CA2042960A1 - Method of inhibiting demineralization of bone - Google Patents

Abstract

Abstract A method of inhibiting bone demineralization comprising administration of an effective amount of a compound having the formula where X is CH or N;
Y is O or S;
R1 is 2-propenyl, C1-C3 alkyl. benzyl or substituted benzyl where the substitutents are one or two of the same or different and are selected from methyl, ethyl, methoxy, ethoxy, hydroxy, chloro, bromo, or fluoro;
R2 is C2-C3 alkyl, allyl or cyclopropylmethyl;
R3 and R4 are both hydrogen or combine to form a carbon-carbon bond; and pharmaceutically acceptable acid addition salts thereof to a mammal requiring bone demineralization inhibition.

Description

Title METfIOD OF INHIBlTlNG DE~lINERALlZATlON OF BONE

S The present invention relates to the use of certain ergoline analogues to inhibit bone deminera1ization.

Backgroulld of the Invention Mammalian bones serve two different and at times incompatible functions. Bones must be strong, light, and be capable of repair and remodeling in response to changing stress.
Bones also serYe as a metabolic reservoir ~or most of the calcium and phosphorous in the body, along wi~h substantial amounts of magnesium, sodium, and carbonate.
Bone is a highly specialized connective tissue with unique properties derived ~rom its extensive matrix structure. A
network of fibrous bundles composed of the protein collagen is presumed to provide the tension-resistant behavior of bone. In 2 0 addition, other material including proteoglycans, noncollagenous proteins, lipids, and acidic proteins associated with a mineral ; compo1md consisting primarily of hydroxyapatite, Calo(P04) 6(OH)~ are deposited in the extensive matrix architecture of bone. Bone tissue is continuously renewed and 2 S remodeled throughout the li~e of mammals.
The processes of bone formation and renewal are carried out by specialized cells. Osteo~enesis is presumably carried ou~ by the "osteoblasts" (bone-forming cells). Remodeling of bone is apparently brought about by an interplay between the '`' ., .

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X-7020A -2- ~29~

activities of bone-resorbing cells called "osteoclasts" and the bone-forming osteoblasts. The bony skeleton is thus not only an architectural structure with a mechanical function but also is a living tissue capable of growth~ modeling, remodeling and repair.
Since these processes are car~ed out by specialized living cells, chemical (pharmaceutical/hormonal), physical and physiochemical alterations can affect the quality, quantity, and shaping of bone tissue.
As part of their storage function~ bones provide calcium and phosphorous when the supply of these elements is deficient. The sodium, phosphorous, ~nd carbonate are also released to buffer excess acid in the diet. These responses preempt the structural func~ions of growth and remodeling such that bone mass rnay decrease due to calcium or phosphorous l S deficiency or with chronic acidosis.
"E~one demineralization," includes loss of both mineral and protein matrix components, resulting in a reduction in bone mass without a reduction in bone volume. Such deminerali~ation occurs in a wide range of subjects, including post-menopausal 2 0 women, paeients who are undergoing or have undergone long-term administration of corticosteroids, patients suffering from Cushing's syndrome, and in patients having gonadal dysgenesis.
Unchecked, the bone demineralization process leads to osteoporosis~ a condition characterized by decrease in bone mass 2 5 (decreased density and enlargement of bone spaces) without a reduction in bone volume producing porosity and fragility. It is estimated that thirty-five percent of wome~ over the age of 65 suffer from osteoporosis. One of the chief rationales ~r estrogen-replacement therapy in post-menopausal women is 3 0 avoidance of, or at lea~t slowing down, the bone demineralization process and the onset of osteoporosis. Calcium therapy has been advoca~ed for treatment of osteoporosis, and some steroids related to vitamin D are alleged to reduce the rate of bone resorption. See U.S. Patenl Nos. 4,448,721 and 4,2û1,881.
The ergoline ring is a te~racycle having the following structure:

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20~X9~0 ' ' ~ I' HN

Certain substituted ergolines are known to be D-2 dopamine a~onists having the ability to inhibit the secretion of prolactin and to af~ect favorably the symptoms of Parkinson's Syndrome. For example. in the foregoing structure when R is n-propyl, Rl is methylthiomethyl, and R" is H, the substituted ergoline has been given the generic name pergolide, which is disclosed in U.S. Patena No. 4~166,182. Pergolide has been proven to be effective in the treatment of some symptoms of Parkinsonism, and is being developed as the mesylate salt.
An object of this invention is to provide a novel l ~ therapeutic approach to the inhibition of bone demineralization ! ~ ~ and, by consequence. of osteoporosis~ by administering certain ergoline compounds.

