PH27078A - Pharmaceutical compositions containing geminal diphosphonates - Google Patents

Description

PHARMACEUTICAL COMPOSITIONS CONTAINING §

GEMINAL DIPHOSPHONATES 5

TECHNICAL FIELD &

This invention relates to pharmaceutical compositions con’; taining compounds which are useful in treating or prevent ing ddséases characterized by abnormal calcium and phos- - phate metabolism, in particular those which =re character] ized by abnormal bone metabolism. This invention further § relates to a method of treating or preventing diseases i . i characterized by abnormal calcium an phosphate netabolion using pharmaceutical compositions of the present. invention; \ I ~. 5

BACKGROUND OF THE INVENTION : 1

A number of pathological conditions which can afflict wk blooded animals involve abnormal calcium and phosphate met; - x bolism. Such conditions may be divided into two broad cath- el gories. £ Co oe cry) ’ , . oNAL f

BAD ORI os 1. conditions which are characterized by anomalous mobi- lization of calcium and phosphate leading to general or ; specific bone loss or excessively high calcium and phosphate levels in the fluids of the body. Such conditions are somé- times referred to herein as pathological hard tissue demi-. neralizations. : 2. conditions which cause or result from deposition of . calcium and phosphate anomalously in the body. These con-" ditions mre sometimes referred to herein as pathological i calcifications, t

The first category includes osteoporosis, a condition in i which bone hard tissue islost disproportionately to the £ development of new hard tissue. Marrow and bone spaces i become larger, fibrous binding decreases, and compact boned becomes fragile. Osteoporosis can be subclassified as & menopausal, senile, drug induced (e.g. adrenocorticoid, & as can occur in steroid therapy), disease induced (e.g.. by arthritic and tumor), etc., however, the manifestations # are essentially the same. Another condition in the first § category is Paget's disease (osteitis deformans). In this} disease, dissolution of normalbbone occurs which is then 1 haphazardly replaced by soft, poorly mineralized tissue ; such that the bone becomes deformed from pressures of weight bearing, particularly in the tibia and femur. Hyperpara- 7 thyroidism, hypercalcemia of malignancy. and osteolytic ; bone metastases are conditions also included in the eiret category. ; :

The second category, involving conditions manifested by anomalous caleium anddphosphate deposition, includes myo- & sitis ossificans progressiva, calcinosis universalis, ana

CTE

BAD ORIGINAL :

oo pets auch afflictions as arthritis, neuritis, bursitis, tendo- nitis and other inflammatory conditions which predispose : involved tissue to deposition of calcium phosphates. x polyphosphonic acids and their pharmaceutically-acceptable salts have been proposed for use in the tireatment and prof phylaxis of such conditions. In particular aiphosphonates; like ethane-1l-hydroxy-1,1-diphosphonic acid (EHDP), propane- 3-amino-1l-hydroxy-1,l-diphosphonic acid (APD), and dichloro- methane diphosphonic acid (cl,MDP) have been the subject of considerable research efforts in this area. Paget's disease and heterotopic ossification are currently success= fully treated with EHDP. The diphosphonates tend to inhi-. ) bit the resorption of bone tissue, which is beneficial to % patients suffering from excessive bone loss. However, ;

EHDP, APD and many other prior art diphosphonates have eng propensity of inhibiting bone mineralization when adminis¥ "tered at high dosage levels.

It is believed that mineralization inhibition is predomi-} ¥ nantly a mass related physico-chemical effect, whereas i - 20 resorption inhibition results from a biological interaction with the cells. It im therefore desirable to develop nocd bisdlogically potent diphosphonate compounds that can be acl ministered at low dosage levels which cause little or no g mineralization inhibition, thereby resulting in a wider i margin of safety. Lowdidosage levels are also desirable 3 avoid the gastro-intestinal discomfort (like diarrhea) softe- times associated with oral administration of large quantities of diphosphonates. E

It is therefore an object of thsiinvention to provide Ng potency compositions for the treatment and prophylaxis o ao BAD ORIGINALE : TE gr abnormal calcium and phosphate metabolism. It is a still ff further object of this invention to provide an improved 5 method for treating diseases characterized by abnormal i calcium and phosphate metabolism. $ > BACKGROUND ART 4 2

U.S. Patent 3,683,080, issued August 8, 1972, to Francis, ¥ discloses compositions comprising polyphosphonates, in i particular diphosphonates, and their use in inhibiting : anomalous deposition and mobilization of calcium phosphate in animal tissue. ’ ¥

Japanese Patent 80-98,193, issued July 25, 1980, to Nissabh

Kygaku Kagyo K.K. discloses pyridyl ethane diphosphonic 4 acid, S-(pyridyl)-thiomethane diphosphonic acid, and the W derivatives with halogen or alkyl group substitution on i the pyridyl ring. These compounds are used as post-emer-§ gence herbicides. £

Japanese Patent 80-98,105, issued July 25, 1980, to

Chemical Industries, discloses —— - diphosphonic acid, and the derivatives with halogen or all kyl group substitution on the pyridyl ring, for use as i herbicides. Various N-(pyridyl)-aminomethane aiphospho- § nates are also disclosed in West German Patent 2,831,578,§ issued February 1, 1979 to Fumio, for use as herbicides. y

European Patent Application 100,718 (Sanofi SA), published

February 15, 1984, discloses varidusalkyl aiphosphonates § : which are —-substituted by a sulfide attached to a 5- ork 6-membered nitrogen- or sulfur-containing heterocycle. :

THese compounds are used as anti-inflammatory and anti- B rheumatic drugs. I

DAD ORIGINALE

- 4 = oe oo 2

British Patent Application 2,004,888, published April 11, ¥ 1979, discloses N-(3-methyl-2-picolyl)-aminomethane and . related compounds for use in herbicidal compositions. :

W. Plogegret al.. z. Anorg. Allg. Chem., 389, 119 (1972), discloses the synthesis of N-(4-pyridyl)-aminomethane di- . phosphonic acid. No properties ox utility of the compound are disclosed.

SUMMARY OF THE INVENTION

The present invention relates to pharmaceutical compositions comprising: 4 (a) from about 0.001 mg P to about 600 mg P of a ge- minal diphosphonic acid compound, or its pharmaceutically- acceptable salt or ester, in which the diphosphonic acid- 4 : containing carbon is linked direci.ly, or via a chain of § length from 1 to about 5 atoms, to a 6-membered aromatic ¥ ring containing one or more nitrogen atoms with the parts : of said compound being comprised as follows: i - said ring may be unsubstituted &ér substituted with one or more substituents selected from the group X consisting of substituted and ussubstituted alkyl § {saturated or unsaturated) having from 1 to about : 6 carbon atoms, substituted and unsubstituted aryl, > substituted and unsubstituted benzyl, hydroxy, nalod gen, carbonyl, alkoxy, nitro, amido, amino, substi-1, tuted amino, carboxylate, and combinations - said lingking chain may be all carbon atoma, a nite gen atom or nitrogen~containing thain, an oxygen 5 atom or oxygen-containing chain, or a selenium atom or selenium-containing chain, with said chain being® unsubstituted or substituted on the nitroyen and/or \ gE \ewo ORINAL = 3 : ks

. Yr & carbon atoms, independently, with one or more subs- tituted or unsubstituted alkyl (saturated or unsa- turated) having from 1 to about 4 carbon atoms, and - said nitrogen atom also. may be substituted with an - acyl group: -said diphosphonate-containing carbon may be unsubs- tituted or substituted with substituted pr unsubsti- tuted alkyl (saturated or unsaturated) having from t 1 to about 6 carbon atoms, substituted or unsubsti- 2 i tuted aryl, substituted or unsubstituted benzyl, ~. amino, substituted amino, amido, hydroxy, alkoxy, , halogen or carboxylate, except where said diphos- ; phonate-containing carbon is directly bonded to a : nitrogen, selenium, or oxygen atom in the linking * chain, then the substituents may be substituted or i unsubstituted alkyl (saturated or unsaturated) eb ing from 1 to about 6 carbon atoms, substituted or i unsubstituted aryl, or substituted or unsubstituted benzyl; and i (b) a pharmaceutical carrier. i :

B The invention further encompasses a method 6f treating di- ! seases characterized by abnoxmal calcium and phosphate { metabolism, comprising administering to a human or animal { in need of such treatment a safe and effective amount of a ¥ diphosphonic acid-containing composition of the present in § vention. k be

This invention relates to pharmaceutical compositions, i erably in unit dosage form,comprising a pharmaceutical cor- § rier and a safe and effective amount of geminal diphospho- ji

Ed ~

————— -YY —_ oo ster nic acid compounds, Or their pharmaceutically-acceptable salts and esters, in which the diphosphonic acid-contain- ) ing carbon is linked to a 6 membered aromatic ring con- - taining one or more nitrogen atoms. preferred rings are pyridine, pyridazine, pyrimidine, and pyrazine. Most preferred are pyrimidine, and especially pyridine. The # rings may be unsubstituted or substituted with one or . more substituents selected from the group consisting of i} substituted and unsubstituted alkyl (saturated or unsatu- - rated) having from 1 to about 6 carbon atoms, substituted: and unsubstituted aryl (e.g.. phenyl and naphthyl), subs- tituted and unsubstituted benzyl, hydroxy. halogen, car- y bonyl (e.g., —CHO and -COCH3) alkoxy (e.dg.. methoxy and : - ethoxy), nitro, amido (e.g... ~NHCOCH;) . amino, substi tuted amino (e.g.. dimethylamino, methylamino, and siothylaninl. carboxylate (e.g... ~OCOCH,) , and combinations thereof. md rings may be fused with other rings, e.g... benzene fused ¢ with pyridine (e.g., quinoline), and cyclohexane fused with pyridine (e.g., 5,6,7.8-tetrahydroquinoline). padi tionalt substituents could be substituted or unsubstituted sulfide, sulfoxide, sulfate, or sulfone. ;

The linkage from the diphosphonic acid-containing carbon; to the ring may be direct through a single bond, or by ni chain of length of from 1 to about 5 atoms. The chain ny be all carbon atoms, a nitrogen atom or witzogen-contaiil ing chain, an oxygen atom or oxygen-containing chain, A a selenium atom or selenium-containing chain. \ghe carnch “ and nitrogen atoms in the chains may, oy. be ¥' : unsubstituted or substituted with one (or one or two in F the case of carbon atoms) substituted or unsubstituted x alkyl (saturated or unsaturated) having from 1 to about 4 ty ‘BAD SRIGINAL i :

. . F ; 4 carbon atoms (methyl and ethyl being preferred). The nitrogen atoms in the chains may also be substituted with an acyl group (e.g.. -COCHY) . Unsubstituted carbon and { nitrogen atoms in the chain are preferred. Also preferred are chains one atom in length, i.e., -CH,=~, =NH-, and —0-.

