WO2001068074A2 - Pharmaceutical compositions of conjugated estrogens and methods of analyzing mixtures containing estrogenic compounds - Google Patents
Pharmaceutical compositions of conjugated estrogens and methods of analyzing mixtures containing estrogenic compounds Download PDFInfo
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- WO2001068074A2 WO2001068074A2 PCT/US2001/006884 US0106884W WO0168074A2 WO 2001068074 A2 WO2001068074 A2 WO 2001068074A2 US 0106884 W US0106884 W US 0106884W WO 0168074 A2 WO0168074 A2 WO 0168074A2
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- sulfate
- salt
- dihydroequilenin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
- A61K31/565—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids not substituted in position 17 beta by a carbon atom, e.g. estrane, estradiol
- A61K31/568—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids not substituted in position 17 beta by a carbon atom, e.g. estrane, estradiol substituted in positions 10 and 13 by a chain having at least one carbon atom, e.g. androstanes, e.g. testosterone
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
- G01N33/743—Steroid hormones
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
- A61K31/565—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids not substituted in position 17 beta by a carbon atom, e.g. estrane, estradiol
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
- A61K31/565—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids not substituted in position 17 beta by a carbon atom, e.g. estrane, estradiol
- A61K31/566—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids not substituted in position 17 beta by a carbon atom, e.g. estrane, estradiol having an oxo group in position 17, e.g. estrone
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P15/00—Drugs for genital or sexual disorders; Contraceptives
- A61P15/02—Drugs for genital or sexual disorders; Contraceptives for disorders of the vagina
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P15/00—Drugs for genital or sexual disorders; Contraceptives
- A61P15/12—Drugs for genital or sexual disorders; Contraceptives for climacteric disorders
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
- A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
- A61P19/10—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
Definitions
- the invention generally relates to pharmaceutical compositions exhibiting estrogenic activity, along with methods of administering and making the same.
- conjugated estrogens e.g., Premarin ® (conjugated estrogens, USP) made available commercially by Wyeth-Ayerst Laboratories of Philadelphia, Pennsylvania
- Premarin ® conjugated estrogens, USP
- USP conjugated estrogens
- Premarin ® conjuggated estrogens, USP
- Premarin ® conjuggated estrogens, USP
- Premarin ® conjuggated estrogens, USP
- the methods utilized to collect this urine have recently come into question. Animal activists have begun to protest these methods and call for a ban on Premarin ® (conjugated estrogens, USP) despite its apparent utility in treating the aforementioned diseases.
- Premarin ® conjuggated estrogens, USP
- Premarin ® conjuggated estrogens, USP
- the essential estrogenic compounds present in Premarin ® conjuggated estrogens, USP
- the essential estrogenic compounds present in Premarin ® have been determined for the first time.
- These essential estrogemc compounds have been determined to consist of the salts of conjugated estrone, conjugated equilin, conjugated ⁇ 8 ' 9 -dehydroestrone, conjugated 17 ⁇ -estradiol, conjugated 17 ⁇ -dihydroequilin, conjugated 17 ⁇ - dihydroequilin, conjugated 17 ⁇ -estradiol, conjugated equilenin, conjugated 17 ⁇ - dihydroequilenin, and conjugated 17 ⁇ -dihydroequilenin.
- the present invention provides a composition of matter.
- the composition of matter comprises a mixture of estrogenic compounds.
- the mixture may be present in chemically pure form.
- the mixture may include salts of conjugated estrone, conjugated equilin, conjugated ⁇ 8 ' 9 -dehydroestrone, conjugated 17 ⁇ - estradiol, conjugated 17 ⁇ -dihydroequilin, conjugated 17 ⁇ -dihydroequilin, conjugated 17 ⁇ -estradiol, conjugated equilenin, conjugated 17 ⁇ -dihydroequilenin, and conjugated 17 ⁇ -dihydroequilenin.
- the mixture may include the same essential estrogenic compounds present in naturally derived equine conjugated estrogens.
- the mixture may include the salts of estrone sulfate, equilin sulfate, ⁇ 8 ' 9 -dehydroestrone sulfate, 17 ⁇ -estradiol sulfate, 17 ⁇ -dihydroequilin sulfate, 17 ⁇ -dihydroequilin sulfate, 17 ⁇ -estradiol sulfate, equilenin sulfate, 17 -dihydroequilenin sulfate, and 17 ⁇ -dihydroequilenin sulfate.
- the mixture may include the sodium salts of conjugated estrone, conjugated equilin, conjugated ⁇ ' - dehydroestrone, conjugated 17 -estradiol, conjugated 17 ⁇ -dihydroequilin, conjugated 17 ⁇ -dihydroequilin, conjugated 17 ⁇ -estradiol, conjugated equilenin, conjugated 17 -dihydroequilenin, and conjugated 17 ⁇ -dihydroequilenin.
- the mixture may include the sodium salts of estrone sulfate, equilin sulfate, ⁇ 8,9 -dehydroestrone sulfate, 17 ⁇ -estradiol sulfate, 17 ⁇ -dihydroequilin sulfate, 17 ⁇ -dihydroequilin sulfate,
- the invention provides a method of treating mammals in need of treatment.
- the method comprises administering an effective amount of a composition of matter.
- treatments that are addressed by the compositions of the invention include vasomotor symptoms, atrophic vaginitis, and osteoporosis.
- the present invention provides a method of analyzing conjugated estrogen constituents.
- the method includes the steps of preparing a solution containing conjugated estrogens and analyzing the conjugated estrogens solution utilizing an HPLC system.
- the conjugated estrogens solution includes a mixture of conjugated estrogens, and a mobile phase having an organic portion that includes between about 0.1% and about 30% of a protic solvent by volume of organic portion and between about 70% and about 100% of a polar aprotic solvent by volume of organic portion, and an aqueous diluent.
- FIG. 1 is a chromatogram of Premarin ® (conjugated estrogens tablets, USP) according to the prior art.
- FIG. 2 is a chromatogram of an embodiment of a conjugated estrogens composition according to the present invention. Detailed Description of the Preferred Embodiments
- the invention relates to a composition of matter.
- the composition of matter comprises a mixture of estrogenic compounds.
- the mixture is present in chemically pure form and includes salts of conjugated estrone, conjugated equilin, conjugated ⁇ 8 ' 9 -dehydroestrone, conjugated 17 ⁇ -estradiol, conjugated 17 ⁇ - dihydroequilin, conjugated 17 ⁇ -dihydroequilin, conjugated 17 ⁇ -estradiol, conjugated equilenin, conjugated 17 ⁇ -dihydroequilenin, and conjugated 17 ⁇ -dihydroequilenin.
- the mixture contains the same essential estrogenic compounds present in naturally derived equine conjugated estrogens. All of the essential estrogenic compounds may be combined as part of the mixture. Alternatively, a portion of the essential estrogenic compounds maybe combined as part of the mixture and some or all of these or other compounds may degrade to some extent resulting in a mixture containing the same essential estrogenic compounds present in naturally derived equine conjugated estrogens.
- Naturally derived equine conjugated estrogens may be defined as a drug product (i.e. Premarin ® (conjugated estrogens tablets, USP)) containing a mixture of estrogens obtained exclusively from natural sources and blended to represent the average composition of material derived from pregnant mares' urine.
- Epmarin ® conjuggated estrogens tablets, USP
- Estradial estrogenic compounds may be defined as estrogenic compounds that are consistent and controlled (i.e. less than +/- 50% variation between lots), are present in concentrations >0.1 % by weight of the mixture of estrogenic compounds, and have a chemical structure that has the potential to have a meaningful estrogenic activity (i.e.
- “chemically pure form” means substantially devoid of impurities present in naturally derived equine conjugated estrogens products, more preferably substantially devoid of indican, sulfated benzyl alcohol, hippuric acid, benzoic acid, and creatinine.
- the essential estrogenic compounds present in naturally derived equine conjugated estrogens have now been determined for the first time.