Summar~ of the Invention :~:
In fulfillment of the above and other objects, this 2 0 invention provides a method of inhibiting bone demineralization, which comprises administering to a mammal in need of treatment, a bone demineralization inhibiting dose of a compound selected from: 8~-~(methoxy or methylthio)methyl]-8-ergolines : and 8,0-[(methoxy or methylthio)methyl]-8-ergolenes and their 2-~: 2 5 aza analogues and pharmaceutically acceptable acid addition salts thereof.
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'~' , ' '' ~' ' X-7020A 4 20~1?~6(3 Detailçd_De~ of the Invention Ergot compounds are well known in the art and have been identified as possessing a number of pharmacological properties including inhibition of fertility, uterotonic activity, influence on body tempe~ature, emetic activity, dopaminergic activity, inhibition of prolactin secre tion and the like. Ergot compounds can be divided into four main structural groups:
clavine alkaloids~ Iysergic acids, Iysergic acid amides, and peptide alka1Oids. See 13erde and Schild, Eds. "Ergot Alkaloids and Related Compounds" Sprirlger-Verlag, New York, 1978, pp. 1-61.
Pergolide is the generic name that has been given to the substituted ergoline D^6-n-propyl-8~-methylthiomethylergoline which is disclosed in U.S. Patent No.
I 5 4,166.182. Example 1. U.S. Patent 4.166~182 is incorporated by reference herein in its entirety. Pergolide has been proven to be effective in the treatment of some symptoms of Parkinsonism~
and is being developed as the mesylate salt.
The method of treatment provided by this invention is ~0 practiced by administer~ng to a mammal in need of bone demineralization inhibition a dose of a compound selected from 8~-[(methoxy or methylthio)methyl]ergolines and 8~-[(methoxy or methylthio)methyll-8~ergolenes and 2-aza analogues of said ergolines and ergolenes and pharmaceutically acceptable acid 2 5 addition salts thereof, that is effective to inhibit bone demineralization. The bone demineralization inhibition contemplated by the present method includes both medical therapeutic and/or prophylactic treatment, as appropriate.
Generally, the compound is formulated with common excipients, 3 0 diluents or carriers~ and compressed into ~ablets, or formulated as elixirs or solutions for convenient oral administration. It can also be formulated for parerlteral administration, for instance by the intramuscular or intravenous routes. The compounds can additionally be administered transdermally, and are well suited 3 5 to formulation as sustained release dosage forms and the like.
Pharmaceutically aceeptable acid addition salts of the compounds employed in this invention include salts derived from non-toxic inorganic acids such as: hydrochloric acid, nitric acid, ''' , .

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X-7020A 5~ 2~29~

phosphoric acid. sulfuric acid~ hydrobrornic acid~ hydroiodic acid.
and others. as well as salts derived from non-toxic organic acids such as aliphatic mono and dicarboxylic acids, phenyl-substituted alkanoic acids. hydroxyalkanoic and hydroxyalkandioic acid~
5 aromatic acids, aliphatic and aromatic sulfonic acids. Such pharmaceutically acceptable salts thus include sulfate, pyrosulfate~ bisulfate. sulfite, bisulfite, nitrate, phosphate.
monohydrogenphosphate, dihydrogenphosphate, metaphosphate~
pyrophosphate, chloride, bromide, iodide, acetate, propionate, 10 caprylate, acrylate, formate, isobutyrate, caprate, heptanoate, propiolate, oxalate, malonate, succi~ate, suberate. sebacate, fumarate, maleate. mandelate, butyne- I ,4-dioate, hexyne- 1,6-dioate, benzoate, chlorobenzoate~ methylbenzoate, dinitrobenzoate. hydroxybenzoate, methoxybenzoate, phthalate, 15 terephthalate, benezenesulfonate, toluenesulfonate.
chlorobenezenesulfonate. xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, laetate~ ,B-hydroxybutyrate, glycollate, malate, naphthalene- I -sulfonate9 naphthalene-2-sulfonate and mesylate. In addition, some of these 2 0 salts may form solvates with water or organic solvents such as ethanol. l~hese solvates are also included as compounds of this invention .
The compounds employed in the method of the present inveneion comprises 8~-1(methoxy or methylthio)methyl3ergolines 2 5 or 8,B-~(methoxy or methylthio)methyl]-8-ergolenes and their 2 aza derivatives having the formula R3 ~,CH2~ CH3 R4 >~
r `' 1' R1_N~ X