The carbon atom: whichthas the phosphonate groups attacheal to it may be unsubstituted (i.e., a hydrogen atom), or sult stituted with amino, substituted amino, amido, hydroxy, al- 2 koxy, halogen, carboxylate, substituted or unsubstituted ; alkyl (saturated or unsaturated) having from 1 to about 3 carbon atoms, substituted or unsubstituted aryl, or subs- _ tituted or unsubstituted benzyl. For the compounds in iy which the phosphonate-containing earbon is linked to the } ring via an oxygen, selenium, or nitrogen-containing chai ) and that oxygen, selenium, or nitrogen atom is bonded sf rectly to the phosphonate containing carbon, then the subf- tituent on the phosphonate-containing carbon may be abe ¥ tituted or unsubstituted alkyl (saturated or aneatozatear§ having from 1 to about 6 carbon atoms, substituted or un=; } 20% substituted aryl, or substituted or unsubstituted “of

Thus, diphosphonic acid compounds to be included in the § pharmaceutical compositions of the present invention havey the structure: | g i 2 fa T 03H; §

Ry — Z c A Q —+ f ¢ — POH, $ ey

Rola © \R2fn By § BAD ORIGINAL ’ ~ 3 ee emake wherein Q is oxygen, ~NR ,~, selenium, or a single ond, { preferred being oxygen, -NR,-, or a singlé bond; m +n 3? is an integer from 0 to about 5, with m + n= 0 or 1 pre i ferred for Q being oxygen, selenium, or -NR,-., and ‘m + n | on “ :

A

Co é 1 or 2 preferred otherwise: 2 is a ring selected from the group consisting of pyridine, pyridazine, pyrimidine, and pyrazine, with preferred being pyrimidine, and especially i pyridine; Ry is hydrogen, substituted or unsubstituted amino, amido, hydroxy, alkoxy, halogen, carboxylate, subs=, tituted or unsubstituted alkyl {saturated or unsaturated) having from 1 to about 6 carbon atoms, substituted or un- substituted aryl, or substituted or unsubstituted benzyl. except than when n = 0 and Q is oxygen, selenium, or eB then Ry is hydrogen, substituted or unsubstituted alkyl : (saturated or unsaturated) having from 1 to about 6 carboft atoms, substituted or unsubstituted aryl, or substituted : or ynsubstituted benzyl, with Ry being hydrogen, chloro, : amino. methyl, or hydroxy preferred: each R, is, indepen-¥ gently, hydrogen, or substituted or unsubstituted alkyl £ (saturated or unsaturated) having from 1 to about 4 car- § ee with R, being hydrogen preferred; Rg is one # ox ore substituents selected from the group consisting i of drogen, substituted or unsubstituted alkyl (satura- cod x unsaturated) having from 1 to about 6 carbon atoms] sub ti tuted and unsubstituted aryl, substituted! and un- ; subshituted benzyl, hydroxy, halogen, carbonyl, alkoxy, : nitxr , amido, amino, substituted amino, carboxylate. and i combihations thereof, with preferred being nydrogen, ‘me- i or Thy. famine. chloro, methoxy, nitro, hydroxy and combi § nation thereof: R, is hydrogen, substituted or | ynsube- 8 . alkyl (saturated or unsaturated) having ‘Eom 1 # to abo t 4 carbon atoms, or acyl (i.e., the amidé of eho} nitrogen), with preferred being hydrogen, methyl, or 4 ‘ ethyl; and pharmaceutically-acceptable salts and ooters I~. oo of these compounds.

Finally, for any of the Ry.

Ry. "sg Oo” - 9 - i % ¢ a, oo §o7e7 y or R, substituents which are themselves substituted, the substitutién on these substituents may be any one or more of the above substituents, preferred being methyl, ethyl amino, chloro, nitro, methoxy, hydroxy, acetamido, and ; acetate, 2

More specifically, the diphosphonic acid compounds, and . their pharmaceutically-acceptable salts and esters, to be included in the pharmaceutical compositions of the ; i present invention are of the structure: n i 12 1032

R, _—Z Cc Cc — FOH, 5 Or join -

Rf Ry > : t fe 2 I 03H, i

R, — 2 C N c C — PO4H, oF

Ls | BN |),

R, R, R, Ry i 3 i : i i. f2 12 103" ¥ _— — EY

Rq Zz T Oo | T POH, : ;

Lh Ry Ry) Ra % wherein m + n, 2, Ry. Ry. Rg, ahd R, are as described be j = v above. £ i IA 8 & \ bi

Generplly preferred diphosphonic acid compounds, and thelr ; pharmaceutically acceptable salts and esters, to be fneln- ded in) the pharmaceutical.icompositions of the present io v #} ventiqn are of the structure: » o \ 3% ‘ GNP 2 H POH ££ app ji | 32 A © or fl Ry O i § POH, : or 4 a

N i od H OR,

R—F0 ¢ T — PO4H, 4 m+n 5 § 1 ; wherein for both structures above m + n = 1 or 2: Ry ie . hydrogen, chloro, amino, or hydroxy: Rg is one or more substituents selected from the group consisting of hydro- by gen, methyl, amino, chloro, nitro, methoxy, hydroxy, and . combinations thereof: or : 103%

Ro | | | — PO,H, ; or :

O R uN R

N 4 1 i > . g

H POH, i

N

R O N Cc C -—— POH, : Or 5 3 372 }

MN | | n | § ;

R, H Ry KX i § | /

H POH 2 / 372 3 / *% i ry {Qo ¢ ¢ — POH, ; or ; | // ly

Hi Ry A | / i : / { i

CN 1 [032 ro — . io rsfO 4-0 C Cc POH, i or §

N | : | 5 2 {

Hh Ry . i 5

AY ® i It \ & i - wherein for the four preceding structures n = 0 or 1 ; : , ’

A §

Ry is hydrogen, chloro, amino, or hydroxy when n = 1; anlg ]

Ry is hydrogen when n = 0; Rq is one or more substituents§ | P ‘

CONG ” Nae selected from the group consisting of hydrogen, methyl, po °° amino, chloro, methoxy, nitro, hydroxy, and combinations g rd qd thereof; and Ry is hydrogen, methyl, or ethyl. : 3 i co

Specific examples of compounds which may be utilized in ¥% - 11 - OF ¥

EEE i compositions of the present invention include: 3 /

N-(2-pyridyl)-aminomethane diphosphonic acid; /

N-(2-(5-amino)-pyridyl)-aminomethane diphosphonic acid; i

N-(2-(5-chloro)-pyridyl)-aminomethane diphosphonic acid: i

N-(2-(5-nitro)-pyridyl)-aminomethane diphosphonic acid; :

N-(2-(3,5-dichloro)-pyridyl)~aminomethane diphosphonic acla;

N-(4~pyridyl)-N-ethyl-aminomethane diphosphonic acid: :

N-(2-(3-picolyl))-aminomethane diphosphonic acid: }

N-(2-(4-picolyl))-aminomethane diphosphonic acid; k

N-(2-(5-picolyl))-aminomethane diphosphonic acid; )

N-(2-(6-picolyl))-aminomethane diphosphonic acid: &

N-(2-(3,4-1lutidine))-aminomethane diphosphonic acid: i!

N-(2-(4,6-lutidine))-aminomethane diphosphonic acid; i

N-(2-pyrimidyl)~aminomethane diphosphonic acid; :

N-(4-(2,6-dimethyl)~-pyfimidyl)-aminomethane diphosphohic acid; N

N-(2-(4,6-dihydroxy)-pyrimidyl)-aminomethane diphosphonid acid: J Co

N- ( 2 ( 5-methoxy)-pyridyl)-aminomethane diphosphonic aciaf}

N-(2-pyridyl)-2-aminoethane-1,1-diphosphonic acidp ;

N-(2-(3-picolyl))-2-aminoethane-1,1-diphosphonic acid: i}

N-( 3-pyridyl)-2-amino-l-chloroethane-1,1-diphosphonic ada:

Co eaiyly)-2-amino-1-hydzoxy-sthane-L 1-dighosphatic } acid; (2-pyridyl)-methane diphosphonic acid; I i (3-pyridyl)-aminomethane diphosphonic acid: (2-pyridyl)-chloromethane diphosphonic acid: & / (4-pyridyl)-hydroxymethane diphosphonic acid; : 2-(2-pyridyl)-ethane-1,1l-diphosphonic acid; g | \ i on 2-(3-pyridyl)-ethane-1,1l-diphosphonic acid: ’ "op® of. 2-(4-pyridyl)-ethane-1,1-diphosphonic acid; i > 2-(2-pyridyl)-l-amino-ethane-1,1-diphosphonic acid: 3 oo brs : 2-(2-pyrimidyl)-l-hydroxy-ethane-1,1l-diphosphonic acid; & 2-(2~(3-picolyl))-l-chloro-ethane~1,l-diphosphonic acid; : 2-(2-(4-methoxy)~pyridyl)-ethane-1,l-diphosphonic acid; KX 1-(2-pyridyl)-propane-2, 2-diphosphonic acid: k 2-(2-pyridyl)-l-chloro-ethane-1,l-diphosphonic acid: 2-(2-pyridyl)-1-hydroxy-ethane-1, l-diphosphonic acid; . 2-(3-pyridyl)-1l-hydroxy-ethane-1,l-diphosphonic acid; x 2-(4-pyridyl)-1l-hydroxy-ethane-1,l-diphosphonic acid; . 3-(3-pyridyl)-l-hydroxy-propane-1,l-diphosphonic acid; 0-(2-pyridyl)-2-oxa-ethane-1,l-diphosphonic acid: ¥

