Preclinical studies

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PRECLINICAL STUDIES

ACTIONS

[ 1 ~ Pamidronate Improves Trabecular Bone Mass/Strength and in Association with IGF-I Cortical Bone Quality in Ovariectomized Rats

P. Ammann, R. Rizzoli, J - M Meyer. D. Slosman, J-Ph. Bonjour. D/vision of Clinical Pathophysiology, Qepartment of Medicine, and School of Dentistry, University Hospital, 1211 Geneva 4, Switzerland

Bisphosphonates have been shown to increase areal bone mineral density (BMD, g/am 2) at skeletal sites containing trabecular bone but nrJt at sites purely cortical in osteoporotic patients, We assessed efforts of pamidronate (APD) to}lowing a stimulator of bone formation on BMD, mechanical properties and morphology. Eight weeks after OVX or SHAM operation, 6-month old rats were allocated to 5 groups of 9 animals. Two were treated with the bone formation stimulator IGF-I (2 mg/kg/day) by s.c. osmotic minipumps for 4 weeks. Then. one group having received IGF-I and one the vehicle were given 2 courses of 5 daily s.c. injection of the bone resorption inhibitor APD (1 rng P/kg) over 8 weeks. A second cycle of treatment (IGF-I followed by APD) was then administered. BMD of lumbar vertebrate (site containing a large proportion of trabecular bone) and Of midshaft tibia (site with mainly cortical bone) were measured in vivo by DXA. Bone strength (Stre.) of vertebral body for compression and of midshaft tibia for flexion was evaluated in vitro using a material testing machine (Instron 1114). External diameter (ED) and cortical thickness (CT) of tibia were analyzed. Values recorded 40 weeks after OVX were expressed in percent difference from OVX controls. Significance of differences was evaluated with an analysis of variance (*from OVX untreated controls, * * f rom IGF-1 + APD- and * * * from APD-treated OVX rats).

Lumbar Vertebrae Midshaft Tibia Stre BMD Stre. BMD ED CT

APD 38.6* 11.1 * 4.5 0.2 0.3 8.5* IGF-I 12.1 **. *** 3.8 13.3 2.3 3.4 0.9**' +** APD + IGF-L 37.2* 12.6" 18.1" 4,3 5.9 . . . . . 12.4"

In lumbar vertebrate. APD induced a significant increment of both BMD and strength, which was not changed by IGF I treatment preceding APD In mJdshaft tibia leortica! bone), onJy combination of IGF-I and APD incseased significantly bone strength whereas BMD was only slightly affected IGF-I increased external diameter of the midshaft tibia, presumably by enhancing pcriosteal appos~tion, and APD its cortical thickness, possibly through an inhibition of endosteal resorption, In conclusion, APD alone positively influenced bone mass and mechanical properties of vertebral body but not of midshaft tibia in OVX rats. In contrast, APD preceded by the administration of a stimulator of bone formation improves bone mechanical properties of cortical bone These approaches could be promising ir~ the treatment of osteoporosis especially of corbcal osteoporosis.

[ • Clodronate (Cl2MBP) Blocks the Effect of High Doses of 1,25-dihydroxyvitamin Da on Bone Resorption end Osteoid Formation

M.-L. Behrens-Stevens, M Bose. M. Br~utigam, M Haberey. Schezlng Research La#ofatofies, Spezielle Phafmakologie, Berl/n FRG

In a previous study we showed that high doses of 1.25-dihydroxyvitamin 03 for 6 days increases osteoclast number during the first 3 days, followed by a decrease in osteoclast number and bone resorption and a dramatic increase in osteoblast number and osteoid formation, The aim of this study was to investigate how the bisphosphonate Clodronate influences this effect. 3 month old female Wistar rats were randomly divided into 7 groups with 5 animals per group. The groups treated with Clodronate were pretTeated with this substance 48 h before 1.25-dihydroxwitam{n 03 application. The control group received the vehicle solution, group 2 : 0 , 5 /~g 1,25-dihydroxwitamin D3/kg/d for 3 days; group 3 : 0 . 5 •g 1,25-dihydroxwitamin D3/kg/d for 6 days; group 4 :6 .6 mg/d Clodronate

for 5 days; group 5:6.6 mg/d Clodronate for 8 days; group 6 :0 ,5 p,g 1,25- dihydroxyvitamin D3/kg/d for 3 days and 6.6 mg Clodronate/d; group 7:0.5 l~g 1.25-dihydro• D3/kg/d for 6 days and 6.6 mg Clodronate/d. The animals were killed at the end of the 1,25-dihydroxwitamin D 3 treatment The right femurae were dissected, cleaned of soft t}ssue and embedded in methymethacrylate for histomorphometry.

CIodronate inhibited the increase in osteoclast number by 1,25- dihydroxwitamin D 3 treatment in the control range. The treatment with clodronate alone reduced the number of osteoclasts to below control values. The dramatic increase in osteoblast number and osteoid formation after 6 days of 1.25-dihydroxyvitamin D 3 treatment could also be blocked by Clodronate. The treatment with Clodronate alone led to a decrease in osteoblast number and osteoid formation (cf. table). These results show that CPodronate not on~y inhibits bone resorplion and osteocfast formation and also inferes with osteoblast recruitment and bone formation.

Groups N Oc./Md Pm N.Oc F [ A r , Ob.Pm/B.Pm OAr/B Ar (rnm) (%) (%)

Control 2.80i0.5 25.45~ 7,5 2.61• 1 0475 i0.2 Group 2 5.37• 42.96~ 9.8 4.71• 1 48 • Group 3 44910.7 27.76~c 1.9 26.83• 645 • 71 Group4 095J_0.2 863• 1 92 2.94• 117 • Group 5 115J_0.5 930~ 4 1 3.05• 0 60 • Grou~ 6 3 03i0 9 24.21~12.8 2.51• 0 95 • s Group 7 326• 1 27 65J_ 8 0 4.21• 1.07 •

[•] The Effect of Cl2MBP on Mechanical Properties, Mineral Content end Bone Volume In Immobilized Rats

A. Dziedzic-Goclawska *, A Kaminski *, R.R Kusy*, M Yamauchi *. * Dental Research Center+ University of North Carolina at Chapel Hi//, USA; ** ~epaftment of Transplanto/ogy, [nstitute of Biostzucture, School of Medicine, uL Chalubinskiego 5, 02-004 Warsaw, Poland

The purpose of this work was to evaluate the effect of treatment with CI2MBP on bone remodeling during immobilization in rats. Male Sprague- Dowley rats (10 wk old) were divided into three groups. The animals in 2 groups were subjected to one hindlimb immobilization against the abdomen using a few layers of elastic bandage tape. with the hip joint in flexion end the knee and ankle joints in extensior~. Since in the model of one hindlimb immobilization the cantralateral limb is overloaded, the animals ef non-immobilized third group were included as controls. The rats of one immobilized group were injected subcutaneously with CI2MBP (12.5 mg P/kg/day for 6 weeks and 6.25 mg P/kg/day for additional 6 weeks), while the animals of the second immobilized group and those non immobilized ones were injected with a vehicle (0.9% NaCI). The animals were sacrificed 12 weeks after beginning of the experiment. All measurements were done on bones taken from immobilized legs of vehicle and cI2aBP-treated rats and compared to the bones taken from non-immobilized control group. Mechanical properties of fresh femurs: strength, stiffness, toughness and ductility were measured, along with measurements of mineral content in cancellous bone of femur epi and mataphyses and in compact bone of femur diaphyses. Metaphyseal regions of proximal tibiae were used for histomorphometric analysis.

In immobilized vehicle treated animals a significant decrease of femur strength (by 8%) and an increase in ductility (by 16%) accompanied by a slight decrease in mineral content (by 3%) in cancellous bone of femur epi- and metaphyses as compared to the control bones were observed, while in the immobilized CIzMBP-treated group an increase in femur strength (by 10%) accompanied by the increase of mineral content (by 8%) were noted compared to the non-immobilized controls. In all groups no changes in mineral content of compact bone were observed. Because the changes in mechanical properties of bone were relatively larger than in mineral content, therefore the former ones provide more sensitive method to evaluate the quality of bone. There was a significant decrease in trabecular bone volume (TBV) (by 15%)in immobilized tibiae of vehicle-treated animals compared to the control tibiae On the other hand, in C[2MBP-treated rats an increase of TBV (by 50%) in immobilized tibiae were observed compared to the control non-immobilized ones.

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In conclusion, CI2MBP effectively prevented the bone loss due to immobilization.

[• Bone Effects of Etidronate in Ovariectomized Rabbits

B. Grardel, B. Flautre. B. Sutter, R Hardouin./R/VIS,/nstRut Ca/or, 62608 Berck/mer, France

A predictable animal model with skeletal remodeling characteristics similar to those of humans is needed to facilitate the understanding of the mechanism of post menopausal osteoporosis. There is no study examining the effects of ovarian dysfunction on bone in rabbits.

We therefore studied 60 female rabbits for 8 months. They were randomized to either ovariectomy or a sham operation. 2 months after surgery the ovariectomized rabbits received either 0.5 mg/kg either 5 mg/kg of etidronate 5 days every 4 weeks, or a placebo.

Bone mineral density of lumbar spine and whole body displayed the same values between sham-operated and ovariectomized non treated groups during all the study. Histomorphometric measurements of bone taken at the end of the study showed no significant difference in microradiographs parameters.

Between the second month after surgery and sacrifice, both etidronate treated groups showed an increase of cancellous bone volume, without increase of bone density, these results are quite different of these observed in human treated osteoporosis.

Bone response after ovariectomy in the rabbit had never been studied before, but the lack of sizeable responses may limit the utiiity of rabbits for the study of cancellous bone loss in ovarian dysfunction osteoporesis.

[ ] E f f e c t of Continuous HEBP Exposure on Deposition end Mineralization of Rat Incisor Dentin

K, Josephsen, A. Nanci. Royal Dental College, University of Aarhus, Vennelyst Boulevard 9, DK-8000 Aarhus C, Denmark," Faculte de M#decine Dentaire, Universit# de Montr#al, Ou#bec, Canada

A single injection of 1 hydroxyethylidene-l, l-bisphosphonate (HEBP) has been shown to temporarily affect initiation of dentin mineralization. In the present study we have investigated the effects of continuous infusion of HEBP on dentin formation in rat incisors. Rats weighing ~100 g were injected with 10 mg P of HEBP/kg b.wt. and then implanted intraperitoneally with mini osmotic pumps that delivered 20 #g or 40/~g P of HEBP/hr continuously for 7 or 14 d. Normal rats were used as controls. Animals were fixed by perfusion and both calcified and decalcified incisors were embedded in Epon for light and electron microscopic analyses.

In normal rats, the zone of initial predentin formation (Zpd) was 1015 /~m long and attained a thickness of 12 /Lm at its most incisal point where mineralization of mantle predentin occurred, demarcating the start of the zone of mineralized dentin (Zmd). All experimental groups showed a common pattern of dentin disturbances. ] he Zpd increased to double the length and to a four-fold thickness incisally. The collagen fibrils in the forming predentin of this region were larger than in controls, and dead cells were sporadically observed in the odontoblast layer. Based on the morphological characteristics of the extracellular matrix, the Zmd was subdivided into regions where 1) distinct islands of mineralized matrix were present in predentin and 2) the islands fused together to form a continuous, although somewhat irregular, layer of mineralized dentin. In both regions, some odontoblasts were surrounded by unmineralized matrix possibly because their secretory pattern was altered and/or their secretory output was reduced with respect to adjacent ce!ls The dentin matrix appeared heterogenous, being composed of collagen fibrils and non-collagenous matrix with variable texture

These data indicate that continuous exposure to HEBP affects the secretory activity of odontoblasts as well as the mineralization of predentin. The presence of isolated dentin islands despite a continuous infusion suggests that mineralization of predentin was cyclically affected, possibly because the inhibitory effects of HEBP on mineralization were offset by a rhythmic variation in the rate of predentin formation and mineralization.

Supported by Calcinfoeden and the MRC of Canada.

Second Workshop on Bisphosphonates

[ - ~ Bone Effects of Two Bisphosphonates (Clodronste and Etidronate) In Ovariectomized Rats

K. Kippo, M Lepola *. R, Hannuniemi, K. V~in~nen *, L. Lauren, T. Virtamo. R Sellman. Leiras Oy, Biomedical Research Center. P.O.Box 415, 20101 Turku, Finland," * University of Ou/u, Department of Anatomy, Oulu, Finland

Bone histomorphometry and biomechanics were investigated after 12 weeks' treatment with clodronate (C; 1,3, and 25 mg/kg), etidronate (E; 3, and 25 mg/kg), and vehicle (Ve). Sprague Dawley rats (n = 21 O) were either ovariectomized (OVX) or sham operated (SHAM) at the age of 3 months and injected subcutaneously with Ve or bisphosphonates once a week. Before sacrifice, 90 animals received a double fluorochrome labeling, Two-way analyses of variance with contrasts were used for statistical evaluation.

The trabecular bone area (B.Ar/TAr) was decreased in the distal femoral metaphysis (19% in SHAMNe and 8% in OVX/Ve group), and in the fourth lumbar (L4) vertebral body (33% and 28%) at 12 weeks post- ovariectomy, This osteopenia was prevented by C (3 and 25 mg/kg). There was a significant difference in B.Ar/T.Ar of femoral metaphysis between C and E (25 mg/kg) groups, the values of OVX rats being 42% and 29%, respectively. Similar difference was found in the SHAM groups. In contrast, there was no significant difference in B.Arfl~.Ar of L4 between C and E (3 and 25 mg/kg) in OVX and SHAM groups. On the lateral cortex of femoral metaphysis, ovariectomy enhanced the endocortical mineral apposition rate (EcMAR); the values were 1.1 /~m/d in SHAM/Ve and 2.1 /zm/d in OVX/Ve group. In the OVX rats treated with C (1 and 3 mg/kg), the values of EcMAR were 1.5/zm/d and 1.4 #m/d, respectively. There was no significant difference in EcMAR between the C and E groups (3 mg/kg).

Ovariectomy caused a reduction in the maximum load in compression of both the femoral neck (100 N in SHAMNe and 88 N in OVX/Ve group) and the L4 vertebra (643 N and 550 N). These changes were inhibited by C (25 mg/kg). At the same dose, C had a significantly greater effect than E (C vs E) on the maximum load of both the femoral neck (99 N vs 93 N) and L4 (701 N vs 592 N) in OVX groups. Similar difference was found in the SHAM groups

The results indicate that C prevents the development of osteopenia and improves impaired bone strength induced by estrogen deficiency. The lowest effective dose of C was 3 mg/kg. C inhibits ovariectemy-induced osteopenia and improves bone strength more effectively than E.

I 7 - ] Long-Term Effects of Clodronate on Bone Strength

M Lepola, R. Hannuniemi *. K. Kippo *, R Jalovaara **, K. V~in~nen. Department of Anatomy; ** Department of Surgery, Umizerstty of Oulu, Kajaanintie 52,4, 90220 Oulu, Finland," * Leiras Oy, Biomedical Research Center, 20101 Turku, Finland

Bisphosphonates are potent inhibitors of bone resorption and have been shown to be effective in preventing osteopenia. However, it is known that a long-lasting, strong inhibition of bone resorption can lead to increased bone fragility, probably because of some deletorius effects on bone mineralization In view of this, long-term effects of clodronate on rat bone were studied.

Three-month-old Spraque-Dawley rats were administered s.c. for 25 weeks by 1) vehicle, 2) clodronate 4 mg/kg/~veek (=100 mg/kg/25 w). 3) clodronate 12 mg/kg/week ( -300 mg/kg/25 w) or 4) clodronate 50 mg/kg/every 4th week (=300 mg/kg/25 w). Tibia ash weight, torsional strength of tibia, three-point bending strength of femur and compressive strengths of femoral neck and 5th lumbar vertebra were measured (n = 20/group). By histomorphometry, the area of trabecular bone from the non-decalcified sections of the secondary spongiosa of distal metaphysis and 4th lumbar vertebra were measured (n 5/group).

Tibia ash weight in groups receiving 4 mg or 12 mg clodronate/kg/w was increased compared with the vehicle group. No differences between the groups were observed neither with respect to bone mechanical strength in torsion of tibia, three-point bending of femur or compression of femoral neck, nor with the trabecular bone volume at the sites measured. In compression of the vertebra L5, the maximum strength was increased in the group receiving clodronate 4 mg/kg/w compared with the vehicle group.

In conclusion, bone mass was increased during the 25 weeks clodronate treatment in rats. On bone strength, clodronate had an increasing effect in vertebra compression, but only with a smaller dose No deleterious effects by clodronate on bone mass, histomorphometry or strength were observed

Preclinical studies - - A c t i o n s $61

[ ] Anticalcific and Antiproteolytic Effects of Imino-Bis-2-Ethylidene Diphosphonlc Acid on Bioprosthetic Heart Valves

B.R Mishchenko. National Research Center for Endocrinology Russian Academy of Medzcal Sciences, D. Ulyanova 11, 117036, Moscow, Russia

Principal causes of disfunction of bioprosthetic heart valves are calcinosis of xenomaterials and proteolysis of CUSPS resulting in perforation.

We performed controlled study of protective effects of imino-bis- 2-ethylidene diphosphonic acid (ITP) and 3-amino-l-hydroxypropane-1, 1-diphosphonic acid (APP) o1 calcinosis and proteolysis of xenomaterials of bioprosthetic heart valves made from bovine pericardial tissue treated with glutaraldehyde. Diphosphonate fixation on the tissue was performed with sutured reagent 1-ethyl-3 (3-dimethylamino) propyl carbodiimide and 1-hydroxybenzotriazole as accelerator. Concentration of diphosphonates was 1% of tissue dry weight.

Inhibitory effect of diphosphonates fixed on biotissue on calcification was demonstrated in the rat model within 90 days of subcutaneous implantation. We have shown that even small amounts of APP and ITP prevented calcification of bioprosthetic heart valves. Coefficients of inhibition were 7.6 and 11.2 respectively. Analysis of individual indices of calcification revealed that frequency of calcinosts using ITP was significantly lower than with the use of APP

Investigation of collagenase effect (1 KU/ml 10 mM/I Hepes buffer, Ph 7.0, Ca ++ - - 5 mM/I) on native bovine pericardium treated with APP and ITP, showed a 3.1 and 8.8-fold decrease in the accumulation of hydroxyproline in the incubation medium respectively compared with control.

Our data show. that ITP improves biological characteristics of bioprosthetic heart valves, prevents calcinosis and protects valve cusps from destruction. We consider, that these effects are linked to an additional diphosphonic group in the ITP molecule.

•7 Sub-chronic Effects of High Doses of Mildronate on Femur Densitometric (DEXA), Tomographic (pQCT) and Mechanical Properties in Young Rats

N. Mondelo, E. Montuori, V. Peluffo, R,F. Capozza, G.R. Cointry, S. Monllo. J.R. Zanchetta. J .L Ferretti. CEMFoC, Laboratories "Gador" Institute de Investigaciones Metab61icas (IDIM), Llbertad 836, 1012 Buenos Aires, Argentina

Previous studies (JBMR 5: $105, 1990 ~ 6: $128, 1991) showed positive effects of Mildronate (dimethyl-pamidronate, prop, INN. a third generation amino-bisphosphonate} on femur biomeehanies, derived from a strong inhibition of bone resorption without any demineralizing effect even at very high oral doses (up to 200 mg/kg/d). These properties are better described now by means of pQCT determinations. Eight groups of 10 male or 10 female weanling rats received doses up to 90 mg/kg/d Mildronate in the drinking water during 3 months. Their femora were then submitted to DEXA (Norland XR-26), pQCT (Stratec XCT-960 with a special software for geometric studies; distal-metaphiseal and midshaft scans) and mechanical (3-point bending tests) determinations.

Treatment substantially enhanced the BMD (DEXA) and the pQCT- assessed BMD (volumetric BMD, vBMD) both in the whole bone and in the central third of the diaphyses, and the pQCT-determined, cross-sectional area and moment of inertia in every section scanned, independently of gender and dose. These changes were positively correlated (p < 0.001 ) to large increases in diaphyseal load-bearing capacity, stiffness, and energy absorption ability with respect to controls (p < 0.001 ).

These results indicate a positive effect of Mildronate on bone mass and mechanical properties of femur diaphyses in normal rats, derived from a strong action on cross-sectional architecture and vBMD of the newly- added bone, with no apparent reduction of cortical vBMD. The geometric changes were consonant with a threshold effect on the mechanostatic mechanism governing bone modeling as a function of bone stiffness.

Confirming our previous findings, no apparent deterioration but an actual increase of bone mineralization (as assessed by either DEXA-BMD or pQCT-vBMD) was detected even at the high dose levels assayed.

•O] Inhibition by a Alendronate of Bone Resorption Induced by the MBT-2 Tumor of Mice

R. Nemoto. Department of Urology, Tottori Prefectural Central Hospital, Tottori 680, Japan

Tumor-bone interactions were experimentally studied using a bladder tumor in mice (MBT-2). The method consisted of inoculating tumor cells subcutaneously over the calvaria in mice, resulting in a local tumor causing fragmentation of the bone. The tumor-induced osteolysis associated with osteoclasts proliferation was accompanied with reactive new bone formation, The osteoclasts decreased in number when the tumors had grown large enough to envelop the residual bone. However, bone destruction continued and seemed to be mediated by the tumor cells by a mechanism that did not involve the osteoclasts. This osteolysis was evaluated by measuring the increased area of bone resorption in reduced opacity to radiograph and histology.

The effects of several agents were investigated in this model. High doses of calcitonin and eyclosporine reduced the bone resorption, and these agents may be effective in the early phase of bone destruction. A new bisphosphonate. ALENDRONATE, inhibited bone resorption markedly in the early and late phase of bone destruction. This inhibition was obtained with no apparent effect on the growth of the MBT-2 tumo[ Autoradiography using 14C-labeled ALENDRONATE showed the concentration of the isotope at the surface of the bone adjacent to the MBT-2 tumors. These results suggest that bisphosphonate may make bone less susceptible to the actions of osteoclasts and tumor cells.

ALENDRONATE appears to be an interesting new bisphosphonate with possible clinical application.

Cancer 69, 2316-321, 1992; 67,643-648, 1991 ; 62.1310-1316, 1988.

•1-• Tiludronate Increases the Mechanical Competence of Lumbar Vertebral Body Reduced after Ovariectomy in Rats

H. Ohnishi, T. Nakamura, H. Tsurukami, H. Murakami, M, Abe, A. Barbier. Dept. Orthopedics, Univ. of Occupational and Environmental Health, Iqtakyushu, Research Center, Me/ji Seika, Tokyo, Japan," Sanofi Recherche, Montpellier, France

To demonstrate thesequent ia l changes in the bone mass and the mechanical properties after ovariectomy (OVX) and to know the effects of tiludronate, 188 Wistar rats of 13 groups, 6 months of age, were fed for 9 months after surgeries, The agent was given at the doses of O(vehicle), 12,5, 25 and 50 mg/kg/day for the duration of 3 months, starting 3 months after OVX, and then only the vehicle 1or the last 3 months. Animals were sacrificed at the start. 3, 6, and 9 months post surgeries. The 3rd lumbar body (L3) and the right femur were examined for mineral content (BMD) and mechanical properties. Undecalcified sections were prepared thereafter. In the first 3 months, BMD of L3 showed 12% reduction with 26% decrease in the compressive strength after OVX In the next 3 months. L3 in the vehicle group showed only 3% decrease in BMD However. its strength marked 24% reduction (P < 0.05). In the highest dose group, L3 showed 2% increase in BMD with 14% increment for the strength (P < 0.05) at the end of the 3-months treatment period. In the Last 3 months, the vehicle group showed 3% decrease in BMD, but no further reduction was observed in the strength. Three months after stopping treatment. animals in the highest dose group showed more 3% increase (P < 0.05) in BMD and maintained the improved mechanical properties. These data clearly demonstrate that the lumbar compressive strength continues to decrease for the longer period than expected from the bone loss after OVX. The improvement of the strength by tiludronate administration is obvious and the action continues after cessation of dosing.

• i • A Histomorphometric Comparison of the Effects on Rat Bone of Short-Term Treatment with the Two Bisphosphonates CGP 42'446 and Pamidronate: Static Parameters

A, Pataki, J.R. Green, K, M(Jller, R. Schneider, A. Studer, K.A. JaeggL Ciba-Geigy Ltd,, Pharmaceuticals Division, CH-4002 Basel, Switzerland

The bisphosphonate compound CGP 42~446 [2-(imidazol-l-yl)-hydroxyethylidene-l,l-bisphosphonic acid] is a very potent inhibitor of bone

S62 Second Workshop on Bisphosphonates

resorption in several pharmacological models. It is currently in clinical development for the indication osteoporosis. In a previous dose-finding study in rats, we reported the effects of CGP 42~446 on some static parameters of bone histomorphometry [Pataki et al., Osteoporosis 1990, 21038-1041, Osteopress, Copenhagen]. We now report the results of a comprehensive static morphometr ic analysis of rat tibial cancellous bone after treatment with CGP 42~446 and pamidronate.

Groups of young, growing, male rats [n = 8, 190-220 g] were treated with either CGP 42'446 [0.1, 1.0 and 10 nanomoles/kg] or pamidronate [10, 10O, 1000 nanomoles/kg] for 10 days by s.c. injection. One day before starting treatment the animals were labelled with calcine [20 mg/kg i.p.], fo l lowed by demeclocyclin [20 mg/kg i.p.] on days 4 and 11. The proximal tibial metaphyses were collected at autopsy on day 16 and changes in the secondary spongiosa were analyzed by histomorphometry using a fully automated system for digital image analysis [Leica ASBA].

Treatment with both CGP 42'446 and pamidronate induced a marked, dose dependent increase in the fol lowing parameters: mineralized bone area and perimeter; trabecular width and number; number of nodes and terminals. Trabecular separation showed a corresponding decrease. In increasing the amount of cancellous bone, as reflected by these parameters, CGP 42'446 was approximately 100 t imes more potent than pamidronate.

It is concluded that both drugs dose-dependently suppressed physio- logical bone resorption during longitudinal bone growth, resulting in an accumulation of cancellous bone. The dose-dependent increase in trabecular width indicates a positive bone balance which can be explained by a distinct suppression of bone resorption during trabecular remodelling.

•f• Pamidronate (APD) Diminishes Side Effects of the New Vitamin D Analogue EB1089 in the Treatment of the Walker Carclnosarcoma 256 in Rats

T. Schilling, C. Albrecht, B. Kohl, R. Ziegler, F. Raue. Intema/Med. I, University of Heidelberg, Bergheimerst~ 58, D-69115 Heidelberg

Vitamin D analogues (EB1089) are known to inhibit the growth of cancer cells. But the hypercalcemic side effects of EB1089 restrict it f rom the use in tumors causing hypercalcemia of malignancy (HHM). We tested whether pamidrenate (APD) is able to prevent the hypercalcemic side effects of EB1089 in the Walker carcinosarcoma (WCS) 256, a Parathyroid-hormone related Protein (PTHrP) mediated model of HHM.

Female rats (160 200 g) were s.c. inoculated with WCS cells at day 0. 11 animals were treated daily with EB1089 i.p. (0.25/~g/kg) in combination with APD (15 mg/kg). 14 animals served as controls and were treated with vehicle alone.

At day 7 the EB1089+APD treated group had significantly decreased serum calcium levels compared to the EB1089-group and at day 9 compared to the EB1089- and the control-group, The tumor weight was significantly reduced by EB1089. EB1089 and EB1089+APD had no influence on circulating PTHrP levels.

Calcium (mM) Calcium (mM) tumor (g) PTHrP (pmol/I) day 7 day 9

Control 2.8J-0.2 4.1• 2 11 3• 1 494-27 EB1089 4.2:::E0.5 4.0:E0.5 8.39_2.4* 639_28 EB1089 + APD 3.7• 3.3~0.4" * * 7.3• 76•

* p < 0.05 compared to control, ** p < 0 05 compared to EB 1089

APD is able to diminish the hypercalcemic side effects of the new vitamin D analogue EB1089. Thus, by combination of both drugs, the dosage of the anti cancer agent EB1089 can be increased without deterioration of the hypercalcemia.

