HRP20050675A2 - Crystalline non-solavated 1-(4-(2-piperidinylethoxy)phenoxy)-2-(4-methanesulfonylphenyl)-6-hydroxynaphthalene hydrochloride - Google Patents

Description

The application claims priority under US Provisional Patent Application No. 60/450,233, filed on February 25, 2003, and International Patent Application no. PCT/IB03/03349, filed July 16, 2003.

Description of the invention

Background of the invention

Uterine leiomyoma (fibroid disease of the uterus) is a clinical problem that appears under a variety of names, including uterine fibrosis, uterine hypertrophy, myometrial hypertrophy, fibrosis uteri, and fibrotic metritis. Essentially, uterine fibrosis is a condition in which there is an abnormal formation of fibroid tissue on the wall of the uterus. This condition causes dysmenorrhea and infertility in women.

Endometriosis is a condition of severe dysmenorrhea, which is accompanied by severe pain, bleeding into the endometrial mass or abdominal cavity, and often leads to infertility. The cause of the symptoms appears to be ectopic growth of the endometrium, which responds abnormally to normal hormonal control, and is located in inappropriate tissues. Due to the inappropriate mixture for endometrial growth, the tissue appears to induce local inflammatory-like responses causing infiltration of macrophages and a cascade of events leading to the development of a painful response. Evidence suggests that the cause of uterine fibrosis and endometriosis is an inappropriate response of fibroid tissue and/or endometrial tissue to estrogen.

In the last ten years, many publications have presented the newly discovered selective estrogen receptor modulators (eng. selective estrogen receptor modulatos, SERMs), for example, US patents no. 5,484,795, 5,484,798, 5,510,358, 5,998,401 and WO 96/09040. Generally speaking, many of these SERMs have been found to exhibit beneficial estrogen agonist activity in the skeletal and vascular systems, with concomitant beneficial estrogen agonist activity in the breast. A small, particularly useful group of such compounds have also been found to have estrogen agonist effects in the uterus. A compound with this SERM profile is particularly promising in the treatment of uterine fibroids and/or endometriosis.

However, the clinical use of such SERM compounds in the treatment of fibroid disease of the uterus and/or endometriosis, especially in premenopausal women, is prevented due to the tendency of said compounds to have a significant ovarian stimulation effect. Therefore, there is currently a great need for new SERM compounds, which will act as estrogen agonists in the uterus, and at the same time will not significantly stimulate the ovaries.

The essence of invention

This invention relates to crystalline unsolvated 1-(4-2-piperidinylethoxy)phenoxy)-2-(4-methanesulfonylphenyl)-6-hydroxynaphthalene hydrochloride, that is, a compound of the formula:

[image]

hereinafter referred to in the text as F-III; having an X-ray diffraction pattern including the following maxima: 15.2 ± 0.1, 17.6 ± 0.1, 18.6 ± 0.1 and 24.1 ± 0.1° in 2θ; when the sample was obtained from a copper radiation source (CuKα, λ = 1.54056 Å).

This invention also relates to pharmaceutical compositions containing F-III and a pharmaceutical carrier. In another embodiment, the pharmaceutical compositions of the present invention may be adapted for use in the treatment of endometriosis and/or uterine leiomyoma.

The present invention also relates to methods for treating endometriosis and/or uterine leiomyoma, comprising administering an effective amount of F-III to a patient in need thereof.

Additionally, this invention relates to the use of F-III in the treatment of endometriosis and/or uterine leiomyoma. The present invention further relates to the use of F-III in the manufacture of medicaments for the treatment of endometriosis and/or uterine leiomyoma.

Short description of the image

Figure 1 is a representative XRD pattern for F-III.

Detailed description of the invention

Since F-III is an unsolvated crystalline form, it is understood to be the anhydride form of 1-(4-2-piperidinylethoxy)phenoxy)-2-(4-methanesulfonylphenyl)-6-hydroxynaphthalene hydrochloride.

