CA1288695C

CA1288695C - Method for increasing bone mass

Info

Publication number: CA1288695C
Application number: CA000505901A
Authority: CA
Inventors: John T. Potts, Jr.; Robert M. Neer; David M. Slovik
Original assignee: General Hospital Corp
Current assignee: General Hospital Corp
Priority date: 1985-04-04
Filing date: 1986-04-04
Publication date: 1991-09-10
Anticipated expiration: 2008-09-10
Also published as: AU5561686A; ZA862510B; DK172816B1; JP2531505B2; JPS6233A; EP0197514B1; DE3686343D1; DK155686D0; JPH0772138B2; IE860891L; DK155686A; DE3686343T2; IE59620B1; US4698328A; ATE79271T1; EP0197514A1; PH23720A; IL78342A; AU599905B2; JPH07179358A

Abstract

ABSTRACT OF THE DISCLOSURE

A method for increasing bone mass in a human af-flicted with osteoporosis or a similar disease which comprises administering to the human so afflicted an effective amount of a composition comprising a para-thyroid hormone or physiologically active fragment thereof, or equivalents thereof, in combination with either (a) a hydroxylated Vitamin D compound, or a structural or functional analogue thereof, or (b) a dietary calcium supplement. Pharmaceutical composi-tions containing the necessary components are also disclosed.

Description

s TITLE OF THE INVENTION
;
M~T~OD OF I~CR~ASING BONE MASS

; BACKGRoUND O~ THE INVENTION
Field of the Invention:
The present invention relates to a pxocess for increasing bone mass in a human afflicted with osteoporosis.