~ - ~ Alendronate Preserves the Mechanical and Histomorhometric Properties in Vertebrae of Aging Estrogen-Deficient Rats

M. Shea, R. Balena, J.A. Guy, G.J. Seedor, A Markatos, G.A. Rodan, W.C. Hayes. Orthopaedic Biomechan/cs [Jab., Beth Israel Hospital, Boston, /VIA 02215 USA

In experimental animals, Alendronate was shown to prevent bone loss associated with ovariectomy or aging. Our purpose was to determine the efficacy of ALN treatment in animals made osteopenic by estrogen

deficiency. Thirty female Sprague-Dawley rats were ovariectomized at 6.5 months. Six months later, they received injections (s.c.) of either vehicle (Ovx-Veh), Low-dose ALN (1.8 bcg/kg) or High-dose ALN (18/~g/kg) twice weekly for one year. In addition, ten non-ovariectomized rats received vehicle (Non-Ovx) for one year. Prior to sacrifice, the rats were injected intraperitoneally with oxytetracycline and calcein using a 10-day interval between labels. At sacrifice, the lumbar vertebrae were removed. Static and dynamic histomorphometric parameters were measured on 5-10/~m thick methacrylate embedded sections of the L3 vertebrae, using Bioquant system IV Bone morphometry software (RSM Biometrics, Nashville, TN). To determine area fraction of bone, the L6 vertebrae were sectioned along the mid-sagittal plane and analyzed using image processing software (SUN Microsystems, Mountain View, CA). The L4 vertebral bodies were tested in compression until failure and then ashed. Data were analyzed by one-way analysis of variance, and differences between groups were assessed using Fisher PLSD (p < 0.05). Cancellous bone volume was 35% lower in the Ovx-Veh group than in Non-Ovx controls. Low-dose ALN maintained cancellous bone volume at the level of the Non-Ovx controls, while High-dose ALN increased it above Ovx-Veh, but the difference was not significant. The osteoclast surface increased by 372% in the Ovx-Veh group, compared to the Non-Ovx controls, and was reduced to control levels by ALN treatment. Bone turnover, reflected in osteoid surface, was increased in Ovx-Veh rats and was reduced by ALN treatment to Non-Ovx levels (Low-dose) or below (High-dose). Area fraction of bone increased by 16% and 18% in rats receiving Low- and High-dose ALN, respectively, over the Ovx-Veh group. Ovariectomy resulted in a 19% reduction in ash weight compared to controls, and High-dose ALN maintained ash weight at the level of the Non-Ovx controls. Finally, ovariectomy caused a 32% reduction in failure load when compared to Non-Ovx controls. High-dose ALN treatment maintained failure load at the level of controls, resulting in a 40% increase over the Ovx-Veh group. In conclusion, osteopenia characterized by increased bone turnover and osteoclastic bone resorption was present in the lumbar vertebrae of rats 18 months post ovariectomy. This resulted in decreased failure load. ALN maintained bone turnover, bone mass, and failure load at the levels of the Non-Ovx controls. Long term ALN therapy in aging estrogen-deficient rats thus preserves both the mechanical and morphologic properties of lumbar vertebrae.

I ~ Alendronate Preserves Geometric and Tissue Properties in the Femora of Aging Estrogen-Deficient Rats

M. Shea, J.A. Guy, G.J. Seeder, G.A. Rodan, W.C. Hayes. Orthopaedic Biomechanics Laboratory, Beth Israel Hospital, Boston, /VIA 02215 USA

The bisphosphonate Alendronate (ALN) has been shown to prevent bone loss in both aging rats, and when given to adult rats at the t ime of ovariectomy. However. the effectiveness of ALN as a treatment for established estrogen-deficiency induced osteopenia is unknown. Our objectives were to determine if ALN treatment would increase bone mass and strength in aging estrogen-deficient rats above the levels of ovariectomized controls. Forty female Sprague-Dawley rats were ovarieetomized at 6.5 months, and twenty were sham-operated. One group of non-ovariectomized and ovariectomized rats were killed as Time- 0 controls. Six months later, the remaining ovariectomized rats received subcutaneous injections of either vehicle (Ovx-Veh), Low-dose ALN (1.8 #g/kg) or High-dose ALN (18 p,g/kg) twice weekly for one year. The remaining non-ovariectomized rats received vehicle (Non-Ovx-control) for one year, Femoral geometric and tissue properties were analyzed using 3-mm slices removed from the femoral mid-shaft. Images of these slices were digitized to determine cortical and medullary area, and areal moment of inertia. The apparent density and ash content of these slices were then determined. The contralateral femora were scanned using dual- energy x-ray absorptiometry to determine bone mineral density (BMD), These femora were then teated to failure in three-point bending. Data were analyzed by one-way analysis of variance, and post-hoc multiple comparisons were performed using Student-Newman-Keuls analysis (p < 0.05). The medullary cavity was increased by 21% due to ovariectomy (Ovx-Veh vs. T ime-00vx) . Femoral mid-shaft moment of inertia was also increased by 25% in the aging estrogen-deficient rats (Ovx-Veh > Time 0 control and Time 0 0 v x ) . The changes in geometry were prevented by both Low and High-dose ALN, which maintained these properties at the level of both Time 0 groups. Estrogen deficiency decreased cortical ash content by approximately 3% ( T i m e - 0 0 v x > Low-dose ALN and

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Ovx-Veh). Ash content was maintained in the High-dose ALN group, at the level of both Time-0 groups. Similarly, BMD was reduced in the estrogen deficient rats (Time-0 control > Ovx-Veh and T ime-00vx groups), and restored by High-dose ALN treatment (High-dose ALN and Non-Ovx control groups > T ime-00vx and Ovx-Veh groups, by approximately 7%). High-dose ALN treatment also prevented reductions in cortical apparent density (High-dose ALN > T i m e - 0 0 v x and Ovx-Veh groups, by 10% and 5%, respectively), The energy to failure was decreased in the estrogen deficient rats ( T i m e - 0 0 v x > Ovx-Veh and Non-Ovx control). However, there were no effects of aging, estrogen-deficiency, or ALN treatment detected in femoral failure load. Estrogen deficiency in the aging rat resulted in compromised cortical bone composit ion and morphometry. High-dose ALN attenuated these changes, and increased BMD and apparent density above the level of the Time-0 ovariectomized group. ALN therefore effectively reduces the adverse skeletal effects that occur as a consequence of aging and estrogen deficiency in the rat, and restores bone mass lost to estrogen deficiency,

•f• Effects of Blsphosphonates on the Biomechanical Properties of Bone in Experimental Models of Osteoporosis

H.K. V~i~in~nen. Dept. of Anatomy, Univ. of Oulu, Finland

Bone strength and other mechanical properties are probably the most important intrinsic factors to determine the vulnerability of bone to low energy fractures. Development of new treatments to cure osteoporosis and other metabolic bone diseases has increased demands to obtain knowledge, not only of bone structure and composit ion, but also of its mechanical properties. Past experience of the effects of, for instance fluoride, clearly indicates that bone mass and density measurements are not sufficient criteria of bone quality.

Several animal models have been used to study the effects of drugs on bone but so far only a limited amount of data is available about the effects of different bisphosphonates on bone strength and comparative studies between different compounds do not practically exist. All drugs which could potentially alter the internal geometry of trabecular or cortical bone might have harmful effects on bone quality in spite of their capacity to increase bone mass. It is thus important that data about the mechanical consequences of treatment on bone strength and other mechanical properties exist before any treatment or prevention can be accepted into large scale use in osteoporosis.

In this presentation, I will first give an overview on the current literature of the effects of different bisphosphonates on bone strength and then go through some recent data about the effects of clodronate on bone strength in different rat models of experimental osteoporosis. These results show that the decrease in bone mass during immobilization can be prevented by clodronate, which also increases the mechanical strength of bone, both in immobil ized and contralateral bones of casted animals. ]n non-immobil ized rats. c]odronate increases bone mass, but not the breaking strength of tibia.

Long-term effects of clodronate on bone strength were studied by giving different doses of the drug to rats for 25 weeks and measuring bone strength in vertebra, femoral neck, and tibial and femoral diaphyses. Again. bone mass increased due to clodronate treatment. Clodronate increased bone strength in vertebra compression and no deleterious effects were observed in any other bones measured. In an ovariectomy model, clodronate treatment prevented the decrease in the strength of femoral neck, tibial diaphyses, and in vertebrae. In this model we compared the ef for t of clodronate with etidronate and observed clodronate to be more effective in preventing loss of the mechanical bone strength.

• Biaphoaphonatea: A Potential Role in the Prevention of Avian Oateoporosla

Sandra Wilson *, Sally E. Solomon **, Barry H. Thorp *. *AFRC Roslin Institute (Edinburgh), Roslin, Midlothian, EH25 9PS, Scotland, "" Poultry Research Group, Department of Veterinary Anatomy, University of Glasgow, Bearsden, Glasgow, G61 1QH

Osteoporosis in laying hens is associated with the modelling and remodell ing of medullary bone, a type of woven bone which acts as a mineral reservoir for the requirements of egg shell formation. Trabecular bone volume (TBV) decreases initially during medullary bone modelling

(immediately prior to lay) and continues to decrease during subsequent remodelling (throughout the laying period). In an attempt to maintain peak, pro-lay, structural, bone mass the bisphosphonate alendronate was administered to pullets before medullary bone modelling. Treated and control hens were sacrificed when they had produced their first egg and after 20 weeks of egg production. At the onset of lay (first egg), TBV was significantly greater (p < 0.01) in the alendronate group (17.59%) than in controls (13.79%), while medullary bone volume was not significantly affected. After 20 weeks, TBV remained significantly higher (p < 002) in the alendronate group (12.72%) than in controls (9.80%), and MBV was lower in the alendronate group than in the control group, There was no significant difference in egg production or egg shell quality between the control and alendrenate treated groups. However. TBV was reduced and MBV increased in both groups compared with the values at the onset of lay. A|endronate therefore appeared to prevent the bone loss associated with medul law bone modell ing but not that which occurs during remodelling.

• Hyperthyroidism-Induced Bone Changes in Rodents and Effects of Aminobisphosphonate Alendronate

M. Yamamoto. A. Markatos, J.G. Seeder, G.A. Redan. R. Balena. Merck Research Laboratories, West Point, PA 19486, USA

To establish rodent models of hyperthyroidism-associated bone loss and to examine the effects of aminobisphosphonate alendronate (ALN) in these models, we conducted studies of three week duration in mice and rats. [Study 1] Female mice, 8 -9w old, were divided into 4 groups and given triiodothyronine (T3)-containing water (0.0, 0.5. 1.0, and 2.0 ug/ml), T3 at the highest dose induced significant (p < 0.05) decreases in femoral ash weight, cancellous bone volume (BV/TV) and cortical tissue area of the tibia. Serum osteocalcin (OC) showed a tendency to increase with increasing dose of T3 and correlated negatively with femoral ash weight (r = -0.432, p = 0.003). The effect of ALN was not tested in this mouse model of hyperthyroidism, [Study 2] Thyroxine (T4). 250 ug/kg/day, administered by sc injection induced a high turnover cancellous bone loss in the tibia. No bone loss was detected by femoral ash weight measurements, There was a highly significant negative correlation between BV/TV and serum OC (r - -0.753, p = 0.0019), a similar finding to that documented in patients with hyperthyroidism. ALN (1.75 mg/kg, gavage, x2/w) completely inhibited the T4-induced bone loss as well as the increases in serum OC, osteoclast surface and mineral apposition rate; but did not suppress these bone turnover parameters below control levels, [Study 3[ Male rats. ~ 9 w old, were divided into 4 groups and treated with T4 without or with two doses of ALN. T4 (500 ug/kg/day) induced a significant decrease in femoral ash weight. The higher dose of ALN (8,75 mg/kg, gavage, x2/w) inhibited the T4 induced bone loss, the lower dose (0,875 mg/kg) did not. Both doses of ALN effectively inhibited T4-induced cancellous bone loss in the tibia, These studies showed that (1) excess thyroid hormone dose-dependently induces high turnover bone loss in rodents in a manner very similar to human hyperthyroidism, and (2) ALN effectively prevents the bone loss.

• ] Effect of Acute and Ordinary Hypokinesia in the Development of Osteopenia in Bones of Rats

Y.G. Zorbas, Y.F. Federenko, K.A. Naexu. Hypokinetic Physiol. Lab., European Institute of Environmental Cybernetics, GR- 162 32 Athens, Greece

The aim of this study was to evaluate the effect of acute hypokinesia (abrupt restriction of motor activity) and ordinary hypokinesia (HK) in the development of osteopenia in bones of rats. The studies were performed on 96 male Wistar rats with an initial body weight of 380-390 g. They were divided into four equal groups. The 1st group of rats sacrificed prior to the investigation (baseline control animals), the 2nd group placed under ordinary vivarium conditions (vivarium control animals), the 3rd group subjected to acute HK (acute HK animals) and the 4th group submitted to ordinary HK (ordinary HK animals). For the simulation of the hypokinetic effect the 3rd and 4th groups of animals were kept in small individual cages made of wood which restricted their movements in all directions without hindering food and water intake. By the 10th day of the study the animals were weighed and sacrificed by decapitation. Longitudinal serial sections of the proximal part of the tibia and lumbar vertebrae cut parallel


to the frontal plane and transverse sections of the tibial diaphysis were stained with hematoxil in and eosin, picrofuchsin, methyl greenpyronine, alcian blue, tuolidine blue and by the Schmorl method. Exposure to acute and ordinary HK led to the development of osteopenia of the lumbar vertebrae and of the tibial metaphyseal spongiosa, which was more pronounced in animals subjected to acute than ordinary HK. There were not observed any signs of osteopenia in the tibial diaphyses of animals subjected to acute and ordinary HK. Osteopenia of the spongiosa of lumbar vertebrae displayed the animals submitted to acute HK. Exposure to acute HK contributes more than ordinary HK in the development of osteopenia in tibial and lumbar vertebrae of rats.

MECHANISMS OF ACTION

~ 0 ~ Bisphosphonste Action on the Osteoblast is not Mediated Through Changes in Intracellular Cytosolic Calcium

V.N. Antic, V. von Tscharner*, R. Felix, H. Fleisch. Institute of Pathophysiology, University of Berne, Berne, Switzerland," * Theodor Kocher /nstitute, University of Berne, Berne, Switzerland

The mechanism by which bisphosphonates inhibit bone resorption is still not clear. Recently we have shown that the inhibitory action of bisphosphonates on osteoclasts is mediated, at least in part. through the osteoblast (Sahni et al., J. Clin. Invest. 1993; 91:2004-2011). Since this effect occurs after a short incubation t ime (5 min) at concentrations of bisphosphonates as low as 10 -11 M, it may be due to a specific binding of the bisphosphonate to the cell membrane with subsequent activation of intracellular signalling systems.

We have studied the possible effects of bisphosphonates on free cytosolic calcium concentration employing the clonal osteoblastic cells CRP 10.30. The concentration of free cytosolic calcium was measured using the calcium sensitive f luorescent probe, Fura 2. The results show that different bisphosphonates tested at different concentrations (BM 21.0955 10 7-10 5 M, alendronate 10-7-10 5 M and clodronate 10 4 M) did not cause any change in intracellular calcium concentration when added to the cell suspension. In addition we studied the influence of bisphosphonates on the rise of intracellular calcium after addition of ATFt When added 2 minutes prior to ATP (10 -4 M) addition, bisphosphonates did not influence the subsequent response to ATI~ Longer periods of cell incubation (5 min, 30 min and 24 hours) in the presence of bisphosphonates before adding ATP likewise failed to produce any measurable effect.

We conclude that under the conditions applied, bisphosphonates do not have any effect on the intracellular cytosolic calcium level in osteoblastic CRP 10.30 cells, nor do they influence the rise in intracellular calcium induced by ATF~

~ - ~ Wall Thickness and Resorption Depth in Cancellous Bone of Ovariectomized (OVX) Baboons Treated with Alendronate

R. Balena, A. Markatos, M. Yamamoto. G.A. Rodan. Merck Research Laboratories, West Point, PA 19486 USA

Skeletal balance depends (1) on the rate of remodeling activation and (2) on the BMU (basic multicellular unit) bone balance, which is the difference between the amount of bone formed and the amount of bone resorbed in individual remodeling units. We have shown that alendronate (ALN) inhibits tissue level bone turnover, estimated by the extent of osteoid surface and mineralizing surface (Balena. Toolan et al, J. Clin. Invest., 1993). The purpose of this study was to analyze the effects of OVX and 2-year ALN treatment (0.0, O.O5, 0.25 mg/kg i.v. x 2/too) on BMU balance in cancellous bone of the L5 vertebra. Bone balance was calculated by subtracting final resorption depth (R.De, /zm), defined by the presence of osteoid in the resorption cavity, f rom wall thickness (W.Th, /zm). In addition, we estimated the mean depth of erosion lacunae, with or without osteoclasts, f rom the surface area and reconstructed length of the erosion cavity using the rectangular model. Mean W.Th was reduced in untreated OVX baboons by 14% relative to NON-OVX. ALN given at 0.05 and 0.25 mg/kg increased W. Th by 21% and 37% (p < 0.05). respectively, relative to untreated OVX baboons. There were no significant differences in final R.De among the various groups. However, the mean depth of erosion lacunae was significantly greater in OVX (by ~30%) and was maintained at

NON-OVX levels by both doses of ALN. The results f rom this study indicate that (1) OVX caused highly significant negative B M U bone balance. (2) ALN increased W.Th and reversed the negative balance. (3) ALN also decreased the mean depth of resorption lacunae. We conclude that ALN induces a positive skeletal balance in OVX baboons both by reducing remodeling activation and by producing a positive BMU bone balance.

•2] Bisphosphonates Inhibit Interferon Alpha Production in Human Monocytes with the Same Potency Order as In Bone Resorption Assay

M. Bevilacqua, G. Baldi, T. Vago, L. Castelli, G. Norbiato. Servizio di Endocrinologia, Ospedale L. Sacco (Vtalba), Via GB Grass/74, 20157 Milano, Italy

Bisphosphonates (BP) have various biochemical effects on mammalian cells, however the potency ratio of BP in in vitro resorption assay (etidronate < clodronate < alendronate < aminohexane BP) has not been mirrored by any biochemical assay. Human monocytes produce Interferon alpha (INFalpha), a cytokine with anti-viral and anti-neoplastic properties, Interleukin 1 beta (ILl beta) and Tumor Necrosis Factor alpha (TNFalpha). Human monocytes were isolated by normal donors burry- coats by discontinuous Percoll gradient and were 94% pure and 90% viable by Trypan blue exclusion. Monocytes (3 x 106 cells/ml) were incubated with poly-l-poly-C (200 ug/ml) for INFalpha or LPS (1 ug/ml) for ILlbeta and TNFalpha stimulation for about 20 hours and Cytokines were measured in the supernatant medium by radioimmunoassay (Medgenix). BP inhibited the production of INFalpha with the fol lowing potency ratio: Etidronate (about 25%) > clodronate (35%) > alendronate (38%) > aminohexane (50%). Within single BP (0.01-10 uM) Interferon alpha production was dose dependently inhibited. BP did not affect TNFalpha and ILlbeta production and did not affect the viability of the cells. As positive controls we evaluated glucoeorticoids: dexamethasone (0.01-1 uM) inhibited dose-dependently the production of INFalpha its effect being counteracted by mifepristone (1 uM), a specific glucocorticoid antagonist. Inhibitors of prostaglandin synthetase did not inhibit Interferon alpha. Inhibition of Interferon alpha production in human monocytes closely mirrors the ability of BP to affect bone resorption, suggesting that their biochemical effect is similar in monocytes and in osteoclasts.

~-3-~ Quaternary Nitrogen-Containing Derivatives of Risedronste are Potent as Inhibitors of Bone Resorption and as Inhibitors of Growth of Dictyostelium Amoebae

R.J. Brown, M.J. Rogers, J. Carran, G.M. Blackburn, D.J. Watts, R.G.G. Russell, S.M. Dansereau. EH. Ebetino. Dept. Molecular Biology and Biotechnology, University of Sheffield, S 10 2UH, UK; Procter and Gamble Pharmaceuticals Inc,, Cincinnati, Ohio, USA

Studies on the structure-activity relationships of bisphosphonates (BPs) revealed that BPs with alkyl side chains that contain a primary amino group (eg Pamidronate), are more potent inhibitors of osteoclast-mediated bone resorption than BPs with short side chains such as Clodronate. BPs that contain a tertiary nitrogen group (eg DimethylAPD and Risedronate) are amongst the most potent anti-resorptive BPs. These primary and tertiary nitrogen-containing BPs are also potent inhibitors of growth of amoebae of the slime mould Dictyostelium disco/deum. Furthermore. since the order of potency of a wide range of BPs as inhibitors of growth of Dictyostelium closely matches the order of potency of the BPs as anti-resorptive drugs, it appears that BPs have targets which are common to both Dictyostelium amoebae and to osteoclasts,

We have recently found that derivatives of Risedronate. synthesised by alkylating the tertiary nitrogen group in the pyridine ring to form a quaternary nitrogen group, are also potent anti-resorptive agents. We have therefore examined the potency of these compounds as inhibitors of growth of Dictyostelium since, if the targets for BPs are similar in osteoclasts and in Oictyostelium, it would be expected that these novel BPs would also be potent inhibitors of Dictyostelium growth.

The quaternary derivatives of Risedronate are, indeed, potent inhibitors of growth of Dictvostelium. The heptyl derivative was even more potent than Risedronate itself (IC50 - 6 /zM compared wi th 13 /zM for Risedronate). However, the methyl and ethyl derivatives were less potent

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(IC50 21 /~M and 28/~M respectively). The derivative with a mercaptoethyl group was slightly more potent (IC50 - 9 /~M) than .Risedronate. The tetra-isopropyl ester of the ethyl derivative of Risedronate is highly soluble and could therefore also be tested for its ability to inhibit Dictyostelium growth. It proved to have no activity at concentrations up to 500/~M.

Quaternary nitrogen-containing derivatives of Risedronate therefore appear to be a new class of bioactive BPs, the potency of which is dependent on the structure of the group attached to the pyridyl nitrogen. Furthermore, these results again support the v iew that the targets for potent BPs in Dictyostelium are similar to the targets for these BPs in osteoclasts.

[ - ~ Utilization of Bisphosphonete Mechanistic Hypotheses in the Design of Potent New Antiresorptive Agents: Pyrldlnium Alkyl Biephosphonetee

EH. Ebetino. S.M. Dansereau, J.E. McOsker, M.D. Francis. Procterand Gamble Pharmaceuticals, Cincinnati, Ohio

The work of several laboratories has now demonstrated the importance of basic nitrogen moieties in the design of potent antiresorptive agents in the phosphonate series. For example, we have studied the pyridyl class of bisphosphonates in particular depth. Through our successful design of bone active conformationaUy rigid pyrindines (Phosphorus Sulfur and Silicon, 76:151 (1993)), a preferred geometry of the nitrogen function relative to the phosphonate residues is likely. Coupled with growing biochemical evidence, these findings suggest the bisphosphonate antire-sorptive mechanism involves specific stereochemical recognition events.

If such a hypothesis is valid it would be possible that these nitrogen moieties interact in an electrostatically positive form with electrostatically negative residues such as carboxylates at putative binding sites. In order to examine this hypothesis further, we designed several pyridinium analogues with non-ambiguous electrostatically positive nitrogen moieties to optimize this potential interaction. We were gratified to discover that this drug design rationale led to several new highly potent methyl pyridinium bisphosphonates such as NE-10244 and NE-10446. By comparison of these methyl pyridinium analogues to NE-58095 and NE58043 respectively, slight potency increases were observed. Also, a surprisingly good structure-activity correlation to the analogous non-quaternary BP series was noted. For example, the quat NE-10335 and its parent NE-10051 are both relatively non-potent analogues. Thus, preliminary evidence suggests this has been a successful approach towards increasing activity.

:?.o.o ,9.0. ,~.o. (L 4)--- C~C- OH "~ "s-o. ~ , - c - - ~ - .

R I0"0 R 0 0

NE-58095 R - H+ NE-58043 R - H + NE-58051 R = H + NE-10244 R = CH~I - NE-10446 R = CH~I - NE-10335 R= CH~I-

[ - ~ The Interaction of High and Low Bone Affinity Blsphoephonate Analogues with Bone Minerals

Arman Ebrahimpour, A.M. Ruble, F.H. Ebetino, S.M. Dansereau. Procter and Gamble Pharmaceuticals, Inc., Cincinnati, Ohio, 45202, USA

Many bone antiresorptive bisphosphonates (BPs) are known to be capable of improving abnormal bone metabol ism in preclinical models. The application of BPs in ossification and calcification pathologies is partly due to the BPs' physicochemical properties. These properties are to a large extent due to the bone affinity of these compounds which results in targeting of the drug to the sites of action on the bone surfaces. However, it may be possible to produce similar bone mineral hydroxyapatite (HAP) dissolution and growth inhibitions using BP analogues of low bone affinity compared with the high bone affinity BPs. An added advantage of low affinity BP analogues compared with the high affinity BPs is the ability to more easily modulate the on/off processes of the former compounds f rom bone mineral surfaces. In the present in vitro physicochemical investigations, the Langmuir adsorption isotherm model, together with the constant composit ion mineral growth and dissolution experiments were used in order to study the bone affinity and the efficacy of BP

analogues in inhibiting the bone mineral HAP growth and dissolution. NE-58095 (risedronate), NE-10864, and NE-1OS01 were among some of the compounds that we have studied. Our results show that using either the BP "bone hook" or the geminal carbon substituent, ie. the "bioactive moiety", we can vary the bone affinity of the BP analogues. The bone affinity of NE-58095. NE-10501, and NE-10864 decreased, respectively, as the "bioactive moiety" of NE-58095 was restricted (ie. NE-10501 ). or the hydroxyl group on one NE-58095 phosphonate group was replaced with the methyl group (ie. NE-10864). These in vitro f indings also corroborated with other studies showing the inhibition of bone resorption activity of osteoclasts. Furthermore, although the HAP affinities for both NE-10864 and NE-10501 decreased compared with NE-58095, NE-10501 and NE- 58095 were similar in inhibiting the in vitro HAP dissolution and growth rates, while phosphonoalkylphosphinates (eg. NE-10864) generally do not show the same in vitro efficacy in inhibiting these rates. Thus, the nature of the substituent groups and the two phosphonate groups on the geminal carbon of BP analogues effect the inhibition of bone mineral dissolution and growth rates as well as their bone affinity but these two processes do not necessarily fo l low the same trend.

: - .F o-

re . ,o,-o.

NE-58095 NE-10864 NE-10501

~ - ~ Effects of Bisphosphonetes on Cell Proliferation, Differentiation end Cytokines Production in Rat end Human Calvaria Osteoblastic Cells

D. Godet, M. Hott, A.M. Graulet, J. Gu6ris, RJ. Marie. INSERM Unit~ 349, Hopital Lariboisiere, 6, rue Guy-latin, 75010 Paris, France

It has been proposed that bisphosphonates (BP) may inhibit osteoclast activity in part through effects on osteoblasts. To determine the mechanisms of action of BP on osteoblasts, we have compared the effects of Etidronate (Eti) and Tiludronate (Tilu, Sanofi) on parameters of cell proliferation and differentiation, and on cytokines production in osteoblastic (Ob) cells. In newborn rat calvaria Ob cells, Eti (1.5-150 ~,M; 24-96 hrs) had no effect on DNA synthesis, whereas Tilu inhibited cell growth at high concentration (150/~M, 96 hrs). In these rat Ob cells, Eti and Tilu did not affect alkaline phosphatase activity (ALP) or osteocalcin (OC) production, at any concentration or t ime point tested, even in presence of 1,25(OH)2 D3 (10 nM). In Ob cells derived from neonatal human skull, Eti and Tilu (1.5-150 ~M, 96 hrs) had no effect on DNA synthesis in preconfluent cells. However, Eti and Tilu (150/~M, 96 hrs) inhibited DNA synthesis in human Ob cells treated at the onset of culture. At high concentration (150 #M, 96 hrs), Tilu inhibited ALP, whereas Eti had no effect. OC production by these cells was not affected by Eti and Tilu. The effects of Eti and Tilu on Interleukin 6 (IL6), Interleukin 1 (ILl) and Tumor Necrosis Factor c~ (TNF~) production by human calvaria Ob cells were determined by immunoradiometric assays (IRMA). The productions of IL6, ILl and TNF(x expressed in ng/ml, or corrected for cell content, were not significantly affected by Eti and Tilu at any concentration (1.5-150/LM) or duration of treatment (48-96 hrs). The results indicate that: 1 ) Tilu inhibits parameters of osteoblast cell growth and differentiation at higher concentrations than those required to inhibit bone resorption in vitro; 2) In conditions where there is no cell contact/exchange with osteoclasts, these BPs have no effect on IL6, ILl and TNF~ production by human osteoblastic cells.

l • T h e Effect of Etidronate and Organophosphate Insectlclde on Bone Cell Functlon

S.J. Hedges, S. Meghji, S. Petursson, B. Henderson, C. Hopper, M. Harris. Dept, of Maxillofacial Surgery, Institute of Dental Surgery, 256 Gray's Inn Road, London WCIX SLD, UK

Bisphosphonates are emerging as powerful tools in the regulation of metabolic bone diseases. Developed countries are at the same t ime employing increasing amounts of organophosphate insecticides in their agri- cultural industries. Bisphosphonates and organophosphate insecticides

$66 Second Workshop on Bisphosphonates

are structurally related. We have found that paraoxon (PO), an organophosphate insecticide, has potent activities on esteoclast and osteoblast metabolism.

