GB2302021A - Inhibiting bone loss or resorption - Google Patents

Abstract

A method of inhibiting bone loss or resorption in a vertebrate comprising administering to said subject a pharmaceutically effective dose of a compound of formula I wherein R is hydrogen or -OR 4 ; R 1 is optionally substituted C 1 -C 6 alkyl; R 2 is -NH 2 , -N (optionally substituted C 1 -C 6 alkyl) 2 , -NH(optionally substituted C 1 -C 6 alkyl), -NH(OH), NH(aryl), N(aryl) 2 , -SH, -S (optionally substituted C 1 -C 6 alkyl), -S(aryl), OH, or morpholino; R 3 is hydrogen acetoxy, or a hydroxy-protecting group; R 4 is hydrogen, optionally substituted C 1 -C 6 alkyl, aryl, arylaklyl, or optionally substituted C 2 -C 8 acyl; or a pharmaceutically acceptable salt or solvate thereof.

Description

FORMULATIONS AND METHODS FOR INHIBITING BONE LOSS Background of the Invention This invention relates to new methods and formulations for treating vertebrates suffering from diseases associated with increased bone loss or resorption, or fracture repair, involving the therapeutic use of Wortmannin analogs.

The current major diseases or conditions of bone which are of public concern include post-menopausal osteoporosis, hysterectomy patients, senile osteoporosis, patients undergoing long-term treatment of corticosteroids, side effects from glucocorticoid or steroid treatment, patients suffering from Cushings's syndrome, gonadal dysgensis, periarticllar erosions in rheumatoid arthritis, osteoarthritis, Paget's disease, osteohalisteresis, osteomalacia, hypercalcemia of malignancy, osteopenia due to bone metastases, periodontal disease, and hyperparathyroidism.

All of these conditions are characterized by bone loss, resulting from an imbalance between the degradation of bone (bone resorption) and the formation of new healthy bone. This turnover of bone continues normally throughout life and 15 the mechanism by which bone regenerates.

However, the conditions stated above will tip the balance towards bone loss such that the amount of bone resorbed is inadequately replaced with new bone, resulting in net bone loss.

One of the most common bone disorders is postmenopausal osteoporosis which affects an estimated 20 to 25 million women in the United States alone. Women after menopause experience an increase in the rate of bone turnover with resulting net loss of bone, as circulating estrogen levels decrease. The rate of bone turnover differs between bones and is highest in sites enriched with trabecular bone, such as the vertebrae and the femoral head. The potential for bone loss at these sites immediate: allowing menopause is 4-58 per year. The resulting decrease in bone mass and enlargement of bone spaces leads to increased fracture risk, as the mechanical integrity of bone deteriorates rapidly.

At present, there are 20 million people with detectable vertebral fractures due to osteoporosis and 250,000 hip fractures per year attributable to osteoporosis in the U.S. the latter case is associated with a 12% mortality rate within the first two years and 30% of the patients wil require nursing home care after the fracture.

Therefore, bone disorders are characterized by a noticeable mortality rate, a considerable decrease in the survivor's quality of life, and a significant financial burden to families.

Essent-aly all of the conditions listed above would benefit fro treatment with agents which inhibit bone resorption. 3one resorption proceeds by the activity of specialized cells called osteoclasts. Osteoclasts are unique in their ability to resorb both the hydroxyapatite mineral ane organic matrix of bone.

Therapeutic treatments to impede net bone loss include the use of estrogens. Estrogens have been shown clearly to arrest the bone loss observed after menopause and limit the progressior. of osteoporosis; but patient compliance has been poor because of estrogen side-effects. These side effects include resumption of menses, mastodynia, increase in the ris of uterine cancer, and possibly an increase in the risk of breast cancer.

Alterratlvely, calcitonin has been used to treat osteoporotic patients. Salmon calcitonin has been shown to directly ir-ibit the resorption activity of mammalian osteoclasts and is widely prescribed in Italy and Japan.

However, calcitonins are prohibitively expensive to many and appear to be short-lived in efficacy. That is, osteoclasts are able to "escape" calcitonin inhibition of resorption by down-regulating calcitonin receptors.

Therefore, recent clinical data suggests that chronic treatment with calcitonin is ineffective to arrest the post-menopausal loss of bone.

