METHODS OF REDUCING THE BODY WEIGHT OF A SUBJECT BY

ADMINISTERING A FATTY ACID ESTER OF AN ESTROGEN OR ESTROGEN

DERIVATIVE IN AN OIL AND COMPOSITIONS CONTAINING THE SAME

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation of U.S. Patent Application No. 11/475,461, filed June 26, 2006. The entire contents of this application are expressly incorporated herein by reference thereto.

ITELD OF THE INVENTION

[0002] This invention relates generally to compositions and methods of reducing the body weight of a subject. More particularly, the invention is directed to methods of reducing the body weight in a subject comprising administering therapeutically effective amounts of a fatty acid ester of an estrogen or estrogen derivative in an oil. Furthermore the invention is directed to compositions comprising a fatty acid ester of an estrogen or estrogen derivative in an oil.

BACKGROUND OF THE INVENTION

[0003] Treatment of obesity and/or overweight is a therapeutic and/or cosmetic problem of major importance that does not have a satisfactory solution yet. Attempts to solve the problem by physical exercise or reduction of food intake are well known. But also known are the difficulties, limitations and general lack of success of all these approaches. Apparently the sheer complexity of mechanisms involved in the control of body mass allows little room for external manipulation, thus limiting the possible damage to body reserves by increased thermogenic stimulation or diminished energy intake. [0004] In the therapeutic fight against obesity and/or overweight, considerable research has focused on trying to find a signal that informs the brain of the size of fat tissue mass. It is believed that such information is required by the brain to promote either the accumulation of fat reserves or their burning by the thermogenic system, via the natural homeostatic mechanisms set to maintain the body mass stable.

[0005] Estradiol or estra- 1,3,5(10)-triene-3, 17-diol is a natural estrogen widely used in estrogenic hormone therapy. Estradiol esters at C- 17 and C-3 with palmitic, stearic, and oleic acids have been chemically synthesized and their long-term estrogenic responses in

ovariectomized rats have been reported (c/ M. A. Vazquez-Alcantara et ah, J. Steroid Biochem. 33:1111-18 (1989)).

[0006] Alemany disclosed fatty acid esters of estrogens for the treatment of obesity and overweight in U.S. Patent No. 5,798,348, which is incorporated herein by reference in its entirety for all purposes. For instance, the fatty acid monoester estrone monooleate ("oleoyl- estrone"), a powerful slimming agent for the treatment of obesity and overweight, was studied. It is believed that oleoyl-estrone acts as a ponderostat signal informing the body weight control system of the mass of fat reserves held in the body. In the morbidly obese, this signaling is altered. Its main physiological effects are a decrease in voluntary food intake with maintenance of energy expenditure, which creates an energy gap that is fulfilled at the expense of lipid reserves, thus decreasing the body fat mass, sparing protein, largely decreasing the need for carbohydrate and reducing insulin resistance. Furthermore, Girouard in PCT Publication Nos. WO03/018529 and WO2004/045560, as well as U.S. Patent No. 6,821,961 to Girouard, which are herein incorporated by reference in their entirety for all purposes, disclose additional fatty acid esters of estrogens or estrogen derivatives for treating obesity.

[0007] However, many fatty acid esters of estrogens or estrogen derivatives, including oleoyl-estrone, are poorly soluble or insoluble in water. Moreover, manipulation of the pH and also the addition of surfactants may not improve the water solubility of fatty acid esters of estrogens or estrogen derivatives. The insolubility or limited solubility of fatty acid esters of estrogens or estrogen derivatives in water introduces difficulties in formulating such compounds into pharmaceutical compositions. In addition, because of the insolubility or limited solubility of the fatty acid esters of estrogens or estrogen derivatives in water, such compounds will not be able to be absorbed and used by the body unless such compounds are presented in a dissolved state at the site of absorption.

