Intermittent intravenous administration of the bisphosphonate ibandronate prevents bone loss and maintains bone strength and quality in ovariectomized cynomolgus monkeys

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Bone 32 (2003) 45–55

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Intermittent intravenous administration of the bisphosphonate ibandronate prevents bone loss and maintains bone strength and quality in ovariectomized cynomolgus monkeys S.Y. Smith,a R.R. Recker,b M. Hannan,c R. Mu¨ller,c and F. Baussd,* a ClinTrials BioResearch Ltd., 87 Senneville Road, Senneville, Quebec, Canada H9X 3R3 Center for Osteoporosis Research, Department of Internal Medicine, Creighton University, 601 North, 30th Street, Suite 5766, Omaha, NE 68131, USA c Orthopedic Biomechanics Laboratory, Beth Israel Deaconess Medical Center and Harvard Medical School, 330 Brookline Avenue, Boston, MA 02215, USA d Roche Diagnostics GmbH, Pharma Research Penzberg, Nonnenwald 2, D-82377 Penzberg, Germany, and Institute of Pharmacology and Toxicology, Heidelberg University, D-68169 Mannheim, Germany b

Received 4 February 2002; revised 23 September 2002; accepted 30 September 2002

Abstract Using a clinically relevant regimen, this study investigated the effects of treatment with ibandronate, a highly potent nitrogen-containing bisphosphonate, on bone loss, biochemical markers of bone turnover, densitometry, histomorphometry, biomechanical properties, and bone concentration in aged ovariectomized monkeys. Sixty-six female cynomolgus monkeys, aged 9 years and older, were ovariectomized (OVX) or sham operated. Intravenous (iv) bolus injections of ibandronate at 10, 30, or 150 ␮g/kg or placebo were administered at 30-day intervals (corresponding to intervals of 3 months in humans), starting at OVX, for 16 months. OVX significantly decreased bone mass at the lumbar spine, proximal femur, femoral neck, and radius and increased bone turnover in a time-dependent manner, as assessed by dual energy X-ray absorptiometry, peripheral quantitative computed tomography, or histomorphometry. Ibandronate iv bolus injections administered at 30 ␮g/kg every 30 days prevented osteopenia induced by estrogen depletion. OVX-induced increases in bone turnover (as determined by activation frequency, bone formation rate, and biochemical markers of bone turnover, including urinary N-telopeptide and deoxypyridinoline excretion and serum values for osteocalcin and bone-specific alkaline phosphatase) were suppressed on treatment, and bone mass, architecture, and strength were preserved at clinically relevant sites. Treatment with high-dose (150 ␮g/kg/dose) iv bolus injections of ibandronate further increased bone mass and improved bone strength at both the spine and femoral neck, without adversely affecting bone quality. In contrast, treatment with a 10 ␮g/kg/dose only partially prevented the OVX-induced effects. These data support the potential for the long-term administration of ibandronate by intermittent iv bolus injections in humans to prevent osteoporosis and improve bone quality at clinically relevant sites. © 2003 Elsevier Science (USA). All rights reserved. Keywords: Ibandronate; Osteoporosis; BMD; Histomorphometry; Biomechanics; Intermittent iv bolus injection

Introduction Osteoporosis is characterized by low bone mass and microarchitectural deterioration of bone tissue, with a consequent increase in fragility and susceptibility to fractures [8]. Estrogen depletion contributes to the low bone * Corresponding author. Fax: ⫹49-0-8856-60-4067. E-mail address: [email protected] (F. Bauss).

mass characteristic of postmenopausal osteoporosis (PMO). Therefore, estrogen depletion has been used as a bone loss animal model for studying osteoporosis therapies. Bisphosphonates are a class of drugs that have demonstrated efficacy in the prevention and treatment of bone loss due to estrogen depletion [18,29,33,35,37]. However, as oral bisphosphonates have very low bioavailability (usually ⬍ 1%) and have been associated with gastrointestinal intolerability [9,10,19], parenteral

