Treatment of osteoporosis

A method of treating osteoporosis is disclosed by administering orally a single daily dosage of phosphate in the evening in an amount, e.g., about 10 mg/kg body weight or less which is effective to raise serum parathyroid hormone levels of the subject being treated.

FIELD OF THE INVENTION 
The present invention is generally in the field of endocrinology and 
treatment of conditions associated with impairment in normal hormone 
levels. More specifically, the present invention provides a composition 
and method for increasing the level of the parathyroid hormone (PTH) in a 
needy subject. In accordance with a preferred embodiment, the method and 
composition are used for the treatment of osteoporosis. 
PRIOR ART 
The following is a list of prior art believed to be relevant as a 
background of the invention: 
1. Rodan, G. A., Mechanical loading, estrogen deficiency, and the coupling 
of bone formation to bone resorption, J. Bone Miner. Res., 6:527-530, 
1991. 
2. Juppner, H., Abou-Samra, A. B., Freeman, M., Kong, X. F., Schipani, E., 
Richards, J., Kolakowski, L. F., Hock, J., Potts, J. T., Kronenberg, H. 
M., and Segre, G. V., A G-protein-linked receptor for parathyroid hormone 
and parathyroid hormone-related peptide, Science, 254:1024-1026, 1991. 
3. Mosekilde, Li, Danielsen, C. C., Sogaard, C. H., McOsker, J. E., and 
Wronski, T. J., The anabolic effects of parathyroid hormone on cortical 
bone mass, dimensions and strength-assessed in a sexually mature, 
ovariectomized rat model, Bone, 16:223-230, 1995. 
4. Reeve, J., Meunier, P. J., Parsons, J. A., Bernat, M., Bilvoet, O. L. 
M., Courpron, P., Edouard, C., Klenerman, L., Neer, R. M., Renier, J. C., 
Slovik, D., Vismans, F. J. F. E., and Potts, J. T., Anabolic effects of 
human parathyroid hormone fragment on trabecular bone in involutional 
osteoporosis: A multicentre trial, Br. Med. J., 280:1340-1344, 1980. 
5. Dempster, D. W., Cosman, F., Parisien, M., Shen, V., and Lindsay, R., 
Anabolic actions of parathyroid hormone on bone, Endocrine Rev., 
14:690-709, 1993. 
6. Finkelstein, J. S., Klibanski, A., Schaeffer, E. H., Hornstein, M. D., 
Schiff, I., and Neer, R. M., Parathyroid hormone for the prevention of 
bone loss induced by estrogen deficiency, New Eng. J. Med., 331:1618-1623, 
1994. 
7. Prank, S., Nowlan, S. J., Harms, H. M., Kloppstech, M., Brabant, G., 
Hesch, R. D., and Sejnowski, T. J., Time series prediction of plasma 
hormone concentration. Evidence for differences in predictability of 
parathyroid hormone secretion between osteoporotic patients and normal 
controls, J. Clin. Invest., 95:2910-2919, 1995. 
Acknowledgement of the above references herein will be made by indicating 
their number from the above list. 
BACKGROUND OF THE INVENTION 
Osteoporosis results in bone fractures in about 50% of postmenopausal women 
and is a leading cause of disability in an aging population. Current 
therapies include an adequate calcium and vitamin D intake as well as 
specific treatment with compounds such as estrogens, calcitonin and the 
bisphosphonates.sup.(1). However, each of these treatments has either 
troubling side effects or limited efficacy. Women fear the small increase 
in breast cancer due to estrogens despite the dramatic reduction in 
myocardial infarctions and reduction in bone resorption. Calcitonin has a 
limited effect and is a protein and therefore needs to be injected or 
inhaled which is inconvenient. The new bisphosphonates such as alendronate 
have had encouraging results with an increase in bone density and decrease 
in fractures with few side effects. Current research for new compounds has 
concentrated on the systemic administration of bone anabolic compounds 
such as parathyroid hormone (PTH) or fragments of PTH or locally acting 
cytokines or bone growth factors such as bone morphogenic proteins. The 
appeal of PTH is that there are specific PTH receptors in bone.sup.(2) and 
it is well established in both experimental animal.sup.(3) and patient 
studies that intermittent doses of injected PTH is the most effective 
agent known to increase bone formation and bone strength.sup.(4-6). This 
effect is additive to that of estrogens. The problem with the 
administration of PTH is that it is a peptide and must therefore be given 
by injection. Women with osteoporosis have intact but inadequately 
functioning parathyroids and an alternative approach is to stimulate their 
own parathyroids to synthesize and secrete more PTH. It has been evidenced 
in experimental animals that this is eminently practicable and this is the 
basis of this present patent application. 
