TREATING CARTILAGE / BONE CONDITIONS WITH WATER-SOLUBLE STRONTIUM SALTS

Description

Field of the Invention

[0001] The present invention relates to Sr-malonate and use of strontium malonate for the preparation of pharmaceutical compositions for use in the treatment and/or prophylaxis of cartilage and/or bone conditions. The strontium malonate has a water-solubility of from 1 g/l to 100 g/l at room temperature.

Background of the Invention

[0002] Osteoporosis is the most common form of metabolic bone disease in humans. It is a condition, which affects a very large number of people all over the world, and as the number of elderly people is set to rise dramatically in the coming decades in most countries, the prevalence and impact of osteoporosis will also increase. The disease Is characterized pathologically by an absolute decrease in the amount of bone mass and the structural quality of bone, and clinically by increased susceptibility to fractures. In fact, osteoporosis is the most significant underlying cause of skeletal fractures in late middle aged and elderly women.

[0003] In general, there are two types of osteoporosis: primary and secondary. Secondary osteoporosis is the result of an identifiable disease process or agent. However, approximately 90% of all osteoporosis cases are idiopathic primary osteoporosis. Such primary osteoporosis includes postmenopausal osteoporosis, age-associated osteoporosis (affecting a majority of individuals over the age of 70 to 80), and idiopathic osteoporosis affecting middie-aged and younger men and women.

[0004] The mechanism of bone loss In osteoporosis is believed to involve an imbalance In the process of bone remodeling. Bone remodeling occurs throughout life, renewing the skeleton and maintaining the strength of bone. This remodeling is mediated by specialized cells of the bone tissue, called "osteoclasts" and "osteobiasts". Osteoclasts (bone dissolving or resorbing cells) are responsible for the resorption of a portion of bone within the bone matrix, during the resorption process. After resorption, the osteoclasts are followed by the appearance of osteoblasts (bone forming cells), which then refill the resorbed portion with new bone.

[0005] The formation of the two cell types as well as their activity In bone Is usually tightly coupled and well regulated in order to maintain the skeletal balance and structural integrity of the bones. However, In people with osteoporosis an Imbalance In this remodeling process develops, resulting In loss of bone at a rate faster than the accretion of bone.

[0006] The single most Important risk factor for osteoporosis Is oestrogen deficiency occurring naturally at the menopause. The decline in endogenous oestrogen production leads to an elevated metabolic activity in the bone tissue where the increase in osteoclast mediated bone resorption surpasses the more modest increase in bone formation resulting in a net loss of bone. The actual number of people affected will grow at a rate greater than simple population growth rates, because the aging of the population is disproportionately increasing the older segment of the population, while the age for the onset of menopause has remained constant. In the last decades there has also been a substantial advance in the ability to predict and monitor osteoporosis, as methods for measurement of bone mineral density (BMD) has improved and new specific biochemical markers of bone resorption and formation has been developed and made available for routine clinical use. New pharmaceutical agents for treatment and/or prevention of osteoporosis have also been developed. The majority of these treatments are either based on substituting the lost endogenous estrogen either in the form of hormone replacement therapy (HRT) or selective estrogen receptor modulators (SERM), or they belong to the class of compounds called bisphosphonates. SERM's and especially HRT is associated with significant side effects, such as increased risk of cancer and cardiovascular disease, whereas bisphosphonates in addition to a potent antiresorptive effect also decreases bone formation to a similar extent, implying that they loose their therapeutic effect after few years of treatment Thus, there Is a need for agents, which are effective in the treatment and/or prophylaxis of osteoporosis.

Description of the Invention

[0007] Previous studies have shown that various strontium compounds modulate bone loss in osteoporosis when present at levels higher than those required for normal cell physiology. The effect is believed to be due to a stimulatory effect of strontium on pre-osteoblastic cell differentiation and migration, and a direct or matrix-mediated inhibition of osteoclast activity by strontium (Reginster, JY, Curr pharm Des 2002:8 (21):1907-16). In other words, strontium both works as an anti-resorptive and an anabolic agent. Various salts of strontium are known from the prior art, such as, e.g., strontium ranelate (distrontium salt of 2-[N,N-di(carboxymethyl)amino]-3-cyano-4-carboxymethylthlophene-5-carboxylic acid) described In EP-B 0 415 850. The ranelate part of the strontium compound, derived from ranelic acid, is unlikely to have any therapeutic effect towards cartilage or bone conditions per se. Other known strontium salts are e.g., strontium tartrate, strontium phosphate, strontium carbonate, strontium nitrate, strontium sulfate and strontium chloride.

[0008] The naturally occurring salts of strontium, such as the carbonate and sulphate salts, have very low water solubility (0.15 g/l or below at room temperature). In contrast, the other strontium salts, such as strontium chloride, strontium hydroxide, strontium nitrate, strontium oxide and strontium acetate have very high solubilities in the range from 225 - 800 g/l in water. In this respect the strontium salts are very similar to the corresponding magnesium and calcium salts.

[0009] Organic strontium salts have been described, but literature reports of this type of compounds are limited to rather few substances. Again In these cases the physiochemical properties have been reported to be very similar to the corresponding magnesium, calcium and barium salts. Carboxylic acids can form stable crystalline salts with divalent earth metals such as strontium, and especially di-carboxylic adds are interesting, as they can have a partial chelating effect. Such complexation may be important in biological systems, where the alkaline earth metals, especially calcium and magnesium, plays important physiological roles Hence the divalent metal ions may exist in a complex form in the aqueous environment In biological systems, rather than in a free and un-bound ionic form. Complex formation constants with the alkaline earth metals in aqueous solution are higher for amino acids than for hydroxy-carboxylic acids and the related non-carboxylic adds, which suggest that the amino group may play a role in the complex formation. Generally the differences in association constants for the various ligands becomes smaller as the radius of the metal Increases, and thus stability of strontium complexes with di-carboxylic acid is lower than the stability of the comparable complexes with calcium and magnesium.

[0010] For a pharmaceutical application of the strontium salts this is very important as it means that strontium salts of dicarboxylic amino adds may be particularly useful. We have found that such salts, such as strontium glutamate and strontium aspartate are more soluble than other dicarboxylic strontium salts of similar molecular size. In pure aqueous solutions of such salts strontium exists in partly complexed form. However, when administered to an animal such as a mammal, i.e. a rat, dog, monkey or human, ionic strontium as well as strontium complexed to the carboxylic add anion will be taken up from the intestinal lumen by both passive and active transport mechanisms. In this case strontium will be displaced from the complexes by available calcium and magnesium which forms much more stable complexes with the ionized amino acids. It appears that with the heavy group II metals, such as strontium, the amino group in both aspartate and glutamate is much less significant for metal complexation, probably due to the unfavourable chelation of large metals in five- or six-membered rings. Accordingly, dianionic amino-acid salts of strontium, such as strontium aspartate and strontium glutamate may be especially suited for prophylactic and/or therapeutic interventions in bone disease as the amino-acids may act to preferentially bind/complex with available free calcium, thus promoting both the Intestinal uptake of the calcium ion, and physiological action of the ion, in particular its role In regulation of bone turnover. As appears from the above, the known strontium salts that are water-soluble have a water-solubility of at least about 225-800 g/l and the other known strontium salts have solubilities that are very low (below 0.1 g/l at room temperature). The invention relates to use of strontium malonate having a water-solubility from 1 g/l to 100 g/l at room temperature for the preparation of a pharmaceutical composition for the treatment of and/or prophylaxis of osteoporosis, osteopenia, Paget's disease, osteolytic lesions produced by bone metastasis, bone loss due to sex steroid hormone deficiency, bone abnormalities caused by cancer therapeutics, immobilization-induced osteopenia or osteoporosis, glucocorticoid-induced osteopenia or osteoporosis or for the improvement of fracture healing after traumatic or atraumatic fracture in a mammal.

[0011] Furthermore, the present inventors have found an improved method for the preparation of this salt.

[0012] The present inventors have found that the use of strontium malonate has prophylactic and/or therapeutic value in that one or more of the following beneficial effects can be obtained:

i) an improved bioavailability of strontium,

ii) an improved absorption of strontium,

iii) a reduction of side effects,

iv) a flexible dose adjustment of strontium in order to tailor prevention and/or treatment of a specific disease stage,

v) a possible reduction in daily dosage

vi) a possible reduction of the number of different pharmaceutical compositions that a patient must use to achieve a therapeutic effect.

[0013] As mentioned above, the strontium malonate for use according to the invention is water soluble, having a water solubility of at least 1 g/l, such as, e.g., at least 5 g/l, at least 10 g/l, at least 20 g/l, at least 30 g/l, at least 40 g/l, at least 50 g/l, at least 60 g/l, at least 70 g/l, at least 80 g/l, at least 90 g/l or 100 g/l measured at room temperature, i.e a temperature of 20-25°C.