X-ray powder diffraction (XRD) was used to characterize F-III. XRD is a technique that reveals long-range order in a crystalline material. X-ray powder diffraction (XRD) patterns were obtained on a Siemens D5000 X-ray powder diffractometer equipped with a CuKα source (λ = 1.54056 Å) and a Kevex Si(Li) solid-state detector operated at a minimum of 50 kV and 40 mA. The samples were scanned from 4 to 35° in 2θ, with a step size of 0.02° in 2θ and a maximum scan speed of 3.0 seconds per step and with 1 mm divergence and receiving notches and 0.1 mm detector notches. A synthetic fluoropholyte stain (NIST 675) was used as an internal standard, to correct for errors caused by sample drift.

The XRD patterns shown here for F-III present sharp maxima and a flat baseline, indicating a highly crystallized material. The angular positions of the maxima in 2q and the corresponding I/Io data for all maxima with intensities equal to or greater than 10% of the highest maximum, for F-III are shown in Table 1. All data in Table 1 are expressed with an accuracy of ± 0.1 ° in 2q.

Table 1

F-III

[image]

It is well known in crystallography that, for any crystal form, the relative intensities of diffraction maxima can vary due to preferred orientation, which arises from factors such as crystal morphology and orientation. When preferred orientation effects are present, the peak intensities are changed, but the characteristic peak positions of the polymorphs are unchanged. See, eg, The United States Pharmacopeia #23, National Formulary #18, pages 1843-1844, 1995. Furthermore, it is also well known in crystallography, for any crystal form, that the positions of the angular maxima can vary slightly. For example, peak positions may shift due to variations in the temperature at which the sample was analyzed, sample drift, or the presence or absence of an internal standard. In this case, the maximum position variability of ± 0.1° in 2q will account for these potential variations, without interfering with the unambiguous identification of the crystalline salts of this invention.

Cross polarization/magic angle spinning (CP/MAS) 13C NMR (solid state NMR or SSNMR) was also used to characterize F-III. The above spectrum was obtained using a Varian Unity Inova 400 MHz NMR spectrometer operating at a carbon frequency of 100.573 MHz. Data acquisition parameters were as follows: 90° proton r.f. pulse width 4.0 μs, contact time 2.5 ms, pulse repetition time 15 s, MAS frequency 10 kHz, spectrum width 50 kHz, and data collection time 50 ms. Chemical shifts were standardized relative to the methyl group or hexamethylbenzene (δ = 17.3 ppm) by swapping samples. Chemical shift data for F-III are as follows: 20.7, 23.8, 25.3, 38.4, 51.2, 52.4, 56.8, 59.8, 110.8, 113, 3, 114.6, 118.0, 118.3, 122.3, 122.9, 125.4, 127.2, 130.0, 132.0, 137.9, 142.2, 143.1, 147.3, 151.3, 153.8 and 158.0 ppm.

Preparation 1

1-(2-{4-[2-(4-methanesulfonyl-phenyl)-6-methoxy-naphthalen-1-yloxy]-phenoxy}-ethyl)-piperidine hydrochloride

To a 3-necked flask equipped with a reflux condenser and a nitrogen release vent, add 6-methoxytetralone (1.0 eq.), 4-bromophenyl-methyl-sulfone (1.02~1.05 eq.), palladium acetate Pd (OAc)2(0.025 eq.), [(oxidi-2,1-phenylene) bis(diphenylphosphine)] (DPEfos ligand, 0.026 eq.) and toluene 10~12 vol. Add sodium t-butoxide (2.5 eq.) in one portion and allow the mixture to exotherm to ~40°C. Heat to 75°C to 80°C. At the end of the reaction, evaluated by HPLC analysis, cool to room temperature. Add slowly 12 volumes of water maintaining the temperature <40°C. Mix for 2 to 3 hours. Filter through a polypropylene pad and rinse with water (3 x 2 volumes). Dry the filter cake overnight at 50°C to obtain 2-(4-methanesulfonylphenyl)-6-methoxytetralone.