We have investigated the interaction between etidronate and PO on 1,25(OH) 2 vitamin D 3 murine calvarial bone resorption and on alkaline phosphatase activity in murine osteoblast-rich cells harvested from neonatal calvaria,

PO alone inhibits 1,25(OH) 2 vitamin D 3 induced calvaria bone resorption (IC50 10 -6 M). PO significantly augmented the inhibition 1,25(OH) 2 vitamin D 3 induced calvaria bone resorption below that observed with PO alone for all concentrations of etidronate used in this study (0.01-10 #g/ml). Alkaline phosphatase activity in osteoblast rich cell culture was slightly decreased in the presence of etidronate in the concentration range 0.01- 10 t~g/ml. The combination of etidronate and PO (2 /~g/ml) interestingly caused a return to control levels of the alkaline phosphatase activities at all concentrations of etidronate except 0.01 /~g/ml.

Etidronate interacts strongly with the organophosphate insecticide paraoxon, effecting osteoclast activity more than osteoblast activity. We observed an apparent protective action of etidronate on osteoblast alkaline phosphatase activity Further research is needed to investigate the interaction of environmental pollutants on the effects of bisphosphonates on skeletal physiology

Etidronate was a generous gift f rom Proctor and Gamble Pharmaceuticals.

~ - ~ T h e Phosphonate Moieties of Bisphosphonates are More than Just s Targeting Function: Evidence from the Study of Bisphosphonate Analogues In Vitro

K.J. Ibbotson, S.M. D'Souza, A. Bayless, A. Ebrahimpour, EH. Ebetino, F.N. Woodiel *, RE. Fall *. LG. Raisz *. Bone Research, Procter [1 Gamble Pharmaceuticals, Miami Valley Laboratories, Cincinnats~ Ohio, USA," * Division of Endocrinology and Metabolism University of Connecticut Health Center Farmington, Connecticut, USA

Bisphosphonates are a class of drugs with high affinity for calcified matrices such as the hydroxyapatite phase of mineralized bone. ]he function of the bisphosphonate moiety in this class of drugs has typically been regarded as a physico-chemical one, that of targeting the drug to a mineralized (hydroxyapatite) surface, the biological activity being ascribed primarily to the various side chains attached to the geminal carbon. In this abstract we present data on three classes of bisphosphonate analogues with different affinities for hydroxyapatite. These analogues are bisphosphinates, phosphono-alkyl phosphinates, and a phosphone carboxylate. Of these analogues the bisphosphinates have the lowest affinity for hydroxyapatite and are completely devoid of antiresorptive activity in the fetal rat long bone assay at concentrations up to 5 mM. The phospheno-alkyl phosphonates (PAPs) and phosphone-carboxylate are derived f rom parent BPs with equivalent anti-resorptive activities, and have e.~senti~lly equivalent affinities for hydroxyapatite {though still less than typical bisphosphonates), and yet show dramatically different antiresorptive effects in vitro. The PAPs were, like the bisphesphinates. devoid of any anti-resorptive activity at concentrations up to 5 raM. The phosphono-carboxylate, however, showed antiresorptive activity equivalent to that of FHBP (etidronate) with an IC50 of around 30-50 izM. Thus two molecules differing in the structure of the phosphonate moiety but with equivalent affinities for hydroxyapatite and with side chains of equivalent biological activities show markedly different antiresorptive effects. These findings provide additional evidence that the phosphonate moiety, beyond determining the affinity of the molecule for mineral surfaces, plays a role in the biological activity of the whole molecule.

[ ~ HEBP (Etidronste) Sporadically Stimulates Expression of Osteopontin by Chondrocytes In Vivo and Results in an Accumulation of this Protein in Cartilage Matrix

M.D. McKee. K. Josephsen, A. Nanci. Universit~ de Montreal, Faculty of Dentistry, Department of Stomatology, Montreal, OC, Canada, H3C 3J7

The bisphesphonate HEBP (1-hydrexyethylidene-l, l-bisphosphonate; etidronate) is known to affect certain osteogenic processes such that, at higher doses, both bone resorption and mineralization are inhibited. In the present study, we have investigated the effects of continuous infusion of HEBP on growth plate chondrocytes and cartilage in the rat tibia, and ex-

amined the distribution of the non-collagenous bone protein osteopontin (OPN) in this tissue, Rats weighing approximately 100 g were injected subcutaneously with 10 mg of HEBP/kg b. wt. fo l lowed immediately by intraperitoneal implantation of mini-osmotic pumps (Alzet) delivering 20 or 40 /~g P of HEBP/hr continuously for 7 or 14 days. Animals were fixed by vascular perfusion with glutaraldehyde and tibiae were dissected, decalcified in EDTA and embedded in LR White for light microscopy (LM), transmission electron microscopy (TEM) and for immunocytochemical analyses. For post-embedding immunocytochemistry, thin sections of the proximal tibial growth plate were incubated with a polyclonal anti-rat OPN antibody fol lowed by protein A-gold complex. As visualized by LM, the thickness of the growth plate increased markedly and was primarily characterized by a general architectural disturbance in the hypertrophic zone and the zone of vascular invasion. At these sites, cartilage matrix surrounding hypertrophic chondrocytes appeared distorted and lost its normal organization as aligned columns of territorial and interterritorial matrix, As viewed by TEM, and after immunoc~oehemical labeling with anti-OPN, the cartilage appeared to be largely unmineralized, despite the presence of numerous small, osteepontin-containing loci of apparent mineralization in the interterritorial matrix of the distorted hypertrophic zone. In this region, strong labeling for OPN was also observed over various elements of the Golgi apparatus in some chondrocytes, a reaction not observed using these same methodologies on tissue f rom normal rats. In v iew of published reports describing the detection of OPN in normal, untreated chondrocytes, the present data suggest that HEBP sporadically up-regulates OPN gene expression in some of these cells, and that this protein is subsequently secreted and accumulates at small calcification loci presumably representing aborted attempts to mineralize the surrounding cartilage matrix.

Supported by MRC of Canada and Caleinfonden.

[ ' ~ YM175 Prevents Loss of Mechanical Competence In Cancellous Bone Through Inhibition of Perforation on Trabeculae

H. Metoie, H. Kinoshita, H. Kanoh, N. Ouehi, T. Abe, T. Nakamura * Yamanouchi Pharmaceutical Co., Ltd., 21 Miyukigaoka, Tsukuba 305, Japan; * University of Occupational and Environmental Health. ~'takyushu 807, Japan

We have found that ovariectomy (OVX) induces reduction of both bone mineral content in lumbar spine and mechanical competence of cancellous bone in a dog. and YM 175, a bisphosphonate, dose-dependently prevents these changes. Then we conducted further investigation of a relationship between mechanical and morphological properties.

OVXed and sham-operated beagles (21-month-old) were fed a commercial (Ca; >4 g/day) or a Ca-restricted (420 mg/day) diet. and YM175 (0, 0.01, 0.1 and 1 mg/kg po respectively) was given 5 t imes a week for 18 months. After the end of the dosing, lumbar vertebrae (L2-4) were obtained. A cancellous core specimens were excised f rom vertebral bodies of L3 and applied to a compression test. The specimens were embedded in methylmethacwlate after the test and cross sections of the bones were evaluated.

OVXed animals which were fed the Ca-restricted diet (OVX control) showed significantly lower values of trabecular bone area and trabecular thickness, and higher value of trabecular separation in the cancellous bone than those in the sham-operated animals fed the commercial cliet (normal control). It was suggested that OVX induced perforation of trabeculae since degree of decrease in trabecular continuity was about two t imes more greater than that in trabecular thickness in OVX control. This corresponded to the result that ultimate strength both per trabeculsr bone area and per unit trabecula were reduced by OVX + Ca-restriction. YM175 prevented all of these changes in a dose-dependent manner and its effects were significant f rom the dose of 0.1 mg/kg. The values of the bone from the animals treated with 1 mg/kg of YM175 exhibited about the same as those in normal control

These results suggest that OVX+Ca-restriction reduces mechanical competence of cancellous bone through perforation of trabeculae and YM 175 preserves architecture and mechanical competence of cancellous bone by inhibiting the perforation.

P r e c l i n i c a l s tud ie s - - M e c h a n i s m s o f a c t i o n

F3-q Tiludronate Affects Polarized Osteoclasts but not Unpolarized Ones

H. Murakami *, N. Takahashi **, N. Udagawa **, S. Tanaka **, I. Nakamura **, D. Zhang **, T. Suda **. ~MeijiSeika Pharmaceutical Research Center, 760 Morooka-cho, Kouhoku-ku, Yokohama 233; **Department of Biochemistr)~, School of Dentistry, Showa Universi~ 1-5-8 Hatanoda#; Shinagawa-ku, Tol(yo 142, Japan

We previously reported that ti ludronate [(4-chlorophenyl)thiomethylene bisphosphonate, provided by SANOFI Research Center, France] showed no inhibitory effect on osteoclast-like multinucleated cell (MNC) formation induced by l(~,25(OH)2D 3 in co-cultures of mouse osteoblastic cells and bone marrow cells (Osteoporosis 1993. Hong Kong. abstracts p. 141). The MNCs obtained f rom co-cultures treated with tiludronate (10 -4 M) together with 1e,25(OH)2D 3 (10 -8 M) on collagen gel-coated plates showed potent activity to form resorption pits similar to that of MNCs formed in the absence of tiludronate. However, pit formation by MNCs was markedly inhibited when tiludronate (10 4 M) was directly added to the pit formation assay. In the present study, we examined whether the inhibitory effect of ti ludronate depends on the polarized structure of osteoclasts. MNCs obtained from co-cultures were put on either dentine slices or collagen gel-coated plates. MNCs plated on dentine slices formed a ringed structure (clear zone) of F-actin containing numerous dots (podosomes) detected by Rhodamine-conjugated Phalloidin, but MNCs plated on collagen gel-coated dishes failed to form such a ringed structure. This indicates that the former MNCs are polarized, but the latter are not. Treatment with ti ludronate of MNCs plated on dentine slices resulted in a marked disruption of podosomes, which correlated well with a decreased activity to form resorbing pits. No morphological changes were detected in MNCs plated on collagen gel-coated dishes in the presence of tiludronate. In contrast, calcitonin caused marked morphological changes with redistribution of F-actin of MNCs plated not only on collagen gel-coated plates but also on dentine slices. Neither calcitonin nor ti ludronate caused structural changes in F actin stress fibers in osteoblastic cells cultured on both collagen-gel coated dishes and dentine slices. These results indicate that ti ludronate affects only polarized osteoclasts attached to the calcified tissue. Bisphosphonates may be specific inhibitors for functioning osteoclasts.

[ • T h e Inhibitory Effect of Alendronate and Cslcitonin on Bone Resorption In Rats Display Different Patterns and are Additive

R.C. M,",hlbauer, H. Fleisch. Dept. of Pathophysiology, University of Berne, Murtenstrasse 35, 3010 Berne, Switzerland

In the past, we have developed a technique allowing continuous monitoring of bone resorption. It is based on the urinary excretion of [3H] tetracycline ([3H]Te) f rom rats prelabeled with [3H]Tc f rom birth. Using urine collected at 6 hourly intervals, we have previously demonstrated the existance of a marked diurnal rhythm of bone resorption, the peak of resorption occurring 6 hours after the daily food administration. A single administration of 0.1 mg P/kg of alendronate was found to persistently reduce the overall [3H]Tc excretion by about 70%, starting 3 days after bisphosphonate administration. We now report that under bisphosphonates the diurnal rhythm of bone resorption is preserved at a proportional level, In contrast, administration of calcitonin at doses ranging f rom 1.25 to 2.5 IU/kg inhibits almost exclusively the food induced peak of bone resorption, and this in a dose-dependent manner. This effect occurs within hours and is fully reversible within 24 h after the last administration. In rats pretreated with alendronate, calcitonin not only inhibits but abolishes this peak resulting in a lower daily bone resorption than either of the two inhibitors given individually. These results indicate fundamental differences in the mechanism of action of the two inhibitors of bone resorption in vivo. While calcitonin acts directly on the osteoclasts, the bisphosphonates may act through the recently described indirect effect through the osteoblasts. These results also suggest that in man the administration of both inhibitors may be useful in some special cases.

$67

F33--1 Pamidronate but not Clodronate Induces Transient Lymphopenie and Enhances Expression of CD69, an Early Lymphocyte Activation Marker

M. Pecherstorfer, R. Jilch, A. Sauty, I. ZimmeFRoth, C. Cro~,, H. Ludwig, D. Thi6baud, Wilhelminenspital and Lainz Hospital Vienna, Austria and CHUV, Lausanne, Switzerland

The bisphosphonates pamidronate and clodronate are effective in the treatment of enhanced bone resorption in malignant and benign diseases. In contrast to clodronate, the administration of pamidronate can induce an acute phase reaction with fever and lymphopenia. We evaluated the different immunological properties of these substances: 27 normocalcemic and 3 hypercalcemic patients (22 female, 8 male; median age 58 years) with metastatic bone disease received intravenous treatment with 60 mg pamidronate, infused over 3 hours. 10 normocalcemic cancer patients with neoplastic bone involvement (7 females, 3 males; median age 68.5) served as the control group. They were treated with 1500 mg clodronete, also administered over 3 hours. Before the infusion and after 10, 24.48 and 72 hours, serum levels of calcium (Ca) and of the cytokines tumor necrosis factor r (TNF~), interleukin I # and interleukin 6 (IL6) were measured. The percentage of lymphocytes expressing markers of early (CD-69) and late activation (HLA DR) was determined by flowcytometry. In the pamidronate group, we observed a significant increase in body temperature (0 versus 10 hours: p < 0.002) and a significant decrease in the lymphocyte count (0 versus 24, 48, 72 hours: p < 0.0005). Moreover. a significant increase in the number of CD-69 expressing T-cells (p < 0.0001 over the whole observation per iod)was observed. Ca decreased (0 versus 72 hours: p < 0.0001). In the pamidronate group, there were also significant increases in serum levels of TNF~x and IL6, which will be reported elsewhere in this symposium. In contrast, clodronate treatment was associated with changes neither in cytokine concentrations nor in the number of activated T cells. We conclude that the aminobisphosphonate pamidronate but not clodronate leads to an activation of T-cells with subsequent release of the cytokines TNF(~ and IL6.

~ - ] T h e Effect of Liposome-Encapsulated and Free Bisphosphonates on IL-1.8, 11.-6 and TNF~x Secretion from Macrophages

N. Pennanen, T. Nissinen, S. Lapinjoki, A. Urtti, J. M~nkk6nen. University of Kuopio, Department of Pharmaceutical Technology and A.l. Virtanen Institute, RO. Box 1627, FIN-70211 Kuopio, Finland

Liposome-encapsulated bisphosphonates are known to affect macrophages in vivo (van Rooijen and Kors, Calcif. Tissue Int. 45, 163, 1989). In vitro, the growth of macrophage-like RAW 264 cells is inhib ited by bisphosphonates, and liposome-encapsulated drugs are 20-1000 times more potent than free drugs (J. Mbnkk~nen et al. unpublished). These findings suggest that liposomal bisphosphonates are macrophage suppressive agents and, thus, useful in the treatment of autoimmune diseases. We have evaluated the effect of three bisphosphonates on the IL-1/~, IL-6 and TNF(~ generation f rom RAW 264 cells. The cells were incubated overnight with liposome-encapsulated and free drugs and subsequently induced to produce cytokines with 10 /~g/ml of LPS. The cytokine concentrations in the culture supernatants were determined by dissociation-enhanced lanthanide f luoroimmunoassay (DELFIA). As a free drug, pamidronate was the most potent inhibitor of the cytokine production from the cells, while etidronate was the least potent. The IC50 values of free pamidronate were 100 /~M, 70 /~M and 34 /~M for the inhibition of IL-1/L IL-6 and TNF(~, respectively, when for clodronate these values were 825/~M, 155/~M and 580 #,M and for etidronate more than 1000 /~M. When the drugs were encapsulated in DSPG-liposomes (DSPG:cholesterol 67:33), the inhibitory effect of both t:lodronate and etidronate was enhanced by 10-20 times. On the contrary, the potency of pamidronate was not increased by liposome-encapsulation. Liposomal clodronate was the most potent inhibitor of the cytokine production, and the IC50 were 40 /~M, 12 /~M and 46 /~M for IL-1/~, IL-6 and TNF=, respectively. Unloaded DSPG-liposomes did not inhibit cytokine production at the concentrations used for the drug delivery. The inhibition of cytokines by the bisphosphonates was not attributed to cytotoxic effect on maerophages, because the viability of the cells was not affected by the drugs. This study proves that bisphosphonates have non-cytotoxic cytokine-inhibitory effect on macrophages, which might be beneficial in autoimmune diseases.


Acknowledgements: This study was financially supported by Technical Development Centre of Finland (TEKES).

• Studies on the Cellular Uptake of Bisphosphonates by Amoebae of the Slime Mould Dictyostelium Discoideum

X. Ji, M.J. Rogers, X. Xiong, D.J. Watts, R.G.G. Russell, G.M. Blackburn, M.R Wilfiamson, E H. Ebetino. Dept. of Molecular Biology and Biotechnology, University of Sheffield, $10 2UH, UK

We have been investigating the mechanism by which bisphosphonates (BPs) inhibit axenic growth of Dictyostelium discoideum amoebae since, for a wide range of BPs, the order of potency as growth inhibitors of Dictyostelium closely matches the order of potency as inhibitors of bone resorption. Since the axenic lAx-2) strain of Dictyostelium takes up nutrients by fluid-phase pinocytosis, it seemed possible that BPs might also be taken up f rom the growth medium by the same process. Any ability of the amoebae to take up BPs could indicate that BPs have an intracellular site of action.

31p_NMR analysis indicated that BPs were present in cell extracts prepared f rom amoebae that had been incubated with BP for two hours then washed thoroughly to remove extracellular BR [3H]-methylene bisphosphonate (MBP) was also internalized by Dictyostelium amoebae, with an initial influx rate similar to that of FITC-dextran, a marker of fluid-phase pinocytosis. The rate and extent of uptake of [3H]-MBP was higher when amoebae were incubated in nutrient medium than in buffer, an effect also observed with FITC-dextran and other markers of pinocytosis. Furthermore, the uptake of BPs and [3H]-MBP was inhibited when amoebae were incubated in the presence of inhibitors of pinocytosis such as caffeine and orthovanadate, or at 4~ The uptake of [3H]-MBP by Dictyostelium was not affected by the presence of other bisphosphonates such as hydroxymethylene-, dichloromethylene- or 4-amino-l-hydroxybutylidene-bisphosphonate.

The techniques that we have used to measure uptake of BPs do not distinguish between BP that is contained in intracellular vacuoles and BP that is in the cytoplasm. However, since several simple BPs, including MBP and dichloromethylenebisphosphonate, are metabolized by Dictyostelium into methylene-containing analogues of ATP, possibly by a back reaction catalyzed by cytosolic aminoacyl-tRNA synthetase enzymes, this appears to be evidence that at least these BPs are taken up and do enter the cytoplasm in Dictyostelium.

• 6 ] A Mutant Strain of the Slime Mould Dictyostelium Discoideum is Resistant to the Cellular Effects of Bisphosphonates

M.J. Rogers, D.J. Watts, R.G.G. Russell, EH. Ebetino. Dept. of Molecular Biology and Biotechnology, University of Sheffield, $10 2UH, UK

Bisphosphonates appear to inhibit bone resorption by having deleterious effects on osteoclasts that are resorbing BP-coated bone. We have been investigating the mechanism by which BPs have growth inhibitory and cytotoxic effects on Dictyostelium discoideum amoebae since it appears that the targets for BPs in Dictyostelium are similar to those in osteoclasts. In an attempt to identify such a target we have isolated a mutant strain of Dictyosteliurn by growing wild-type Ax-2 amoebae in a concentration of AHBuBP (50-100 /~M) which did not al low growth but which did not cause the cells to lyse immediately. After 3 months of continuous culture, amoebae began to grow in the presence of AHBuBP presumably as a result of a spontaneous mutation which conferred resistance to AHBuBR These amoebae eventually grew in the presence of 100 /~M AHBuBP at the same rate as that of the wild-type amoebae in the absence of BR A stock culture was then grown up f rom a single resistant clone (strain MRI02). The resistance of MR102 to AHBuBP and other BPs was examined by growing the amoebae in various concentrations of BPs and determining the IC50. Amoebae of the MR102 strain were found to be more than 15-fold resistant to AHBuBP, about Z~fold resistant to four other potent BPs, but less than 2-fold resistant, if at all, to MBP, HMBP and CI2MB~

These results suggest that, in Dictyostelium, the potent BPs act on the same target, whereas MBP, HMBP and CI2MBP may act by a different mechanism. In agreement with this, we have previously shown that MBR HMBP and CI2MBP are metabolised by Dictyostelium into an analogue

of ATP, whereas the potent BPs are not. We are currently investigating the basis of the resistance of MR102. It does not appear to be due to a decreased ability of MR102 amoebae to internalise BPs, a process which appears to be essential for the action of BPs on Dictyostelium.

8P IC50 for Ax 2 strain IC50 for mutant MR102 strain

MBP 2 mM 2 mM HMBP 600/~M 600 #M CI2MBP 500/tM 850 #M AHPrBP 180/zM 800 p,M AHBuBP 30/~M 500/~M 3-PHEBP 13/~M 90 #M MePentAPD 12 u.M 50 u.M

[-3--7--] Bisphosphonates Appear to be Metabolised by Aminoacyl-tRNA Synthetase Enzymes in Extracts of Dictyostelium Amoebae and Extracts of Human Cells

M.J. Rogers, R.J Brown, M Hodkin, D.J. Watts. R.G.G. Russell, G.M. Blackburn, EH. Ebetino. Dept. of Molecular Biology and Biotechnology, University of Sheffield, $10 2UH, UK

Bisphosphonates (BPs) are used to inhibit osteoclast-mediated bone resorption in the treatment of a number of disorders of mineral metabolism. The mechanisms of action of these compounds remain unclear, although there is some evidence that some BPs (e.g. dichloromethylenebisphosphonate) may be cytotoxic fol lowing cellular uptake. We have found that BPs with simple side chains (e.g. methylene-, dichloromethylene- and hydroxymethylene-bisphosphonate) are incorporated into AppCp analogues of ATP by amoebae of the slime mould Dictyosteliurn discoideum and by cell-free extracts of mammalian cells. Since the purified lysyl tRNA synthetase enzyme from E. coil has been shown to catalyse incorporation of methylenebisphosphonate into an AppCp nucleotide with concomitant inhibition of synthesis of lysyl-tRNA, we examined whether the incorporation of other BPs into AppCp nucleotides is also catalysed by aminoacyl-tRNA synthetases.

The inhibition by BPs of 20 aminoacyl tRNA synthetase enzymes in a cell-free extract of Dictyostel/um amoebae was assayed by measuring the amount of radiolabelled amino acid that was incorporated into aminoacyl-tRNA in the presence and absence of 500/~M BP. Only seven aminoacyl-tRNA synthetases (specific for the amino acids Asp, Asn, Gly, His, Lys, Phe and Ser) appeared to be significantly affected by BPs. Furthermore, only the BPs that we have previously shown to be metabolised into AppCp nucleotides were able to inhibit any of the seven aminoacyl-tRNA synthetases Enzyme activity was barely affected by any of the potent BPs that are not metabolised.

Cell-free extracts of HL-60 and MG-63 cells were then used as a source of human aminoacyl tRNA synthetases to determine whether BPs could inhibit the same seven synthetases that are inhibited by BPs in Dictyostelium. Once again, only the BPs that are metabolised into AppCp nucleotides were able to inhibit at least one of the seven synthetase enzymes from HL-60 or MG-63 cells. Thus, there was a perfect correlation between the BPs inhibiting the aminoacyl-tRNA synthetases and the BPs incorporated into AppCp nucleotides. This would indicate that incorporation of BPs is probably catalysed by aminoacyl-tRNA synthetases. Since these enzymes are ubiquitous and essential for protein synthesis. these results indicate a possible mechanism by which simple RPs like CI2MBP are cytotoxic to cells such as osteoclasts which are capable of intemalising BPs.

[ - ~ Simple Bisphosphonates are Metabolised into Analogues of ATP by Dictyostelium Amoebae and by Extracts of Human Cells

M.J. Rogers, V. Hodkin, R.J. Brown, M.R Williamson. X. Ji, D.J. Watts, R.G.G. Russell, G.M. Blackburn, EH. Ebetino. Dept. of Molecular Biology and Biotechnology, University of Sheffield, $10 2UH, UK

Bisphosphonates (BPs) are generally considered to be metabolically inert P-C-P analogues of pyrophosphate that do not take part in any cellular reactions. We have been investigating the mechanisms by which BPs are growth inhibitory and cytotoxic to amoebae of the sl ime mould Dictyostelium discoideum, since it appears that Dictyosteliurn may contain targets for BPs that are similar to those in osteoclasts. Following an

Prec l in ica l s tud ies - - M e c h a n i s m s o f act ion S69

earlier report that methylenebisphosphonate can be metabolised by Dictyostelium into methylene-containing analogues of ATP (AppCH2P) and Ap4 A (AppCH2ppA), we have found that Diclyostelium is capable of metabolising certain other BPs into analogues of A]-P and Ap4 A,

Metabolites of BPs were identified by a combination of 31p_NMR, 19F-NMR and ion-exchange FPLC analysis of cell extracts prepared f rom amoebae that had been incubated for 2 h with 5 mM BI~ After screening a wide range of BPs for incorporation into nucleotides, it became clear that only the BPs of low potency, with simple side chains (eg -H, -F, -CI, -OH, -CH 3, =CH2), were metabolised by Dictyostelium into the corresponding AppCp analogue of ATP (eg AppCHFp, AppCCl2p, AppCHOHp, AppCHCH3P). Only methylene-, difluoromethylene~ and possibly hydroxymethylene-bisphosphonate were incorporated into the corresponding AppCppA analogue of AP4A. The more potent BPs, with larger side chains, such as the aminohydroxyalkylbisphosphonates or heterocycle-containing BPs were not metabolised by Dictyostelium. Sim- ilar results were obtained by incubating cell extracts of Dictyostelium (rather than intact cells) with BPs, indicating that lack of incorporation was not due to lack of cellular uptake.

Since BPs affect mammalian cells such as osteoclasts and macrophages, we are currently examining whether the BPs that are metabolised by Dictyostelium are also metabolised by mammalian cells. Intact promyelocytic HL-6O cells and MG-63 osteosarcoma cells do not metabolise any BPs, even after two weeks of culture with 500 /~M BP However, extracts prepared f rom these cells are capable of incorporating into nucleotides the same BPs that are metabolised by Dictyostelium. Furthermore, the BPs that are not metabolised by Dictyostelium are not metabolised by extracts of HL-60 or MG-63 cells. These results indicate that some BPs are not necessarily metabolically inert analogues of pyrophosphate. Human cells appear capable of incorporating some BPs, including Clodronate, into non-hydrolysable analogues of ATR The cellular effects of these BPs may be due to their metabolism but will be limited to those cells, such as osteoclasts, able to internalise BPs.