Summary of the Invention The invention provides formulations for and methods of inhibiting bone resorption, bone loss, and facilitating fracture repair and bone healing in a vertebrate comprising administering to said subject a pharmaceutically effective dose of a compound of formula I:

wherein R is hydrogen or -OR4; R1 is optionally substituted C1-C6 alkyl; R2 is -NH2, -N(optionally substituted Cl-C6 alkyl)2, -NH(optionally substituted C1-C6 alkyl), -NH(OH), NH(aryl), N(aryl)2, -SH, -S(optionally substituted C1-C6 alkyl), S(aryl), OH, or morpholino; R3 is hydrogen, acetoxy or a hydroxy-protecting group; R4 is hydrogen, optionally substituted C1-C6 alkyl, aryl, arylalkyl, or optionally substituted C2-Cg acyl; or a pharmaceutically acceptable salt or solvate thereof.

Detailed Descrintion of the Invention The current invention concerns the discovery that certain Wortmannin analogs of formula I are useful in the inhibition of bone loss/resorption, and facilitation of fracture repair and bone healing.

The term "C1-Cg alkyl" represents a straight or branched alkyl chain having from one to six carbon atoms.

Typical straight or branched C1-C6 alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, secbutyl, t-butyl, n-pentyl, isopentyl, and the like. The above C1-C6 alkyl groups may be substituted by one or two halogen, hydroxy, protected hydroxy, amino1 protected amino,C1 to C7 acyloxy, nitro, carboxy, protected carboxy, carbamoyl, carbamoyloxy, cyano, methylsulfonylamino or Cl- C4 alkoxy groups. The substituted alkyl groups may be substituted once or twice with the same or with different substituents.

Examples of the above substituted alkyl groups include cyanomethyl, nitromethyl, hydroxymethyl, triyloxymethyl, propionyloxymethyl, aminomethyl, carboxymethyl, allyloxycarbonylmethyl, allyloxycarbonylaminomethyl, carbamoyloxymethyl, methoxymethyl, ethoxymethyl, tbutoxymethyl, acetoxymethyl, chloromethyl, bromomethyl, iodomethyl, 6-hydroxyhexyl, 2,4-dichloro(n-butyl), 2amino(iso-propyl), 2-carbamoyloxyethyl and the like.

The term "optionally substituted C2-Cg acyl" represents a C1-C7 alkyl group attached to a carbonyl group. Typical acyl groups include propionyl, butylryl, acetyl, valeryl, and caprolyl. Examples of substituents which may be present in the alkyl portion of the acyl group are halogen atoms, for example, chlorine; amino groups, for example dimethylamino; and alkylidene groups such as methylidene. Typical substituted acyl groups include substituted C2-Cg acyl groups such as N, Ndimethylaminopropionyl, acryloyl and chloroacetyl.

The term "aryl" represents an aromatic moiety, such as phenyl, and polynuclear aromatic moieties, such as naphthyl, fluorenyl, anthracyl and phenanthryl, which may be optionally substituted with one or more moieties chosen from the group consisting of halogen, hydroxy, cyano, nitro, C1-C6 alkyl, C1-C4 alkoxy, carboxy, acetyl, formyl, carboxymethyl, hydroxymethyl, amino, aminoethyl or trifluoromethyl. Examples of substituents aryl groups include 4-methylphenyl, 2-methylphenyl, 4-methoxyphenyl, 4 (i-propyl)phenyl, 4-cyclopentylphenyl, 4-(1,1,4,4 tetramethylbutyl ) phenyl, 4-acetylphenyl, 4trifluoromethylphenyl, 4-chlorophenyl, 2-bromophenyl, 3iodophenyl, 6-bromonaphthyl, 3,4-methylene,-dioxyphenyl, indenyl, 1,2,3,4 tetrahydronaphthyl, and 1,2,4,4, tetramethyl-1,2,3,4,-tetrahydronaphthyl.

The term "arylalkyl" represents a C1-C4 alkyl group bearing an aryl group. Representatives of arylalkyls include benzyl, 1-phenylethyl, 2-phenylethyl, 3phenylpropyl, 4-phenylbutyl, 2-methyl-2 phenylpropyl, 4 (chlorophenyl) methyl, (2,6-dichlorophenyl) methyl, (4 hydroxyphenyl) methyl, (2 , 4-dinitrophenyl ) methyl or the like.