Combine 2-(4-methanesulfonylphenyl)-6-methoxytetralone (1.0 eq.), hyflo (20%/wt), and toluene (7.5 vol). Add PBr3 (1.5~1.75 eq.) in one portion, with stirring at room temperature. Heat the contents to reflux (~110°C) overnight. At the end of the reaction, estimated by HPLC analysis (usually 15 hours), cool the solution to 45°C or 90°C and slowly add 20 volumes of tetrahydrofuran (THF). Mix for 30 minutes at 45°C or 90°C and filter while warm over a hyflo pad. Wash the pad with 2 x 2 volumes of THF at 45°C or 90°C. Concentrate the filtrate to approximately 7 volumes. Add 7.5 volumes of water to the rest of the mixture, keeping the temperature below 40°C. (NOTE: the initial addition of water is very exothermic with strong evolution of HBr). Cool the thick solution to room temperature and stir for 2 to 3 hours. Filter through a polypropylene pad and rinse with 2 x 2 volumes of water. Dry the filter cake overnight at 60°C under vacuum to obtain 1-bromo-2-(4-methanesulfonylphenyl)-3,4-dihydro-6-methoxynaphthalene.

Mix 1-bromo-2-(4-methanesulfonylphenyl)-3,4-dihydro-6-methoxynaphthalene and 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ, 1.8 eq.) in 10 volumes of acetonitrile and 5 volumes of THF. Under a nitrogen atmosphere, heat the contents of the reaction at 73-75°C. Monitor the progress of the reaction by GC analysis until the reaction is complete. Additional DDQ (0.2-0.3 eq.) may be required to complete the reaction. Cool the contents to room temperature and add 10 volumes of 1 N sodium hydroxide. Stir for approximately 1 hour and filter. Wash the filter cake with 2 volumes of water, 3 x 5 volumes of 50% acetonitrile/water and finally 3 volumes of methanol. Dry the filter cake under vacuum at 65°C to obtain 1-bromo-2-(4-methanesulfonylphenyl)-6-methoxynaphthalene.

Charge 1-bromo-2-(4-methanesulfonylphenyl)-6-methoxynaphthalene, 4-(2-piperidinylethoxy)phenol (2.0 eq.), cesium carbonate (2.0-2.1 eq.), and copper chloride ( 0.15eq.) to 12 volumes of «diglyme». Evacuate the vial for ~2 minutes, then purge with nitrogen. Repeat the evacuation/cleaning with nitrogen 3 times. Heat the mixture to 130°C until the end of the reaction according to HPLC analysis. At the end of the reaction, cool the contents to near room temperature and add 12 volumes of ammonium hydroxide and stir for approximately 30 minutes. Filter to remove solids and wash the solids with 9 volumes of 30% MeOH/NH4OH, concentrating the solid on the filter support. Wash the solids with 2 x 9 volumes of 30% MeOH/NH4OH, concentrating the solid on the filter support. Wash with 4 volumes of methanol. Dry the filter cake under vacuum at 60°C to obtain the free base of the title compound. Concentrate the free base in 9 volumes of toluene and heat the thick solution to 70-75°C. Dissolve 1.1 equivalents of hydrogen chloride gas in 2 volumes of ethanol. Add the ethanolic HCl solution to the hot thick toluene solution. Cool the solution to room temperature and stir for 1 to 2 hours. Filter and wash with a small amount of toluene. Dry the filter cake under vacuum at 65°C to obtain the title compound.

Example 1

F-III

Mix 1-(2-{4-[2-(4-methanesulfonyl-phenyl)-6-methoxy-naphthalen-1-yloxy]-phenoxy}-ethyl)-piperidine hydrochloride with 5 volumes of 1,2-dichloroethane (DCE) and cool the mixture to <10°C. Add 5 equivalents of boron trichloride by subsurface addition. Stir at room temperature until the reaction is complete as determined by HPLC analysis. Quench the reaction with 5.6 volumes of 3A-ethanol (ethanol denatured with about 5% methanol) keeping the contents at <50°C. Cool to room temperature and mix for 1 to 3 hours. Filter the solids and wash the filter cake with 3A-ethanol. Dry the "cake" filter under vacuum at 65°C. Dissolve the isolated product in 9.8 volumes of 3A ethanol and 1.5 volumes of deionized water at approximately the reflux temperature of the mixture. Allow the solution to reflux for approximately 30 minutes, and then allow the mixture to cool to room temperature. Once at room temperature, stir the resulting thick solution for 1 to 2 hours at room temperature, then filter and wash the filter cake with 3A ethanol. Dissolve the filter cake in 19 volumes of acetonitrile and 1.4 volumes of deionized water at reflux. Azeotropically remove water by distillation until a total of 12.1 volumes of distillate is removed. Cool the resulting thick solution to room temperature, filter and wash the filter cake with acetonitrile to obtain the title compound.