O-(2-pyridyl )-oxamethane diphosphonic acid: i 0-( 2-pyrimidyl )-oxamethane diphosphonic acid; { 0-(2-(4-amino)-pyridyl)-oxamethane diphosphonic acid;

O-(2-pyrimidyl)-2~oxa-ethane-1,l-diphosphonic acid; i 0-(2-(3-picolyl))-2-oxa-ethane-1,l-diphosphonic acid; & 0-(2-(3-picolyl))=-oxamethane-diphosphonic acid; & 0-(2-pyridyl)-l-hydroxy-2-oxa-ethane-1,l-diphosphonic acills i 0-(4-pyridyl)-l-amino-2~oxa-ethane-1,1l-diphosphonic aciaril and pharmaceutically-acceptable salts and esters chereot 1

Preferred compounds are § . N-(2-(5-amino)~-pyridyl)-aminomethane diphosphonic acid; # oo

N-(2-(5-chloro)-pyridyl)-aminomethane diphosphonic acid; § .

N-(2-(3-picolyl))-aminomethane diphosphonic acid; :

N-(2-(4-picolyl))-aminomethane diphesphonic acid: 4

N-(2-(5-ptcolyl))-aminomethane diphosphonic acid; §

N-(2-(6-picolyl))-aminomethane diphosphonic acid; i -

N-(2-(3,4-lutidine))-aminomethane diphosphonic acid; £

N-(2-pyrimidyl)-aminomethane diphosphonic acid: i }

N-(2-pyridyl)-2-aminocethane-1,l-diphosphonic acid; i am 2-(2-pyridyl)-ethane-1,l-diphosphonic acid; £ WW 2-(3-pyridyl)~ethane~1l,l-diphosphonic a¢id: x o OF° _ gol —

2-(4-pyridyl)-ethane-1,l~-diphosphonic acid; ; ; 7-(2-pyridyl)-l-hydroxy-ethane-1,1-diphosphonic acid; ' 2~(3-pyridyl)-l-hydroxy-ethane-1,l-diphosphonic acid; r 2-(4-pyridyl)-l-hydroxy-ethane~1,l-diphosphonic acid; 5 0-(2~(3-picolyl))=-oxamethane-diphosphonic acid; and ’ : pharmaceutically-acceptable salts and esters thereof. :

The diphosphonate compounds to be included in the phar- 1 maceutical compositions of the present invention can be : made using the synthetic methods disclosed in Japanese x patent 80-98,193 (July 25, 1980, to Nissan Kygygaku Kagyo i

K.¥.), Japanese Patent 80-98,105 (July 25, 1380, to =

Nissan Chemical Industries), West German patent 2,831,5/8g ‘ (February 1, 1979, to Fumio), and W. Ploger et al., Z. E

Anorg. Allg. Chem., 383, 119 (1972), the disclosures of which are incorporated herein by reference. The amino= ethane diphosphonic acid compounds, however, are best ¥ prepared as follows: i synthesis of N= (2-(3-picolyl))aminoethane LP i ne) above-named compound is prepared via a typical vichel xeaceion between tetraethyl vinyldiphosphonate and amin ‘3-pikoline. (See H.O. House, Modern synthetic Reaction ¢ 2nd fe W.A. Benjamin Inc. p. 595-632, the disclosure of which. is incorporated herein by reference.) 1 \ |” co . Fo a solution of 1.62 g (15 mmol) of 2-amino-3-picoline if tetrahydrofuran at 5°c was added 4.50 g (15 mmol) tetra- ethyl vinyldiphosphonate. The reaction mixture was stirred at rom temperature for 16 hours. Evaporaticn of the or vent and chromatography (acetone/hexane, 4/1) of the pro-= eV duct on silica gel gave pure tetraethyl N-(2-(3-picolyl)) ww 2-aminoethane diphosphonate. P-31 NMR of the pure “1

Co - 14 -

A ge ethyl ester in CDCl, shows a resonance at 22.1!ppm. The: s ester was hydrolyzed in refluxing 6N HCl overnight. The it product showed a P-31 NMR signal in D,0 at pH = 12 of 19.0 ppm.

N-(2-pyridyl)-2-aminoethane DP and N-(2-(5-picolyl))-2-ami- noethane DP were prepared in an identical manner. i

Compounds having the general formula * 12 fost :

R4 -_— 7 7 T —— Fo ,H,

R, n OH . (wherein n is an integer of from 1 to about 5, preferably’ n = 1; and zZ, R, and R, are as described hereinbefore, wilh preferred Z being pyrimidine and especially pyridine, pred ferred R, being hydrogen, and preferred R, being one or mre substituents selected from the group consisting of hydroghh, methyl, amino, chloro, nitro, methoxy, hydroxy, and combi nations thereof) are best prepared as follows: ¥

Synthesis of 2-(2-pyridyl)-l-hydroxy-ethane-1, 1-aiphospholiic ) acid: #

A 3-neck round-bottom flask fitted with a reflux condense}. and a magnetic stir bar is charged with 6.94 grams oon § mole) 2-pyridine acetic acid, 9.84 grams (0.14 mole) phosy phorus acid, and 150 ml of chlorobenzene. This reaction iti x ture is heated on a baling water bath, and 16.5 grams ©. mole) phosphorus trichloride is added dropwise with stirding. Py

This reaction mixture is heated for 2-1/2 hours during wilh on time a viscous yellow oil forms. The reaction mixture fo then cooled in an ice bath and the chlorobenzene solutions - decanted off from the solidified product. The reaction &.o containing this solidified product is charged with 150 + . = 15 - E

Co | 27°78 : water and heated in a boiling water bath for several hours.

The hot solution is then filtered through cCelite 545%. 300 ml of methanol is added to the warm filtrate solution, and a precipitate develops. After cooling in ice for 1 hour. the precipitate is filtered off and then wabhed with me tHa- nol/water (1/1 volume/volume), methanol, and ether, and air dried. The product may be recrystallized from hot water.

Yield is approximately 5.9 grams (52%). The sample is cHa- racterized by P-31 and C-13 NMR. .

By "pharmaceutically-acceptable salts and esters" as uscd herein is meant hydrolyzable esters and salts of the diphos- phonate compounds which have the same general pharmacologi- . cal properties as the acid form from which they are derived, and which are acceptable from a toxicity viewpoint. ode ceutically-acceptable salts include alkali metal { sodium Bnd potassium), alkaline earth metal (calcium and magnesium) non-toxic heavy metal (stannous and indium), and — and low molecular weight substituted ammonium (mono-, di and triethanolamine) salts. Preferred compounds are the & sodium, potassium, and ammonium salts. ] = By "pharmaceutical carrier" as used herein is meant one & more compatible solid or liquid filler diluents or encap 7 sulating substances. By "compatikle" as used herein is § meant that the components of the composition are capable of being commingled without interacting in a manner whic # would substantially decrease the pharmaceutical efficacy : of the total composition under ordinary use situations. &

Some examples of substances which can serve as pharmaceu + « en tical carriers are sugars such as lactose, glucose and cub 2 rose: starches such as corn starch and potato starch: cod v lulose and its derivatives, such as sodium 7] - 16 = # . LF

SFo1¢

Co - ¢1°1 . : cellulose, ethylcellulose, cellulose acetate; powdered tragacanth; malt; gelatin; talc; stearic acid; magnesium stearate; calcium sulfate; vegetable oils, such as pea- . nut oil, cottonseed oil, sesame oil, olive oil, corn oil: and oil of theobroma; polyols such as propylene glycol, glycerin, sorbitol, mannitol, and polyethylene glycol; |; agar; alginic acid; pyrogen-free water: isotonic saline: and phosphate buffer solutions, as well as other non-toxle compatible substances used in pharmaceutical formulations.

Wetting agents and lubricants such as sodium lauryl sul-; fate, as well as coloring agents, flavoring agents, lubri- cants, excipients, tableting agents, stabilizers, anti- § oxidants and preservatives, can also be present. Other J - compatibde pharmaceutical additives and actives (e.g., § vitamin D or vitamin D metabolites, and mineral supple- ments) may be included in the pharmaceutical composi tion, of the present invention. !

