Title of Invention	A METHOD FOR SCREENING FOR Ca2+-CHANNEL BLOCKERS INVITRO
Abstract	This invention relates to a method for screening for Ca<sup>2+</sup> -channel blockers invitro comprising the steps: treatment of cells having Ca<sup>2+</sup>-channels with a putative Ca<sup>2+</sup>-channel blocker; contacting the cells with 1-amino-3-(N,N-dimethylamino)propylidene-1,1-bisphosphonic acid, any of its soluble salts or any of its hydrates; measuring a response as a result of the contacting step as herein described and to a method for screening functional analogues of 1-amino-3-(N,N-dimethylamino)propylidene- 1, 1 -bisphosphonic acid, any of its soluble salts or any of its hydrates.

Title of Invention

A METHOD FOR SCREENING FOR Ca2+-CHANNEL BLOCKERS INVITRO

Abstract

This invention relates to a method for screening for Ca2+ -channel blockers invitro comprising the steps: treatment of cells having Ca2+-channels with a putative Ca2+-channel blocker; contacting the cells with 1-amino-3-(N,N-dimethylamino)propylidene-1,1-bisphosphonic acid, any of its soluble salts or any of its hydrates; measuring a response as a result of the contacting step as herein described and to a method for screening functional analogues of 1-amino-3-(N,N-dimethylamino)propylidene- 1, 1 -bisphosphonic acid, any of its soluble salts or any of its hydrates.