Formulation (pharmaceutical preparation)

A compound of the present invention is preferably formulated in dosage unit form, i.e., in a single carrier, for example, a tablet or capsule, prior to administration to a patient receiving therapy. The term "patient" includes female persons and female animals, such as domestic animals (dogs, cats, horses and the like). The preferred patient for treatment is a female person.

These pharmaceutical preparations are prepared by known procedures, using well-known and widely available ingredients. In preparing the formulations of this invention, the active ingredient (F-III) will usually be mixed with a carrier, or diluted with a carrier, or enclosed in a carrier which may be in the form of a capsule, sachet, paper or other packaging. When the carrier serves as a diluent, it may be a solid, semi-solid, or liquid material that acts as a carrier, excipient, or medium for the active ingredient. Thus, preparations can be in the form of tablets, pills, powders, lozenges, sachets, «cachets», elixirs, suspensions, emulsions, solutions, syrups, aerosols (as a solid or in a liquid medium), soft and hard gelatin capsules, suppositories, sterile injectables solutions and sterile packaged powders.

Some examples of suitable carriers, excipients and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, aqueous syrup, methyl cellulose, methyl and propylhydroxybenzoate, talc, magnesium stearate and mineral oil. Formulations may additionally include lubricating agents, humectants, eg polysorbate 80 or lauryl sulfate, emulsifying and suspending agents, preservatives, sweeteners and flavors. The preparations of the invention can be formulated so as to enable rapid, continuous or delayed release of the active compound, after administration to the patient.

Examples of wording

Capsules or tablets of 10 mg

Add about 156 mg of filler (lactose, mannitol or dextrose), about 20 mg of disintegrant (microcrystalline cellulose or starch), about 4 mg of super disintegrant (crospovidone or sodium starch glycolate), about 4 mg of binder (hydroxy-propyl-methyl cellulose or hydroxy -propyl cellulose) and about 10 mg of F-III into the granulator and mix to distribute the powder evenly. Spray an aqueous granulation solution composed of povidone, hydroxy-propyl-methyl cellulose or hydroxy-propyl cellulose (enough to deliver about 2-4% of the dry powder weight) and a humectant, such as polysorbate 80 or sodium lauryl sulfate (enough to deliver between 0.5 and 3% by weight), at a uniform rate to the powder, with mixing. Sift the wet granulated material through a coarse sieve to break up large agglomerates. Dry the filtered granular powder either by fluid bed processing or in a convection oven. Reduce the dried granular powder to a uniform size by passing it through a colloid mill or other suitable device, and then transfer the material to a mixer. Evenly grind the granulated powder with a lubricant (magnesium stearate, or sodium stearyl fumarate in approximately 1% of the weight of the total formulation) and an additional disintegrant (approximately 2-4% of the weight in external powders). Fill the resulting powders into hard gelatin capsules or compress them into tablets (after which the tablets are coated with a film as described below). The total weight of a capsule or tablet prepared in this way is about 200 mg.

Capsules or tablets of 45 mg

Add approximately 162 mg of filler (lactose, mannitol or dextrose), approximately 10 mg of disintegrant (microcrystalline cellulose or starch), approximately 45 mg of F-III to the granulator and mix to distribute the powder evenly. Spray an aqueous granulation solution composed of povidone (about 35% by weight) and polysorbate 80 (about 10% by weight) at a uniform rate onto the powder, while stirring. Sift the wet granulated material through a coarse sieve to break up large agglomerates. Dry the filtered granular powder either by fluid bed processing or in a convection oven. Pass the dried granulated powder through a colloid mill or other suitable device, and then transfer the material to a mixer. Evenly mix the granulated powder with a lubricant (magnesium stearate; approximately 1% by weight of the total formulation) and an additional disintegrant (approximately 2% by weight in external powders). Fill the resulting powders into hard gelatin capsules or compress them into tablets (after which the tablets are coated with a film as described below). The total weight of a capsule or tablet prepared in this way is about 250 mg.

Alternatively, to prepare the tablet, add the filler, disintegrant and F-III to a mixer and mix to distribute the powders evenly. Once the powders are evenly distributed, add the lubricant and mix again. Transfer the mixed material to a tablet compression device to prepare tablets, which are then film-coated using a suitable film-forming agent.