- These essential estrogenic compounds consist of the following 10 compounds, the salts of their conjugates, or mixtures thereof: estrone; equilin; ⁇ ' - dehydroestrone; 17 ⁇ -estradiol; 17 ⁇ -dihydroequilin; 17 ⁇ -dihydroequilin; 17 ⁇ - estradiol; equilenin; 17 ⁇ -dihydroequilenin; and 17 ⁇ -dihydroequilenin.
- the essential estrogenic compounds present in naturally derived equine conjugated estrogens consist of the sodium salts of the conjugates of estrone; equilin; ⁇ ' - dehydroestrone; 17 ⁇ -estradiol; 17 ⁇ -dihydroequilin; 17 ⁇ -dihydroequilin; 17 ⁇ - estradiol; equilenin; 17 ⁇ -dihydroequilenin; and 17 ⁇ -dihydroequilenin.
- the essential estrogenic compounds present in naturally derived equine conjugated estrogens consist of the sodium salts of estrone sulfate; equilin sulfate; ⁇ 8 ' 9 -dehydroestrone sulfate; 17 ⁇ -estradiol sulfate; 17 ⁇ -dihydroequilin sulfate; 17 ⁇ - dihydroequilin sulfate; 17 ⁇ -estradiol sulfate; equilenin sulfate; 17 ⁇ -dihydroequilenin sulfate; and 17 ⁇ -dihydroequilenin sulfate.
- the estrogenic compounds may be present in various forms, including, but not limited to, estrogenic ketones and their corresponding 17 ⁇ - and 17 ⁇ -hydroxy derivatives.
- the estrogenic compounds may include estrone, 17 ⁇ -estradiol, 17 ⁇ -estradiol, equilin, 17 ⁇ - dihydroequilin, 17 ⁇ -dihydroequilin, equilenin, 17 ⁇ -dihydroequilenin, 17 ⁇ - dihydroequilenin, ⁇ 8 ' 9 -dehydroestrone, 17 ⁇ ⁇ 8 ' 9 -dehydroestradiol, 17 ⁇ ⁇ 8 ' 9 - dehydroestradiol, 6-OH equilenin, 6-OH 17 ⁇ -dihydroequilenin, and 6-OH 17 ⁇ - dihydroequilenin.
- the estrogenic compounds may also be present as conjugated estrogens.
- the conjugates may be various conjugates understood by those skilled in the art, including, but not limited to, glucuronide and sulfate. The most preferred conjugate is sulfate.
- the estrogenic compounds may also be present as salts of conjugated estrogens.
- the salts may be various salts understood by those skilled in the art, including, but not limited to, sodium salts, calcium salts, magnesium salts, lithium salts, and amine salts such as piperazine salts. The most preferred salts are sodium salts.
- a synthetic estrogenic compound may be defined as one that is derived or obtained from sources other than natural sources.
- the mixture preferably comprises from about 40 to about 75 percent of an estrone compound, from about 15 to about 40 percent of an equilin compound, from about 2 to about 10 percent of a ⁇ 8 ' 9 -dehydroestrone compound, from about 2 to about 10 percent of a 17oc-estradiol compound, from about 10 to about 20 percent of a 17 ⁇ -dihydroequilin compound, and from about 0.5 to about 5 percent of a 17 ⁇ - dihydroequilin compound.
- the mixture preferably further comprises from about 0.05 to about 3.5 percent of a 17 ⁇ -dihydroequilenin compound, from about 0.05 to about 3 percent of a 17 ⁇ -dihydroequilenin compound, from about 0.05 to about 6 percent of a equilenin compound, and from about 0.05 to about 2.5 percent of a 17 ⁇ -estradiol compound.
- the estrogen compounds are preferably present as salts of the conjugated compounds, most preferably sodium salts of the estrogen sulfates.
- the sum percent of the estrone compound and the equilin compound preferably ranges from about 70 to about 95 percent, more preferably from about 75 to about 95 percent.
- the ratio of the percent of the equilin compound to percent of the estrone compound is preferably from about 0.25 to about 0.75, more preferably from about 0.3 to about 0.7, and most preferably from about 0.35 to about 0.65.
- percent is to be understood to mean the percent by weight based on the labeled content of conjugated estrogens.
- Estrogenic compounds and/or mixtures thereof are commercially available from various suppliers including Berlichem, Inc. of Montville, New Jersey;
- the composition of the invention may include at least one additional pharmaceutically active ingredient.
- additional active ingredients include, but are not limited to, androgens, progestins, calcium salts, and vitamin D and its derivatives such as calcitriol.
- additional active ingredients include, but are not limited to, androgens, progestins, calcium salts, and vitamin D and its derivatives such as calcitriol.
- androgens include, without limitation, methyltestosterone; fluoxymesterone; oxandrolone; oxymetholone; stanozolol; 7 ⁇ -methyl- 19-nortestosterone; testosterone; testosterone cypionate; testosterone enanthate; testosterone propionate; danazol; 5 ⁇ -androstan-3 ⁇ -ol-16-one; 5 ⁇ -androstan-3 ⁇ ,16 ⁇ -diol; 5 -androstan-3 ⁇ ,16 ⁇ -diol; and 5 -androstan-3 ⁇ ,17 ⁇ - diol.
- progestins are set forth in U.S. Patent No. Re. 36,247 to Plunkett et al., the disclosure of which is incorporated herein in its entirety.
- Examples include, but are not limited to, desogestrel; dydrogesterone; ethynodiol diacetate; medroxyprogesterone acetate; levonorgestrel; medroxyprogesterone acetate; hydroxyprogesterone caproate; norethindrone; norethindrone acetate; norethynodrel; allylestrenol; 19-nortestosterone; lynoestrenol; quingestanol acetate; edrogestone; norgestrienone; dimethisterone; ethisterone; cyproterone acetate; chlormadinone acetate; megestrol acetate; norgestimate; norgestrel; deso
- Calcium salts may include, without limitation, organic acids salts of calcium such as calcium citrate, calcium lactate, calcium fumurate, calcium acetate, and calcium glycerophosphate, as well as inorganic salts such as calcium chloride, calcium phosphate, calcium sulphate, and calcium nitrate.
- organic acids salts of calcium such as calcium citrate, calcium lactate, calcium fumurate, calcium acetate, and calcium glycerophosphate
- inorganic salts such as calcium chloride, calcium phosphate, calcium sulphate, and calcium nitrate.
- compositions of matter defined herein may be incorporated into various known estrogen-containing drug products such as, Premarin ® made commercially available by Wyeth-Ayerst Laboratories of Philadelphia, Pennsylvania.
- the composition of matter of the invention may also be employed as part of a continuous estrogen-progestogen therapy regimen such as that described by U.S. Patent No. Re. 36,247 to Plunkett et al. and is present commercially as Prempro ® and Premphase ® made available by Wyeth-Ayerst Laboratories, the disclosure of which is incorporated herein by reference in its entirety.
- a preferred mixture of active estrogenic compounds of the present invention may be illustrated by the chromatogram of FIG. 2.
- the present invention also encompasses pharmaceutically acceptable drug products comprising a composition of matter of the present invention and at least one pharmaceutically acceptable carrier, diluent, or excipient, the selection of which are known to the skilled artisan.
- the drug product formulations can be in the form of tablets; effervescent tablets; pills; powders; elixirs; suspensions; emulsions; solutions; syrups; soft and hard gelatin capsules; transdermal patches; topical gels, creams and the like; suppositories; sterile injectable solutions; and sterile packaged powders.
- the drug product is present in a solid pharmaceutical composition that may be suitable for oral administration.
- a solid composition of matter according to the present invention may be formed and may be mixed with an excipient, diluted by an excipient or enclosed within such a carrier which can be in the form of a capsule, sachet, tablet, paper, or other container.
- the excipient serves as a diluent, it may be a solid, semi-solid, or liquid material which acts as a vehicle, carrier, or medium for the composition of matter.