,, ' X-7020A -6- 20429fi)~

where X is CH or N:
Y is O or S:
R l is 2-propenyl, C 1- C 3 alkyl, benzyl or substituted benzyl where the substitutents are one or two of the same or different and are selected from methyl, ethyl, methoxy, ethoxy, hydroxy, chloro, bromo. or fluoro;
R2 is C~C3 alkyl, allyl or cyclopropylmethyl;
R3 and R4 are both hydrogen or combine to form a l O carbon-carbon bond; and pharmaceutically acceptable acid addition salis thereof.
Preferred compounds of Formula I are those where:
X is C~;
Y is S:
Rl is hydrogen or isopropyl;
R.2 is n-propyl;
R3 and R4 are both hydrogen: and pharmaceutically acceptable acid addition salts thereof.
Methods of preparing the compounds of Folmula I and 2 0 their precursors are taught in U.S. Patent Nos. 4,166,182 and 4,675,322, and in Bach, et al., J ed._Chem., 23, 492-494 (1980) all of which are incorporated by re~erence herein in their entirety. Modifications to the above methods may be necessary to accommodate reactive functionalities of particular substituents.
Such modifications would be both apparent, and known or readily ascertained, by those skilled in the art.
The pharmaceutically aceeptable acid addition salts of the invention are typically ~ormed by reacting a compound of Formula I with an equimolar or excess amount of acid. The 3 0 reactants are generally combined in a mutual solvent such as diethyl ether or benzene, and the salt normally precipitates out of solution within about one hour to 10 days, and can be isolated by f~ltration or the solvent is stripped o~f by conventional means.
Pharmaceutical formulations useful with compounds of Formula I
are disclosed in U.S. Patent No. 4,166,182.
l'he particular dosage of a compound of Formula I
required to treat or inhibit bone demineralization according to this invention will depend upon the severity of the disease, its .. ,~ . ~ . . . . .

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roule of administration, and related factors that will be decided by the attending physician. Generally, accepted and ef~ective doses will be from about 0.01 to about 10 mg, and more typically from about 0.1 to about S mg. Suc;h dosages will be administered 5 to a subject in need of treatment from once to about four times each day, or more often as needed to ef~ectively inhibit the bone demineralization process.
As used herein~ the term "inhibiting bone demineralization" should be understood as meaning prev~nting the 10 decrease in bone mass witll a static bone volume. This prevention may comprise a complete arresting of the demineralization process or a decrease in the progression of the demineralization process.
Pergolide was evaluated for inhibition of bone 15 demineralization in the following test:
Pergolide, was added to a rat diet at the .001% by weight level. Groups of nine or ten rats, age three months, were put on the following diets, one group per diet: (I) a pergolide containing diet, ad libitum; (2) an L-DOPA containing diet ad 20 libitum; and (3) pair fed, i.e., same feed consumption as pergolide-containing diet. After 18 months, although there were insufflcient numbers of rats evaluated upon which to base statistically significant conclusions, pergolide appeared to be inhibiting bone demineralization; that is, there was higher bone 2 5 density (glcm~ (obtained by dividing bone mineral content (g/cm) by bone width (cm)), between drug treated and the control group (Table 2). At 24 months, however, statistically significant differences were obtained, as set forth in Table 1. In Table 1, column I gives the diet; column 2, bone mineral content (BMC);
3 0 column 3, bone width (BW); and column 4, bone density (BMCIBW), ~column 2 divided by column 3).

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Diet ~C E~W BMC/BW

Pair fed .07656 ~ .00419 .3770 i .0088.2033 * .0129 control .û01% .08733 + .00100.3703 :~ .0116 .2368 -~ .0346 pergolide L-DOPA .0822 + .00691 .3792 i .0210 .2173 :t .0215 l O
Table 2 below gives the test results of the above evaluation conducted with pergolide where the measuremen~s were made after 18 months oi age. The data. particularly Table 1, establishes the ability of the ergot compound pergolide to l 5 inhibit bone demineralization.

