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Methods for inhibiting bone loss |
| RE39049 |
Methods for inhibiting bone loss
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| Patent Drawings: | |
| Inventor: |
Black, et al. |
| Date Issued: |
March 28, 2006 |
| Application: |
10/375,341 |
| Filed: |
February 27, 2003 |
| Inventors: |
Black; Larry J. (Franklin, IN)
Cullinan; George J. (Trafalgar, IN)
|
| Assignee: |
Eli Lilly and Company (Indianapolis, IN) |
| Primary Examiner: |
Webman; Edward J. |
| Assistant Examiner: |
|
| Attorney Or Agent: |
Woodard, Emhardt, Moriarty, McNett & Henry LLP |
| U.S. Class: |
514/333; 514/422; 514/443; 514/448; 514/578 |
| Field Of Search: |
514/333; 514/337; 514/443; 514/448 |
| International Class: |
A61K 31/44; A61K 31/40; A61K 37/06 |
| U.S Patent Documents: |
4133814; 4185108; 4380635; 4418068; 4729999; 4894373; 4970237; 5011853; 5075321; 5118667; 5208031; 5393763; 5395842; 5441947; 5445941; 5457116; 5457117; 5461065; 5462949; 5464845; 5468773; 5472962; 5478847; 5482949; 5510370; 5534527; 5567713; 5591753; 5624940; 5629425; 5641790; 5646137; 5731327; 5747510; 5811120; 5972383; 6087378; 6096781; 6156786; 6395769; 6458811; 6713494 |
| Foreign Patent Documents: |
0062505; 068563; 0605193; 2097788; 304924; WO93/10113; WO98/10113; WO 93/10741 |
| Other References: |
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Black et al., "Distinct, Structure-Related Profiles of Estrogenic and Anti-Estrogenic Activity in the Tamoxifen and LY117018 Series;" The Endocrine Society, Abstract 1982. cited by other.
Black et al., "Uterine Bioassay of Tamoxifen, Trioxifene, and New Estrogen Antagonist (LY117018) in Rats and Mice," Life Sciences, 26:1980, 1453-1458. cited by other.
Black et al., "Differential Interaction of Antiestrogens with Cytosol Estrogen Receptors," Molecular and Cellular Endocrinology, 22:1981, 95-103. cited by other.
Black et al., "Evidence for Biological Action of the Antiestrogens LY117018 and Tamoxifen by Different Mechanisms," Endocrinology 109; 1981, 987-989. cited by other.
Black, L. J. "Biological Actions and Binding Properties of a New Estrogen Antagosist LY117018," In: Hormone Antagonists, 129-45, 1982 (M. K. Agarwal ed.) Walter de Gruyter and Co., Berlin N.Y. cited by other.
Black et al., The Antiestrogenic Action of LY139481: Species Uniformity Duration of Action and Kinetics of 3H-LY139481 Distribution In Vivo. Sixty-fifth Annual Meeting of the Endocrine Society, San Antonio, Tex., Jun. 8-10, 1983, abs. 93. cited byother.
Black et al., The Relationship of the Antiestrogenic Efficacy of LY156758 to its Pharmacokinetics and Metabolism Following Oral Administration to Adult Ovariectomized Rats, Seventh International Congress of Endocrinology, Quebec City, Canada, Jul.1-7, 1984, abs. 323. cited by other.
Black et al., Synthesis and Antiestrogenic Activity of [3,4-Dihydro-2(4-methoxypehnyl)-1-napthalenyl][4-[2-pyrrolidinyl)ethoxyl]- -phenyl] methanone, methanesulfonic acid salt, Journal of Medicinal Chemistry 22; 1979, 962-966. cited by other.
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Barr Laboratories' Second Supplemental Responses to Eli Lilly's Interrogatories No. 5, 6 and 9 (served on Sep. 20, 2004). cited by other. |
|
| Abstract: |
The current invention provides methods and pharmaceutical formulations that are useful for inhibiting the loss of bone. These methods and formulations can be used without the associated adverse effects of estrogen therapy, and thus serve as an effective and acceptable treatment for osteoporosis. |
| Claim: |
We claim:
1. A method of inhibiting post-menopausal bone loss in a postmenopausal woman in need of treatment to prevent or to treat post-menopausal osteoporosis comprising administering to ahuman said woman in need of said treatment an amount of a compound of Formula I ##STR00011## wherein X is a bond, CH.sub.2, or CH.sub.2CH.sub.2; R and R.sup.1, independently, are hydrogen, hydroxyl, C.sub.1-C.sub.6-alkoxy, C.sub.1-C.sub.6-acyloxy,C.sub.1-C.sub.6-akoxy-C.sub.2-C.sub.6-acyloxy, R.sup.3-substituted aryloxy, R.sup.3-substituted aroyloxy, R.sup.4-substituted carbonyloxy, chloro, or bromo; R.sup.2 is a heterocyclic ring selected from the group consisting of pyrrolidino, piperidino,and hexamethyleneimino; R.sup.3 is C.sub.1-C.sub.3-alkyl, C.sub.1-C.sub.3-alkoxy, hydrogen, or halo; and R.sup.4 is C.sub.1-C.sub.6-alkoxy or aryloxy; or a pharmaceutically acceptable salt thereof, that is effective to inhibit post-menopausal boneloss in a human said woman.
2. A method of claim 1 wherein the human woman has been diagnosed as suffering from post-menopausal osteoporosis.
3. A method of claim 1 wherein the human is a post-menopausal female.
4. A method of claim 1 wherein the human is a male.
5. A method of claim 1 wherein the compound is administered prophylactically.
6. A method of claim 1 wherein X is a bond.
7. A method of claim 1 wherein R.sup.2 is piperidino or pyrrolidino.
8. A method of claim 6 wherein R.sup.2 is piperidino or pyrrolidino.
9. A method of claim 6 wherein R.sup.2 is piperidino.
10. A method of claim 6 wherein R.sup.2 is pyrrolidino.
11. A method of claim 1 wherein R and R.sup.1 are both hydroxyl, C.sub.1-C.sub.6-acyloxy, C.sub.1-C.sub.6-alkoxy-C.sub.2-C.sub.6-acyloxy, R.sup.3-substituted aroyloxy, or R.sup.4-substituted carbonyloxy.
12. A method of claim 11 wherein R and R.sup.1 are both hydroxyl.
13. A method of claim 11 wherein R and R.sup.1 are both C.sub.1-C.sub.6-acyloxy.
14. A method of claim 13 wherein R and R.sup.1 are both n-butanoyloxy, 2,2-dimethylpropanoyloxy, or 3,3-dimethylbutanoyloxy.
15. A method of claim 11 wherein R and R.sup.1 are both R.sup.4-substituted carbonyloxy.
16. A method of claim 15 wherein R and R.sup.1 are both methoxycarbonyloxy or phenyloxycarbonyloxy.
17. A method of claim 11 wherein R and R.sup.1 are both R.sup.3-substituted aroyloxy.
18. A method of claim 17 wherein R and R.sup.1 are both benzoyloxy, methylbenzoyloxy, or naphthoyloxy.
19. A method of claim 8 wherein R and R.sup.1 are both hydroxyl, C.sub.1-C.sub.6-acyloxy, C.sub.1-C.sub.6-alkoxy-C.sub.2-C.sub.6-acyloxy, R.sup.3-substituted aroyloxy, or R.sup.4-substituted carbonyloxy.