Synthesis of strontium salts

[0014] Organic strontium salts of carboxylic add anions can be synthesized by a number of different pathways. A conventional method for preparation of such organic strontium salts is to utilise the reaction between and organic acid and strontium hydroxide in an aqueous solution. This neutralisation reaction of, e.g. fumaric acid and strontium hydroxide salt follows the following scheme:

        Sr²⁺(aq)+ 2OH^- (aq)+ HOOCCHCHCOOH(aq)→ Sr(OOCCHCHCOO)(aq)+2H₂O(l)

[0015] The suspension of dissolved strontium fumarate can then be Induced to precipitate by sublimation of water and subsequent up-concentration of the salt. Crystals will slowly form and precipitate from the solution.

[0016] An alternative approach is to utilise the sodium or potassium salt of the appropriate carboxylic acid anion and strontium chloride. As all organic strontium salts will be less soluble than the highly soluble chloride salt, the organic strontium salt will precipitate under these conditions leaving NaCl and excess SrCl₂ in the solution. The equation below exemplifies this reaction scheme using as an example the reaction between SrCl₂ and sodium-fumarate.

        Sr²⁺(aq)+ 2Cl^- (aq)+ 2Na⁺ (aq) + C₄H₂O₄^2-(aq)→ Sr(OOCCHCHCOO)(aq)+Cl^-(aq)+Na⁺(aq)

[0017] The present inventors have found that different strontium salts requires different synthesis pathways, and for some strontium salts we have identified optimized synthesis and manufacturing procedures. It has been found that synthesis of strontium salts of the di-carboxylic aminoacids aspartate and glutamate (in either D- or L- form) is very difficult when following these conventional reaction pathways, and generally results in low yields and purity of the obtained crystalline salt. In order to facilitate large-scale manufacture of pure strontium salts of dicarboxylic amino adds to carry out the pharmaceutical use according to the present invention, the present inventors have studied various synthesis pathways of these particular strontium salts. Thus, It has surprisingly been found that synthesis of well defined and pure strontium glutamate in hexahydrate form is most convenient carried out with the free add form of glutamate and strontium hydroxide and requires elevated temperatures, such as temperatures above 80°C, or more preferred 100°C or even 920°C or most preferred more than 130°C. Furthermore, we have found that addition of small volumes of alcohol can accelerate the crystal-formation of dissolved aqueous organic strontium salts. The inventors have also found that synthesis of the reference compound strontium L-glutamate from L-glutamlc add and SrCl₂ results In a new hexahydrarte crystalline form distinct from the previously described strontium L-glutamate hexahydrate.

[0018] Examples of these synthesis procedures for organic strontium salts of relevance for the provision of strontium malonate for the treatment and/or prophylaxis of the specific bone diseases of the present invention are provided in the examples herein.

[0019] A method for the preparation of strontium salts including strontium malonate is described herein.

[0020] As mentioned above strontium is believed to have an effect on cartilage and/or bone conditions and/or other conditions, thus the salt may be used for the preparation of a pharmaceutical composition for the treatment and/or prophylaxis of a cartilage and/or a bone condition Including the ones mentioned above. The pharmaceutical composition of strontium malonate may further comprise one or more physiologically acceptable excipients.

[0021] For the treatment and/or prophylaxis of a bone disease as mentioned above in a mammal, the possibility of administering various amounts of strontium may be desired. The amount of strontium in a pharmaceutical composition used in the invention may be adjusted by adding an additional amount of strontium in the form of a strontium-containing compound to the composition.

[0022] In certain cases it may be beneficial to further add one or more active substances to a pharmaceutical composition as described herein. The one or more active substances may have a therapeutic and/or prophylactic effect on a cartilage and/or a bone disease and/or other conditions such as those mentioned above. The term "active substance having a therapeutic and/or prophylactic effect on diseases and conditions affecting metabolism and structure integrity of cartilage and/or bone" includes active substances that can attain a particular medical result, such as, e.g., reduce the incidence of bone fracture, increase bone density and/or improve healing of bone or decrease or halt the degradation of articular cartilage or promote formation of new cartilage or prevent or decrease progression of radiological evident joint damage. Examples of such substances are bone antl-resorptive and/or anabolic agents. However, one or more active substances having other effects than those mentioned above may also be included in a pharmaceutical composition of the invention. Such active substances could be e.g. disease-modifying anti-rheumatic drugs, or other anti-rheumatic drugs.

[0023] Specific examples of active substances, which may be used In a pharmaceutical composition as described herein are calcium-alpha-ketoglutarate, calcium and/or salts thereof, vitamin D such as, e.g., vitamin D3 and/or functional equivalents of vitamin D3, glucagon-like peptide-2, glucagon-like peptide-2 releasing compositions, bisphosphonates including ibandronate, zoledronate, alendronate, risedronate, ethidronate chlodronate, tiludronate and pamidronate; selective estrogen receptor modulators (SERMs) including raloxifene, arzoxifene, droloxifene, tamoxifen, 4-hydroxy-tamoxifen, 4'-iodotamoxifen, toremifene, (deamlnohydroxy)-toremifene, chlomiphene, levormeloxifene, ormeloxifene, chroman derivatives, coumarin derivatives, idoxifene, nafoxidine, TAT-59, LY-353381, CP-336156, MDL-103323, EM-800, ICI-182,ICI 183,780, ICI 164,384, ICI 183,780, ICI 164,384, diethylstilbesterol, genistein, nafoxidine, nitromifene citrate, moxesterol, diphenol hydrochrysene, erythro-MEA, allenolic acid, equilin-3-sulphate, cyclophenyl, chlorotrianisene, ethamoxytriphetol, lasofoxifene, bazedoxifene, genistein, tibolone, ospemifene, tesmilifene, droloxifene, panomifene, zindoxifene, meproxifene and faslodex; calcitonin, parathyroid hormone, parathyroid hormone related peptide, glucosamine sulphate, glutamic acid and/or salts thereof, aspartic add and/or salts thereof, proline, glutamine and hydroxyproline.

[0024] As mentioned above, the compounds and compositions of the present invention are used for the preparation of a pharmaceutical composition for the treatment and prophylaxis of various conditions as specified on page 4a.

[0025] The subject may be a mammal, such as, e.g. a human or a domestic animal, such as, e.g., a cat, a dog, a horse, a cow or a sheep.

[0026] In one aspect, the invention relates to use of strontium malonate as described above wherein the daily dose of strontium to be administered to the mammal in a one day period may be at least 0.01 g, such as, e.g., at least 0.025 g, at least 0.050 g, at least 0.075 g, at least 0.1 g, at least 0.2 g, at least 0.3 g, at least 0.4 g or at least 0.5 g or from 0.01 g to 2 g such as, e.g., from 0.1 g to 2 g, from 0.3 g to 2 g or from 0.3 g to 1 g.

[0027] The invention also relates to a use wherein the strontium salt is administered in the form of a pharmaceutical composition as described above.

[0028] The invention further describes a use wherein the administration may take place one or more times daily, such as from 1 to 5 times daily.

[0029] The invention also describes a use wherein the administration may take place one or more times weekly, such as from 1 to 3 times weekly.

[0030] As described above one or more active substances may be added to a pharmaceutical composition as described herein, administered as part of the same treatment as the administration of strontium malonate. One example of such an active substance is Vitamin D. Vitamin D plays a major role in Calcium absorption, since activated vitamin D₃ (1,25-dihydroxycholecalaferol) and to a smaller extent other active forms of vitamin D, acts to increase the calcium absorption from the small intestine. Vitamin D₃ acts to Increase the entry of calcium through the plasma membrane Into the enterocytes, and is capable of reducing the excretion of calcium to urine by increasing the reabsorbtion of calcium In kidneys. It is likely that vitamin D has the same effect on strontium absorption as it has on calcium absorption.

[0031] Vitamin D is activated in e.g. the liver and kidneys. High levels of calcium are having a reducing effect on activation of vitamin D, and high levels of strontium will probably have the same effect as calcium on the activation of vitamin D.

[0032] Thus, the administration of an amount of vitamin D together with strontium-malonate will most likely have a beneficial effect on the uptake of strontium.

[0033] Vitamin D may be vitamin D₃, and the daily dose of vitamin D₃ may be at least 1 µg, such as, e.g. at least 1.25 µg, at least 1.50 µg, at least 2 µg, at least 3 µg, at least 4 µg, at least 5 µg, at least 10 µg, at least 15 µg, at least 20 µg, at least 25 µg, at least 30 µg, at least 40 µg or at least 50 µg or from 1 µg to 50 µg such as, e.g., from 1.50 µg to 40 µg, from 2 µg to 30 µg, from 3 µg to 30 µg, from 4 µg to 30 µg, from 5 µg to 30 µg, from 10 µg to 30 µg, from 10 µg to 20 µg or from 15 µg to 25 µg.

[0034] More specifically, the daily dose of vitamin D₃ may be from 5 µg to 30 µg, such as, e.g., from 10 µg to 20 µg.