SUMMARY OF THE INVENTION

[0008] Since it is of great interest to provide satisfactory new approaches for the treatment of obesity or overweight, the present invention relates generally to compositions and methods for reducing the body weight of a subject, such as a human. More particularly, the invention is directed to methods for reducing the body weight in a subject comprising administering a composition comprising a therapeutically effective amount of a fatty acid ester of an estrogen or estrogen derivative in an oil, wherein the fatty acid ester of an estrogen

or estrogen derivative is soluble in the oil. Furthermore the invention is directed to compositions comprising a fatty acid ester of an estrogen or estrogen derivative in an oil. [0009] In this specification, the term "estrogen" refers to the substances tending to promote estrus and stimulate the development of female secondary sex characteristics. This term comprises natural, semisynthetic and synthetic estrogens, both steroidal and nonsteroidal, such as estrone, diethylstilbestrol, estriol, estradiol and ethinyl estradiol. [0010] The term "estrogen derivative" refers to a compound that is derived from estrogen and usually maintains its general structure. Estrogen and estrogen derivatives are substances that induce biological responses linked to the stimulation of estrogen receptors and other biological systems that result in biological actions similar to those of estradiol and estrone.

[0011] In this specification, the phrase "fatty acid" refers to the carboxylic acids that are components of natural fats, such as, without limitation, oleic, linoleic, linolenic, stearic, palmitic, palmitoleic, arachidonic, eicosenoic, docosenoic, and tetracosenoic acids. [0012] The term "fatty acid ester" or "fatty acid ester of an estrogen or estrogen derivative" refers to any compound containing one or more fatty acids bonded to an estrogen or estrogen derivative molecule.

[0013] In this specification, the term "oil" refers to a wide range of substances that in general contain fats. Oils can be derived from minerals, animals, plants, plant seeds or nuts and are usually composed largely of glycerides or fatty acids, usually oleic, palmitic, stearic and linoleic. Examples of mineral oils include but are not limited to, petroleum oils that are aliphatic, wax, aromatic, asphalt or mixtures thereof. Examples of oils derived from animals include, but not limited to, fats such as tallow, lard and stearic acid, fish oils, fish-liver oils, oleic acid and sperm oil. Examples of oils derived from plants include oils derived from nuts, seeds, fruits and vegetables, including but not limited to, corn, linseed, tung, oiticica, soybean, olive, cottonseed, castor, coconut, safflower and palm oil. Other examples of oils derived from plants are essential oils which are complex volatile liquids derived from flowers stems and leaves and often the entire plant. Though the composition of the present invention is described and claimed as comprising "an oil," the phrase "an oil" is to be defined as either a single oil or an oil blend comprising more than one oil. [0014] As used herein, the terms "subject," "patient" and "animal" are used interchangeably. The terms "subject" and "patient" refer to an animal (e.g., a bird such as a chicken, quail or turkey, or a mammal), preferably a mammal including a non-primate (e.g., a cow, pig, horse, sheep, rabbit, guinea pig, rat, cat, dog, and mouse) or a primate (e.g., a

monkey, chimpanzee and a human), and more preferably a human. In one embodiment, the subject is a non-human animal such as a farm animal (e.g., a horse, cow, pig or sheep), or a pet (e.g. , a dog, cat, guinea pig or rabbit). In a preferred embodiment, the subject is a human. [0015] In certain embodiments of the present invention the methods include, a method of reducing body weight in a human by orally administering a composition that includes a therapeutically effective amount of a fatty acid ester of an estrogen or estrogen derivative in an oil, wherein the oil is derived from a plant such as, safflower oil, canola oil or soybean oil.

[0016] Also, in certain embodiments the compositions of the present invention for reducing body weight in a subject. For example the compositions of the present invention include a composition for reducing body weight in a subject having a therapeutically effective amount of a fatty acid ester of an estrogen or estrogen derivative in an oil, wherein the composition is suitable for oral administration, such as in a capsule and wherein the oil, is derived from a plant such as, safflower oil, canola oil or soybean oil.