Full Text	The present invention relates to a method for screening for Ca2+-channel blockers invitro condition. The compound l-amino-3-(N,N-dimthylamino)-propylidene-l,l-bisphosphonic acid, or any of its soluble salts or any of its hydrates, belongs to the group of bisphosphonates which are compounds containing two phosphonate groups bound to a carbon and two additional groups R1and R2, respectively, which bind strongly to calcium crystals, inhibit their growth, suppress bone resorption and are being used in the treatment of a vaR1ety of disorders of bone metabo¬lism. l-amino-3-(N,N-dimethylamino)-propylidene-l,l-bisphosphonic acid is the amino-substituted form of olpadronate (OPD) and is hereafter sometimes referred to as NH2-OPD. Bisphosphonates are recognized potent inhibitors of bone resorption and are successfully used in the treatment of common bone disorders such as osteoporosis and tumoral bone diseases, amongst others. Structurally, bisphosphonates are deR1vatives of methylene bisphosphonate in which the two hydrogens on the geminal carbon atom are replaced by two groups, namely R1 and R2, which usually are not identical In many cases the R1 moiety is a hydroxyl group, be¬cause of its ability to form a tR1dentate structure together with the two phosphonate groups to bind bone mateR1al. Extensive studies have been carR1ed out on the effects of bisphosphonates on bone cells, in particular those related to their anti-resorptiye potency. The molecular mechanisms involved in such actions, however, are not clear. The anti-resbrptive activity and its utility in the treat¬ment of osteoporoses and related bone diseases have, for example, been reported and de¬scR1bed m the following patents: WO 94/00129 to Frances M. et al., WO 93/11786 to Gueddes A. et al., WO 92/14474 to McOskar et al., US Patent No. 4,942,157 to Gall R. et al., and in US Patent No. 3,962,432 to Schmidt-Dunker M. Although it appears that the nature of the R2 moiety of bisphosphonates determines their anti-resorptive activity, replacement of the hydroxyl group in R1 by an NH2 group in some bisphosphonates (e.g. olpadronate and pamidronate) markedly-reduces their anti-resorptive activity. This decrease in anti-resorptive efficacy of the amino deR1vatives is thought to be due to substitution-dependent changes in the cellular effects of the compounds, since their affinity for bone mineral is not significantly affected. This lack of anti-resorptive activity is, for ex¬ample, disclosed in vanBeek et al., 1996, Journal of Bone and Mineral Research, vol. 11, no. 10, pp. 1492-1497, and international patent application PCT/EP96/02981, the latter disclosing the use of l-amino-3-(N,N-dimethyIamino)-propyhdene-l,l-bisphosphonic acid for the treatment of osteoporoses, arthR1tis and peR1odontal diseases. The structure and stability of skeletal bones is - to a high degree - influenced and determined by an equilibR1um between the activities of o.geo.blMts. and_osteoclaste,J.^ the cells responsi¬ble for mineraUzatioR1 and resorption of bone structure, respectively. Therefore, bones are subject to a continuous restructuR1ng process involving anabolic and catabolic reactions. In human beings, as of the age of 30, the catabolic processes prevail such that there will be a net loss in total bone substance naturally. Consequently, the rate at which the renewal of the bone tissue takes place will be lowered with increasing age. Object of the present invention is to provide means for prevention of the onset of clinically pathological bone-conditions. Another object of the present invention is to avoid a predisposition to bone disfunctions or bone diseases. Yet a further object of the present invention is to optimize the physiological bone functions in patients. The present invention in its vaR1ous aspects and embodiments disclosed hereafter solves all these objectives and provides further advantages. Summary of the invention The present invention is related to" novel uses of l-amino-3-(N,N-dimethylamino)-propylidene-l,l-bisphosphonic acid and/or any of its soluble salts and/or any of its hydrates. For the purposes of the following specification, it is intended that the term l-amino-3-(N,N-dimethylamino)-propylidene-l,l-bisphosphonic acid also encompasses any of its soluble salts and/or any of its hydrates. BR1efly stated, the present invention provides novel uses of l-amino-3-(N,N-dimethylamino)-propylidene-l,l-bisphosphonic acid, medicaments compR1sing l-amino-3-(N,N- • dimethylamiiio)-propylidene-l,l-bisphosphonic acid and screening methods involving 1-amino-3-(N,N-dimethylamino)-propylidene-1,1 -bisphosphonic acid. Within one aspect of the present invention, there is provided the use of l-amino-3-(N,N- dimethylamino)-propylidene-1,1-bisphosphonic acid for the manufacture of a medicament for selective modulation of osteoblasts. Furthermore, there is provided the use of l-amino-3- (N,N-dimethylamino)-propylidene-1,1-bisphosphonic acid for the manufacture of a medica- ment for the maintenance of a healthy bone structure. There is also provided the use of l-amino-3-(N,N-dimethylamino)-propylidene-l,l-bisphosphonic acid for the manufacture of a medicament for the prevention of osteopathies in healthy patients. The invention also provides for the use of l-amino-3-(N,N-dimethylamio)-propylidene-l,l- bisphosphonic acid for the manufacture of a medicament for the treatment of patients who , have recently undergone treatment with corticosteroids. Additionally there is provided the use of l-amino-3-(N,N-dimethylamino)-propylidene-l,l-bisphosphonic acid for the manufacture of a medicament for post-treatment of osteopathies wherein an anti-resorptive activity is not desired. The invention also provides for the use of l-amino-3-(N,N-dimethylamino)-propyIidene-l,l-bisphosphonic acid for the manufacture of a medicament for the treatment of children having an osteopathy.. In another aspect of the invention, there is provided the use of l-amino-3-(N,N-dimethylamino)-propylidene-l,l-bisphosphonic acid for the manufacture of a medicament for the stimulation of both signalling cascades and reaction mechanisms mediating the action of l-amino-3-(N,N-dimethylamino)-propylidene-l,l-bisphosphonic acid which can be blocked by Ca2+ -channel blockers. In yet another aspect of the present invention, there is provided the use of l-ainino-3- (N,N-dimethylamino)-propylidene-l,l-bisphosphonic acid for the manufacture of a medicament for the mobilization of Ca2+-ions from IP3 sensitive stores. In yet another aspect of the invention, there is provided a method for screening for Ca2+- charmel blockers compR1sing the steps: treating of cells having Ca2+-channels with a putative Ca2+-channel blocker; contacting the cells with l-amino-3-(N,N-dimethylamino)-propylidene-l,l- bisphosphonic acid, any of its soluble salts or any of its hydrates; measuR1ng a response as a result of the contacting step. It is envisioned that the treatment-step and the contacting-step occur sequentially with the treatment-step preceding the contacting-step or vice versa, or that they occur simultaneously. In another aspect, the invention also provides a method for screening for functional analogues of l-amino-3-(N,N-dimethylamino)-propylidene-l,l-bisphosphonic acid, any of its soluble salts or any of its hydrates compR1sing the steps; 2+ 2+t- treating of cells having Ca -channels with Ca -channel blockers; contacting the cells with the putative functional analogue which, in the absence of any Ca2+-channel blockers, is known to cause a Ca2+-ion influx into the cells; measuR1ng a response as a result of the contacting step. It is envisioned that the treatment-step and the contacting-step occur sequentially with the treatment-step preceding the contacting-step or vice versa, or that they occur simultaneously. In the present invention there is also disclosed a medicament compR1sing l-amino-3-(N,N-dimethylamino)-propylidene-l,l-bisphosphonic acid and at least one physiologically accept¬able carR1er, for the selective modulation of the osteoblasts, for the maintenance of a healthy bone structure, for the prevention of osteopathies or for the post-treatment of osteopathies where an anti-resorptive activity is not desired. In one aspect of the present invention it provides a medicament compR1sing l-amino-3-(N,N-dimethylamino)-propylidene-l,l-bisphosphonic acid, which is to be applied in healthy pa¬tients or in patients at or above 40 years of age. The present invention also-discloses a method for the selective modulation of osteoblasts and/or for the maintenance of a healthy bone structure and/or for the prevention of osteopa¬thies in healthy patients and/or for the treatment of patients who have recently imdergone treatment with corticosteroids, and/or for post-treatment of osteopathies where an anti-resorptive activity is not desired, and/or for the treatment of children having an osteopathy and/or for the stimulation of those signaling cascades and reaction mechanisms mediating the action of l-amino-3-(N,N-dimethylamino)-propylidene-l,l-bisphosphonic acid or any of its soluble salts or any of its hydrates, which can be blockedby Ca^t-channel blockers,and/or for the mobilization of C+2ions fromIP3-sensitive stores, comprising administeR1ng l-amino-3-(N,N-dimethylamino)-propylidene-l,l-bisphosphonic acid or any of its soluble salts or any of its hydrates alone or in combination with a pharmaceutical carR1er to a patient, the l-amino-3-{N,N-dimethylamino)-propylidene-l,l-bisphosphonic acid or any of \| its soluble salts or any of its hydrates being administered in doses of O.I to 1000 mg/oral ap¬plication or 0.02 to 200 mg/parenteral application. It is preferred that the term "selective modulation of osteoblasts" compR1ses a stimulation of the cellular activities of the osteoblasts, more preferably an influence on the Ca2+-homeostasis of the osteoblasts. A preferred embodiment of "selective modulation" compR1ses a transient increase of the Ca2+"-levels in the osteoblasts. A selective modulation of osteoblasts may also encompass and manifest itself in the synthesis of osteocalcine and/or osteopontin, osteonectin, calprotectin, fibronectin, matR1x Gla protein and/or bone sialoprotein. It is intended in the present invention that the term "maintenance of a healthy bone structure" also encompass a prevention of clinically pathological conditions or diseases. Within one aspect of the present invention it is preferred that where l-amino-3-(N,N-dimethylamino)-propyHdene-l,l-bisphosphonic acid is used for the manufacture of a me¬dicament for the maintenance of healthy bone structure, that the medicament is applied in healthy patients, more preferably patients without osteopathies. The term "healthy bone structure" is meant to include any bone structure characteR1zed by a balanced equilibR1um between catabolic and anabolic processes resulting in a structure capable of withstanding me¬ chanical stress that occurs as a result of the organism"s activities, as well as a structure that is enable of remodelling itself by aforementioned equilibR1um and repaiR1ng itself by the re¬moval of weakened or worn-out parts and the build-up of strong parts, thereby avoiding mi-crocracks and parts prone to fracture. It has been foimd that, especially with older people, the bones have a less strong structure because of their reduced rate of renewal. Furthermore, older people may get weaker, more fragile bones due to sedentary lifestyle, muscular weakness and insufficient nutR1tion. As a result the affected bones, im fact, comprise ""unfit" mineralized structures and therefore be¬come bR1ttle and unstable which very often is accompanied by a tendency for fractures. These "unfit" structures have not manifested themselves yet as clinically pathological conditions but are less than ideal. They can also be found- apart from in people at or above the age of 40 years - in people recently treated with corticosteroids or in people recently treated with anti- osteoporotic agents such as fluoR1ne and common bisphosphonates such as etidronate and chlodronate. The term " patient" is meant to encompass human beings as well as any vertebrate animal, for example cats, dogs, cows, horses and other domestic animals. The term "osteopathy" designates any pathological condition of the bone resulting in a weak¬ened or irregular or abnormal bone structure. Within the present invention it is used synony¬mously with "bone disease". Preferably the term "osteopathy" refers to a cUnically pathologi¬cal condition selected from the group compR1sing osteoporosis, Paget"s disease, arthR1tis, peR1odontal osteopenia, adolescent scoliosis, fracture, disuse osteopenia, post-transplant os¬teopenia, hyper-parathyroidism-associated osteopenia, drug-induced osteopenia, nutR1tional osteopenia, metabolic bone disease, osteopenia of prematuR1ty and ossification disorder. Where the term "patient" is referR1ng to a human being, in one embodiment it is preferred that the medicament is applied in human beings at or above the age of 40 years, in another em¬bodiment it is preferred that the medicament is applied in children. Preferably, the term "chil¬dren" is meant to encompass individuals who are from 0 to 16 years old. Where l-amino-3-(N,N-dimethylamino)-propylidene-l,l-bisphosphonic acid is used for the manufacture of a medicament for the prevention of osteopathies in healthy patients or for the manufacture of a medicament for the treatment of children having an osteopathy it is pre¬ferred that the osteopathy is selected from the group compR1sing osteoporosis, Paget"s disease, arthR1tis, peR1odontal osteopenia, adolescent scoliosis, fracture, disuse osteopenia, post-transplant osteopenia, hyper-parathyroidism-associated osteopenia, drug-induced osteopenia, nutR1tional osteopenia, metabolic bone disease, osteopenia of prematuR1ty and ossification disorder. In one embodiment it is preferred that l-amino-3-(N,N-dimethylamino)-propylidene-l,l-bisphosphonic acid - after the application of the