The choice of a pharmaceutical carrier to be used in con® junction withi the diphosphonates of the present conposichons is basically determined by the way the diphosphonate is E be administered. If the compound is to be a preferred pharmaceutical carrier is sterile, physioclogic8l saline, the pH of which has been adjusted to about 7a. fou ever, the preferred mode of administering the a nates of the present invention is orally, and tho\ prefer al unit dosage form is therefore tablets, capsules and the Yike, comprising from about 0.1 mg P to about 600 mg P of ve di phosphonic acid compounds described herein. Pharmaceut {fal a carriers suitable for the preparation of unit dosage sor ORC for oral administration are well known in the art. eid selection will depend on secondary considerations like B 5 taste, cost, shelf stability, which are not critical fork % g oo - ) ofg the purposes of the present invention, and can be made i out difficulty by a person skilled in the art. The pharm&® ceutical carrier employed in conjunction with the diphosplb- nates of the present invention is used at a concentration; sufficient to provide a practical size to dosage relation® ship. Preferably, the pharmaceutical carrier comprises J from about 0.01% to about 99.99% by weight of the total composition. ;

EXAMPLE I :

Capsules are prepared by conventional methods, comprised 1 follows: )

Ingredient Mg per capsule % : N~(2-(3-picolyl)) aMDP 100 (as mg P) i

Starch 55.60 7

Sodium lauryl sulfate 2.90 i

The above capsules administered orally twice daily for 6 : months substantially reduce bone resorption in a patient 3 weighing approximately 70 kilograms afflicted with osteo- & porosis. Similar results are obtained when the N-(2-(3- i picolyl))-aminomethane diphosphonic acid, or its pharma- ceutically-acceptable salt or ester, in the above-describet capsules is replaced with i

N-(2-(5-amino)-pyridyl)-aminomethane diphosphonic acid; 8

N-(2-(5-chloro)-pyridyl)-aminomethane diphosphonic acid; E 28 N-{(2-(4-picolyl))-aminomethane diphosphonic acid; § - QP

N-(2-(5-picolyl))-aminomethane diphosphonic acid; feo

N-(2-(6-picolyl) )-aminomethane diphosphonic acid; 8 Co

N-(2-{(3,4-1lutidine) )~aminomethane diphosphonic acid; &

N-(2-pyrimidyl)-aminomethane diphosphonic acid; 2

N-(2-pyridyl)-2-aminoethane-1, l1-diphosphonic acid: §

Pi: - 18 - i y 2-{2-pyridyl)-ethane-1,1l-diphosphonic acid; . 2-(3-pyridyl)-ethane-1, 1-diphosphonic acid; : } 2-(4-pyridyl)-ethane-1,1-diphosphonic acid; 4 2-(2-pyridyl)-1-hydroxy-ethane-1,1-diphosphonic acid: 5 2-(3-pyridyl)-1-hydroxy-ethane-1,l-diphosphonic acid; > 2-(4-pyridyl)-l-hydroxy-ethane-1, 1-diphosphonic acid;

O-(2-(3-picolyl))-oxamethane-diphosphonic acid: or the pif maceutically-acceptable salts or esters thereof. L

Ph

EXAMPLE 11 §

Tablets are prepared by conventional methods, formulated as follows: 5 - Ingredient mg per tablet i

N~{2-pyrimidyl) AMDP 25.00 b

Lactose 40.00 t

Starch 2.50 5

Magnesium stearate 1.00 {

The above tablets administered orally. twice daily for 6 ’ months substantially reduce bone resorption in a patient ' weighing approximately 70 kilograms afflicted with osteo- ! porosis. Similar results are obtained when the N-(2-pyri midyl) AMDP, or its pharmaceutically-acceptable salt or » ester, in the above-described tablets is replaced with %

N-(2-(5-amino)-pyridyl)-aminomethane diphosphonic acid; i

N-(2-(5~chloro)-pyridyl)-aminomethane diphosphonic acid: #

N-(2-(3xpicolyl))-aminomethane diphosphonic acid; §

N-(2-(4-picolyl))-aminomethane diphosphonic acid; y A

N-(2-(5-picolyl) )~-aminomethane diphosphonic acid: 5 es

N-(2-(6-picolyl))-aminomethane diphosphonic acid; } ® -

N-(2-(3,4-lutidine))-aminomethane diphosphonic acid; :

Bn 30 N-(2-pyridyl)-2-aminoethane-1, l1-diphosphonic acid; i - 19 - T oo Ses 2-(2-pyridyl)-~-ethane-1, 1-diphosphonic acid: : 2~(3-pyridyl)-ethane-1,l-diphosphonic acid; : 2—-(4-pyridyl)-ethane-1, 1-diphosphonic acid; : 2-(2-pyridyl)-l-hydroxy-ethane-1, l1-diphosphonic acid; 2-(3-pyridyl)-1-hydroxy-ethane-1, l-diphosphonic acid; 2-(4-pyridyl )-l-hydroxy-ethane-1, l-diphosphonic acid; 0-(2-(3-picolyl))-oxamethane~dphosphonic acid; or the phar- maceutically~acceptable salts or esters thereof. ;

EXAMPLE I11 .

Injectable solutions are prepared by conventional methods using 1.0 ml of either physiological saline or water solu= _ tion and 3.5 mg of 2-(2-pyridyl)-ethane-1,1-diphosphonic acid, adjusted to pH = 7.4. i

One injection, one time daily for 4 days results in appr dt ciable alleviation of hypercalcemia of malignancy in patients weighing approximately 70 kilograms. ¥

Similar results are obtained when the 2-(2-pyridyl)-ethane- 1,1-diphosphonic acid in the above-described treatment i. replaced with i

N-(2~(5-amino)-pyridyl)-aminomethane diphosphonic acid: x

N-(2-(5~chloro)-pyridyl)-aminomethane diphosphonic acid: &

N-(2-(3-picolyl))-aminomethane diphosphonic acid: i

N-(2~(4~picolyl))-aminomethane diphosphonic acid; §

N~(2-(5-picolyl))-aminomethane diphosphonic acid; i

N-(2-(6-picolyl))-aminomethane diphosphonic acid; 2

N=(2-(3,4~-lutidine))-aminomethane diphosphonic acid: 7 Js

N~-(2-pyrimidyl)-aminomethane diphosphonic acid; : a

So ORC

N-(2-pyridyl)-2-aminoethane-1,l-diphosphonic acid; @ ob® _ 2-(3-pyridyl)-ethane-1,1-diphosphonic acid; 3 wn 2-(4-pyridyl)-ethane-1, l-diphosphonic acid; 2 - 20 - 5 ~ §

CT oPT¢ 2-(2-pyridyl)-l-hydroxy-ethane-1, l-diphosphonic acid; 2-(3-pyridyl) ~l-hydroxy-ethane-1, l-diphosphonic acid; = 2-(4-pyridyl)-l-hydroxy-ethane-1, l-diphosphonic acid; 5 0-(2-(3-picolyl) )-oxamethane-diphosphonic acid; or pnarm- § ceutically-acceptable salts or esters thereof. :

The compositions of the present invention are useful in the. treatment ol abnormal calcium and phosphate metabolism. by other diphosphonic acids aud their phasmscout ics ty-ocecr able salts have been proposed for use in the treatment anf prophylaxis of such conditions. In particulary, ecthane-1- i hydroxy-1,l-diphosphonic acid (EHDP). propanc-3-aminoe-—1- : hydroxy=-1, l-diphosphonic acid (AFD), and dichlicromethane i . diphosphonic acid (CLEP) have been the subject of consi 1 derablyu research efforts in this area. i

However, the compositions of the present invention are gendt erally more biologically potent in inhibiting bone I tion than the art-disclosed diphosphonates. ‘thus, the cond positions of the present invention may provide one or wr of the following advantages over the art-disclosed diphos-4 phonates of (1) being more potent in inhibifing bone recht tion: (2) possessing less potential for inhibition of ol mineralisation, since mineralization inhibition is believe 1 to be predominantly a mass related physico-chemical effect ; (3) having yenerzclly a wider margin of safety (i.e., widcr dosing interval between the lowest effective antiresorptiv } dose and the lowest dose producing mineralization inhibi ti ns (4) allowing lower oral dosages to be administered, there of a avoiding the gastro-intestinal discomfort (like diarrhea) & “0 ’ sometimes associated with higher dosages of diphosphonates i : and (5) having potential for flexibility of dosing method dE &

Another aspect of this invention is a method for treating £ or preventing diseases characterized by abnormal calcium } ome gn oo es and phosphate metabolism, in particiilar those which are : characterized by abnormal bone metabolism, in persons at ) ‘risk to such disease, comprising the step of administering to persons in need of such treatment a safe and effectives amount of a diphosphonic a¢id-containing composition of i the present invention. 2

The preferred mode of administration is oral, but other modes of administration include, without limitation, trans- dermal, mucosal, sublingual, intramuscular, intravenous, intraperitoneal, and subcutaneous administration, as well! as topical application. 3 ~ By "abnormal calcium and phosphate metabolism" as used : herein is meant (1) conditions which are characterized by anomalous mobilization of calcium and phosphate leading ob general or specific bone loss, or excessively high calci \ and phosphate levels in the: fluids of the body; and (2) 1 conditions which cause or result from deposition of calcidh and phosphate anomalously in the body. The first category includes, but is not limited to, osteoporosis, Pagets sf sease, hyperparathyroidism, hypercalcemia of malignancy, i and osteolytic bone metastases. The second category inc1idfes, but is not limited to, myositis ossificans progressiva, cdl- cinosis universalis, and such afflictions as arthritis, ke ritis, bursitis, tendonitis and other inflammatory condi- § tions which predispose involved tissue to deposition of 4 a calcium phosphates. : ae $o,00 ral : By "person at risk", or "person in need of such treatment} as used herein is meant any human or lower animal which 3 suffers a significant risk of abnormal calcium and phos- 3 phate metabolism if left untreated, and any human or lowe x animal diagnosed as being afflicted with abnormal calcium §

EE S11 and phosphate metabolism. For example, postmenopausal women; persons undergoing certain steroid therapy: personb on certain anti-convulsant drugs; persons diagnosed as hav- ing Pagets disease, hyperparathyroidism, hypercalcemia of malignancy, or osteolytic bone metastases; persons diag- - 43 nosed as suffering from one or more of the various forms of osteoporosis; persons belonging to a population group f known to have a significantly higher than average chance of developing osteoporosis, e.g... postmenopausal women, men over age 65, and persons beng treated with drugs knowh to cause osteoporosis as a side effect: persons diagnosed’ as suffering from myositis ossificans progressiva or calei- nosis universalis: and persons afflicted with arthritis, ! neuritis, bursitis, tendonitis and other inflammatory con © 15 ditions which predispose involved tissue to diposition of calcium phosphate. ¥ *