BRIEF DESCRIPTION OF THE DRAWINGS ⁽

[0017] Figure 1 shows an embodiment of a manufacturing process for the preparation of composition in accordance with the present invention.

DETAILED DESCRIPTION OF THE INVENTION

[0018] As noted above, the present invention relates to methods and compositions for reducing body weight in a subject, preferably a human, comprising administering a therapeutically effective amount of a fatty acid ester of an estrogen or estrogen derivative in an oil.

[0019] In some embodiments, the estrogen of the fatty acid ester of an estrogen or estrogen derivative comprises estrone, (i.e. 3-hydroxyestra-l,3,5(10)-trien-17-one); diethylstilbestrol, (i.e. 4,4'-(l,2-diethyl-l,2-ethenediyl)-bisphenol); estriol, (i.e. estra- l,3,5(10)triene-3,16,17-triol), ethinyl estradiol, (i.e. 19-nor-17a-pregna-l, 3,5(10)-trien-20- yne-3,17-diol); or estradiol. The estrogen preferably comprises an estrone. Further, in some embodiments, the estrogen derivative of the fatty acid ester of an estrogen or estrogen derivative preferably comprises 2 hydroxyestrone or 2 hydroxy-β-estradiol. [0020] In some embodiments, the fatty acid can comprise, for instance, oleic acid, linoleic acid, linolenic acid, stearic acid, palmitic acid, palmitoleic acid, arachidonic acid, eicosenoic acid, docosenoic acid, tetracosenoic acid or, any other aliphatic acid with a

straight, branched, substituted, saturated, or unsaturated chain. Preferably, the fatty acid comprises an oleic acid.

[0021] In some embodiments, the fatty acid includes an acyl group and the estrogen is steroidal and has a steroid ring system with a C-3 position and a hydroxyl group at the C-3 position. In these embodiments, the acyl group of the fatty acid is attached to the hydroxyl group at the C-3 position of the steroid ring system in the fatty acid ester.

[0022] In other embodiments, the fatty acid includes an acyl group, wherein the estrogen comprises estrone, diethylstilbestrol, estriol or ethinyl estradiol, and wherein the fatty acid comprises an oleic acid, linoleic acid, linolenic acid, stearic acid, palmitic acid, palmitoleic acid, arachidonic acid, eicosenoic acid, docosenoic acid, or tetracosenoic acid; and with the proviso that, when the estrogen is steroidal and has a steroid ring system with a

C-3 position and a hydroxyl group at the C-3 position, the acyl group of the fatty acid is attached to the hydroxyl group at the C-3 position of the steroid ring system in the fatty acid ester.

[0023] The fatty acid ester of estrogen or estrogen derivative preferably comprises a fatty acid monoester, such as, for example, estrone monooleate ("oleoyl-estrone"), diethylstilbestrol monooleate, estrone monoeicosenoate or diethylstilbestrol monoeicosenoate. Oleoyl-estrone or estrone monooleate has the chemical formula:

[0024] which is also known as [3(Z)]-3-[(l-oxo-9-octadecenyl)oxy]-estra-l,3,5(10)- trien-17-one.

[0025] In certain embodiments, the fatty acid ester of an estrogen or estrogen derivative is substantially pure before being added to the oil.

[0026] The amount of the fatty acid ester of an estrogen or estrogen derivative in the composition of the present invention can range from about 0.01% to about 99% by weight of the composition ("by weight"). The amount of fatty acid ester of an estrogen or estrogen derivative can also range from about 0.1% to about 45% by weight, from about 0.1% to about

40% by weight, from about 0.1% to about 35% by weight, from about 0.1% to about 30% by

weight. In certain embodiments, the fatty acid ester of an estrogen or estrogen derivative can comprise about 1% to about 25% by weight or about 1% to about 20% by weight. In one embodiment the percent of the fatty acid ester of an estrogen or estrogen derivative is about 1% to about 15% by weight.