Description of the Prior Art:
Osteoporosis is a generic description for a group of diverse diseases which are characterized by a reduction in the mass of bone per unit volume with a histologically normal ratio of osteoid to bone. The effects of these diseases are particularly severe when the mass per unit volume decreases to a level below that required for adequate mechanical support.
Osteoporosis is a particularly important cause of morbidity in the elderly. The most frequent symptoms are back pain and deformity of the spine resulting from a collapse of the vertebrae, especially in the lumbar and thoracic spine regions.
The problem presented worldwide by osteoporosis is truly staggering. It is estimated that in the United States alone, there are millions who symptoma-tically exhibit this disease. The disease appears to , be an invariable accompaniment of aging, particularly among womenr and the incidence is increasiny with the age expectancy of the population. Osteoporosis often I occurs when there is malnutrition, as a complication I due to immobilization and paralysis, as a result of j liver, kidney, or intestinal disease, and also after ~ or during various forms of endocrine or rheumatic I diseases. As improved public health measures increase I life expectancy in third world nations and under-developed countries, the incidence of osteoporosis is beginning to be recognized as a significant problem in these countries as well. For these reasons, a treatment that can arrest the course of this widely present, destructive disease represents a significant medical advance.
Various~ methods have been discussed in the prior art for increasing bone mass in humans with osteopo--rosis. These treatments include administration of sodium fluoride, androgens, biologically active forms l of parathyroid hormone alone, calcitonin, and calci-¦ tonin in combination with high dietary phosphate.
Except for treatment with sodi~n fluoride, the effects of these treatments are modest. Sodium , fluoride treatment increases trabecular bone in some j patients but has uncertain effects on total bone mass and bone strengtht a high risk of osteomalacia, as well as other unpleasant side effects.
In addition to these methods of treating osteo-porosis by increasing bone mass, other methods are known which are designed to preserve existing bone mass. These methods involve the use of estrogens or , -:. .
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calcium alone, or l-hydroxy vitamin D3 or 1,25-dihy-droxy vitamin D3 alone.
Typical publications disclosing and discussing these prior art methods include the following.
Brugger et~al., U.S. Patent 3,956,260, discloses the preparation and use of a synthetic polypeptide for I the treatment of osteoporosis. This polypeptide is ! unrelated to that of the present invention. Christie et al~, U.S. Patent 4,241,051l teaches topical , application of the hormone calcitonin for the j treatment of diseased bone in the ear.
Reeve et al., British Medical Journal, 280:1340 (1980), describes the results of a multicenter trial j evaluating the effect of a fragment of human para-thyroid hormone (hPTH (1-34)) on osteoporosis in ¦ humans. The authors report that this regimen resulted in a considerable increase in axial trabecular bone, but calcium retention, and hence, total bone mass, improved in only about half of these patients. In ¦ other patients calcium retention worsened and there-j fore, total bone mass decreased. The authors specu-late that hPTH(1~34) might be most effective if administered in combination with estrogen, calci-tonin, a diphosphonate, or some other agent that would limit resorption while allowing bone formation to continue.
Reeve et al., in Monoclonal Antibodies And Developments In _Immunoassay, p. 239, published by Elsevier/North-}lolland Biomedical Press (1981), report their progress towards answering some of the questions raised in the above study. The authors s~ate that their studies of intestinal calcium absorption point to a possible defect in osteoporosis and further speculate that it may be necessary to circumvent this defect with, for example, 1,25-~OH)2 vitamin D3 in modest dosage, given during intervals when no hPTH injection is administered.
Hefti et al~, Clinical Science, 62:389 (1982), describes studies using a high calcium diet supple-mented with either parathyroid hormone or 1,25-(OH)2 vitamir? D3 using normal and osteoporotic adult rats.
The authors report that, although these studies show-ed an increase of whole-body calcium and skeletal mass, there was no restoration of individual trabecu-lae lost during the development of osteoporosis. Endo et al., Nature, 286:262 (1980), discuss the use of metabolites of vitamin D in conjunction with para-thyroid hormone (PT~) to stimulate bone formation in vitro. However, these treatments with PTH and 1,25-(OH)2 vitamin D3 were no more effective than PTH
alone in stimulating re-calcification of bone.
Rader et al., Calcified Tis~ue International, 29(1):21 (1979), describes the treatment of thyro-parathyroidectomized rats with dietary calciurn and intraperitoneal injection of a parathyroid extract.
Although this treatment stimulated 1,25-(OH)2 vitamin D3 production and effected a marked increase in bone mineralization, it was also found to produce bone resorption as evidenced by the appearance of cavities in the cortical bone. There was no effect on rates of bone formation, or bone matrix apposition. Wong et al.~ Surqical Forum, 30:100 (1979), teach the administration to thyroparathyroidectomized do~s of daily intramuscular parathyroid extract or oral 1,25-~OH)2 vitamin D3 simultaneously with thyroid replacement therapy. The effect of these treatments on absorption of dietary calcium is discussed in the context of parathyroidism although not in the context of osteoporosis.
¦ Peacock et al., Vitamin D Proceedin~s Workshop., I E. Norman, Ed., p. 411 (1977)/ disclose the inhibi-tion by calcitonin and steroid sex hormones of the resorptive effect of vitamin D metabolites and para-thyroid hormone on mouse calvaria bone iD tissue ¦ culture. Pechet et al., American Journal of Medi-, cine, 43~5):696 (1967), teach that minimum levels of ¦ parathyroid hormone are necessary in order for ¦ vitamin D to exert its effects on bone resorption rather than bone formation. In Mahgoub et al., Biochemical and BiophYsical Research CommunicationSt 62:901 (1975)~ the authors describe experiments and ¦ state that active vitamin D metabolites (~5-o~
vitamin D3 and 1,25-(OH)2 vitamin D33 potentiate the ability of parathyroid hormone to elevate the cyclic AMP levels of cultured rat fetal bone cells.
None of these methods, however, have provided a clinically useful techni~ue for treating osteoporosis and related disorders and often cause undesirable side effects. As a consequence, there is still a need for a generally effective treatment having minimal side effects.