[ - ~ Alterations to the Phosphonete Groups of Bisphoephonatea Decrease their Potency as Inhibitors of Bone Resorption and as Inhibitors of Dictyostelium Growth

M.J. Rogers, X. Xiong, R.J. Brown, D.J. Watts, R.G.G. Russell, A3Z Bayless, A. Ebrahimpour, Ell. Ebetino. Dept. of Molecular Biology and Biotechnology, University of Sheffield, $10 2UH, UK, Procter ~ Gamble Pharmaceuticals Inc., Cincinnati, USA

The inhibitory effect of bisphosphonates (BPs) on bone resorption in rive appears to be due to a combination of the targeting of BPs to bone mineral and to a subsequent cellular effect on bone-resorbing osteoclasts. The high affinity of BPs for hydroxyapatite is a property of the two phosphonate groups (P-C-P), whi le the cellular effects of BPs appear to be dependent on the structures of the side chains attached to the germinal carbon atom. BPs also have growth-inhibitory and cytotoxic effects on amoebae of the sl ime mould Dictyostelium discoideum, For a wide range of BPs the structure-activity relationship as growth inhibitors closely matches that for inhibition of bone resorption. The cellular targets for BPs therefore appear to be similar in Dictyostelium amoebae and osteoclasts.

Alterations to the phosphonate groups of BPs have a marked effect on their affinity for bone mineral and hence anti-resorptive potency. For example, bisphosphinic acid analogues of BPs have recently been found to be inactive as inhibitors of bone resorption in an in vivo resorption assay, presumably because they have negligible affinity for bone mineral. Hybrid compounds, the phosphonoalkylphosphinates (PAPs). retain some of the affinity of the parent BPs for bone mineral but have somewhat less anti-resorptive potency.

Since the cellular effects of BPs on Dictyostelium amoebae occur in the complete absence of any mineral phase, we examined whether alterations to the phosphonete groups affected potency as growth inhibitors of Dictyostelium. Bisphosphonates and hybrid PAPs derived from a variety of BPs had exactly the same order of potency as inhibitors of Dic~yostelium growth and as inhibitors of bone resorption. Thus, the parent BPs were the most potent growth inhibitors, the hybrid PAPs were much less potent, and the bisphosphonates were barely growth-inhibitory if at all.

These results suggest that the decrease in, or loss of, anti-resorptive potency of the PAPs and bisphosphonates respectively is not due solely to decreased affinity for bone mineral, and imply that the phosphonate

groups as well as the side-chains of BPs play an important role in producing a cellular effect on osteoclasts. This could involve the binding of the phosphonate groups to complementary sites on a target protein such as an essential enzyme.

~ - ~ Structure-Activity Relationships of Bisphosphonates in Dictyoetelium Diecoideum, e Novel Model for Identifying the Cellular Mechanisms of Action of Bisphoephonates

M.J, Rogers, X. Ji, X. Xiong, R.J. Brown, D.J. Watts, R,G.G. Russell, GM, Blackburn, A.M Bayless, S.M. Dansereau, F.H. Ebetino. Dept Molecular Biology and Biotechnology, University of Sheffield, Sl O 2UH, UK; Procter

Gamble Pharmaceuticals Inc., Cincinnati, USA

The relationship between the anti-resorptive potency of bisphosphonates (BPs) and the structure of the two side chains attached to the geminal carbon atom has been the subject of study for over 20 years. Although much progress has been made towards defining structures which have high potency (such as the presence of a basic nitrogen atom at specific positions along an alkyl chain or within a heterocyclic group) the rational design of BPs with maximum potency is difficult since the targets for BPs in osteoclasts have not been identified. This is largely because of the difficulty in isolating sufficient osteoclasts for biochemical studies, and the lack of suitable in vitro models in which the potency of BPs closely matches their potencyin vivo,

BPs are also inhibiters of growth of amoebae of the slime mould Oictyostelium discoideum, At sufficiently high concentrations, BPs are cytotoxic and the amoebae lyse, We have used values of IC50 to rank BPs in order of potency as growth inhibitors and hence examine the structure- activity relationship of BPs in Dietyoste/ium. The order of potency of BPs as growth inhibitors of Dictyostelium closely matches the order of potency of the BPs as inhibitors of bone resorption. Hence, BPs with simple side chains such as CI2MBP and HEBP are poor growth inhibitors. The aminohydroxyalkylBPs are more potent, depending on the length of the alkyl chain, while akylated derivatives of AHPrBP such as Me2AHPrBP and MePentAHPrBP are more potent than AHPrBP itself. BPs having a nitrogen at specific positions within a heterocyclic group (eg CGP42446 and 3- PHEBP) are amongst the most potent growth inhibitors. Furthermore, for pairs of heterocycle-containing BPs that differ only slightly in structure (eg in the length of the carbon 'spacer' chain between the geminal carbon atom and the heterocyclic group, or in the position of a methyl group in the ring) the changes to the structure of the side chain which dramatically increase or decrease anti-resorptive potency also affect growth inhibitory potency similarly, It therefore appears that the side chain structures which define anti-resorptive potency also define potency as growth inhibitors of Dictyostelium. This suggests that the targets for BPs in Dictyostelium may be similar to those in osteoclasts. Since Dictyostelium can be easily grown in large numbers, it may prove to be a novel and useful model with which to identify the cellular targets for BPs.

~ - ~ Serum Levels of TNFa and 11.-6 in Patients Treated by Pamidronate, Clodronate end BM 21.0955

A. Sauty *, M. Pecherstorfer **, P Fioroni *, L. Juillerat *, Pb. Leuenberger *, P Burekhardt *, D. Thi~baud *. "Dpt of Internal Medicine, University Hospital, 1011 CHUV-Laosanne, Switzerland," * *Dpt of Oncolegy, Lainz Hospital, Wolkersbergenstrasse 1, A-1130 Vienna, Austria

Biphosphonates have been shown to induce mild fever in some patients, and haematological and biochemical modifications compatible with an acute phase reaction. We recently described the increase of TNF~ levels in serum of 21 patients, 24 h after an infusion of pamidronate (APD), whereas IL-6 levels showed a trend to increase, and IL-1/~ level were not influenced. Moreover, in an in vitro experiment on whole blood, an increase of TNF~ and IL-6 levels were also shown in response to APD (A. Sauty et el. Calcif. Tissue Int 52; $76, 1993).

In the present study, serum levels of immunoreaetive IL-6 and TNF(x were measured at O, 10, 24, 48 h and 72 h in 9 additional patients treated with 60 mg APD (making a total of 30 patients), 9 patients with elodronate (1500 rag) and 6 patients with BM 21.0955 (2 rag) all given intravenously.

These results confirmed our previous report that, compared to mean basal value (12.9 :L 8.0 [• pg/ml), mean serum level of TNF~ increased


significantly 24 h after APD infusion (25.2 4- 15.6 pg/ml, p < 0.00002) and was still elevated at 48 h (27.0 + 18.0 pg/mf). From 48 to 72 h, serum TNFc~ levels decreased to levels close to pretreatment values. Similarly, after APD treatment, mean IL-6 level was increased at 24 h compared to basal value (respectively 53.5 4- 75.9 and 27.7 -I- 37.4 pg/ml, p < 0.03). However, the range of values was wide. On the opposite, neither clodronate nor BM 21.0955 induced an increase in serum levels of TNF~ or IL 6. Moreover, whi le blood lymphocyte count was significantly reduced 24 to 48 h after treatment with APD, it was increased in patients treated with clodrenate.

In conclusion, APD but not elodronate or BM 21,0955, at doses used for treatment, induced a transient increase in serum TNF~ and IL-6 levels. The source of these proinflammatory cytokines and their clinical role are still unknown.

[ • T h e Effects of Bone-Bound Bisphosphonates on the Resorption Cycle of Isolated Rat Osteoclasts

K, Se]ander, K. V~n~nen+ Department of Anatomy, University of Ou/u, Kajaanintie 52 A, 90220 Oulu, Finland

The function of isolated osteoclasts has been shown to alter between migration and resorption phases in vitro. Cytoskeletal reorganization is needed when the cell enters either one of these steps: formation of sealing zone and ruffled border is essential before osteoclasts can resorb bone. The effects of bone-bound clodronate (CI2MBP), etidronate (EHDP) and pamidronate (APD), on isolated rat osteoclasts were studied in the pit formation assay using immunofluorescence and h~stochemical techniques. Bone-bound bisphosphonates did not interfere with the organization of cytoskeleton in osteoclast during the early moments of the resorption phase. After longer incubation t ime the number of these attachment structures increased strongly on the control slices, but remained low on the bisphosphonate-groups studied. At this t ime the actin and vinculin staining started to exhibit abnormal, more diffuse and blurred appearance. The number of osteoclasts diminished from bisphosphonate- covered bone slices as a function of t ime, the effect being most impressive in the CI2MBP group. Most osteoclasts that remained attached to the APD-, CI2MBP- or EHDP coated bone slices exhibited also remarkable cytoplasmic retractions, as detected by TRAP-staining. The other cell types present showed no signs of cellular damage. Total area of resorption remained at significantly lower level on the bisphosphonate-covered bone slices and this was due to decrease in both the number and the size of individual resorption pits. Large, united resorption lacunae, which reflect the ability of osteoclasts to expand the original resorption pit, were a typical f inding on the control slices but were never detected from the APD- or CI2MBP-covered bones.

~ - ~ Inhibition of Growth of Dictyostelium Amoebae is Dependent on the Cellular Uptake of Bisphosphonates by Pinocytosis

X. Xiong, M.J. Rogers, X. Ji, R.G.G. Russell, D.J. Watts, F.H. Ebetino, Dept. of Molecular Biology and Biotechnology, University of Sheffield, $10 2UH, UK

Bisphosphonates (BPs) are inhibitors of osteoclast-mediated bone resorption although it is unclear whether their effects on osteoclasts are mediated extra- or intra cellularly. BPs also have growth inhibitory and cytotoxic effects on amoebae of the Ax-2 strain of Dictyostelium discoideum grown in a simple, semi-defined (axenic) medium. The amoebae take up dissolved nutrients by pinocytosis, the process by which the amoebae also appear to take up BPs, Dictyostelium amoebae may also be grown with suspensions of the bacterium Klebsiella aerogenes, The amoebae then feed on bacteria by phagocytosis, and pinocytosis is inhibited. If BPs have an intracellular site of action in Dictyostelium, it would be expected that inhibiting the pinocytic uptake of BPs by growing the amoebae on bacteria would make the amoebae resistant to the effects of BPs,

Uptake of BPs by amoebae was monitored by fol lowing accumulation of labelled BP in the amoebae, and by using ion-exchange FPLC to assay the incorporation of methylenebisphosphonate into AppCH2P and AppCH2ppA nucleotides as a result of intracetlular metabotism.

It was found that amoebal uptake of BPs was markedly reduced when the amoebae were grown on a bacterial substrate instead of axenically. Furthermore, amoebae grown on bacteria were partially resistant to

inhibition of growth by BPs, Thus. to obtain similar levels of growth inhibition in the two culture conditions, it was necessary to have final concentrations of BPs in bacterial cultures that were five- to twenty- fold higher than in axenic cultures. These results suggest that the internalization of BPs by pinocytosis is essential for the inhibitory effects of BPs on Dictyostelium growth and that the site of action of BPs in Dictyostelium is intracellular. Since the targets for BPs in Dictyostelium appear to be similar to those in osteoclasts, BPs probably act intracellularly on bone-resorbing osteoclasts.

We have also isolated a mutant strain of Dictyostelium. MR102, which is resistant (when grown axenicalty) to the growth inhibitory effects of potent BPs such as Alendronate. When both the wild-type Ax-2 and MR102 strains were grown on a bacterial substrate, amoebae of the MR102 strain were even more resistant to the effects of BPs than amoebae of the Ax-2 strain. This is consistent with the conclusion reached previously that the resistance of the MR1O2 strain is not due to a decreased ability to take up BR but is due to mutation of the intracellular target for BPs.

• Alendronate Effects on NH~ Permeability and Intracellular pH Regulation in Mammalian Osteoclastic Cells

Z. Zimolo. G. Wesolowski, G.A. Redan. Department of Bone Biology end Osteoporosis Research, Merck Research Laboratories, West Point, PA 19486, USA

The aminobisphosphonate alendronate (ALN) is a potent inhibitor of bone resorption in rive andin vitro, The biochemical mode of action of ALN and other bisphosphonates is unknown. Previous findings indicate that ALN causes disappearance of osteoclastic ruffled border membrane and alters membrane fluxes of NH~ and H +. The goal of this study was to further investigate osteoclastic handling of NH~ and the recovery of intracellular pH (pH i) after intraceltular acidification induced by an NH~ pulse, pH i was measured utilizing microfluorometry with pH i indicator BCECF and monitored with a photomultiplier.

Osteoclasts were isolated f rom neonatal rats' long bones and plated on glass in the presence of 10% serum. When exposed to 20 mM NH~,, NH~ permeability of osteoclastic cells, estimated by measuring pH i changes. varies widely for unknown reasons (0.017-2.330 pH units/rain). On glass, most (> 95%) cells display low (< 0.1 pH units/rain) NH~ permeability and no Na+-independent phi recovery after acidification. Other cells have high NH~ permeability and exhibit bafilomycin A 1 (10 ~,M) inhibitable, Na + independent H + extrusion (0,1-1.4 pH units/min), which is most likely due to a H + pump. This latter phenotype which was rare on glass, was present in cells plated on thin (0.2 mm) bone slices, as well as in 50% of cells plated on neutralized bovine collagen type I. ALN (0.5 mM), studied so far only in cells on glass, converted the high NH~ permeabil ity and Na+-independent H + extrusion to low permeabil ity and inhibited H + extrusion.

In summary, osteoclasts exhibit two types of pH i handling: (A) low NH~ permeability without Na+-independent acid extrusion (H + pump); (B) increased permeability to NR~ accompanied with Na + independent R + extrusion. ALN converts type B pH i regulation into type A.

P H A R M A C O K I N E T I C S A N D

T O X I C O L O G Y

[ ~ Pharmacokinetics of Pamidronate in Patients with Tumor-induced Osteolysis

J.J. Body, J.C, Dumon, J. Ford, G. Flesch. Bone Metabolism Unit, lnstitutJ. Bordet, Universit# lJbre de Bruxelles, 1000 Brussels, Belgium," Cibe-Geigy Co, Basel, Switzerland

We have treated 36 patients with tumor-induced osteolysis (TIe) by pamidronate (Pam., Aredia| given at 15 mg (n = 5), 30 mg (n = 12), 60 mg (n = 10), 90 mg (n = 3), or 120 mg (n = 6) at a constant infusion rate of 15 mg/hour. Patients did not receive any other systemic antineoplastic therapy during the trial. Tumor types consisted of 24 breast cancers. 3 myetomas, 3 prostate and 3 miscellaneous tumors. Urines were collected at 12-hour intervals during 48 hours for pare. determination (Flesch. J Chrom 1989). There was a significant correlation between the amount of pare. retained in the organism, supposedly in the skeleton, and the

Preclinical studies ~ Pharmacokinetics and toxicology S 71

dose received (r = 0.87; P < 0.001 ). The mean (4- SEM) skeletal retention of pam., expressed as a percentage of the dose administered, was 67 4- 3% (range, 30-94%). It was not significantly influenced by the dose administered, 69 4- 9%, 73 4- 4%, 66 4- 5%, 68 4- 7%, and 54 -I- 7%, after 15, 30, 60. 90, and 120 mg, respectively Skeletal retention was not a function of the tumor type (65 4- 4% for breast and 68 4- 5% for other tumors). However, a preliminary analysis suggests a relationship between retention and the number of metastatic skeletal sites, as evaluated by bone scintigram (n = 24; r = 0,50, P < 0.05), and the bone turnover, as evaluated by fasting urinary hydroxyproline excretion (n = 34; r = 0.39, P < 0.05). The correlation with fasting urinary calcium excretion was, however, not significant (r = 0.15). Urinary excretion of pam. was rapid, as 69 4- 4% of unretained pam. was excreted within the first 12 hours and 92 4- 3% within 24 hours. In summary, skeletal pamidronate retention increased linearly with the dose administered, from 15 to 120 mg, although the percentage retained was slightly less at that high dose level. The retention varied between 30% and 94% (mean 67%) of the dose administered and was correlated with the number of bone metastases and with baseline hydroxyproline excretion.

~ - ~ T h e Intestinal Tolerability of the Bisphosphonate CGP 42'446: Effects In Vitro on Ceco-2 Cell Monolayers and In Vivo on Perfused Rat Ileal Loops

J.R. Green *, I. Hassan **, K.A. Jaeggi *. * Ciba-Geigy Ltd., Basel, CH-4002, Switzerland, ** Horsham, RH12 4AB, England

The heterocyclic bisphosphonate CGP 42'446 is a very potent inhibitor of bone resorption which is in clinical development. As concern has been expressed about the gastrointestinal tolerability of bisphosphonates in man, we have investigated whether the increased osteotropic potency of this new compound is paralleled by increased gut intolerability. Therefore, the acute effects of CGP 42'446 and pamidronate have been compared in vitro using the human intestinal adenocarcinoma cell line Caco-2 and in vivo and in a perfused ileal loop model in anaesthetized rats.

Caco-2 cells were grown on permeable supports by standard methods to produce a confluent monolayer with several properties characteristic of epithelial cells in the distal ileum. Toxic effects of test compounds on the monolayer were assessed by monitoring paracellular permeability with [14C]mannitol. At concentrations of 0.1-50 mM, both bisphosphonates caused a dose-dependent increase of [14C]mannitol transport across the cell monolayer over 2 hr. The effect of CGP 42'446 was 10-fold less than that of pamidronate, indicating that CGP 42'446 is less toxic than pamidronate to the cell monolayer in this in vitro system.

In the in vivo model, rats were labelled intravenously with 10/zCi of the metabolically inert sugar [3H]raffinose and its urinary excretion was prevented by ligation of the renal vasculature. A 10 cm ileal loop was perfused in situ with bisphosphonate [1-30 mM] in isotonic saline and mucosal damage was assessed by leakage of label into the peffusate. For both CGP 42'446 and pamidronate, a 4 hr exposure to luminal concentrations of 1-10 mM had no effect whereas 30 mM bisphosphonate resulted in marked damage to the mucosal permeability barrier.

Although CGP 42'446 is 100-1000 times more potent than pamidronate as an inhibitor of bone resorption [Green et al., J Bone Min Res, in press], there is no evidence of a parallel increase in its potential to damage the intestinal epithelium in vitro or in vivo. It is concluded that, in comparison to pamidronate, CGP 42'446 has an improved preclinical therapeutic ratio that should result in a better gastrointestinal tolerability profile in man.

[ - ~ Recovery from Pamidronate a Longterm Study in the Dog

M.D Grynpas *, M Kasra *, M. Dumitriu *, J .M Very**, B.R Mertz **. * Department of Pathology, Samuel Lunenfeld Research Institute, Mount Sinai Hospital and University of Toronto, 600 University Avenue, Toronto, Ontario, M5G 1)(5, Canada; **Ciba-Geigy, Basle, Switzerland

The goal of this study was to find out if bone can recover after Iongterm administration of bisphosphonate. Disodium pamidronate (APD) was given orally by gavage to mature beagle dogs at doses of 0, 2.5, 12.5, and 25 mg/kg/day for one year (0.1% concentration) and the animals were allowed to recover for another year. At sacrifice, the os ilium was used to determine bone mineralization profile and, subsequently, each density fraction was analyzed chemically The ribs were used to determine the lattice parameters and the size of the apatite crystals of bone. The sternum was used to determine selected morphometric parameters using image analysis of specimen X-ray films and, subsequently, to determine bone mechanical properties using a 3 point bending technique. We found that the 12.5 and 25 mg/kg/day doses exhibit a significant shift towards greater mineralization versus control, while the lower dose (2.5 mg/kg/day) was indistinguishable from the controls. The lattice parameters and crystal size of bone apatite remained unchanged, The image analysis shows a dose-related increase in trabecular volume and thickness, The connectivity increased with dose while the anisotropy of bone remained unchanged. Both, the elastic modulus and the maximum stress of bone remain unaffected byAPD. We conclude that when dogs are treated with APD for one year. their bones can re-establish their physical-chemical characteristics (mineralization profile, chemistry and crystal size/strain) after one year of recovery, provided that the treatment dose is 2.5 mg/kg/day In addition, the mechanical properties of the bone remained unaffected and the gains in trabecular volume and thickness are maintained.

[• ~mTc-Methylene Diphosphonate (~MTc-MDP) - - A Potential Prognostic Agent for Assessing Bone Turnover

DN. Pahuja, O.P.D. Noronha. Radiation Medicine Centre, (B.A.R.C.), T.M.C. Annexe, Pare/, Bombay-400012, India

Biphosphonates are used for suppressing the osteoclast mediated hypercalcemia. However, they also inhibit new bone formation. It is therefore necessary to know when to stop/restart the treatment. To gain an insight we have employed 99rnTc-MDP tracer to assess the calcemic status/bone turnover rates in different groups of rats with secondary hyperparathyroidism due to vitamin D (vit. D)-deficiency induced hypocalcemia.

Age-paired rats (70-80 g) were pair-fed on three different diets: (1) Normal-vit. D-repleted diet with Ca-0.4% (+D); (2)vit. D-deficient diet with Ca-0.4% (-D); (3) vit. D-deficient diet for 1st 16 weeks followed by high Ca (2.0%) diet ( -D +Ca).

Gradual rise in the skeletal uptake of 99mTc-MDP was observed in the 2rid group of rats after ~5-6 weeks peaking to ~80% by 16 weeks vs. 35% in that of 1st group. In contrast, there was gradual decrease in the skeletal uptake of 99mTc-MDP in 3rd group of rats after 10 weeks ( -D +Ca), which returned to normal by 18-20 weeks. This correlated well with the serum levels of Ca, P, Mg and alkaline phosphatase and bone Ca-content.

The results suggest that 99mTc-MDP bone uptake could serve as a good screening test in hypercalcemic patients to ascertain their rate of bone- turnover and to decide when to stop/restart biphosphonate treatment.

S72

C L I N I C A L S T U D I E S

O S T E O P O R O S I S

[ - ~ Long-Term Effects In Poatmenopausal Osteoporosis of a Treatment Course with Alendronate

S. Adam• M, Rossini, N. Zamberlan. E Bertoldo, D. Gatti. V Braga, Istituto di Semeiotica e Nefrologia Medica, University of Verona, Italy

Alendronate administration induces a long lasting inhibition of bone turnover in patients with Paget's disease and increases bone mass in patients with post-menopausal osteoporosis. Aim of this study was to investigate the duration of the effects on bone density and bone turnover of a short-term treatment course of postmenopausal osteoporosis with oral alendronate. Thirty postmenopausal women (age range 59-69 years) with severe osteopenia were given, in a double blind, random• trial. either Alendronate (Ist. GentiB, Pisa, Italy; 15 patients) 20 mg/day orally, or placebo, for 6 months but the follow-up continued for further 12 months. All patients had calcium supplements up to a daily calcium intake of 1200 mg and this intake was kept for the 12 months of post-treatment follow-up. In the table are listed the main changes (% of initial values 4- S.D.) observed in the treated patients for the following parameters: lumbar spine bone mineral density (BDL24, Sophos L-XRA), serum bone alkaline phosphatase (sbALP), urinary hydroxyproline (Ur.OHP), urinary pyridinoline (Ur.PYR).

Parameters 5 months ON 3 months OFF 6 months OFF 12 months OFF

BDL24 104 (2) *** 105 (2)*** 105 (3)*** sb.ALP 53 (23)*** 70 (26)*** 104 (22) 106(39) ur.OHP 62 (25)*** 92 (21) 89 (22) 90 (22) ur.PYR 72 (22*** 83 (22)** 77 (34)* 88 (28)

*p < 0.05, **p < 0001, ***p < 0.001 versus baser•

In the placebo group the markers of bone turnover did not change, whereas a significant decrease of BDL24 ( - 2 % 4- 2; p < 0.001) was observed 12 months after treatment withdrawal.

Conclusions: 20 mg oral Alendronate given for 6 months remarkably increased vertebral density; 12 months after treatment withdrawal the indices of bone turnover rose back to initial values but this was not associated with the expected decrease in bone density. This might reflect a persistent positivization of bone balance which counteracts the increases in remodelling space.

[ • Long-term Persisting Action of Pamidronate on BMD in Osteoporosis

J.R Devogelaer, R. Triki, G. Depresseux, C. Nagant de Deuxchaisnes. Department of Rheumatologz St-Luc University Hospital, avenue Hippocrate 10, B-12QO Brussels, Belgium

We have previously shown shown that cyclical intermittent pamidronate administration was able to induce a significant increase in BMD in the two first years of therapy, with a levelling-off effect later on. The aim of the present study was to determine whether after the drug was discontinued, the bone loss resumed. We have followed 19 patients for up to 30 4_ 2 months after withdrawal from pamidronate. Their lumbar and hip BMD was measured regularly by DXA (QDR-1000, Hologic Inc.). At the lumbar spine, a non-significant loss ( -0 .10 =E 0,47% per year) was observed, whereas at the total hip. the non-significant loss amounted to 1.6 4- 0,64% per year. The same proved true for the hip subregions, the results are summarized in the table.

Femoral neck Ward's tr iangle T rochan te r Intertroch.

0.54 --0.77 -0.50 -- 1.22 =E0.72 4-091 4-0.02 4-0.78

In conclusion, after pamidronate therapy withdrawal, there is a residual protecting effect which lasts for at least 30 months, more marked at the spine.

[ • Biochemical Effects of a Single Pamidronate Infusion In Postmenopausal Osteoporosis

J.C. Dumon, A. Peretz, S. Rozenberg, J.J. Body. Inst. J. Bordet, Hop. Univ St Pierre and Brugrnann, 1000 Brussels, Belgium

Intravenous bisphosphonates are contemplated for the treatment of postmenopausal osteoporosis, but little is known about optimal therapeutic schemes and the biochemical effects of a single infusion. We have eval uated in 43 osteoporotic women (lumbar Z score -1 .52 4- 0.13, mean 4- SEM) the effects of a single 30-rag pamidronate (Aredia | infusion on vaF ious biochemical parameters of bone metabolism. Fasting urinary calcium excretion (uCa, NI < 0.21 mg/mg Creat) was determined every 2 weeks, the patients being retreated when uCa had reached baseline levels or after 3-4 months in any case. Mean uCa fell from 0.165 4- 0,019 to a nadir of 0.091 4- 0.012 on d21-28, the decrease remaining significant (P < 0.05) up to d77-84. The patients whose baseline uCa was above the median value of the group (i.e. 0.12) had a significant fall in uCa levels also up to d77-84, whereas the fall was not significant in the other half of the patients (0.073 4- 0.007 to 0.060 4- 0.011 mg/mg Creat). Fasting urinary hydroxyproline excretion (n = 38) fell significantly up to d21-28, reaching 71 4- 5% of baseline values (P < 0.05), There was no correlation between the changes in uCa and hydroxyproline. Blood parameters were measured before each infusion in all patients and every week during one month in 7 of them. Serum Ca (NI 8.5--10,3 mg/dl) fell from 9.6 4- 0.1 to a nadir of 9.1 4- 0.1 on d7 (range 8.7-9.6; P < 0.05), concomitantly with a peak in intact PTH levels (35 4- 5 to 71 4- 12 pg/ml, P < 0.05). Serum PTH remained elevated up to d28 and could have contributed to the fall in uCa. Before the second course, all parameters had returned to baseline levels, except osteecalcin (BGP, Incstar assay) which fell from 3.4 4- 0.2 to 2.5 4- 0.2 ng/ml (P < 0.001 ). In summary, a single 30-mg pamidronate infusion had a prolonged effect on parameters of bone turnover. There was a transient, slight and asymptomatic fall in Ca levels accompanied by a sharp increase in PTH secretion.