20. A method of claim 9 wherein R and R.sup.1 are both hydroxyl.
21. A method of claim 10 wherein R and R.sup.1 are both hyroxyl.
22. A method of claim 8 wherein R and R.sup.1 are both C.sub.1-C.sub.6-acyloxy.
23. A method of claim 22 wherein R and R.sup.1 are both n-butanoyloxy, 2,2-dimethylpropanoyloxy, or 3,3-dimethylbutanoyloxy.
24. A method of claim 8 wherein R and R.sup.1 are both R.sup.4-substituted carbonyloxy.
25. A method of claim 24 wherein R and R.sup.1 are both methoxycarbonyloxy or phenyloxycarbonyloxy.
26. A method of claim 8 wherein R and R.sup.1 are both R.sup.3-substituted aroyloxy.
27. A method of claim 26 wherein R and R.sup.1 are both benzoyloxy, methylbenzoyloxy, or naphthoyloxy.
28. A method of claim 1 wherein the compound of formula I is administered in an amount of 0.1 to 1000 mg.
29. A method of claim 1 wherein the compound of formula I is administered in an amount of 200 to 600 mg.
30. A method of claim 1 wherein the compound of formula I is administered in an amount of 600 to 1000 mg.
31. A method of claim 20 wherein the human woman has been diagnosed as suffering from post-menopausal osteoporosis.
32. A method of claim 20 wherein the human is a post-menopausal woman.
33. A method of claim 20 wherein the compound is administered prophylactically.
34. A method of claim 1 wherein the administration of a compound of formula I does not significantly affect the primary six target tissues.
35. A method of claim 1 wherein a compound of formula I is administered in combination with estrogen. |
| Description: |
BACKGROUND OF THE INVENTION
This invention relates to the discovery that a group of 2-phenyl-3-aroylbenzothiophenes is useful in the prevention of bone loss.
The mechanism of bone loss is not well understood, but in practical effect, the disorder arises from an imbalance in the formation of new healthy bone and the resorption of old bone, skewed toward a net loss of bone tissue. This bone lossincludes a decrease in both mineral content and protein matrix components of the bone, and leads to an increased fracture rate of, predominantly, femoral bones and bones in the forearm and vertebrae. These fractures, in turn, lead to an increase ingeneral morbidity, a marked loss of stature and mobility, and, in many cases, an increase in mortality resulting from complications.
Bone loss occurs in a wide range of subjects, including post-menopausal women, patients who have undergone hysterectomy, patients who are undergoing or have undergone long-term administration of corticosteroids, patients suffering from Cushing'ssyndrome, and patients having gonadal dysgensis dysgenesis.
Unchecked, bone loss can lead to osteoporosis, a major debilitating disease whose prominent feature is the loss of bone mass (decreased density and enlargement of bone spaces) without a reduction in bone volume, producing porosity and fragility.
One of the most common types of osteoporosis is found in post-menopausal women affecting an estimated 20 to 25 million women in the United States alone. A significant feature of post-menopausal osteoporosis is the large and rapid loss of bonemass due to the cessation of estrogen production by the ovaries. Indeed, data clearly support the ability of estrogens to limit the progression of osteoporotic bone loss, and estrogen replacement is a recognized treatment for post-menopausalosteoporosis in the United States and many other countries. However, although estrogens have beneficial effects on bone, given even at very low levels, long-term estrogen therapy has been implicated in a variety of disorders, including an increase inthe risk of uterine and breast cancer, causing many women to avoid this treatment. Recently suggested therapeutic regimens, which seek to lessen the cancer risk, such as administering combinations of progestogen and estrogen, cause the patient toexperience regular withdrawal bleeding, which is unacceptable to most older women. Concerns over the significant undesirable effects associated with estrogen therapy, and the limited ability of estrogens to reverse existing bone loss, support the needto develop alternative therapy for bone loss that generates the desirable effects on bone but does not cause undesirable effects.
Attempts to fill this need by the use of compounds commonly known as antiestrogens, which interact with the estrogen receptor, have had limited success, perhaps due to the fact that these compounds generally display a mixed agonist/antagonisteffect. That is, although these compounds can antagonize estrogen interaction with the receptor, the compounds themselves may cause estrogenic responses in those tissues having estrogen receptors. Therefore, some antiestrogens are subject to the sameadverse effects associated with estrogen therapy.
The current invention provides methods for inhibiting the loss of bone without the associated adverse effects of estrogen therapy, and thus serves as an effective and acceptable treatment for osteoporosis.
The 2-phenyl-3-aroylbenzothiophene compounds that are the active component in the formulations and methods of this invention were first developed by C. David Jones and Tulio Suarez as anti-fertility agents (see U.S. Pat. No. 4,133,814, issuedJan. 9, 1979). Certain compounds in the group were found to be useful in suppressing the growth of mammary tumors.
Jones later found a group of related compounds to be useful for antiestrogen and antiandrogen therapy, especially in the treatment of mammary and prostatic tumors (see U.S. Pat. No. 4,418,068, issued Nov. 29, 1983). One of these compounds,the compound of formula I wherein X is a bond, R and R.sup.1 are hydroxyl, and R.sup.2 is a piperidino ring, was clinically tested for a brief time for the treatment of breast cancer. That compound is called raloxifene, formerly keoxifene.
SUMMARY OF THE INVENTION
This invention provides new methods for the treatment of bone loss comprising administering to a human in need of treatment an effective amount of a compound of formula I ##STR00001## wherein
X is a bond, CH.sub.2, or CH.sub.2CH.sub.2;
R and R.sup.1, independently, are hydrogen, hydroxyl, C.sub.1-C.sub.6-alkoxy, C.sub.1-C.sub.6-acyloxy, C.sub.1-C.sub.6-akoxy-C.sub.2-C.sub.6-acyloxy, R.sup.3-substituted aryloxy, R.sup.3-substituted aroyloxy, R.sup.4-substituted carbonyloxy,chloro, or bromo;
R.sup.2 is a heterocyclic ring selected from the group consisting of a pyrrolidino, piperidino, or hexamethyleneimino;
R.sup.3 is a C.sub.1-C.sub.3-alkyl, C.sub.1-C.sub.3-alkoxy, hydrogen, or halo; and
R.sup.4 is C.sub.1-C.sub.6-alkoxy or aryloxy; or
a pharmaceutically acceptable salt thereof.
The invention also provides pharmaceutical formulations for inhibiting bone loss comprising a compound of formula I, wherein R, R1, R2, and X are as defined above in an amount that increases or retains bone density, together with apharmaceutically acceptable carrier.
DETAILED DESCRIPTION OF THE INVENTION
The current invention concerns the discovery that a group of 2-phenyl-3-aroylbenzothiophenes (benzothiophenes) of formula I are useful in the treatment of osteoporosis. The benzothiophenes of formula I inhibit the loss of bone that results froma lack of endogenous estrogen such as occurs in women following cessation of menstruation due to natural, surgical, or other processes. The reduction of bone density and mass that more rarely occurs in men is also tied to the loss of hormonal regulationand is therefore also a target for therapy according to the methods of the current invention.