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SUMMARY OF THE INVENTION
Accordingly, it is an object of this invention to provide a method for increasing bone mass in a human afflicted with osteoporosis.
It is a further object of this invention to pro-vide a pharmaceutical composition which can be used for increasing bone mass in a human afflicted with osteoporosis.
These and other objects of the invention, as will hereinafter become more readily apparent, have been accomplished by providing a method for increasing bone mass in a human which comprises administering to said human a parathyroid hormone or physiologically active fragment thereof, or equivalent thereof, in combination with (a) a hydroxylated vitamin D com-pound, or a structural or functional analogue thereof, or ~b) a dietary calcium supplement.

DESCRIPTION OF THE DRAWINGS
Figure 1 shows the changes in trabecular bone density accompanying therapy according to the invention in five adult males with idiopathic osteoporosis.

DESCRIPTION OF THE PREFERRED_EMBODIMENTS
The inventors have devised a method of increasing bone mass which represents a significant improvement over the prior art methods intended to accomplish this effect. The present invention comprises the administration, to a human afflicted with osteoporo-sis, of a combination of a parathyroid hormone or physiologîcally active fragment thereof, with either a hydroxylated vitamin D derivative, or a structural :

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or functional analogue thereof, or a dietary calcium supplement. The invention also comprises pharmaceuti-cal compositions intended for use in this method.
The parathyroid hormone appears to stimulate bone formation. The hydroxylated vitamin D component seems to act both to increase the intestinal absorp-tion of calcium and to positively affect bone meta-bolîsm either directly or through its effects on cal-cium absorption from the intestines. Alternatively, a high calcium dietary supplement appears to increase calcium absorption from the intestines, thereby shifting the equilibrium of bone formation and bone resorption in favor of bone formation. The synergis-tic effects of these treatments are demonstrated hereinafter in clinical studies.
The present invention is intended to be used in all diseases classified as osteoporosis, particularly post-menopausal osteoporosis, senile osteoporosis, idiopathic osteoporosis, immobilization osteoporosis, post-partum osteoporosis, juvenile osteoporosis, and osteoporosis secondary to gonadal insufficiency, malnutrition, hyperprolactinemia, prolactinoma, dis-orders of the gastrointestinal tract, liver, or kid-neys, and osteoporosis that is a sequella of prior osteomalacia, chronic acidosis, thyrotoxicosis, hyperparathyroidism, glucocorticoid excess or chronic disorders involving the bone marrow, and heritable fonns of osteoporosis such as osteogenesis imperfecta and its variants, and other heritable disorders of connective tissue.
The first component involved in the method of the invention is a "parathyroid hormone~, n or fragment thereof héreafter abbreviated "PTHF." PT~F may . : :

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consist of the flrst twenty-six, twenty-eight, thirty-four or any other physiologically active - number of amino acids (numbered from the amino `1 terminal) of a parathyroid hormone obtainable from a ¦ human or other vertebrate.
- The term "obtainable" is intended to indicate that the PTHF is not necessarily deriv~d from animal-produced parathyroid hormone but may be synthetically made, based on a natural model.
The term "fragment" is not intended to eliminate compounds larger or smaller khan those specifically shown, but is intended to include all components obtainable from naturally occurring parathyroid hormones, up to and including or beyond the natural compounds themselves.
PTHF also- encompasses chemically modified para-thyroid hormone fragments which retain the activity associated with parathyroid hormone. The necessary activity is the stimulation of bone formation. Modi-fications that may be considered include:
(1) PT~F with carboxyl amino acid extensions beyond position 34 up to or beyond position ! 84 of the human PTH moleculel or amino-terminal extensions, or amino acid substitu tions that produce other desirable features, such as an alpha-carboxyl amide at the carboxyl terminus. A desirable modification should delay metabolism and/or enhance activity ln vlvo;

(2) PTHF extended to include amino acids 1~38, which would enhance receptor binding and hence the activity per mole;

(3) PTHF~with D-amino acid substitutions so as to delay metabolism and thereby enhance potency in vivo;