F5-2-] Assessment of 8 Markers of Bone Turnover in Elderly Osteoporotic Women Treated with Alendronate (ALN)

R Garnero, W.J. Shih, E. Gineyts. D.B. Karpf, P.D. Delmas. INSERM Unit 234, H&pital E. Herriot, Pay. F,, 69437, Lyon, France and Merck ,9 Co., Inc, Bahwaz N J, USA

In order to test the sensitivity and the clinical usefulness of various markers of bone turnover, we assessed 4 markers of bone formation [intact human osteocalcin (I-OC, ELSA-OST-NAI'", Cis biointernational), I-OC+N terminal Mid fragment (T-OC, ELSA-OSTEO'", Cis), bone alkaline phosphatase (BAR Ostase". Hybritech), carboxyterminal propeptide of type I collagen (PICP'", Farmos Diagnostica)] and 4 markers of bone resorption [total urinary deoxy pyridinoline (D-Pyr) by HPLC, urinary free Pyr (F Pyr, Pyrilinks'", Metra Bio systems), urinary crosslinked N-telopeptide of type I collagen (NTX, Osteomark'", Osteomark" , Ostex), serum crosslinked-C-telopeptide ( IcrP ' - , Farmos)] in 84 late postmenopausal (pMP) women (mean age 63. >5 years pMP) with low bone mass enrolled in a placebo-controlled trial of the bisphosphonate ALN (5 and 10 mg/d for 24 months). Baseline measurements of bone turnover were compared with those of 46 healthy premenopausal (Pre-MP) women (mean age: 40 years).

Bone formation markers (mean 4- SE)

l-OC T-OC BAP PICP ng/ml ng/ml ng/ml ng/ml

PreMP 103• 21.6• 6.2=1:03 109• pMP 15.4• 30.7• 1 0 16.7:1_06 11Bi3 A% 50%* 41%* 104% ~ 8%

Bone resorption markers (mean i SE)

D-P,/r F-Pyr NTX ICTP nM/mM Cr nM/mM Cr /~BCE1/mM Cr ng/ml

PreMP 5.44-0.2 42.3t l 30,4• 3.33:E0.08 pMP 6.8• 77 5 t 3 3 82 54-46 3.424-01 z&% 63* 83* 171" 3%

1 BCE: bone collagen equivalent. *p < 0001

C l i n i c a l s t u d i e s - - O s t e o p o r o s i s S 7 3

The long term precision, assessed in the placebo group by 7 measurements over 15 months, was high and better for the 4 serum formation markers (12.5 to 17.4%) than for urinary markers of bone resorption (24 to 29%). Upon treatment, resorption markers decrease earlier than markers of formation (1 vs 3 mths) and a dose response effect between 5 and 10 mg was seen only for I-OC, T-OC, BAP and NTX. The % change from baseline after 3 months of ALN 10 mg/d were: I-OC - - 40% +, T-OC - - 37% +, BAP - - 37% +. PICP - - 38% +, D-Pyr - - 38%*, F-Pyr - - 0%, NTX - - 71%*. ICTP - - 10% (vs placebo: +p < 0.01. * p < 0.001). In response to ALN, bone markers (except F-Pyr) returned to premenopausal levels and this steady state was maintained for the duration of the study. Highly significant correlations were observed between % change in bone markers at 6 mths end % change in spine BMD after 18 mths for I-OC (r = -0.54), T-OC (r = -0.59), BAP (r = -0.70) PICP (r = -0.69) and NTX (r = -0 .57) (all p < 0.001 ). The correlation was modest for ICTP (r = -0.26, p < 0.05) and was not significant for F-Pyr (r = -0.08). In conclusion: (1) Our study shows a marked increase of bone turnover in osteoporotic late PMP women, for which the most sensitive markers were F-Pyr and NTX for resorption, BAP and OC for formation. (2) Bone turnover was normalized by ALN, and the response to therapy could be effectively monitored with NTX for resorption, BAP and OC for formation.

~ - ~ Results of the treatment with dlphosphonates and 1-alpha-colecalciferolo on patients affected by senile osteoporosis

E. Grisostomi, E. Gerber, C. Grisostomi, I~ Scartozzi. G. Pelliccia, R. Fortune, T. Vitali, L. Giorgi. Department of Orthopaedy of United Hospitals of Fermo and Porto San Giorgio -U.S.L. 2 I- Via della Misericordia, 63023 Fermo (A.l~ Italy

Ninety patients aged between 69 and 89, affected by senile osteoporosis, have been studied; the illness had been diagnosed by emato-chemical exams and bone densitometn/. Patients accused an increase of dorsal ciphosis, vertebral squeezing and high back pain that heavily limited their movement autonomy in daily life. Among these 90 patients, 60 have been treated according to a program that included the supply of 300 mg of Clodronate disodium in 100 cc of phisiological solution by intravenous infusion twice per week, for 4 weeks, together with 0 5 mcg of 1-alpha-cholecalciferol per day. Lately the home therapy included an intermuscular injection of 100 Clodronate disodium per week for three months, associated with 0.5 mcg of 1-alpha cholecalciferol per day, followed by an arrest of therapy of three months.

The remaining 30 patients formed a control group that has been treated with 500 mg of calcium per os. associated with 1-alpha-cholecalciferol per day, for three months, followed by an arrest of the therapy for the same period.

All patients have been checked by ematochemical exams for the calcic metabolism (calcemia. phosphoremia, alkaline phosphatase, osteocalcina. hydrossiprolinuria according to Nordin test), at the beginning as well as at the end of the treatment. At the end of the therapeutical program it has been given a questionary for subjective evaluation of pain and functional recovering.

Patients have been re-checked after six months by same questionary and blood exams. A significant improvement of almost all parameters in the treated group respect to the control group has been noted, for calcic metabolism as well as for pain and functionality subjective evaluation.

synthesis, the carboxyterminal propeptide of type I procollagen (PICP) in 60 osteopenic, postmenopausal women. All had experienced a distal forearm fracture, and all had vertebral bone mineral density below mean peak bone mass -1 SD (0.9 g/cm2).

In a double blind study patients were randomized to receive either pamidronate 150 rag/day (N = 20), 75 rag/day (N = 20), or placebo (N = 20) for four weeks. Biochemical markers of bone turnover (S- osteocalcin. S-alkaline phosphatase, fU hydroxyproline/creatinine and fU- calcium/creatine ratio) were measured weekly during four weeks of treatment. Results are given as medians and ranges.

Pretreatment values of ICTP and PICP were 2.1 (1.2-8.8)/~g/L and 101 (39-210) /~g/L respectively. Both markers were significantly decreased compared to healthy adults, but no difference between groups was observed. ICTP correlated positively with osteocalcln (p < 0.02), and PICP (p < 0.02), but we found no correlation to the urinary excretion of neither hydroxyproline nor calcium. PICP was positively correlated to osteocalcin (p < 0.03). alkaline fosfatase (p < 0.001), fU-hydroxyproline/-creatinine ratio (p < 0.001) and fU-calcium/creatine ratio (p < 0.05).

During treatment with pamidronate ICTP and PICP were reduced (p < 0.02), but only at 75 mg/day. This was in contrast to the effect of treatment on excretion of hydroxyproline (only at 150 mg/day) and calcium (in both groups).

In conclusion we found a decreased type I collagen metabolism in postmenopausal, osteopenic women. Correlation between ICTP and PICP to other markers of bone metabolism were satisfactory. Only pamidronate in a dose of 75 mg/day seems to be effective in decreasing bone resorption according to ICTR

This study was supported by grant from Danish Rheumatism Association.

~ - ~ The Effect of Nasal Calcltonln vs. Clodronate on Bone Mass in Poetmenopaueal Women. Preliminary Report of e 6 Month's Treatment

A. Kocijan~i~. J. Pre~elj. Medical Centre Ljubljana, Dept. of Endocrinology, Zalo,~ka 7, 61 000 Ljubljana, Slovenia

Twenty postmenopausal women participated in the study. They were divided into two groups due to randomly allocated treatment. Group A (n = 9) was treated by ealeitonin nasal spray 100 IU/day and calcium carbonate (600 mg calcium daily) and group B (n = 11) had two cycles of the following regime: phosphate 1 g twice daily for three days before commencing 400 mg disodium clodronate per day for a fortnight followed by calcium 600 mg daily to the end of the third month.

The two groups were comparable as to the age (63.0 :E 8.1 vs. 61.3 -I- 10.3 yrs), body mass index (23.0 • 3.9 vs. 23.5 4- 2.1 kg/m2), time since menopause (16.5 _E 9.8 vs. 19.7 i 7.9 yrs) and the initial lumbar spine (L 2 L4) mineral density (0.817:1:0.08 vs. 0.807 -E 0.08 g/cm2).

Results: BMD (g/cm 2)

before TH after 6 mo p Group A 0.817i0.08 0 811 i0.09 0.595 Group B 0.807• 0 839i0.08 0.004

Though both substances are known to inhibit bone resorption, calei- tonin only prevented further bone loss while clodronate even induced an increase in bone mass.

• Effects of Different Dosages of Pamidronate on Type I Collagen Metabolism in 60 Poetmenopeueel Women

M. Hansen, H.A. Sorensen, IRE Jensen, "E Storm, L. Risteli, J. Risteli, L. Hyldstrup. Dept. of Rheumatology and Endocrinology, Hvidovre Hospital, Kettegaard Alle 30, 01(-2650 Hvidovre, Denmark

Type I collagen accounts for more than 90% of the organic bone matrix and consequently markers of type I collagen metabolism are therefore potential markers of bone metabolism.

A new markerfortype I collagen degradation, the carboxyterminal crosslinked telopeptide region of type I collagen (ICTP) has been introduced as a specific marker in serum for bone resorption. Hence, treatment with pamidronate disodium is expected to decrease serum levels of ICTR

The aim of the study was to assess the dose-response effect of pamidronate on ICTP together with a marker of type I collagen

•6] Recovery of Serum Calcium Concentrations Following Acute Hypocalcaemia in Osteoporotic Patients on Long-Term Therapy with Oral Pemidronete

J.O. Landman, D.H. Schweitzer, N.A.T. Hamdy, S.E. Papapoulos. Department of Endocrinology, University Hospital, Rijnsburgerweg 1 O, 2333 AA Leiden, The Netherlands

Bisphosphonates have a long skeletal residence time and there are concerns that their long-term uninterrupted administration may be associated with cumulative, unfavorable effects on the skeleton. We have previously shown that oral pamidronate therapy (150 mg/d orally) given for five years to patients with osteoporosis is not associated with adverse skeletal effects or with a cumulative effect on bone metabolism. In the present study we examined whether this regimen may affect the recovery of serum calcium concentrations following an acute hypocalcaemic challenge. For


this, two groups of osteoporotic patients (10 patients per group) were given short (5 min) EDTA infusions (10 mg/kg BW in NaCI 0,9%) and serum calcium and PTH values were fol lowed for 24 hours. The first group (control) comprised 10 patients never treated with bisphosphonates whi le the 10 patients of the second group (APD) had been treated with oral pamidronate 150 mg/d for at least five years. In both groups EDTA infusions induced a similar decrease in serum calcium (0.211-1-0.05 and 0.2194-0.03 mmol/1 in the control and APD groups, respectively) 5 to 10 minutes after the infusion; 9 of the 10 patients of the control group and all patients of the APD group became hypocalcaemic. Thereafter, serum calcium concentrations increased similarly in both groups and returned to basal within 12 hours. Analysis of the calcium changes during the whole 24 hr period revealed no differences between the two groups. In response to the decrease in serum calcium, plasma PTH increased in both groups within 5 minutes. The peak increase was 14.24-2.5 pmol/I in the control and 14.24-3.2 pmol/I in the APD group, PTH values returned to basal 60 min after the EDTA infusions in both groups. Thereafter, they remained wi th the reference range in all patients but the A mean values of the APD group were slightly but significantly higher than those of the control group. Our results demonstrate that chronic uninterrupted administration of pamidronate to patients with osteoporosis does not impair the recovery of serum calcium concentrations fol lowing an acute hypocalcaemic challenge.

5•-1 Continuous Therapy with Oral Pamidronate is not Associated with Chronic Stimulation of PTH Secretion in Patients with Osteoporoeie

J.O. Landman, S.E. Papapoulos. Department of Endocrinology, University Hospital, Rijnsburgerweg 10, 2333 AA Leiden, The Netherlands

We have previously reported that uninterrupted treatment of patients with osteoporosis with oral pamidronate (150 mg/day) is associated with a continuous increase in BMC of the lumbar spine. These changes in bone mass cannot be only explained by the antiresorptive action of the drug and raised the possibility of an anabolic effect of this treatment on skeletal tissue. It is well known that administration of suppressive doses of pamidronate are associated with transient decreases in serum calcium concentrations and subsequent increases in plasma PTH levels. As PTH is known to have anabolic effects on bone we examined whether treatment with pamidronate may lead to a sustained increase in PTH secretion, For this, 33 patients with vertebral osteoporesis treated with 150 mg pamidronate daily were fol lowed 6-monthly for two years. Serum alkaline phosphatase activity and urinary hydroxyproline excretion decreased to 82 4- 4% and 75 -E 5% of initial values, respectively within one year and remained at these levels during follow-up. BMC of the lumbar spine (measured either by DPA or by DEXA) increased by 5.3 =1- 1.5% and 4.6 4- 1.3% the first and the second year of treatment, respectively. Initial serum calcium concentrations were 2.38 4- 0.15 mmol/I and did not change with treatment (the values were: 6 months 2.39 d: 0.01, 12 months 2.38 �9 0.01, 18 months 2.37 • 0.03, 24 months 2.39 4- 0.01 mmol/I). Similarly plasma intact PTH (initial value 2.37 =E 0.23 pmolfl) did not change significantly during the period of fol low-up (the values were: 6 months 2.48 4- 0.20, 12 months 2.46 4- 0.26, 18 months 2.27 4- 0.20, 24 months 2.43 =E 0.25 pmol/I). When the results were analyzed according to the use or not of calcium and/or vitamin D supplements (16 patients with, 17 patients without} there were again no differences in calcium or PTH values. Our study suggests that a potential anabolic effect of uninterrupted treatment with pamidronate on bone mineral content in patients with osteoporosis is not due to chronic hypersecretion of PTH. It does not, however, exclude possible occurrence of day to day subtle changes in PTH secretion with treatment.

[ - ~ ICT Etidronate for Osteoporosis: 5-Year Results

P.D. Miller. R.D. Jackson, RD. Ross, R.D. Wasnich, H.K. Genant, S.T. Harris, A.A. Licata. N.B. Watts, C.H. Chestnut. University of CO Health Science Center, Lakewood CO 80214

To determine long-term safety and efficacy of intermittent cyclical therapy (ICT) with etidronate, a 2-year open-label fol low-up to a 3-year placebo- controlled study was conducted in 277 postmenopausal women. ICT consisted of etidronate (400 mg) for 14 days fol lowed by calcium carbonate t 250 mg (containing 500 mg elemental calcium) for the next 76

days. Clinical parameters included bone mass of the spine and hip, and spinal fracture rates. For the latter, data were calculated for all patients and for a subgroup at higher risk (i.e., baseline spinal bone mass below the 50th percentile and more than 2 spinal fractures).

During the 2-year fol low-up study, patients receiving a total of 5 years of ICT maintained bone mass of the spine and hip achieved during the first 3 years of treatment, Former Non-Etidronate patients receiving ICT in the 2-year study had increases in bone mass of the spine (4.3%), femoral neck (2.9%) and trochanter (2,2%) after 2 years. Spinal fracture rates (per 1000 patient years) were lower in patients who received ICT in the first 3 years than in those who did not (86 vs 117) and remained low in the subsequent 2 years when both groups received ICT (59 vs 68, respectively). Spinal fracture rates for the higher risk subgroup of patients receiving ICT in the first 3 years were significantly lower (p < 0.05) compared to those that did not (228 vs 412, respectively); in the 2-year fol low-up period, lower rates were observed both in the group that previously received ICT (174) and in the group that had not (233).

We conclude that ICT etidronate is an effective and well-tolerated treatment for postmenopausal osteoporosis for up to five years,

[ • - ] Cyclical Etldronate (CE) Increases Bone Mass in Patients with Established Steroid-Induced Osteoporosis (SLOP)

RD. Miller, D.L. Hamilos, D.C. Mcintyre, M.J. Yanover, M.S. Anger, M.N. Harrison. D.M. Gillum, E.M. Fish, A.L. Erickson. University of Colorado, Health Sciences Center, 1750 Pierce St., Lakewood, CO 80214, USA

Chronic glucocorticoid administration has been shown to decrease bone mineral density (BMD). CE therapy increases BMD in patients with postmenopausal osteoporosis. In this study the effects of CE in patients with SlOP was examined. Eighteen patients (6 men and 12 women, ages 21-80 y), with a variety of diseases requiring chronic steroid administration, received 5-30 mg/d of prednisone (mean: 13 rag/day) for 0.25 to 20 y (mean: 6 y). CE (400 mg OHS x 14 d repeated every 74 d) was given along with elemental calcium (1500 mg/d) and Vitamin D (400 IU/d). Thirty-three age-matched (22-62 y) control subjects receiving a comparable steroid dose (mean: 13 rag/d) were given calcium and Vitamin D only. BMD, measured by DEXA, increased f rom a mean of 0.730 • 0.098 to 0.7804-0.118 g/cm 2 (% change +6.8%. p - 0.0056) during the first year of the CE therapy, BMO increased during the second year in 12 CE treated objects to 0.821 4-0.12 g/cm 2, +5.1%. The BMD in the control subjects did not change (0.872 to 0.878 g/cm 2, +0.7%, NS) during the first year of observation. However, during the second year, nine control subjects decreased their BMD by 5.1% (0.878 to 0.833 g/cm2). In conclusion, CE increases BMD in patients with SlOP whereas control subjects maintain or lose BMD. The increase in BMD continues through the second year in patients receiving CE. Therefore, CE may be an effective therapy for SIOR

[•-• Long Term Effects of Continuous Biephosphonate Therapy in Children with Vertebral Osteoporosls

S.E. Papapoulos. N.A.T. Hamdy, V Papapoulou, R. Valkema. Departments of Endocrinology, Pathology and Nuclear Medicine, University Hospital, Rijnsburgerweg 10, 2333 AA Leiden, The Netherlands

Bisphosphonates have a long skeletal half-life and there are concerns that their long-term uninterrupted administration may induce unfavorable effects as a result as a result of their accumulation in the skeleton. We have used various approaches to investigate this possibility in vivo in man. In the present study we examined the effects of long-term therapy wi th nitrogen-containing bisphosphonates on the growing skeleton. During growth the skeleton is particularly sensitive to factors which affect adversely bone metabolism and potential deleterious effects of long-term bisphosphonate therapy may thus best be identified in children. We studied 7 children aged between 10 and 17 years with spinal osteoporosis of varying aetiologies who were treated with pamidronate (n = 5) or dimethyI-APD (n = 2) for a min imum of 2 and a max imum of 6 years (mean 4.1 years). All patients tolerated treatment very well. Linear growth was not affected and continued along the percentile at the start of treatment, Bone mineral density which was below 2 SD of the normal mean for age. increased impressively with treatment. When bisphosphonate was given before puberty the increase in BMD had a cleady different slope

C l i n i c a l s t u d i e s - - O s t e o p o r o s i s S 7 5

f rom that of healthy children and there was a tendency to catch up with normal values. There were parallel increases in the BMD of the femoral neck indicating that the gain was not due to redistribution of calcium in the skeleton. Calcium balance measured in 4 patients was 0.07 4- 2.4 mmol td before treatment, rose to 8.7 4- 0.32 mmol/d after 10 to 15 days and remained similarly positive for the fol lowing 12 to 18 months (7.8 + 1,2 retool/d). The striking radiological changes have previously been described (Radiology 1992; 184: 249). During the period of observation no new fractures occurred except in a child with juvenile osteoporosis who suffered new peripheral fractures after adequate trauma (automobile accident). Bone biopsies taken f rom 2 patients after 6 years of uninterrupted bisphosphonate therapy showed bone of lamellar structure with no evidence of a mineralization defect. These studies provide further reassurance that long-term continuous administration of nitrogen- containing bisphosphonates induce no unwanted effects on the skeleton. They further suggest that this type of treatment may be beneficial to children with vertebral osteoporosis in whom very limited therapeutic options are currently available.

~ - ~ Effect of Cyclic Treatment with Pamidronate on Bone Remodeling and Vertebral Density in Postmenopaueel Osteoporoeie

A. Peretz. J.C. Dumon, S. Rozenberg, V. Siderova, P. Bergmann. J.J, Body. Hhpitaux Univ. Saint-Pierre, Brugmann; Institut J. Bordet` Universit~ Libre de Bruxelles, Brussels, Belgium

The effects of cyclic pamidronate treatment on biochemical parameters of bone turnover and vertebral bone mineral density (BMDL) were assessed in 35 women with postmenopausal osteoporosis (T score < -2.0). They received at least 6 iv courses of 30 mg of pamidronate infused over 2 to 4 hours. Intervals between courses were defined according to the fasting urinary calcium excretion (uCa, mg Ca/rag Creat) which was measured before treatment and every 1-2 weeks after the first course. The patients were retreated when uCa had reached baseline levels or after 3-4 months in any case. The mean interval between treatments was 99 days (range 19-172), and complet ion of the therapeutic regimen occurred after 493 (129-860) days. Serum Ca, Pi, and intact PTH levels were back to baseline levels before each course. On the contrary, parameters of bone turnover decreased with t ime: osteocalcin (BGP) to 70% of baseline values (P < 0.05) after the first course with a nadir at 60% (P < 0.01), and at 75% for ALP (NS) after the 4th course. Hydroxyprolinuria changed in a similar manner, reaching a nadir at 65% of baseline values. After each course, uCa reached a nadir at around 60% of baseline after 1-3 weeks fol lowed by a return to baseline values. The response of patients with high or low bone turnover, as defined by baseline uCa above or below the median value of the group (i.e. 0.15) were not significantly different. The median gain in BMDL f rom baseline was 5% (range. -5 .6 to 14.5%) after one year and similar in both groups. The treatment was clinically well tolerated. The absence of sustained modifications in calcium and PTH serum levels, the decrease in bone turnover and the increase in BMDL suggest that pamidronate is an effective and safe therapeutic approach in postmenopausal osteoporosis.

~ - ] Clodronate in the Treatment of Corticosteroid-lnduced Osteoporoala among Asthmatic Patients

H, Puoliioki, J. Herrala. K, Liippo, M. Raitio, O. Impivaara. M.M. Nieminen, E. Tale. Departments of Pulmonary Diseases, Turku and Tampere Universify Hospitals and Social Insurance Institution Rehabilitation Research Centre, Turku, Finland

In spite of the development of inhaled corticostereids a number of patients with asthma or chronic bronchitis must still use oral corticosteroids regularly. The risk of osteoporosis in these patients is clearly increased in long term. In this double-blind, placebo-controlled study 74 (33 male) such adult patients having a long history of small dose oral corticosteroid therapy were randomized to receive oral clodronate (Bonefos| for one year either 800, 1600, 2400 mg/day or identical placebo. All patient groups were comparable in age, body mass index, corticosteroid usage duration and ventilation function parameters. The main criteria for efficacy was the measurement of bone mineral density (BMD) and content (BMC) values by Norland XR26 DEXA.

The result showed that clodronate had a statistically significant

increasing effect both in the BMD and BMC parameters in femoral neck measurements, p values being e.g. 0.0037 and 0.042, respectively. between the groups on 2400 mg and placebo. Same kind of trend was seen in the trochanter region measurements.

Both the biochemical and histological f indings have suggested that long-term glucocorticoid therapy causes a reduction in bone turnover, The results demonstrate that continuous oral clodronate is effective in stopping bone loss or increasing bone mass even in those cortieosteroid treated patients having a long history of steroid administration.

~ ] Continuous Therapy with Pamldronate In Postmenopausal Oateoporosis

I.R Reid, D.J. Wattle, M.C. Evans, G.D. Gamble, J.R Stapleton, J. Cornish. Department of Medicine, Universily of Auckland, New Zealand

There is a need for an effective and acceptable therapy for postmenopausal osteoporosis. The bisphosphonates show promise in this role. though the only long-term, controlled trials in women with established osteoporosis have used etidronate, a relatively weak inhibitor of bone resorption which is given intermittently because continuous administration interferes with bone mineralization. The newer bisphosphonates, such as pamidronate, might be more effective because of their greater anti-resorptive potency and their lack of effect on bone mineralization.

We performed a two-year, randomized, double-blind, placebo-controlled trial of pamidronate 150 mg/day in 48 postmenopausal osteoporotir women. Bone mineral density of the total body. lumbar spine and proximal femur was measured every six months by dual-energy x-ray absorptiometry.

Bone mineral density increased progressively in the total body (1.9 4- 0.7%. P < 0.01), lumbar spine (7.0 =1: 1.0%, P < 0.0001) and femoral trochanter (5.4 4- 1.3%, P < 0.001 ) in those receiving pamidronate but did not change significantly in those receiving placebo. There were significant falls in bone density at both the femoral neck (P < 0.02) and at Ward's triangle (P < 0.01) in those taking placebo, which did not occur in the pamidronate group. The differences between the treatment groups were significant at all sites (0.0001 < P < 0.05) except Ward's triangle. Vertebral fracture rates were 13/100 patient-years in the pamidronate group and 23/100 patient years in those receiving placebo (NS) and height loss tended to be less in those receiving pamidronate (NS),

It is concluded that pamidronate is an effective therapy in postmenopausal osteoporosis.

~ 4 ] The Effects of Cyclical Treatment with Clodronate and Monofluorophosphate on Biochemical Markers of Bone Remodeling and Bone Mass in Patients with Corticoid Induced Osteoporosis

H. Resch, I~ Pietschmann, F. Pietschmann, E. Neiger. R Bemecker, J.L. Baumann, E. Krexner, A. Battmann, R. Willvonseder. KH Barmherzige Brs Medical Department, Gr. Mohrengasse 9, A 1020 Vienna

Although the biochemistry of glucocorticoid induced bone loss remains to be determined, the mechanism is thought to involve a combination of effects. Suggested mechanisms include direct effects on bone cells, alterations in calcium absorption and renal handling of calcium and abnormalities in the secretion of sex hormones. Although both bone formation and bone resorption are negatively affected by glucocorticoids. both classes of effects may be corrected by therapeutic interventions, Bisphosphonates as antiresorbers and inhibitors of increased bone resorption have been reported to counteract bone loss in cortison treated patients. Fluorides are believed to increase bone mass by acting directly on osteoblasts to promote differentiation and proliferation to counteract the corticoid induced catabolic effect on the osteoblasts. As a consequence the aim of our study was to assess the effects of clodronate and fluoride treatment on biochemical markers of bone remodeling and axial (QCT) and peripheral bone density (SPA) in patients with corticoid induced osteoporosis and acute vertebral fractures. 21 Patients were treated cyclical with 1600 mg clodronate (n = 11) 2 out of 10 weeks or monofluorophosphate (MFP, n = 10) 76 mg twice daily continuously in a prospective, randomized study. The treatment with clodronate decreased urinary markers of bone resorption (hydroxyproline/erea and calcium/crea; p < 0.05) and serum markers of bone formation (total alkaline phosphatase, procollagen 1 and osteocalcin; p < 0.05). In the

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fluoride group biochemical markers for bone formation tended to be increased, however, the differences were not statistically significant; urinary ma~kers remained unchanged. Both treatments clodronate and MFP caused a slatistically significant increase in spinal (axial) bone density after 6 months (p < 0.05). In a preliminary observation the increase in spinal bone density can be maintained over 12 months under both therapies. No changes could be seen in peripheral bone density. The significance of our finding is. that short term cyclical treatment with clodronate can stop bone resorption and induce even an increase in bone mass. whereas MFP seems to stimulate primarily bone formation. As a mechanism of action clodronate seems to cause a decrease in resorption depth while keeping mean wall thickness (the amount of new bone) normal and achieving a net gain in trabecular bone.