By "human or lower animal afflicted with or at risk to ost teoporosis” as used herein is meant a subject diagnosed i suffering from ore or more of the various forms of onved porosis, or a subject belonging to a group known to have a significantly higher than average chance of developing od ooo porosis, e.g., postmenopausal women, men over the age or ks, and persons being treated with drugs known to cause orth porosis as a side effect (such as adrenocorticoid). 8

By "safe and effective amount" as used herein is meant ad amount of a compound or composition high enough to, signifi y cantly positively modify the condition to be treated; ~f low enough to avoid serious side effects (at a reasonab ld! 7 \. benefit/risk ratio), within the scope of sound medical dae Lo ment. The safe and effective amount of diphosphonates A vary with the particular condition being treated, the 2d ¥ ks : C3 | § »

CW

Loge oo ? and physical condition of the patient being treated, the ; severity of the condition, the duration of treatment, the nature of concurrent therapy, and the specific diphospho- " } nate employed. However, single dosages can range from : about 0.001 mg P to about 3500 mg P, or from about 0.1 : micrograms P/ky oi body weight to about 500 mg P/kg of body weight. Preferrea single dosages are from about 0.1] y mg bP to about 000 mg P, or from about 0.01 to about 50 mu :

P/ky ol body weight. Up to about four single dosages per t day may be administered. baily dosages greater than abou th i, 2000 my I'/kg ave nol required to produce the desired of (ct and may preduce undesirable side effects. The higher oral ; ges within this range are, of course, required in the cos 2, of oral aduwinistration because of limited absorption. {

Schenl, todel f

The compounds wore evaluated for in vivo bone resorption =» iniibition and mineralization inhibition in an animal me IF system known in the field of bone metabolism as the Scheni i

Model. The general principles of this model system are d ii closed in shinoda et al., Calcif. Tissue Int., 35, 87-99 ae (1983): and in Schenk et al., calcif. Tissue Res. 11, 196-ZU4 (1973), the disclosures of which are incorporated herein bh reference.

Materials and HMethods: k

Animals §

Preweaning l7-day-old (30 grams) male Sprague Dawley rats § . (Charles River Rreeding Laboratories) were shipped with # hy a their mothers and placed in plastic cages with their mothers - 3 upon arrival. At 21 days of age, pups receiving Rat Chow Eo wo and water ad libitum were randomly allocated into trea tue th - 7 groups comprising [ive animals per group, except for con- 3 ~

J | Fr trol animals receiving saline vehicle which had 10 rats # per group. On day O and again on day 1 all animals were given a subcutaneous injection of calcein (Sigma) as a 1 solution in 0.9% Nacl solution to label the skeleton.

Dose Solutions and Dosing Procedure

All solutions were prepared for subcutaneous injection in 0.9% normal saline and adjusted to pH 7.4 using NaOH and/or

HCl. Dose solution calculation was made by considering the mass of powder (based on molecular weight, hydration) of § the active material in mg/kg (body weight) that correspoids to mgP/kg. Concentrations were based on dosing 0.2 m1/10b g body weight. Initially, all compounds were adninisterdd at 0.1, 1.0 and 10.0 mg P/kg/day for 7 days. Compounds i showing activity at 0.1 mg P/kg/day were then tested at g logarithmic decrements down to 0.001 mg P/kg/day. aed ments in dosage based on changes in body weight were nade) on a daily basis. b

Necropsy, Tissue Processing and Histomorphometry : on day 8 after the start of dosing, all animals were sac . 20 rificed by co, asphyxiation. Tibias were dissected frec | ; and placed in 70% ethyl alcchol. Cne tibia was dehydra-¥ ted in graded ethanol solutions and embedded in methyl methacrylate using a rapid procedure described in Boyce # at al., Lab. Investig., 48, 683-689 (1983), the disclosu es of which are incorporated herein by reference. The tibia} was sectioned longitudinally through the metaphyseal are} (LeitzR saw microtome at 150 mn). Specimens were stained ff ~N on one surface with silver nitrate and mounted on micros & Po cope slides for evaluation with a Quantimet Image AnalyzdF oo (cambridge Instruments, Inc.) using both incandescent and} oo rT , _ 25 - i

: ultraviolet illumination. Metaphyseal trabecular bone con> tent was measured in the region between the fluorescent label and the growth plate: expressed as percent of total . area (bone + marrow). Epiphyseal growth plate width was 5 obtained as the mean value of 10 equally-spaced measure- ments across the section.

Statistical evaluation of data was made using parametric and non-parametric analysis of variance and wilcoxons rank sum test to determine a statistically significant effect compared to control animals. "

The Schenk model provided data for in vivo bone resorption! inhibition by the compounds. The lowest effective (anti- y resorptive) dose ("LED") for representative compounds Ley ted, as determined by the Schenk model, are provided in if

Table I. q y a 3

TABLE 1 t.

Lowest Effective (Antiresorptive) Dose i : Schenk " yl

Diphosphonate Compound LED (mg P/kg) +

EHDP 1.0 E a Eh.

C1 ,MDP 1.0 J. 3

APD 0.1 i

N

N-(2-pyridyl) AMDP* 0.01 &

N-(2~(5-chloro)-pyridyl) aMDP* 0.01. 4]

Ay

N-(2-(3-picolyl)) AMDP* 0.001 £

N-(2-(4-picolyl)) AMDP* 0.001 &

N-(2-(5-picolyl)) AMDP* 0.001 PB

N-(2-(6-picolyl)) AMDP* 0.001 kJ i 4 eT

N-(2-pyrimifiyl) AMDP* 0.001 & Ca . 8 AG

N-(4-pyridyl)-N-ethyl AMDP* 0.1 jop0 © ps

WT

- 26 - pe i bone - continued - .

Schenk

Diphosphonate Compound LED (mg P/kg) . 2-(2-pyridyl) EDP* 0.01 ¥ 2-(3-pyridyl) EDP* 0.01 1-(2-pyridyl) propyl DP* 10

EHPP = ethane-l-hydroxy-1,1-DP

Cl,MDP = dichloromethane DP )

APD = 3-aminopropane-l-hydroxy-1,1-DP

AMDP = aminomethane diphosphonic acid, where the ring is attached to the amine. * = compounds included in pharmaceutical compositions of the present invention. )

EDP & ethane-1,l-diphosphonic acid, where the ring is at- tached at the 2 position of the ethane.

Propyl DP = propane-2, 2-diphosphonic acid i

Diphosphonate compounds which have a bone mineralization inhibiting effect cause widening of the epiphyseal growth” plate, since matrix production continues but nineralizatidh is impeded. The widening of the epiphyseal growth plate h observed in the Schenk model is, therefore, a measure of the mineralization inhibiting effect of the diphosphonate conghund tested. 4

The lowest tested dosages producing a statistically sd ficant widening of epiphyseal growth plate of compounds teés- ted are given in Table II. 3 i ~

TABLE 11 i Lov .

Mineralization Inhibition (Schenk Model) [oo

Lowest tested dosagl} > : 30 producing a statistically significant widenin¥ of

A

- 27 - t oo 797

Co ! - continued - 3

Diphosphonate epiphyseal growth plate compound (mg P/kg) : _

EHDP 10 ’ 5 APD 10 cl ,Mpp —

N-(2-pyridyl) AMDP* 0.1 ;

N-(4-pyridyl)-N-ethyl AMDP* -- 1) ¥

N-(2-(3-picolyl)) AMDP* -- 1)

N-(2-(4-picolyl)) AMDP 0.1 :

N-(2-(5~picolyl)) AMDP* 0.1

N-(2-(6~-picolyl)) AMDP* -- 1) a . N-(2-pyrimidyl) AMDP* 1.0 .

N-(2-(5-chloro)-pyridyl) AMDP* -= 1) § 2-(3-pyridyl) EDP* - ¥ 2-(2-pyridyl) EDP* \ -- 1) : - = No plate widening observed at highest dose tested (high- est dose tested is 10 mg P/kg/day unless otherwise indicacha) 1. Highest dose evaluated is 1 mg P/kg/day (compound Jothal1ly toxic at 10 mg p/kg/day) i

EHDP = ethane-l-hydroxy-1,1-DP £

APD = 3-aminopropane-l-hydroxy-1,1-DP ¥

Cl MDP = Dichloromethane DP i

BMDP = aminomethane diphosphonic acid, where the ring is % attached to the amine #

EDP = ethane-1,l-diphosphonic acid, where the ring is at- tached at the 2 position of the ethane K * = Compounds included in pharmaceutical compositions of ¥ —% the present invention ¥ eV eh? pre

Thyroparathyroidectomized (TPTX) Rat Model i . -

co | 21078

The compounds were evaluated for in vivo bone resorption § inhibition potency by an animal model system known as the ill thyroparathyroidectomized (TPTX) rat model. The general ¥ principles of this model system are disclosed in Russell et al., calcif. Tissue Research, 6, 183-196 (1970), and ink

Muhlbauer and Fleisch, Mineral Electrolyte Metab., 5, 296% 303 (1481), the disclosures of which are incorporated herd® in by reference. The basic biochemical concept of the ro system is inhibition of the parathyroid hormone (PTH) - id duced rise in scrum and ionized calcium levels by the res-g pective bone active polyphosphonates. R

Materials and Methods: 2 . -

Materials

Low calcium and low phosphorous diets used were prepared » by Tekladl Test Diets (Harlan Industries, Madison, Wisconge sin 53711; Order #TDB82195) in a pellet form of approxiniat dy 0.18% calcium and 0.22% phosphorous. The diets contained B all the essential vitamins and minerals required for the B . rat, with the exception of calcium and phosphorous. The calcium and phosphorous levels of the pellets were veriti dB - analytically (Procter & Gamble Co., Miami valley Laborato ries, Cincinnati, Ohio).