Terms such as "protected hydroxy" and "hydroxy protecting group", means hydroxy moieties bonded to conventional groups stable to the reaction conditions in the process aspect of the instant invention. Such groups include the formyl group, the benzhydryl group, the trityl group, the trimethylsilyl group, and the like. Similar hydroxy-protecting groups such as those described by C. B.

Reese and E. islam in "Protective Groups in Organic Chemistry" J. F. W. McOmie, Ed., Plenum Press, New York, N.Y., 1973, Chapters 3 and 4, and T. W. Greene, "Protective Groups in Organic Synthesis", John Wiley and Sons, New York, N.Y., 1981, Chapter 2 shall be recognized as suitable. All that is further required of these groups is that one skilled in the art is able to substitute and remove them from the hydroxy group(s) without disrupting the remainder of the molecule.

Table 1 illustrates a few of the compounds of formula I.

Table 1 Formula Deszanation E R1 R2 R-3 I acetoxy methyl NEt2 H II acetoxy methyl NH2 H III acetoxy methyl NHMe H IV acetoxy methyl NHEt H V acetoxy methyl NHPr H VI acetoxy methyl NHCH2Ph H VII acetoxy methyl NHPh H VIII acetoxy methyl NHOH H IX acetoxy methyl SBu H The use of the compounds of formula I for the described methods is surprising in that, as compared to other Wortmannin analogs, they have relatively low activity as to inhibiting phosphatidylinesitol-3-kinase (PI3kinase), as set out below: Table 2 Compound ICSO(aM) I 80 II > 500 III > 500 IV > 500 V > 500 VI > 500 VII > 500 VIII 152 IX 52 PI3-kinase activity was assayed using purified bovine brain kinase.Inhibitor candidates were initially disolved in DMSO and then diluted 10-fold with 50 mM HEPES buffer, pH 7.5, containing 15 mM MgC12 and 1 mM EGTA. Ten microlitres of this solution was incubated with enzyme (9p1) and Pl (5p1 of a 2 mg/ml stock solution in 50 mM HEPES buffer, pH 7.5 containing 1 mM EGTA). The final reaction mixture contained IC or 250g/ml of compound and 3% DMSO (v:V).

This concentration of DMSO had no effect on Pl 3-kinase activity; control reaction mixtures contained 3% DMSO (v:v) without inhibitor. Reactants were preincubated 5 min at room temperature and then the enzyme reaction was started upon addition of 1 pl[y~32PJATP(2mCi/ml, 500WM stock solution; 0.03 mci/ml, 20AM final concentration). The reaction was allowed to proceed 10 min at room temperature with frequent mixing, after which time the reaction was quenched by addition of 40 Ml of 1N HC1. Lipids were extracted with addition of 80p1 CHCl3:MeOH (1:1, v:v).The samples were mixed and centrifuged, and the lower organic phase was applied to silica gel TLC plate which was developed in CHCl3:MeOH:H2O:NH4OH (45:35:8.5:1.5, v:v).

Plates were dried, and the kinase reaction visualized by autoradiography. The P1 3-monophosphate region was scraped from the plate and quantitated using liquid scintillation spectroscopy. The level of inhibition was determined as the percentage of [32P]-counts per minute compared to controls.

With the low PI3-kinase inhibition activity, not only was the anti-tumor activity thought to be low, but also the usefulness as a bone loss/resorption inhibitor. However, it is believed that the compounds of formula I, upon contacting or being exposed to the acidic environment of bone, have the open furan ring re-formed.

The biosynthetic production of wortmannin is well known in the art and the analogs are synthesized from wortmannin. Typically, wortmannin is produced by the fermentation of any one of a number of previously disclosed and publicly available microorganisms such as Talaromyces wortmannin [Nakanishi, et al., J. Biol. Chem., 267 (4): 2157-2163 (1992)]; and Penicillium wortmannii, Myrothecium roridium, and Fusariwn oxysporum [Abbas, et al., Appl.

Environ. Mcrobiol., 54(5): 1267-1274 (1988)]. Following fermentation, Wortmannin is extracted and purified via known methods.