Postmenopausal osteoporotic women do not have the appropriate increase in 
nocturnal serum PTH levels. In osteoporotic women, careful and repeated 
measurements of serum PTH, and analysis by time series prediction of 
plasma hormone concentration has shown that there are differences in the 
predictability of parathyroid hormone secretion between postmenopausal 
osteoporotic patients and postmenopausal non-osteoporotic 
controls.sup.(7). 
SUMMARY OF THE INVENTION 
It is the object of the present invention to provide a method and 
composition for increase in PTH levels in needy subjects. 
It is an object in accordance with a preferred embodiment of the invention 
to provide such a method allowing to restore a circadian rhythm of PTH 
serum levels similar to that existing in normal individuals. 
It is an objection in accordance with another preferred embodiment of the 
invention to provide a composition and method for the treatment of 
osteoporosis. 
These and other objects of the invention will become clear from the 
description below. 
In accordance with the present invention, it has been found that 
administration of phosphate results in an increase in PTH levels. 
Furthermore, in accordance with the invention, the phosphate is preferably 
administered to individuals in the evening or at night, giving rise to 
increase in PTH levels during night time, as is the case in normal 
individuals. 
The present invention thus provides novel means for the treatment of 
individuals suffering from conditions resulting from impairment in PTH 
levels, by the administration of phosphate. The treatment has utility both 
in human and veterinary medicine. 
The present invention thus provides a method of treatment of conditions 
resulting from impairment in parathyroid hormone (PTH) levels, comprising 
administering to a subject in need an effective amount of phosphate. 
The present invention also provides a composition for use in treatment of 
conditions in subjects resulting from impairment in parathyroid hormone 
(PTH) levels, such as osteoporosis comprising an effective amount of 
phosphate together with a physiologically acceptable carrier. 
The present invention still further provides use of phosphate for the 
preparation of compositions for the treatment of conditions in subjects 
resulting from impairment in PTH levels. 
In accordance with a preferred embodiment of the invention, the phosphate 
is administered to individuals at night, and this administration forms 
part of a combined treatment involving also a timed administration of an 
effective amount of an auxiliary agent, e.g. calcium, which is capable of 
decreasing the PTH level during day time. Such an agent may typically be 
administered to the individual in the morning, and also once again during 
the day. As a result of such a combined treatment, PTH will have a low 
level during day time and a higher level during night time, giving rise to 
a circadian profile of PTH levels, similar to that existing in normal 
individuals. 
The term "effective amount" used above, should be understood as meaning an 
amount of an active ingredient, i.e. phosphate or an auxiliary agent, 
which is capable of exerting a desired therapeutic effect. In the case of 
phosphate, an effective amount is an amount sufficient to cause an 
increase in the PTH levels; in the case of said auxiliary agent, an 
effective amount is an amount sufficient to cause a decrease in PTH 
levels. As will be clear, an effective amount may at times vary between 
different groups of individuals depending on the factors such as age, type 
of treated condition, etc., as will no doubt be clear to the artisan. 
DETAILED DESCRIPTION OF THE INVENTION 
The present invention provides novel means for the treatment of conditions 
associated with reduction in PTH levels. Such conditions include in 
particular osteoporosis. In the following, the invention will be described 
with specific reference to the treatment of osteoporosis, it being 
understood that it applies, mutatis mutandis, also to treatment or other 
conditions. 
Osteoporosis, as noted above, is a condition which appears primarily in 
postmenopausal women, and at times also in men, which results, primarily, 
from decrease in PTH levels. In addition to reduction in the PTH level, 
osteoporosis is also associated with impairment of the normal circadian 
rhythm of PTH--low levels during day time and higher levels at night. 
In accordance with the invention, phosphate is used for increasing the 
level of PTH in needy individuals. The phosphate may be administered to 
individuals in the form of a salt, e.g. a sodium or a potassium salt, 
either in their neutral or acid forms. The phosphate may be administered 
in the form of pills, capsules, a liquid, drinkable solution, etc. At 
times, the phosphate may also be administered parenterally, e.g. within a 
saline solution, although it will be appreciated that such form of 
administration, particularly since it is needed on a daily basis, is less 
physiologically tolerable. 
An effective amount of phosphate is typically within the range of about 
2-10 mg/kg body weight, preferably with the range of about 3-6 mg/kg body 
weight. 
In accordance with a preferred embodiment of the invention, phosphate is 
administered to needy individuals in the evening or night, e.g. prior to 
bedtime. 
In accordance with a specifically preferred embodiment such a treatment is 
accompanied by the administration of said auxiliary agent, e.g. calcium 
during day time. Calcium can be administered for example, once in the 
early morning hours and once during mid-day. Such administration of 
calcium will maintain a relatively low level of PTH during day time. An 
effective amount of calcium may for example be about 5-20 mg/kg body 
weight, preferably about 6-15 mg/kg body weight and most desirably about 
6-10 mg/kg body weight. Calcium may be administered to individuals, as 
known, per se, in the form of a pill, in the form of a drink, e.g. prior 
prepared from an effervescent calcium preparation, etc. 