TablQ 2 Diet~ 8MIC BW BMCIBW
Pair fed .08025 i .00096 .3745 i: .0075.2145 i .0019 control .001% .08700 + .00572 .3605 i .0088.2415 i .0195 pergolide 2 5 L-DOPA .07900 i .00424 .3540 i .0137.2235 ~t .0095 It is apparent from the above data in Table 1 that rats ~: on the pergolide-containing diet had statistically significant (95~ confidence level or better) greater bone clensity than pair-3 0 fed control rats.
Pergolide, was administered to the rats at a rate of 0.5 mg/kg of mammalian body weight. For a 5(~-100 kg human, the colTesponding dose level would be about 25-50 mg/day.
However, since humans are more sensitive to the pharmacologic 3 5 actions of pergolide than are rats, dose levels in the range 0.1-10 mg of pergolide per day by the oral route would, in most instances, preven~, 03 a least slow the process of, bone : :
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X-7020A -9- 20~2.~60 demineralization. a universal part of the aging process in mammals.
The compound D-l-isopropyl-6-n-propyl-8,B-methylthiomethyl ergoline was evaluated for itS ability to inhibit bone demineralization in an ovariectomized rat model according to the following procedures. Briefly, this model uses ovaricctomized rats which show a significant degree of trabecular bone loss at four weeks following the removal of the ovaries. Typically, ~-estradiol will completely protect the bones from the ovar~ectomy-induced lesion.
Animals. Seventy-five day old, female Sprague Dawley rats (weight range of 225 to 275 g; Charles River, Portage, Ml) were used in these studies. Ovar~ectomies (or a sham surgical procedure for controls) were performed by the vendor. The animals were shipped one week post-surgery and housed in hanging wire cages in groups of four. Room temperature was maintained at 22.2 + 1.7C with a minimum relative humidity of 40%. The photoperiod in the room was 12 hours light and 12 hours dark, with light onset at 0600 hour. The animals had ad lib 2 0 access to food (Teklad diet, TD 89222, 0.5% calcium, 0.4%
phosphorous; Madison, Wl) and wa~er. The animals were allowed one week to acclimate to these conditions prior to experimental rnanipulation.
Materials. Suspensions of D-l-isopropyl-6-n-propyl-2 5 8~-methylthiomethyl ergoline as the hydrochloride salt were prepared in 1% carboxymethylcellulose (CMC). One percent CMC
injections were used as the control vehicle. ,B-Estradiol (obtained from Sigma Chemical Co.; St. Louis, MO) was suspended in a 20% polyethylene glyeol vehicle and was employed as an 3 0 internal standard for these studies.
Protocol. At the end of the one week acclimatization period (therefore, two wee~s post-ovariectomy), dosing with test compounds was initiated. Subcutaneous injections of 1% CMC, D-l-isopropyl-6-n-propyl-8,B-methylthiomethyl ergoline (0.1 to 3 3 5 mg/kg) or ,B-estradiol (100 llg/kg) were delivered daily for two consecutive weeks. On the day following the fourteenth dose (four weeks post-ovarigctomy), the animals were asphyxiated '~ ~ , ., . . . ~ ,.

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X-7020A - l 0-with carbon dioxide~ body weight and uterine weight were recorded~ and the left femur was removed and frozen (-20C)~
Bone Assays. Single photon absorptiometry was perfonned on the left femur using a ~lorland 2780 x-ray 5 densitomer. Triplicate readings were made at the proximal aspect of the patellar groove. Bone mineral density was calculated as a i~unction of the bone mineral content and bone width at the point of measurement.
Statistics. Each experimental group consisted of 8-I O 16 animals. Data for control and treated rats were compared by one way analysis of variance (ANOV~). When statistical significance was indicated (p c 0.05) post-hoc range testing was performed (Scheffe F test).

1 5 Table_3 Group Bone Mineral Densitva Control 100.0 i 7.6 2 0 Ovariectomy 0.6 i S.3 ~-Estradiol (100 ~lg/kg)70.8 ~t 8.8b 0.01 mg/kg 29.2 :: 10.7 0.03 mg/kg 37.8 i 7.3b 2 5 0.1 mg/kg 37.9 :t 5.6b 0.3 mglkg 50.3 :t 3.7b 1.0 mg/kg 53.4 ~ 8.6b 3.0 mg/kg 47.8 i 6.7b 30 a Bone mineral density values are presented as a percent protection (i Standard Error of Mean (SEM)) from loss induced four weeks after ovariectomy. ~ protection =
[(bmctesr~m~ov~dl(bmccontro~bmcov~] x 100 3 5 b p < 0.05 vs. ovariectQmy group It is advantageous to administer a compound of Formula I by the oral route to an aging mamrnal (e.g., a pos~-.