PTH was acquired as a powdered bovine extract (Sigma CheniEal cc., P. O. Box 14508, st. Louis, Missouri, order #pP-0892,

Lot #72F-9650) at an activity of 138 USP units per my. PRE was prepared in 0.9% saline such that the final concentraifon Pe was 100 U.S.P./ml. All solutions were filtered through ag4 oN whatman Filter paper and refiltered through a 0.45 um ne Mp0 OF cel R filter. f

Dose Solutions and Dosing Procedure £ - 29 - v ¢ oo - } fot? : All solutions of compounds to be tested for bone Bn inhibition potency were prepared for subcutaneous inject id in 0.9% normal saline and adjusted to pH 7.4 using NaOH afiu/- : or HCl. Dose solution calculation was made by consideringj the mass of powder (based on molecular weight, hydration) of the active material in mg/kg (body weight) that corres ponds to mg P/kg. Concentrations were based on dosing 0. % ml/100 yrams of body weight. Initially, all compounds wen administered at 0.0), 0.1, and 1.0 mg P/kg/day for 4 days h where necessary the test was repeated, whereby the animal@ were administered with 0.5 LED in order to refine the wed mination of LED. Adjustments in dosage based on changes Ar body weight were made on a daily basis. £ ~. 3

Animals

In this study 50 male wistar rats weighing approximately Ji 150-160 grams were thyroparathyroidectomized surgically HO the breeder (Charles River Breeding Laboratories). All r®ts were double housed on arrival in suspended cages with Purigha

Laboratory Rodent Chow! and tap water ad libitum. After acclimation to the laboratory environment for 3-5 days, JR ~ rats were placed on a low calcium, low phosphorous (0.16708 0.22%) diet (TekladR) and given 2% (W/V) calcium gluconat\ supplemented deionized water via water bottles. ' ' ' !

Method 3 on day four of low-calcium diet all rats were anesthetir ff with Ketaset® (Ketamine Hydrochloride, 100 mg/ml, Bristo WB oo Myers), 0.10 ml/100 grams of body weight, weighed and thd oF : bled from the retro-orbital venous plexus for serum totals > } calcium analysis using Flame Atomic Absorption (FAA). A » : : 30 rats weighing less than 180 grams were eliminated from tHE Co

E071 ! study. Animals were then randomized statistically such A that the mean total serum calcium for each group was the E same. Only rats deemed hypocalcemic (total serum calcium? - < 8.0 mg/dl) were placed in study groups comprising six ! animals per group. v ;

Treatments with the various experimental compounds com- 7 menced on day 6 and lasted through day 9 of the study (ot" 1:00 P.M. each day). Nose solutions were prepared to be ’ given at a constant rate of 0.2 ml/100 grams of body weight subcutaneously in the ventral skin flap where the hind leg meets the torso. All rats were weighed and dosed daily.

A 25 gauge 5/8" needle was used to administer drug, alter= nating dose sites daily. On day 8, animals were changed i to deionized, distille. water via water bottles. On day EB 9 all rats were fasted in the afternoon at approximately § 4:00 P.M. On day 10 of study no treatment was given. Inf the morning a 600 ul sample of whole blood was collected §- from each rat in Microtainer (B-D#5060) serum separater B tubes for serum total calcium (FAA). Two 125 nl samples of heparinized whole blood were also collected to be used® » for ionized calcium analysis. Immediately following bloo collection all rats were weighed and injected with wove - parathyroid hormone subcutaneously at a rate of 75 USP (£11- tered) per 100 grams of body weight. Blood sampling for } total and ionized calcium was repeated three and one-half} . hours post-PTH injection. 8

All pre- and post-PTH total and ionized calciums were cad Th tistically analyzed for significance compared to PTH sland es (control) using Students t-test, amdysis of variance, and oh® their non-parametric equivalents. The post minus pre- change and % change were also determined on calcium level

, £1090 oo ¥ and pre-drug vs post-drug body weights. ‘

The physiological effect of the PTH challenge is a rise in . serum calcium level, with peak activity observed at three ; and one-half hours. Since the hormonal and dietary controls of calcium metabolism are minimized in the TPTX model, an. observed increase in serum calcium level is presumably the result of resorption of bone material. Since polyphospho= nates tend to inhibit resorption of bone materials, the ’ animals pretreated with polyphosphonate showed a rise in 1 serum calcium level after PTH challenge which was less thah that found in control animals which had been treated with . saline vehicle instead. The lowest dose at which the poly

BE phosphonate is capable of inhibiting bone resorption, as : : evidenced by a decreased rise in serum calcium upon I'T'H § challenge, is a measure of the bone resorption inhibition y potency of the polyphosphonate. The LED values of the R .. bone resorption inhibition potency of representative com- & pounds as determined by the TPTX rat model are presented : . 5 in Table III. &

TABLE 111 ir

Lowest Effective (Antiresorptive) Dose #

TPTX 8

Diphosphonate Compound LED (mg F/kg) i ;

EHDP | 1.0 ¥

C1 ,MDP 1.0 § / © APD 0.1 g Co

N-(2-pyridyl) AMDP* } 0.01 F eV

N-(2-(5-amino)-pyridyl) pMDP* 0.01 fro

N-(2-(5-chloro)-pyridyl) AMDD* 0.01 &

N-(2-(5-nitro)-pyridyl) AMDP* 0.1 §

N=-(2-(5-carboxy)~-pyridyl) AMDP N £

. f : - continued -

TETX ‘

Diphosphonate Compound LED (mg P/kg) : N-(2-(3,5-dichloro)-pyridyl aMDp* 1.0

N-(4-pyridyl)-N-ethyl AMDP* 0.1 .

N= (2-(3-picolyl)) AMDE* 0.002 ’

N-(2-(4~picolyl)) AMDP* 0.001

N-(2-(5-picolyl)) AMDP* 0.001 .

N-(2-(6~picolyl)) AMDP* 0.01

N-(2-(3,4-lutidine)) AMDE* 0.01

N-(2-(4,6-1lutidine) AMDP* 0.01) {

N-(2-pyrimidyl) AMDP* 0.01 ;

N-(4-(2,6-dimethyl)-pyrimidyl) AMDP* 1.0 ! . N-(2-(4,6-dihydroxy)-pyrimidyl) AMDP* 0.011) ¥ ’ 15 N-{2-pyridyl) AEDLP* 0.01 : ’ N-(2-(3-picolyl) AEDP* 10 : 2-(2-pyridyl) EDP* 0.01 5 2-(3-pyridyl) EDP* 0.01 k 2-(4-pyridyl) EDP* } 0.1 y 1-(2-pyridyl) propyl DP* 1.0 2 2-(2-pyridyl)-1l-chloroethane DP* 0.1 b - O-(2-pyridyl)-oxamethane DP* 1.0 i 0-(2-(3-picolyl))-oxamethane DP* 0.1 .

N = no activity at any of the dosage levels tested r

EHDP = ethane-l-hydroxy-1,1-DP i

Cl,MDP = dichloromethane DP p

APD = 3-aminopropane-l-hydroxy-l,1-DP %

AMDP = aminomethane diphosphonic acid, where therxing is od oo tached to the amine 8 _ wb iy ore

AEDP = 2-aminoethane-1,l-diphosphonic acid, where the BS is attached to the amine 8 v i

TT fete { ;

EDP = ethane-l,l-diphosphonic acid, where the ring is at- tached at the 2 position of the ethane propyl DP = propane-2,2-diphosphonic acid - * = compounds included in pharmaceutical compositions of 3 the present invention iy 1) = activity level questionable due to lack of dose response

EXAMPLE LV {

Patients weighing approximately 70 kilograms who are clini- cally diagnosed as suffering from hypercalcemia of malignancy are administered 0.7 mg P of 2-(2-pyridyl)-ethane-1,1-diphos- phonic acid, or its pharmaceutically-acceptable salt or ester. by a 2-1/2 hour intravenous infusion one time daily for 4 days. This treatment results in an appreciable allevatiof of the hypercalcemia of malignancy. 5

J

Similar results are cbtained when the Si 1,1l-diphosphonic acid in the above-described treatment io replaced with ;

N-(2-(5-amino)-pyridyl)-aminomethane diphosphonic acid; !

N-(2-(5-chloro)-pyridyl)-aminomethane diphosphonic acid; § .. 20 N-(2-{3-picolyl))-aminomethane diphosphonic acid; ;

N-(2~(4-picolyl))-aminomethane diphosphonic acid;

N-(2-(5-picolyl))-aminomethane diphosphonic acid; ¥

N-(2-(6-picolyl}))-aminomethane diphosphonic acid; y

N-(2-(3,4-lutidine))-aminomethane diphosphonic acid: ; 25° N-(2-pyrimidyl)-aminomethane diphosphonic acid; § ‘N-(2-pyridyl)-2-aminoethane-1,l-diphosphonic acid; i Be 2-(3-pyridyl)-ethane-1,l-diphosphonic acid; i ON 2-(4-pyridyl)-ethane-1,1l-diphogphonic acid: wos 2-(2-pyridyl)-1-hydroxy-ethane-1, l-diphosphonic acid; Te 2-(3-pyridyl)-1-hydroxy-ethane-1, l-diphosphonic acid; '

oo | OFF 4 27078 bk, § (i pyridyl)-1nyaroxy-sthane-1. 1-diphosphonic acid; freon mtn sn acid; or # harmaceutically-acceptable salts or esters thereof.