[0035] Another active form of vitamin D is vitamin D₂. The dally dose of vitamin D₂ may be at least 1 µg, such as, e.g. at least 1.50 µg, at least 2 µg, at least 3 µg, at least 4 µg, at least 5 µg, at least 10 µg, at least 15 µg, at least 20 µg, at least 25 µg, at least 30 µg, at least 40 µg, at least 50 µg, at least 60 µg, at least 70 µg, at least 80 µg, at least 90 µg, at least 100 µg, at least 110 µg, at least 120 µg or at least 125 µg or from 1 µg to 125 µg such as, e.g., from 1.50 to 120 µg, from 2 µg to 110 µg, from 3 µg to 100 µg, from 4 µg to 90 µg, from 5 µg to 80 µg, from 5 µg to 125 µg, from 10 µg to 70 µg, from 10 µg to 60 µg, from 10 µg to 50 µg, from 10 µg to 40 µg, from 10 µg to 30 µg, from 10 µg to 20 µg, or from 15 µg to 25 µg.

[0036] More specifically, the daily dose of vitamin D₂ is from 5 µg to 125 µg, such as, e.g., form 10 µg to 20 µg.

[0037] Other functional equivalents of vitamin D₃ and D₂, such as alphacalcidol, calcitriol or dihydrotachysterol, may also be administered. Alpha-calcidiol, 1α-hydroxy-cholecalciferol, may be administered in amounts of 0.2-3 µg/day, preferably 0.25-2 µg/day. Calcitriol, 1,25-dihydroxy-cholecalciferol, may be administered in amounts of 0.1-10 µg/day, preferably 0.125-2 µg/day and dihydrotachysterol, a vitamin D₂ analogue, may be administered In amounts of 0.1-3 mg/day, preferably 0.2-0.6 mg/day.

[0038] The administration of the strontium component, and vitamin D may take place simultaneously.

[0039] The administration of the strontium component, and/or vitamin D may take place sequentially.

[0040] Calcium is another example of an active substance that may be administered as part of the same treatment as the administration of strontium malonate. Calcium is the most abundant mineral in the body, and a major constituent of bone and teeth as calcium phosphate and calcium carbonate. Calcium is also essential in intra- and extracellular fluid exchange, blood dotting, and in maintaining a regular heartbeat. It is also important in the initiation of neuromuscular as well as metabolic functions. Most of the calcium in the body is stored in the bones.

[0041] Thus, calcium is an important participant in many processes in the body, and administration of calcium may have a therapeutic and/or prophylactic effect on many of the diseases and conditions mentioned above.

[0042] The daily dose of calcium may be from 0.5 g to 2 g such as, e.g., 0.5 g to 1.5 g, from 0.5 g to 1 g and from 1 g to 1.5 g.

[0043] The strontium component may be administered in a dose corresponding to a daily dose of from 0.3 g to 1 g, and the dose of calcium corresponds to a daily dose of from 0.5 g to 1 g.

[0044] The administration of the strontium malonate, and calcium may take place simultaneously, either In a single administration form or in separate administration forms for simultaneous administration as described above.

[0045] Preferentially, the strontium malonate and calcium may be administered sequentially.

[0046] Studies have shown that strontium is a full agonist of the calclum-sensing receptor (CaR). Even though the role of the CaR In regulating bone cells is not fully investigated, it appears that strontium and calcium may exert their effect on bone metabolism via the same receptor.

[0047] Accordingly, it may be beneficial not to administer the strontium malonate and calcium at the same time.

[0048] Calcium may be administered after the administration of strontium, i.e. at least 0.5 h, such as, e.g., at least 1 h, at least 2 h, at least 3 h, at least 4 h, at least 5 h, at least 6 h, at least 7 h, at least 8 h, at least 9 h, at least 10 h, at least 11 h or at least 12 h after the administration of the strontium malonate.

[0049] Calcium may be administered before the administration of strontium, i.e. at least 0.5 h, such as, e.g., at least 1 h, at least 2 h, at least 3 h, at least 4 h, at least 5 h, at least 6 h, at least 7 h, at least 8 h, at least 9 h, at least 10 h, at least 11 h or at least 12 h before the administration of strontium malonate.

[0050] The strontium malonate may be administered simultaneously and calcium may be administered at least 1 h, such as, e.g., at least 2 h, at least 3 h, at least 4 h, at least 5 h, at least 6 h, at least 7 h, at least 8 h, at least 9 h, at least 10 h, at least 11 h or at least 12 h after the administration of strontium malonate.

[0051] The strontium malonate and the alpha-ketoglutarate or amino add component, if relevant, may be administered simultaneously and calcium may be administered at least 1 h, such as, e.g., at least 2 h, at least 3 h, at least 4 h, at least 5 h, at least 6 h, at least 7 h, at least 8 h, at least 9 h, at least 10 h, at least 11 h or at least 12 h before the administration of strontium malonate.

[0052] Calcium and vitamin D may be administered simultaneously at least 1 h, such as, e.g., at least 2 h, at least 3 h, at least 4 h, at least 5 h, at least 6 h, at least 7 h, at least 8 h, at least 9 h, at least 10 h, at least 11 h or at least 12 h before the simultaneously administration of strontium malonate and vitamin D.

[0053] Calcium and vitamin D may be administered simultaneously In the morning, and a-strontium malonate and vitamin D may be administered simultaneously in the evening.

[0054] A further example of an active substance that may be administered as part of the same treatment as the administration of strontium is parathyroid hormone. Parathyroid hormone is composed of 84 amino acid residues and is released in vivo in response to a decrease in the level of extra cellular calcium. Administration of PTH or fragments thereof in a pharmaceutically relevant dose is known to stimulate bone formation, produce a robust increase in bone mineral density and substantially reduce the occurrence of vertebral and non-vertebral fractures. Parathyroid hormone acts directly on the kidney to diminish urinary calcium, and increases bone resorption via an indirect mechanism involving osteoblasts. Parathyroid hormone also increases the activation of vitamin D by stimulating the activity of 1α-hydroxylase enzyme in the kidney, subsequently leading to a better absorption of calcium and, possibly, strontium.

[0055] A commercially available parathyroid hormone containing drug comprises the 34 N-terminal amino adds region of human parathyroid hormone, which is believed to be the biologically active region.

[0056] Accordingly, an amount of parathyroid hormone, or a fragment or analogue thereof, or a parathyroid hormone related peptide, or a fragment or analogue thereof, may be administered as part of the same treatment as the administration of strontium salt. In the following the term "PTH" covers parathyroid hormone, fragments, analogues and functional analogues thereof together with parathyroid related hormone and fragments, analogues and functional analogues thereof. PTH may be used as a combined or sequential administration with strontium and, if relevant, alpha-ketoglutarate.

[0057] The dally dose of PTH, when calculated as recombinant human parathyroid hormone (1-34), may be at least 1 µg, such as, e.g. at least 2 µg, at least 3 µg, at least 4 µg, at least 5 µg, at least 10 µg, at least 15 µg, at least 20 µg, at least 25 µg, at least 30 µg, at least 35 µg, at least 40 µg, at least 50 µg, or at least 60 µg, or from 1 µg to 60 µg such as, e.g., from 2 to 50 µg, from 3 µg to 40 µg, from 4 µg to 40 µg, from 5 µg to 40 µg, from 10 µg to 40 µg, from 10 µg to 35 µg, from 10 µg to 30 µg, from 10 µg to 25 µg, from 10 µg to 20 µg, from 15 µg to 40 µg, from 20 µg to 40 µg or from 20 µg to 30 µg.

[0058] More specifically, the daily dose of PTH, when calculated as recombinant human parathyroid hormone (1-34), may be from 10 µg to 40 µg, such as, e.g., from 10 µg to 30 µg, from 10 µg to 20 µg, from 20 µg to 40 µg or from 20 µg to 30 µg.

[0059] The medical treatment according to the invention may comprise administration of a dally dose of bisphosphonate from about 0.1 mg to 60 mg, such as from 0.2 mg to 30 mg, from 0.2 mg to 20 mg or from 0.2 mg to 10 mg.

[0060] In a combination treatment in which strontium malonate is given in combination with one or more SERM's, the SERM should be used in a dose as determined previously from clinical investigation of the given SERM.

Pharmaceutical compositions

[0061] The pharmaceutical compositions used in the invention normally comprise the specific compounds together with one or more physiologically acceptable excipients, i.e. a therapeutically inert substance or carrier.

[0062] The carrier may take a wide variety of forms depending on the desired dosage form and administration route.

[0063] The pharmaceutically acceptable excipients may be e.g. fillers, binders, disintegrants, diluents, glidants, solvents, emulsifying agents, suspending agents, stabilizers, enhancers, flavors, colors, pH adjusting agents, retarding agents, wetting agents, surface active agents, preservatives, antioxidants etc. Details can be found in pharmaceutical handbooks such as, e.g., Remington's Pharmaceutical Science or Pharmaceutical Excipient Handbook.