- suitable excipients will be understood by those skilled in the art and may be found in the National Formulary 19, pages 2404-2406 (2000), the disclosure of pages 2404 to 2406 being incorporated herein in their entirety.
- the drug product formulations may include lubricating agents such as, for example, talc, magnesium stearate and mineral oil; wetting agents; emulsifying and suspending agents; binding agents such as starches, gum arabic, microcrystalline cellulose, cellulose, methylcellulose, and syrup; anticaking agents such as calcium silicate; coating agents such as methacrylates and shellac; preserving agents such as methyl- and propyl hydroxybenzoates; sweetening agents; or flavoring agents.
- lubricating agents such as, for example, talc, magnesium stearate and mineral oil
- wetting agents such as starches, gum arabic, microcrystalline cellulose, cellulose, methylcellulose, and syrup
- anticaking agents such as calcium silicate
- coating agents such as methacrylates and shellac
- preserving agents such as methyl- and propyl hydroxybenzoates
- sweetening agents or flavoring agents.
- Polyols, buffers, and inert fillers may also be used.
- polyols examples include, but are not limited to, mannitol, sorbitol, xylitol, sucrose, maltose, glucose, lactose, dextrose, and the like.
- Suitable buffers encompass, but are not limited to, phosphate, citrate, tartarate, succinate, and the like.
- Other inert fillers which may be used encompass those which are known in the art and are useful in the manufacture of various dosage forms.
- the solid formulations may include other components such as bulking agents and/or granulating agents, and the like.
- the drug products of the invention may be formulated so as to provide quick, sustained, or delayed release of the active ingredient after administration to the patient by employing procedures well known in the art.
- composition of matter of the present invention may be mixed with a solid, pulverant carrier such as, for example, lactose, saccharose, sorbitol, mannitol, starch, amylopectin, cellulose derivatives or gelatin, as well as with an antifriction agent such as, for example, magnesium stearate, calcium stearate, and polyethylene glycol waxes.
- a solid, pulverant carrier such as, for example, lactose, saccharose, sorbitol, mannitol, starch, amylopectin, cellulose derivatives or gelatin
- an antifriction agent such as, for example, magnesium stearate, calcium stearate, and polyethylene glycol waxes.
- the mixture may then be pressed into tablets. Tablets for oral use may also be prepared in the following manner, although other techniques may be employed.
- the solid substances are ground or sieved to a desired particle size, and the binding agent is homogenized and suspended in
- the resulting mixture is moistened to form a uniform suspension.
- the moistening typically causes the particles to aggregate slightly, and the resulting mass is pressed through a stainless steel sieve having a desired size.
- the layers of the mixture are then dried in controlled drying units for determined length of time to achieve a desired particle size and consistency.
- the granules of the dried mixture are sieved to remove any powder.
- disintegrating, anitfriction, and anti-adhesive agents are added.
- the mixture is pressed into tablets using a machine with the appropriate punches and dies to obtain the desired tablet size.
- the operating parameters of the machine may be selected by the skilled artisan.
- the above prepared cores maybe coated with a concentrated solution of sugar, which may contain gum arabic, gelatin, talc, titanium dioxide, or with a lacquer dissolved in volatile organic solvent or mixture of solvents. Additionally, coating may be carried out in aqueous or nonaqueous media using various excipients including, but not limited to, dispersed methylcellulose, dispersed ethylcellulose, dispersed methacrylates or mixtures thereof. To this coating various dyes may be added in order to distinguish among tablets with different active compounds or with different amounts of the active compound present. Additionally, active ingredients may be added to the coatings. In a particular embodiment, the active ingredient may be present in a core surrounded by one or more layers including sustained release coating layers.
- Hard gelatin capsules may be prepared in which capsules contain a mixture of the active ingredient and vegetable oil.
- Hard gelatin capsules may contain granules of the active ingredient in combination with a solid, pulverulent carrier, such as, for example, lactose, saccharose, sorbitol, mannitol, potato starch, corn starch, amylopectin, cellulose derivatives, or gelatin.
- a solid, pulverulent carrier such as, for example, lactose, saccharose, sorbitol, mannitol, potato starch, corn starch, amylopectin, cellulose derivatives, or gelatin.
- the formulation is in the form of orally- administered tablets which contain the composition of matter of the present invention as set forth herein along with the following inactive ingredients: calcium phosphate tribasic, calcium sulfate, carnauba wax, cellulose, glyceryl monooleate, lactose, magnesium stearate, methylcellulose, pharmaceutical glaze, polyethylene glycol, stearic acid, sucrose, and titanium dioxide.
- Such ingredients may be present in amounts similar to those present in Premarin ® (conjugated estrogens tablets, USP) made commercially available by Wyeth-Ayerst Laboratories of Philadelphia, Pennsylvania. Tablets employing the active ingredients of the invention may contain excipients similar to those contained in the 0.3 mg., 0.625 mg., and 1.25 mg tablets of Premarin ® (conjugated estrogens tablets, USP).
- Liquid preparations for oral administration may be prepared in the form of syrups or suspensions, e.g., solutions containing an active ingredient, sugar, and a mixture of ethanol, water, glycerol, and propylene glycol. If desired, such liquid preparations may contain coloring agents, flavoring agents, and saccharin. Thickening agents such as carboxymethylcellulose may also be used.
- such a formulation may comprise sterile aqueous injection solutions, non-aqueous inj ection solutions, mixtures of aqueous and non-aqueous injection solutions, or dry sterile lyphilized cake for reconstitution comprising compositions of matter of the present invention.
- aqueous injection solutions When aqueous injection solutions are prepared, the composition of matter may be present as a water soluble pharmaceutically acceptable salt.
- Parenteral preparations may contain anti-oxidants, buffers, bacteriostats, and solutes which render the formulation isotonic with the blood of the intended recipient.
- Aqueous and non-aqueous sterile suspensions may include suspending agents and thickening agents.
- the formulations may be presented in unit-dose or multi-dose containers, for example sealed ampules and vials.
- Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.
- the drug product of the present invention is in the form of an injectable solution containing a predetermined amount (e.g., 25 mg) of the composition of matter in a sterile lyphilized cake which also contains lactose, sodium citrate, and simethicone.
- a predetermined amount e.g. 25 mg
- lactose, sodium citrate, and simethicone e.g., lactose, sodium citrate, and simethicone.
- the pH of a solution containing the above ingredients may be adjusted using a suitable agent (e.g., sodium hydroxide or hydrochloric acid).
- Reconstitution may be carried out according to known methods, e.g., using a sterile diluent (5 mL) containing 2 percent benzyl alcohol in sterile water.
- a preferred injectable solution is similar to Premarin ® Intravenous made commercially available by Wyeth-Ayerst Laboratories.
- the composition of matter also may be formulated such that it is suitable for topical administration (e.g., vaginal cream). These formulations may contain various, excipients known to those skilled in the art.
- Suitable excipients may include, but are not limited to, cetyl esters wax, cetyl alcohol, white wax, glyceryl monostearate, propylene glycol monostearate, methyl stearate, benzyl alcohol, sodium lauryl sulfate, glycerin, mineral oil, water, carbomer, ethyl alcohol, acrylate adhesives, polyisobutylene adhesives, and silicone adhesives.
- the drug product is in the form of a vaginal cream containing the composition of matter as set forth herein present in a nonliquefying base.
- the nonliquefying base may contain various inactive ingredients such as, for example, cetyl esters wax, cetyl alcohol, white wax, glyceryl monostearate, propylene glycol monostearate, methyl stearate, benzyl alcohol, sodium lauryl sulfate, glycerin, and mineral oil.
- Such composition may be formulated similar to Premarin ® Vaginal Cream made commercially available by Wyeth-Ayerst Laboratories.
- Dosage units for rectal administration may be prepared in the form of suppositories which may contain the composition of matter in a mixture with a neutral fat base, or they may be prepared in the form of gelatin-rectal capsules which contain the active substance in a mixture with a vegetable oil or paraffin oil.