Calcium and phosphate preparations for administration to individuals may be 
provided together in a single package, at times accompanied by 
instructions for use in an administration regimen in accordance with the 
combined treatment of the invention. Typically, such a package will 
comprise daily dosage forms of each of these active agents. For example, 
the package may comprise two calcium tablets and one phosphate tablet for 
each day, the two calcium tablets intended and marked so as to be taken by 
the individual in the morning and at mid-day, and the phosphate pill 
intended and marked so as to be taken by the individual prior to bed time.

METHODS 
I. Animals 
Weanling male Hebrew University strain rats were maintained for 3 weeks on 
the following diets (Teklad, Ill.): Low phosphate, normal calcium (0.02% 
phosphate, 0.6% calcium); normal phosphate, normal calcium (0.3% 
phosphate, 0.6% calcium); high phosphate, high calcium (1.2% phosphate; 
1.2% calcium); vitamin D deficient, low calcium (0.02% calcium). After 1 
to 21 days the thyroparathyroid tissue was excised under pentobarbital 
anesthesia, and blood samples taken. All rat surgery was performed at 9-10 
a.m. The excised tissue was immediately frozen in liquid nitrogen and 
stored at -70.degree. C. 
II. Measurement of cellular mRNA levels 
RNA was extracted from rat thyroparathyroid tissue and the levels of PTH 
mRNA were measured by Northern blots after extraction with TRI Reagent 
(Molecular Research Center Inc., Cincinnati, Ohio). RNA was denatured and 
ethidium bromide was added to each sample at a concentration of 0.1 mg/ml. 
The samples were size-fractionated by electrophoresis on 1.25% agarose 
gels containing formaldehyde and transferred to Hybond filters (Amersham, 
England) by diffusion blotting. The integrity of the RNA and the 
uniformity of RNA transfer to the membrane were determined by UV 
visualization of the ribosomal RNA bands of the gels and the filters. The 
filters were fixed by UV cross-linking and hybridized as previously 
described (Naveh-Many et al., J. Clin. Inves., 90:2434-2438 (1992); Silver 
et al., J. Clin. Inves., 78:1296-1301 (1986)). Hybridization was to a 
random primed rat PTH cDNA (a gift of H. Meyer, GBF, Braunschweig, 
Germany) and 18S RNA (gift of Mr. A. Levine, Baltimore, Md.). 
III. Immunohistochemistry 
Proliferating cell nuclear antigen (PCNA). Paraffin tissue blocks were cut 
to 4-6 mm-thick sections, deparaffinized in xylene and alcohols, and 
placed for 15 mins. in alcohol-H.sub.2 O.sub.2, 3%, for blocking 
endogenous peroxidase. To reveal masked antigens in formalin-fixed, 
paraffin-embedded tissue sections, slides were placed in citrate buffer 
(pH 6.0) and treated in the microwave at 92.degree. C. for 10 mins. After 
removing container from the microwave and cooling for 15 mins, slides were 
placed in PBS (pH 7.6). Sections were then treated with Bovine Serum 
Albumin (BSA) to prevent background staining, and incubated for one hour 
with the primary antibody PCNA-PC-10 (Zymed Laboratories, Inc., San 
Francisco, Calif.) at room temperature in a humidified chamber (Okazaki et 
al., J. Biol. Chem., 269:27855-62 (1994); Peer et al., Ophthalmology, 
101:56-62 (1994)). Slides were rinsed with PBS for 3-4 mins. and incubated 
with the biotinylated linked antibody for 30 mins. and with the labeling 
reagent peroxidase conjugated streptavidin for 30 mins. (Bio Genex 
Laboratories, San Ramon, Calif.). After rinsing, the peroxidase label was 
demonstrated using 3-amino-9-ethyl carbazole (AEC) for 15 mins., and 
counter stained with Mayer Hematoxylin. AEC produces a red end-product 
that is soluble in alcohol and is used with an aqueous mounting media 
(Kaiser's glycerol gelatin). A negative control was run using the same 
technique but omitting the primary antibody and adding the 
streptavidin-biotin complex. PCNA positive cells were counted per 
microscope field with the PT section completely filling the microscope 
field. For each rat four microscope fields were counted and the mean used. 
The variation amongst sections in each rat was always &lt;10%. Each group 
represents the mean.+-.SEM of 4-5 rats. 