[0064] Above are mentioned specific examples of the amounts of compounds administered. However, it will be understood that the amount of the compounds actually administered will be determined by a physician in light of the relevant circumstances including the condition to be treated, the choice of compounds to be administered, the age, weight, and response of the individual patient, the severity of the patient's symptoms and the chosen route of administration. While the present compounds are preferably administered orally, the compounds may also be administered by any other suitable route, such as e.g. parenteral.

[0065] In a specific embodiment, the pharmaceutical composition is designed for oral administration, and the peak concentration of strontium malonate is reached at least 2.5 hours after oral administration.

[0066] The pharmaceutical composition comprising a compound according to the invention may be in the form of a solid, semi-solid or fluid composition.

[0067] The solid composition may be in the form of tablets such as, e.g. conventional tablets, effervescent tablets, coated tablets, melt tablets or sublingual tablets, pellets, powders, granules, granulates, particulate material, solid dispersions or solid solutions.

[0068] In one embodiment of the invention, the pharmaceutical composition may be in the form of a tablet. The tablet may be coated with a coating that enables release of at least part of the salt in the proximal part of the small intestine, such as e.g. the duodenum and/or the proximal jejunum, such as at least 50% w/w, at least 60% w/w, at least 65% w/w, at least 70% w/w, at least 80% w/w or at least 90% w/w of the total amount of the salt contained in the tablet.

[0069] The tablet may have a shape that makes it easy and convenient for a patient to swallow. The tablet may thus e.g. have a rounded or a rod-like shape without any sharp edges. Furthermore, the tablet may be designed to be divided In two or more parts.

[0070] A semi-solid form of the composition may be a paste, a gel or a hydrogel.

[0071] The fluid form of the composition may be a solution, an emulsion Including nano-emulsions, a suspension, a mixture, or a syrup. The solution, suspension or emulsion may be designed for intravenous, intramuscular, intraartricular eg subcutaneous injection.

[0072] Other suitable dosages forms of the pharmaceutical compositions according to the invention may be capsules, sachets, troches, devices etc. The pharmaceutical compositions may be prepared by any of the methods well known to a person skilled In pharmaceutical formulation.

Other aspects of the Invention

[0073] As mentioned above, use of strontium malonate according to the invention may lead to improved fracture healing after traumatic or atraumatic fracture, where the fracture e.g. may be one of the following traumatic or atraumatic fractures: fracture to the distal radius, such as e.g. a Colle's fracture or a Smiths fracture, a fracture of the femur, such as e.g. the proximal femur, such as e.g. a cervical fracture, a trochanteric fracture or a subtrochanteric fracture.

[0074] The improved fracture healing may be defined In terms of a reduction of the time a patient will require a plaster, reduction of the time to healing as defined on a X-ray, reduction in the time to fracture stability, improvement of callus formation as viewed by X-ray, reduction in time before appearance of callus formation as viewed by X-ray and/or reduction in time for regaining full or near-full mobility or physical activity level.

[0075] Other embodiments of the invention appear from the appended claims. The details and particulars described above and relating to the compounds and compositions according to the invention apply mutatis mutandis to the other aspects of the invention.

Legends to figures

Reference Figure 1. X-ray diffractogram of crystals of the reference compound strontium glutamate hexahydrate prepared by the method as described in example 5.

[0076] Figure 2. X-ray diffractogram of crystals of strontium malonate prepared by the method as described in example 5. The malonate salt of strontium has not previously been characterized and comprise a new crystallographic structure, but it is apparent from the stable baseline, and well defined spacing of diffraction peaks, that the crystal form of the malonate salt is homogeneous and pure.

Reference Figure 3. Results of the optimisation reference experiments for strontium glutamate synthesis outlined in table 4. The influence on the yield of the synthesis of the reference compound strontium glutamate was investigated by varying four parameters. (Yields above 100% indicate incomplete drying).

[0077] Figure 4: Plot of serum strontium concentrations measured in rats given a single dose of strontium as indicated in the upper part of each panel. The data points represent mean and standard deviation for each measuring point. Pre-dosis represent corresponding samples taken from animals treated with vehicle alone, (Example 6).

Examples

Example 1 - Reference example

[0078] General method for preparation of crystalline salts of strontium by precipitation from dissolved strontium chloride and dissolved sodium salts of the appropriate carboxylic anions

[0079] In a glass-beaker of 100 mL volume, 5 g of the sodium salt of the carboxylic acid was dissolved in a small volume of water that was slightly heated at temperatures not greater than 30-50 °C. The final volume was 25-50 mL. In another beaker 10 g of SrCl₂ (SrCl₂ hexahydrate, Sigma-Aldrich 43,966-5) was dissolved in 100 mL of water. This latter solution was slowly decanted Into the first solution of the dissolved sodium salt. The transfer continued until an initial cloudiness was observed, which resulted in a total volume of 50-100 mL. The solution was allowed to rest at room temperature (22-24 °C) for several days until significant amounts of crystallized precipitate of the organic strontium salt appeared.

[0080] The reaction that proceeds is exemplified by the reaction between strontium ions and sodium fumarate (reaction schemes (a) and (b)):

        NaOOCCHCHCOONa(s)+H₂O(l)→^-OOCCHCHCOOH(aq)+2Na⁺(aq)+OH^-(aq)     (a)

        ^-OOCCHCHCOOH(aq)+Sr²⁺ (aq)→ Sr(OOCCHCHCOO)(aq)+ H⁺(aq)     (b)

[0081] In order to accelerate the crystallisation, we have found that addition of small volumes of ethanol, such as from 5 - 10 vol/vol % to 50 - 60 % vol/vol induces a significant acceleration of the precipitation of the desired strontium salt. Addition of ethanol is of special importance in the synthesis of strontium salts with solubility exceeding 2 g/l at room temperature (22-24 °C), and will thus provide a substantial benefit for the synthesis of strontium salts of L-aspartate, L-glutamate and lactate. In order to reach the required product within a short period, it was essential to observe an initial crystallisation or an initial dimness in the solution right from the first stage.

[0082] After the precipitation, the solution was filtered on a Büchner funnel using a suction flask and the crystals were flushed in small volumes of ethanol. Crystals of some of the salts were very soluble, so in order to improve the yield of crystals, the solution was allowed to rest longer, such as at least 30 - 60 min. Repeated crystallisation resulted in yields of approx. 50%. Strontium salts of L-aspartate and of lactate were very soluble, with solubility exceeding 25 g/l in water at room temperature.

[0083] The lactate and L-glutamate salts of strontium were precipitated from solutions with an excess of strontium chloride and large crystals of the lactate salt were achieved by slow evaporation of the solvent.

Example 2 - Reference example

General method for preparation of crystalline salts by neutralisation of carboxylic acids with strontium hydroxide

[0084] A small amount of the organic acid proper (0.75 - 3 g, see table below) was dissolved in water by heating to temperatures between 30°C - 50 °C. Then, strontium hydroxide (Sigma Aldrich, Sr(OH)₂*8H₂O, MW 265.71, CAS no. 1311-10-0, approx. 10 g/L) was slowly added. Then, a magnetic stirring rod was added and the stirring and gentle heating (i.e. 30 - 50°C) of the suspension was started. After some time, the solution clarifies and all the solid material dissolves. The heating is maintained, and after three hours of incubation, the solution is filtered while hot on a Büchner funnel. Very small amounts of impurities were left in the filter.

[0085] The filtrate was subsequently allowed to cool at room temperature overnight, which resulted in growth of fine-powdered crystals of the desired strontium salt. Further purifications of the salts can be performed by repeated re-crystallizations (table 1).

Table 1: Amounts of start reagent used for organic strontium salt synthesis and recoveries in the synthesis of eight specific organic strontium salts following the general reaction pathway with free-acid forms of the anion, and strontium hydroxide

Strontium salt of (free acid used):	Sr(OH)2 *8H2O	Free acid	Amount obtained	Recovery*	Melting Temp.	Solubility	Crystal structure
Fumarate1	2.044 g	1.140 g	0.999 g	99 %	>380°C	Yes	No
α-ketoglutarate2	2.017 g	1.441 g	0.828 g	72 %	>380°C	Yes	No
succinate	2.098 g	1.177 g	0.958 g	92 %	230°C	Yes	Yes
L-Ascorbate3	2.094 g	1.805 g	2.005 g	15 %	>380°C	Yes	No
L-Glutamate	2.017 g	1.453 g	0.175 g	15 %	>380°C	Yes	Yes
Citrate	2.057 g	1.918 g	1.123 g	48 %	>380°C	Yes	Yes
D-Aspartate	2.190 g	1.316 g	0.167 g	14 %	>380°C	No	No
Tartrate	2.070 g	1.502 g	2.005 g	129%	>380°C	Yes	Yes

Notes *) Recovery calculated in·% of the strontium content in Sr(OH)2*8H2O. 1) Fumaric acid is insoluble in water, and ethanol is added to the suspension until complete solubilization is achieved. The synthesis is continued with this material. 2) The strontium-AKG salts has a slight brownish appearance and a melting temperature % 3) In addition to the indicated amounts of strontium hydroxides and L-ascorbate an additional 4.087g SrCl2*6H2O solubilized in water is added to the reaction mixture.