WHAT IS CLAIMED 1S: 5 . 2% A pharmaceutical composition comprising: u (a) from about 0.001 mg P to about 600 mg P of a % geminal diphosphonic acid compound, or a pharmaceuti~ # cally-acceptable salt or ester thereof, having the § structure: 4 R, RoN\ { R.,—/8 Z l Q ! c= 1032 . I 3 ' ~N LOH, ji R R KR 2/0 2 f 1 i wherein 2 is a ring selected from the group consisting ot § eiaine, pyridazine, pyrimidine and pyrazine; (Q is oxygen, 4 ~NR, =, Or a single bond; m + n is an integer from U to about 4 53 provided that when Q is a single bond, m + n is 0 or 1, i Ry is hydrogen, substituted or unsubstituted amino, amido, i hydroxy, Cy=Cyq alkoxy, halogen, carbuxylate, substituted or : unsubstituted, saturated or unsaturated alkyl having trom "1 to aboul 6 carbon atoms, substituted or unsubstituted phe- nyl or napthyl, or substituted or unsubstituted benzyl, ex-— » cept that when n = 0 and ( is oxygen or nitrogen, then Ky 4 is hydrogen, sulstituted or unsubstituted, saturated or un- : : saturated alkyl having from 1 to about 6 carbon atows, sabs- 3 tituted or unsubstituted phenyl or napthyl, or substituted 3 or unsubstituted benzyl; R, is hydrogen, or substituted or i unsubstituted, saturated or unsaturated alkyl having from 3 1 to about 4 carbon atoms; Rg is one or more substituents 4 selected From the group consisting of hydrogen, substituted wn . # and unsubstituted, saturated or unsaturated alkyl having ~ \ 1 4 oo

Claims (1)

1. Co ir t from 1 to about 6 carbon atoms, substituted and unsubsti tus ted phenyl or napthyl, substituted and unsubstituted ben- ' zyl, hydroxy, halogen, carbonyl, C,-Cy alkoxy, nitro, ami df. ‘ amino, substituted amino, carboxylate, and combinations ¥ thereof; R, is hydrogen, substituted or unsubstituted, sa turated or unsaturated alkyl having from 1 to about 4 carbn atoms, or acyl wherein R,. R,., Rj and R,., if substituted, i are substituted with a moiety selected from the group condbt- i ing of methyl, ethyl, amino, chloro, nitro, methoxy, hydraky, po acetamido, and acetate; and (b) a pharmaceutical carrier. ; Pe

   2. pa pharmaceutical composition according to Claim 1, cont - prising: ! . (a) from about 0.001 mg P to about 600 mg P of - 8 geminal diphosphonic acid compound, or a pharmaceut i cally-acceptable salt or ester themof, having the si. 2hc- ture: : 12 : 173% Ry-—— & i ¢ —_— POH, ; t R, on Ry ! wherein 7 is a ring selected from the group coniisting -§ pyridine, pyridazine, pyrimidine and pyrazine: m + n is ard] integer from UO to about 5; Ry is hydrogen, substituted 4 unsubstituted amino, amido, hydroxy, Cy -Cyalkoxy. halogen 2 25 carboxylate, substituted or unsubstituted, saturated or oo saturated alkyl having from 1 to about 6 carbon atoius, 4 tituted or unsubstituted phenyl or napthyl, or substitut: dy i unsubstituted benzyl: R, is hydrogen, or substituted or ui substituted, saturated or unsaturated alkyl having from 1 § . to about 4 carbon atoms; R, is one or more substituents qf- Pi \ TA rd - 36 - x i \

   ected from the group consisting of hydrogen, substituted 3 & and unsubstituted, saturated or unsaturated alkyl having i from 1 to about 6 carbon atoms, substituted and unsubsti-§ - tuted phenyl or napthyl, substituted and unsubstituted f benzyl, hydroxy, halogen, carbonyl, C,~-Cjalkoxy, nitro, ; amido, amino, substituted amino, carboxylate, and combi- 5 nations thercor wherein Ry: R, and Rg, if substituted, ard substituted with a moiety selected from the group consis ch ing of methyl, ethyl, amino, chloro, nitro, methoxy, byron XY acetamide and acetate; and i (b) a pharmaceutical carrier. ’

   3. A pharmaceutical composition according to Claim 1, 4 wo comprising: 3 ] (a) from about 0.001 mg P to about 600 mg P of a geminal diphosphonic acid compound, or a pharmaceut iB cally-acceptable salt or ester thereof, having the 5 structure: } i wpe k Ry— 7. 4—C y § ¢ —— POH, §

   20 . R, R, Ry 3 m n 2 wherein 2 is a ring selected from the group consisting oti pyridine, pyridazine, pyrimidine and pyrazine; m + n is 3 integer from 0 to about 5; Ry is hydrogen, substituted orl unsubstituted amino, amido, hydroxy, C,-Cjalkoxy, halogen carboxylate, substituted or unsubstituted, saturated or ulf- saturated alkyl having from 1 to about 6 carbon atoms, sulas- tituted or unsubstituted phenyl or napthyl, or cupsti tut or unsubstituted benzyl, except that when n = 0, then . PP hydrogen, substituted or unsubstituted, saturated or uns on turated alkyl having from 1 to about 6 carbon atoms, sub go

   Co ) 1 tituted or unsubstituted phenyl or napthyl, or substituted’ or unsubstituted benzyl: R, is hydrogen, or substituted of unsubstituted, saturated or unsaturated alkyl having from : } 1 to about 4 carbon atoms: Rs is one or more substituents i selected from the group consis ting of hydrogen, cubstitut and unsubstituted, saturated or: unsaturated alkyl having £ from 1 to about 6 carbon atoms, substituted and um ted phenyl or napthyl, substituted and unsubstituted bon hydroxy, halogen, carbonyl, Cy-Cjalkoxy, nitro, amido, ihe substituted amine, carboxylate, and combinations thereof: L is hydrogen, substituted or unsubstituted, saturatod or isa- turated alkyl having from 1 to about 4 carbon atoms, 1 A wherein Ris Koy Ry and Ey if substituted, are cust tuted] ~~ with a noiety selected from the group consisting of wethy ll ; 15 ethyl, amino, chloro, nitro, methoxy, hydroxy, acetamido 8 acetate; and x (b) a pharmaceutical carrier. §

   4. pa pharmaceutical composition according to Claim 1 cond prising: i 2 (a) from about 0.001 mg P to about 600 mg Pp of 8 geminal diphosphonic acid compound, or a pharmaceut i8f - ally-acceptable salt or ester thereof, having the ¥ structure: 72 2 1°32 2 Ry— ’ 0 ‘ ¢ —— PO,H, 4 26 R, R, Ry = m n = wherein 2 is a ring selected from the group consisting «fj pyridine, pyridazine, pyrimidine and pyrazine: m + n is afl§ Pp ~ integer from 0 to about 5; R, is hydrogen, substituted or} onan unsubstituted amino, amido, hydréxy. C,-Czalkoxy, watose GPO carboxylate, substituted or unsubstituted, saturated or of saturated alkyl having from 1 to about 6 carbon atoms, cull - 38 - T

   Co 711} ED : 3 : cp . a Sagi tituted or unsubstituted phenyl oF napthyl. ©F substituted y | ne or unsubstituted penzyl. except that when I = 0, then Ry ved is hydrogen. gubstituted or unsubstituted. saturated of un- { pas i saturated alkyl having from 1 to about 6 carbon atoms. subs- 8 - 5 tituted OF unsubstituted phenyl or napthyl. or substituted bo or unsubstituted penzyl: Ro is hydrogen. or substituted or unsubstituted saturated or unsaturated alkyl having from - : al 1 to about 4 carbon atoms: Rg is one OF more substituents i selected fyom the group consisting of hydrogen: substituted and unsubstituted: saturated ox unsaturated alkyl having BB from 1 tO about © carbon atoms, substituted and ansubstitul ted phenyl OF napthyl. substituted and unsubstituted | ) hydroxy. halogen, carbonyl. c,-Cqalkoxy: nitro, amido. amigb } substituted amino, carboxylate: and combinations thereof

   = . 15 wherein Ry+ Ry and Ry» if substituted: are substituted will oo a moiety selected from the group consisting of methyl. e L1, : - oo amino, chloro: nitro, methoxy. hydroxy: _cotamido and acd bl : : . tate; and 8 so (pb) a pharmaceutical carrier. 2 7

   5." BA pharmaceutical composition according to claim 2, wig e~ - in 7. is pyridine. -

   6. A pharmaceutical composition according to claim ,, eres in z is pyrimidine. : x 3 :

   7. a phapmaceutical composition accordind to ‘Claim 3, where- in 7 18 pyridine. on J aT, 3 ° ¥

   8. A pharmaceutical composition according to: Claim 2, ere- in z 18 pyrimidine. 3

   - 9. A pharmaceutical composition according to claim Bee in Z is pyridine. 3 i

   10. A pharmaceutical composition according to cain where . - 39 - | iY hIGINAL J re — a —— rT od ar AS —— . . 1 0 lak jn 2 is pyrimidine. boa Ls Foal il. A pharmaceutical composition accordind to Claim 2, wheres § nh inm+ n= l. Poa - Boom po 3 Tl . 12. A pharmaceutical composition according to claim 2 Loo wherein m + n= 2. Co Lo

   13. A pharmaceutical composition accordind to Claim 3, HE, wherein 1m + n= 0. Vo

   14. DN pharmaceutical composition according to claim 3. { wherein m + n= 1. ~ Lo

   15. A pharmaceutical composition according to claim 4, So wherein it + n=0- - L dr in - nol ’ 16. A pharmaceutical composition according to claim 4, [ Lo EE wherein m + 1 = 1. i § en . ir ' i Fa EE a 17. A pharmaceutical composition according to claim 5. 1 oo #15 wherein @ + n= 1. a : A