[-~-1 Mechanism of Low Dosage Bisphosphonates Influence on Bone Tissue Remodelling in Systemic Osteoporosis

S.S. Rodionova. M N. Shvets. Central Institute of Traumatology and Orthopaedics, Priorov Str. 10, Moscow 125299, Russia

45 patients with systemic osteoporosis and low level of remodelling were treated with Xydiphone (2% solution, 5-6 mg/kg body weight) within 1 year. Drug regimen was the following: 2 months administration, 2 months interval. After 1 year we noted in some patients either the increase of bone mass or the cessation of its loss. Histomorphometry of bioptate from the upper flaring portion of the • showed the increase of osteoblasts number in spongy and cortical bone. appearance of connection lines and partial restoration of osteoid tissue volume, i.e. the appearance of bone formation increase signs. We detected the correlation between the intensity of bone formation under Xydiphone influence and the number of osteoclasts in the initial bioptates. Thus, in cases when their marked depression was noted at the beginning the ability of Xydiphone to increase the osteoblasts pool was not extended to the increase of bone formation. Our data corraborate the fact that bone formation is possible only in sites where resorption takes place. Besides. we have obtained the evidence that bisphosphonates in dosages 5-6 mg per kg of body weight, in contrast to Iow ones, do Hot possess "ant~res~rpt~ve" effect. Tt~e main mechanism of their action is the influence on the processes of bone formation.

• ] Effect of Xydiphone Treatment on Established Osteoporosis

L. Ya. Rozhinskaya, EJ. Marova, B,I~ Mishchenko, G.S. Kolesnikova, L.E. K'~rpatovskaya. National Research Center for Endocrinology, Russian Academy of Meclical Sciences, D. Uljanova, 11, Moscow, 117036, Russia

Xydiphone is the potassium and sodium salt of hydroxyethilidendiphos- phonic acid. It is character• by poor intestinal absorption but marked inhibitory effects on bone resorption.

We treated 60 patients, who received xydiphone (6-8 mg/kg), calcium 400 mg and vitamin D 3 (1r OHD 3 - - 0.5 mkg daily for 8 week. followed by a 4 week "free" period. Our patients received such treatment for two years. Group "~ consisted of 10 patients with postmenopausat osteoporosis (90~,. Group 2 - - 10 men with idiopathic osteoporosis (age 30 to 40 years). Group 3 - - 25 patients with remission of Cushing's disease, Group 4 - - 15 persons with Cushing's disease after bilateral adrenalectomy, who received 5-15 mg of prednisolone. Group 5 was control - - 12 patients after remission of Cushing's disease treated only with calcium and vitamin

D 3 . All patients showed decrease back pain. New vertebral fractures took

place in 2 patients with PO and in 3 patients in control group. We could see positive changes in bone mineral density, measured by DEXA in lumbar spine in groups 3 and 4 (4% and 2% respectively), In groups 3 and 4 calciuria and hydroxyprolinuria (OPRU) decreased significantly after 1 year of treatment. In group 30PRU decreased from 7.8 -4- 0.9 to 4.9 -4- 1.2, p < 0.01; and in group 4 - - from 8.5 =E 1.02 to 5.1 =E 0.98, p < 0.02. Osteocalcin level increased only in patients of group 3 (from 2.4 • 0.7 to 4.8 :E 0.5 ng/ml, p < 0.02). In control group we noted a decrease of back pain, increase of blood calcium level and increase of OPRU excretion.

We conclude, that treatment with xydiphone in established osteoporosis has significant positive clinical effect, preventing new vertebral fractures especially in steroid osteoporosis. Xydiphone decreases parameters of bone resorption.


[-6---~ Dose Response with Intravenous Pamidronate (APD) in Postmenepausal Osteoporosis: A Comparison of 30 mg and 60 mg doses

D. Thi~baud, J.M. Bigler, M,A. Krieg. O. Lamy, P, Burckhardt. Opt. Int. Medicine, University Hosp;tal, CH- 1011 Lausanne, Switzerland

To compare two doses of intravenous pamidronate (APD) in the therapy of postmenopausal osteoporosis, 30 osteoporotic women received either 30 mg or 60 mg APD mg iw over 1 hour every 3 months (group 30 rag: n = 16. mean age 65, initial mean of 2.7 fractures/patient; group 60 mg: n = 14, 68 yrs. 1 .B ~x/pat) in a randomized open study. Both groups received 1 g Calcium and 1000 U Vii.D/day, but no estrogens or drugs acting on bone. Results (mean + SEM) for lumbar spine and hip bone mineral density (BMD, g/cm2), urinary hydroxyproline/creatinine (OHR/zmol/mmol), serum osteocalcin (BGP, ng/ml) and parathormone (PTH, pg/ml), all measured before the infusions of APD, are shown in the table:

Months 0 (3 12 18 % (0-18)

APe 30 mg BMD spine 0.63• 0+66• 0.67~E*~ 03 0.6B**• +B

fern neck 0.56• 0.57• 0.57• 058*• +4 OHP/Creat 24.8• 16 7**• 14.2"'113 15.8"*• -36 BGP 32.4• 17.7+*• 16.2"*• 16.1"*:1:2.4 -50 PTH 38.9• 35.1• 34.4• 33.1• -15

APD 60 mg BMD spine 0.65• 0.68":E0.04 0.69**• 0.73"*~0,05 + 11

fern neck 0.55• 0,56• 0.55• 056&0.02 +2 OH P/Croat 22.8• 13.6~ J~ 1.5 135"*• 0 15 1**• --34 BGP 328• 18.2"~177 16.4~176177 15.5"• --53 P3-H 28.4• 33.5• 33.4• 368• +30 "p < D 05, ** p < 001 in comparison with day 0 (paired t test).

Plasma calcium, croat• PTH and urinary calcium/creat did not change in any group. BGP and urinary OHP decreased with both doses of APD. There were opposite trends in PTH levels ( - 1 5 % with 30 mg and +30% with 60 rag). In both groups, spine BMD increased signif icantly(8% with 30 mg, and 11% with 60 mg) at 18 months, Spine BMD increased earlier with 60 mg. Femoral neck BMD increased only with 30 mg, at 18 months. 2 and 5 new vertebral fractures occurred in the 30 mg and 60 mg groups respectively. Side effects were a transient fever with flu-like symptoms in 5 patients after 30 mg and 3 patients after 60 mg (first infusion). Mild phlebitis occurred in 1 patient after 30 rag, and in 3 patients after 60 mg. A recurrent phlebitis led to stop treatment in 1 patient with 60 rag.

In postmenopausal osteoporosis, intermittent intravenous treatment with 30 mg of pamidronate (APD) increased spinal and hip BMD, whereas 60 mg increased spinal BMD only. The 30 mg dose of APD was better tolerated than 60 mg of APD,

T U M O R B O N E D I S E A S E S

[-68--8--~ Pamidronate for Therapy of Breast Cancer-Induced Osteolysis: A Biochemical Dose-Response Study

J,J, Body, J.C. Dumon, J, Ford, Bone Metabolism Unit, lnstitutJ, Border, Brussels, Belgium ; Ciba-Geigy` Basel, Switzerland

Bisphosphonates are increasingly used in normocalcemic patients for tumor-induced osteolysis (TIe) but little is known about their metabolic effects and the adequate therapeutic schemes. We determined in 21 patients with breast cancer and TIe the biochemical effects of a single infusion of pamidronate, given at 30 mg (n = 5), 60 mg (n - 5), 90 mg (n = 5) or 120 mg (n = 6). Patients did not receive any other systemic antineoplastic therapy dunng the trial. We selected patients with baseline fasting urinary Ca/Croat > 0,105 rag/rag (median value of our normal range) and they were followed weekly to be retreated when Ca/Creat had reached baseline levels. The biochemical effects were maximal at d7. Mean (=E SEM) Ca/Croat levels fell, for the whole group, from 0.208 i 0.018 to 0.048 • 0.008 mg/mg on d7 and remained significantly (P < 0 01) lower than baseline up to d56. For the close-response analysis, we pooled the groups 3 0 4 0 mg and 90-120 rag. The fall in CalCreat was dose-related+ whether in intensity (d7) or in duration, Hydroxyproline excretion fell less, from 7.0 • 1.2 to 4.0 =E 0 3 mg x 100/mgCreat, and mean values remained significantly lower than baseline up to d49 The decrease in hydroxyproline was only significant in the group 90-120 rag.

Clinical s tudies - - T u m o r b o n e disease $77

Serum Ca levels fell f rom 9.3 =1:0.1 to 8.7 4- 0.1 mg/dl on d7, but the decrease was significant only after the close of 120 mg and no patient developed symptomatic hypocalcemia. There was a slight increase in Mg levels and a clearcut fall in Pi levels, more marked after 90-120 rag. Intact PTH levels increased f rom 29 4- 4 to 91 5- 13 pg/ml. They remained significantly elevated up to d28 and probably contributed to the fall in Ca/Creat. BGP concentrations did not change significantly. In summary, single pamidronate infusions, given at doses f rom 30 to 120 mg, induced expected, transient and dose-related changes in parameters of calcium metabol ism in patients with TIO. The dose of 90 mg could be the most adequate to inhibit bone resorption in this population.

• ] Treatment of Tumor-Induced Hypercalcemia with Pamidronate: What is the Optimal Therapeutic Scheme

J.J. Body, J.C. Dumon. Bone Metabolism Unit, Institut ,Z Bordet, Universit~ Libre de Bruxelles, 1000 Brussels, Belgium

We have reviewed our experience with pamidronate in 160 patients with tumor-induced hypercalcemia (TIH). Serum Ca was normalized in 92% of the cases (87% when Ca was corrected for protein levels). A mult iparameter regression analysis revealed that the hypocalcemic response to pamidronate was significantly (P < 0.01 ) influenced by initial Ca levels up to day 5-7 and, thereafter, only by the dose received, but not by the tumor type or by the presence of bone metastatic involvement. To confirm the dose effect, we divided the patients into three groups according to the median dose received, namely 0.5 mg/kg (n - 35), 1.0 mg/kg (n = 52), and 1.5 mg/kg (n - 73). The differences between the three groups were significant (P < 0.05) f rom day 5-7 until the end of the evaluation (day 22-26). Similarly, the success rate, considering Corr.Ca levels, was 80% for the groups 0.5 and 1.0 mg/kg combined, compared to 94% for the group 1.5 mg/kg (P < 0.05). The duration of normocalcemia was also more prolonged in the latter group, but the influence of the dose received could not be detected in patients with Ca levels <<12 mg/dl. The dose response relationship was present only in patients with an elevated index of tubular calcium reabsorption, explaining why patients with HHM respond indeed less well to relatively low doses of pamidronate. On the contrary, at the doses that we have used, the decrease in Ca levels or in fasting urinary calcium excretion were not influenced by the primary tumor site or the presence of bone metastatic involvement. In summary, our study establishes the existence of a dose response relationship for pamidronate in TIH over an efficient calcium-lowering dose range, at least in patients with Ca levels above 12 mg/dl. We recommend a dose of 1.5 mg of pamidronate/kg for the treatment of TIH, thus 90 mg for most individuals, except for patients with mild hypercalcemia for whom a dose of 1 mg/kg appears to be sufficient.

•0-• Comparative Evaluation of Markers of Bone Resorption After a Single Dose of Pamidronate in Patients with Breast Cancer-induced Osteolysis

J.J. Body, J.C. Dumon, E. Gineyts, ~D. Delmas. InstitutJ. Bordet, 1000 Brussels, Belgium, INSERM Unit 234, Lyon, France

We have evaluated markers of bone resorption in 19 patients with breast cancer-induced osteolysis who had received a single dose of pamidronate (Aredia| given at 30 mg (n - 4), 60 mg (n - 5). 90 mg (n - 5), or 120 mg (n = 5). We measured serum Ca, Pi, intact PTH (NI 10-50 pg/ml) and fasting urinary calcium (uCa, NI < 0.21 mg/mg Creat), hydroxyproline (Hypro, NI < 4.7 mgxlO0/mgCreat), pyridinoline (Pyr, NI < 78 nmol/mmol Creat), and deoxyPyr (DPyr, NI < 12 nmol/mmol Creat). Crosslinks were measured by f luorometry after extraction, cellulose chrom, and HPLC. All parameters were measured two t imes before therapy and then at weekly intervals for 10 (4-10) weeks. Baseline uCa was elevated in 6/19 patients, compared to 12/19. 17/19. and 16/19 for Hypro, Pyr, and DPyr, respectively. Correlations between markers of bone matrix resorption were significant (D-Pyr vs Pyr. rs - 0.71, P < 0,005; D-Pyr vs Hypro, rs - 0.64, P < 0.01 ) but this was not the case with uCa. Maximal effects were observed at d7 for all parameters. Mean ( i SEM) uCa decreased to 26% of baseline levels, f rom 0.20 4- 0.02 to 0.05 4- 0.01 mg/mg Creel, and remained significantly lower than baseline up to d56. The relative fall in markers of the resorption of bone matrix was less (50-76% of baseline), as Hypro fell f rom 7.3 :E 1.7 to 3. 7 =t. 0.6 mgxlOO/mg Creat, Pyr f rom 147 :t: 17 to 114 4- 17 and DPyr f rom 21.1:1:2.8 to 11.3:1:2.2 nmol/mmol Creel. The fall in Hypro

and DPyr remained significant up to d42, but only up to d14 for Pyr. Intact PTH levels increased from 29 4- 4 to 93 =E 13 pg/ml and remained higher than baseline up to d28. This marked and sustained increase in PTH levels could have contributed to the fall in uCa, and thus par[ly explain why uCa fell relatively more than markers of bone matrix. Hypro and DPyr appear to be the most adequate markers to monitor inhibition of osteolysis in breast cancer, but DPyr is more sensitive and specific than hydroxyproline.

~ - ~ Comparative Evaluation of Various Markers of Bone Formation in Tumor-Induced Hypercalcemia

J.C. Dumon, J.M. des Grottes, A. Cleeren, J.J. Body. Bone Metabolism Unit, Institut J. Bordet, Universit~ Libre de Bruxelles, 1000 Brussels, Belgium

Despite the recent introduction of new markers, the biochemical evaluation of bone formation (BF) remains difficult, especially after bisphosphonate therapy, notably because of the coupling between BF and bone resorption (BR). Tumor-induced hypercalcemia (TIH) is an interesting model for evaluating these markers, because of the reported uncoupling between BF and BR. We review here the evaluations that we performed in TIH, before and after successful therapy with pamidronate, of various markers of BE namely BGR AIk Phos and its bone isoenzyme (BAP), and PICP None of them correlated with markers of BR (fasting urinary calcium and hydroxyproline). Serum BGP was measured in 16 patients with TIH by two commercial assays, using bovine or human standards, bBGP (NI 0.8-5.1 ng/ml) or hBGP (NI 3.2-6.7 ng/ml). Scatter of baseline BGP levels was marked, but mean values were not different than NI and not significantly influenced by the presence of bone metastases (BM+). After pamidronate, bBGP fell f rom 4.4 4- 0.5 to 3.9 4- 0.7 and hBGP from 7.0 :E 1.2 to 6.0 4- 1.2 ng/ml (P < 0.05). AIk Phos and BAP were evaluated in 37 patients with TIH. Baseline BAP levels were increased compared to NI, and higher in patients BM+ than in patients BM-, 16.7 (9.4-47.1) vs 10.3 (6.9-18.5) p,g/L (P < 0.05). After therapy. BAP levels increased f rom 16.3• [12.3 (6.9-47.1)] to 22.2 :s 3.5 [12.7 (7.1-116)] p,g/L (P < 0.05). Lastly, PICP levels, measured in 22 patients with TIH, were also higher in patients BM+ than in patients BM-, 234 (106-5670)vs 145 (82-213)/~g/L (P < 0.05), but they did not increase significantly after therapy. In summary, these markers of BF were not lower than NI in TIH, and were differently affected by the presence of bone metastases and by bisphosphonate therapy. BGP could be more influenced by bone turnover than BAP or PICP, and the increase in BAP levels after therapy could reflect some effects of bisphosphonates on osteoblasts.

~ - ~ Bisphosphonates for Bone Metastases of Prostate Cancer: Evaluation of the Acute Biochemical Effects of Pamidronate

J.C. Dumon. M Oleffe, J.J. Body. Bone Metabolism Unit, InstitutJ. Bordet, Universit~ b'bre de Bruxelles, 1000 Brussels, Belgium

Bisphosphonates are used in patients with prostate cancer metastatic to bone and favorable effects on bone pain have been reported. However, the biochemical effects of bisphosphonates on calcium metabolism in these patients with essentially blastic metastases have been little studied. We report here a preliminary evaluation of the short-term effects of pamidronate infusion (15-90 mg infused over 1~6 hours) in 6 patients with prostate cancer metastatic to bone. Parameters were measured before treatment and after 1 week (d7), which corresponds to the t ime of maximal biochemical effects of pamidronate in tumor-induced osteolysis. Serum Ca (NI 8.5-10.3 mg/dl) fell f rom 8.1 :t= 0.3 (range 7.1-8.9) to 7.6 =h 0.6 (5.3-8.7) mg/dl. Ca corrected for protein levels (Corr.Ca) fell similarly f rom 8.9-t-0.2 (range, 8.4-9.3) to 8.1 4- 0.7 mg/dl (range, 6.0-8.8) but no patient presented a specific symptomatology of hypocalcemia. Intact PTH levels (NI 10-50 pg/ml) increased f rom 56 + 13 (range 18-110)to 102 :E 23 (58-147) pg/ml (P < 0.05). Serum phosphate levels did not change significantly (2.9 =t: 0.3 to 2.8 :E 0.3 mg/dl). The concentrations of 1,25 (OH) 2 vit.D 3 (NI 20-60 pg/ml) increased f rom 24 :E 6 to 42 • 10 pg/ml. Serum osteocalcin (BGR NI 1.1 5.1 ng/ml) increased from 3.9:1:1.0 (range 0.7 7.1 ) to 7.1 :E 2.0 ng/ml (range 1.0-10.4). BGP levels correlated with fasting urinary hydroxyproline excretion (Hypro, NI < 4.7 mg x 100/mg Creat) at baseline (r - 0.88; P < 0.05) but no longer at d7, as Hypro fell f rom a median value of 15.3 (range 2.2-46.0)to 10.2 (1.4-18.4). Fasting urinary calcium (uCa, NI < 0.21 mg/mg Creat) fell only slightly f rom 0.05 (0.01 0.28) to 0,02 (0.01-0.05). In

S 7 8 Second Workshop on Bisphosphonates

summary, pamidronate infusion in patients with osteoblastic metastases from prostate cancer can lower parameters of bone resorption. However, this therapy must be used cautiously, as it can markedly aggravate the tendency to hypocalcemia and secondary hyperparathyroidism consecutive to the pathologically increased bone formation.

• Efficacy of Pamidronate for Recurrences of Tumor-Induced Hypercalcemla According to the Tumor Type

I Louviaux, JC, Demon, J.J. Body. Bone Metabolism Unit, InstitutJ, Bordet, Universit# IJT~re de Bruxelle& 1000 Brussels, Belgium

Tumor-induced hypercalcemia (TIH) is now successfully treated by bisphosphonates in most cases. However, TIH often recurs and limited data indicate that retreatment is less successful than the initial administration. We have compared the responses to two therapeutic courses (A and B) of pamidronate (median dosage per course, 1 mg/kg; range. 0 5-2.0) in 27 patients retreated after 43 (14-340) days. Tumor types consisted of 11 breast. 3 head and neck, 3 lung, and 10 miscellaneous tumors. Mean (4- SEM) serum Ca fell f rom 12.9 4- 0.3 lo 9.3 • 0.2 mg/dl on d6 (median day of the nadir) after A, and from 13.2 4- 0.3 to 10.2 4- 0.3 mg/dl after B. The fall in Ca was significantly lower after B than after A (P < 0.05 from d2 to d6). However, this lower response was not significant in patients with breast cancer (12.4 _L 0.4 to 9.2 4- 0.2 mg/dl after A. and 13.9 4- 0.6 to 10.0 4- 0.6 mg/dl after B) but was evident in patients with other tumors (13,2 4- 0,5 to 9,4 4- 0.4 mg/dl after A, and 12.6 4- 0 4 to 10.3 4- 0.3 mg/dl after 8}. Thirteert patients were retreated by a third course (C~ of pamidronate (median dose, 1.5; range, 1.5-2.0 mg/kg 14 (8-17) days after B. Patients with breast cancer still responded, f rom 14.0 4- 0.9 to 11.0 4- 0.5 mg/dl, but the response in other tumors was weak, from 13.7 4- 0.5 to 12.4 4- 0.6 mg/dl. Fasting hydroxyproline excretion (Hypro; NI < 4.7 mg x 100/mgCreat) fell after A f rom 7.5 4- 0.8 to 4.8 4- 1.1 and after B f rom 9.6 4- 1.6 to 5.6 4- 0.8 (P < 0.05 vs A), but C had no effect (12.5 4- 2.0 to 13.4 4- 2.7). The increase in baseline Hypro levels was not accompanied by an increase in Ca excretion, suggesting an increased tubular reabsorption. In summary, the response to pamidronate for recurrences of T~H was progressively less, but the lower response was actually mainly observed in patients with tumors other than breast cancer. Our findings on uCa and Hypro suggest that this reduced response is due to increased bone resorption and increased tubular reabsorption of calcium.

[ - ~ Treatment of Metastatic Disease in Osteosercoma Patients, New Applications of Radiolabelled Poly- and Bisphosphonates

O. Bruland, M. Aas, ~.P. Solheim, M. Windern, J. Hole. Department of Medical Oncology and Radiotherapy, The Norwegian Radium Hospital, Montebello, 0310 Oslo, Norway

In the treatment of osteosarcoma, early detection, Iocalisation and estimation of the exact number of metastatic lesions is crucial since surgica~ removal of pulmonay metastases, usually the first and only site of detectable melastatic spread, may cure e significant perl ion of selected patients. In most osteosarcoma metastases, primitive bone matrix is abundant. Bone seeking radiopharmaceuticals may therefore be used to detect and treat such lesions.

In attempts to localize subclinical lung metastases peroperatively we have evaluated a hand-held gamma counter (Neoprobel000-system) in osteosarcoma patients selected for thoracotomy, 99mTc-methylene diphosphonate (MOP), used for conventional bone scanning was injected 14-16 hrs prior to surgery in 6 patients. Metastases less than one cm were detected using the probe. However, the ability to detect subclinical metastases requires either a focusing coll imator optimized tot the photon energy emitted f rom 99mTc, or the use of 1251 labelled bisphosphonates. At present we explore these strategies, both experimentally and clinically

The radioactivity content in metastases and various normal tissues was measured in a well counter. Tumor/normal bone ratios of 5-10 and tumor/lung ratios of 100-300 were obtained using 99m Tc-M DP These ratios are considerably higher than those previously obtained with radiolabelled tumor-associated mo~oclo~al antibodies. Further optimalization of this approach may be useful in two respects: Firstly, it may facildate a radical surgical procedure which is mandatory for successful t reatment Secondly, it may identify those patients who have widespread small metastatic foci,

rendering s.urgery in vain, In the latter group of patients, the selection of a suitable radionuclide conjugated to a bone seeking molecule, such as 153Sm-EDXMP, is an attractive treatment strategy to explore. A case where this approach was used will be presented.

•5] Aredia Infusions in Breast Cancer: A Randomised Phase III Trial to Assess Delay in Progression of Bone Metastases

PE Conte, J, Latreille. L. Mauriac, L. Koliren, J.M. Ford. Unite Operatzve Oncologia Medico, Opedale S. Chiara, via Rome 65, 56100 Pisa, Italy

To evaluate the efficacy and tolerability of Aredia (pamidrorate d/sodium), 295 breast cancer patients (pts) with lytie or mixed lyric/sclerotic bone metastases (BM) were randomised to receive Aredia 45 mg i,v. over 1 hour every 3 weeks plus standard chemotherapy or chemotherapy alone. 43 centres participated in 7 countries. Placebo infusions were not given to control pts,

The primary end-point was t ime to progression of disease (PD) in bone on serial X-rays and bone scans (imaging studies), To minimise bias, this endpoint was determined by 'blind' observers during an "extra-mural review" (EMR) of al~ imaging studies performed during the trial. Secondary end-points included the complications of BM - - pain, analgesic and radiotherapy requirements, pathological fracture and hypercalcaemia.

The first patient was enrolled Feb 90. Median r ime since the diagnosis of BM was 3 months, indicating early disease in many pts. The final analysis of t ime to PD in bone was performed on all data collected until 31.1.93, as showtt 1n the fe~owing table:

Aredia Control

No. of Pts random/sod 142 153 No. of Pts evaluated by EMR 116 108 No of Pts with PD in bone 72 69 Median time to PD in bone (days) 249 168

Time to PD in bone was increased by 48% in the Aredia group and the differer~ce between the groups was statistically significant {p = 0.02 Wilcoxon test). A marked improvement in bone pain was observed in 44% of Aredia pts and in 30% of controls (p = O.05 Chi-square). Median t ime to first radiotherapy was 697 and 571 days in the Aredia and control groups, respectively Aredia was well tolerated with no major toxicities reported. This treatment regimen comprising 3-weekly infusions of Aredia 45 mg over 1 hour significantly delays the progression of skeletal disease in breast cancer pts.

~ - - ~ Bone Gla-protain as Marker in Bone Metastases

G. Franc/n/, R. Petrioli, S. Mars/l/, A. Aquino. Medical Ontology Division - University of Siena -Italy

Bone Gla-protein (BGP) is the most abundant non collagenous protein in bone tissue, but its exact role is yet undefined,

One hundred and five patients with bone metastases were evaluated: ostee~ytic metastases (n -69 ; 12 with hypercaicemia~, osteob~astic or mixed bone metastases (n=36). The fol lowing markers of bone turnover were measured, serum bone Gla protein (BGP), serum alkaline phosphatase (AIk,Ph.), 99 mTc whole body retention (WBR%), 24-hour urinary hydroxyproline/creatinine ratio (UHOP/Cr) and 24-hour urinary calcium/croat/nine ratio (UCa/Cr).

All of the patients received 300 mg of dichloromethylene bisphosphonate (CI2MDP) i.v. for 7 days, Biochemical analyses were performed at basal conditions and on days 3, 7, 14, and 21 after start of parenteral CIzMDP administration,

In osteolytic metastases BGP was elevated in 10 cases (7 with hypercalcemia). A significant correlation between BGP and AIk.Ph., BGP and UHOP/Cr, and between BGP and WBR% was observed.

In mixed and osteoblastie bone metastases BGP was elevated in 33 cases with a significant correlation between BGP and WBR%. During parenteral treatment with CI 2 MDP, BGP showed a fast significant increase while AIkPh. decreased significantly at the beginning and increased thereafter.

These data indicate that BGP can be considered a good marker o1 osteoblastic activity. The absence of a significant correlation between BGP and Alk.Ph. in mixed and osteoblastic metastases and their different trend

Cl in ica l s tudies - - T u m o r b o n e disease $7 9

during the CI2MDP treatment can be considered as an expression of two different stages of osteoblastic activity

[-7-7-] Treatment of Hypercalcemia with YM175, a New Bisphosphonate: Elevated Threshold for Parathyroid Hormone Secretion in Hypercalcemia Patients

S. Fukumoto, ]~ Matsumoto, K. Takebe, ]~ Onaya, S. Etoh, H. Nawata, E. Ogata. Internal Medicine IV, University of Tokyo, Tokyo 112, Japan," Internal Medicine Ill' Hirosaki Universi~ Japan; Internal Medicine III, Yamanashi Universi~ Japan," Internal Medicine l' University of Occupational (1 Environmental Health, Japan; Japanese Foundation for Cancer Research, Tokyo 170, Japan

YM175 is a third generation bisphosphonate with a potent inhibitory effect on osteoclastic bone resorption in vivo and in vitro. Previous clinical studies in malignancy-associated hypercalcemia (MAH) patients have shown that serum parathyroid hormone (PTH) increases inappropriately for serum Ca during the course of treatment with bisphosphonates. However, the mechanism whereby PTH secretion is regulated in these patients remains unknown. The present study was undertaken to examine the clinical effectiveness of YM175 as well as to clarify the relationship between serum Ca and PTH secretion in MAH patients. Seventy-nine MAH patients were given a single infusion of either 2.5, 5 or 10 mg of YM175. Serum Ca levels reached a nadir between 4 and 6 days after treatment, and increased gradually thereafter. The effect of YM175 on serum Ca was dose-dependent, and YM175 was estimated to be about 6 t imes more potent than AHPrBR To examine the relationship between serum Ca and PTH level in detail, we divided 23 patients treated with 10 mg of YM175 into two groups: a group with 12 patients whose serum intact PTH level remained subnormal throughout the study period, and another group with 11 patients who showed an increase in intact PTH into normal or supranormal levels at somet ime during the follow-up. Serum Ca reached a nadir on day 6 in both groups, and the magnitude and velocity of reduction in serum Ca was not different between the two groups. However, serum Ca levels both before and 6 days after treatment were significantly higher in suppressed PTH group. Furthermore, there was a clear relationship between serum Ca and intact PTH levels: intact PTH started to increased when serum Ca decreased to about 11 mg/dl, whereas set point for the suppression of PTH level during the later increasing serum Ca phase was normal in these patients. These results demonstrate that YM 175 is a highly effective bisphosphonate for the treatment of MAH, and that serum PTH starts to increase at higher serum Ca levels than in normocalcemic subjects. The elevated threshold for PTH secretion observed in MAH patients is similar to the "hysteresis" phenomenon between serum Ca and PTH secretion previously reported in Ca-loaded normal subjects.