- the present invention relates to methods of treating mammals (e.g., man) in need of treatment.
- the methods include administering an effective amount of a composition of matter as defined herein to the mammal in need of treatment.
- the methods may be used for a number of treatments such as, but not limited to, vasomotor symptoms; atrophic vaginitis; osteoporosis; hypoestrogenism due to hypogonadism, castration, or primary ovarian failure; breast cancer in selected persons with metastatic disease; advanced androgen-dependent carcinoma of the prostate; abnormal uterine bleeding; and kraurosis vulvae.
- the administration may be cyclic, occurring for one or more short periods of time or courses of treatment (i.e. short-term use).
- the administration may be continuous, occurring over extended periods of time (i.e. long-term use).
- long-term use would be from the onset of menopause until death.
- Cyclic and continuous administration may be either interrupted or uninterrupted.
- Uninterrupted administration occurs one or more times daily such that there is no break in treatment.
- Interrupted administration occurs other than daily, for example a repeated course of treatment including three weeks of daily treatment followed by one week of no treatment.
- the present invention relates to a method of analyzing a mixture containing estrogenic compounds.
- the method comprises the steps of preparing a solution containing estrogenic compounds and analyzing the estrogen containing solution utilizing an HPLC system.
- the estrogenic compounds to be analyzed preferably include a mixture of conjugated estrogen compounds. More preferably, the mixture is present in Premarin ® (conjugated estrogens tablets, USP) or in the composition of matter of the present invention.
- the estrogen containing solution includes a mobile phase.
- the mobile phase may include an organic portion, an aqueous portion, and one or more diluents.
- the organic portion and the aqueous portion act as diluents.
- the estrogen containing solution may be prepared in various ways.
- the mixture of estrogenic compounds may first be mixed with the organic portion, the aqueous portion, or both followed by mixing the resulting organic portion with the resulting aqueous portion.
- the organic portion and the aqueous portion may first be mixed together to form a mobile phase followed by mixing the mixture of estrogenic compounds with the mobile phase.
- the mixture of estrogenic compounds is preferably first mixed with the organic portion followed by mixing the resulting organic portion with the aqueous portion.
- the mobile phase includes preferably between about 55 and about 90 percent, more preferably between about 65 and about 80 percent, and most preferably between about 70 and about 75 percent aqueous portion.
- the mobile phase also includes preferably between about 10 and about 45 percent, more preferably between about 20 and about 35 percent, and most preferably between about 25 and about 30 percent organic portion, where percent organic or aqueous portion is measured as a percent volume of the mobile phase.
- the pH of the mobile phase is preferably between about 2.5 and about 7, more preferably between about 2.5 and about 3.5, and most preferably between about 2.8 and about 3.2.
- the organic portion may comprise between about 0 and about 30 percent protic solvent and between about 70 and about 100 percent polar aprotic solvent, where percentages are percent by volume organic diluent.
- the organic portion preferably comprises between about 5 and about 25 percent protic solvent and between about 75 and about 95 percent polar aprotic solvent, more preferably comprises between about 10 and about 20 percent protic solvent and between about 80 and about 90 percent polar aprotic solvent, and most preferably comprises between about 10 and about 15 percent protic solvent and between about 85 and 90 percent polar aprotic solvent.
- the protic solvent preferably includes lower alkyl alcohols, and more preferably is methanol.
- the polar aprotic solvent preferably includes lower alkyl nitriles, and more preferably is acetonitrile.
- the organic portion may also include an ion-pairing agent.
- the ion-pairing agent may preferably have a concentration of between about 0.5 and about 2 millimoles/liter organic portion (mM), more preferably between about 0.5 and about
- the ion-pairing agent is preferably tert-butyl ammonium hydroxide, although other agents may be employed as will be understood by those skilled in the art.
- the organic portion may also include various components known by one skilled in the art including, but not limited to, buffer salts, acids, and bases.
- the aqueous portion may include an ion-pairing agent.
- the aqueous portion may preferably have an ion-pairing agent concentration of between about 0.5 and about 2 millimoles/liter aqueous portion (mM). More preferably, the aqueous portion has an ion-pairing agent concentration of between about 0.5 and about 1.5 mM, and most preferably between about 0.5 and about 1.0 mM.
- the ion-pairing agent is preferably tert-butyl ammonium hydroxide, although other agents may be employed as will be understood by those skilled in the art.
- the aqueous portion has a pH of preferably between about 2.5 and about 7, more preferably between about 2.5 and 3.5, and most preferably between about 2.8 and about 3.2.
- the aqueous portion may also include various components known to one skilled in the art including, but not limited to, buffer salts, acids, and bases. Preferable buffer salts are phosphate salts.
- the analyzing step may be performed utilizing an HPLC system known to one skilled in the art.
- the HPLC system may preferably include a reversed phase column.
- the column has a length and an inside diameter.
- the length of the column may be between about 5 and about 100 cm.
- the length of the column is preferably between about 5 and about 30 cm, more preferably between about 10 and about 20 cm, and most preferably 15 cm.
- the inside diameter of the column may be between about 2 and about 100 mm, and is preferably between about 3 and about 50 mm, more preferably between about 3 and about 20 mm, and most preferably between about 3 and about 10 mm.
- the column is preferably packed with an alkyl based stationary phase.
- the alkyl based stationary phase is preferably a C 18 stationary phase.
- the particle size of the stationary phase is preferably between about 2 and about 20 ⁇ m, and is more preferably between about 2 and about 10 ⁇ m.
- the HPLC system may include one or more suitable detectors.
- the detectors preferably include fluorescence and ultraviolet (UN) detectors.
- the UV detector preferably includes a diode array.
- the UV detector detects at a wavelength preferably between about 190 and about 400 nanometers (nm), more preferably between about 200 and about 300 nm.
- the fluorescence detector preferably has an excitation between about 250 and about 310 nm, more preferably between about 260 and about 300 nm, and most preferably between about 270 and about 290 nm.
- the fluorescence detector preferably exhibits emissions between about 300 and about 320 nm and between about 395 and 415 nm, more preferably between about 305 and about 315 nm and between about 400 and about 410 nm.
- the HPLC system may have certain operating parameters including column flow rate and column temperature.
- the column flow rate is preferably between about 0.1 and about 10 milliliters/minute (mL/min), more preferable between about 2 and about 5 mL/min, and most preferably about 3 mL/min.
- the column temperature may be preferably between about 10 and about 35°C, more preferably between about 15 and about 30°C, and most preferably about 25°C. While the foregoing parameters may be preferred, other parameters may be employed as will be understood by those skilled in the art.
- the analyzing step may further comprise the step of collecting the peaks of interest and preferably comprises the step of fraction collecting the peaks of interest.
- the fraction collecting step is preferably performed using a multi-channel fraction collector.
- Methods of the present invention may be performed as a part of a method for characterizing naturally derived equine conjugated estrogens (i.e. Premarin ® (conjugated estrogens tablet ' s, USP)) to determine the essential estrogenic compounds thereof. Methods for characterizing Premarin ® (conjugated estrogens, USP) will now be described in the following Examples. In the Examples, “mL” means milliliter, “°C” means degrees Celcius, “mM” means millimoles/liter, “M” means moles/liter,
- the second criterion may be the most critical: the assignment of potential active ingredients requires a definition of esfrogenicity. A structure-function approach to defining esfrogenicity was taken. According to Goodman & Gilman's The Pharmacological Basis of Therapeutics (9th Ed., 1996, p. 1412), estrogenic activity is controlled by the presence of the phenolic A ring (at carbon 3) and a ⁇ - hydroxyl or ketone group in position 17 of the D ring. The reference states that "the phenolic A ring is the principal structural feature responsible for selective, high- affinity binding to estrogen receptors.
- Probe temperature 280°C b. Liquid Chromatography/Mass Spectrometry (LC-MS)
- Drug Substance may be prepared as follows. Weigh an appropriate amount to yield 200 mL of solution. Dissolve this amount in 61 mL of organic diluent and mechanically shake for 10 minutes. Add about 100 mL of aqueous diluent and mechanically shake for 10 minutes. Dilute to volume with aqueous diluent and mix well. Filter a portion of the solution through a 0.45 ⁇ m PTFE filter.