The benzothiophenes of formula I are a series of nonsteroidal compounds that exhibit high affinity for conventional estrogen receptors in primary sex target tissues. However, they elicit minimal estrogenic responses in those tissues, andactually serve as potent antagonists of natural estrogens such as estradiol. In contrast to the report of Feldmann, S. et al., "Antiestrogen and antiandrogen administration reduce bone mass in the rat", Bone and Mineral, 7:245 (1989), thebenzothiophenes of formula I are able to antagonize classical estrogenic responses in primary sex target tissues without significantly reducing bone density when given to intact or estrogen treated animals, and they prevent bone loss in estrogendeficient animals. This dichotomy indicates selective agonist/antagonist actions on specific target cells which would appear to be highly desirable in treatment of the menopausal syndrome. Accordingly, the real benefit of the current discovery is thatthe benzothiophenes of formula I inhibit the loss of bone but do not elicit significant estrogenic responses in the primary sex target tissues. Thus, the current invention provides a method of inhibiting bone loss comprising administering to a human inneed of treatment an amount of a compound of formula I that inhibits bone loss but does not significantly affect the primary sex target tissues. This combination of features allows for long-term treatment of the chronic ailment with a diminished risk ofdeveloping the undesirable effects of customary estrogen replacement therapy.
The biological action of the benzothiophenes of formula I is complex and may be unrelated to the detectable presence of the parent compound in the blood. Following oral administration of a preferred benzothiophene of this invention, raloxifene(raloxifene hydrochloride), to human subjects in the clinic, the parent compound was not detected in the serum of those subjects. It was determined that following oral administration, the compound was extensively conjugated to the glucuronidated formand cleared quickly from the bloodstream. Although no biological endpoints were measured in the human recipients, there was concern that the compound was not bioavailable.
Experiments were undertaken to address the bioavailability issue in laboratory animals where biological activity could be assessed. The animal studies indicated that raloxifene was maximally active in inhibiting both uterine uptake oftritiated-estradiol and the normal uterotrophic response to estradiol even under conditions where raloxifene was extensively conjugated in the plasma of the animals. Moreover, the conjugate, isolated from the urine of human subjects treated withraloxifene, displayed significant antiestrogenic/antiuterotrophic activity when administered intravenously to rats, and inhibited the interaction of tritiated-estradiol with rat uterine estrogen receptors in a manner similar to the parent compound. These studies suggested the conjugated compound may have been converted to the parental form at the site of action, presumably by the action of .beta.-glucuronidase. Such conversion may contribute to the activity of the compound. .beta.-Glucuronidaseis fairly ubiquitous and is thought to be active in the resorption process of bone remodeling, and would presumably be available for converting the conjugated compound to the parental form if required for activity. Therefore, conjugation of thebenzothiophenes of formula I is not considered to be necessarily detrimental to their bioavailability as an inhibitor of bone loss.
Thus, the method of treatment provided by this invention is practiced by administering to a human in need of inhibition of bone loss, a dose of a compound of formula I or a pharmaceutically acceptable salt thereof, that is effective to inhibitbone loss. A particular benefit of this method is that it avoids potentially harmful and unacceptable estrogenic side effects. The inhibition of bone loss contemplated by the present method includes both medical therapeutic and/or prophylactictreatment, as appropriate.
The method also includes the administration of a compound of formula I given in combination with estrogen. The term estrogen as used herein refers to any compound which approximates the spectrum of activities of the naturally acting moleculewhich is commonly believed to be 17.beta.-estradiol. Examples of such compounds include estriol, estrone, ethynyl estradiol, Premarin (a commercial preparation of conjugated estrogens isolated from natural sources--Ayerst), and the like. Again, due tothe selective agonist/antagonist properties of the compounds of the formula I, this combination provides for the full benefits of estrogen therapy without the concomitant adverse effects associated with estrogen therapy alone.
The general chemical terms used in the description of a compound of formula I have their usual meanings. For example, the term "C.sub.1-C.sub.3-alkyl" includes such groups as methyl, ethyl, propyl, and isopropyl.
The term "C.sub.1-C.sub.6-alkoxy" includes such groups as methoxy, ethoxy, propoxy, butoxy, pentyloxy, and hexyloxy and also includes branched chain structures such as, for example, isopropoxy and isobutoxy.
The term "C.sub.1-C.sub.6-acyloxy" includes methanoyloxy, ethanoyloxy, propanoyloxy, butanoyloxy, pentanoyloxy, hexanoyloxy, and the like and also includes branched chain structures such as, for example, 2,2-dimethylpropanoyloxy, and3,3-dimethylbutanoyloxy.
The term "C.sub.1-C.sub.6-alkoxy-C.sub.2-C.sub.6-acyloxy" contemplates, for example, methoxyethanoyloxy, methoxypropanoyloxy, methoxybutanoyloxy, methoxypentanoyloxy, methoxyhexanoyloxy, ethoxyethanoyloxy, ethoxypropanoyloxy, ethoxybutanoyloxy,ethoxypentanoyloxy, ethoxyhexanoyloxy, propoxyethanoyloxy, propoxypropanoyloxy, propoxybutanoyloxy, and the like.
It should also be understood that as used herein, references to alkyl and alkoxy structures also include cycloalkyl and cycloalkoxy groups where the number of carbons within the structure is at least 3.
The terms "R.sup.3-substituted aryloxy" and "R.sup.3-substituted aroyloxy" include such groups as phenyloxy, thienyloxy, furyloxy, naphthyloxy, benzoyloxy, thienoyloxy, furoyloxy, naphthoyloxy, and the like, where the R.sup.3 substitution groupmay be hydrogen, hydroxyl, C.sub.1-C.sub.3-alkyl, C.sub.1-C.sub.3-alkoxy, or halo.
The term "R.sup.4-substituted carbonyloxy, where the R.sup.4 substitution group may be C.sub.1-C.sub.6-alkoxy or aryloxy, includes carbonate structures such as methoxycarbonyloxy ethoxycarbonyloxy, propoxycarbonyloxy, butoxycarbonyloxy,pentyloxycarbonyloxy, hexyloxycarbonyloxy, phenyloxycarbonyloxy, thienyloxycarbonyloxy, furyloxycarbonyloxy, and naphthyloxycarbonyloxy.
Preferred methods of this invention comprise the use of compounds of formula I wherein R and R.sup.1 are other than hydrogen, alkoxy, aryloxy, chloro, or bromo and therefore represent ester and carbonate configurations. Other preferred methodsinclude the use of formula I compounds wherein R and R.sup.1 are the same as one another. Certain R.sup.2 groups also demonstrate preferable characteristics when used in the methods of this invention. For example, preferred methods of this inventioninclude the use of formula I compounds wherein R.sup.2 is piperidino or pyrrolidino, especially piperidino. A further preferred subgroup of the preferred piperidino and pyrrolidino compounds include compounds wherein R and R.sup.1 are other thanhydrogen and, in particular, those wherein R and R.sup.1 are hydroxyl.
All of the compounds used in the methods of the current invention can be made according to established procedures, such as those detailed in U.S. Pat. No. 4,133,814 and U.S. Pat. No. 4,418,068. In general, the process starts with abenzo[b]thiophene having a 6-hydroxyl group and a 2-(4-hydroxyphenyl) group. The starting compound is protected, alkylated, and deprotected to form the formula I compounds wherein R and R.sup.1 are both hydroxy. The formula I compounds that are ethers,esters, and carbonates may then be formed if desired. Examples of the preparations of such compounds are provided in the U.S. patents discussed above. Specific preparations of yet other derivatized compounds useful in the current invention areoutlined in the Preparations sections below. Modifications to the above methods may be necessary to accommodate reactive functionalities of particular substituents. Such modifications would be both apparent to, and readily ascertained by, those skilledin the art.