[-7--~ Mildronete in Paget's Disease. Assessment of its Therapeutic Efficacy in Partial Responders to Pamidronate

D. Gonz~lez, M. Pastrana, C. Mautalen. Secci&n Osteopat/as M~dicas, Hospital de Cl/nicas, C&rdoba 2351. (1120) Buenos Aires, Argentina

The use of oral bisphosphonates have been a step forward in the treatment of Paget's disease. However in patients with a extensive bone disease it has been difficult to achieve complete biochemical remissions of the serum alkaline phosphatase (SAP) and total urinary hydroxyproline (THP).

Ten patients (average age 70 ~ 5 years)who had been treated previously with oral Pamidronate wi thout reaching a complete biochemical remission were treated with Mildronate (IG-8801) a third generation dimethylated aminobisphosphonate. Patients had received Pamidronate during 38 �9 28 months divided in I to 7 cycles of 4 to 12 months. Average dose was 550 mg with a range of 400 to 800 mg per day. Mildronate was given in a dose of 100 to 200 mg per day during 6 :E 3 months. Four patients received two cycles of treatment.

"rhe results were as fol lows:

Parnidronate Mildronate Bass] Lowest Final Basal Final

SAP (UKA) 81+42 25• 33• 50• 16+ 6* THP (mg~4 h) 214+97 58• 98:555 101• 29+13 *,~

* p < 0.005 vs post Pamldronate; ~ p < 0.005 vs lowest during Pamidronate.

After Pamidronate therapy 3/10 patients attained normal SAP values and 4/10 reached normal THP values. After Mildronate 6/10 patients reached normal SAP values and 9/10 achieved normal THP values.

In addition Mildronate was given to seven patients who had not been treated before. All patients reached normal values of THP: basal 75:1: 52, final 17 ~: 3 mg/24 hs and SAP: basal 45 • 22, final 12 -{- 3 (UKA) after 3 months of treatment except one patient who normalized SAP values 6 months after stopping therapy.

Oral Mildronate appears to be a very effective therapy for Paget's disease, active on Pamidronate partial responders, probably due to its better gastric tolerance that allows to use a relatively higher dose.

•7-• Value of the Bisphosphonete DimethyI-APD in the Management of Symptomatic Prostatic Carcinoma Metastatic to the Skeleton

N.A.T. Hamdy, R.C.M. Pelger, A.A.B. Lycklama ~ Nijeholt, A.H. Zinderman, S E. Papapoulos. Departments of Endocrinology and Urology, University Hospital' Rijnsburgerweg 10, 2333 AA, Leiden, The Netherlands

Prostatic carcinoma (PC) is associated with osteoblastic bone metastases, but increased bone resorption has been consistently demonstrated on bone biopsies. Bisphosphonates have been given to patients with PC metastatic to the skeleton with variable success. We studied clinical and biochemical effects of the short and long-term administration of the new potent bisphosphonate, dimethyI-APD (d-APD) in 28 patients with hormone refractory PC and bone metastases (T2_4NxM+GI_3). All patients received 4 mg d-APD intravenously (iv) daily for 5 days and in 16 of the patients treatment was continued with oral d-APD 200 mg daily. Follow-up was for a min imum of 3 months (mean 7.8 4- 1.1 months). At the start of treatment all patients had severe intractable bone pain and 82% and 36% were using NSAID and/or opiates respectively. Serum alkaline phosphatase activity (ALP) was elevated in 92% of the patients and was positively correlated to urinary hydroxyproline excretion (r = 0.811, p < 0.0001), suggesting a coupling between bone formation and resorption.

A rapid significant suppression of bone resorption was observed in all patients after ivtreatment. This was sustained for 4-6 weeks in all patients, but reversed thereafter in patients not receiving oral therapy. During the first 3 months, there were no significant changes in biochemical indices of bone formation in either group. A significant decrease in bone pain was observed in the majority of patients (76%) during iv therapy, in whom characteristic night pain disappeared and the reduction in the consumption of NSAID and opiates was marked; in 30% of patients the use of opiates could be discontinued and in 50% the pain could be controlled with a reduced dose. At 3 months this response was significantly sustained in patients on continuous oral therapy (69%), compared to those treated only iv (p = 0.046). Treatment with bisphosphonates had no effect on long term survival (p = 0.27).

Our findings suggest, that intravenous d-APD fol lowed by oral main- tenance therapy provides a useful tool in the palliative management of symptomatic prostatic carcinoma metastatic to the skeleton.

[ 8 0 • E f f e c t s of Clodronete in Myelometosis

J.A. Kanis, E.~ McCIoskey, I. MacLennan, J. Orgee, M.N.C. Beneton. WHO Collaborating Centre for Metabolic Bone Disease, University of Sheffield, Sheffield S I 0 2RX,, UK

Accelerated bone destruction due to focal disease or diffuse osteoporosis is a common complication of myelomatosis. It gives rise to bone pain, fracture and hypercalcaemia. The mechanisms for increased bone resorption involve several disturbances of the normal remodell ing mechanism of the skeleton, principally by the activation of osteoclasts.

Despite recent advances in cytotoxic treatment, skeletal complications are important causes of morbidity in patients with myeloma. In addition, increased bone resorption persists despite the attainment of chemotherapy-induced plateau. For this reason, there has been a great deal of interest in the use of clodronate since it is a specific inhibitor of osteoclast mediated bone resorption. Unlike etidronate, it does not impair the mineral•177 of bone and is suitable for long-term use. There is now good evidence that the use of intravenously administered clodronate is useful in the management of hypercalcaemia, and indirect evidence to indicate that its calcium lowering effect is associated with a decrease in bone resorption. Moreover, the effect of clodronate appears to persist for

$ 8 0 Second Workshop on Bisphosphonates

the duration of exposure. For this reason clodronate has been assessed for the long-term control of hypercalcaemia, bone pain and pathological fractures. A long term double blind placebo controlled study from the Finnish myeloma group indicates that clodronate significantly decreases the appearance and rate of progression of osteolytic lesions. Our own larger study in the UK has shown that clodronate significantly decreases the rate of vertebral fracture, back pain and hypercalcaemia. These effects are masked at the t ime of initial chemotherapy due to the large beneficial effect of chemotherapy. They become apparent and significant at the time of progression of disease. For these reasons, clodronate provides a useful adjunct to the management of patients with myelomatosis.

~ - ~ Histomorphological Changes of Trabecular Bone in Patients with Primary Breast Cancer After Continuous Long Term Oral Treatment with ClaMBP

B. Krempien. I. Diel. Department of Pathology, University of Heidelberg, INF 220, D69120 Heidelberg

Iliac creast biopsies and bone marrow aspirates were obtained in 200 patients with breast cancer at time of primary surgery in order to detect micrometastatic tumor cells within the bone marrow.

Patients were then given CI2MBP orally (1600 mg/d) for 2 years. After this period a second biopsy was taken and both biopsies were analysed by histomorphometry. In 30 patients (average age 57 yr), who as so far have undergone a second biopsy, we found a highly significant increase of bone mass (increase of volumetric density, of trabecular thickness, decrease of intertrabecular distance).

Osteoid volume had increased slightly, Trabecular interconneetivity did not change, No bone marrow metastases or tumor osteopathy could be seen in spite of tumor cell positivity of the initial marrow aspirate of 35%.

Our data show that continuous oral administration of CI2MBP will markedly increase bone mass in peri- and postmenopausal women. The increase of trabecular bone was accompanied by a small increase of osteoid. Two years after surgery of the primary tumor no bone marrow metastases and tumor osteopathy were detectable histologically. These findings might support our hypothesis that an early and continuous oral treatment with CI2MBP will decrease the incidence of bone marrow metastases and tumor osteopathy. However, this hypothesis can only be proven, once more patients will have undergone their second biopsy after treatment.

[ - ' ~ Effect of Clodronate on Biochemical Parameters of Bone Metabolism in Women with Breast Cancer

M,E. Martinez, F. Pastrana *, C.M. Jariego, M,J. Sanchez-Cabezudo. Ginecology Services, Hospital La Paz, Madrid, Spain," * Biochemistry, Hospital La Paz, Madrid, Spain

The efficacy of bisphosphonate in the treatment of bone resorption in breast cancer has been proven. A difference between acute and mid term effects on biochemical markers of bone metabolism has been observed.

In order to evaluate the short term and mid term effects of clodonatre on biochemical markers of bone and on ealcidiol serum levels, we studied 19 selected women with breast cancer and bone metastasis. All were normocalcemic and had normal renal function throughout the study. 12 women were treated with sodium clodronate (mebonate) (300 mg IV during five days and 1600 mg oral during six months). 7 women were treated with calcitonin (control).

Blood levels of ionized calcium, PTH, calcidiol (CD), osteocalcin (OC) and urinary hidroxyproline (HP) were measured in basal conditions and at 2, 4, and 6 months.

Results: Months Ionized Ca PTH CD OC HP

B 1 25 36 18 3.8 36 2 1.23" 47" 15" 3.3* 20" 4 1,24" 57" 12" 4.1 24* 6 1 25 44" 10 w 4.2 24"

~ p < 0.o5 vs Basal HP and OC decreased in control group.

At short term (2 m), clodronate induced a decrease in osteoblastic (OC) and osteoclastic (HP) biochemical markers. Lower ionized calcium and secondary increased PTH serum levels were also found.

Later, ionized calcium and OC levels returned to baseline values but HP

remained lower despite higher PTH serum levels. Calcidiol serum levels decreased during the study.

Conclusion: Continuous clodronate administration induced in the short term (2 months) bone resistance to PTH induced osteoblastic and osteoclastic activities. Osteoblastic function is later (6 m) compensated. Clodronate induced a decrease in vitamin D levels and evendepletion in some patients.

•3] Oral Clodronate Reduces Skeletal Morbidity in Multiple Myelomatosis

E.V. McCIoskey, I. MacLennan, M. Grayson, K. Chapman, J.A. Kanis. WHO Collaborating Centre for Metabolic Bone Disease, University of Sheffield, Sheffield $I0 2R)(, UK

We have examined the effect of oral clodronate on the incidence of skeletal morbidity in a double-blind placebo controlled trial forming part of the Vlth MRC Myelomatosis Trial. A total of 609 patients (255 women. 354 men, mean age 61.6 years, range 32-75) were randomized at the time of diagnosis to receive either elodronate 1600 mgs daily (n = 304) or an identical placebo. (n = 305) in addition to chemotherapy and/or radiotherapy. Sixty one patients (10%) never received treatment with either cledronate or placebo and are excluded from this analysis.

There were no significant differences in survival, t ime to plateau or time to progression between patients receiving clodronate or placebo. Chemotherapy and/or radiotherapy at diagnosis were associated with a large, significant reduction in skeletal morbidity. For example, the prevalence of back pain decreased from 49% at entry to 17% at 6 months in the placebo wing (p < 0.0001). There was no significant difference in the initial response of skeletal symptoms in the clodronate and placebo- treated patients. In contrast, at the time of disease progression, the proportion of patients experiencing a deterioration in performance status was significantly lower in those receiving clodronate (23% vs 46%. p - 0.005). The proportion of patients experiencing increased back pain at progression (as judged by an increase in back pain score of 2 or more) was also significantly reduced by clodronate (7.5% vs 21.4%, p < 0.05). A similar trend was observed in the incidence of rib pain (4.0% vs 11.8%).

The rates of new vertebral fractures in the first year of treatment were similar in placebo and clodronate-treated patients (84 vs 94 vertebral fractures/100 patient years). However, the proportion of patients experiencing new vertebral fractures after the first year of study was significantly lower in the clodronate wing (35% vs 49%, p = 0.07) with fracture rates of 33 and 54 new fractures/100 patient years respectively (p < 0.003).

We conclude that long-term oral clodronate modifies the progression of skeletal disease in multiple myeloma with a consequent decrease in the associated morbidity. It provides a useful adjunct to the clinical management of patients with myelomatosis.

[ - - ~ Tumour-lnduced Hypercalcaemia; Relationship Between Mechanisms of Hypercalcaemia and Response to Pamldronate Treatment

R. Murray *, P Ibarra-Palacios **, V Grill *** . A. Kandra **, S. Meilenbrock**, • Pitt*, J. Martin *** . *PeterMacCa//um Cancer Institute, Me/bourne, Australia; ** C/ba, Medical Department, Basle, Switzerland," *** St. Vincent's Institute of Medical Research and St. Vincent's Hospital, ['itzroy, Australia

Turnout-induced hypercalcaemia (TIH) is primarily due to an increase in bone resorption and to a lesser degree, in some tumours, to increased tubular reabsorption of calcium especially in squamous cells. Pamidronate is a powerful inhibitor of bone resorption. A prospective, open study was performed to assess the relationship between the pathophysielogical mechanisms of TIH and the calcium lowering response after a single infusion of 45 mg Aredia, The main objective was to assess the relationship between the responders and non-responders to the pretreatment mechanisms of ] IH determined on the basis of tubular maximum for calcium reabsorption corrected for sodium excretion (TmCa;NaCorr) and secon- darily, to measure the renal phosphate threshold (TMP04/GFR), cyclic AMP (cAMP) and urinary cAMP (UcAMP), serum parathyroid (iPTH) and PTHrR The study group consisted of 80 patients with histologically proven malignancy and TIH with corrected serum calcium > 2 8 0 mmol/L after rehydration. Serum calcium and biochemical parameters were measured

C l i n i c a l s tud ie s - - T u m o r b o n e d i sease S81

daily, iPTH and PTHrP were measured on Day 0 and Day 6. 76 patients were eligible in the efficacy analysis. Patients were retrospectively divided into three groups according to their response in terms of corrected serum calcium to pamidronate with an overall response rate of 91% [67% corn plete responders (CR) and 24% were partial responder (PR)]. Correlation between the pretreatment biochemical parameters and post-treatment response were assessed using the Spearman Coefficient. An increase in mean baseline TmCa:NaCorr and a low TmP04/GER were observed in the three categories groups independent of the response to treatment with Aredia. For all individual parameters whether univariate or multivariate, a low correlation coefficient was shown in relation to treatment with Aredia and/or achieving normocalcaemia. Among these variables, UcAMP, TmCa Corr and TraP04 were better correlated than PTH and PTHrI~ Elevated PTHrP (> 3 pmmol/L) was observed in 69%, 78% and 71% of the CR, PR and NR groups, respectively. No change in PTHrP was found over t ime in the three response groups. Pretreatment PTH was suppressed in the R and PR groups and a rapid rise in PTH concentration was observed in these two groups, demonstrating a rapid recovery of PTH secretion, even fol lowing prolonged suppression.

~ - I A Randomlaed Double-Blind Comparison of Single Infusions of Pamidronate or Clodronete for Hypercalcaemla of Malignancy

O.P. Purohit *. C. Anthony *, J. Owen *, J. Kanis **, R. Coleman *. * YCRC Dept. of Clinical Oncology, Weston Park Hospital Sheffield, $10 2S J, England," **Human Metabolism Et Biochemistr)~, Royal Hallamshire Hospital, Sheffield, SlO 2S J, England

In conjunction with rehydration, the bisphosphonates are the treatment of choice for hypercalcaemia of malignancy. Single infusions of either pamidronate or clodronate are usually effective but a direct comparison of the two agents given at the highest doses commonly used has not been performed. 41 patients (15 breast, 12 squamous carcinomas, 4 Iymphomas, 4 bladder, 2 prostate and 4 others) with hypercalcaemia of malignancy (corrected serum calcium >2.7 retool/I) persisting after 48 hours of saline rehydration were randomly allocated to receive a four hour intravenous infusion of either pamidronate 90 mg or clodrenate 1600 mg. No other systemic anticancer treatment was prescribed.

There were no significant differences in the post-hydration serum calcium values (mean 3.17 mmol/I for pamidronate and 3.06 mmol/I for clodronate), tumour type or frequency of bone metastases between the two treatments, One patient on each treatment died within 2 days and was not assessable for response. 19/19 (100%) patients achieved normocalcaemia fol lowing pamidronate and 16/20 (80%) with clodronate. The median t ime to achieve normocalcaemia was 4 days (range 2-14) for pamidronate and 3 days (range 2-6) with clodronate. The median duration of normocalcaemia was 28 days (range 10-28+ days) after pamidronate and 14 days after clodronate (range 7-21 days) (p <.01). Two patients who failed to respond to clodronate were successfully treated with pamidronate and achieved normocalcaemia for 14 and 28+ days respectively. 2 patients experienced fever after pamidronate but no significant toxicity was observed with either treatment.

We conclude that both agents are effective in the management of hypercaleaemia of malignancy. At the doses studied, the effects of pamidronate are more complete and longer lasting than those of clodronate,

We would like to thank all the Consultant Medical Staff at Weston Park Hospital for referring patients for inclusion in this clinical trial.

~ - ~ The Effect of a Single High Dose Intravenous Treatment with Pamidronate on Metastatic Bone Pain and Quality of Life

O.P. Purohit, C. Anthony, R. Coleman. YCRC Dept. of Clinical Oncology, Weston Park Hospital Sheffield, England

The bisphosphonates are able to relieve pain f rom metastatic bone disease and, when given intravenously, may promote bone healing of lytic metastases. In this study, the aim was to assess the acute effects of a single "high dose" intravenous treatment with pamidronate on pain. mobility, analgesic consumption and quality of life (QOL). 34 normocalcaemic patients with painful progressing bone metastases (22

from breast, 5 prostate and 7 others) received a single intravenous infusion of 120 mg pamidronate as palliative therapy No other systemic therapy or drugs known to influence bone metabolism were administered during the study. Patients subjective response to treatment was assessed weekly with a pain questionnaire recording a composite of pain intensity, mobility, performance status and analgesic consumption, In addition, patients completed the Rotterdam Symptom Check List (RSCL) for measurement of QOL.

The mean reduction in the pain questionnaire score (recorded on at least two occasions) was 25% (s.e.m. 3%. range 0-78%). 20 patients (59%) showed a >>20% improvement and were classified as responders. The median duration of symptomatic response was 12 (range 4-24+) weeks. The responding patients showed a reduction in RSCL score ( improvement in e e L ) f rom 35% before treatment to 27% at 6 weeks but no significant improvement was noted in non-responders. 21 patients were treated with pamidronate when their symptoms deteriorated again. 8/15 responders showed a second reduction in pain score of >>20% but this was not seen in any of the 6 non responders. 5 patients have remained well with no additional treatment for their disease other than repeat infusions of pamidronate every 3 6 months. Treatment was well tolerated, 8 (24%) experienced fever after the first treatment only and 4 had asymptomatic, biochemical evidence of hypocalcaemia.

The acute inhibition of osteoclastic bone resorption induced by a single high dose treatment with pamidronate can provide useful palliation for patients with bone metastases. Responding patients may be retreated as symptoms dictate to good effect.

We would like to thank all the Consultant Medical Staff at Weston Park Hospital for referring patients for inclusion in this clinical trial.

• 7 Effect of a Single High Dose Treatment with Pamidronate on Markers of Bone Resorption in Patients with Bone Metastases

O.R Purohit *, I. Dickson **, R. Coleman *. * YCRC Dept. of Clinical Oncology, Weston Park Hospital, Sheffield, England," **Dept. of Biochemistry, Brunel Universi~', Middlesex, England

30 patients with painful progressing bone metastases received a single intravenous infusion of 120 mg pamidronate as palliative therapy. No other systemic therapy or drugs known to influence bone metabol ism were administered during the study. The effect of treatment on various markers of bone resorption was measured every two to four weeks until additional bisphosphonate or anticancer treatments were required. Urinary excretion of calcium (UCCR), hydroxyproline (OH-Pr/Cr), hydroxylysyl- pyridinoline (HP/Cr) and lysyl-pyridinoline LP/Cr) were measured fol lowing collection of the fasting second voided early morning sample of urine. HP and LP were measured by high performance liquid chromatography. Serum was also collected for routine biochemistry and measurement by radio-immunoassay of carboxyterminal cross-linked telopeptide of type I collagen (ICTP). Over a twelve week period of evaluation, the median % of baseline values for the different resorption markers were:

Week 0 2 4 6 8 12 UCCR 100 23 30 41 50 79 OH-Pr/Cr 100 63 63 77 86 90 HPICr 100 65 72 76 - LP/Cr 100 52 60 76 - ICTP 100 105 117 104 -

Pamidronate induced a >10% reduction in UCCR in 29/30 (97%). OH-Pr/Cr in 26/30 (87%), HP/Cr in 16/21 (76%), LP/Cr in 20/21 (95%) and ICTP in 10/21 (45%). An increase in the HP/LP ratio f rom a mean of 7.4 (s.c. 0.64) at baseline to 13.5 (s.e. 2.93) after 2 weeks suggests that in the context of bone metastases, HP is a less specific marker of bone resorption.

For assessing the biochemical effects of bisphosphonates in metastatic bone disease, the urinary excretion of calcium and lysylpyridinoline appear to be the most useful markers of bone resorption. However, none of the changes in resorption markers were able to predict which patients would derive symptomatic benefit with pain relief after pamidronate.

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~ 8 ~ Clodronate has Effects on Osteoblasts and Osteoclasts. Monitoring the Effect Using PINP and ICTP Assays

L. Risteli *, J. Risteli *, M. Laakso **, R. Lahtinen **, R Virkkunen *** , I. Elomaa *** . * Departments of Medical Biochemistry and Clinical Chemistry, University of Oulu, Finland, ** Department of Medicine, University of Kuopio, Finland; *** Department of Radiotherapy and Oncology, University of Helsink~ Finland

In a recent trial we reported that clodronate reduced the incidence of bone lesions, fractures, pain and hyperealcaemie in multiple myeloma. All patients received melphalan-prednisolon therapy and were randomized to use either elodronate or placebo for two years one month after starting MP [1]. Then we showed in 244 patients of this trial that serum assay for cross-linked carboxyterminal telopeptide of type / collagen (ICTP) was a good follow-up marker for osteolysis and the best prognostic factor in multiple myeloma [2].

Here we determined serum assay of aminoterminal propeptide of type / procollagen (PINP) [3] from the same patients, since it has been assumed that the effect of clodronate on the osteoblasts osteoclasts is mediated through the osteoblasts [4]. PINP is the synthesis product of osteoblast which is produced earlier than the other osteoblast products like AP and osteocalcin [5]. Our results indicate that PINP and ICTP levels decreased only in the clodronate group. The significant difference between the study groups was seen earlier in the PINP than ICTP levels, suggesting that clodronate have actions not only on osteoclasts but also on osteoblasts (Table):

Time Placebo Clodronate (me) ICTP PINP ICTP PINP

rnean• rnean4-SEM n @ mean• mean4-SEM n @

0 7.844-0.53 61.54-3.2 119 8,38+0.80 68.94- 44 125 1 # 7.904-1.51 74.64-7.2** 47 7.134-0.80 95.95:14 1 " * 66 4 7.61:1-0.88 76.2• 71 6.56• 73.4+10.1 72 7 6.75:1:0.70 75.94-5.0 *** 71 5,614-0.37"* 53.44- 5.1"* 72

25 6.95• 69.34-7.5 43 4584-032** 37.2J- 35*** 47

# Time of randomization to placebo or clodronate. Statistical testing: *Wilcoxon test within the groups ( ' *p < 0.01; ***p < 0,001). @ Mann-Whitney U-test between the groups: ICTP: (~7 me p = NS; 25 me p < 0.01. PINF~" 0-1 me p = NS; 4 me p < 0.01; 7 mop < 0.001; 25 me p < 0.001

[1] Lahtinen et al, Lancet 1992; 340: 1049-52. [2] Risteliet el, J Bone Min Res 1993; 8(Suppl. 1): 826. [3] Kauppila et al Calcif Tiss Int 1993; 52(Suppl. 1): 362. [4] Fleisch, In Bisphosphonates in bone diseases, 1993, p. 43. [5] Steinetal. FASEBJ1990; 4: 3111-23.

~ 9 ~ Single Infusions of DimethyI-APD in Malignancy-Associated Hypercslcaemia; Factors Affecting the Calcium-Lowering Response

D.H. Schweitzer. N.A.T. Hamdy, S.E. Papapoulos. Department of Endocrinology, University Hospital, Rijnshurgerweg 1 O, 2333 AA Leiden, The Netherlands

It has been reported that the calcium-lowerlng response to bisphosphonates may be impaired in patients with high plasma PTHrP levels. We recently reported that serum 1.25-dihydroxyvitamin D (1,25-DHD) concentrations are not suppressed or elevated in about 25% of patients with solid tumours. In the present study we examined the calcium-lowering effect of dimethyI-APD and the relationship between this response and initial concentrations of PTHrP and 1,25-DHD in 27 patients with malignancy- associated bypercalcaemia. 20 patients participated in a randomized double-blind, study comparing the effects of a single infusion of 10 vs. 20 mg d-APD whereas the rest were treated with a single infusion of 20 mg d-APD.4 patients were treated for a second hypercalcaemic episode. Evaluation was performed one day after normalization of adjusted serum calcium concentration or 5 days after the administration of the bisphosphonate. In the whole group the initial mean serum calcium, PTHrP and 1.25-DHD concentrations were 3.49 4- 0.08 mmol/I, 5.46 4- 0.86 pmol/I and 63 4- 7 pmol/1, respectively. After treatment these values were 2.50 4- 0.04 (p < 0,001 ), 4.69 4- 1.09 (ns)and 92 4- 12 (p < 0.05). In 21/27 patients serum calcium concentration normalized within 5 days while in the remaining 6 patients it decreased from a mean of 3.86 to a mean of 2,86 mmol/I.


Single infusions with 10 mg d-APD were equally effective as infusions with 20 rag, There was a significant correlation between the serum calcium concentrations after treatment and initial plasma PTHrP (r - 0.55 p < 0.001) No such relation with initial serum 1,25-DHD concentrations was found. These results demonstrate that a single infusion of 10 mg d-APD is very effective in the treatment of malignancy-associated hypercalcaemia. Our studies further confirm the relation between initial plasma PTHrP values and response to bisphosphonate treatment. Although there was no relation between the calcium-lowering response and initial values of 1.25- DHD in the whole group, the patients less likely to respond adequately to bisphosphonate treatment are those with high plasma PTHrP and serum 1,25-DHD concentrations before t reatment

P A G E T ' S D I S E A S E

Fg-0-] Comparative Biological and Radiological Responses to Oral Etidronate and Tiludronste in Paget's Disease of Bone

J.R Devogelaer. R Stasse, J. Malghem, B. Maldague, C. Nagant de Deuxchaisnes. Departments of Rheumatology and Radiology, St-Luc University Hospital, avenue Hippocrate 1 O, B-1200 Brussels, Belgium

In a double-blind study, we have assessed the action of daily etidronate (E : 400 mg) and tiludronate (T : 400 mg) given orally for 6 months to patients with active Paget's disease of bone (serum alkaline phosphatase (SAP) at least twice the upper limit of normal (60 IU/I)), and with assessable radiological lesions. 12 patients (9 M, 3 F, aged 66.0 4- 2.3) received blindly either E (n = 5) or T (n = 7). The initial characteristics of the patients were similar. No patient weighed less than 50.5 kg. The biological response was similar in both therapy groups as far as serum calcium, SAP and fasting urinary Ca/creat ratios were concerned. Serum phosphate was slightly but significantly more elevated after 3 and 6 months of therapy in the E group (p < 0.05). The ~ray films were read blindly by 2 radiologists (JM, BM) prior to and after 6 months of therapy Their judgment was concordant. 19 bones could be evaluated, 3 with predominantly lytic (L) lesions, 4 with mixed L + condensing (C) lesions, 9 with predominantly C lesions, and 3 with purely C lesions. After 6 months, 7 bones appeared denser, 5 showed questionable densification, 3 remained unchanged, 2 showed some questionable increased resorption and 2 a frank resorption. After breaking the code, all the negative evolutions were confined to the E group, and all the positive evolutions to the �9 group. One patient from the E group developed symptomatic microfissures of her pagetic tibia.