[0027] The dose of the fatty acid ester of an estrogen or estrogen derivative in the composition is dependent upon a number of factors, including, the nature of the severity of the condition to be treated, the particular compound in the composition, the route of administration and the age, weight, and response of the individual patient. The dose of the fatty acid ester of an estrogen or estrogen derivative can generally range from about 0.000005 mg/kg/day to about 20 mg/kg/day. Preferably, the therapeutically effective amount of the fatty acid ester of an estrogen or estrogen derivative comprises an amount of about 0.00005 mg/kg/day to about 10 mg/kg/day or the therapeutically effective amount of the fatty acid ester of an estrogen or estrogen derivative comprises an amount of about 0.0005 mg/kg/day to about 2 mg/kg/day. More preferably, the therapeutically effective amount of the fatty acid ester of an estrogen or estrogen derivative comprises an amount of about 0.001 mg/kg/day to about 1 mg/kg/day.

[0028] The composition of the fatty acid ester of estrogen or estrogen derivative in an oil can be administered as a single daily dose or multiple daily doses or administered in doses over various other time periods. The daily dose can be between about 0.001 mg/day to about 1000 mg/day. Preferably, the therapeutically effective amount of the fatty acid ester of an estrogen or estrogen derivative comprises an amount of about 0.01 mg/day to about 500 mg/day or the therapeutically effective amount of the fatty acid ester of an estrogen or estrogen derivative comprises an amount of about 0.1 mg/day to about 100 mg/day. More preferably, the therapeutically effective amount of the fatty acid ester of an estrogen or estrogen derivative comprises an amount of about 1 mg/day to about 50 mg/day. In some cases it may be necessary to use dosages outside of these ranges. In certain embodiments, the fatty acid ester of an estrogen or estrogen derivative can be administered in an amount of about 0.3 mg, 1 mg, 10 mg or 50 mg in a capsule, such as a once-a-day capsule. [0029] The compositions of the present invention also comprise an oil. Preferably, oils used in the present invention are edible. Oils suitable for the present invention can be triglycerides; such as, medium chain triglycerides, Captex ^® 300-6C and Captex ^® 300 EP; fats; oils derived from animal or plant oils; such as vegetable and fruit oils. Examples of such oils derived from plants include without limitation, canola oil; safflower oil; soybean oil; corn oil; olive oil; sesame oil; rapeseed oil; sunflower oil; rice oil; palm oil; cottonseed oil; linseed

oil; tung oil; oiticica oil; castor oil; perilla oil; coconut oil; and oils such as Labrasol ^® , Labrafil ^® M 125CS, Labrafac ^® CC, and blends thereof. The preferred oils of the present invention include canola oil, safflower oil, soybean oil, corn oil, olive oil, sesame oil and the medium chain triglycerides Captex ^® 300-6C, Captex ^® 300 EP.

[0030] Oils derived from animals and mineral oils may also be used in the present invention. Examples of such oils derived from animals include without limitation, fats such as tallow, lard and stearic acid, fish oils, fish-liver oils, oleic acid and sperm oil. Examples of mineral oils include without limitation, petroleum oils which are aliphatic, wax, aromatic, asphalt or mixtures thereof.

[0031] The amount of oil used depends on various factors, such as the type of oil used, the amount of fatty acid ester of an estrogen or estrogen derivative used and the type of dosage form used. The preferred amount of oil is the amount that is required to suitably dissolve the fatty acid ester of an estrogen or estrogen derivative. The solubility of a fatty acid ester of an estrogen or estrogen derivative, such as oleoyl-estrone in an oil such as, safflower oil, in terms of milligrams of the fatty acid ester in grams of oil can be between about 0.001 mg/g to about 500 mg/g when the solute is gravimetrically measured. In some embodiments the solubility is at least 25 mg/g, 50 mg/g or 200 mg/g when the solute is measured gravimetrically.