- a free stock steroid solution of estrone, equilin and 17 ⁇ -dihydroequilin in methanol at 0.2 mg/mL may be prepared for each free estrogen.
- a resolution solution containing about 0.03 mg/mL often-component conjugated estrogens reference standard and approximately 0.006 mg/mL each of 17 ⁇ -dihydroequilin, equilin and estrone as for the standard solution may be prepared.
- a volume of 100 mL of resolution solution is prepared, mixed and filtered as for the standard solution.
- a phosphate buffer solution may be prepared.
- the phosphate buffer solution is preferably a 50 mM potassium phosphate buffer solution.
- TBAH with a volumetric ratio of 277:0.9 may be prepared.
- the pH of the solution may be adjusted to 3.0 ⁇ 0.1 with phosphoric acid.
- An organic diluent containing a solution of acetonitrile and methanol with a volumetric ratio of 26.5:4 may be prepared.
- a mobile phase may be prepared by mixing a solution of organic diluent and aqueous diluent with a volumetric ratio of 30.5:69.5.
- Samples to be analyzed may be prepared. First, tablets are prepared by washing 10-20 tablets in water to remove the outer coating then blowing them to dryness under a nitrogen purge. The tablets are then ground to a fine powder using either a mortar and pestle or the AAI grinder at 450 RPM for 1 minute. The ground tablets are then mixed with the mobile phase to form a mobile phase containing conjugated estrogens.
- the equivalent weight often tablets is placed into a 200 mL volumetric flask.
- 61 mL of organic diluent are added to the flask and the flask is mechanically shaken for ten minutes.
- about 100 mL of aqueous diluent is added to the flask and the flask is mechanically shaken for ten minutes.
- the resulting solution is then diluted to volume with aqueous diluent and mixed. A portion of the solution is filtered through a 0.45 ⁇ m PTFE filter.
- the equivalent weight often tablets is placed into a 400 mL volumetric flask.
- Intravenous Lyophilized Cake When 25 mg Intravenous Lyophilized Cake is to be analyzed, the vial is opened, and about 5 mL of mobile phase is added to the vial. The vial is then shaken briefly until the cake has visibly dissolved. The solution is quantitatively transferred to a 200 mL volumetric flask, diluted to volume with mobile phase, and mixed. 5.0 mL of this solution are pipetted to a 10 mL volumetric flask, diluted to volume with mobile phase, and mixed well.
- An example of a chromatographic procedure is as follows: 100 ⁇ L of resolution solution and a diluent blank were separately injected into the chromatograph. No interferences were observed at the relative retention times (RRTs) for any of the known estrogen peaks in the blank injection. Equal volumes of the standard solution and the sample preparations were separately injected into the chromatographic systems. Each peak was integrated and evaluated based on the peak area responses for the ten known estrogen peaks.
- a Semi-Preparative HPLC Method employed a mobile phase A and a mobile phase B.
- Mobile phase A was an aqueous phase that included 0.9 mM TBAH and 0.075% 12.1 N HC1, with all percentages by volume mobile phase A.
- Mobile phase B was an organic phase that included 0.9 mM TBAH, 13.1% MeOH, 86.9% ACN and 0.075% 12.1 N HC1, with all percentages by volume mobile phase B.
- Samples to be analyzed may be prepared. First, about 100-200 tablets are washed in water to remove the outer coating then blown to dryness under a nitrogen purge. The tablets are then ground in the AAI grinder at 450 RPM for 1 minute. A solution having a concentration of 0.31 mg/mL conjugated estrogens maybe prepared as for the analytical standard solution described above. For example, the equivalent weight of 100 1.25 mg tablets are placed in a 400 mL volumetric flask. 122 mL of organic diluent is added to the flask and the flask is mechanically shaken for ten minutes. About 200 mL of aqueous diluent is then added to the flask and the flask is again mechanically shaken for ten minutes. The resulting solution is then diluted to volume with aqueous diluent and mixed. A portion of the solution is filtered through a 0.45 ⁇ m PTFE filter.
- An example of a chromatographic procedure is as follows: 1.0-1.5 mL of the sample was injected multiple times on the chromatographic system and the peaks of interest were fraction collected using a multi-channel fraction collector. Cross over of the peak identity to the analytical procedure was performed using diode array comparison to follow peak movement. Once all of the fractions were collected, each fraction was passed through an appropriate ion-exchange column to remove the ion- pairing reagent. The fractions were concentrated using a rotary evaporator under vacuum to an appropriate volume and set aside to allow crystal growth or perform analyses by other means for identification.
- Premarin ® tablets are Premarin ® vaginal cream and Premarin ® intravenous (IN). Premarin ® intravenous lots were also examined to ensure that peaks observed from Premarin ® tablets come from Conjugated Estrogens,
- the selected tablet lots allowed direct comparison of Premarin ® lots from various countries of origin, the most popular tablet strengths, and aged samples of 1.25 mg tablets covering lots with expiration dates at three-year intervals. From these samples the characterization of the components of Premarin ® was carried out by closely examining each individual peak by the HPLC Chromatographic Assay Method described above. Components which were present at consistent levels greater than 0.1 % in the lots examined were considered for investigation as potential essential estrogenic compounds. Consistency was one of the elements considered when determining if a compound was a potential essential estrogenic compound, and was defined as a variation of the amount found in the current Premarin ® lots of less than ⁇ 50%.
- Each sulfated ketone can have a corresponding sulfated 17oc- and 17 ⁇ - hydroxy derivative; each of these can then hydrolyze to form the non-sulfated species.
- the fluorescence detector was introduced in series into the chromatographic system and the excitation and emission wavelengths were optimized to enhance the signals of the chromatographic peaks at 312 nm and 405 nm in Premarin ® . Overlays of the two fluorescence chromatograms with the UV chromatogram were used to examine the peaks for spectral characteristics of potential estrogenic components. The ten USP-defined sodium estrogen sulfates all exhibit fluorescent emissions at 312 nm and most also show the emission at 405 nm. From these spectral overlays, 11 additional peaks were observed which exhibited a fluorescence emission at one or both of the emission wavelengths at a specific retention time where UV absorption was not detectable.
- Fluorescence spectral information also can be used in the characterization of Premarin ® for screening the chromatographic peaks and eliminating them as non- estrogenic. If peaks did not exhibit fluorescence at one of the two wavelengths, they were considered non-estrogenic due to the lack of the required phenolic A ring responsible for specific estrogen receptor binding. There were a total of 22 peaks that did not fluoresce. Eight of these peaks were > 0.1 %, and one of these has been identified as the mobile phase peak. Therefore, seven non-estrogenic peaks > 0.1% (RRT values of 0.102, 0.121, 0.198, 0.441, 0.647, 0.705, and 1.045) present in Premarin ® were identified as non-estrogenic by this criterion.
- the ten USP-defined conjugated estrogens consist of the sulfated forms of estrone, equilin, and equilenin present in both the ketone and the 17 ⁇ and 17 ⁇ - hydroxy forms to give the first nine components and ⁇ 8 ' 9 -dehydroestrone sulfate as the tenth. Overlays of the diode array spectra of the three sulfated forms of estrone, equilin, and equilenin were prepared. Each set of diode array spectra all exhibited similar spectral characteristics.
- Each of these 12 sulfated compounds can also undergo metabolism or degradation by hydrolysis to desulfonate the compound.
- Samples of non-sulfated estrone, equilin, 17 ⁇ -dihydroequilin, 17 ⁇ -estradiol, 17 ⁇ -dihydroequilenin, and equilenin were available to examine Premarin ® for the presence of non-sulfated forms.
- the diode array spectra of each of these six samples were compared to the corresponding sulfated ketone spectra.