The compounds used in the methods of this invention form pharmaceutically acceptable acid and base addition salts with a wide variety of organic and inorganic acids and bases and include the physiologically acceptable salts which are often usedin the pharmaceutical chemistry. Such salts are also part of this invention. Typical inorganic acids used to form such salts include hydrochloric, hydrobromic, hydroiodic, nitric, sulfuric, phosphoric, hypophosphoric and the like. Salts derived fromorganic acids, such as aliphatic mono and dicarboxylic acids, phenyl substituted alkanoic acids, hydroxyalkanoic and hydroxyalkandioic acids, aromatic acids, aliphatic and aromatic sulfonic acids, may also be used. Such pharmaceutically acceptable saltsthus include acetate, phenylacetate, trifluoroacetate, acrylate, ascorbate, benzoate, chlorobenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, methylbenzoate, o-acetoxybenzoate, naphthalene-2-benzoate, bromide, isobutyrate, phenylbutyrate,.beta.-hydroxybutyrate, butyne-1,4-dioate, hexyne-1,4-dioate, caprate, caprylate, chloride, cinnamate, citrate, formate, fumarate, glycollate, heptanoate, hippurate, lactate, malate, maleate, hydroxymaleate, malonate, mandelate, mesylate, nicotinate,isonicotinate, nitrate, oxalate, phthalate, teraphthalate, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate, propiolate, propionate, phenylpropionate, salicylate, sebacate, succinate, suberate, sulfate, bisulfate,pyrosulfate, sulfite, bisulfite, sulfonate, benzene-sulfonate, p-bromophenylsulfonate, chlorobenzenesulfonate, ethanesulfonate, 2-hydroxyethanesulfonate, methanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, p-toluenesulfonate,xylenesulfonate, tartarate, and the like.
In addition, some of the formula I compounds may form solvates with water or organic solvents such as ethanol. These solvates are also contemplated for use in the methods of this invention.
The pharmaceutically acceptable acid addition salts are typically formed by reacting a compound of formula I with an equimolar or excess amount of acid. The reactants are generally combined in a mutual solvent such as diethyl ether or benzene. The salt normally precipitates out of solution within about one hour to 10 days and can be isolated by filtration or the solvent can be stripped off by conventional means.
Bases commonly used for formation of salts include ammonium hydroxide and alkali and alkaline earth metal hydroxides, carbonates and bicarbonates, as well as aliphatic and aromatic amines, aliphatic diamines and hydroxy alkylamines. Basesespecially useful in the preparation of addition salts include ammonium hydroxide, potassium carbonate, sodium bicarbonate, calcium hydroxide, methylamine, diethylamine, ethylene diamine, cyclohexylamine and ethanolamine.
The pharmaceutically acceptable salts generally have enhanced solubility characteristics compared to the compound from which they are derived, and thus are often more amenable to formulation as liquids or emulsions.
The current invention also provides useful pharmaceutical formulations for inhibiting bone loss comprising a formula I compound plus one or more pharmaceutically acceptable excipients. Pharmaceutical formulations can be prepared by proceduresknown in the art. For example, the compounds can be formulated with common excipients, diluents, or carriers, and formed into tablets, capsules, suspensions, powders, and the like. Examples of excipients, diluents, and carriers that are suitable forsuch formulations include the following: fillers and extenders such as starch, sugars, mannitol, and silicic derivatives; binding agents such as carboxymethyl cellulose and other cellulose derivatives, alginates, gelatin, and polyvinyl pyrrolidone;moisturizing agents such as glycerol; disintegrating agents such as agaragar, calcium carbonate, and sodium bicarbonate; agents for retarding dissolution such as paraffin; resorption accelerators such as quaternary ammonium compounds; surface activeagents such as cetyl alcohol, glycerol monostearate; adsorptive carriers such as kaolin and bentonire; and lubricants such as talc, calcium and magnesium stearate, and solid polyethyl glycols.
The compounds can also be formulated as elixirs or solutions for convenient oral administration or as solutions appropriate for parenteral administration, for instance by intramuscular, subcutaneous or intravenous routes. Additionally, thecompounds are well suited to formulation as sustained release dosage forms and the like. The formulations can be so constituted that they release the active ingredient only or preferably in a particular part of the intestinal treat, possibly over aperiod of time. The coatings, envelopes, and protective matrices may be made, for example, from polymeric substances or waxes.
The particular dosage of a compound of formula I required to treat or inhibit bone loss according to this invention will depend upon the severity of the disease, its route of administration, and related factors that will be decided by theattending physician. Generally, accepted and effective doses will be from about 0.1 to about 1000 mg, and more typically from about 200 to about 600 mg. Such dosages will be administered to a subject in need of treatment from once to about three timeseach day, or more often as needed to effectively inhibit the bone loss process.
It is usually preferred to administer a compound of formula I in the form of an acid addition salt, as is customary in the administration of pharmaceuticals bearing a basic group such as the piperidino ring. It is also advantageous to administersuch a compound by the oral rote to an aging human (e.g. a post-menopausal female or a male showing evidence of bone loss by X-ray analysis). For such purposes the following oral dosage forms are available.
FORMULATIONS
In the formulations which follow, "Active ingredient" means a compound of formula I.
Formulation 1: Gelatin Capsules
Hard gelatin capsules are prepared using the following:
TABLE-US-00001 Ingredient Quantity (mg/capsule) Active ingredient 0.1-1000 Starch, NF 0-650 Starch flowable powder 0-650 Silicone fluid 350 centistokes 0-15
The ingredients are blended, passed through a No. 45 mesh U.S. sieve, and filled into hard gelatin capsules.
Examples of specific capsule formulations containing raloxifene that have been made include those shown below:
Formulation 2: Raloxifene Capsule
TABLE-US-00002 Ingredient Quantity (mg/capsule) Raloxifene 1 Starch, NF 112 Starch flowable powder 225.3 Silicone fluid 350 centistokes 1.7
Formulation 3: Raloxifene Capsule
TABLE-US-00003 Ingredient Quantity (mg/capsule) Raloxifene 5 Starch, NF 108 Starch flowable powder 225.3 Silicone fluid 350 centistokes 1.7
Formulation 4: Raloxifene Capsule
TABLE-US-00004 Ingredient Quantity (mg/capsule) Raloxifene 10 Starch, NF 103 Starch flowable powder 225.3 Silicone fluid 350 centistokes 1.7
Formulation 5: Raloxifene Capsule
TABLE-US-00005 Ingredient Quantity (mg/capsule) Raloxifene 50 Starch, NF 150 Starch flowable powder 397 Silicone fluid 350 centistokes 3.0
The specific formulations above may be changed in compliance with the reasonable variations provided.
A tablet formulation is prepared using the ingredients below:
Formulation 6: Tablets
TABLE-US-00006 Ingredient Quantity (mg/tablet) Active ingredient 0.1-1000 Cellulose, microcrystalline 0-650 Silicone dioxide, fumed 0-650 Stearate acid 0-15
The components are blended and compressed to form tablets.