[0032] The amount of oil can range from about 1% to about 99% by weight of the composition ("by weight"). The amount of oil can also range from about 10% to about 99% by weight, from about 20% to about 99% by weight, from about 25% to about 99% by weight, from about 30% to about 99% by weight, from about 40% to about 99% by weight. In certain embodiments, the oil can comprise about 50% to about 99% by weight or about 75% to about 99% by weight. In one embodiment percent of the oil is about 80% to about 99% by weight.

[0033] In the methods and compositions of the present invention, the fatty acid ester of an estrogen or estrogen derivative in an oil can be administered in a single composition. The compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations and the like, depending on its intended route of administration.

[0034] Suitable routes of administration include parenteral (e.g., subcutaneous, intramuscular, intraorbital, intracapsular, intraspinal, intrasternal, intravenous, intradermal, intraperitoneal, intraportal, intra-arterial, intrathecal, transmucosal, intra-articular, and

intrapleural,), transdermal (i.e., topical), epidural, and mucosal (e.g., intranasal) injection or infusion, as well as oral, inhalation, pulmonary, and rectal administration.

[0035] For oral administration, the compositions may be formulated as tablets, pills, dragees, troches, capsules, liquids, elixirs, solutions, mouthwash, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated. Preferred types of capsules include, but are not limited to, Capsugel, Licaps or Type 0 capsules. When the composition of the present invention is formulated as capsules, the capsules can further be secured by any methods known in the art including sealing and banding methods.

[0036] For topical administration, the compositions may be formulated as solutions, gels, ointments, salves, creams, suspensions, etc. as are well-known in the art.

[0037] For administration by inhalation, the compositions may be formulated as an aerosol spray from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the composition and a suitable powder base such as lactose or starch.

[0038] The parenteral preparation can be enclosed in ampules, disposable syringes or multiple dose vials made of glass or plastic.

[0039] The composition may also be formulated in rectal or vaginal compositions such as suppositories or retention enemas that contain conventional suppository bases such as cocoa butter or other glycerides.

[0040] The compositions and the compositions used in the methods of the present invention can further include at least one pharmaceutically acceptable excipient. As used herein, the term "pharmaceutically acceptable" means suitable for use in, or in preparing a pharmaceutical product or composition. The term "excipient" refers to a diluent, adjuvant

(e.g., Freund's adjuvant (complete and incomplete)), filler, carrier, or vehicle with which the therapeutic is administered.

[0041] Such pharmaceutically acceptable excipients include, but are not limited to, water, salt solutions, alcohol, silicone, waxes, oils, polyethylene glycols, propylene glycol, liposomes, sugars, gelatin, surfactants, celluloses, polyvinylpyrollidones, or a combination thereof.

[0042] For oral administration, the composition can include excipients such as sugars, e.g., lactose, sucrose, mannitol and sorbitol; cellulose preparations such as maize starch,

wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or polyvinylpyrollidone (PVP); fats, oils, granulating agents; and binding agents such as microcrystalline cellulose, gum tragacanth or gelatin; disintegrating agents, such as cross-linked polyvinylpyrollidone, agar, alginic acid or a salt thereof such as sodium alginate, Primogel, or corn starch; lubricants, such as magnesium stearate or Sterotes; glidants, such as colloidal silicon dioxide; a sweetening agent, such as sucrose or saccharin; or flavoring agents, such as peppermint, methyl salicylate, or orange flavoring. If desired, solid dosage forms may be sugar-coated or enteric-coated using standard techniques.

[0043] In certain embodiments during the sealing and banding process, preferred excipients include ethyl alcohol, polysorbate, gelatin and water.

[0044] For parenteral administrations, the composition comprises one or more of the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerol, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose.

[0045] Also for parenteral administrations, the compositions may be formulated in solutions, preferably in physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiological saline buffer. The solution may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. In a preferred embodiment, the compositions are formulated in sterile solutions.