medicament in a patient - will be present at extracellular concentrations in a range between 10-8 and 10-1010M, more preferably lO-7M and 10""M, most preferably at an extracellular concentration of about lO-7M. In the embodiment where l-amino-3-(N,N-dimethylamino)-propylidene-l,l-bisphosphonic acid is used for the manufacture of a medicament for the stimulation of those signaling cas¬cades and reaction mechanisms mediating the action of l-amino-3-(N,N-dimethylamino)-propylidene-1,1 -bisphosphonic acid, which can be blocked by Ca2+-channel blockers it is pre¬ferred that the Ca 2+-channel blockers are selected from the group compR1sing nifedipine and verapamil. In one embodiment it is preferred that l-amino-3-(N,N-dimethylamino)-propylidene-l,l-bisphosphonic acid be used in doses of 0.01 to 1000 mg/oral application, more preferably 12.5 to 75 mg/oral application. The term "oral application" is meant to include solid or solu¬ble liquid pharmaceutical formulations, gels, soft capsules, tablets, capsules containing solid preparations, soluble liquid forms or suspensions, and pills. Within one aspect of the present invention it is envisioned that l-amino-3-(N,N-dimethylamino)-propylidene-l,l-bisphosphonic acid be used also for topical administration to be applied on skin and/or muco¬sae. Forms of application useful for this purpose compR1se ointments, creams, sprays, sup¬positoR1es and gels. In another embodiment of the present invention it is preferred that l-amino-3-(N,N-dimethylamino)-propylidene-1,1-bisphosphonic acid be used in doses of 0.02 to 200 mg/parenteral application, more preferably 2.5 to 15 mg/patenteral application. The tenn\| "parenteral application" is meant to include any application that avoids the gastro-intestinal tract, for-€xample by sub-cutaneous, intra-muscular or intra-venous injection or infusion. The term "IPs-sensitive stores" refers to those intracellular"Stores of Ca2+-ions which, upon the presence of IP3 (= inositol tR1phosphate) rapidly release Ca2+ions. Such IPs-sensitive stores oicompass for example the endoplasmic reticulum and, in smooth muscle cells, the sarcoplasmic reticulum. It is to be noted that according to the present invention the influence exerted by l-amino-3-(N,N-dimethylamino)-propylidene-l,l-bisphosphomc acid on the Ca2+ homeostasis of the osteoblasts, in particular an increase of the Ca2+Mevels in the osteoblasts, can occur in at least two different ways: Ca2+"-ions can be mobilized from endogenous stores, such as those which are IPa-sensitive or thapsigargin-senstitive. Secondly, there can also occur a Ca2+-ioh-mflux from extracellular stores. This may for ex¬ample occur through voltage-dependent Ca2+-channels (VDCC), but other forms of Ca2+-channels, e.g. ligand-gated channels, can be envisioned, too. It is within the scope of the present invention that l-amino-3-(N,N-dimethylamino)-propylidene-l,l-bisphosphonic acid also be used for screening for Ca^"^-channel blockers. The term "putative Ca -channel blocker" is meant to designate a compound to be tested for its Ca -channel-blocking capabilities. The term "putative fimctional analogue" of l-amino-3-(N,N-dimethylamino)-propylidene-l,l-bisphosphonic acid designates any compound that is known to cause a Ca2+-ion influx into the cells in the absence of any Ca2+-channel blockers and is therefore mimicking l-amino-3-(N,N-dimethylamino)-propyUdene-l,l-bisphosphonic acid in its influence on Ca^-homeostasis of the cells. The term "measuR1ng a response" is also meant to include the meas¬urement or detection of the absence of a response. Within the present invention, the term "re-sponse" is preferably meant to designate a change in cytosolic Ca -levels. It is also within the scope of the present invention to combine l-amino-3-(N,N-dimethylamino)-propylidene-l,l-bisphosphomc acid in its novel uses together with at least one other compound/substance, in synergistic combinations. The term "combination" is meant to include the incorporation of l-amino-3-(N,N-dimethylamino)-propylidene-l,l-bisphosphonic acid together with another (other) substance(s) into the same medicament, as well as the administeR1ng of a medicament compR1sing l-amino-3-(N,N-dimethylamino)-propylidene-l.1-bisphosphonic acid to a patient withtaother: (other) substance - isteiing this (these) other substance(s) in a physically separated, i.e. spatially or temporalIy separated manner from the medicament. In the case where the substance(s) is (are) adminis¬tered in a separated manner from the medicament compR1sing l-amino-3-(N,N-dimethylamino)-propylidene-l,l-bisphosphomc acid, it is preferred that the l-amino-3-(N,N-dimethylamino)-propylidene-l,l-bisphosphonic acid-compR1sing medicament is administered with the other substance(s) in a single-step (concerted intake, infusions, injections), sequen¬tial-step (one after the other) or cyclic-step fashion. In one embodiment it is preferred that the medicament compR1sing 1 -amino-3-(N,N-dimethylamino)-propylidene-1,1 -bisphosphonic acid is applied before, duR1ng or after treatment with other amino-substituted bisphosphonates. For example, a sequential administeR1ng of the l-amino-3-(N,N-dimethylamino)-propylidene-1,1-bisphosphonic acid-compR1sing medicament with other amino-substituted bisphospho¬nates may be indicated, since other amino-substituted bisphosphonates exert a partial anti-resorptive effect. So a patient may need a l-amino-3-(N,N-dimethyIammo)-propylidene-l,l-bisphosphonic acid-compR1smg medicament for bone formation due to the selective modulat¬ing effect of l-amino-3-(N,N-dimethylamino)-propyndene-1,1-bisphosphonic acid on the osteoblasts, and at the same time another amino-substituted bisphosphonate for a "tunable" degree of mhibition of bone resorption, depending on his/her individual requirement. As another example a patient might first require a high degree of inhibition of bone resorp¬tion, continue then with low or none and then change again afterwards, according to mdivid-ual requirements which can be determined by biochemical, densitometR1cal, X-ray or other measurements performed on the bones of the individual. Thus l-amino-3-(N,N-dknethylamino)-propylidene-l,l-bisphosphonic acid is an ideal candi¬date forming the basis for an individually tunable therapy of conditions where bones are weak and/or fragile. This substance/these substances/compounds of the aforementioned synergistic combinations is/are selected from the group compR1sing calcium salts, calcium citrate and calcium carbon¬ate, other amino-substituted bisphosphonates, pharmaceutically active fluoR1ne-containing salts, vitamins of the D-group and their metabolites, cholecalciferol, calcifediol, calcitR1ol, ergocaldferol, PTH, anabolic hoimones, such as estrogens, substances with estogenic activ; ity on the bone, progestogens, androgens, growth hormones, peptides with growth hormone activity, selective modulators of the estrogenic receptor, raloxifene and others. The term "amino-substituted bisphosphonate" preferably designates a bisphosphonate compound that has an amino group at the 1-position. Other substitution positions, however, can be envi¬sioned, too. Where the "aminodeR1vative" of a 1-hydroxy bisphosphonate is mentioned here, this is meant to encompass molecules having a structure in which the hydroxy-group is re¬placed by an amino group. It is one of the surpR1sing advantages" of the present invention that l-amino-3-{N,N-dimethylamino)-propylidene-l,l-bisphosphonic acid acts to avoid the formation of the afore¬mentioned "unfit" bone structures and aids in removing them once they have been formed due to its selective modulating capabilities with respect to the osteoblasts. Therefore, unfit mineralized structures in the bone are prevented and removed respectively. For example, these structures can aR1se in older people, but have not manifested themselves in •clinically pathological conditions yet. Another important example where these unfit structures may occur is the growing skeleton, i.e. children. These may be healthy children or children having an osteopathy. The present invention provides means for removing these unfit struc¬tures or preventing them in the first place, thus contR1buting to a healthy bone structure which is capable of withstanding the mechanical stress exerted upon the bone through daily usage. These and other aspects of the present invention will become evident upon reference to the following examples and attached fi.gures. In addition, vaR1ous references are set forth below which descR1be in more detail certain procedures or expeR1mental details and are therefore incorporated by reference in their entirety. De-scR1ption of the figures Figure 1 shows the chemical structures of the Rl and R2 chains of the bisphosphonates used in the present studies, i.e. etidronate (EHDP), paihidronate (APD), olpadronate (OPD) and NHa-oIpadronate (NH2-OPD) (=l-amino-3-(N,N-dimethylamino)-propylidene-l,l- bisphosphoilic^d).; ,-. .. Figure 2 shows the induction of osteocalcin (OC) synthesis by bisphosphonates in cultured osteoblasts: Cells were grown by 72 hours in the absence (control) or presence of the indicated concentra¬tions of la, 25-dihydroxy-vitamin D3 [l,25(OH)2D3], EHDP, APD, OPD or NH2-OPD. Then osteocalcin (OC) released into the medium was quantitated as descR1bed in Example 4. Data are the average ± SD of 3 independent expeR1ments performed by tR1plicate. Figure 3 shows the rapid actions of bisphosphonates on cytosolic Ca2+ in osteoblasts: Fura-2 loaded cells were exposed to the indicated concentrations of la, 25-dihydroxy-vitamin D3 [l,25(OH)2D3], EHDP or APD (Panel A) and OPD or NH2-OPD (Panel B), and cytosoUc calcium levels were then monitored as descR1bed under Example 3. Shown are time-traces representative from at least 4 independent recordings. Figure 4 shows the dose dependency of the rapid actions of OPD and NH2-OPD on cytosolic Ca2+ in osteoblasts: Fura-2 loaded cells were treated with vehicle (buffered saline solution. Basal), or the indicated concentrations of OPD or NH2-OPD, and intracellular Ca^" concentration ([Ca2+]i) was meas¬ured as descR1bed. [Ca2+]! values were collected at the peak of the BP-induced Ca2+ response. Data are the average of 4 independent [Ca2+li recordings ± sd. p values (p Figure 5 shows the kinetics of the bisphosphonate-dependent cytosolic Ca2+ change relative to 1,25(0H)2D3 effect: Fura-2 loaded cells were exposed to l,25(OH)2D3 (lOnM), or 10 nM of either"EHDP, APD, OPD or NH2-OPD, and cytosolic calcium was momtored,as descR1bed. A) Both the magni-vtude of change at maximum jpeak reached, as well as the corresponding time-to-peak were-registered and compared to those for the steroid. Shown are results repiesentatiye fiom 4 in¬dependent expeR1ments, B) The early phase (0-2 min) of Ca2+ increment in response to 10 nM BP or l,25(OH)2D3 was monitored and plotted as change in Ca2+i vs. Minutes of exposure. Figure 6 shows cytosolic calcium-levels in Fura-2 loaded cells that had been treated with (Thp-treated) or without (Control) 1 (μiM thapsigargin and then exposed to OPD (10 nM) or NH2-OPD (10 nM). Cytosolic calcium levels were monitored as descR1bed under Example 3. Shown are time-traces representative froni at least 3 independent recordings. Example 1 Bisphosphonates (BP) have the following general structure: wherein R1- and R2-chains can have vaR1ous identities. The structures of the Rl and R2 chains of the bisphosphonates used in this study are shown in Figxu-e 1. All of them were from Gador S.A. (Buenos Aires, Argentina). Fura-2/pentaacetoxymethyl ester (Fura-2/AM), pluronic F-127, nifedipine, verapamil, neomycin, Dulbecco"s modified Eagle"s mediimi and fetal bovine serum were from Sigma Chemical Co. (St.Louis, MO, USA): U73122 (l-(6-((17p-3-methoxyesfra-l,3,5(10)-tR1en-17-yl)amino)hexyl)-lH-pyrrole-2,5-dione) and U73343 (l-(6-((17P-3-methoxyestra-l,3,5(10)-tR1en-17-yl)amino)hexyl)-2,5-pyrrolidine) were from Biomol Research LaboratoR1es Inc. (Plymouth Meeting, PA, USA). All other reagents used were of analytical grade. • Example 2 Cell culture Osteoblast-like rat osteosarcoma cells (ROS17/2.8) were cultured as monolayers at 37"C in Dulbecco"s modified Eagle"s medium (DME) containing 10% fetal bovine serum imder hu¬midified air (5.5% CO2). After 48 hours, medium was changed to DME containing 1% fetal bovine serum. Unless otherwise stated, cells were allowed to grow until confluence (4-5 days after plating). Example 3 Intracellular calcium measurements intracellular Ca2+ changes were monitored by using the Ca^"^-sensitive fluorescent dye Fura-2 as previously descR1bed (Vasquez et al., 1998, J. Biol. Chem. 273, 33954-33960). Cell dye loading was achieved by incubating the cells in buffer A containing (in mM): 138 NaCI, 5 KCl, 1 MgCl2, 5 glucose, 10 HEPES (pH 7.4), 1.5 CaCl2, plus 0.1% bovine serumalbumin (BSA), 2 \xM of the pentaacetoxymethylester deR1vative (membrane permeable) Fura-2/AM and 0.012% pluronic F-127, in the dark duR1ng 40 min at room temperature (20-25°C) in order to minimize dye compartmentalization. Unloaded dye was washed out and cells were stored in buffer B (buffer A without BSA, Fura-2/AM and pluronic F-127) in the dark (room tem¬perature) by at least 30 min pR1or to use, to allow for complete intracellular dye deesteR1fica-tion. For fluorescence measurements the coverslips containing dye-loaded cells were moimted into quartz cuvettes and introduced into the thermostatized (37°C) sample compartment of a SLM Aminco 8100 spectrofluoR1meter. Fura-2 intracellular fluorescence intensity was moni¬tored at an emission wavelength of 510 imi (8 run bandpass) by alternating with an electroni¬cally controlled chopper (300 Hz) the excitation wavelength between 340 and 390 nm em¬ploying a dual excitation monochromator at 4 imi bandpass. Signals from short and long wavelength were ratioed (R = 340/390) thus making the measurement independent of vaR1a¬tions in cellular dye content, dye leakage or photobleaching. Calibration of Fura-2 signal to calculate [Ca^"^J; values was performed for each coverslip as follows: maximal (Rmax) and minimal (Rmin) intracellular dye fluorescence signals were-determined by adding 5 μM ionoinycin plus 3 mM Ca2+ and 10 mM EGTA(pH 7X)) plus 10 mM TR1s-base (P)H 9.0), re¬spectively. Under these conditions of measurement (BT"C, cytosolic environment for the dye), the dissociation constant (Kd) for the Ca^"^-Fura-2 complex is generally assumed to be 225 nM (13), and [Ca2+] deR1ves from: [Ca2+] = Kd (R - Rmin)/(Rmax - R) x β where R is the ratio of Fura-2 fluorescence at the selected wavelengths, Rmax and Rmin rep¬resent ratios from Ca2+ saturated and Ca2+ free infracellular dye, respectively, and p is the ratio between the specific fluorescence of the Ca2+ free and Ca^"" bound forms of the dye at the longer wavelength (Sf2/Sb2). Example 4 Determination of Osteocalcin synthesis Osteoblasts grown by 48 hours in multiwells were changed to Dulbecco"s modified Eagle"s mediimi containing 1% fetal bovine serum plus the indicated concenfration of BP, 10"^ M l,25(OH)2D3 (positive confrol), or vehicle alone, and then allowed to grow for an additional peR1od of 72 hours. The medium from each well was collected and stored frozen at -80°C until use for osteocalcin measurements. Osteocalcin (OC) was quantitatively measured by radio¬immunoassay using a commercially avalaible kit (Osteocalcin DSL-6900, Diagnostic Systems LaboratoR1es Inc., Webster, Texas, USA) according to manufacturer"s instructions. Osteocal¬cin values were corrected for total cellular protein content. Example 5 Statistical Analysis Statistical significance of data was evaluated using Student"s Mest (14) and probability values below 0.05 (p Example 6 Synthesis and release of the bone matR1x protein osteocalcin in response to the action of olpa-dronate and NH2-OPD. We first examined the action of both olpadronate (OPD) and NH2-OPD (see Figure 1 for chemical structures) on the synthesis and release of the bone matR1x protein osteocalcin (OC), ■ and compared it with that of the bisphosphonates pamidronate (APD) and etidronate (EHDP), both being shown in figure 1. In osteoblasts incubated duR1ng 72 hours in the presence of 10"^ M l,25(OH)2D3, release of OC into the culture medium was increased 100% respect to con¬ trol, untreated cells (Figure 2). Under the same incubating conditions OPD and NH2-OPD markedly stimulated OC synthesis in a dose-dependent fashion, as EHDP and APD did. Both APD and OPD were more effective than the steroid hormone whereas the action of NH2-OPD was lower than for the secosteroid, being comparable to EHDP. At 10"^ M, the order of po¬ tency relative to l,25(OH)2D3 was: OPD (1.9) > APD (1.1)> l,25(OH)2D3 (1.0) > EHDP = NH2-OPD (0.85). At 10"" M the induction of OC synthesis was significantly reduced, but the order of potency among the bisphosphonates remained the same. No significant differences with respect to basal OC synthesis was detected when cells were exposed to concentrations of BPs below 10" M. In order to detemune the possibihty that, as for other anaboUc parameters, short term alterations of intracellular Ca2+ regulation could be related to the action of these bisphosphonates on OC induction, we used fluoR1metry to monitor changes in osteoblast Ca2+i levels. The secosteroid l,25(OH)2D3 acts as a Ca2+ mobilizing hormone in rat osteoblasts, its response being well documented and characteR1zed in Farach-Carson et al., 1998, Am.J.Kidney Dis. 31, 729-742. Thus, the steroid also represents a good positive reference for evaluation of BP effects on Ca i in these cells. In Fura-2 loaded cells, similarly to l,25(OH)2D3, EHDP, APD and OPD induced a rapid (30-60 sec) and sustained (>5 min) in¬ crease, in [Ca^]i with a biphasic time-course profile, suggesting contR1bution by both release of Ca^t-fiom endogenous stores and cation influx fi-om the outside (Figure 3). NH2-OPD, generated a rapid (60 sec) but transient Ca2+ R1se, with a marked down-turn phase after peak returning to near basal levels within 1-2 min (Figure 3B). For OPD and its aminodeR1vative, increments in [Ca^"]i become detectable fi-om 10-10 M (1.1-1.2 fold over basal levels, p : but maximal difference with respect to basal were reached at 10 M (1.4-1.6 fold stimula- - ;- tibn,p hormone (80 vs. 90 seconds, for either APD and NH2-OPD vs. l,25(OH)2D3, respectively) whereas EHDP exhibited a highly delayed kinetics (time-to-peak = 120 seconds). The kinetic analysis of the initial ^hase of Ca2+ R1se induced by these compounds revealed that, as noted above, EHDP exhibited the slowest rate of Ca2+ elevation (Figure 5B). In ROS 17/2.8 cells the rapid, non-genomic Ca2+, response to l,25(OH)2D3 is composed of an • • • • 9-1- imtial fast sterol-mduced Ca release from endogenous thapsigargin-sensitive stores which is followed by cation influx from the outside (see Khoury et al., 1995, J.Nufr. 125, 1699S-1703S). This cation entry pathway accounts for the sustained Ca^"^j phase, which has been shown to be contR1buted by L-type voltage-dependent (VDCC) Ca^* channels. As NH2-OPD has been shown to be absolutely devoid of antiresorptive properties (Van Beek E. et al., 1996, J. Bone Miner. Res. 11, 1492-1497), particularly at doses at which, according to the present invention, some cellular effects on both osteocytes and osteoblasts are preserved it is here proposed to be a selective modulator of the osteoblast. Thus, our attention was focused on OPD and its amino-substituted analog and expeR1ments were performed to determine if simi¬lar Ca2+ routes were involved in the effect of these compounds on [Ca2+]i reported here. As for the steroid, pretreating the cells with the VDCC blockers nifedipine (2 ^M) or verapamil (5 μM) only partially (70%) reduced the [Ca2+]i increase induced by OPD, but almost com¬pletely abolished the action of NH2-OPD (see Example 7). hi the case of OPD, the effect of VDCC blockade was particularly evident at the influx phase of the response, while the early Ca i transient remained imaltered (not shown), hi order to evaluate if the fast, early phase of Ca^* increase by BPs involves activation of the phosphoinositide-specific phosphoUpase C (PLC) pathway, the action of two structurally and mechanistically unrelated enzyme inhibi¬tors on BP-induced early Ca2+ response was assayed. The rapid (1 min) OPD- and NH2-OPD-dependent increase* in cytosoUc Ca^* was totally blocked by pretreatment with the PLC in¬hibitors U73122 (2 \iM) or neomycin (0.5 mM) (see Example 8) but not by 1173343 (not shown), an analogue of U73122 devoid of effect on PLC(Vazquez G.et al, 1998,J Biol Chem. 273, 33954-33960). When intracellular muscle Ca2+ stores were phaimacologically depleted by inhibition of the sarcoplasmic reticulum Ca2+-ATPase with 1 μiM thapsigargin, the response to either OPD or NH2-OPD, was completely blocked (Figure 6). Example 7 Effects of nifedipine and verapamil on the Ca2+ response of osteoblasts to OPD and NH2-OPD Fura-2 loaded osteoblasts were treated with vehicle (buffered saline solution, Basal), OPD (10"* M) or NH2-OPD (10"* M) and intracellular Ca2+ concentration ([Ca2+];) was measured as descR1bed. Given are [Ca2+]i values corresponding to the plateau phase (5 min after BP expo¬sure; see Figure 3) of the BP-induced Ca^"^ response. When used, both nifedipine (2 \iM) and verapamil (5 \|xM) were added 3 min before stimulation. Data are the average of 5 independ¬ent [Ca^"Ji recordings ± sd. p values (p Example 8 Effects of phospholipase C inhibition on OPD and NH2-OPD induced Ca2+i responses in osteoblastic cells. Fura-2 loaded cells were treated with vehicle (buffered saline solution, Basal), OPD (10-8 M) or NH2-OPD (10-8 M) and intracellular Ca2+ concentration ([Ca2+];) was measured as de¬scR1bed. Unless otherwise indicated, [Ca2+]! stimiilation was evaluated at the plateau phase of the BP-induced response (see Figure 3). When used, the PLC inhibitors U73122 (2 μM) or neomycin (0.5 mM, data in parentheses) were added into the measurement cuvette 3 min be-fore stimulation. In the PLC-inhibition assay [Ca2+ ]i values measured at 1- and 5-min. after stimulation are given. Results are expressed as percent of control (100%) to allow compaR1son among different assay conditions, and are the average of 3 independent expeR1ments ± sd. *p Example 9 The present in vitro studies were conducted in order to evaluate the effects of the olpadronate and its analog NH2-OPD on intracellular Ca2+ levels in cultured osteoblasts. It is well docu¬mented that replacing the -OH group at Rl in BP with a -NH2 group induces deep changes in the biological properties of the BP, the magnitude of which depends on the natiire of the pa¬rental molecule. Indeed, replacement of the hydroxyl group of etidronate by an amino group produces no detectable effect on antiresorptive efficacy of this compound, whereas the equivalent substitution in OPD leads to complete loss of antiresorptive activity (see van Beek E. et al, 1996, J. Bone Miner. Res. 11,1492-1497). The observed profile for OPD Ca2+i response was highly similar to that of l,25(OH)2D3, in-volving an initial rapid BP-induced Ca mobilization from thapsigargin-sensitive endogenous stores, followed by cation influx from the extracellular millieu which finally accounts for the sustained Ca2+i phase. The concept that, similarly to l,25(OH)2D3, the rapid BP-induced Ca2+i transient is due to mobilization of the cation from IPs-sensitive stores, is strongly supported by the blocking effect of the PLC inhibitors U73122 and neomycin, both acting at different sites of PLC activity. In ROS17/2.8 cells, l,25(OH)2D3 induces a fast (30-60 seconds) and monophasic generation of IP3 (see Lieberherr, M., 1987, J. Biol. Chem. 262, 13168-13173; Civitelli et al., 1990, EndocR1nology 127, 2253-2262). Although the present data suggest that a similar mechanism of endogenous Ca2+ mobilization-might operate for these two related BPs, the effects of both OPD and NH2-OPD on phospholipid metabolism in these cells re¬mains to-be investigated. As polyphosphoinositide turnover could be directly or mdirectly involved in the control of Ca2+ influx from outside the cell (see Irvine, RF., 1992, FASEB J. 6, 3085-3091), it appears that differences in the extent of inositol phosphate liberation accoimt -for the greater stimulation of Ca2+ entry through Ca2+ channels by OPD and NH2-OPD here reported. We observed that the Ca2+ influx phase of the Ca2+ response to OPD and NH2-OPD was, al¬though at different extent, abolished by VDCC blockers, suggesting that modulation of volt¬age-dependent Ca2+ channels is involved in the non-genomic action of both BPs in osteoblas¬tic cells. The events by which BPs exert genomic (e.g., induction of OC synthesis) and non-genomic (e.g., rapid activation "of the calcium message) actions are certainly both temporally and, probably, spatially separated duR1ng physiological bone remodeling. Many studies, particu¬larly those relating membrane-initiated and nuclear receptor-mediated pathways in l,25(OH)2D3 actions on bone, have clearly established that the induction of the Ca2+ signal is not always necessary for activation of the nuclear processes in osteoblasts (see Khoury et al., 1994, EndocR1nology 135, 2446-2453). However, Ca2+ signals have systematically been asso¬ciated with changes in the expression level of osteocalcin and osteopontin (see Farach-Carson et al., Am. J. Kidney Dis. 31, 729-742, and those references therein). We observed here that NH2-OPD exhibits a diminished efficacy relative to that of the parental molecule OPD to in¬duce OC synthesis, whereas the magnitude of changes in cytosolic Ca2+ are also significantly lower in response to NH2-OPD than for OPD, with highly different profiles. Thus, it suggests the existence of a direct correlation between BP potency to induce OC synthesis and their ability to generate rapid changes in cytosolic Ca2+. Tnis observation is valid for all of the other BPs assayed in the present studies. On this basis, it seems as for the secosteroid hor¬mone l,25(OH)2D3, the rapid, non-genomic actions of BPs on the osteoblast Ca2+ signalling system could tR1gger the BP genomic effect, thus affecting osteogenesis. Although the struc¬tural change introduced in OPD could be alteR1ng its interaction with its cellular target/s, the observed differences between OPD and NH2-OPD action on Ca2+ regulation could be related to differential signalling cascades and/or mechanisms mediating the action of each of these BPs. At the doses used in the present study NH2-OPD has been shown to act as a selective modulator of the osteoblast. The possibility then R1ses that the differential effects of OPD and NH2-OPD on the Ca2+ homeostasis of the osteoblast could explain their differences in antire-sorptive potency. These results show that NH2-OPD is a selective modulator of the osteoblast, particularly in the above-mentioned novel uses. The"mventive features disclosed in the prweding descR1ption as well as in the claims, tables and figures can be essential to the realization of the invention in its vaR1ous embodiments, either singly or in the form of random combinations. WE CLAIM: 1. A method for screening for Ca2+-channel blockers invitro comprising the steps: treating of cells having Ca2+-channels with a putative Ca2+-channel blocker; contacting the cells with l-amino-3-(N,N-dimethylamino)-propylidene- 1,1-bisphosphonic acid, any of its soluble salts or any of its hydrates; measuring a response as a result of the contacting step as herein described. 2. A method for screening for functional analogues of l-amino-3-(N,N- dimethylamino)-propylidene-l,l-bisphosphonic acid, any of its soluble salts or any of its hydrates, invitro, comprising the steps: treating of cells having Ca2+-channels with Ca2+-channel blockers; contacting the cells with the putative functional analogue which, in the absence of any Ca2+-channel blockers, is known to cause a Ca2+-ion influx into the cells; measuring a response as a result of the contacting step as herein described.