(4) PTHF chemically modified so as to enhance its absorption through the skinr mucous membranesr or gastro-intestinal tract to avoid thereby the necessity of parenteral injection; and (5) physiologically acceptable salts and esters of PTHF.
A PTHF obtainable fxom a mammal (PTHF 1-34) is generally preferred over other types of parathyroid hormone fragments, such as derivatives. Use of a PTHF consisting of the first thirty-four amino acid residues of human parathyroid hormone (hereafter abbreviated~"hPTHF 1-34"~ is especially preferred for use in humans. Other preferred PTHF's are those which display some or all of the following desirable features: increased potency with regard to the neces-sary activityt increased ease of administration, increased selectivity to decrease potential side effects, and decreased antigenicity in humans to avoid an adverse immune response. PTHF molecules having the following formulas are particularly preferred:

H2N-Ser-Val-Ser-Glu-Ile-Gln-Leu--Met-His-Asn-Leu-Gly-Ly~s-His-Leu-Asn-Ser-Met-Glu-Arg-Val-Glu-Trp--Leu-Arg-Iys-Lys-Leu-Gln-Asp-Val-His-Asn-Phe-COOH

, ' Ranges of administration of hPTHF 1-34, for exam-ple, are 100-700 units/day, more preferably 200-600 units/day, and most preferably 400-500 units/day, wherein 'lunits" are defined in terms of the Inter-national Reference Preparation of hPTHF 1-34 and comparative bioassays in one of the established PTH
bioassays. Potency ratios o different PT~ analogues differ in different assays. The "units" are expressed in the chick hypercalcemic assay.
For other PTHF molecules, the ranges of admin-istration are those high enough to stimulate bone remodeling in humans, yet not so high as to produce net bone resorption nor enough bone mineral mobili-zation to produce hypercalcemia or hypercalciuria. For compounds other than hPT~ 1-34, dosage can be quanti-tated on a weight basis, or in terms oE an appropri-ately established reference standard.
~ ydroxylated vitamin D compounds of the invention include l-alpha-hydroxy vitamin D3 and l-alpha,25-di-hydroxy vitamin D3 and their vitamin D2 analogues:
l-alpha-hydroxy vitamin D2 and 1-alpha,25-dihydroxy vitamin D~. These molecules have the following formulas:
X
~æ
~/ ~OH

cP a~
Jl Jl ~, CH ! ~ CH2 H~ oH HO ~
l-alpha-hydroxy l-alpha,25-dihydroxy vitamin D vitamin D
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wherein the bond between C-22 and C-23 may be single or double and wherein X may be H or -CH3.

Chemical variations which retain the characteris-tics of these hydroxylated vitamin D molecules are contemplated as ecluivalents. Preferred are those like the glucuronide and sulfate derivatives of the above, dihydrotachysterol, 5,6-trans forms of vitamin D, and all other forms of vitamin D capable oE stimu-lating the intestinal absorption of calcium without the necessity for prior hydroxylation at carbon 1 by the patient. Most preferred are compounds which also stimulate absorption of calcium for brief periods of time (days rather than weeks) after their aclministra-tion is discontinued. These latter compounds are useful because they permit rapid reduction in bio logieal effeet in the event of overdose. Very large doses of vitamin D and 25-OH vitamin D stimulate the intestinal absorption of caleium without prior hy-droxylation at earbon 1, but the resultant biologieal effeets often last or weeks or months after adminis-tration of the drug has been halted, thus exposing the patient to a risk of toxieity.
The dose ranges for the aclministration of the vitamin D eomponent are those large enough to produee the eharaeteristic effeets of vitamin D, partieularly an enhaneed intestinal absorption of ealeium in osteoporotie patients, but not so large as to produee hypercalciuria or hypercaleemia. Possible dose ranges of various vitamin D analogues are illustrated in Table 1.