Example 3

Determinations of solubility of organic strontium salts

Synthesis of strontium salts

[0086] The great majority of strontium salts could be obtained by reacting the sodium salt of the organic acid with strontium chloride following the general synthesis method described in example A. However, the reference compounds, strontium citrate, strontium tartrate, strontium succinate and strontium α-ketoglutarate for the solubility investigations were obtained by synthesis from the free acid forms of the carboxylic acid and strontium hydroxide as described in comparison example 2. The reference compound strontium glutamate was obtained by using an incubation temperature of 100°C and using strontium chloride and L-glutamic acid for the synthesis for obtaining pure and homogeneous hexahydrate crystals of strontium glutamate. The strontium glutamate salt obtained by this method is distinct from a previously described form of crystalline strontium L-glutamate. Detailed investigations of solubility were carried with the strontium salts listed in table 2:

Table 2: Overview of strontium salts used in investigation of solubility. MW indicates the molecular weight of the homogeneous crystalline form of the salt with the indicated amount of crystal water and % Sr gives the molar percentage that strontium constitutes of this crystalline form

Strontium salt	MW	% Sr
Sr-ranelate (*7H2O) -ref. compound	639.6	27.4
SrCl2 (*6H2O) -ref. compound	266.6	32.9
Sr-fumarate (*6H2O) -ref. compound	309.7	28.3
Sr-L-glutamate (*6H2O) -ref. compound	340.7	25.7
Sr-α-ketoglutarate (*6H2O) -ref. compound	339.7	25.8
Sr-aspartate (*3H2O) -ref. compound	272.7	32.1
Sr-succinate (*6H2O) -ref. compound	311.7	28.1
Sr-ascorbate (*6H2O) -ref. compound	545.8	16.1
Sr-malenate (*6H2O) -ref. compound	309.7	28.3
Sr-malonate (*1H2O)	207.7	42.2
Sr-pyruvate (*6H2O)-ref.compound	369.7	23.7
Sr-tartrate (*6H2O) -ref. compound	343.7	25.5
Sr-citrate(*6H2O) -ref. compound	749.1	35.1

[0087] The solubility of the organic carboxylic acid strontium salts were measured in water. The solubility of these salts was also measured as a function of temperature. This was performed by incubating the saturated solutions of the salts in temperature-controlled incubators. Furthermore, the solubility of the salts was studied in pure distilled water as well as a 0.05 M ammonium carbonate buffered solutions, with a physiological pH of 7.5.

[0088] The buffered solutions were immersed into a bath of water temperature controlled at either room temperature (22 - 24 °C), at 30 °C or at 40 °C. The test tubes were stirred and the solutions were subsequently incubated in an incubater with constant temperature for 24 hours. In order to eliminate any reminiscent strontium chloride influence on the determination of solubility, all the precipitate was collected at the bottom of the test tubes and the solutions above the precipitate were carefully removed and substituted by fresh solutions. After substitution of the solutions, the test tubes were stirred again and allowed to rest for another 24 hours. From these solutions, the dissolved proportions of the strontium salt were collected in volumes of 1 mL at the specified temperature. The solutions were diluted to 50 mL before analysis by Flame Atomic Absorption Spectrometry (F-AAS). Before subsequent series of sampling, the solutions were equilibrated at the next temperature for 24 hours.

Analysis of Strontium by flame atomic absorption spectrometry F-AAS

[0089] Two methods were used for quantification of strontium in solutions: Flame Atomic Absorption Spectrometry (F-AAS), and the more sensitive inductively-coupled-plasma-mass spectrometry (ICP-MS). For most investigations, the F-AAS method had sufficient sensitivity.

[0090] Some of the very soluble strontium salts were further diluted before analysis by F-AAS. The measurements were performed by using a Perkin-Elmer 2100 equipped with a hydrogen lamp for correction of the background signal. Strontium was measured at a slit with of 0.2 nm, the wavelength was 460.8 nm operated at an energy of 58 and a current of 8 mA.

Temperature and pH influence on organic strontium salt solubility

[0091] For the majority of the organic strontium salts listed in table 2, temperature changes in the interval from 20 - 40 °C had only little influence on solubility (table 3). However, for the ref-compound strontium L-glutamate a significant influence of temperature on solubility was observed in the range between 20 °C and 40 °C. The solubility of this salt increased more than threefold in the investigated interval in contrast to most other salts. It is noted, that the solubility under physiological conditions (37 °C), is of relevance for the pharmaceutical use of the substances, and thus the surprising increase in strontium glutamate solubility at higher temperature may have great potential therapeutic implications.

[0092] The solubility of the strontium salts in an ammonium carbonate buffered solution of pH 7.5 was generally higher than the solubility determined in pure water (table 3). However, there were some notable exceptions, such as the reference compound strontium maleate, which had decreased solubility in the buffered solution. Accordingly, it was found most relevant to compare the solubility of the strontium salts by comparing the values obtained in water, as shown in table 3.

Relative solubility

[0093] The water-solubilities of the organic strontium salts at room temperature and at 40°C, are listed in table 3. The strontium salts of L-aspartate and of lactate (reference compounds) had solubilities exceeding 50 g/l hampering exact determination of solubility with the employed experimental procedures.

[0094] The results correspond to the observations during the synthesis experiments where the citrate, the fumerate and the tartrate precipitated instantly when synthesized by the production procedures described in comparison examples 1 and 2. This is indicative of a poor solubility of these strontium salts, as apparent by the lower solubility of these salts compared to the other organic strontium salts at both 22 °C and 40 °C.

[0095] The glutamate salt (reference compound) showed a higher solubility than the other salts, especially at a temperature of 40 °C. During the synthesis of this salt, it was necessary to add alcohol to the solution, to initiate crystal growth, indicative of relatively high water solubility. The other studied strontium salts only precipitated after evaporation of the solvent for a few days at room temperature, but addition of alcohol was not required to initiate crystal formation and precipitation.

Table 3. Relative solubility in water buffered solutions at pH 7.5 at 40°C and room temperature (22 - 24°C) of the investigated Strontium-salts, as determined by F-AAS.

STRONTIUM SALT	SOLUBILITY AT ROOM TEMPERATURE (22 - 24°C) (mg/L)		SOLUBILITY AT 40°C (mg/L)
Anion	In water	pH 7.5	In water	pH 7.5
Malonate**	1474	2816	1441	2127
L-glutamate** -ref. compound	2111	3022	7093	7195
L-aspartate** -ref. compound	4200		7900
Pyruvate* -ref. compound	2204	1946	1929	1829
α-ketoglutarate** -ref. compound	1316	2252	3534	3809
fumarate** -ref. compound	571	1215	444	977
Maleate** -ref. compound	3002	1680	2527	1457
Tartrate** -ref. compound	883	1831	1028	1400
Ranelate**** -ref. compound	760	890	1450	1970
Succinate** -ref. compound	1137	926	1116	2233
Citrate*** ref-compound	107	388	147	430

*) Mono-carboxylic acid **) Di-carboxylic acid ***) Tri-carboxylic acid ****) Quattro-carboxylic acid

Example 4

Preparation of strontium malonate monohydrate by synthesis at 100 °C

[0096] Initially, a suspension of malonic acid (white colored) is prepared by adding 100 mL of millipore water to 10.406 g (0.1 moles) of solid malonic acid (Fluka,, MW 104.06 g/mole, CAS no. 141-82-2, lot. no. 449503/1, filling code 44903076) in a 250 mL beaker. To this suspension was added 26.571 g (0.1 moles) of solid strontium hydroxide (Sigma Aldrich, Sr(OH)₂*8H₂O, MW 265.71, CAS no. 1311-10-0). Then, a magnetic stirring rod was added and the stirring and heating was started to the point of boiling of the suspension. The final suspension is also white colored and the stirring was sustained by maintaining a medium rotation rate of the stirring apparatus. In order to prevent carbon dioxide from entering the solution, the beaker was covered by a covering glass.

[0097] After some minutes of boiling and stirring, the solution clarified and all the solid material dissolved. The boiling was maintained, and additional water was added when required, as to replace the water lost by boiling. After three hours of boiling, the solution was filtered white boiling on a Büchner funnel. Very small amounts of impurities were left In the filter. The filtrate was subsequently allowed to cool to room temperature, which resulted In growth of fine-powdered crystals of strontium malonate. Precipitation of the final product progressed rapidly during filtration and the majority of the product was found in the filter (unheated). Only In rare instants, the precipitation progressed in the filtrate. The product was filtered and dried at 110 °C In an oven for ½ hour followed by drying 12 hours In a dessicator over silica orange. Before analysis by x-ray crystallography and by FAAS, the salts were ground to fine powder by a mortar.

[0098] The total yield of strontium malonate was approximately 98% before recrystallisation, and the majority of impurities consisted of reminisces of the reagents and of strontium carbonate. The product was unambiguously identified as strontium malonate by x-ray crystallography and comparing the data to results of the Cambridge Crystallographic Database.