IV. DNA nick end labeling of tissue sections 
This was performed essentially as described (Gavrieli et al., J. Cell 
Biol., 119:493-501 (1992)). Tissue sections were treated with proteinase 
K, washed four times, treated with 2% H.sub.2 O.sub.2, rinsed and immersed 
in buffer with biotinylated dUTP and rinsed. The sections were covered 
with Extra-avidin peroxidase (BioMakor, Rehovot, Israel), washed and 
stained with AEC for 30 mins. (Gavrieli et al., stipra). 
V. Serum measurements 
Serum calcium and phosphate were measured in a Roche autoanalyzer. Serum 
1,25(OH).sub.2D 3, levels were measured by a radioreceptor assay (Incstar, 
Minneapolis, Minn.). Serum iPTH levels were measured with a rat 
immunoradiomimetric assay (Nichols, San Clemente, Calif.). Statistical 
analysis was performed on the Macintosh program Statview 512+, using 
Student's unpaired two-tailed t test. The results are presented as the 
mean.+-.SEM. 
EXAMPLE 1 
Serum Biochemistry of Animals Fed with Different Diets 
The biochemistry of serum calcium, serum phosphate and serum 1,25(OH).sub.2 
D.sub.3 of animals fed with low calcium, low phosphate and high phosphate 
is shown in Table 1. 
TABLE 1 
______________________________________ 
The effects of 3 weeks of diet given to weanling rats 
Serum 
Serum calcium 
Serum phosphate 
1,25(OH).sub.2 D.sub.3 
(means .+-. SE) 
(mean .+-. SE) 
(means .+-. SE) 
Diet mg/dl mg/dl pg/ml 
______________________________________ 
Control 10.6 .+-. 0.6 
9.8 .+-. 1.2 
68.0 .+-. 28.3 
Low calcium 
.sup. 6.8 .+-. 0.1.sup..dagger. 
9.9 .+-. 0.5 
&gt;400.sup..dagger. 
Low phosphate 
.sup. 12.6 .+-. 0.6.sup..dagger-dbl. 
.sup. 4.0 .+-. 0.4.sup..dagger. 
&gt;400.sup..dagger. 
High phosphate 
11.1 .+-. 0.6 
9.4 .+-. 0.8 
30.0 .+-. 5.9.sup..dagger-dbl. 
______________________________________ 
Four Rats in each group: 
.sup..dagger-dbl. P &lt; 0.05 and 
.sup..dagger. P &lt; 0.01 compared with control diet. 
As can be seen serum calcium was decreased in the rats fed a low calcium 
diet and increased in rats fed a low phosphate diet. Serum phosphate only 
changed in the rats fed a low phosphate diet where it was decreased. Serum 
1,25(OH).sub.2 D.sub.3 was markedly increased in rats fed both the low 
calcium and the low phosphate diets, and decreased by the high phosphate 
diet. 
EXAMPLE 2 
PTH mRNA Levels of Animals Fed with Different Diets 
Weanling animals were fed diets for three weeks containing low phosphate 
(0.02%), control normal diet; high phosphate (1.2%) and low calcium. The 
results are shown in FIG. 1. As can be seen, PTH mRNA levels were 
increased in the rats fed the low calcium diet and markedly decreased in 
the rats fed a low phosphate diet with no changes in a control gene 18 S 
RNA (not shown). 
EXAMPLE 3 
PCNA of Parathyroid Tissue from Rats Fed with Different Diets 
Parathyroid tissue was obtained from four control untreated rats (1 FIG. 
3); rats fed with low calcium (0.02%) for 10 days (2 FIG. 2); low 
phosphates (0.02%) for 21 days (3 FIG. 2); or rats injected i.p. w.t. 
1,25(OH).sub.2 D.sub.3 (25 pmol/d for 3 days) (4 FIG. 2) and the 
proliferating cell nucleus antigen (PCNA) was determined as described in 
(III) above. 
The results are shown in FIG. 2 (PCNA staining) or FIG. 3 (PCNA counts of 
positive cells per field). As can be seen, PCNA staining of 
thyroparathyroid tissue showed that there were .about.10 PCNA positive 
cells per microscope field in the PTs of a control rat 10 days after 
weaning and there was a six fold increase in weanling rats fed a low 
calcium diet for 10 days (FIGS. 2 and 3). After 21 days of diet there was 
a smaller number of PCNA positive cells in the control rats (P&lt;0.05) as 
compared with rats at 10 days after weaning and those rats on a low 
calcium diet had a 3.6-fold increase in PCNA positive cells (FIGS. 2 and 
3). After a low phosphate diet for 21 days there were no PCNA positive 
cells at all (FIGS. 2 and 3) as well as a marked decrease in PTH mRNA 
levels (FIGS. 1 and 3). Rats fed a high phosphate diet had a moderate 
increase in PTH mRNA levels (FIGS. 1 and 3) and more PCNA positive cells 
than the controls, but not nearly as much as rats fed a low calcium diet 
(FIG. 3).