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Tab~e~l . RANGEa Vitamin D Mbst Analo~ue Broad Preferred Preferred l-alpha,25-(OH)2 Vit D3 or q.05-2.0 0.1-1.0 0.25-0.50 l-alpha,25-(OH)2 Vit D2 l-alpha-OH Vit D3 or . . 0.05-3.0 0.1-1.5 0.25-0.75 l-alpha-OH Vit 25-C~I Vit D
or 3 10-150 20-100 20-50 25-OH Vit D2 Vit D 1250 ncg. 1250 ~cg 1250 mcg or 3 every 3 days on alternate 3 x Vit D to 3750 mcg/ & ys to weekly 2 day. 2500 mcg/day.

.
dihydrotachysterol 0.2-1.2 0.2 0.6 0.2-0.4 mg/da~ ng/day mg/day a Units in mcg/day unless other~ise note~.

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By "dietary calcium supplement" as used in this invention is meant supplementing the normal diet with calcium at a level greater than that level which is recommended as the daily dietary allowance. In normal adult humans, the recommended daily allowance is 20-25 millimoles calcium/day and slightly higher in postmenopausal women, yet the customary intake of calcium among adults in the ~.S. is only 12-18 millimoles/day. Since in many osteoporotic humans, the intestines are inefficient in absorbing calcium, this sub-optimal calcium diet merely serves to aggra-vate their osteoporosis. Accordingly, a dietary calcium supplement for an adult would involve the administration of sufficient calcium to increase the total oral intake of diet plus supplement to 38-50 millimoles/day. When a dietary calcium supplement is used, the calcium is administered in a non-~oxic form. The dosage rates mentioned herein refer to the amounts of calcium present, and the dosage rate of the actual compound used can be easily calculated therefrom using the formula weight of the compound being administered. Milk or any non-toxic salt of calcium may be utilized provided that the counter ion is not toxic to the human in which it is being admin-istered. Typical suitable non-toxic counter ions include carbonate, citrate, phosphate, gluconate, lactate, chloride, and glycerol phosphate. The upper limit o the dietary calcium supplement is determined by the toxic effects of calcium, which varies slight-ly from patient to patient, as is well understood by those skilled in the art. Typically, in humans, the maximum allowance per day is 2000 mg calcium per day.
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Use of the method of the invention is aided by pharmaceutical combinations comprising the ingredi--ents intended for use in the method of the invention.
Such pharmaceutical combinations were not suggested by the prior art since the method of the invention, which involves using specific combinations of ingre-dients, was unknown to the~prior art.
By "pharmaceutical combination" as used herein is meant to include intimate mixtures of the two compo-nents of the invention, as in classical compositions, and also non-mixed associations, such as those found in kits or pharmaceutical packs.
A typical pharmaceutical mixed composition of the invention would contain PTHF in combination with a hydroxylated vitamin D compound or calcium in a form suitable for use as a dietary calcium supplement. The composition may additionally contain a pharmaceuti-cally acceptable carrier and, if intended for oral administration, may comprise the PT~F in a liposome to protect this component.
The components can be administered parenterally by injection, rapid infusion, nasopharyngeal absorp-tion, dermal absorption, and orally. Preparations for parenteral administration include sterile or aqueous or non-aqueou8 solutions, suspensions, and emulsions. Examples of non-aqueous solvents are pro-pylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Carriers or occlusive dressings can be used to increase skin permeability and enhance drug absorption. Liquid dosage forms for oral admin-istration may generally comprise a liposome solution "

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containing the liquid dosage form. Suitable forms Eor suspending ~he liposomes include emulsions, sus-pensions, solutions, syrups, and elixirs containing inert diluents commonly used in the art, such as purified water. Besides the inert diluents, such com-positions can also include adjuvants, such as wetting agents, emulsifying and suspending agents, and sweet-ening, flavoring, and perfuming agents.
The invention also relates to a method for preparing a medicament or pharmaceutical composition comprising the components of the invention, the medicament being used for increasing bone mass.
The materials for use in the administration of the invention are ideally suited for the preparation of a kit. Such a kit may comprise a carrier means being compartmentalized to receive in close confine-ment one or more container means such as vials, tubes and the like, each of said container rneans comprising one of the separate elements to be used in the method as well as such means as syringes and needlesr sprays or dermal patches for administration of said elements.
For example, one of the container means may contain parathyroid hormone fragment ~1-34) in lyophilized form or in solution. A second container may comprise a hydroxylated Vitamin D compound or a dietary calcium supplement in tablet form or in solu-tion. The carrier means may also contain a third container means comprising a buffer solution for rehydrating lyophilized components present in the kit.