[0099] Further improvements of the synthesis may include degassing by nitrogen or by argon of the water and of all aqueous solutions, which prevents contact to carbon dioxide that eventually may lead to formation of impurities of strontium carbonate. It follows that a person skilled in the art will easily be able to adapt the procedure to proceed under an inert gas atmosphere.

Example 5

Methods of manufacture of water soluble strontium salts of dicarboxylic acids using temperatures above 100 °C

[0100] According to methods developed previously and described in examples 2-4, synthesis of strontium salts of dicarboxylic organic acids, and especially strontium salts of amino adds can be difficult to produce in larger scale (i.e. > 1 kg) due to low yields and difficulties in separating the desired reaction products from contaminants. Strontium salts of carbonate are of special concern as they will form as impurities when the reaction is occurring in atmospheric air containing normal levels of carbon dioxide. We have described in example 4 that the total yield of the product when strontium malonate is manufactured from the free acid form of the anion, and strontium hydroxide depends on temperature and on time of synthesis, In order for the reaction to reach completion, the mixture of the amino add proper and strontium hydroxide needs boiling In water for three hours, allowing ample time for strontium in the reaction mixture to react with carbon dioxide in the air. In this example we disclose methods of improving the synthesis further by providing optimized reaction conditions, where temperature is increased above 100°C in a dosed container, and where reaction times are significantly reduced.

[0101] The present example provides representative data from the optimization of conditions for synthesis of the reference compound strontium glutamate in an autoclave system. Strontium glutamate is used as a reference example, but it the optimizations described in the example is also applicable for the synthesis of other strontium salts, where the exact reaction conditions can be optimized as disclosed In this example. The reaction temperatures must be maintained below the melting point or below the temperature of decomposition of the organic anion moiety of the desired strontium salt. As an example, malonic add decomposes at 132-134 °C, and thus synthesis of strontium malonate must be performed at temperatures below 132°C.

[0102] Strontium L-glutamate was used as a reference strontium compound in the optimisation experiments. The purity of the product was monitored by comparing to crystallographic data and by measuring the content of strontium. Ideally, the content of strontium is 25.7% In strontium L-glutamate hexahydrate, which is the product formed In these experiments, It follows that other soluble strontium salts may be prepared by similar methods with high yield and purity.

Experimental

[0103] Preparation of solutions: A suspension of glutamic add (white coloured) is prepared by adding 100 mL of millipore water to 14.703 g (0.1 moles) of solid L-glutamic acid (Sigma Aldrich, C₅H₉NO₄, MW 187.14 g/mole, CAS no. 142-47-2. lot. no. 426560/1, filing code 43003336) In a 250 mL beaker. To this suspension was added 22.257 g, 26.571 g or 31.885 (0.08 moles, 0.1 moles or 0.12 moles) of solid strontium hydroxide (Sigma Aldrich, Sr(OH)₂*8H₂O, MW 265.71, CAS no. 1311-10-0).

Optimisation experiments

[0104] After preparation of the salts, the nine optimisation experiments were performed according to the settings of reference table 4.

Reference table 4. Parameters and main results of the optimisation procedure for synthesis of strontium glutamate. The pressure was monitored but not used in the optimisation process. The strontium content (%Sr) was measured by FAAS but not used as quality parameter. The yield (%) was applied as the quality parameter.

Experiment no.	Autoclave temperature (°C)	Time of synthesis (min.)	Base-acid ratio	Total volume (ML)	Autoclave pressure (bar)	Yield%	%SR (AAS)
1	125	15	0,8	50	1,55	94	25
2	124	30	1	75	1	112	22
3	124	60	1,2	100	1,6	121	21
4	127	15	0,8	100	1,2	118	22
5	132	30	1	50	1,55	120	25
6	132	60	1,2	75	1,6	50	22
7	134	15	0,8	75	1,65	108	24
8	134	30	1	100	1,65	76	14
9	132	60	1,2	50	1,65	82	24

Procedure

[0105] 

1 The calculated amount of add was weighed and transferred to a bluecap autoclave bottle and the Millipore water was added. The bottle was closed and shaken, in order to obtain a finely grained suspension.

2 The calculated amount of strontium hydroxide octahydrate was weighed and added to the acid solution of (1) and the bottle was vigorously wortexed until all coarse lumps of material were transformed Into fine-grained powder.

3 The bottle was placed in the autoclave and the temperature was set. While in the autoclave no additional stirring was carried out.

4 At t = 100° C the valve of the autoclave was closed and the timing was started.

5 During the autoclaving were monitored the actual temperature and the actual pressure.

6 After the time of autoclaving ended, the steam was let out, as soon as possible, with due respect to safety precautions.

7 At approx. 110° C the autoclave was opened and the solution was recovered. Again, the bottle was shook, as to obtain a high degree of mixing.

8 The solution was Immediately filtered hot on a Büchner funnel after autoclaving, which left only traces of carbonate in the filter. The product precipitated from the solution during cooling to room temperature.

9 After precipitation, the product was filtered and dried in an oven for ½ an hour at 110° C.Then, it was dried in an dessicator over silica-gel orange. Finally, the product was ground to fine powder in a mortar.

10 The product was weighed after grinding and the total yield calculated.

Preparation of strontium malonate according to the Invention

[0106] In order to confirm the applicability of the disclosed high temperature synthesis method for other strontium sails than strontium L-glutamate, strontium malonate was prepared. Basically the reaction conditions found for preparation of strontium L-giutamate was employed. A suspension of malonic acid (white coloured) is prepared by adding 100 mL of millipore water to 10.41 g (0.1 moles) of solid malonic acid (FLUKA 63290, MW 104.1) In a 250 mL beaker. To this suspension was added 22.257 g, 26.571 g or 31.885 (0.08 moles, 0.1 moles or 0.12 moles) of solid strontium hydroxide (Sigma Aldrich, Sr(OH)₂*8H₂O, MW 265.71, CAS no. 1311-10-0). The reaction procedure described above was follower, and the temperature was maintained below 130°C to avoid decomposition of malonic add, while the reaction time was maintained at 15 min.

Content of strontium (% Sr):

[0107] A sample of 0.2 g was dissolved in 100 mL 0.1 M HNO₃ prepared In Millipore water. This solution was further diluted by a factor of 500 by a solution of 1% KCl, and the content of strontium was determined by FAAS. The measurements were performed by using a Perkin-Elmer 2100 equipped with a hydrogen lamp for correction of the background signal. Strontium was measured at a slit with of 0.2 nm, the wavelength was 460.8 nm operated at an energy of 58 and a current of 8 mA.

X-ray crystallography

[0108] A second check of purity was performed by powder x-ray crystallography using a Huber G670 diffractometer. A characteristic diffractogram of the reference compound strontium glutamate is shown in reference fig. 1. An X-ray diffractogram of strontium malonate obtained by the high temperature synthesis method disclosed in the present example is shown in fig. 2. The double peak on the low angle side of the peak of maximum intensity observed In both figure 1 and 2 is an artefact of the instrument.

Results and discussion of the synthesis of reference compounds

[0109] In table 4, it is observed that some of the synthesis conditions resulted in relatively low yield and in strontium glutamate of low purity as apparent from the molar % of strontium In the reaction product The product of experiment no. 8 was produced in relatively low yield, and it did not contain the expected 25.7% of strontium, which was also confirmed by the x-ray analysis. Despite this outlier, in general, the outcome of the optimisation experiments is dose to the expected products. Incomplete reaction provides a product of too low content of strontium while formation of strontium carbonate during the synthesis gives a too high value of the strontium content. Conditions employed in experiments 1 and 5 gave the strontium content in best agreement with the expected value. Of notice, it is also apparent although the product of experiment no. 6 was produced in low yield; it contained an amount of strontium that corresponded to the expected value.

[0110] By studying the influence of the individual parameters on the total yield (table 4 and reference fig. 3), it becomes clear that temperature, time of autoclaving and base-acid ratio are Important for the synthesis while total volume is less Important. A yield higher than 100%, which is observed In experimental conditions 2, 3, 4, 5 and 7 originates from incomplete drying, but this effect is almost eliminated when the average values are considered, as in reference fig. 3. Thus, the maximum yield was obtained by using a high temperature (133 °C), a short time of autoclaving (15 min.) and a surplus of strontium hydroxide. Accordingly, temperature is more important than time but it compares In importance to the base-to-acld ratio. However, great care must exerted as to not exceed the temperature of decomposition In the synthesis of other strontium salts, which for, e.g., the malonate is 132-134 °C. A 10^th experiment of control of optimisation was performed, as to confirm the maximum yield of the optimisation experiments.