In conclusion, E and T are able to produce similar biological responses in Paget's disease of bone. However, when assessing the bone quality, the biological response has to be compared with the radiological response. The only positive radiological modifications were observed in patients on T, while a clearcut worsening was observed exclusively in patients on E.

[ ' ~ Combined Dldronel and Colchicin Treatment in Paget's Disease

J. Don&th, M. Szil~gyi, E. Piroska, B. Fornet*. Nationallnstitute of Rheumatology and ~ Physiotherapy and International Medical Center, Budapest, Hungary

Paget's disease of bone often causes pathological fractures. There is no formal evidence that medical treatment, with the calcitonins, the diphosphonates or any other agent, significantly alter the natural history of fractures.

According to our own observations eight of 40 patients with Paget's disease had fractures. Here we present a particularly florid case with incomplete fracture of the tibia developing during a long lasting treatment of calcitonin. In contrast, due to the insufficiency on the results obtained with calcitonin, a combined treatment with low doses of Didronel (5 mg/kg daily) and Colchicin (2 x 0.5 mg daily) for six months had beneficial effects. There was a remarkable decrease of pains evaluated on a visual analogue scale, the serum alkaline phosphatase activity has been normalized. As the consequence of the applied medication the bone scans have shown decreased activity of process, while the CT depicted the sclerotisation of the fractured tibia, The beneficial clinical and biochemical responses to this combined treatment suggest that it may be used to suppress very active Paget's disease, with pathological fractures.

C l i n i c a l s t u d i e s - - V a r i o u s d i s e a s e s $83

[ 9 ~ Intravenous Pamidronate in Paget's Disease; Severity-Dependent Biochemical and Radiographic Responses

D.H. Gutteridge, G.O. Stewart, B.G.A. Stuckey, R.L. Prince, G.N. Kent, C.I. Bhagat, R.W. Retallsck, R.I. Price, L.C. Ward, R.I. Thompson, G.C. Nicholson. Sir Charles Gairdner Hospital Nedlands, 6009, West Australia 8 Fremantle Hospital, Fremantle, WA

Severity-based daily dosage was used to treat 71 patients (pts). Mild (fasting hydroxyproline excretion (HypE) < 5.0 umol/IGF) had total 120 mg (Gp I); moderate (HypE 5.00-9.99) 180 mg (Gp II); severe (HypE >> 10) 240 mg. Serial studies for 2 yrs included pain scores, plasma and urine bone turnover indices, bone densitometry at 3 sites, and x-rays of lytic lesions in long bones and skull.

Increasing dosage (and severity) produced increasing mean % fall in HypE at 6 me - - Gp I 45%; Gp II 32%, Gp Ill 23%. However. increasing severity was associated with increasing resistance to normalization of HypE at 6 me - - Gp I 88%. II 40%, Ill 0%. Bioehem. relapse (pl. alk. phos. > 150% of 6 me level) was severity dependent in spite of increasing dosage - - at 2 yrs Gp I 17%, Gp II 27%, Gp III 62%. Of 22 pts with lytic lesions, all were in remiss!on at 3 me; relapse at 2 yrs was again severity-dependent - - Gp 113%. Gp II 43%, Gp Ill 57%. In 6 out of 9 pts in lyric relapse, lyric preceded biochemical relapse,

Skeletal pain of three types (Pagetic bone, Pagetic joint or unrelated) at 6 me showed a sign decrease in all 3 types. Pagetic bone showed the lowest scores and longest persistence of relief.

Bone density at the ultradistal (60% trabecular) forearm showed a severity dependent response - - the greatest loss being in Group III with 7.8 -~ 1.7% (M 4- SEM) decrease at 6 me and later improvement to a 1.2 4- 1.8% decrease. At a more proximal cortical site mean decline at 8 months was 5.3 4- 1% with persistence of loss. There was no bone loss in Group I; Group II was intermediate. At the hip sites in non-Pagetic bone there were no significant changes.

Pamidronate is safe, well tolerated and effective. In spite of higher dosage in more severe disease, increasing severity had a higher incidence of earlier biochem and radiol relapse. Higher initial dosages in Gps II ~ III are proposed for future studies.

•9• Utility of Galactosyl Hydroxylysine for Assessing Pamidronate Treatment of Paget's Disease of Bone

M. Michalsky. J. Stepan. H. Wilczek, Z. Rosenova, I. More. 3rd Department of Internal Medicine, Charles University Faculty of Medicine, Unemocnice 1, CZ 128 O0 Prague, Czech Republic, and University of Trieste, Italy

The urinary excretion of the collagen degradation product galactosyl hydroxylysine (GHL) has been proposed as specific and quantitative index of bone resorption. We compared the value of the morning urinary excretion of GHL to that of hydroxyproline (OHP) in 23 patients with Paget's disease of bone. GHL excretion rate was significantly correlated with that of OHP (r = 0.95. P < 0.001) and with the activity of bone isoenzyme of serum alkaline phosphatase (BALP) (r - 0.90, P < 0.001). The rate and degree of suppression of bone resorption were monitored in 10 of the patients treated with intravenous pamidronate at a dose of 30 rag/day for 3 days by measurements of GHL, OHFt Treatment with pamidronate resulted in a decrease in both indices of bone resorption in the Z-score f rom baseline values (P < 0.001) was similar for both indices (GHL 9.8 4- 2.3 and 5.5 + 2.3, OHP 10.1 -4- 2.4 and 5.1 4- 2.4) and it occurred in all the patients. All patients showed a marked improvement in clinical symptomatology (pain and mobility). The results suggest that (1) pamidronate is an effective alternative for treatment of patients with Paget's disease of none, (2) GHL is a parameter of bone resorption a~ sensitive as OHP in Paget's disease if bone, (3) GHL is a marker of disease activity in Paget's disease of bone, (4) the assay of GHL is easier, faster, and of lower cost that the OHP determination. It is concluded that this convenient GHL should be valuable for the clinical investigation of bone resorption in Paget's disease of bone.

~ - ~ Evidence that oral tiludronate has a highly effective therapy of Paget's disease of bone

J.Y. Reginster, C. Roux, C. Picot, I~ Franchimont, M. Dougados. Centre Universitaire d'lnvestigation du M~tabolisme Osseux et du Cartilage Articulaire, University of IJ'~ge, Ll~ge, Belgium; Service de Rhumatologie, HSpital Cochin, Paris, France," Sanofi Recherche, Paris, France

Tiludronate (Chloro-4-phenyl-thiomethylene bisphosphonate), a third generation bisphosphonate, is able to reduce bone turnover wi thout causing side effects or mineralisation impairment. Its efficacy and safety in Paget disease of bone were evaluated in 3 clinical trials.

(a) 149 patients were included in a double-blind randomized placebo- controlled trial and randomly assigned to a daily dose of 100, 200, 400 or 800 mg of oral ti ludronate (capsule) or a placebo. Beginning at a dosage of 200 mg per day, there was a direct dose-dependent effect on the reduction of serum alkaline phosphatase activity (SAP) and fasting urinary excretion of hydroxyproline/creatinine (OH-Cr). Because of its significant better antiresorptive effects and greater analgesic properties combined with an excellent clinical and biochemical tolerance, the 800 mg daily dose of tiludronate appears to be optimal for the treatment of Paget's disease of bone.

(b) 128 patients were included in an open label uncontrolled trial where they received a daily dose of 400 mg of oral ti ludronate (using tablets with a bio-availability twice higher than the capsules), There was a reduction of initial SAP activity after 3 months ( -47.2 :L 2.2%) and 6 months ( -58.3 4- 2.3%). We concluded that the dose of 400 mg daily (tablets) of this new formulation appears to be a satisfactory ti ludronate regimen for the treatment of Paget's disease of bone.

(c) 234 patients were enrolled in a double-blind, randomized, multicenter comparison of 6 months where they were randomly allocated into one of three treatment groups: ti ludronate (tablets) 400 mg/d for 3 months, then placebo for 3 months (group 1 ), ti ludronate 400 mg/d for 6 months (group 2) or etidronate 400 mg/d for 6 months (group 3). At 3 months, there were significantly more responders with ti ludronate (57.4%) than with etidronate (13.9%) (p < 0.0001). At 6 months there was no statistically significant difference between the two tiludronate groups but the number of responders at 6 months was significantly higher with ti ludronate for either 3 (60.3%) or 6 months (70.1%) than with etidronate (25.3%).

The results of these three studies indicate that ti ludronate is highly useful in the therapy of Paget disease of bone and that the daily dose of 400 mg for either 3 or 6 months appears to be an optimal approach of this disease.

V A R I O U S D I S E A S E S

~ Clinical and Biochemical Responses to Single Infusion of Pamidronate in Active Rheumatoid Arthritis; A Double-Blind, Placebo-Controlled Study

E Eggelmeijer, S.E. Papapoulos, B.A.C. Dijkmans, F.C. Breedveld. Departments of Rheumatology and Endocrinology, University Hospital Rijnsburgerweg 10, 2333 AA Leiden, The Netherlands

Pamidronate (APD) suppresses bone resorption but it has also been reported to have anti-inflammatory properties. We studied the clinical and biochemical responses to two different doses of pamidronate given as single iv infusions to patients with rheumatoid arthritis (RA). Thirty patients with active RA were admitted to a rheumatology clinic and were randomly allocated to receive a single infusion of placebo, 20 or 40 mg of pamidronate in 500 ml normal saline over three hours. Patients were fol lowed for 3 weeks. Pamidronate treatment induced an immediate reduction in urinary calcium and hydroxyproline excretions (corrected for creatinine excretion) to a maximum of 29% and 64% of initial values, respectively. This reduction was sustained during the 3-week fol low-up and did not differ between the two groups who received pamidronate. There were no significant changes in either of these parameters in the placebo-treated patients. Compared to placebo, pamidrenate treatment improved also significantly clinical parameters of disease activity. In the patients treated with 20 mg iv there was a significant decrease in the number of swollen joints and in the Ritchie index. In the patients treated with 40 mg iv apart f rom significant changes in these parameters there were additional significant decreases in ESR (from 82 :E 24 to 69 :E 29 mm; mean -I-SD; p < 0.01) and in serum C-reactive protein (from 68 5:25 to 53 • 26 mg/I; p < 0.01 ).

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A single intravenous infusion of pamidronate to patients with active RA suppresses bone resorption effectively and reduces significantly disease activity after 3 weeks. The latter effect appears to be dose- dependent and suggests that for the induction of cellular responses with pamidronate doses higher than those required for suppression of bone resorption should be given. Thus bisphosphonates may not only prevent the bone loss associated with active RA but may also affect directly the inflammatory process. Effective treatment schedules need, however, to be established,

~-6~ Fibrous Dysplasia in Children: Response to Pamidronate Infusion

F.H. Glorieux, G. Lanoue, R. Travers. McGi//University and the Genetics Unit, Shriners Hospital for Crippled Children, 1529 Cedar Ave, Montreal (Qu6bec) Canada H3G 1A6

Polyostotic fibrous dysplasia is a sporadic crippling condition characterized by disturbance of skeletal growth, frequent bone fractures and ensuing bone deformities. The anatomical lesions include trabeculae made of woven bone, high remodeling and intense marrow fibrosis. These histologic changes are akin to those seen in Paget's disease. By analogy with the latter, an attempt at decreasing bone turnover with intermittent I.V. administration of pamidronate (APD) was initiated in three male patients (ages 4, 12 and 17 years) with severe polyostotic fibrous dysplasia. APD (1 rag/k/d) was given for a 3-day course that was repeated 4 times over a 12 month period, The decision to repeat the treatment was based on the changes in serum alkaline phosphatase (ALP) activity. Short-lived decreases in serum calcium and phosphorus, BGP, TRAP and TmP/GFR occurred at times of infusion. The most striking effect was a rapid fall in ALP already evident at the end of each infusion session. This effect was sustained for periods of 1-3 months. There was a concomitant but less impressive decrease in cAMP and hydroxyproline excretion (D-Pyr excretion values available later). Each patient noted an impression of better being, and there was a decrease in fracture rate. Radiologic changes suggest an improvement in bone tissue organization and there was no evidence of rickets after the 12 month period. Control bone biopsies (after one year, in two cases) are currently under analysis.

The changes in ALP (and BGP), evident within 4 hours from initiation of therapy, point to the osteoblasts as the target cells of the bisphosphonate in mediating the bone response.

These preliminary results indicate that APD could be an efficacious therapeutic agent in polyostotic fibrous dysplasia, a crippling condition for which no satisfactory medical treatment is yet available.

~ - ~ Successful Treatment of Multifocal Eosinophilic Granuloma of the Bone with Oral Clodronate

Cs. Horv~t, J, Demeter, M, Szendr6i./. Dept. Medicine anld Dept. Orthopaedic Surg., Semmelweis Univ. Med. School Kor~nyi 2/a, H-1083 Budapest, Hungary

A 47 ys old man was referred us because of pains in his pelvis and right shoulder and disability to walk without help. We found lyric lesions on X-ray of the involved bones and multifocal eosinophilic granuloma by histology. Cytostatic chemotherapy was poorly tolerated (etoposide). To prevent hip fracture an operation was also made on the left femur.

At the end of the second year after diagnosis oral clodronate (1600 mg/day) therapy was introduced. After sic months treatment the pain disappeared and the patient regained his abiliW for doing hard work. No new lesions were seen on bone X-ray or on bone scan. The mineral density measured by single and dual photon absorptiometry (SPA and DEXA) remained constant in spine and radius and increased in the femoral neck - in parallel with appearance of sclerotization int he area of the previous osteolysis shown by X-ray. Biochemical parameters of bone metabolism were also controlled.

Discontinuing the clodronate therapy a recurrence of pain was observed without new bone lesions to be proven. Repeated administration of 1600 mg/d oral clodronate for 5 weeks resulted in complete disappearance of symptoms.

No side-effects of clodronate were observed.


[ - - ~ Clinical, Scintigraphic and Laboratory Evaluation of Pamldronate as a Possible Treatment in Two Cases of Diaphysal Dysplasias: Ribbing's and Cammuratti- Engelmann's Diseases

Z. Rubin, G. Ghiringhelli, J.L. Mansur, J. Somoza. Seccion Osteopatias Medicas, Hospital De Clinicas, U.B.A. Cordoba 2351, Buenos Aires, Argentina

Engelmann's Disease or Progressive Diaphysal Dysplasia (PDD) is a rare autosomal dominant disorder, sometimes non hereditary, which begins in childhood, and is characterized, by symmetrical excess of osseous apposition, in dyaphisis and metaphisis of long bones. In severe cases skull and vertebrate could be involved. Clinically, patients refer limb pain, muscular weakness and atrophy, easy fatigability and waddling gait.

Later, Ribbing described an illness, that he thought was a separate entity, with sclerosis and enlargement of dyaphisis of femora and tibiae, which begins after puberty, is less extensive, not always symmetric and without gait or neurological involvement. Some authors think it is the adult form of the PDD.

We report one case of each entity: (1) A white female, 69 years old, with clinic and radiology of Ribbing's disease, had positive scintigraphy in the affected areas and elevated bone markers: Alcaline phosphatase (AI Ph): 57 UKA, Total Urinary Hydroxiproline (THP): 60 mg/24 hs, Bone-Gla Protein, (BGP): 40 ng/ml. Considering the high bone turnover, treatment with pamidronate, 400 mg/day and Ca 1 g/day was start. After 2 months, pain almost disappeared, and THP became normal: 14 rag/24 hs. with normalization of BGP: 8 ng/ml, slightly elevation of AI Ph: 21 UKA, and a decrease of MDP uptake by affected bones, after one year treatment.

(2) Because of this results we decided to begin treatment in a white female, 17 years old, 32 kg, weight, 1.47 m height, with PDD characteristics and also a high turnover, (THP 95 mg/24 hs; AIPh 32 UKA). After six months of Ca 1 g/day by meals and Pamidronate 100 mg/day, she became painless with normal strength and gait, almost normalization of THP (48 rag/24 hs) although a small decrease of AI Ph. and no changes in scintigraphy.

Pamidronate could possibly be useful in treatment of dysplasias with high bone turnover.

[ - ~ Aminohydroxy-Propylidene Bisphosphonate (APD) Treatment Improves the Clinical Skeletal Manifestations of Gaucher's Disease

R. Samuel, K. Katz, S.E. Papapoulos, Z. Yosipovitch, R. Zaizov, U.A. Liberma n. Metabolic Diseases, Department of Orthopedics and Sambur Center of Pediatric Hematology/Oncology, Beilinaon Medical Center, Petah 17qva 49100, Sackler Faculty of Medicine, Tel A viv Universi~ Tel Aviv, Israel

Gaucher's disease is an autosomal recessive disorder. Episodes of "bone crisis" are among the most troublesome manifestations of this disease, presenting clinically as acute, severe pain, local and systemic inflammatory signs and osteonecrosis. Pathological fractures tend to develop in the weakened osteonecrotic region. We evaluated the long-term effects and safety of APD treatment on the frequency and severity of the clinical skeletal manifestations. Five adolescent patients with Gaucher's disease were treated with APD, 150 to 300 mg per day for 14 to 66 months. During the 5 years before treatment, the patients suffered from 7 to 14 bone crisis episodes, or 1.4 to 2.8 episodes per patient's year. Three patients were free of bone crisis episodes during 14 to 32 months of treatment, while 2 patients had 2 such episodes during 54 and 66 months of APD treatment (a decrease from 1 .B and 2.8 per year to 0.5 and 0.4, respectively). Four patients suffered 1 to 3 atraumatic bone fractures during the 5 years preceding treatment, only 1 patient sustained a fracture on APD (190 months' treatment). Use of APD was not associated with any clinical or biochemical aberrations. In all patients, a band-like metaphyseal sclerosis appeared on long bone radiography. However, APD did not interfere with bone growth. In conclusion, the marked improvement in the clinical skeletal manifestations of Gaucher's disease and the absence of toxic side effects in adolescent patients treated with APD, support previous findings in 3 adult patients on the efficacy of APD and indicate possibilities for its use in inducing prolonged remissions in affected patients.

C l i n i c a l s tu d i e s - - M i s c e l l a n e o u s $ 8 5

MISCELLANEOUS

~ 0 - ~ The Effects of Calcium on Pyridinium Croaslink Excretion In Healthy Young Women

L.M. Demers, J. Chambers, L. Gaydos, L. Curley. 1: Lloyd, A. Lipton. The M.S. Hershey Medical Center, The Pennsylvania State Universi~ Hershey, PA. 17033

As part of an osteoporosis prevention study in young women, we examined bone mass accrual and the influence of dietary calcium supplementation in a cohort of young girls during a period of rapid adolescent growth (11-13 years). To bioehemically assess bone turnover, we determined the excretion of the bone collagen crosslinks Pyridinoline (PYD) and Deoxypyridinoline (DPD) every 6 months over a 24 month period and compared results to the development of bone density in 38 subjects receiving a 500 mg daily calcium supplement and 41 age matched subjects receiving placebo. Both PYD and DPD have been identified as sensitive and s{aecific markers of increased bone resorption in patients with metabolic bone disease. Crosslink measurements were determined by HPLC from a 24 hr urine collection and results were normalized to creatinine excretion. After 18 months of study, the calcium supplemented group showed a significant increase in bone density over the placebo population (Placebo 0.94 + 0.8 g/cm 2 vs calcium 0.97 + 0.7, P < .001). This was accompanied by a significant suppression in PYD and DPD excretion over the course of 18 months (PYD:Placebo, 92.8 :~ 7.5 vs calcium, 75.6:1: 6.6, P < 0.05; DPD:Placebo, 26.8 + 2.1 vs calcium 22.5 + 1.9/~M/M creatinine, P < 0.05). These findings support the beneficial effects of additional dietary calcium on bone mass development in young girls as reflected by bone density measurements and urine pyridinium crosslink levels. These studies also support the clinical utility of urine crosslink measurements for assessing bone turnover in healthy subjects.

~ - ~ A Comparison of HPLC and ELISA for the Measurement of Urine Pyridinium Crosslinks

L.M. Demers. L. Gaydos. L. Curley. A. Lipton. The M,S. Hershey Medical Center of The Pennsylvania State Universi~ Hershey, PA. 17033

Pyridinoline (PYD) and deoxypridinoline (DPD) are crosslink factors found in mature bone collagen that are released into the circulation with bone resorption. Recent studies have suggested that the measurement of these collagen crosslinks in urine is a sensitive and specific marker of bone loss in patients with metabolic bone disease. HPLC methods were initially developed to determine these crosslinks in urine based on ion pairing and the natural f luorescence that PYD and DPD possess. HPLC, however, is a tedious method to perform and it normally takes several days to analyze for the crosslinks. Recently, polyclonal and monoclonal antibodies have been raised to PYD and DPD by Metra Biosystems, Inc. (Palo Alto, CA) and formatted into an ELISA for direct measurement of free pyridinoline in urine. The immunoassay method is based on a competit ive assay where free PYD from the sample competes with PYD coated on a microtiter plate for binding to a rabbit anti-PYD antibody and a goat anti rabbit IgG-alkaline phosphatase conjugate with p nitrophenol color development from the substrate p-nitrophenyl phosphate. We have evaluated ELISA reagents for PYD and have compared results to our established HPLC method. The correlation f rom 121 patient samples over a range f rom 10 to 3500 nanomoles of PYD was 0.892 with a regression line of y - 1.005 x -20.4. The antibody for PYD cross reacts with DPD thus the assay appears to be a total pyridinoline assay. The method appears valid and useful for the measurement of pyridinoline crosslinks in urine of patients with metabolic bone disease.

F I ~ - I Absence of Renal Toxicity in Cancer Patients with Bone Metastases Receiving Rapid Intravenous Infusions of Aredia (Pamidronate Disodium)

J.M. Ford. C. Tyrrell. E. Madson, A. Harris. Ciba-Geigy Ltd., 4002 Basle, Switzerland

Oral administration of bisphosphonates (BP) is associated with low bioavailability - - especially if taken with food - - and GI intolerance in some patients (pts) leading potentially to a loss of compliance and marked variations in the daily dose absorbed. This is of major concern when

treating the aggressive osteolysis which characterises malignancy. The repeated intravenous (IV) infusion of BP represents an attractive alternative provided the infusion duration is acceptably short without compromising pt safety.

Animal studies have clearly demonstrated that kidney damage is dose-limiting when BP are given IM Renal failure has also been reported fol lowing the clinical use of BP and for this reason infusions have traditionally been given over several hours. In 2 separate trials we performed detailed tests of renal function in cancer pts with bone metastases (BM) fol lowing repeated 'rapid' infusions of Aredia 90 mg given according to the fol lowing schedules:

Trial 1 - - 2 infusions over 90 mins, separated by a 3-week interval (10 pts)

Trial 2 - - 4 infusions over 60 rains, separated by 1-week intervals (12 pts)

Renal function was monitored by measuring serial renal 51Cr_EDTA clearances, excretion of urinary enzymes (NAG and fl2-microglobulin), serum creatinine and urea. The infusions were well tolerated and no significant changes were seen in any of the renal function parameters.

In 3 additional trials, 260 pts with breast or prostate cancer have been treated with 45-60 mg infusions given over 1 hour every 2-3 weeks (range 1-39 infusions per pt). A single pt developed WHO Grade 1 elevation of serum creatinine without an obvious clinical explanation after receiving 45 mg/hr every 3 weeks for 20 infusions. Despite the lack of short-term toxicity of the 90 mg/hr infusions, we currently recommend an infusion rate of 45 mg/hr for long-term infusional therapy with Aredia in BM, at least until additional data are available f rom a larger number of pts treated for longer periods of time.

I l i A Novel Polyclonal Antibody Against Deoxy-pyrldlnoline to be Used in a Radio Immuno Assay

M.J.C. van Hoek, M.F.W.C. Martens, R. W~lchli *, R. Gamse *, F.M.A. Rosmalen. Nichols Institute Diagnostics B. ~, Nieuweweg 172, 6603 BT, Wijchen, The Netherlands," * Sandoz Pharma, CH-4002 Basel, Switzerland

Early diagnosis and the fo l low up on drug therapy is very important in patients with metabolic bone disease. Some of the promising markers for bone resorption are the pyridinium crosslinks, especially deoxy pyridinoline (DPD) which only occurs in bone and dentine. A disadvantage of the current measurement of pyridinium crosslinks is that they are usually measured by HPLC, which is t ime consuming and difficult to perform.

To establish a RIA against DPD we raised antibodies, in sheep, against a synthetic DPD molecule, coupled to BSA. In the assay system we use an iodinated form of DPD as tracer. DPD used in the assay for standard is also of synthetic origin. By using this synthetic DPD we are able to detect as little as 6 pg DPD/tube. By measuring 1 /~1 of urine or 100 ~,1 of human serum, in the assay, detection limits of 6 ng/ml and 60 pg/ml, respectively, will be achieved.

The preliminary assay protocol is as fol lows: - 100 #1 standard, serum or diluted urine sample/20 /~1 diluted

antiserum/100/~1 tracer (I 125-DPD) - 16-18 hours incubation 4~ - add 1 ml HAS/PEG (precipitating reagent), 30 min. inc. - centrifugation 30 min. - count pellet The cross reactivity of the polyclonal antibody against synthetic

pyridinoline (PYD) is < 1.0%. Preliminary correlation studies on urine samples with the established HPLC method (performed by: Nichols Institute Reference Laboratory, San Juan Capistrano, CA) revealed a significant correlation.

It is believed that this new, specific polyclonal antibody against DPD can be used in a radio immuno assay for measurement of DPD in urine and/or serum.

[ ~ - - 1 Side-Effects of Clodronate (Bonefoe| as Evaluated by Literature and Post-Marketing Survey

K. Laitinen, K. Aranko. Leiras Oy ̀PO Box 325, FIN-O0101 Helsinki, Finland

Clodronate has been subject to clinical investigation for more than 10 years. Both the oral and intravenous formulations have been studied,


primarily in the setting of increased bone resorption associated with malignancy. Up till now, the efficacy of the drug has been documented in over 50 clinical studies involving more than 1000 patients treated with clodronate. Bonefos | by Leiras Oy has been marketed in Finland since February 1985 and later in other European countries and outside Europe, e.g. in Canada. Totally Bonefos | has been registered in more than 30 countries.

According to production figures from Leiras, between 1980 and 1992 the amount of use of oral clodronate comprises 17,000 patient years (based on a daily dose of 1600 mg), and the drug has been given to thousands of patients. Additionally, over 40,000 packages (5 x 5 ml) of Bonefos| have been used. No serious side-effects associated with clodronate use have been recorded. However, the expected survival

of most patients treated with clodronate is short because of the underlying disease.

Gastrointestinal disturbances are clinically important side-effects with clodronate. Nausea, vomiting or diarrhoea have been reported in about 10% of patients with high oral doses of clodronate. Although intravenous clodronate may cause renal side-effects when injected rapidly in large doses, the administration of clodronate is generally associated with improved renal function, and in patients with benign disorders, e.g. Paget's disease, no adverse effect on glomerular filtration rate has been noted. A reported concern associated with bisphosphonates, drug- induced leukemia, has subsequently been considered as coincidental. Recently, there was a case report of erythroderma associated with clodronate use.

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