Full Text

The present invention relates to a method for screening for Ca2+-channel blockers invitro condition.
The compound l-amino-3-(N,N-dimthylamino)-propylidene-l,l-bisphosphonic acid, or any of its soluble salts or any of its hydrates, belongs to the group of bisphosphonates which are compounds containing two phosphonate groups bound to a carbon and two additional groups R1and R2, respectively, which bind strongly to calcium crystals, inhibit their growth, suppress bone resorption and are being used in the treatment of a vaR1ety of disorders of bone metabo¬lism.
l-amino-3-(N,N-dimethylamino)-propylidene-l,l-bisphosphonic acid is the amino-substituted form of olpadronate (OPD) and is hereafter sometimes referred to as NH2-OPD.
Bisphosphonates are recognized potent inhibitors of bone resorption and are successfully used in the treatment of common bone disorders such as osteoporosis and tumoral bone diseases, amongst others. Structurally, bisphosphonates are deR1vatives of methylene bisphosphonate in which the two hydrogens on the geminal carbon atom are replaced by two groups, namely R1 and R2, which usually are not identical In many cases the R1 moiety is a hydroxyl group, be¬cause of its ability to form a tR1dentate structure together with the two phosphonate groups to bind bone mateR1al.
Extensive studies have been carR1ed out on the effects of bisphosphonates on bone cells, in particular those related to their anti-resorptiye potency. The molecular mechanisms involved in such actions, however, are not clear. The anti-resbrptive activity and its utility in the treat¬ment of osteoporoses and related bone diseases have, for example, been reported and de¬scR1bed m the following patents: WO 94/00129 to Frances M. et al., WO 93/11786 to Gueddes A. et al., WO 92/14474 to McOskar et al., US Patent No. 4,942,157 to Gall R. et al., and in US Patent No. 3,962,432 to Schmidt-Dunker M.

Although it appears that the nature of the R2 moiety of bisphosphonates determines their anti-resorptive activity, replacement of the hydroxyl group in R1 by an NH2 group in some bisphosphonates (e.g. olpadronate and pamidronate) markedly-reduces their anti-resorptive activity. This decrease in anti-resorptive efficacy of the amino deR1vatives is thought to be due to substitution-dependent changes in the cellular effects of the compounds, since their affinity for bone mineral is not significantly affected. This lack of anti-resorptive activity is, for ex¬ample, disclosed in vanBeek et al., 1996, Journal of Bone and Mineral Research, vol. 11, no. 10, pp. 1492-1497, and international patent application PCT/EP96/02981, the latter disclosing the use of l-amino-3-(N,N-dimethyIamino)-propyhdene-l,l-bisphosphonic acid for the treatment of osteoporoses, arthR1tis and peR1odontal diseases.
The structure and stability of skeletal bones is - to a high degree - influenced and determined by an equilibR1um between the activities of o.geo.blMts. and_osteoclaste,J.^ the cells responsi¬ble for mineraUzatioR1 and resorption of bone structure, respectively. Therefore, bones are subject to a continuous restructuR1ng process involving anabolic and catabolic reactions. In human beings, as of the age of 30, the catabolic processes prevail such that there will be a net loss in total bone substance naturally. Consequently, the rate at which the renewal of the bone tissue takes place will be lowered with increasing age.
Object of the present invention is to provide means for prevention of the onset of clinically pathological bone-conditions.
Another object of the present invention is to avoid a predisposition to bone disfunctions or bone diseases.
Yet a further object of the present invention is to optimize the physiological bone functions in patients.
The present invention in its vaR1ous aspects and embodiments disclosed hereafter solves all these objectives and provides further advantages.

Summary of the invention
The present invention is related to" novel uses of l-amino-3-(N,N-dimethylamino)-propylidene-l,l-bisphosphonic acid and/or any of its soluble salts and/or any of its hydrates. For the purposes of the following specification, it is intended that the term l-amino-3-(N,N-dimethylamino)-propylidene-l,l-bisphosphonic acid also encompasses any of its soluble salts and/or any of its hydrates.
BR1efly stated, the present invention provides novel uses of l-amino-3-(N,N-dimethylamino)-propylidene-l,l-bisphosphonic acid, medicaments compR1sing l-amino-3-(N,N- • dimethylamiiio)-propylidene-l,l-bisphosphonic acid and screening methods involving 1-amino-3-(N,N-dimethylamino)-propylidene-1,1 -bisphosphonic acid.
Within one aspect of the present invention, there is provided the use of l-amino-3-(N,N-
dimethylamino)-propylidene-1,1-bisphosphonic acid for the manufacture of a medicament for
selective modulation of osteoblasts. Furthermore, there is provided the use of l-amino-3-
(N,N-dimethylamino)-propylidene-1,1-bisphosphonic acid for the manufacture of a medica-
ment for the maintenance of a healthy bone structure.
There is also provided the use of l-amino-3-(N,N-dimethylamino)-propylidene-l,l-bisphosphonic acid for the manufacture of a medicament for the prevention of osteopathies in healthy patients.
The invention also provides for the use of l-amino-3-(N,N-dimethylamio)-propylidene-l,l-
bisphosphonic acid for the manufacture of a medicament for the treatment of patients who
, have recently undergone treatment with corticosteroids.
Additionally there is provided the use of l-amino-3-(N,N-dimethylamino)-propylidene-l,l-bisphosphonic acid for the manufacture of a medicament for post-treatment of osteopathies wherein an anti-resorptive activity is not desired.

The invention also provides for the use of l-amino-3-(N,N-dimethylamino)-propyIidene-l,l-bisphosphonic acid for the manufacture of a medicament for the treatment of children having an osteopathy..
In another aspect of the invention, there is provided the use of l-amino-3-(N,N-dimethylamino)-propylidene-l,l-bisphosphonic acid for the manufacture of a medicament for the stimulation of both signalling cascades and reaction mechanisms mediating the action of l-amino-3-(N,N-dimethylamino)-propylidene-l,l-bisphosphonic acid which can be blocked by Ca2+ -channel blockers.
In yet another aspect of the present invention, there is provided the use of l-ainino-3- (N,N-dimethylamino)-propylidene-l,l-bisphosphonic acid for the manufacture of a medicament for the mobilization of Ca2+-ions from IP3 sensitive stores.
In yet another aspect of the invention, there is provided a method for screening for Ca2+- charmel blockers compR1sing the steps:
treating of cells having Ca2+-channels with a putative Ca2+-channel blocker;
contacting the cells with l-amino-3-(N,N-dimethylamino)-propylidene-l,l-
bisphosphonic acid, any of its soluble salts or any of its hydrates;
measuR1ng a response as a result of the contacting step.
It is envisioned that the treatment-step and the contacting-step occur sequentially with the treatment-step preceding the contacting-step or vice versa, or that they occur simultaneously.
In another aspect, the invention also provides a method for screening for functional analogues of l-amino-3-(N,N-dimethylamino)-propylidene-l,l-bisphosphonic acid, any of its soluble salts or any of its hydrates compR1sing the steps;
2+ 2+t-
treating of cells having Ca -channels with Ca -channel blockers;

contacting the cells with the putative functional analogue which, in the absence of any Ca2+-channel blockers, is known to cause a Ca2+-ion influx into the cells;
measuR1ng a response as a result of the contacting step.
It is envisioned that the treatment-step and the contacting-step occur sequentially with the treatment-step preceding the contacting-step or vice versa, or that they occur simultaneously.
In the present invention there is also disclosed a medicament compR1sing l-amino-3-(N,N-dimethylamino)-propylidene-l,l-bisphosphonic acid and at least one physiologically accept¬able carR1er, for the selective modulation of the osteoblasts, for the maintenance of a healthy bone structure, for the prevention of osteopathies or for the post-treatment of osteopathies where an anti-resorptive activity is not desired.
In one aspect of the present invention it provides a medicament compR1sing l-amino-3-(N,N-dimethylamino)-propylidene-l,l-bisphosphonic acid, which is to be applied in healthy pa¬tients or in patients at or above 40 years of age.
The present invention also-discloses a method for the selective modulation of osteoblasts and/or for the maintenance of a healthy bone structure and/or for the prevention of osteopa¬thies in healthy patients and/or for the treatment of patients who have recently imdergone treatment with corticosteroids, and/or for post-treatment of osteopathies where an anti-resorptive activity is not desired, and/or for the treatment of children having an osteopathy and/or for the stimulation of those signaling cascades and reaction mechanisms mediating the action of l-amino-3-(N,N-dimethylamino)-propylidene-l,l-bisphosphonic acid or any of its soluble salts or any of its hydrates, which can be blockedby Ca^t-channel blockers,and/or for the mobilization of C+2ions fromIP3-sensitive stores, comprising
administeR1ng l-amino-3-(N,N-dimethylamino)-propylidene-l,l-bisphosphonic acid or any of its soluble salts or any of its hydrates alone or in combination with a pharmaceutical carR1er to a patient, the l-amino-3-{N,N-dimethylamino)-propylidene-l,l-bisphosphonic acid or any of |

its soluble salts or any of its hydrates being administered in doses of O.I to 1000 mg/oral ap¬plication or 0.02 to 200 mg/parenteral application.
It is preferred that the term "selective modulation of osteoblasts" compR1ses a stimulation of the cellular activities of the osteoblasts, more preferably an influence on the Ca2+-homeostasis of the osteoblasts. A preferred embodiment of "selective modulation" compR1ses a transient increase of the Ca2+"-levels in the osteoblasts. A selective modulation of osteoblasts may also encompass and manifest itself in the synthesis of osteocalcine and/or osteopontin, osteonectin, calprotectin, fibronectin, matR1x Gla protein and/or bone sialoprotein.
It is intended in the present invention that the term "maintenance of a healthy bone structure" also encompass a prevention of clinically pathological conditions or diseases.
Within one aspect of the present invention it is preferred that where l-amino-3-(N,N-dimethylamino)-propyHdene-l,l-bisphosphonic acid is used for the manufacture of a me¬dicament for the maintenance of healthy bone structure, that the medicament is applied in healthy patients, more preferably patients without osteopathies. The term "healthy bone structure" is meant to include any bone structure characteR1zed by a balanced equilibR1um between catabolic and anabolic processes resulting in a structure capable of withstanding me¬ chanical stress that occurs as a result of the organism"s activities, as well as a structure that is enable of remodelling itself by aforementioned equilibR1um and repaiR1ng itself by the re¬moval of weakened or worn-out parts and the build-up of strong parts, thereby avoiding mi-crocracks and parts prone to fracture.
It has been foimd that, especially with older people, the bones have a less strong structure because of their reduced rate of renewal. Furthermore, older people may get weaker, more fragile bones due to sedentary lifestyle, muscular weakness and insufficient nutR1tion. As a result the affected bones, im fact, comprise ""unfit" mineralized structures and therefore be¬come bR1ttle and unstable which very often is accompanied by a tendency for fractures. These "unfit" structures have not manifested themselves yet as clinically pathological conditions but are less than ideal. They can also be found- apart from in people at or above the age of 40 years - in people recently treated with corticosteroids or in people recently treated with anti-