[0111] Furthermore,an additional experiment was performed to validate the applicability of the high temperature synthesis method for the preparation of other organic strontium salts than strontium L-glutamate. Strontium malonate was chosen. This salt may be considered especially difficult to prepare under the high temperature conditions due to the low dissociation temperature of the malonic acid anion. However, as shown in figure 2, crystalline pure and well defined strontium malonate could easily be obtained. The crystal structure of the compound has not been completely resolved as it is a new structure not previously described, but the data shows that the high temperature method is likely to be applicable for many other organic strontium salts.

[0112] Further improvements of the synthesis include introduction of Inert atmospheres to the synthesis environment, as well as degassing of all solutions by either nitrogen gas or by argon gas, as to reduce the formation of strontium carbonate.

Conclusion

[0113] The optimisation experiments show that it is possible to synthesize the reference compound strontium glutamate in high yields by elevating the temperature to values above 100°C, and by using a short time (15 min.) in the autoclave. Also, a 20% surplus of strontium-hydroxide also improves the total yield without compromising the purity of the synthesized strontium salt. A slightly more vigorous drying than sllica-gel orange should be applied to the drying procedure in order to obtain completely dried product Examples of more potent drying agents are concentrated sulphuric add or calcium oxide, but also conventional lyophilization or other mechanic treatments may be applicable for this procedure.

Example 6

Pharmacokinetic properties of dicarboxylic strontium salts

[0114] The aim of this experiment was to assess the bioavailability of dicarboxylic strontium salts including the compound used in the present invention, strontium malonate, compared with strontium chloride and strontium ranelate. The bioavailability was assessed by determination of serum strontium concentration at regular Intervals over a 24 hour period and calculating AUC.

[0115] The experiment was performed with female SPF Wistar rats of the strain HanTac:WH (GALAS) from Taconic M&B A/S, Ejby, DK-4623 Lille Skensved, Denmark. At the start of the acclimatisation period, the rats were approximately 9 weeks old with a weight of approximately 200-250 g. The animals were housed in a room provided with filtered air at a temperature of 21 °C ± 3°C and relative humidity of 55% ± 15% and a ventilation system providing 10 air changes per hour. The room was Illuminated to give a cycle of 12 hours light and 12 hours darkness. The rats were fed a complete pelleted rodent diet "Altromin 1314" (Chr. Petersen A/S, DK-4100 Ringsted, Denmark). The rats had free access to bottles with domestic quality drinking water acidified with hydrochloric acid to pH 2.5 In order to prevent microbial growth.

[0116] The rats were randomly allocated randomly in seven groups of 9 animals treated as indicated in the table below. The groups, dose levels, animal numbers were as listed in table 5:

Table 5: The 7 treatment groups of the pharmacokinetic experiment. The doses administered in the group are listed in the fist column, and salt, MW and Sr content in the middle columns.

Dose1 (mg /kg)	Group	Strontium salt	MW	% Sr	Dose Equivalent1 (Amounts In mg)	Animal No's
Vehicle	Control	Vehicle (0.5 % CMC)	-	-	-	1-9
500	B	Sr-ranelate (*7H2O) -ref. compound	639.6	27.4	500 = 137 mg Sr++	10-18
416	C	SrCl2 (*6H2O)*	266.6	32.9	137 mg Sr++ = 416	19-27
533	D	Sr-glutamate * (*6H2O)	340.7	25.7	137 mg Sr++ = 533	28-36
427	E	Sr-aspartate* (*3H2O)	272.7	32.1	137 mg Sr++ = 427	37-45
484	F	Sr-malonate* (*6H2O)	309.7	28.3	137 mg Sr++ = 484	46-54
325	G	Sr-malonate (*1H2O)	207.7	42.2	137 mg Sr++ = 325	55-63

* reference compound 1 Doses are adjusted to provide equimolar strontium dose as 500 mg/kg Strontium-ranelate (heptahydrate)(group B).

[0117] The test article (strontium salt) was given once by oral gavage according to the most recent body weight data. The control group was dosed with the vehicle alone (0.5% carboxy methyl cellulose, CMC). The vehicle was prepared with de-ionized water for all treatment groups including controls. The test substances (strontium salts) were solubilized/suspended in a volume corresponding to 5 ml/kg body weight. In order to keep the compounds in suspension, the formulations were kept on a magnetic stirrer before and during dosing.

Blood samples for toxicokinetics

[0118] On the day of treatment (Day 1), blood samples were taken from all animals. Blood samples were collected from 3 animals per group at the following time points: Pretreatment, and 30 min, 1, 1.5, 2, 4, 8 and 24 hours post-treatment, so that three animals from each group had samples taken at time 0,1.5 and 6 hours, 3 other rats at time 0.5, 2, 8 hours and the remaining three animals in the group had samples taken at 1, 4 and 24 hours.

[0119] Approximately 0.5 - 0.6 ml blood was obtained at each time point from the orbital venous plexus into plain tubes for serum. The blood was kept at room temperature for 30 to 60 minutes and until centrifugation (10 min, 1270 G, +20 °C). The serum was transferred to Nunc cryotubes (Nunc, Denmark) and frozen at -18°C for subsequent analysis of strontium content by graphite -furnace atomic-absorption spectrometry (GF-AAS).

Graphlte-fumace atomic-absorption spectrometry (GF-AAS)

[0120] Concentrated HCl was added to the serum samples to a final concentration of 0.2% HCl and the samples were then subjected to analysis using a Perkin-Elmer 2100 equipped with a hydrogen lamp for correction of the background signal. Strontium was measured at a slit with of 0.2 nm, the wavelength was 460.8 nm operated at an energy of 58 and a current of 8 mA.

Results of the pharmacokinetic study of strontium salt absorption

[0121] In figure 4, the serum concentration measured in the six groups treated with strontium salts are plotted as a function of the time after administration of the compounds. It is apparent that administration of the strontium salts results In a rapid and highly significant increase in serum strontium concentrations. When comparing the pharmaco kinetic properties of different salts, it is apparent that both the highly soluble strontium chloride as well as the relatively poorly soluble strontium ranelate (see example 3), is rapidly absorbed, reaching a maximum serum concentration after approximately 2 hours.

[0122] The di-carboxylic adds with higher solubility, and especially the reference strontium salts of the amino acids L-aspartate and L-glutamate reach the maximal serum concentration with a slower kinetic rate and, with maximal concentration reached after approximately 8 hours. Furthermore, the serum strontium concentration in the time interval from 0 - 8 hours after the administration of the test substance appears more stable, at least for some of the di-carboxylic adds such as the aspartate and malonate salts of strontium. This pattern of two distinct peaks of maximal serum concentration is also apparent in the group treated with strontium malonate. It is likely to indicate that the strontium ion is taken up by two distinct absorption mechanisms, and that the highly soluble strontium salts according to the present invention may have particular potential to exploit the biphasic nature of the strontium uptake mechanism, and thus proved an overall benefit apparent as higher bioavailability of the strontium.

[0123] When AUC calculations were performed the general course of the curves, as evidenced by average values in fig. 4, was best described by modelling the response/pharmacokinetic curves in a specially developed mathematical model. In the initial step, it assumes that the strontium is not metabolised but simply transferred from the stomach/upper digestive tract of the rat into epithelial cells by an active transport mechanism. Also without metabolism, the strontium ion is then transferred from the stomach/upper digestive tract where it is simultaneously released to the blood vessels. Only during the circulation of strontium through the veins, the strontium is dispersed and metabolised by the body tissue. This credible but simplified description, thus includes a two-step mechanism of absorption of ionic strontium after oral administrations of the strontium ions. After the strontium dose was administered to the rats, a characteristic time of uptake was found as t = 12 min. The maximum content of strontium In the serum was observed after approx. 30 min. The characteristic time value of 12 min. is interpreted as the duration of strontium ions being taken up by the active transport mechanism from the intestinal lumen and secreted into circulation. The time of strontium transfer between the stomach and the blood vessels is initiated almost instantly, while the time of transfer between the guts and the blood vessels proceeds at a later stage that depends on the type of salt Investigated. The malonate, In particular, exhibits a peak In the uptake-versus time from the guts to the blood vessels at t = 360 min., as seen In fig. 4. Thus, the time of body metabolism of the malonate is very long, as compared to that of the other salts. For all salts, however, the strontium content levels out after approx. 1750 min. (29 hours) and approaches the natural level corresponding to the pre-dose level.

[0124] The model calculations (not shown) were applied to the determination of the areas under the curve that are shown in table 6. The standard deviations of the AUC values correspond to the general uncertainty on the measurements of fig. 4, and their magnitude does not allow for a significant discrimination between the salts. The AUC values of the salts are much higher than the AUC value of the pre-dose samples.

Table 6. Determination of the area under the curve according (AUC) to the model calculations.