Alternatively, tablets each containing 0.1-1000 mg of active ingredient are made up as follows:
Formulation 7: Tablets
TABLE-US-00007 Ingredient Quantity (mg/tablet) Active ingredient 0.1-1000 Starch 45 Cellulose, microcrystalline 35 Polyvinylpyrrolidone 4 (as 10% solution in water) Sodium carboxymethyl cellulose 4.5 Magnesium stearate 0.5 Talc 1
The active ingredient, starch, and cellulose are passed through a No. 45 mesh U.S. sieve and mixed thoroughly. The solution of polyvinylpyrrolidone is mixed with the resultant powders which are then passed through a No. 14 mesh U.S. sieve. The granules so produced are dried at 50.degree.-60.degree. C. and passed through a No. 18 mesh U.S. sieve. The sodium carboxymethyl starch, magnesium stearate, and talc, previously passed through a No. 60 U.S. sieve, are then added to the granuleswhich, after mixing, are compressed on a tablet machine to yield tablets.
Suspensions each containing 0.1-1000 mg of medicament per 5 mL dose are made as follows:
Formulation 8: Suspensions
TABLE-US-00008 Ingredient Quantity (mg/5 ml) Active ingredient 0.1-1000 mg Sodium carboxymethyl cellulose 50 mg Syrup 1.25 mg Benzoic acid solution 0.10 mL Flavor q.v. Color q.v. Purified water to 5 mL
The medicament is passed through a No. 45 mesh U.S. sieve and mixed with the sodium carboxymethyl cellulose and syrup to form a smooth paste. The benzoic acid solution, flavor, and color are diluted with some of the water and added, withstirring. Sufficient water is then added to produce the required volume.
Illustrative compounds that can be used in the formulations and methods of this invention are shown in Table 1.
TABLE-US-00009 TABLE 1 Compound No. X R and R.sup.1 R.sup.2 Form 1 bond ##STR00002## piperidino base 2 bond ##STR00003## piperidino HCl 3 bond ##STR00004## piperidino base 4 bond ##STR00005## piperidino HCl 5 bond --OC(O)CH.sub.2CH.sub.2CH.sub.3piperidino base 6 bond --OC(O)CH.sub.2CH.sub.2CH.sub.3 piperidino HCl 7 bond --OC(O)C(CH.sub.3).sub.3 piperidino base 8 bond --OC(O)C(CH.sub.3).sub.3 piperidino HCl 9 bond --OC(O)CH.sub.2C(CH.sub.3).sub.3 piperidino base 10 bond--OC(O)CH.sub.2C(CH.sub.3).sub.3 piperidino HCl 11 bond ##STR00006## piperidino HCl 12 bond ##STR00007## piperidino base 13 bond --OC(O)OCH.sub.2CH.sub.2CH.sub.2CH.sub.3 piperidino base 14 bond --OC(O)OCH.sub.2CH.sub.2CH.sub.2CH.sub.3 piperidino HCl 15bond ##STR00008## piperidino base 16 bond ##STR00009## piperidino HCl 17 bond ##STR00010## piperidino base 18 bond --OC(O)CH.sub.2CH.sub.2OCH.sub.3 piperidino base --OC(O)CH.sub.2OCH.sub.3 19 bond --OC(O)CH.sub.2CH.sub.2OCH.sub.3 piperidino HCl--OC(O)CH.sub.2OCH.sub.3 20 bond OH piperidino base 21 bond OH piperidino HCl 22 bond H piperidino base 23 CH.sub.2 OH piperidino HCl 24 CH.sub.2CH.sub.2 OH piperidino HCl 25 CH.sub.2 H piperidino HCl 26 bond OH pyrrolodino base 27 bond OH pyrrolodinoHCl 28 CH.sub.2 OH pyrrolodino HCl 29 CH.sub.2CH.sub.2 OH pyrrolodino HCl 30 bond H pyrrolodino HCl 31 bond OH hexamethyleneimino HCl 32 CH.sub.2 OH hexamethyleneimino HCl 33 CH.sub.2CH.sub.2 OH hexamethyleneimino HCl 34 bond OCH.sub.3 piperidino HCl
In the following Preparations, the compound numbers correspond to those given in Table 1
Preparation 1
Preparation of Compound 1
6-(4-Fluorobenzoyloxy)-2-[4-(4-fluorobenzoyloxy)phenyl]benzo[b]thien-3-yl-- [4-[2-(piperidin-1-yl)ethoxy]phenyl]methanone
Raloxifene, 6-hydroxy-2-(4-hydroxyphenyl)benzo[b]thien-3-yl-4-[2-(piperidin-1-yl)etho- xyphenyl]methanone hydrochloride, (5.1 g, 10 mmol) was suspended in 250 mL of dry tetrahydrofuran (THF) and 7.1 g (70 mmol) of triethylamine, and approximately10 mg of 4-(N,N-dimethylamino)pyridine were added. The suspension was cooled in an ice bath and placed under an atmosphere of nitrogen. 4-Fluorobenzoyl chloride (4.75 g, 30 mmol), dissolved in 20 mL of dry THF, was slowly added over a twenty minuteperiod. The reaction mixture was stirred and allowed to slowly warm to room temperature over a period of eighteen hours. It was then filtered, and the filtrate was evaporated to a gum in vacuo. The crude product thus obtained was dissolved in a smallvolume of chloroform and chromatographed (HPLC) on a silica gel column eluted with a linear gradient of solvent, starting with chloroform and ending with a mixture of chloroform-methanol (19:1 (v/v)). The fractions containing the desired product asdetermined by thin layer chromatography (silica, chloroform-methanol (9:1)) were combined and evaporated to a gum. The final product was crystallized from ether to give 3.21 g of compound 1.
PMR: consistent with the structure FDMS: m/e=717 M+
Elemental Analysis for C.sub.42H.sub.33F.sub.2NO.sub.6S: Theor: C, 70.29; H, 4.60; N, 1.95 Found: C, 70.05; H, 4.60; N, 1.89 Mol. Wt.: 717.
PREPARATION 2
Preparation of Compound 2
6-(4-Fluorobenzoyloxy)-2-[4-(4-fluorobenzoyloxy)phenyl]benzo[b]thien-3-yl-- [4-[2-(piperidin-1-yl)ethoxy]phenyl]methanone hydrochloride
Compound 1 (5.15 g, 7.18 mmol) was dissolved in 25 mL THF, and 150 mL ether was added. Dry HCl gas was bubbled into the solution, and a white gummy precipitate formed. The liquid was removed by decanting, and the residue was crystallized fromethyl acetate with a small amount of ethanol added to effect solution. The product was filtered, washed with ether, and dried to give 4.41 g of Compound 2 as a white powder.
PMR: consistent with the structure
Elemental Analysis for C.sub.42H.sub.34ClF.sub.2NO.sub.6S: Theor: C, 66.88; H, 4.54; N, 1.86 Found: C, 66.59, H, 4.39; N, 1.60 Mol. Wt.: 753.5.
PREPARATION 3
Preparation of Compound 3
6-(Cyclopropylcarbonyloxy)-2-[4-(cyclopropylcarbonyloxy)phenyl]benzo[b]thi- en-3-yl-[4-[2-(piperidin-1-yl)ethoxy]phenyl]methanone
The title compound was prepared using procedures analogous to those in Preparation 1, but using cyclopropylcarbonyl chloride, except that the product was not crystallized. Yield=2.27 g.