osteoporotic agents such as fluoR1ne and common bisphosphonates such as etidronate and chlodronate.
The term " patient" is meant to encompass human beings as well as any vertebrate animal, for example cats, dogs, cows, horses and other domestic animals.
The term "osteopathy" designates any pathological condition of the bone resulting in a weak¬ened or irregular or abnormal bone structure. Within the present invention it is used synony¬mously with "bone disease". Preferably the term "osteopathy" refers to a cUnically pathologi¬cal condition selected from the group compR1sing osteoporosis, Paget"s disease, arthR1tis, peR1odontal osteopenia, adolescent scoliosis, fracture, disuse osteopenia, post-transplant os¬teopenia, hyper-parathyroidism-associated osteopenia, drug-induced osteopenia, nutR1tional osteopenia, metabolic bone disease, osteopenia of prematuR1ty and ossification disorder.
Where the term "patient" is referR1ng to a human being, in one embodiment it is preferred that the medicament is applied in human beings at or above the age of 40 years, in another em¬bodiment it is preferred that the medicament is applied in children. Preferably, the term "chil¬dren" is meant to encompass individuals who are from 0 to 16 years old.
Where l-amino-3-(N,N-dimethylamino)-propylidene-l,l-bisphosphonic acid is used for the manufacture of a medicament for the prevention of osteopathies in healthy patients or for the manufacture of a medicament for the treatment of children having an osteopathy it is pre¬ferred that the osteopathy is selected from the group compR1sing osteoporosis, Paget"s disease, arthR1tis, peR1odontal osteopenia, adolescent scoliosis, fracture, disuse osteopenia, post-transplant osteopenia, hyper-parathyroidism-associated osteopenia, drug-induced osteopenia, nutR1tional osteopenia, metabolic bone disease, osteopenia of prematuR1ty and ossification disorder.
In one embodiment it is preferred that l-amino-3-(N,N-dimethylamino)-propylidene-l,l-bisphosphonic acid - after the application of the medicament in a patient - will be present at extracellular concentrations in a range between 10-8 and 10-1010M, more preferably lO-7M and 10""M, most preferably at an extracellular concentration of about lO-7M.

In the embodiment where l-amino-3-(N,N-dimethylamino)-propylidene-l,l-bisphosphonic acid is used for the manufacture of a medicament for the stimulation of those signaling cas¬cades and reaction mechanisms mediating the action of l-amino-3-(N,N-dimethylamino)-propylidene-1,1 -bisphosphonic acid, which can be blocked by Ca2+-channel blockers it is pre¬ferred that the Ca 2+-channel blockers are selected from the group compR1sing nifedipine and verapamil.
In one embodiment it is preferred that l-amino-3-(N,N-dimethylamino)-propylidene-l,l-bisphosphonic acid be used in doses of 0.01 to 1000 mg/oral application, more preferably 12.5 to 75 mg/oral application. The term "oral application" is meant to include solid or solu¬ble liquid pharmaceutical formulations, gels, soft capsules, tablets, capsules containing solid preparations, soluble liquid forms or suspensions, and pills. Within one aspect of the present invention it is envisioned that l-amino-3-(N,N-dimethylamino)-propylidene-l,l-bisphosphonic acid be used also for topical administration to be applied on skin and/or muco¬sae. Forms of application useful for this purpose compR1se ointments, creams, sprays, sup¬positoR1es and gels.
In another embodiment of the present invention it is preferred that l-amino-3-(N,N-dimethylamino)-propylidene-1,1-bisphosphonic acid be used in doses of 0.02 to 200 mg/parenteral application, more preferably 2.5 to 15 mg/patenteral application. The tenn| "parenteral application" is meant to include any application that avoids the gastro-intestinal tract, for-€xample by sub-cutaneous, intra-muscular or intra-venous injection or infusion.
The term "IPs-sensitive stores" refers to those intracellular"Stores of Ca2+-ions which, upon the presence of IP3 (= inositol tR1phosphate) rapidly release Ca2+ions. Such IPs-sensitive stores oicompass for example the endoplasmic reticulum and, in smooth muscle cells, the sarcoplasmic reticulum.
It is to be noted that according to the present invention the influence exerted by l-amino-3-(N,N-dimethylamino)-propylidene-l,l-bisphosphomc acid on the Ca2+ homeostasis of the osteoblasts, in particular an increase of the Ca2+Mevels in the osteoblasts, can occur in at least

two different ways: Ca2+"-ions can be mobilized from endogenous stores, such as those which are IPa-sensitive or thapsigargin-senstitive.
Secondly, there can also occur a Ca2+-ioh-mflux from extracellular stores. This may for ex¬ample occur through voltage-dependent Ca2+-channels (VDCC), but other forms of Ca2+-channels, e.g. ligand-gated channels, can be envisioned, too.
It is within the scope of the present invention that l-amino-3-(N,N-dimethylamino)-propylidene-l,l-bisphosphonic acid also be used for screening for Ca^"^-channel blockers. The term "putative Ca -channel blocker" is meant to designate a compound to be tested for its Ca -channel-blocking capabilities.
The term "putative fimctional analogue" of l-amino-3-(N,N-dimethylamino)-propylidene-l,l-bisphosphonic acid designates any compound that is known to cause a Ca2+-ion influx into the cells in the absence of any Ca2+-channel blockers and is therefore mimicking l-amino-3-(N,N-dimethylamino)-propyUdene-l,l-bisphosphonic acid in its influence on Ca^*-homeostasis of the cells. The term "measuR1ng a response" is also meant to include the meas¬urement or detection of the absence of a response. Within the present invention, the term "re-sponse" is preferably meant to designate a change in cytosolic Ca -levels.
It is also within the scope of the present invention to combine l-amino-3-(N,N-dimethylamino)-propylidene-l,l-bisphosphomc acid in its novel uses together with at least one other compound/substance, in synergistic combinations. The term "combination" is meant to include the incorporation of l-amino-3-(N,N-dimethylamino)-propylidene-l,l-bisphosphonic acid together with another (other) substance(s) into the same medicament, as well as the administeR1ng of a medicament compR1sing l-amino-3-(N,N-dimethylamino)-propylidene-l.1-bisphosphonic acid to a patient withtaother: (other) substance
-
isteiing this (these) other substance(s) in a physically separated, i.e. spatially or temporalIy separated manner from the medicament. In the case where the substance(s) is (are) adminis¬tered in a separated manner from the medicament compR1sing l-amino-3-(N,N-dimethylamino)-propylidene-l,l-bisphosphomc acid, it is preferred that the l-amino-3-(N,N-dimethylamino)-propylidene-l,l-bisphosphonic acid-compR1sing medicament is administered

with the other substance(s) in a single-step (concerted intake, infusions, injections), sequen¬tial-step (one after the other) or cyclic-step fashion. In one embodiment it is preferred that the medicament compR1sing 1 -amino-3-(N,N-dimethylamino)-propylidene-1,1 -bisphosphonic acid is applied before, duR1ng or after treatment with other amino-substituted bisphosphonates. For example, a sequential administeR1ng of the l-amino-3-(N,N-dimethylamino)-propylidene-1,1-bisphosphonic acid-compR1sing medicament with other amino-substituted bisphospho¬nates may be indicated, since other amino-substituted bisphosphonates exert a partial anti-resorptive effect. So a patient may need a l-amino-3-(N,N-dimethyIammo)-propylidene-l,l-bisphosphonic acid-compR1smg medicament for bone formation due to the selective modulat¬ing effect of l-amino-3-(N,N-dimethylamino)-propyndene-1,1-bisphosphonic acid on the osteoblasts, and at the same time another amino-substituted bisphosphonate for a "tunable" degree of mhibition of bone resorption, depending on his/her individual requirement.
As another example a patient might first require a high degree of inhibition of bone resorp¬tion, continue then with low or none and then change again afterwards, according to mdivid-ual requirements which can be determined by biochemical, densitometR1cal, X-ray or other measurements performed on the bones of the individual.
Thus l-amino-3-(N,N-dknethylamino)-propylidene-l,l-bisphosphonic acid is an ideal candi¬date forming the basis for an individually tunable therapy of conditions where bones are weak and/or fragile.
This substance/these substances/compounds of the aforementioned synergistic combinations is/are selected from the group compR1sing calcium salts, calcium citrate and calcium carbon¬ate, other amino-substituted bisphosphonates, pharmaceutically active fluoR1ne-containing salts, vitamins of the D-group and their metabolites, cholecalciferol, calcifediol, calcitR1ol, ergocaldferol, PTH, anabolic hoimones, such as estrogens, substances with estogenic activ; ity on the bone, progestogens, androgens, growth hormones, peptides with growth hormone activity, selective modulators of the estrogenic receptor, raloxifene and others. The term "amino-substituted bisphosphonate" preferably designates a bisphosphonate compound that has an amino group at the 1-position. Other substitution positions, however, can be envi¬sioned, too. Where the "aminodeR1vative" of a 1-hydroxy bisphosphonate is mentioned here,

this is meant to encompass molecules having a structure in which the hydroxy-group is re¬placed by an amino group.
It is one of the surpR1sing advantages" of the present invention that l-amino-3-{N,N-dimethylamino)-propylidene-l,l-bisphosphonic acid acts to avoid the formation of the afore¬mentioned "unfit" bone structures and aids in removing them once they have been formed due to its selective modulating capabilities with respect to the osteoblasts.
Therefore, unfit mineralized structures in the bone are prevented and removed respectively. For example, these structures can aR1se in older people, but have not manifested themselves in •clinically pathological conditions yet. Another important example where these unfit structures may occur is the growing skeleton, i.e. children. These may be healthy children or children having an osteopathy. The present invention provides means for removing these unfit struc¬tures or preventing them in the first place, thus contR1buting to a healthy bone structure which is capable of withstanding the mechanical stress exerted upon the bone through daily usage.
These and other aspects of the present invention will become evident upon reference to the following examples and attached fi.gures. In addition, vaR1ous references are set forth below which descR1be in more detail certain procedures or expeR1mental details and are therefore incorporated by reference in their entirety.
De-scR1ption of the figures
Figure 1 shows the chemical structures of the Rl and R2 chains of the bisphosphonates used
in the present studies, i.e. etidronate (EHDP), paihidronate (APD), olpadronate (OPD) and
NHa-oIpadronate (NH2-OPD) (=l-amino-3-(N,N-dimethylamino)-propylidene-l,l-
bisphosphoilic^d).; ,-. ..
Figure 2 shows the induction of osteocalcin (OC) synthesis by bisphosphonates in cultured osteoblasts:

Cells were grown by 72 hours in the absence (control) or presence of the indicated concentra¬tions of la, 25-dihydroxy-vitamin D3 [l,25(OH)2D3], EHDP, APD, OPD or NH2-OPD. Then osteocalcin (OC) released into the medium was quantitated as descR1bed in Example 4. Data are the average ± SD of 3 independent expeR1ments performed by tR1plicate.
Figure 3 shows the rapid actions of bisphosphonates on cytosolic Ca2+ in osteoblasts:
Fura-2 loaded cells were exposed to the indicated concentrations of la, 25-dihydroxy-vitamin D3 [l,25(OH)2D3], EHDP or APD (Panel A) and OPD or NH2-OPD (Panel B), and cytosoUc calcium levels were then monitored as descR1bed under Example 3. Shown are time-traces representative from at least 4 independent recordings.
Figure 4 shows the dose dependency of the rapid actions of OPD and NH2-OPD on cytosolic Ca2+ in osteoblasts:
Fura-2 loaded cells were treated with vehicle (buffered saline solution. Basal), or the indicated concentrations of OPD or NH2-OPD, and intracellular Ca^" concentration ([Ca2+]i) was meas¬ured as descR1bed. [Ca2+]! values were collected at the peak of the BP-induced Ca2+ response. Data are the average of 4 independent [Ca2+li recordings ± sd. p values (*p Figure 5 shows the kinetics of the bisphosphonate-dependent cytosolic Ca2+ change relative to 1,25(0H)2D3 effect:
Fura-2 loaded cells were exposed to l,25(OH)2D3 (lOnM), or 10 nM of either"EHDP, APD, OPD or NH2-OPD, and cytosolic calcium was momtored,as descR1bed. A) Both the magni-vtude of change at maximum jpeak reached, as well as the corresponding time-to-peak were-registered and compared to those for the steroid. Shown are results repiesentatiye fiom 4 in¬dependent expeR1ments, B) The early phase (0-2 min) of Ca2+ increment in response to 10 nM BP or l,25(OH)2D3 was monitored and plotted as change in Ca2+i vs. Minutes of exposure.

Figure 6 shows cytosolic calcium-levels in Fura-2 loaded cells that had been treated with (Thp-treated) or without (Control) 1 (μiM thapsigargin and then exposed to OPD (10 nM) or NH2-OPD (10 nM). Cytosolic calcium levels were monitored as descR1bed under Example 3. Shown are time-traces representative froni at least 3 independent recordings.
Example 1
Bisphosphonates (BP) have the following general structure:

wherein R1- and R2-chains can have vaR1ous identities. The structures of the Rl and R2 chains of the bisphosphonates used in this study are shown in Figxu-e 1. All of them were from Gador S.A. (Buenos Aires, Argentina). Fura-2/pentaacetoxymethyl ester (Fura-2/AM), pluronic F-127, nifedipine, verapamil, neomycin, Dulbecco"s modified Eagle"s mediimi and fetal bovine serum were from Sigma Chemical Co. (St.Louis, MO, USA): U73122 (l-(6-((17p-3-methoxyesfra-l,3,5(10)-tR1en-17-yl)amino)hexyl)-lH-pyrrole-2,5-dione) and U73343 (l-(6-((17P-3-methoxyestra-l,3,5(10)-tR1en-17-yl)amino)hexyl)-2,5-pyrrolidine) were from Biomol Research LaboratoR1es Inc. (Plymouth Meeting, PA, USA). All other reagents used were of analytical grade.
•
Example 2
Cell culture
Osteoblast-like rat osteosarcoma cells (ROS17/2.8) were cultured as monolayers at 37*"C in Dulbecco"s modified Eagle"s medium (DME) containing 10% fetal bovine serum imder hu¬midified air (5.5% CO2). After 48 hours, medium was changed to DME containing 1% fetal

bovine serum. Unless otherwise stated, cells were allowed to grow until confluence (4-5 days after plating).
Example 3
Intracellular calcium measurements
intracellular Ca2+ changes were monitored by using the Ca^"^-sensitive fluorescent dye Fura-2 as previously descR1bed (Vasquez et al., 1998, J. Biol. Chem. 273, 33954-33960). Cell dye loading was achieved by incubating the cells in buffer A containing (in mM): 138 NaCI, 5 KCl, 1 MgCl2, 5 glucose, 10 HEPES (pH 7.4), 1.5 CaCl2, plus 0.1% bovine serumalbumin (BSA), 2 \xM of the pentaacetoxymethylester deR1vative (membrane permeable) Fura-2/AM and 0.012% pluronic F-127, in the dark duR1ng 40 min at room temperature (20-25°C) in order to minimize dye compartmentalization. Unloaded dye was washed out and cells were stored in buffer B (buffer A without BSA, Fura-2/AM and pluronic F-127) in the dark (room tem¬perature) by at least 30 min pR1or to use, to allow for complete intracellular dye deesteR1fica-tion. For fluorescence measurements the coverslips containing dye-loaded cells were moimted into quartz cuvettes and introduced into the thermostatized (37°C) sample compartment of a SLM Aminco 8100 spectrofluoR1meter. Fura-2 intracellular fluorescence intensity was moni¬tored at an emission wavelength of 510 imi (8 run bandpass) by alternating with an electroni¬cally controlled chopper (300 Hz) the excitation wavelength between 340 and 390 nm em¬ploying a dual excitation monochromator at 4 imi bandpass. Signals from short and long wavelength were ratioed (R = 340/390) thus making the measurement independent of vaR1a¬tions in cellular dye content, dye leakage or photobleaching. Calibration of Fura-2 signal to calculate [Ca^"^J; values was performed for each coverslip as follows: maximal (Rmax) and minimal (Rmin) intracellular dye fluorescence signals were-determined by adding 5 μM ionoinycin plus 3 mM Ca2+ and 10 mM EGTA(pH 7X)) plus 10 mM TR1s-base (P)H 9.0), re¬spectively. Under these conditions of measurement (BT"C, cytosolic environment for the dye), the dissociation constant (Kd) for the Ca^"^-Fura-2 complex is generally assumed to be 225 nM (13), and [Ca2+] deR1ves from: [Ca2+] = Kd (R - Rmin)/(Rmax - R) x β

where R is the ratio of Fura-2 fluorescence at the selected wavelengths, Rmax and Rmin rep¬resent ratios from Ca2+ saturated and Ca2+ free infracellular dye, respectively, and p is the ratio between the specific fluorescence of the Ca2+ free and Ca^"*" bound forms of the dye at the longer wavelength (Sf2/Sb2).
Example 4
Determination of Osteocalcin synthesis
Osteoblasts grown by 48 hours in multiwells were changed to Dulbecco"s modified Eagle"s mediimi containing 1% fetal bovine serum plus the indicated concenfration of BP, 10"^ M l,25(OH)2D3 (positive confrol), or vehicle alone, and then allowed to grow for an additional peR1od of 72 hours. The medium from each well was collected and stored frozen at -80°C until use for osteocalcin measurements. Osteocalcin (OC) was quantitatively measured by radio¬immunoassay using a commercially avalaible kit (Osteocalcin DSL-6900, Diagnostic Systems LaboratoR1es Inc., Webster, Texas, USA) according to manufacturer"s instructions. Osteocal¬cin values were corrected for total cellular protein content.
Example 5
Statistical Analysis
Statistical significance of data was evaluated using Student"s Mest (14) and probability values below 0.05 (p Example 6
Synthesis and release of the bone matR1x protein osteocalcin in response to the action of olpa-dronate and NH2-OPD.

We first examined the action of both olpadronate (OPD) and NH2-OPD (see Figure 1 for
chemical structures) on the synthesis and release of the bone matR1x protein osteocalcin (OC), ■
and compared it with that of the bisphosphonates pamidronate (APD) and etidronate (EHDP),
both being shown in figure 1. In osteoblasts incubated duR1ng 72 hours in the presence of 10"^
M l,25(OH)2D3, release of OC into the culture medium was increased 100% respect to con¬
trol, untreated cells (Figure 2). Under the same incubating conditions OPD and NH2-OPD
markedly stimulated OC synthesis in a dose-dependent fashion, as EHDP and APD did. Both
APD and OPD were more effective than the steroid hormone whereas the action of NH2-OPD
was lower than for the secosteroid, being comparable to EHDP. At 10"^ M, the order of po¬
tency relative to l,25(OH)2D3 was: OPD (1.9) > APD (1.1)> l,25(OH)2D3 (1.0) > EHDP =
NH2-OPD (0.85). At 10"" M the induction of OC synthesis was significantly reduced, but the
order of potency among the bisphosphonates remained the same. No significant differences
with respect to basal OC synthesis was detected when cells were exposed to concentrations of
BPs below 10" M. In order to detemune the possibihty that, as for other anaboUc parameters,
short term alterations of intracellular Ca2+ regulation could be related to the action of these
bisphosphonates on OC induction, we used fluoR1metry to monitor changes in osteoblast Ca2+i
levels. The secosteroid l,25(OH)2D3 acts as a Ca2+ mobilizing hormone in rat osteoblasts, its
response being well documented and characteR1zed in Farach-Carson et al., 1998,
Am.J.Kidney Dis. 31, 729-742. Thus, the steroid also represents a good positive reference for
evaluation of BP effects on Ca i in these cells. In Fura-2 loaded cells, similarly to
l,25(OH)2D3, EHDP, APD and OPD induced a rapid (30-60 sec) and sustained (>5 min) in¬
crease, in [Ca^*]i with a biphasic time-course profile, suggesting contR1bution by both release
of Ca^t-fiom endogenous stores and cation influx fi-om the outside (Figure 3). NH2-OPD,
generated a rapid (60 sec) but transient Ca2+ R1se, with a marked down-turn phase after peak
returning to near basal levels within 1-2 min (Figure 3B). For OPD and its aminodeR1vative,
increments in [Ca^"*]i become detectable fi-om 10-10 M (1.1-1.2 fold over basal levels, p : but maximal difference with respect to basal were reached at 10 M (1.4-1.6 fold stimula-
- ;-
tibn,p
hormone (80 vs. 90 seconds, for either APD and NH2-OPD vs. l,25(OH)2D3, respectively) whereas EHDP exhibited a highly delayed kinetics (time-to-peak = 120 seconds). The kinetic analysis of the initial ^hase of Ca2+ R1se induced by these compounds revealed that, as noted above, EHDP exhibited the slowest rate of Ca2+ elevation (Figure 5B).
In ROS 17/2.8 cells the rapid, non-genomic Ca2+, response to l,25(OH)2D3 is composed of an
• • • • 9-1-
imtial fast sterol-mduced Ca release from endogenous thapsigargin-sensitive stores which is followed by cation influx from the outside (see Khoury et al., 1995, J.Nufr. 125, 1699S-1703S). This cation entry pathway accounts for the sustained Ca^"^j phase, which has been shown to be contR1buted by L-type voltage-dependent (VDCC) Ca^* channels. As NH2-OPD has been shown to be absolutely devoid of antiresorptive properties (Van Beek E. et al., 1996, J. Bone Miner. Res. 11, 1492-1497), particularly at doses at which, according to the present invention, some cellular effects on both osteocytes and osteoblasts are preserved it is here proposed to be a selective modulator of the osteoblast. Thus, our attention was focused on OPD and its amino-substituted analog and expeR1ments were performed to determine if simi¬lar Ca2+ routes were involved in the effect of these compounds on [Ca2+]i reported here. As for the steroid, pretreating the cells with the VDCC blockers nifedipine (2 ^M) or verapamil (5 μM) only partially (70%) reduced the [Ca2+]i increase induced by OPD, but almost com¬pletely abolished the action of NH2-OPD (see Example 7). hi the case of OPD, the effect of VDCC blockade was particularly evident at the influx phase of the response, while the early Ca i transient remained imaltered (not shown), hi order to evaluate if the fast, early phase of Ca^* increase by BPs involves activation of the phosphoinositide-specific phosphoUpase C (PLC) pathway, the action of two structurally and mechanistically unrelated enzyme inhibi¬tors on BP-induced early Ca2+ response was assayed. The rapid (1 min) OPD- and NH2-OPD-dependent increase* in cytosoUc Ca^* was totally blocked by pretreatment with the PLC in¬hibitors U73122 (2 \iM) or neomycin (0.5 mM) (see Example 8) but not by 1173343 (not shown), an analogue of U73122 devoid of effect on PLC(Vazquez G.et al, 1998,J Biol Chem. 273, 33954-33960).
When intracellular muscle Ca2+ stores were phaimacologically depleted by inhibition of the sarcoplasmic reticulum Ca2+-ATPase with 1 μiM thapsigargin, the response to either OPD or NH2-OPD, was completely blocked (Figure 6).