- Premarin ® was examined for the non-sulfated ⁇ 8 ' 9 -dehydroestrone peak.
- the position of the double bond at the 8,9 position may allow more extended conjugation than is present in estrone and equilin, but less than in equilenin, so the UV shift was predicted to be somewhere in between, with little or no shift observed.
- the predicted region was examined for the non-sulfated ⁇ 8,9 -dehydroestrone, and the peak was found at an RRT value of 1.589, which matched the expected diode array spectrum with essentially no UV shift.
- the resultant material tended to be yellowish viscous oils, rather than dry solid materials.
- the yellowish viscous oil was caused by the presence of polyethylene glycol and other excipients in the Premarin ® formulation which leached out during the HPLC analysis, raising the baseline of the chromatogram and contaminating each of the fractions. This viscous material hindered crystallization and ultimate purification of the fractions.
- Peaks 6 and 7 were thought to be sulfated and will be discussed first. Peaks 4 and 8 appeared to be non-sulfated carboxylic acids and were investigated next followed by peak 1 that appeared non-sulfated also. Over the course of the investigations, multiple fraction collections and purifications were performed. The following individual synopses for each peak represent multiple individual runs and experiments.
- the original peak 6 fraction from the fraction collector was light blue in color, unlike all the other fractions that were colorless to pale yellow.
- Peak 6 was extracted from the dry rotary evaporator mixture of buffer and compound using ethanol. This solution was then concentrated on the rotary evaporator to produce a dark yellow viscous oil. When this yellow oil was removed from the rotary evaporator vacuum, it began to rapidly change colors. It changed from yellow to red, to violet, to blue in a matter of minutes, and after several days to weeks changed to brown in color. HPLC chromatography of the colored fraction confirmed that peak 6 in the chromatograms remained unchanged. The color change appeared to be an air oxidation of some minor component present with peak 6. When the yellow oil was isolated and stored under dry nitrogen gas, little or no color change was observed. A portion of this fraction was set aside to attempt crystallization and a portion was sent for LC-MS analysis.
- FAB-MS FAB-MS and showed a single, weak, sample related molecular ion (M-H)- at m/z 212. Positive ion FAB showed no significant sample related peaks. Negative ion ESI-MS showed an intense molecular ion (M-H)- at m/z 212 consistent with the FAB-MS data. The negative ion ESI-MS/MS spectrum also confirmed the parent ion at 212 m/z. This spectrum showed a number of daughter ions including the ion at 80 m/z consistent with an SO 3 ⁇ fragment and an ion at 1 32 m/z consistent with the non- sulfated molecular ion. These results are all consistent with a molecular mass of 213Da and the presence of a sulfate moiety.
- Peak 7 was extracted from the dry rotary evaporator mixture of buffer and compound using ethanol. This solution was then concentrated on the rotary evaporator to produce a yellow viscous oil. Portions of this fraction were set aside to attempt crystallization and for submission for LC-MS analysis.
- Yellow cube shaped crystals were also grown from the yellow solution containing peak 7.
- One of these of about 0.12 x 0.12 x 0.12 mm was selected and mounted on the end of a glass fiber.
- a SEM/EDX analysis showed chlorine as expected, but revealed copper or zinc present as the metal ion. This was determined to be an organometallic salt and would not be considered an estrogenic component of Premarin ® .
- the viscous yellow oil was dissolved in met ⁇ -nitro benzyl alcohol matrix and run under positive ion FAB-MS, which generated a major molecular ion (M+H) + at a m/z of 242.
- the intensity of this peak was sufficient to permit an HR-MS analysis to generate a molecular composition and an EI-MS to study the fragmentation pattern.
- HR-MS was used and generated an accurate molecular weight of 242.2841. Based upon that mass, the molecular composition was determined to be C 16 H 6 ⁇ .
- a strong EI-MS spectrum was only achieved at relatively high probe temperatures near 280°C and showed strong signals at m z 100, 142, and 185.
- the negative ion ESI-MS/MS spectrum also confirmed the parent ion at 187 m/z.
- This spectrum showed a number of major daughter ions including the ion at 80 m z consistent with an SO 3 - fragment and an ion at 107 m/z consistent with the non- sulfated molecular ion.
- the ion at m/z 92 is consistent with a fragment of toluene. These results are all consistent with a molecular mass of 188Da and the presence of a sulfate moiety.
- a fraction of this peak was collected from the LC-MS and confirmed by HPLC. The fraction was run on the gradient HPLC system and was shown to be pure, matching the retention time of peak 7 in the Premarin ® chromatogram. Overlays of the diode arrays of the two peaks appeared identical confirming the identity of peak 7.
- the mass spectral data indicate the molecular formula for peak 7 is C 7 H 8 SO 4 .
- This peak contains a sulfate and an aromatic ring, which generates a structure of either a sulfated benzyl alcohol or cresol. Authentic standards of these compounds were not commercially available, so direct injection in the HPLC system as confirmation was not possible. The most likely chemical structure for peak 7 is sulfated benzyl alcohol.
- Peak 4 was extracted from the dry rotary evaporator mixture of buffer and compound using ethanol. This solution was concentrated on the rotary evaporator to produce a yellow viscous oil. Portions of this fraction were set aside to attempt crystallization and for use in other physical testing to characterize the material.
- Crystallization was attempted utilizing a wide variety of techniques including slow evaporation.
- Peak 8 was extracted from the dry rotary evaporator mixture of buffer and compound using ethanol. This solution was then concentrated on the rotary evaporator to produce a yellow viscous oil. Portions of this fraction were set aside to attempt crystallization and for submission for LC-MS analysis.
- Crystallization attempts utilizing a wide variety of techniques produced several crystals from peak 8 for examination by single crystal analysis. The first of these were blue-green prisms. A crystal was chosen and mounted on a glass fiber for
- ammonium copper(II) chloride is formed from the evaporation of a solution of ammonium chloride and copper(II) chloride and as the dihydrate form are blue to bluish-green tetragonal rhombododecahedral crystals.
- the fraction was neutralized with ammonium hydroxide and allowed to form by slow evaporation indicating that the fraction probably contained some copper(II) chloride that reacted to form these crystals upon evaporation. Based upon all this, the X-ray structure solution was not completed. SEM/EDX analysis was performed on the crystal and verified the presence of copper and chlorine in the crystal.
- a crystal was selected and mounted on a glass fiber for X-ray analysis.
- the molecule is believed to contain about 33 non-hydrogen atoms in the asymmetric unit if it is an acentric space group and about 16-17 non-hydrogen atoms if it is centric.
- a search of the ICDD database did not reveal any structures with similar cell constants.
- the data set was collected and based upon the systematic absences, the space group #51 (Pnna) was selected.
- the structure was refined to R factors of 6.8 and 7.7 percent and revealed that the molecule sat on a special position (2-fold axis) such that only one-half of the molecule needed to be found and refined.
- the final structure was shown to be a tertiary butyl amine of the general formula [NBu ][MCl 4 ], where M is a first row transition element in a +3 oxidation state, and is possibly Fe. It appears this complex was formed from the interaction of the mobile phase ion-pairing agent with a metal salt. Since this was not a compound of interest the structure delineation was stopped at this point.
- Chromatographic UN analysis during the LC-MS analysis shows a single large peak for this fraction.
- examination of the ESI-MS data recorded throughout the chromatogram did not reveal any significant ion that could be assigned to the UN response.
- a number of weak signals throughout the run were detected but these were polymeric ethoxylates (from the PEG excipient) and were not consistent with the major UN peak.
- This single major peak in the LC-MS was run under negative ion FAB-MS and showed a possible very weak, sample related molecular ion (M-H)- at m/z 377.
- Positive ion FAB showed a weak sample related molecular ion (M+ ⁇ a) + at m/z 401.