PMR: consistent with the structure FDMS: m/e=610 M.sup.+
PREPARATION 4
Preparation of Compound 4
6-(Cyclopropylcarbonyloxy)-2-[4-(cyclopropylcarbonyloxy)phenyl]benzo[b]thi- en-3-yl-[4-[2-(piperidin-1-yl)ethoxy]phenyl]methanone hydrochloride
Compound 4 was prepared from Compound 3 as described in Preparation 2.
PREPARATION 5
Preparation of Compound 5
6-(n-Butanoyloxy)-2-[4-(n-butanoyloxy)phenyl]benzo[b]thien-3-yl-[4-[2-(pip- eridin-1-yl)ethoxy]phenyl]methanone
Compound 5 was prepared using the method of Preparation 1, but starting with n-butanoyl chloride, to give 4.12 g of final product as an oil.
PMR: consistent with the structure FDMS: m/e=614 (M.sup.+1)
Preparation 6
Preparation of Compound 6
6-(n-Butanoyloxy)-2-[4-(n-butanoyloxy)phenyl]benzo[b]thien-3-yl-[4-[2-(pip- eridin-1-yl)ethoxy]phenyl]methanone hydrochloride
Compound 5 (4.12 g) was dissolved in ethyl acetate (50 mL), and a solution of HCl in ether was added until the precipitation stopped. The liquid was decanted off, and the white, gummy residue was triturated with diethyl ether and filtered. Theresidue was dried to give 1.33 g of Compound 6.
PMR: consistent with the structure
Elemental Analysis of for C.sub.36H.sub.40ClNO.sub.6S: Theor.: C, 66.50; H, 6.20; N, 2.15 Found: C, 66.30; H, 6.28; N, 1.98 Mol. Wt.: 650.24.
PREPARATION 7
Preparation of Compound 7
6-(2,2-Dimethylpropanoyloxy)-2-[4-(2,2-dimethylpropanoyloxy)phenyl]benzo[b- ]thien-3-yl-[4-[2-(piperidin-1-yl)ethoxy]phenyl]methanone
Compound 7 was prepared using the procedure of Preparation 1, but using 2,2-dimethylpropanoyl chloride.
PREPARATION 8
Preparation of Compound 8
6-(2,2-Dimethylpropanoyloxy)-2-[4-(2,2-dimethylpropanoyloxy)phenyl]benzo[b- ]thien-3-yl-[4-[2-(piperidin-1-yl)ethoxy]phenyl]methanone hydrochloride
Compound 8 was Prepared from Compound 7, as described in Preparation 2.
FDMS: m/e=641 (M-HCl-1)
Elemental Analysis of C.sub.38H.sub.44ClNO.sub.6S: Theor.: C, 67.29; H, 6.54; N, 2.07 Found: C, 67.02; H, 6.54; N, 1.90 Mol. Wt.: 678.29.
PREPARATION 9
Preparation of Compound 9
6-(3,3-Dimethylbutanoyloxy)-2-[4-(3,3-dimethylbutanoyloxy)phenyl]benzo[b]t- hien-3-yl[4-[2-(piperidin-1-yl)ethoxy]phenyl]methanone
Compound 9 was prepared using the procedures of Preparation 1, but with 3,3-dimethylbutanoyl chloride.
Preparation 10
Preparation of Compound 10
6-(3,3-Dimethylbutanoyloxy)-2-[4-(3,3-dimethylbutanoyloxy)phenyl]benzo[b]t- hien-3-yl[4-[2-(piperidin-1-yl)ethoxy]phenyl]methanone hydrochloride
Compound 10 was prepared from Compound 9 as described in Preparation 2.
FDMS: m/e=669 (M-HCl-1)
Elemental Analysis of C.sub.40H.sub.48ClNO.sub.6S: Theor.: C, 68.02; H, 6.85; N, 1.98 Found: C, 67.75; H, 6.83; N, 2.04 Mol. Wt.: 706.35.
PREPARATION 11
Preparation of Compound 11
6-(4-Methylbenzoyloxy)-2-[4-(4-methylbenzoyloxy)phenyl]benzo[b]thien-3-yl[- 4-[2-(piperidin-1-yl)ethoxy]phenyl]methanone hydrochloride
Compound 11 was prepared from the free base using a procedure similar to that of Preparation 2.
FDMS: m/e=710 (M-HCl-1)
Elemental Analysis of C.sub.44H.sub.40ClNO.sub.6S: Theor.: C, 70.81; H, 5.39; N, 1.88 Found: C, 71.10; H, 5.39; N, 1.94 Mol. Wt.: 746.33.
PREPARATION 12
Preparation of Compound 12
6-Benzoyloxy-2-[4-benzoyloxy)phenyl]benzo[b]thien-3-yl[4-[2-(piperidin-1-y- l)ethoxy]-phenyl]methanone
Compound 12 was prepared from the appropriate acid chloride as described in Preparation 1.
FDMS: m/e=682 (M+1)
Element Analysis of C.sub.42H.sub.35NO.sub.6S: Calc: C, 73.80; H, 5.14; N, 2.05 Found: C, 73.27; H, 5.27; N, 1.94 Mol. Wt.: 681.8.
PREPARATION 13
Preparation of Compound 13
6-(n-Butoxyoyloxy)-2-[4(n-butoxyoyloxy)phenyl]benzo[b]thien-3-yl[4-[2-(pip- eridin-1-yl)ethoxy]phenyl]methanone
Compound 13 was prepared in a manner analogous to that described in Preparation 1, except that n-butylchloroformate was used in place of the acid chloride. Yield=6.13 g in form of oil.
PMR: consistent with structure FDMS: m/e=674 (M+1)
PREPARATION 14
Preparation of Compound 14
6-(n-Butoxycarbonyloxy)-2-[4(n-butoxycarbonyloxy)phenyl]benzo[b]thien-3-yl- [4-[2-(piperidin-1-yl)ethoxy]phenyl]methanone hydrochloride
Compound 13 was converted to the hydrochloride salt in a manner analogous to that described in Preparation 6.
PMR: consistent with structure
Elemental Analysis of C.sub.38H.sub.44ClNO.sub.8S: Calc: C, 64.26; H, 6.24; N, 1.97 Found: C, 63.97; H, 6.34; N, 1.98 Mol. Wt.: 710.29.
PREPARATION 15
Preparation of Compound 15
6-(Phenyloxycarbonyloxy)-2-[4-(phenyloxycarbonyloxy)phenyl]benzo[b]thien-3- -yl[4-[2-(piperidin-1-yl)ethoxy]phenyl]methanone
This compound was prepared in a manner analogous to that described in Preparation 13, but using the appropriate acyl ester. Yield=3.59 g of final product as a tan amorphous powder.
PMR: consistent with structure FDMS: m/e=713 (M+)
PREPARATION 16
Preparation of Compound 16
6-(Phenyloxycarbonyloxy)-2-[4-(phenyloxycarbonyloxy)phenyl]benzo[b]thien-3- -yl[4-[2-(piperidin-1-yl)ethoxy]phenyl]methanone hydrochloride
Compound 15 was converted to the hydrochloride salt in a manner analogous to that described in Preparation 6.
PMR: consistent with structure
Elemental Analysis of C.sub.38H.sub.44ClNO.sub.8S: Calc: C, 67.24; H, 4.84; N, 1.87 Found: C, 66.94; H, 4.96; N, 1.84 Mol. Wt.: 750.27.