Preferably, wortmannin is microbially synthesized and isolated in substantially pure form from a fermentation culture identified as A24603.1.

Wortmannin is produced by culturing the abovereferenced A24603.1 strain under submerged aerobic conditions in a suitable culture medium until a recoverable amount of wortmannin is produced. Wortmannin can be recovered using various isolation and purification procedures understood in the art.

The medium used to grow the A24603.1 culture can be any one of a number of media. For economy in production, optimal yield, and ease of product isolation, however, preferred carbon sources in large-scale fermentation are glucose and soluble starch such as corn starch. Maltose, ribose, xylose, fructose, galactose, mannose, mannitol, potato dextrin, methyl oleate, oils such as soybean oil and the like can also be used.

Preferred nitrogen sources are enzyme-hydrolyzed casein and cottonseed flour, although pepsinized milk, digested soybean meal, fish meal, corn steep liquor, yeast extract, acid-hydrolyzed casein, beef extract, and the like can also be used.

Among the nutrient inorganic salts which can be incorporated in the culture media are the customary soluble salts capable of yielding calcium, magnesium, sodium, ammonium, chloride, carbonate, sulfate, nitrate, zinc, and like ions.

Essential trace elements necessary for the growth and development of the organism also should be included in the culture medium. Such trace elements commonly occur as impurities in other substituents of the medium in amounts sufficient to meet the growth requirements on the organism.

For production of substantial quantities of wortmannin, submerged aerobic fermentation in stirred bioreactors is preferred. Small quantities of wortmannin may be obtained by shake-flask culture. Because of the time-lag in production commonly associated with inoculation of large bioreactors with the spore form of the organism, it is preferable to use vegetative inoculum. The vegetative inoculum is prepared by inoculating a small volume of culture medium with the spore form or mycelial fragments of the organism to obtain a fresh, actively growing culture of the organism. The vegetative inoculum medium can be the same as that used for larger fermentations, but other media are also suitable.

Wortmannin is produced by the A24603.1 organism when grown at temperatures between about 23 and 29 C. Optimum temperature for wortmannin production appears to be about 25 C.

As is customary in submerged aerobic culture processes, sterile air is blown into the vessels from the bottom while the medium is stirred with conventional turbine impellors. In general, the aeration rate and agitation rate should be sufficient to maintain a level of dissolved oxygen of at least 45% of air saturation with an internal vessel pressure of about 5 atmospheres.

Following its production, wortmannin can be recovered from the fermentation medium by methods used in the art.

The Wortmannin produced during fermentation of the A24603.1 organism occurs mainly in the broth.

Typically, Wortmannin can be recovered from the biomass by a variety of techniques. A preferred technique involves filtering whole fermentation broth with a ceramic filter. The filtrate is eluted with an organic solvent such as ethyl acetate and concentrated. The concentrate is suspended in alcohol until crystallization occurs and the solution is filtered, washed and dried. For confirmation, the crystalline material is dissolved in an organic solvent and chromatographed on a reverse-phase silica gel absorbent (C8 or C18). Fractions are eluted in an organic-aqueous buffer such as 60% acetonitrile.

Procedure for the Preparation of Compounds of formula I The chemistry used to prepare the compounds of formula I is known to one skilled in the art, and can be found in Haefliger et al., Helv. Chin. Acta., 56, 2901(1973); Haefliger et al., Helv. Chim. Acta., 58, 1620(1975); and Haefliger et al., Helv. Chim. Acta., 58, 1629(1975).

1.00g (2.33 mmole) of Wortmannin was dissolved in 40 mL methylene chloride and stirred at 250C as 2.56 mmoles of the amine was added dropwise. The reaction turned dark orange immediately and was concentrated in vacuo. The resulting orange amorphous solid was recrystallized from 50% ethyl acetate-isooctane to give yellow-orange crystalline solids, whose structures were assigned on the basis of their spectroscopic and analytical data. This procedure was used for the preparation of diethylamino adduct (I), methylamino adduct (II), ethylamino adduct (IV), and n-propylamino adduct (V). Each of these compounds gave acceptable spectroscopic and analytical data (1H NMR, IR, MS, elemental analysis). The spectroscopic data was nearly identical to (VI) in all of these cases.