Biological tests

Ishikawa cell proliferation test:

This assay measures cell proliferation (using alkaline phosphatase), both agonistically, in the presence of a compound of the present invention alone, and antagonistically, where the ability of a compound of the present invention to block growth stimulation by estradiol is measured.

Human endometrial tumor Ishikawa cells are maintained in MEM (minimum essential medium, with Earl's salts and L-glutamine, Gibco BRL, Gaithersburg, MD), supplemented with 10% fetal bovine serum (FBS) (v/v), (Gibco BRL). One day before the test, the culture medium was replaced with test medium, DMEM/F-12 (3:1) (Dulbecco's Modify Eagle Medium: Nutrient Mixture F-12, 3:1 mixture, no phenol red, Gibco BRL) supplemented with 5% dextran-coated activated charcoal-purified fetal bovine serum (DCC-FBS) (Hyclone, Logen UT), L-glutamine (2 mM), MEM sodium pyruvate (1 mM), HEPES (N-[2-hydroxyethyl )]piperazine-N'-[2-ethanesulfonic acid] 2 mM) all from Gibco BRL). After overnight incubation, Ishikawa cells are washed with Dulbecco's phosphate-buffered saline (1x) (D-PBS) without Ca2+ and Mg2+ (Gibco BRL) and trypsinized using 0.25% trypsin/EDTA, without phenol red (Gibco BRL), for 3 min. The cells are resuspended in the test medium and the concentration is adjusted to 250,000 cells/ml. Approximately 25,000 cells in 100 μl of medium are added per well of a 96-well flat-bottom microtiter plate (Costar 3596) and incubated at 37°C in a humidified incubator with 5% CO2 for 24 hours. The next day, serial dilutions of the compounds (up to 6 times the final concentration in the test) are prepared in the test medium. The test is performed in two ways, agonistic and antagonistic.

For the agonist mode, 25 μl/well of assay medium, followed by 25 μl/well of the diluted compound of this invention (at 6x final concentration) are applied to the plates. For antagonist mode, plates are coated with 25 μl/well of 6 nM E2 (β-estradiol, Sigma, St. Louis, MO), followed by 25 μl/well of a diluted compound of this invention (at 6x final concentration). After an additional incubation of 48 hours, at 37°C in a humidified incubator with 5% CO2, the medium is removed from the wells and 100 μl of fresh assay medium is added to each microculture. Serial dilutions of compounds are prepared and added to the cells as described above. After an additional incubation of 72 hours, at 37°C in a humidified incubator with 5% CO2, the assay is stopped by removing the media and washing the plates twice in Dulbecco's phosphate-buffered saline (1x) (D-PBS) (Gibco BRL). The plates are dried for 5 min and frozen at -70°C for at least 1 hour. The tiles are then removed from the freezer and left at room temperature to thaw. 100 μl of 1-StepTM PNPP (Pierce Chemical Company, Rockford, IL) was added to each well. After incubation for 20 min, the plates are read on a spectrophotometer at 405 nm.

Data are linearly interpolated to obtain EC50 (for agonist mode) or IC50 (for antagonist mode) values. For antagonistic mode, % efficacy for each compound was calculated relative to E2 alone (1 nM). For the agonist mode, the % efficacy for each compound was calculated according to the response to tamoxifen.

In agonist mode, F-III has been tested and is less stimulatory than tamoxifen. In antagonist mode, F-III inhibited the response to 1 nM estradiol by more than 70%.

Ten-day rat hormone test (ovarian stimulation):

The initial first ovarian toxicity study was conducted via a 10-day hormone monitoring rat in which estradiol and luteinizing hormone levels were measured after administration of F-III GYN SERM. This study was conducted by administering the compound orally for 10 days to mature (9-10 weeks old) F344 female rats. For assessment of LH and estradiol levels, carcass blood was collected by rapid decapitation approximately 2 hours after the tenth dose. Serum, obtained after centrifugation, was removed and stored frozen below -60°C until analysis. Serum levels of LH and estradiol were measured using a radioimmunoassay (RIA).