PREPARATION 17
Preparation of Compound 17
6-(Naphthoyloxy)-2-[4(1-naphthoyloxy)phenyl]benzo[b]thien-3-yl[4-[2-(piper- idin-1-yl)ethoxy]phenyl]methanone
Compound 17 was prepared as described in Preparation 1 using the appropriate acid halide. Yield=3.5 g of a white amorphous powder.
PMR: consistent with structure FDMS: m/e=781 (M+)
Elemental Analysis of C.sub.50H.sub.39NO.sub.6S: Calc: C, 76.80; H, 5.03; N, 1.79 Found: C, 76.53; H, 5.20; N, 1.53 Mol. Wt.: 781.94.
PREPARATION 18
Preparation of Compound 18
6-(Methoxyethanoyloxy)-2-[4(methoxyethanoyloxy)phenyl]benzo[b]thien-3-yl[4- -[2-(piperidin-1-yl)ethoxy]phenyl]methanone
Compound 18 was prepared as described in Preparation 1 using the appropriate acid halide. Yield=3.61 g of a gummy solid.
PRM: consistent with structure FDMS: m/e=618 (M+1)
PREPARATION 19
Preparation of Compound 19
6-(Methoxyethanoyloxy)-2-[4(methoxyethanoyloxy)phenyl]benzo[b]thien-3-yl[4- -[2-(piperidin-1-yl)ethoxy]phenyl]methanone hydrochloride
Compound 19 was prepared from 3.5 g of Compound 18 as described in Preparation 2. Yield=1.65 g of amorphous white powder.
PMR: consistent with structure FDMS: m/e=618 (M+1)
Elemental Analysis of C.sub.34H.sub.36NO.sub.8S: Calc: C, 62.43; H, 5.55; N, 2.14 Found: C, 62.23; H, 5.63; N, 2.15.
The following nonlimiting examples illustrate the methods and formulations of this invention.
EXAMPLE 1
In the examples illustrating the methods, a model of post-menopausal osteoporosis was used in which effects of different treatments upon femur density were determined.
Seventy-five day old female Sprague Dawley rats (weight range of 225 to 275 g) were obtained from Charles River Laboratories (Portage, Mich.). They were housed in groups of 3 and had ad libitum access to food (calcium content approximately 1%)and water. Room temperature was maintained at 22.2.degree..+-.1.7.degree. C. with a minimum relative humidity of 40%. The photoperiod in the room was 12 hours light and 12 hours dark.
One week after arrival, the rats underwent bilateral ovariectomy under anesthesia (44 mg/kg Ketamine and 5 mg/kg Xylazine (Butler, Indianapolis, Ind.) administered intramuscularly). Treatment with vehicle, estrogen, or a compound of formula Iwas initiated on the day of surgery following recovery from anesthesia. Oral dosage was by gavage in 0.5 mL of 1% carboxymethylcellulose (CMC). Body weight was determined at the time of surgery and weekly thereafter and the dosage was adjusted withchanges in body weight. Vehicle or estrogen treated ovariectomized (ovex) rats and non-ovariectomized (intact) rats were evaluated in parallel with each experimental group to serve as negative and positive controls.
The rats were treated daily for 35 days (6 rats per treatment group) and sacrificed by decapitation on the 36th day. The 35 day time period was sufficient to allow maximal reduction in bone density, measured as described herein. At the time ofsacrifice, the uteri were removed, dissected free of extraneous tissue, and the fluid contents were expelled before determination of wet weight in order to confirm estrogen deficiency associated with complete ovariectomy. Uterine weight was routinelyreduced about 75% in response to ovariectomy. The uteri were then placed in 10% neutral buffered formalin to allow for subsequent histological analysis.
The right femurs were excised and scanned at the distal metaphysis 1 mm from the patellar groove with single photon absorptiometry. Results of the densitometer measurements represent a calculation of bone density as a function of the bonemineral content and bone width.
Influence of Raloxifene on Bone Density
The results of control treatments from five separate experiments are accumulated in Table 2. In summary, ovariectomy of the rats caused a reduction in femur density of about 25% as compared to intact vehicle treated controls. Estrogen,administered in the orally active form of ethynyl estradiol (EE.sub.2), prevented this loss of bone in a dose dependent manner, but it also exerted a stimulatory action on the uterus resulting in uterine weights approaching that of an intact rat whenadministered at 100 .mu.g/kg. Results are reported as the mean of measurements from thirty rats .+-.the standard error of the mean.
In these studies, raloxifene also prevented bone loss in a dose dependent manner; however, only minimal increase of uterine weight over the ovariectomized controls was present in these animals. The results of five assays using raloxifene arecombined in Table 3. Accordingly, each point reflects the responses of thirty rats and depicts a typical dose response curve for raloxifene in this model. Results are reported as the mean + the standard error of the mean.
TABLE-US-00010 TABLE 2 Bone Density Uterine Weight (mg/cm/cm) (mg) ovariectomy control 170 .+-. 3 127 .+-. 5 (0.5 mL CMC oral) Intact control 220 .+-. 4 545 .+-. 19 (0.5 mL CMC oral) EE.sub.2 100 .mu.g/kg, oral 210 .+-. 4 490 .+-. 11
TABLE-US-00011 TABLE 3 Bone Density Uterine Weight (mg/cm/cm) (mg) ovariectomy control 171 .+-. 3 127 .+-. 5 (0.5 mL CMC oral) Intact control 222 .+-. 3 540 .+-. 22 (0.5 mL CMC oral) raloxifene 0.01 mg/kg, oral 176 .+-. 3 150 .+-. 5raloxifene 0.10 mg/kg, oral 197 .+-. 3 196 .+-. 5 raloxifene 1.00 mg/kg, oral 201 .+-. 3 199 .+-. 5 raloxifene 10.00 mg/kg, oral 199 .+-. 3 186 .+-. 4
EXAMPLE 2
Raloxifene was administered alone or in combination with ethynyl estradiol. Rats treated with raloxifene alone had uterine weights which were marginally higher than the overiectomized controls and much less than those of ethynyl estradioltreated rats, which approached those of the intact controls. Conversely, raloxifene treatment significantly reduced bone loss in ovariectomized rats, and when given in combination with ethynyl estradiol it did not appreciably reduce the protectiveeffect of the estrogen on bone density. The results are shown in Table 4.