X-7020A ~ 2 0 ~ S O

menopausal female) mammal or to any aging male showing evidence of bone demineralization by x-ray. For such pulposes.
the following oral dosage forms are available. "Active ingredient" means a compound of Formula 1.
S Hard gelatin capsules are prepared using the followirlg ingredients:

~)uantlty (mg/capsule!

Ac~ive ingredient 0.01-10 mg Starch dr~ed 200 Magnesium s~earate 10 The above ingredien~s are mixed and filled into hard 15 gelatin capsules.
A table formulation is prepared using the ingredients below:

Ouantity (rn~/capsule) Active ingredient 0.01-10 mg Cellulose. microcrystalllne400 Si1icon dioxide, fumed 10 Steara~e acid 5 The components are blended and compressed to form tablets.
Alternatively, tablets each containing .1-10 mg of active ingredient are made up as follows:

3 0 Active ingredient 0.01-10 mg S tarc h 45 Il~g Microcyrstalline cellulose 35 mg Polyvinylpyrrolidone (as 10% solution in water) 4 mg 3 5 Sodium carboxymethyl starch 4.5 mg Magnesium stearate 0.5 mg Talc 1 mg .

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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.
5 The granules so produced are dried at 50-60C and passed through a No. 18 mesh U.S. sieve. The sodium carboxymethyl starch, magnesium stearate and talc, previously passed through a No. 60 mesh U.S. sieve. are then added to the granules which, after mixing, are compressed on a tablet machine to yield tablets.
(:apsules each containing 0.01-10 mg of medicament are made as follows:

Active ingredient 0.01-10 mg S tarc h 59 mg Microcrystalline cellulose 59 mg Magnesium stearate The active ingredient, cellulose, starch~ and magnesium stearate are blended, passed through a No. 45 mesh 2 0 U.S. sieve, and filled into hard gelatin capsules.
Suspensions each containing 0.01-10 mg of medicament per 5 ml dose are made as follows:

Active ingredient 0.01-10 mg 2 5 Sodium carboxymethyl cellulose Sû 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 carboxymethylcellulose and syrup to form a smoo~h paste. The benzoic acid solution, flaYor, 3 5 and color are d;luted with some of the water and added, with stirring. Sufflcient water is then added to produce the required volume.

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X-7020A -13- 2~42960 It should be understood that the instant specification and examples are set forth by way of illus~ration and not limitation~ and that various modifications and changes may be made without departing from the spirit and scope of the present S invention as defined by the appended claims.

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Claims (7)

1. A process for preparing a pharmaceutical formulation adapted for inhibiting bone demineralization in mammals comprising admixing a pharmaceutically acceptable carrier, diluent or excipient with a compound having the formula ¦

where X is CH or N;
Y is O or S;
R1 is 2-propenyl, C1-C3 alkyl, benzyl or substituted benzyl where the substitutents are one or two of the same or different and are selected from methyl, ethyl, methoxy, ethoxy, hydroxy, chloro, bromo, or fluoro;
R2 is C2-C3 alkyl, allyl or cyclopropylmethyl;
R3 and R4 are both hydrogen or combine to form a carbon-carbon bond or pharmaceutically acceptable acid addition salts thereof.

2. The process according to Claim 1 where X is CH;
Y is S;
R1 is hydrogen or isopropyl;
R2 is n-propyl; R3 and R4 are both hydrogen, or pharmaceutically acceptable acid addition salts thereof.

X-7020A - (EPO) -15-

3. The process according to Claim 2 in which the mammal has been diagnosed as suffering from osteoporosis.

4. The process according to Claim 3 in which the mammal is a post-menopausal female mammal.

5. The process according to Claim 3 in which the dose of said compound is 0.01 to 10 mg/day.

6. The process according to Claim 2 where said compound is pergolide or a pharmaceutically acceptable acid addition salt thereof.

7. The process according to Claim 2 where said compound is D-1-isopropyl-6-n-propyl-8.beta.-methylthiomethyl ergoline or a pharmaceutically acceptable acid addition salt thereof.