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-1.6-The above disclosure generally descrlbes the present invention. A more complete understand-iny can be obtained by reference to -the following specific examples which are provided herein for purposes of illustration only and are not intended to be limiting unless otherwise specified.

The effects of the methods and composi-tions of the invention were investigated in human patients. Three adult males afflicted with osteoporosis were given 500 units of a human parathyroid hormone fragment (hPTHF 1-34) and 0.25 ug of 1,25-dihydroxy vitamin D3 (1,25-(OH)2 D3) daily for a period of six to twelve months while consuming a normal calcium diet (15-20 mmoles/day)~ A fourth patient was given the same amount of parathyroid hormone fragment while receiving a high calcium intake (to-tal intake more than 50 mmoles Ca/day).
The effects of these treatments on calcium and phosphorus balances are shown in Table 2. The effec-t of these -trea-tments on bone density is shown in Table 3.
The balance measuremen-ts involve complete collection and chemical analysis of all excreta for 18 days, during which the patient ate exactly the same foods each day. The mineral excreted were compared to those inges-ted to determine the total body retention, or "balance," for each patien-t.
Cortical bone density was determined in the Eorearm by measuring the attenuation of radioac-tive 1 5I
photons by the sha:Et o:E -the radius. Trabecular bone density was determined in the spine by quantitative computed - ~

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x~ray tomography measurements of the lumbar ~ertebral bodies. These techniques are widely used for such purposes by those of skill in the art.

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An improvement in calcium balance can be noted in Table 2. Failure to observe consistent improvement in calcium balance was one negative factor previously experienced with parathyroid hormone when it was administerea without effort to au~ment intestinal absorption of calcium beyond provision of a diet containing the recommended dietary allowance for calcium.
Serial maintenance of cortical bone density in the forearm of three of these same patients are shown in Table 3. The control measurements were made over several months for each individual, and the values on treatment also reflect measurements made at intervals at sev~ral mo~ths each.

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Computerized tomography was used to measure bone density in the spine of patient 4. These measure-ments were performed on three lumbar vertebral (Ll through L3). A single 1 cm thick section was obtained through the mid-vertebral body using the scout view for localization. After the first series of scans, the patient was allowed to move freely before the duplicate set of measurements was taken. The results of the scans are shown in Table 4.

Table 4 Bone Density After 11 Months Of Treatment Readin~ 1 Readinq 2 Ll 91.1 90.7 L2 88.0 . 90.2 L3 83.2 90.7 Bone Densit After 17 Months Of Treatment y Ll111 107 Bone Density_at End of Treatment ; Ll104 109 : L2110 104 ~3106 107 The bone densities shown in Table 4 which were taken after eleven months of treatment are approxi-mately 2 standard deviations below the mean for a man , , .

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-22 ~2~ 5 of this patient's age. By comparison, bone density measurements made at -the end of treatment according to the method of the invention show an incre~se in bone density over the nine-mon-th treatment interval of approximately 20~.