- Negative ion ESI-MS showed a weak response with high background including the one indicated in FAB-MS analysis at m/z 377. Analysis by ESI-MS/MS was not possible since the ESI-MS spectrum did not reveal a molecular ion. The conclusions of these studies indicated a molecular mass of 378Da. Further analyses were performed to confirm and verify any of these results. To improve sensitivity for the MS analyses, the single peak was collected from the LC- MS and lyophilized to concentrate it for use in subsequent analyses. The negative ion ESI-MS spectrum showed a possible molecular ion (M-H) at m/z 371.
- peak 8 was suspected to be an aromatic carboxylic acid. This information was used to try to find an organic carboxylic acid that matched the specific retention time and diode array.
- Benzoic acid, sodium salt was readily available from Chem Service and was analyzed by HPLC. Overlays of the chromatograms and the diode array specfra of the benzoic acid and Premarin showed matching peak shape and retention times and identical diode array spectra for the peaks confirming the identity of peak 8 as benzoic acid. According to The Merck Index, benzoic acid occurs in the urine of vertebrates.
- Peak 1 was extracted from the dry rotary evaporator mixture of buffer and compound using ethanol. This solution was then concentrated on the rotary evaporator to produce a yellow viscous oil. Portions of this fraction were set aside to attempt crystallization and for use in other physical testing to characterize the material.
- Peak 9 was extracted from the dry rotary evaporator mixture using ethanol and concentrated on the rotary evaporator to produce a yellow viscous oil. This was set aside to crystallize and after several days, a single small clear plate of dimensions
- Conjugated Estrogens USP to determine the steroidal components present. It was reported that Conjugated Estrogens, USP is a mixture of 17 estrogen sulfates, three progestins, four progestin sulfates, and four androgens. In addition to the ten USP-def ed sulfated estrogens,
- esters #1 and #2 The presence of the 17 ⁇ - and 17 ⁇ -hydroxy derivatives of ⁇ 8 ' 9 -dehydroestrone sulfate (estrogens #1 and #2) have been confirmed and are described above.
- estrogen #4 An authentic standard of estrogen #4 was not commercially available, but a standard of 1, 3,5(10)-estratrien-2,3-diol-17-one-2-methyl ether (non-sulfated derivative of estrogen #4) was injected to evaluate the possible presence of estrogen
- Estrogen #4 contains a phenolic A ring substitution which, like estrogen #3, is known to obviate binding to estrogen receptors. The presence or absence of these estrogens should not contribute significantly to estrogenic activity in Conjugated Estrogens, USP.
- a peak for estrogen #7 should have been observed based upon the amount Wyeth-Ayerst reported present from the GC acid hydrolysis experiments. In their report, Wyeth-Ayerst claimed that the 10 estrogens compose about 13.3% by weight of the drug substance, and that esfrogen #7 (estradiene) is the fourth largest estrogen present. A peak of this magnitude should have been detected by the chromatographic methods if it was present. The presence or absence of these non-estrogenic compounds are not expected to impact the estrogenic activity of Premarin ® (conjugated estrogens, USP).
- Progestin #3 was investigated using HPLC by changing the UN detection to 205 nm and altering the mobile phase composition to a 50:50 ratio of organic:aqueous.
- the standard was prepared to represent about a 1% level in Premarin ® and was observed as a small distinct peak at a retention time of about 15.11 minutes. Examination of an overlay of this chromatogram with a Premarin ® tablet injection under the same conditions did not reveal any corresponding peak in Premarin ® .
- androgen #1 was investigated using HPLC by changing the UN detection to 205 nm and altering the mobile phase to a 40:60 ratio of organic: aqueous. The standard was prepared to represent about a 1% level in Premarin ® and at that level no distinct peak was detected.
- Estriol (l,3,5(10)-estratrien-3,16 ⁇ ,17 ⁇ -triol), which according to The Merck Index is usually the predominant estrogenic metabolite in urine, was expected to be present.
- Samples of both the sulfated and non-sulfated forms of estriol were injected in the HPLC system. The sample peaks in the chromatograms matched up well with the retention times of unknowns in Premarin ® at RRT values of 0.121 and 0.174 for the sulfated and non-sulfated forms. However, when the diode array spectra were overlaid, it was determined that neither of the pairs of two peaks were the same components and that estriol sulfate and estriol were not present in Premarin ® tablets.
- Premarin ® intravenous lots were also examined by the same HPLC method used for Premarin ® tablets.
- Premarin ® intravenous was chosen since it contains the same Conjugated Estrogens, USP, drug substance, as Premarin ® tablets, but does not contain the excipients that had caused leaching problems in the chromatography for tablet samples.
- the chromatograms were examined to investigate the consistency of the Conjugated Estrogens, USP, drug substance from product to product.
- the three Premarin ® intravenous lots were examined by the HPLC Chromatographic Assay Method described above. The three lots yielded similar chromatographic patterns. An overlay of one of the intravenous lots with a Premarin ® tablet lot, however, showed substantial differences in the peak patterns observed in the first seven minutes.
- the essential estrogenic compounds in Premarin ® were determined to be salts of conjugated estrone, conjugated equilin, conjugated ⁇ 8 ' 9 -dehydroestrone, conjugated 17 ⁇ - estradiol, conjugated 17 ⁇ -dihydroequilin, conjugated 17 ⁇ -dihydroequilin, conjugated 17 ⁇ -esfradiol, conjugated equilenin, conjugated 17 ⁇ -dihydroequilenin, and conjugated 17 ⁇ -dihydroequilenin.
- TBAH Tetrabutylammonium hydroxide
- aqueous aqueous
- a 50 mM phosphate buffer solution may be prepared by dissolving approximately 40.8 g of potassium phosphate monobasic in 6000 mL of water and filtering the resulting solution through an HNLP 0.45 ⁇ m filter.
- An organic diluent containing a solution of acetonitrile and methanol with a volumetric ratio of 24.5:4.5 may be prepared by combining 1225 mL of acetonitrile and 225 mL of methanol, mixing well, and letting the solution equilibrate to room temperature.
- An aqueous diluent containing the phosphate buffer and 0.4 M TBAH solution with a volumetric ratio of 71 :0.17 may be prepared by combining 3550 mL of the phosphate buffer and 8.5 mL of 0.4 M TBAH solution and mixing well.
- the mobile phase for separation A includes a solution of organic diluent and aqueous diluent with a volumetric ratio of 29:71. This mobile phase may be prepared by combining 1160 mL of organic diluent and 2840 mL of aqueous diluent, mixing well, and degassing the resulting solution.
- the mobile phase for separation B includes a solution of acetonitrile, methanol, phosphate buffer, and 0.4 M TBAH solution with a volumetric ratio of 6:38:56:0.4.
- This mobile phase may be prepared by first combining 2240 mL of phosphate buffer with 16 mL of 0.4 M TBAH solution, and the adjusting the pH of the resulting solution to 3.0 ⁇ 0.1 with 85% phosphoric acid. To this mixture, 240 mL of acetonitrile and 1520 mL of methanol are added. The resulting solution is then mixed well and degassed.
- a blank injection solution may be prepared using the same diluents that are used to prepare samples.
- the blank injection solution may be prepared by adding 29 mL of the organic diluent into a 100 mL volumetric flask, diluting the organic diluent to volume with aqueous diluent, and mixing well.
- a standard solution containing about 0.03 mg/mL of conjugated estrogens may be prepared by weighing approximately 160 mg of the 10-component conjugated estrogens drug substance (label claim 37.5 ⁇ g/mg) and transferring the weighed drug substance to a 200 mL volumetric flask. A volume of 100 mL of Mobile Phase A is added to the flask and the flask is mechanically shaken for 15 minutes. The resulting solution is then diluted to volume with Mobile Phase A and mixed well. The standard solution maybe stable for 13 days at ambient conditions. A portion of the solution is filtered through a 0.45 ⁇ m PTFE filter with a 1 ⁇ m pre-filter, discarding the first 3 mL.
- a Sensitivity Solution A containing about 0.048 ⁇ g/mL solution (equivalent to 0.1% label claim relative to the total conjugated estrogens) of conjugated estrogens in Mobile Phase A may be prepared.