   18. A pharmaceutical composition according to Claim 7. - I wherein m + D7 Oe Ce : - a RS cy

   19. A pharmaceutical composition according to Claim ‘s Co wherein m + D7 0. i LL ! C an

   20. A pharmaceutical composi tion: according to: claim A . comprising: EI . ’ : R ir ov Co (a) from about 0.001 mg p to about 600 mg P Ea geminal aiphosphonic acid compound. or a .pharnac Li Ce : cally-acceptable salt or ester thereof , /fiavingd > 2 nin CL 25 structure: wo . A vol . . B . v St Rj N I 0 POs, LN oo CEL sa , Si B fn Ry ayy pC: N, BAD ORIG . - 40 - ) | AL . , a ER oo i A wherein m + n = 0 or 1. R, is hydrogen, chloro, amino, § or hydroxy: Ry is one or more substituents selected £ from the group consisting of hydrogen, methyl, amino, ? ’ chloro, methoxy, hydroxy, nitro, and combinations ; thereof: and (b) a pharmaceutical carrier. 4

   2l. A pharmaceutical composition according to Claim 2, ti comprising: ) (a) from about 0.001 mg P to about 000 wy I of a geminal diphosphonie acid compound, or a pharmaceuti- cally-accepltable salt or ester therof, having Lhe structure: . H [Oats . . R3 N i [ — FO3H, : ie 135 H fuen Ry S wherein m + n = 1 or 2: Ry is hydrogen, chloro, amino, i Or hydroxy: Ry is one or more substituents selected from 2 the group consisting of hydrogen, methyl, cmino, chloro, : methoxy, hydroxy, nit ro, and combinations thereof: and § 0 (b) a pharmaceutical carrier. 3

   22. A pharmaceutical composition acoording to Claim 3, { comprising: 2 (a) from about 0.001 mg P to about 600 wy P of a © geminal diphosphonic acid compound, or a pharumaceuti- 5 cally-acceptable salt or ester thereof, having the ; structure: : 103 { r—FOF— | —— (cH,) —— i — bon, ; § R, Ry AY 2 wherein n = 0 or 1: Ry, is hydrogen, chloro, amino, or i J BAD ORIGINAL

   . + ; v hydroxy when n = 1, and R, is hydrogen when n = 0; Rg ¥ is one or more substituents selected from the group : consisting of hydrogen, methyl, amino, chloro, methoxy, © - nitro, hydroxy, aud combinations thereof; and R, is i hydrogen, methyl, or ethyl: and & (b) a pharmaceutical carrier, .

   23. A pharmaceutical composition according to Claim 3, § comprising: 3 (a) from about 0.001 ng P to about 600 my p of a Y geminal diphosphonic acid compound, or a pharmaceuti- ; / cally-acceptable salt or ester thereof, having the i structure: . I ERE : } fo —_ x — (CH,) —— ¢ —— Log, ; R, Ag ¥ i wherein n = 0 or 1: Ry is hydrogen, chloro, amino, or i hydroxy when n = 1, and Ry is hydrogen when n :: 0; Ry ! is one or morc substituents selected from the group ¥ consisting of hydrogen, methyl, amino, chloro, methoxy, i nitro, hydroxy, and combinations thereof: and Ky is i ) - hydrogen, methyl, or ethyl: and % Bh (b) a pharmaceutical carrier. %

   24. A pharmaceutical composition according to Claim 4, 8 ~ comprising: | 8 B (a) from about 0.001 mg P to about 600 my I' of a ¥ geminal diphosphonic acid compound, or a pharmaceuti- " cally-acceptable salt or ester thereof, having the struc- i ture: i of AE £ Ry —foy 0 —— (CH,) —— i —— POH, £ w Ry 8 — ond § oO £ vo

   CE Bote wherein n = 0 ox 1; Ry is hydrogen, chloro, amino, . hydroxy when n = 1, and R, is hydrogen when n = 0; i is ome or more substituents selected from the group; - consisting of hydrogen, methyl, amino, chloro, met hbxy., nitro, hydroxy, and combinations thereof: amd ; (b) a pharmaceutical carrier. :

   25. A pharmaceutical composition according to Claim 4, . oe comprising: : (a) Crom about 0.001 mg P to about 600 mg P of B & geminal diphosphonic acid compound, or a pharmaceu ti cally-acceptable salt or ester thereof, having ene 3 structure: i £4 _ re JH, % iy —fo— 0 —— (CH,) —— i — PO, 4 Ry 3 oo wherein n = 0 or 1; Ry is hydrogen, chloro, amino, Br - hydroxy when n = 1, and Ry is hydrogen when n = 0; &, “ is one or more substituents selected from the group E consisting of hydrogen, methyl, amino, chloro, met Rixy nitro, hydroxy, and combinations thereof; and & ~ (b) a pharmaceutical carrier. | 3

   26. An pharmaceutical composition according to Claim 1, comprising: } & (a) from about 0.001 mg P to about 600 mg Pp or B geminal diphosphonic acid compound, or a pharmaceu di cally-acceptable salt or ester thereof, selected i Jn group consisting of ¥ N-(2-(5-amino)-pyridyl)-aminomethane diphosphonic ai a; N-(2-(5-chloro)-pyridyl)-aminomethane diphosphonic fio. N-(2-(3-picolyl))-aminomethane diphosphonic acid: ; N-(2-{(4-picolyl))-aminomethane diphosphonic acid: N-(2-(5-picolyl))-aminomethane diphosphonic acid; ; ae eo - 43 - i oo = £ N-{(2-(6-picolyl))-aminomethane diphosphonic acid; E N-(2-(3,4-luftidine))-amincinethane diphosphonic acids} » N-{2-pyrimidyl)-~aminomethane diphosphonic acid; 5 - N-(2-pyridyl)-2-aminoethane-1, 1-diphosphonic acid; 8 2-(2-pyridyl)-ethane-1, 1-diphosphonic acid; ’ 2-(3-pyridyl)-ethane-1,l-diphosphonic acid; 2-(4-pyridyl)-ethane-1,1-diphosphonic acid: i O=(2-(3-picolyl))-oxamethane-diphosphonic acid; and 5 {(b) a pharmaceutical carrier. :

   27. A pharmaceutical composition according to Claim 1, ; comprising: (a) from about 0.001 mg P to about 600 my I of : we i= (2={3-picolyl))-aminomethane diphosphonic aciu, ork a pharmaceutically-acceptable salt or ester enc f and id (b) a pharmaceutical carrier. k : E

   28. A pharmaceutical composition according to Claim 1, ¥ comprising: ly 3 (a) from about 0.001 mg P to about 600 wg F of ; 20 2-(2-pyridyl)-ethane-1, l-diphosphonic acid, or a Eg pharmaccutically-acceptable salt or ester thereof; Ha (b) a pharmaceutical carrier.

   29. A method of treating diseases characterized by abn. rfial : calcium and phosphate metabolism, comprising niece to a person in need of such treatment a safe and effectiyl & amount of a composition of Claim 1. E ’ Fo

   30. A method of treating diseases characterized by abnodlal, calcium and phosphate metabolism, comprising administer: : to a person in need of such treatment a safe and eflectiv amount of a composition of Claim 2. i ae po” - 44 - | Ae m+ eT . Lie T : LL CO ~ Lo CAE - Val

   31. DB method of treating diseases characterized py abnormse 3 calcium and phosphate metabolism, comprising administering pe to a person in need such treatment a safe and effective Co ’ amount of 2 composition of claim 3. - oo oo 5 32. A method of treating diseases characterized by abnordBl oo calcium and phosphate metabolism, comprising aninistering : to a person in need such treatment 2a safe and effective 4 amount of a composition of claim 4. 3

   33. NB method of treating diseases characterized by abn. Mal oo calcium and phosphate metabolism, comprising sdminister ing to a person in need of such treatment a safe and effect ile . : oo amount of a composition of claim 5. 5 Cy 34. A method of treating diseases characterized by abndnal calcium and phosphate metabolism, comprising aminister ig to a person in need of such treatment 2a safe and effective amount of a composition of claim 21. i :

   35. Bb method of treating giseases characterized by abnbrnal calcium and phosphate metabolism, comprising inisteidng : h to a person in need of such treatment 2a safe and rece amount of 2a composition of claim 22.

   36. A method cf treating diseases characterized by abfiorinal calcium and phosphate metabolism, comprising administeling : to a person in need of such treatment a safe and eticdbive . amount of 2 composition of claim 23. $ gE

   37. A method of treating diseases characterized by $buormal calcium and phosphate metabolism, comprising ministering : to a person in need of such treatment a safe and oor ive amount of a composition of claim 24. # :

   38. bn method of treating diseases characterized by ¢ normal. ; 30 calcium and phosphate metabolism, comprising adninisgering

   \ .. | ¥ BAD ORIGHAL 9

   They J nk eel . 79 P Cod 7 Lo i Fo p . Cw ’ ! eT ¥ . .. i

   . to a person in need of such treatment a safe and effectiVt { ; \ amount of a composition of Claim 25. i :

   39. A method of treating diseases characterized by abnotmal ir & : calcium and phosphate metabolism, comprising aaministerigs to a person in need of such treatment a safe and effective ! amount of a composition of Claim 26. i

   40. A method of treating diseases characterized by I» calcium and phosphate metabolism, comprising administerigg

   ' . to a person in need of such treatment a safe and ef tectife . » yy amount of a composition of Claim 27. 4

   41. A method of treating diseases characterized by abnormal Se calcium and phosphate metabolism, comprising administer ing * % to a person in need of such treatment a safe and el fective a.wount of a composition of claim 28. 3 : fi

   42. A method of treating diseases characterized by mofnal calcium and phosphate metabolism, comprising administer 2 to a person in need of such treatment a safe and citectile amount of a composition of Claim 29. & XN - * * * * * * * * * * * * * * % * % / James J. BENEDICT k- bo Christopher M. PERLINGE : Inventors Hl : NL. i ‘ $4 ! : } 3 oo 7 } BAD ORIGINAL 9