Compound II. A solution of 25 mg (0.058 mmole) of Wortmannin, 9.0 mg (0.116 mmole) of ammonium acetate and 16 mg (0.116 mmole) of anhydrous powdered potassium carbonate in 2 mL 50% THF-water was stirred at 250C for 30 min. The reaction was poured into 10 mL methylene chloride and the organic phase washed once with water, dried over sodium sulfate and concentrated in vacuo. The resulting solid was recrystallized from 50% ethyl acetate-isooctane to give 21 mg (81%) of a yellow solid (mp 140-1430C).

1H NMR (300 MHz, CDCl3) 60.82(s, 3H), 1.55 (s, 3H), 1.88 (dd, 1H, J=15.1 and 3.3 Hz), 2.03 (s, 3H), 2.20-2.40 (m, 3H), 2.55 (m, 1H), 2.82-2.98 (, 2H), 3.19 (m, 2H), 3.26 (s, 3H), 4.34 (dd, 1H, J=7.2 and 1.2 HZ), 5.80 (bs, 1H), 5.99 (dd. 1H, J=7.9 and 3.2 Hz), 7.14 (s, 1H), 8.61 (dd, 1H, J=15.1 and 8.3 Hz), 9.36 (m, 1H). IR (KBr) 1222, 1580, 1627, 1642, 1687, 1743, 3367 cm-l. MS (FAB) 446 (M++1).

Anal. (C23H27NOg), C, H. N.

Compound fX. To a solutoin of Wortmannin (200 mg, 0.47 mmole) in methylene chloride (2ml) was added n-butanethiol (0.09 ml. 0.75 mmole) and 1 drop of triethylamine. After stirring overnight under nitrogen, another drop of triethylamine was added and the reaction stirred an additional hour. The volatiles were removed in vacuo and the residue chromatographed by radial chromatography (silica gel, 1:1 EtOAc/hexanes) to give 186 mg (77%) of product as a bright yellow-orange solid (mp 88-900C).

1H NMR (300 MHz, CDCl3) 60.82 (s, 3H), 1.55 (s, 3H), 1.88 (dd, 1H, J=15.1 and 3.3 Hz), 2.03 (s, 3H), 2.20-2.40 (m, 3H), 2.55 (m, 1H), 2.82-2.98 (m,2H), 3.19 (m,2H), 3.26 (s,3H), 4.34 (dd, 1H, J=7.2 and 1.2 Hz), 5.80 (bs, 1H), 5.99 (dd, 1H J=7.9 and 3.2 HZ), 7.14 (s, 1H), 8.61 (dd, 1H, J=15.1 and 8.3 Hz), 9.36 (m, 1H). IR (CHCl3) 1197, 1317, 1421, 1625, 1743, and 2976 cm-l. MS (FD) m/e 518 (M+).

Anal. (C27H34OgS), C, H.

For therapeutic treatment of the specified indications, a compound of formula I, may be administered as such, or can be compounded and formulated into pharmaceutical compositions in unit dosage form for parenteral, transdermal, rectal, nasal or intravenous administration or, preferably, oral administration. Such pharmaceutical compositions are prepared in a manner well known in the art and comprise at least one active compound selected from the group consisting of compounds of formula I, ssociated with a pharmaceutically carrier. The term *active compound', as used throughout this specification, refers to at least one compound of formula I or pharmaceutically acceptable salts or solvates thereof.

The compounds are effective over a wide dosage range and, for example, dosages per day will normally fall within the range of from .001 to 10 mg/kg, more usually in the range of from .01 to 1 mg/kg. However, it will be understood that the effective amount administered will be determined by the physician in the light of the relevnt circumstances including the condition to be treated, the choice of compound to be administered, and the chosen route of administration, and therefore the above dosage ranges are not intended to limit the scope of the invention in any way.

In such a composition, the active compound is known as "active ingredient". In making the compositions, the active ingredient will usually be mixed with a carrier, or diluted by a carrier, or enclosed within a carrier which may be in the form of a capsule, sachet, paper or other container. When the carrier serves as a diluent, it may be a solid, semisolid, or liquid material which acts as a vehicle, excipient of medium for the active ingredient.

Thus, the composition can be in the form of tablets, pills, powders, lozenges, sachets, cachets, elixirs, emulsions, solutions, syrups, suspensions, soft and hard gelatin capsules, sterile injectable solutions, and sterile packaged powders.