- a 2.0 mL volume of the standard preparation is pipetted into a 100 mL volumetric flask.
- the standard solution is then diluted to volume with Mobile Phase A and mixed well.
- 2.0 mL of the resulting solution is then pipetted into a 25 mL volumetric flask, diluted to volume with Mobile Phase A, and mixed well.
- the Sensitivity Solution A may be stable for 4 days at ambient conditions.
- a stock free steroids solution containing a 0.2 mg/mL solution of estrone RS, equilin RS, and 17 ⁇ -dihydroequilin RS in methanol may be prepared by weighing approximately 10 mg of 17 ⁇ -dihydroequilin, equilin, and estrone and quantitatively transferring these into a 50 mL volumetric flask. The flask is filled to approximately half- volume with methanol. The flask is then sonicated until the solids have dissolved
- a resolution solution containing about 0.03 mg/mL of 10-component conjugated estrogens and approximately 0.006 mg/mL each of 17 ⁇ -dihydroequilin, equilin, and estrone may be prepared by weighing approximately 80 mg of 10- component conjugated estrogens drug substance (label claim 37.5 ⁇ g/mg) and transferring the resulting solution to a 100 mL volumetric flask.
- Samples to be analyzed may be prepared.
- Premarin ® tablets about 10-20 tablets are washed in water to remove the outer coating then blown to dryness under a nitrogen purge. The tablets are then ground in a grinder at 450 RPM for 1 minute.
- a grinder at 450 RPM for 1 minute.
- Premarin ® an amount of washed and ground tablets equivalent to 10 average tablet weight (ATW)) are placed in a 100 mL volumetric flask. A 29 mL volume of organic diluent is added and the flask is mechanically shaken for 10 minutes. About 50 mL of aqueous diluent is added to the resulting solution and the flask is again mechanically shaken for 10 minutes. The resulting solution is then diluted to volume with aqueous diluent and mixed. A portion of the solution is filtered through a 0.45 ⁇ m PTFE filter with a 1 ⁇ m pre-filter, discarding the first 3 mL. To prevent evaporation, the filtrate is preferably placed promptly into the injection vials.
- ATW average tablet weight
- Chromatographic separation B is used for identifying and quantifying the equilin sulfate and ⁇ 8 ' 9 -dehydroestrone sulfate peaks. Chromatographic separation B is used for identifying and quantifying the peaks of the remaining estrogens and unknown impurities.
- the mobile phase robustness of Separation A was investigated by making slight deliberate changes to the mobile phase composition. Nariations to the amount of acetonitrile in the mobile phase (+/- 1%), to the amount of methanol (+/- 1% absolute value), to the amount of tetrabutyl ammonium hydroxide (TBAH) (+/- 10% absolute value), to the buffer pH (+/- 0.2 units), and to the concentration of the buffer (+/- 10%) were made.
- AEDS 17 ⁇ -estradiol sulfate
- EQNS equilenin sulfate
- compositions of matter and drug products of the present invention may for the first time provide a mixture of synthetic estrogenic compounds where the mixture contains the same essential estrogenic compounds present in Premarin ® (conjugated estrogens tablets, USP).
- compositions of matter and drug products of the present invention may provide a synthetic alternative to naturally derived Premarin ® (conjugated estrogens tablets, USP). This may reduce or eliminate what has been perceived to be pest to animals while still providing drug products useful in treating diseases and medical conditions such as severe vasomotor symptoms associated with menopause, atrophic vaginitis, osteoporosis, hypoestrogenism due to hypogonadism, castration, or primary ovarian failure, breast cancer in selected persons with metastatic disease, and advanced androgen-dependent carcinoma of the prostate.
- premarin ® conjuggated estrogens tablets, USP
Abstract
Description
Claims
Priority Applications (6)
Application Number | Priority Date | Filing Date | Title |
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JP2001566638A JP2004500396A (en) | 2000-03-10 | 2001-03-05 | Pharmaceutical compositions of conjugated estrogens and methods for analyzing mixtures containing estrogen compounds |
EP01918326A EP1267852A2 (en) | 2000-03-10 | 2001-03-05 | Pharmaceutical compositions of conjugated estrogens and methods of analyzing mixtures containing estrogenic compounds |
MXPA02008856A MXPA02008856A (en) | 2000-03-10 | 2001-03-05 | Pharmaceutical compositions of conjugated estrogens and methods of analyzing mixtures containing estrogenic compounds. |
AU4541701A AU4541701A (en) | 2000-03-10 | 2001-03-05 | Pharmaceutical compositions of conjugated estrogens and methods of analyzing mixtures containing estrogenic compounds |
CA002402559A CA2402559A1 (en) | 2000-03-10 | 2001-03-05 | Pharmaceutical compositions of conjugated estrogens and methods of analyzing mixtures containing estrogenic compounds |
AU2001245417A AU2001245417B2 (en) | 2000-03-10 | 2001-03-05 | Pharmaceutical compositions of conjugated estrogens and methods of analyzing mixtures containing estrogenic compounds |
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US09/524,132 US6855703B1 (en) | 2000-03-10 | 2000-03-10 | Pharmaceutical compositions of conjugated estrogens and methods of analyzing mixtures containing estrogenic compounds |
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EP (1) | EP1267852A2 (en) |
JP (2) | JP2004500396A (en) |
KR (2) | KR100804224B1 (en) |
AU (2) | AU2001245417B2 (en) |
CA (1) | CA2402559A1 (en) |
MX (1) | MXPA02008856A (en) |
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EP1971325A2 (en) * | 2005-12-27 | 2008-09-24 | Duramed Pharmaceuticals, Inc. | Conjugated estrogen compositions, applicators, kits, and methods of making and use thereof |
EP2289486A1 (en) * | 2005-12-27 | 2011-03-02 | Teva Women's Health, Inc. | Conjugated estrogen compositions, applicators, kits, and methods of making and use thereof |
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US8217024B2 (en) * | 2005-12-27 | 2012-07-10 | Teva Women's Health, Inc. | Conjugated estrogen compositions, applicators, kits, and methods of making and use thereof |
US8247393B2 (en) | 2005-12-27 | 2012-08-21 | Teva Women's Health, Inc. | Conjugated estrogen compositions, applicators, kits, and methods of making and use thereof |
US20110021981A1 (en) * | 2006-05-22 | 2011-01-27 | Chaudry Irshad H | Methods And Compositions For Treating Trauma-Hemorrhage Using Estrogen And Derivatives Thereof |
US9301970B2 (en) * | 2006-05-22 | 2016-04-05 | The Uab Research Foundation | Methods and compositions for treating trauma-hemorrhage using estrogen and derivatives thereof |
EP2381947A1 (en) * | 2008-12-23 | 2011-11-02 | Quest Diagnostics Investments Incorporated | Mass spectrometry assay for estrogenic compounds |
EP2381947A4 (en) * | 2008-12-23 | 2012-08-22 | Quest Diagnostics Invest Inc | Mass spectrometry assay for estrogenic compounds |
US10837971B2 (en) | 2008-12-23 | 2020-11-17 | Quest Diagnostics Investments Incorporated | Mass spectrometry assay for estrogenic compounds during hormone replacement therapy |
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CN109632982A (en) * | 2018-12-05 | 2019-04-16 | 华南理工大学 | A kind of method of quick measurement natural estrogen combination |
Also Published As
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JP2011256181A (en) | 2011-12-22 |
JP2004500396A (en) | 2004-01-08 |
AU2001245417B2 (en) | 2005-10-06 |
AU4541701A (en) | 2001-09-24 |
KR20070045364A (en) | 2007-05-02 |
WO2001068074A3 (en) | 2002-03-21 |
KR20030064614A (en) | 2003-08-02 |
CA2402559A1 (en) | 2001-09-20 |
KR100804224B1 (en) | 2008-02-18 |
EP1267852A2 (en) | 2003-01-02 |
US6855703B1 (en) | 2005-02-15 |
MXPA02008856A (en) | 2004-10-14 |
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