ANION OF Sr-SALT	AUC mg/L·min	STDDEV mg/L·min
α-ketoglutarate -ref. compound	9000	1600
Aspartate -ref. compound	7000	1700
Chloride - ref. compound	7300	2000
Glutamate -ref. compound	10100	3100
Malonate	15000	8500
Pre-dose	168	67
Average	6800	5400

[0125] These effects of delayed uptake of strontium and serum levels in a sustained level over longer time periods observed with strontium salts with di-carboxylic organic anions may enhance the pharmacological properties of the compounds. The delayed attainment of C_max may be an advantage for the use of the strontium compound In the treatment of diseases and conditions affecting bone metabolism. In these cases it is often an advantage to administer the compound in the evening before bedtime, as this would allow the compound to act at night, when resorption of bone is occurring at the highest rate. Furthermore, the administration before bedtime minimizes the potential interference from calcium in the normal diet, as the pharmaceutical preparation of the strontium salt would be taken after the last meal. This is in contrast to administration during the day, where the calcium content of normal meals would have the potential to Interfere and reduce the uptake of strontium. The gradual increase in serum strontium concentration over 4 - 8 hours after administration of the compound would comply well with evening administration of the compound and appears well suited to maximize the therapeutic effect of the strontium compound on bone metabolism.

Claims

1. Use of strontium malonate having a water-solubility at room temperature in a range from 1 g/l to 100 g/l for the preparation of a pharmaceutical composition for the treatment of and/or prophylaxis of osteoporosis, osteopenia, Paget's disease, osteolytic lesions produced by bone metastasis, bone loss due to sex steroid hormone deficiency, bone abnormalities caused by cancer therapeutics, immobilization-induced osteopenia or osteoporosis, glucocorticoid-induced osteopenia or osteoporosis or for the improvement of fracture healing after traumatic or atraumatic fracture in a mammal.

2. Use according to claim 1, wherein the total dose of strontium to be administered to the mammal in a one day period is at least 0.01 g, such as, e.g. at least 0.025 g, at least 0.050 g, at least 0.075 g, at least 0.1 g, at least 0.2 g, at least 0.3 g, at least 0.4 g or at least 0.5 g or from 0.01 g to 2 g such as, e.g., from 0.1 g to 2 g, from 0.3 g to 2 g or from 0.3 g to 1 g.

3. Use according to claim 1 or 2, wherein the pharmaceutical composition is designed for oral or parenteral administration.

4. Use according to claim 3, wherein the pharmaceutical composition is designed for oral administration, and the peak concentration of strontium is reached at least 2.5 hours after the oral administration.

5. Use according to claim 3 or 4, wherein the pharmaceutical composition is in the form of tablets, capsules, sachets, powders, pellets, granules, granulates, mixtures, syrups, solutions, suspensions, emulsions, or the like, for oral administration.

6. Use according to claim 3, wherein the pharmaceutical composition is in the form of a solution, suspension or emulsion, or the like, for intraveneous, intramuscular, intraartricular or subcutaneous injection.

7. Use according to claim 3 or 4, wherein the pharmaceutical composition is in the form of a toothpaste or a mouthwash intended for application to the teeth or to the oral mucosa.

8. Use according to any of the preceding claims wherein the mammal is a human, such as e.g., a human female or male adult, adolescent or a child.

9. Use according to any of claims 1-7, wherein the mammal is a domestic animal, such as, e.g. a cat, a dog, a horse, a cow or a sheep.

Ansprüche

1. Verwendung von Strontiummalonat mit einer Wasserlöslichkeit bei Raumtemperatur in einem Bereich von 1 g/l bis 100 g/l zur Herstellung einer pharmazeutischen Zusammensetzung zur Behandlung und/oder zur Prophylaxe von Osteoporose, Osteopenie, der Pagetschen Krankheit, durch Knochenmetastase verursachten osteolytischen Läsionen, eines Knochenschwundes aufgrund eines Mangels an Sexualsteroidhormonen, durch Krebstherapien verursachten Knochenanomalien, einer durch Immobilisierung induzierten Osteopenie oder Osteoporose, einer durch Glucocorticoide induzierten Osteopenie oder Osteoporose oder zur Verbesserung des Heilens von Frakturen nach einer traumatischen oder atraumatischen Fraktur bei einem Säuger.

2. Verwendung nach Anspruch 1, wobei die Gesamtdosis des einem Säuger während eines eintägigen Zeitraums zu verabreichenden Strontiums wenigstens 0,01 g, wie beispielsweise wenigstens 0,025 g, wenigstens 0,050 g, wenigstens 0,075 g, wenigstens 0,1 g, wenigstens 0,2 g, wenigstens 0,3 g, wenigstens 0,4 g oder wenigstens 0,5 g oder von 0,01 g bis 2 g, wie beispielsweise von 0,1 g bis 2 g, von 0,3 g bis 2 g oder von 0,3 bis 1 g beträgt.

3. Verwendung nach Anspruch 1 oder 2, wobei die pharmazeutische Zusammensetzung zur oralen oder parenteralen Verabreichung vorgesehen ist.

4. Verwendung nach Anspruch 3, wobei die pharmazeutische Zusammensetzung zur oralen Verabreichung vorgesehen ist und die Höchstkonzentration des Strontiums wenigstens 2,5 h nach der oralen Verabreichung erreicht wird.

5. Verwendung nach Anspruch 3 oder 4, wobei die pharmazeutische Zusammensetzung in Form von Tabletten, Kapseln, Beutelchen, Pulvern, Pellets, Körnchen, Granulaten, Mischungen, Sirupen, Lösungen, Suspensionen, Emulsionen oder dergleichen zur oralen Verabreichung vorliegt.

6. Verwendung nach Anspruch 3, wobei die pharmazeutische Zusammensetzung in Form einer Lösung, Suspension oder Emulsion oder dergleichen zur intravenösen, intramuskulären, intraartikulären oder subkutanen Injektion vorliegt.

7. Verwendung nach Anspruch 3 oder 4, wobei die pharmazeutische Zusammensetzung in Form einer Zahnpasta oder Mundspülung vorliegt, die zum Auftragen auf die Zähne oder auf die Mundschleimhaut vorgesehen ist.

8. Verwendung nach einem der vorhergehenden Ansprüche, wobei der Säuger ein Mensch wie z.B. ein menschlicher weiblicher oder männlicher Erwachsener, ein(e) Jugendliche(r) oder ein Kind ist.

9. Verwendung nach einem der Ansprüche 1 - 7, wobei der Säuger ein Haustier wie beispielsweise eine Katze, ein Hund, ein Pferd, eine Kuh oder ein Schaf ist.

Revendications

1. Utilisation de malonate de strontium ayant une solubilité dans l'eau à la température ambiante comprise dans une gamme de 1 g/l à 100 g/l pour la préparation d'une composition pharmaceutique destinée au traitement et/ou à la prophylaxie de l'ostéoporose, de l'ostéopénie, de la maladie de Paget, de lésions ostéolytiques produites par des métastases osseuses, d'une perte d'os due à une déficience en hormone stéroïde sexuelle, d'anomalies des os provoquées par des agents de traitement d'un cancer, d'une ostéopénie ou d'une ostéoporose induite par une immobilisation, d'une ostéopénie ou d'une ostéoporose induite par des glucocorticoïdes ou pour l'amélioration de la guérison de fractures après une fracture traumatique ou atraumatique chez un mammifère.

2. Utilisation selon la revendication 1, dans laquelle la dose totale de strontium à administrer au mammifère en une période d'un jour est d'au moins 0,01 g, comme par exemple, au moins 0,025 g, au moins 0,050 g, au moins 0,075 g, au moins 0,1 g, au moins 0,2 g, au moins 0,3 g, au moins 0,4 g ou au moins 0,5 g ou de 0,01 g à 2 g comme par exemple, de 0,1 g à 2 g, de 0,3 g à 2 g ou de 0,3 g à 1 g.

3. Utilisation selon la revendication 1 ou 2, dans laquelle la composition pharmaceutique est conçue pour une administration orale ou parentérale.

4. Utilisation selon la revendication 3, dans laquelle la composition pharmaceutique est conçue pour une administration orale, et la concentration de pic du strontium est atteinte au moins 2,5 heures après l'administration orale.

5. Utilisation selon la revendication 3 ou 4, dans laquelle la composition pharmaceutique est sous la forme de comprimés, de gélules, de sachets, de poudres, de granulés, de granules, de granulats, de mélanges, de sirops, de solutions, de suspensions, d'émulsions ou similaires, pour une administration orale.

6. Utilisation selon la revendication 3, dans laquelle la composition pharmaceutique est sous la forme d'une solution, d'une suspension ou d'une émulsion ou similaires, pour une injection intraveineuse, intramusculaire, intra-articulaire ou sous-cutanée.

7. Utilisation selon la revendication 3 ou 4, dans laquelle la composition pharmaceutique est sous la forme d'une pâte dentifrice ou d'un bain de bouche conçus pour l'application aux dents ou aux muqueuses orales.

8. Utilisation selon l'une quelconque des revendications précédentes, dans laquelle le mammifère est un humain, comme par exemple, un adulte, un adolescent ou un enfant humain de sexe féminin ou masculin.

9. Utilisation selon l'une quelconque des revendications 1 à 7, dans laquelle le mammifère est un animal domestique, comme par exemple, un chat, un chien, un cheval, une vache ou un mouton.

Drawing