Some examples of suitable carriers, excipients, and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate alginates, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, tragacanth, gelatin, syrup, methyl cellulose, methyl- and propylhydroxybenzoates, talc, magnesium stearate, water, and mineral oil. The formulations can additionally include lubricating agents, wetting agents, emulsifying and suspending agents, preserving agents, sweetening agents or flavoring agents.

The compositions may be formulated so as to provide quick, sustained, or delayed release of the active ingredient after administratio to the patient by employing procedures well known in the art.

For oral administration, a compound can be admixed with carriers and diluents, molded into tablets, or enclosed in gelatin capsules. The mixtures can alternatively be dissolved in liquids such as 10% aqueous glucose solution, isotonic saline, sterile water, or the like, and administered intravenously or by injection.

The compositions are preferably formulated in a unit dosage form, each dosage containing from about 0.04 to about 900 mg and, more frequently, from about 1 to about 500 mg of the active ingredient. The term "unit dosage form" refers to physically discreet units suitable as unitary dosages for human subjects and other mammals, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, in association with the required pharmaceutically acceptable carrier. By "pharmaceutically acceptable", it is meant the carrier, diluent or excipient must be compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.

The following formulation examples are illustrative only and are ot intended to limit the scope of the invention in any way. The meaning of the term "active ingredient" is as defined above.

Formulation 1 Hard gelatin capsules are prepared using the following ingredients: Quantity (ma/caDsule) Active ingredient 250 Starch, dried 200 Magnesium stearate 10 Total 460 mg Formulation 2 A tablet is prepared using the ingredients below: Quantity (ma/capsule) Active ingredient 250 Cellulose, microcrystalline 400 Silicon dioxide, fumed 10 Stearic acid Total 665 mg The components are blended and compressed to form tablets each weighing 665 mg.

Formulation 3 An aerosol solution is prepared containing the following components: Weiaht Active ingredient 0.25 Ethanol 25.75 Propellant 22 (Chlorodifluoromethane) 70.00 Total 100.00 The active compound is mixed with ethanol and the mixture added to a portion of the propellant 22, cooled to -300C and transferred to a filling device. The required amount is then fed to a stainless steel container and diluted with the remainder of the propellant.The valve units are then fitted to the container Formulation 4 Tablets, each containing 60 mg of active ingredient, are made as follows: Active ingredient 60 mg Starch 45 mg Microcrystalline cellulose 35 mg Polyvinylpyrrolidone (as 10% solution in water) 4 mg Sodium carboxymethyl starch 4.5 mg Magnesium stearate 0.5 mg Talc 1 ma Total 150 mg The active ingredient, starch and cellulose are passed through a :;o. 45 mesh U.S. sieve and mixed thoroughly. The aqueous scution containing polyvinyl- pyrrolidone is mixed with the resultant powder, and the mixture then is passed through a No. 14 mesh U.S. sieve. The granules so produced are dried at 500C 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 each weighing 150 mg.

Formulation 5 Capsules, each containing 80 mg of active ingredient, are made as follows: Active ingredient 80 mg Starch 59 mg Microcrystalline cellulose 59 mg Magnesium stearate 2 ma Total 200 mg The active ingredient, cellulose, starch and magnesium stearate are blended, passed through a No. 45 mesh U.S. sieve, and filled into hard gelatin capsules in 200 mg quantities.

Formulation 6 Suppositories, each containing 225 mg of active ingredient, are made as follows: Active ingredient 225 mg Saturated fatty acid 2.000 ma glycerides Total 2,225 mg The active ingredient is passed through a No. 60 mesh U.S. sieve and suspended in the saturated fatty acid glycerines previously melted using the minimum heat necessary. The mixture is then poured into a suppository mold of nominal 2 g capacity and allowed to cool.

Formulation 7 Suspensions, each containing 50 mg of active ingredient per 5 ml dose, are made as follows: Active ingredient(s) 50 mg Sodium carboxymethyl cellulose 50 mg Syrup 1.25 mL Benzoic acid solution 0.10 mL Flavor q.v.

Color q.v.

Purified water to total 5 mL The active ingredient 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 a portion of the water and added, with stirring. Sufficient water is then added to produce the required volume.