Five adul-t males with idiopathic os-teoporosis were treated wi-th hPTHF(1-34) and 1,25-(OH)2 D3 (patients 5-8) or with hPTHF(1-34) and calcium (patient 4) using dosages as described in Example 1. The results of this study are presented graphically in Figure 1. The trabecular bone density measurements in individual lumbar vertebral bodies are expressed as K2HPO4 equivalents. All patients showed a significant increase in vertebral trabecular bone density during treatment.
Patient 4 showed a steady increase in bone densities until the twentieth month of therapy, at which time treatment was discontinued. When bone densities were measured fourteen months after the cessation of therapy, this patient's bone density had again declined. This further indicates the effectiveness of the combined therapy according to the invention in reversing the effects of osteo-porosis on trabecular bone density of -the vertebrae.
In patients 7 and 8, it was impossible to measure several of the vertebrae because of vertebral Eractures preceding treatment. These figures denote a progressive and consistent improvement in the -trabecular bone clensity of these patients.
Cortical bone density was measured in the forearm of these same patients prior to treatment and every ,~

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three months during treatment. The density measure-ments showed no consistent change, The invention now being fully described, it will be apparent to one of ordinary skill in the art that many chang,es and modifications can be made thexeto without departing from the spirit or scope of the invention as set forth herein.

Claims

1. A pharmaceutical combination useful for the for the treatment of osteoporosis in humans comprising a parathyroid hormone or physiologically active fragment thereof, in combination with:
(a) a hydroxylated Vitamin D compound, or (b) a non-toxic calcium salt, and a carrier for said combination which is pharmaceutically acceptable to humans.

2. The combination of claim 1, wherein said parathyroid hormone fragment is a peptide consisting of the first 34 amino acid residues of a parathyroid hormone obtainable from a human or animal.

3. The combination of claim 2, wherein said hormone fragment is obtainable from a human.

4. The combination of claim 1, wherein said fragment has the amino acid sequence:

5. The pharmaceutical combination of Claim 1, wherein said combination comprises from 100 to 700 units of said parathyroid hormone fragment.

6. The pharmaceutical combination of Claim 2, wherein said combination comprises from 200 to 600 units of said parathyroid hormone fragment.

7. The pharmaceutical combination of Claim 3 and 4, wherein said combination comprises from 400 to 500 units of said parathyroid hormone fragment.

8. The combination of Claim 1, wherein said combination comprises 1-hydroxy Vitamin D or 1,25-di-hydroxy Vitamin D.

9. The combination of Claim 1, wherein said Vitamin D compound is 1,25-dihydroxy Vitamin D.

10. The combination of Claim 1, wherein said combination contains from 0.05 to 150 mcg of said Vitamin D compound.

11. The combination of Claim 8, wherein said combination contains from 0.05 to 3.0 mcg of said Vitamin D compound.

12. The combination of Claim 9, wherein said combination contains from 0.05 to 2.0 mcg of said Vitamin D compound.

13. The combination of Claim 1, wherein said combination comprises calcium coupled to a non-toxic counter ion.

14. The combination of Claim 13, wherein said non-toxic counter ion is carbonate, citrate, phosphate, gluconate, lactate, chloride and glycerol phosphate.

15. The combination of Claim 1, wherein said combination contains from 12.5 to 50 mmoles of said calcium salt.

16. The combination of Claim 14, wherein said calcium salt is calcium carbonate.

17. A kit useful for administering a bone mass increasing composition comprising a carrier being compartmentalized to receive in close confinement therein one or more containers wherein:
(a) a first container or series of con-tainers contain parathyroid hormone obtainable from a human, or other animal;
(b) a second container contains a hydroxylated Vitamin D compound or a non-toxic salt, and (c) a third container contains a buffer for reconstituting or diluting com-ponents of said kit, said kit con-taining at least one container of each of the three caterogies (a), (b) and (c).

18. The kit of claim 17, wherein said kit contains means for administering said first agent and said second agent.

19. The kit of claim 17, wherein said first container contains a peptide fragment of parathyroid hormone.

20. The kit of claim 19, wherein said peptide fragment consists of the first 34 amino acid residues from the amino terminal of parathyroid hormone.

21. The kit of claim 17, wherein said synergistic agent is a hydroxylated Vitamin D compound.

22. The kit of claim 21, wherein said compound is 1-hydroxy Vitamin D.

23. The kit of claim 21, wherein said compound is 1, 25-dihydroxy Vitamin D.

24. The kit of claim 17, wherein said synergistic agent is a dietary calcium supplement.