Rat primary antibody to LH and reference preparations (rat LH:RP-3) were obtained from Dr. A.F. Parlow, Director, Pituitary Hormones and Antisera Center, Harbor-UCLA Medical Center, Torrance, CA. The upper limits of detection in the LH test were 30 ng/ml, and the lower limits of detection were 0.1 ng/ml for 100 μl of sample.

E2 clinical tests. DiaSorin s.r.l., Saluggia (Vercelli), Italy. The upper limit of detection is 1000 pg/ml, and the lower limit of detection is 5 pg/ml. F-III has been tested in the above assays and does not significantly raise circulating estradiol and LH levels.

Advantages

As an estrogen antagonist in breast and uterine tissue, F-III is useful in the treatment of conditions for which estrogen has been shown to be the main cause. As an estrogen agonist in the skeletal and vascular systems, F-III is useful in the treatment of conditions for which estrogen has been shown to have beneficial effects.

The terms "treating" and "treating", as used herein, include their generally accepted meanings, i.e. alleviating, ameliorating, controlling, preventing, preventing, restraining, slowing, stopping or reversing the progression or severity of the pathological condition described herein or its consequences. The term "preventing" refers to reducing the likelihood that a recipient of a compound of the present invention will be exposed to or develop any of the pathological conditions described herein or their consequences.

Diseases, disorders, or conditions for the treatment of which a compound of the present invention is useful include, but are not limited to, (1) uterine and/or breast cancer; (2) endometriosis; (3) uterine leiomyomas; and (4) osteoporosis. Treating uterine leiomyoma as described here can also reduce the symptoms that are present, such as pain, frequency of urination and uterine bleeding.

Dose

As used herein, the term "effective amount" means an amount of F-III capable of treating the conditions described herein or their adverse effects.

The specific dose applied is determined according to certain circumstances present in a particular situation. These circumstances include the route of administration, the recipient's previous medical history, the pathological condition or symptom being treated, the severity of the condition/symptom being treated, and the age and gender of the recipient. The receiving patient's physician should determine the administered therapeutic dose in light of the relevant circumstances.

In general, the effective minimum daily dose of F-III will exceed approximately 5 mg. Typically, the effective maximum daily dose will not exceed 350 mg. The exact dose can be determined, in accordance with standard practice in the medical profession of "dose titration" of the recipient; that is, a low dose of the compound is initially administered, and the dose is gradually increased until the desired therapeutic effect is observed.

Method of application

F-III can be administered by a variety of routes, including intramuscular, intranasal, intravaginal, intravenous, oral, rectal, subcutaneous, topical, and transdermal administration. The preferred method is oral administration.

Combined therapy

F-III may be used in combination with other drugs used in the treatment of diseases or conditions for which these compounds are beneficial. Such other drugs can be administered, in the manner and in the amount in which they are usually used, simultaneously or sequentially with the salt of this invention. When F-III is used concurrently with one or more other drugs, the preferred dosage is in the form of a pharmaceutical unit containing such other drugs in addition to this compound. Accordingly, the pharmaceutical compositions of the present invention include those containing one or more other active ingredients. One example of another active ingredient that can be combined with a compound of the present invention, either administered separately or in the same pharmaceutical preparation, includes agents used in hormone replacement therapy (HRT).

Claims (5)

1. Crystalline unsolvated 1-(4-2-piperidinylethoxy)phenoxy)-2-(4-methanesulfonylphenyl)-6-hydroxynaphthalene hydrochloride characterized by having an X-ray diffraction pattern comprising the following maxima: 15.2 ± 0.1 , 17.6 ± 0.1, 18.6 ± 0.1 and 24.1 ± 0.1° in 2θ; when the sample was obtained from a copper radiation source (CuKα, λ = 1.54056 Å). 2. The salt of claim 1, characterized in that the X-ray diffraction pattern further comprises the following maxima: 16.9 ± 0.1, 19.2 ± 0.1, 21.3 ± 0.1 and 21.6 ± 0 ,1° in 2θ. 3. A method for treating endometriosis, characterized in that it comprises administering to a patient in need of it an effective amount of the compound of claim 1 or 2. 4. A method for treating uterine leiomyoma, characterized in that it comprises administering to a patient in need of it an effective amount of the compound of claim 1 or 2. 5. The compound of claim 1 or 2, characterized in that it is used for the treatment of endometriosis and/or leiomyoma of the uterus.