Formulation 8 An intravenous formulation may be prepared as follows: Active ingredient 100 mg Isotonic saline 1,000 mL Drugs which prevent bone loss and/or add back lost bone may be evaluated in the ovariectomized rat. This animal model is well established in the art (see, for example, Wronski, et al. (1985) Calcif. Tissue Int 37:324328; Kimmel, et al. (1990) Calcif. Tissue Int. 46:101-110; and Durbridge, et al. (1990) Calcif. Tissue Int. 47:383387; these references are hereby incorporated in their entirety). Wronski, et al. ((1985) Calcif. Tissue Int.

43:179-183)) describe the relationship between bone loss and bone turnover in the ovariectomized rat model.

Also established in the art is the acute rat in vivo model assay (Thompson et al., Endocrinology, 97 pp. 283-289 (1975); Rasmussen, Calc. Tiss. Res., 23, pp. 87-94 (1977); Sammon et al., Am. J. of Physiology; 218, No. 2, pp. 479485 (1970); Hargis et al., Endo. 94, No. 6 pp. 1644-1649 (1974); Thomas et al., Bone and Mineral, 4, pp. 73-82 (1988); and Au et al., Am. J. Physiol., 209, No. 3, pp.

637-642 (1955)).

Osteoclast precursors were isolated from the medullary bone of egg-laying hens (dekalb XL) which were maintained on a calcium deficient diet for 2-4 weeks (Alvarez et al., 1991, Endocrinology 128:2324-2335). The bone cell suspension was passed through a ll0im nylon mesh, and incubated in erythrocyte lysing buffer (Sigma Chemical Co., St. Louis, MO) for 1 min to lyse red blood cells. Cells were collected by centrifugation at 350xg for 70 min at 40C, layered over a discontinuous Nycodenz gradient (Accurate chemical & Scientific Corp., Westbury, NY) (1.073, 1.099, 1.143 g/cm3), and then centrifuged at 350xg for 20 min at 40C.Cells from the monocyte band were pooled and resuspended in a-MEM, pH 7-7.2, 10% serum, antibiotics, and 5pg/ml cytosine-1-B-D-arabinofuranoside (Sigma Chemical Co., St. Louis, MO) at 40C and plated onto serum coated dishes (Costar, Cambridge, Mass.). After 24 hr culture at 390C andf 5% CO2/air, non-adherent cells were removed by washing with PBS, while adherent monocytes were harvested with cell dissociation buffer (Gibco Laboratories). Monocytes were re-plated at 3 x 105/cm2 onto culture pates (Costar) and cultured for an additional 6 days.

Bone resorbing activity was measured by quantitating the [3H] release into the media from bone particles (20-53 Rm) labeled with [3H] proline (Amersham Corp., Arlington Heights, IT), as described earlier (Blair et al., 1986, J.

Cell Biol 102:1164-72). Hen monocytes were plated on 48well dishes (Costar) or glass (Nunc) and incubated between days 5-7 with 100 Rg/cm2 bone particles, that were labeled in vivo with [3H] proline as detailed previously (Blair et al., 1986, J. Cell Biol 102:1164-72). Radioactivity released into the media was measured by liquid scintillation counting (LKB Instruments, Inc., Gaithersburg, MD). Standard curves to convert dpm to Fg bone were generated by measuring radioactivity in samples ashed in a Packard 306 oxidizer (Packard Instruments, Sterling, VA).

Table 3 Effect of Compounds A and B on Bone Resorption Activity Compound A Compound B Control 100 100 10-6 23+1 29+2 10-7 38i2 43+6 Concentration: 10-8 68+4 76+5 10-9 ~ 93t4 Compound A and Compound B have the following structures:

Resorption activity is expressed as percent control, with control leels (1008) corresponding to 23-31Rg of bone resorbed. Data are meant standard deviation for n=3-6.

These resorption experiments with isolated osteoclasts were shown to be integral to a mechanistic understanding of another resorption inhibitor, biosphosphonates (Fleisch, H.

