound of claim 1; one or more pharmaceutical carriers, diluents or excipients; and estrogen. The composition of claim 3, wherein the estrogen is Premarin ^® . 5. The composition of claim 2 comprising the crystalline compound of claim 1; one or more pharmaceutical carriers, diluents or excipients; and progestin. 6. A composition according to claim 5, wherein the progestin is selected from the group consisting of norethylnodrel and norethindrone. 7. The composition of claim 2 comprising the crystalline compound of claim 1; one or more pharmaceutical carriers, diluents or excipients; and an AChE inhibitor. 8. The composition of claim 7, wherein the AChE inhibitor is selected from the group consisting of physostigmine salicylate, tacrine hydrochloride and donepezil hydrochloride. The composition of claim 2 comprising the crystalline compound of claim 1; one or more pharmaceutical carriers, diluents or excipients; estrogen; and progestin. 10. A process for the preparation of a compound according to claim 1 comprising crystallizing 6-hydroxy-3- [4- [2- (piperidin-1-yl) ethoxy] phenoxy] -2- (4-methoxyphenyl) benzo [b] thiophene hydrochloride from tetrahydrofuran. The compound of claim 1, which is prepared by the method of claim 10.