[0046] Specifically for intravenous administration, suitable excipients include physiological saline, bacteriostatic water, Cremophor® EL (BASF; Parsippany, NJ) or phosphate buffered saline (PBS). The excipient can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. In all cases, the composition must be sterile and should be injectable through a syringe. The compositions should be stable under the conditions of manufacture and storage and preserved against the contaminating action of microorganisms such as bacteria and fungi. The proper fluidity can be maintained by the maintenance of the required particle size by the use of a coating in the case of dispersion and by the use of surfactants in the case of a solution. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for

example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, and sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin. [0047] For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art for transmucosal administration and include, but are not limited to, detergents, bile salts, and rusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories.

[0048] In addition to the formulations described previously, the composition may also be formulated as a depot preparation. Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the therapeutic agents may be formulated with suitable polymeric or hydrophobic materials (for example as a solution or emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt. [0049] Additionally, the composition can be delivered using a sustained-release system, such as semi-permeable matrices of solid polymers containing the composition. Various forms of sustained-release materials have been established and are well known by those skilled in the art. Sustained-release capsules may, depending on their chemical nature, release the composition for a few hours, days, weeks, months, up to over 100 days. Depending on the chemical nature and the biological stability of the composition, additional strategies for stabilization may be employed.

[0050] The compositions of the present invention can also be incorporated into liposomes. Such compositions can be incorporated into liposomes using known techniques. In one embodiment, the compositions of the present invention can be formulated a lipidic suspension. Such lipidic suspensions include lipoprotein suspensions and liposome suspensions, preferably obtained by addition of soy oil and egg phospholipids. The formulation should be substantially isotonic with the blood of the treated subject, and it should contain the composition in the form of a stable lipidic suspension, i.e., in the form of finally divided particles incorporated in suspended microdrops with protecting layers of lipids, these lipids being of lipoproteins or of any common constituents of liposomes. [0051] The compositions are preferably pharmaceutically suitable for administration.

The compositions may be manufactured by means of conventional mixing, dissolving,

granulating, and dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.

Examples Example 1

[0052] An oil evaluation was done to determine the solubility behavior of oleoyl- estrone as a function of type of oil, concentration and temperature. The solubility of oleoyl- estrone was evaluated at concentrations of 60 mg/g and 200 mg/g. For the 60 mg/g solutions, the solubility was observed for 96 hours at room temperature, then the temperature was cycled to 5°C for 96 hours, after which the solution was returned to room temperature. For the 200 mg/g solutions, the solubility was observed for 96 hours at 40 ⁰ C, then the temperature was cycled to room temperature for 24 hours, then stored at 5°C for 96 hours before being returned to room temperature. Table 1 and Table 2 show the oils tested and the test results.

TABLE 1

TABLE 2

[0053] On the basis of the data from the single oil evaluation, oleoyl-estrone was found to be soluble and thermally stable, in among other oils, safϊlower oil, soybean oil, canola oil and the medium chain triglycerides. These four types of oils were then evaluated in binary blend systems with an oleoyl-estrone concentration of 200 mg/g at room temperature for 24 hours and 96 hours. Table 3 shows the oils tested and the tests results from the tests performed.

TABLE 3

Example 2

[0054] The compatibility of oleoyl-estrone in various oil systems was also assessed.

The respective stability of oleoyl-estrone in various oils was assessed following storage at (A) 25°C/60% and relative humidity (RH), (B) 40°C/75% RH, (C) 50 ⁰ C and (D) 60 ⁰ C in canola oil, safflower oil, Captex and a 2:1 Captex: Labrafil mixture. The stability test methods include appearance/color, potency, and related substances (e.g. impurities or degradant in the oleoyl-estrone and oil mixture). The results of the three week tests are set forth in Tables 4-7.

TABLE 4

TABLE 5

TABLE 6

TABLE 7

[0055] After three weeks of storage at (A) 25°C/60% RH, (B) 40°C/75% RH, (C) 5O ⁰ C and (D) 6O ⁰ C, there was no measurable degradation of oleoyl-estrone in canola oil, safflower oil or Captex. There was measurable degradation of oleoyl-estrone in the mixture of Captex and

Labrafil stored at 50 ⁰ C and 60 ⁰ C. In these samples, the levels of estrone were found to have increased significantly.