1987, Bone 8(S1):S23-S28; Sato and Grasser, 1990, J. Bone Min. Res. 5:31-40 Sato et al., J. Clin. Invest. 88:20952105). Biosphosphonates, and in particular amino hydroxybutylidene Bisphosphonate, were shown to be therapeutically efficacious in treating Paget's disease (Pedrazzoni et al., 1989, Bone Miner. 7:301-307; O'Doherty et al., 1990, J. Bone Miner. Res. 5:483-491); hypercalcemia of malignancy (Ralston, et al., 1989, Lancet ii:1180-1182; Bilezikian J, 1992, N Engl J Med 326:1196-1203); osteolytic lesions due to metastases (Attardo-Parrinello et al., 1987, Arch. Intern. Med. 147:1629-1633); steriod and glucocorticoid induced osteoporosis (Reid, et al., 1988, Lancet i:143-146; Reid et al., 1990, J. Bone Miner. Res.

5:619-623); and postmenopausal osteoporosis (Watts et al., 1990, N Engl J Med 323:73-79).

Six month old, virgin Sprague-Dawley female rats (Harlan, IN) weighing about 270g are maintained on a 12 hr light/dark cycle at 220C with ad lib access to food (TD 89222 with 0.5% Ca and 0.4% P, Teklad, Madison, WI) and water. Bilateral ovariectomies are performed, except for SHAM controls, at 6 months of age. Rats are grouped into treatment units of n=6 and orally dosed daily for 35 days (from day 4-38 post-surgery) to include: 1) sham-operated control (SHAM), 2) ovariectomized control (OVX), 3) OVX treated with the Compound A or B. SHAM and OVX control rats are administered 100 ul/lOOg body weight of 20% w/v hydroxypropyl-S-cyclodextrin (Aldrich Chemical Co., Milwaukee, WI) by gavage.Treated rats are given agents dissolved in 20% hydroxypropyl-B-cyclodextrin in a total volume of 100 ul/lOOg of body weight at doses ranging from 0.001-1 mg/kg,'day by gavage. All animal procedures are reviewed before implementation by an internal animal welfare committee, to ensure compliance with NIH guidelines.

After 35 days of treatment, rats are anesthetized with ketamine HCL (120 mg/kg): xylazine HC1(24 mg/kg) and blood is collected by cardiac puncture. The animals are then asphyxiated by C02 inhalatoin. Uteri, and femora are rapidly removed. A 960 pQCT (Norland/Stratec, Ft. Atkinson, WI) is used to analyze distal and mid femora cross-sectionally. Tissue parameters of cross-sectional area (X-Area, mm2), volume, mineral content (TMC, CaP04 content in mg), and mineral density (TMD, CaP04 concentration in mg/cm3) are quantitated for intact femora, using Dichte software version 5.1. Condyles are used as positioning aids for the distal femora, and a voxel size of 0.149 x 0.149 x 1.0 mm is used. Using hydroxyapatite phantoms and different regions of a COMAC European Forearm phantom, an accuracy of 9% (TMD, mg/cm3 hydroxyapatite) is derived for this instrument, due to an internal, constant offset value of 59.3 mg/cm3. However, a precision of 1% (TMD) is calculated for the QCT by averaging the coefficient of variation (variability) as defined by standard deviation/mean for repositioned femora.

Claims (5)

We claim:

1. The use of a compound of formula I

wherein R is hydrogen or -OR4; R1 is optionally substituted C1-C6 alkyl; R2 is -NH2, -N (optionally substituted C1-C6 alkyl)2, -NH(optionally substituted C1-C6 alkyl)1 -NH(OH), NH(aryl), N(aryl)2, -SH, -S(optionally substituted C1-C6 alkyl), -S(aryl), OH, or morpholino; R3 is hydrogen, acetoxy, or a hydroxy-protecting group; R4 is hydrogen, optionally substituted C1-C6 alkyl, aryl, arylalkyl, or optionally substituted C2-Cg acyl; or a pharmaceutically acceptable salt or solvate thereof for the manufacture of a medicament for inhibiting bone loss or resorption in a vertebrate.

2. The use of Claim 1 wherein said subject is human.

3. The use of Claim 2 wherein said human has or is susceptible to osteoporosis.

4. The use of Claim 1 wherein R is acetoxy; R1 is methyl; R2 is -NH2 or -NHCH2; and R3 is hydrogen.

5. A pharmaceutical formulation comprising a compound of formula 1, and optionally one or more excipients diluents, or carriers.