TABLE-US-00012 TABLE 4 Bone Density Uterine Weight (mg/cm/cm) (mg) Experiment A Ovariectomy control 162 .+-. 4 142 .+-. 18 (0.5 mL CMC oral) Intact control 219 .+-. 5 532 .+-. 49 (0.5 mL CMC oral) EE.sub.2 100 .mu.g/kg, oral 202 .+-. 6 450.+-. 17 EE.sub.2 100 .mu.g/kg + 204 .+-. 2 315 .+-. 10 raloxifene 0.10 mg/kg, oral EE.sub.2 100 .mu.g/kg + 200 .+-. 5 250 .+-. 21 raloxifene 1 mg/kg, oral Experiment B Ovariectomy control 165 .+-. 8 116 .+-. 6 (0.5 mL CMC oral) Intact control 220.+-. 4 605 .+-. 69 (0.5 mL CMC oral) EE.sub.2 100 .mu.g/kg, oral 215 .+-. 11 481 .+-. 24 raloxifene 1 mg/kg + 197 .+-. 7 263 .+-. 17 EE.sub.2 100 .mu.g/kg, oral raloxifene 1 mg/kg 198 .+-. 11 202 .+-. 5
EXAMPLE 3
The ability of raloxifene to inhibit bone loss was compared to that of tamoxifen (SIGMA, St. Louis, Mo.). Tamoxifen, a well known antiestrogen currently used in the treatment of certain cancers, has been shown to inhibit bone loss (see forexample, Love, R., et al. 1992 "Effects of tamoxifen on bone mineral density in post-menopausal women with breast cancer", N Eng J Med 326:852; Turner, R., et al. 1988 "Tamoxifen inhibits osteoclast-mediated resorption of trabecular bone in ovarianhormone-deficient rats", Endo 122:1146). A relatively narrow range of doses of raloxifene and tamoxifen was administered orally to ovariectomized rats as in the previous example. Although both of these agents displayed the ability to prevent reductionof femur density while evoking only modest uterotrophic activity, as identified by gains in uterine weight (Table 5), a comparison of several histological parameters demonstrated a marked difference between the rats treated with these agents (Table 6).
Increases in epithelial height are a sign of estrogenicity of therapeutic agents and may be associated with increased incidence of uterine cancer. When raloxifene was administered as described in Example 1, only at one dose was there anystatistically measurable increase in epithelial height over the ovariectomized controls. This was in contrast to the results seen with tamoxifen and estrogen. At all doses given, tamoxifen increased epithelial height equal to that of an intact rat,about a six-fold increase over the response seen with raloxifene. Estradiol treatment increased epithelial height to a thickness greater than intact rats.
Estrogenicity was also assessed by evaluating the adverse response of eosinophil infiltration into the stromal layer of the uterus (Table 6). Raloxifene did not cause any increase in the number of eosinophils observed in the stromal layer ofovariectomized rats while tamoxifen caused a significant increase in the response. Estradiol, as expected, caused a large increase in eosinophil infiltration.
Little or no difference was detectable between raloxifene and tamoxifen effects on thickness of the stroma and myometrium. Both agents caused an increase in these measurements that was much less than the effect of estrogen.
A total score of estrogenicity, which was a compilation of all four parameters, showed that raloxifene was significantly less estrogenic than tamoxifen.
TABLE-US-00013 TABLE 5 Bone Density Uterine Weight (mg/cm/cm) (mg) Ovariectomy control 171 .+-. 5 126 .+-. 17 (0.5 mL CMC oral) Intact control 208 .+-. 4 490 .+-. 6 (0.5 mL CMC oral) EE.sub.2 100 .mu.g/kg, oral 212 .+-. 10 501 .+-. 37raloxifene 1 mg/kg, oral 207 .+-. 13 198 .+-. 9 tamoxifen 1 mg/kg, oral 204 .+-. 7 216 .+-. 18
TABLE-US-00014 TABLE 6 Epithelial Stromal Myometrial Stromal Height Eosinophils Thickness Expansion Ovariectomy control 1.24 1.00 4.42 10.83 (0.5 mL CMC oral) Intact control 2.71 4.17 8.67 20.67 (0.5 mL CMC oral) EE.sub.2 100 .mu.g/kg, oral 3.425.17 8.92 21.17 raloxifene 1 mg/kg 1.67 3.17 5.42 14.00 tamoxifen 1 mg/kg 2.58 2.83 5.50 14.17
EXAMPLE 4
Other compounds of formula I were administered orally in the rat assay described in Example 1. Table 7 reports the effect of a 1 mg/kg dose of each compound in terms of a percent inhibition of bone loss and percent uterine weight gain.
TABLE-US-00015 TABLE 7 Compound % Inhibition % Uterine Number of Bone Loss.sup.a Weight Gain.sup.b 2 26 26 6 24 19 8 66 24 10 52 24 11 26 28 12 60 15 14 121 32 16 108 25 18 21 17 27 25 1 34 26 -6 .sup.aPercent inhibition of bone loss = (bonedensity of treated ovex animals - bone density of untreated ovex animals) - (bone density of estrogen treated ovex animals - bone density of untreated ovex animals) .times. 100. .sup.bPercent uterine weight gain = (uterine weight of treated ovexanimals - uterine weight of ovex animals) - (uterine weight of estrogen treated ovex animals - uterine weight of ovex animals) .times. 100.
EXAMPLE 5
Fracture rate as a consequence of osteoporosis is inversely correlated with bone mineral density. However, changes in bone density occur slowly, and are measured meaningfully only over many months or years. It is possible, however, todemonstrate that the formula I compounds, such as raloxifene, have positive effects on bone mineral density and bone loss by measuring various quickly responding biochemical parameters that reflect changes in skeletal metabolism. To this end, in acurrent test study of raloxifene at least one hundred-sixty patients are enrolled and randomized to four treatment groups: estrogen, two different doses of raloxifene, and placebo. Patients are treated daily for eight weeks.
Blood and urine are collected before, during, and at the conclusion of treatment. In addition, an assessment of the uterine epithelium is made at the beginning and at the conclusion of the study. Estrogen administration and placebo serve as thepositive and negative controls, respectively.
The patients are healthy post-menopausal (surgical or natural) women, age 45-60 who would normally be considered candidates for estrogen replacement in treatment for osteoporosis. This includes women with an intact uterus, who have had a lastmenstrual period more than six months, but less than six years in the past.
Patients who have received any of the following medications systematically at the beginning of the study are excluded from the study: vitamin D, corticosteroids, hypolipidemics, thiazides, antigout agents, salicylates, phenothiazines, sulfonates,tetracyclines, neomycin, and antihelmintics. Patients who have received any estrogen, progestin, or androgen treatment more recently than three months prior to the beginning of the study; patients who have ever received calcitonin, fluoride, orbisphosphonate therapy; patients who have diabetes mellitus; patients who have a cancer history anytime any time within the previous five years; patients with any undiagnosed or abnormal genital bleeding; patients with active, or a history of,thromboembolic disorders; patients who have impaired liver or kidney function; patients who have abnormal thyroid function; patients who are poor medical or psychiatric risks; or patients who consume an excess of alcohol or abuse drugs.
Patients in the estrogen treatment group receive 0.625 mg/day and the two raloxifene groups receive dosages of 200 and 600 mg/day, all groups receiving oral capsule formulations. Calcium carbonate, 648 mg tablets, is used as calcium supplementwith all patients taking 2 tablets each morning during the course of the study.
The study is a double-blind design. The investigators and the patients do not know the treatment group to which the patient is assigned.
A baseline examination of each patient includes quantitative measurement of urinary calcium, creatinine, hydroxyproline, and pyridinoline crosslinks. Blood samples are measured for serum levels of osteocalcin, bone-specific alkaline phosphatase,raloxifene, and raloxifene metabolites. Baseline measurements also include examination of the uterus including uterine biopsy.
During subsequent visits to the investigating physician, measurements of the above parameters in response to treatment are repeated. The biochemical markers listed above that are associated with bone resorption have all been shown to beinhibited by the administration of estrogen as compared to an untreated individual. Raloxifene is also expected to inhibit the markers in estrogen deficient individuals as an indication that raloxifene is effective in inhibiting bone loss from the timethat treatment is begun.
Subsequent longer term studies can incorporate the direct measurement of bone density by the use of a photon absorptiometry and the measurement of fracture rates associated with therapy.
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