[0056] The following examples include pharmaceutical compositions comprising capsules containing oleoyl-estrone and safflower oil, and methods of making them.

Example 3

[0057] Capsules containing 1 mg, 10 mg and 50 mg of oleoyl-estrone are prepared according to the formulations shown in Table 8 and according the method shown in Figure 1.

TABLE 8

[0058] Figure 1 is a flow diagram that illustrates the manufacturing process of making the oleoyl-estrone capsules as discussed herein. Each of the three batch formulations were manufactured by dissolving the indicated amount of oleoyl-estrone in safflower oil (Step A of Figure 1). To facilitate dissolution, heat was applied to maintain a temperature of 4O ⁰ C. Then gelatin capsules are filled with each batch of oleoyl-estrone and safflower oil and then placed upright in a capsule holder (Step B of Figure 1). The fill weights of the 1 mg, 10 mg, and 50 mg dosage strength capsules are provided in Table 9.

TABLE 9

[0059] After the capsules are filled they are sealed or banded. A mixture of ethyl alcohol and water is used to seal the capsules. The mixture is prepared by combining 50 ml of alcohol and 50 ml of water in a 200 ml beaker and stirred for two minutes (Step C of Figure 1). The sealing mixture is applied to the top circumference of each capsule body. The cap is then placed on the capsule body and snapped closed (Step D of Figure 1). The capsules are placed under an

air dryer set at 4O ⁰ C to 45 ⁰ C for 30 seconds to allow the water and alcohol solution to dry (Step

E of Figure 1).

[0060] In order to band the capsules, a banding mixture of water, gelatin and Polysorbate

80 is prepared by mixing the water, gelatin and Polysorbate 80 together while being stirred gently. Table 10 shows the weight percentage of the water, gelatin and Polysorbate 80 in the mixture.

TABLE 10

[0061] The banding mixture is allowed to swell for 1-2 hours at room temperature. The mixture is then placed in an oven overnight at 55 ⁰ C ± 1O ⁰ C. The capsules are banded using a bench-top capsule banding machine with appropriate capsule holding slats. Once the capsules are banded the capsules are allowed to dry for 5-7 minutes or until they become tack-free. [0062] Following drying, the capsules are placed overnight on paper lined trays. The capsules may be turned once by rolling them during storage to facilitate detection of leak. The next day capsules are examined for leaks or defects. Any leaking or defective capsules are discarded and accounted for in the final capsule reconciliation (Step F of Figure 1). [0063] Stability tests were preformed on 1 mg, 10 mg and 50 mg capsules comprising oleoyl-estrone and safflower oil. Appearance; composite assay with HPLC (e.g. to determine the amount of the oleoyl-estrone in the capsules); percent of related substances/degradation products; and dissolution were tested at time zero, 1 month, 3 months, 6 months, 9 months and 12 months. The results of the stability testing are shown in Tables 11-13.

TABLE 11 Results of Stability Tests on 1 mg Capsules

TABLE 12 Results of Stability Tests on 10 mg Capsules

TABLE 13 Results of Stability Tests on 50 mg Capsules

[0064] While the foregoing description and drawings may represent preferred embodiments of the present invention, it should be understood that various additions, modifications, and substitutions may be made therein without departing from the spirit and scope of the present invention as defined in the accompanying claims. In particular, it will be clear to those skilled in the art that the present invention may be embodied in other specific forms, structures, arrangements, and proportions, and with other elements, materials, and components, without departing from the spirit or essential characteristics thereof. One skilled in the art will appreciate that the invention may be used with many modifications of structure, arrangement, proportions, materials, and components and otherwise, used in the practice of the invention, which are particularly adapted to specific environments and operative requirements without departing from the principles of the present invention. The presently disclosed embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims and not limited to the foregoing description. Furthermore, all references mentioned herein are incorporated by reference in their entirety for all purposes.