Example 7
Effects of nifedipine and verapamil on the Ca2+ response of osteoblasts to OPD and NH2-OPD
Fura-2 loaded osteoblasts were treated with vehicle (buffered saline solution, Basal), OPD (10"* M) or NH2-OPD (10"* M) and intracellular Ca2+ concentration ([Ca2+];) was measured as descR1bed. Given are [Ca2+]i values corresponding to the plateau phase (5 min after BP expo¬sure; see Figure 3) of the BP-induced Ca^"*^ response. When used, both nifedipine (2 \iM) and verapamil (5 |xM) were added 3 min before stimulation. Data are the average of 5 independ¬ent [Ca^"Ji recordings ± sd. p values (*p

Example 8
Effects of phospholipase C inhibition on OPD and NH2-OPD induced Ca2+i responses in osteoblastic cells.
Fura-2 loaded cells were treated with vehicle (buffered saline solution, Basal), OPD (10-8 M) or NH2-OPD (10-8 M) and intracellular Ca2+ concentration ([Ca2+];) was measured as de¬scR1bed. Unless otherwise indicated, [Ca2+]! stimiilation was evaluated at the plateau phase of the BP-induced response (see Figure 3). When used, the PLC inhibitors U73122 (2 μM) or neomycin (0.5 mM, data in parentheses) were added into the measurement cuvette 3 min be-fore stimulation. In the PLC-inhibition assay [Ca2+ ]i values measured at 1- and 5-min. after stimulation are given. Results are expressed as percent of control (100%) to allow compaR1son among different assay conditions, and are the average of 3 independent expeR1ments ± sd. *p

Example 9
The present in vitro studies were conducted in order to evaluate the effects of the olpadronate and its analog NH2-OPD on intracellular Ca2+ levels in cultured osteoblasts. It is well docu¬mented that replacing the -OH group at Rl in BP with a -NH2 group induces deep changes in the biological properties of the BP, the magnitude of which depends on the natiire of the pa¬rental molecule. Indeed, replacement of the hydroxyl group of etidronate by an amino group produces no detectable effect on antiresorptive efficacy of this compound, whereas the equivalent substitution in OPD leads to complete loss of antiresorptive activity (see van Beek E. et al, 1996, J. Bone Miner. Res. 11,1492-1497).
The observed profile for OPD Ca2+i response was highly similar to that of l,25(OH)2D3, in-volving an initial rapid BP-induced Ca mobilization from thapsigargin-sensitive endogenous stores, followed by cation influx from the extracellular millieu which finally accounts for the sustained Ca2+i phase. The concept that, similarly to l,25(OH)2D3, the rapid BP-induced Ca2+i transient is due to mobilization of the cation from IPs-sensitive stores, is strongly supported by the blocking effect of the PLC inhibitors U73122 and neomycin, both acting at different sites of PLC activity. In ROS17/2.8 cells, l,25(OH)2D3 induces a fast (30-60 seconds) and monophasic generation of IP3 (see Lieberherr, M., 1987, J. Biol. Chem. 262, 13168-13173; Civitelli et al., 1990, EndocR1nology 127, 2253-2262). Although the present data suggest that a similar mechanism of endogenous Ca2+ mobilization-might operate for these two related BPs, the effects of both OPD and NH2-OPD on phospholipid metabolism in these cells re¬mains to-be investigated. As polyphosphoinositide turnover could be directly or mdirectly involved in the control of Ca2+ influx from outside the cell (see Irvine, RF., 1992, FASEB J. 6, 3085-3091), it appears that differences in the extent of inositol phosphate liberation accoimt -for the greater stimulation of Ca2+ entry through Ca2+ channels by OPD and NH2-OPD here reported.
We observed that the Ca2+ influx phase of the Ca2+ response to OPD and NH2-OPD was, al¬though at different extent, abolished by VDCC blockers, suggesting that modulation of volt¬age-dependent Ca2+ channels is involved in the non-genomic action of both BPs in osteoblas¬tic cells.

The events by which BPs exert genomic (e.g., induction of OC synthesis) and non-genomic (e.g., rapid activation "of the calcium message) actions are certainly both temporally and, probably, spatially separated duR1ng physiological bone remodeling. Many studies, particu¬larly those relating membrane-initiated and nuclear receptor-mediated pathways in l,25(OH)2D3 actions on bone, have clearly established that the induction of the Ca2+ signal is not always necessary for activation of the nuclear processes in osteoblasts (see Khoury et al., 1994, EndocR1nology 135, 2446-2453). However, Ca2+ signals have systematically been asso¬ciated with changes in the expression level of osteocalcin and osteopontin (see Farach-Carson et al., Am. J. Kidney Dis. 31, 729-742, and those references therein). We observed here that NH2-OPD exhibits a diminished efficacy relative to that of the parental molecule OPD to in¬duce OC synthesis, whereas the magnitude of changes in cytosolic Ca2+ are also significantly lower in response to NH2-OPD than for OPD, with highly different profiles. Thus, it suggests the existence of a direct correlation between BP potency to induce OC synthesis and their ability to generate rapid changes in cytosolic Ca2+. Tnis observation is valid for all of the other BPs assayed in the present studies. On this basis, it seems as for the secosteroid hor¬mone l,25(OH)2D3, the rapid, non-genomic actions of BPs on the osteoblast Ca2+ signalling system could tR1gger the BP genomic effect, thus affecting osteogenesis. Although the struc¬tural change introduced in OPD could be alteR1ng its interaction with its cellular target/s, the observed differences between OPD and NH2-OPD action on Ca2+ regulation could be related to differential signalling cascades and/or mechanisms mediating the action of each of these BPs. At the doses used in the present study NH2-OPD has been shown to act as a selective modulator of the osteoblast. The possibility then R1ses that the differential effects of OPD and NH2-OPD on the Ca2+ homeostasis of the osteoblast could explain their differences in antire-sorptive potency. These results show that NH2-OPD is a selective modulator of the osteoblast, particularly in the above-mentioned novel uses.
The"mventive features disclosed in the prweding descR1ption as well as in the claims, tables and figures can be essential to the realization of the invention in its vaR1ous embodiments, either singly or in the form of random combinations.

WE CLAIM:
1. A method for screening for Ca2+-channel blockers invitro comprising the steps:
treating of cells having Ca2+-channels with a putative Ca2+-channel
blocker;
contacting the cells with l-amino-3-(N,N-dimethylamino)-propylidene-
1,1-bisphosphonic acid, any of its soluble salts or any of its hydrates;
measuring a response as a result of the contacting step as herein
described.
2. A method for screening for functional analogues of l-amino-3-(N,N-
dimethylamino)-propylidene-l,l-bisphosphonic acid, any of its soluble salts or
any of its hydrates, invitro, comprising the steps:
treating of cells having Ca2+-channels with Ca2+-channel blockers;
contacting the cells with the putative functional analogue which, in the
absence of any Ca2+-channel blockers, is known to cause a Ca2+-ion
influx into the cells;
measuring a response as a result of the contacting step as herein described.

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Patent Number

216434

Indian Patent Application Number

IN/PCT/2001/579/CHE

PG Journal Number

13/2008

Publication Date

31-Mar-2008

Grant Date

13-Mar-2008

Date of Filing

25-Apr-2001

Name of Patentee

GADOR S.A

Applicant Address

Darwin 429, C1414CUI Buenos Aires,

Inventors:

#	Inventor's Name	Inventor's Address
1	ROLDAN, Emilio, J., A	Gador S.A., Darwin 429, 1414 Buenos Aires,
2	PEREZ-LLORET, Anibal	Gador S.A., Darwin 429, 1414 Buenos Aires,
3	VAZQUEZ, Guillermo	Dpto. de Biologica, Bioquimica y Farmacia, Universidad Nacional del Sur, San Juan 670, 8000 Bahia Blanca,
4	BOLAND, Ricardo	Dpto. de Biologica, Bioquimica y Farmacia, Universidad Nacional del Sur, San Juan 670, 8000 Bahia Blanca,
5	PAPAPOULOS, Sokrates, E	University of Leiden, Rijnsburgerweg 10, NL-2300 RC Leiden,

PCT International Classification Number

A61K 31/663

PCT International Application Number

PCT/EP1999/008269

PCT International Filing date

1999-10-29

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1	P980105446	1998-10-30	Argentina