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S.Y. Smith et al. / Bone 32 (2003) 45–55

administration has potential for improved patient acceptance and compliance. Ibandronate is a highly potent nitrogen-containing bisphosphonate that can be administered as an intravenous (iv) bolus injection. In animal studies ibandronate was more potent than risedronate (2⫻), alendronate (10⫻), pamidronate (50⫻), and clodronate (500⫻) in the prevention of bone destruction [26]. The lowest dose that completely prevented ovariectomy-induced bone loss in rats and dogs was 1 ␮g/kg/day [3,5,24]. Preclinical studies have shown that the total cumulative dose of ibandronate seems to be more important than the treatment schedule, at least within two bone remodeling period [3,5,25]. Clinically, ibandronate has proven efficacy in hypercalcemia of malignancy [13] and in osteoporosis [28,30,34]. Ibandronate is presently the only bisphosphonate with ongoing oral and iv clinical development programs in the treatment and prevention of PMO. Following surgical ovariectomy, cynomolgus monkeys show changes in bone mass and bone turnover similar to those observed in postmenopausal women, thus providing a suitable large-animal model for studying the efficacy of novel antiosteoporotic drugs [16]. According to available guidelines for the development of antiosteoporotic drugs, the ovariectomized nonhuman primate is the preferred model in which to study effects on bone remodeling, particularly with regard to bone mass, architecture, and strength [7,12,40]. These guidelines suggest that treatment schedules used in primates should be as close as possible to the clinical application of the respective drugs. However, the treatment regimens used in studies reported thus far on bisphosphonates in ovariectomized monkeys reflected neither the regimen of the later approved drug nor those regimens currently under clinical investigation [1,6,36]. Bolus iv injections of ibandronate every 3 months are currently being studied for the treatment of women with PMO [34]. This dosing interval of 3 months corresponds to the average bone remodeling period (or sigma) in humans. Since the bone remodeling period in monkeys is about one third of that in humans [20], we used a dosing interval of 30 days in this present study. In addition, a total exposure period of 16 months was selected, which would correspond to 4 years in humans. Importantly, therefore, this is the first nonhuman primate study to use a dosing schedule that mimics a clinical regimen of a bisphosphonate in order to investigate its effect on bone mineral density (BMD), histomorphometry, bone strength, and biochemical markers of bone formation and resorption. The doses selected for this study were derived from optimal doses that prevented bone loss in ovariohysterectomized dogs and ovariectomized rats [2]. From these data, the optimal dose of ibandronate in cynomolgus monkeys was estimated to be 30 ␮g/kg. A dose of five times the proposed optimal dose (150 ␮g/kg) was tested to evaluate safety, as specified in the FDA guidelines [12]. In addition, a low dose of ibandronate was investigated (10 ␮g/kg). This

dose range was selected in the context of the narrow dose response relationship in dogs and rats.

Materials and methods The study was performed according to the protocol and Standard Operating Procedures established at CTBR, Quebec, Canada, and Creighton University, Omaha, Nebraska, USA (histomorphometry), Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts, USA (biomechanics), and Roche Diagnostics GmbH, Pharma Research, Germany (ibandronate concentration in bones). Animals and experimental design Sixty-six nonpregnant female cynomolgus monkeys (Macaca fascicularis), aged 9 years or older (mean age 12–14 years at baseline based on information provided by the animal supplier), were received from reputable North American suppliers, where they had been reared or maintained in captivity for several years. All animals had a history of at least one offspring and were a minimum of 11 months postpartum or postlactation when they entered the study. Animals were socially housed in wall-mounted cages (2 animals/cage) equipped with an automatic watering system. All animals had access to PMI Fiber-Plus Monkey Diet Jumbo 5050 daily, containing 0.9% calcium, 0.7% phosphorus, 6.6 IU of vitamin D/g, and food supplements of Primatreats and/or fresh fruit. The animal room environment was controlled, with settings targeted at temperature 22 ⫾ 3 °C, humidity 50 ⫾ 20%, photoperiod 12 h light and 12 h dark, air changes 12/h. Animals were randomized by lumbar spine (L1–L4) BMD [dual energy X-ray absorptiometry (DXA)] into five groups: two control groups (n ⫽ 15 per group) and three ibandronate treatment groups (n ⫽ 12 per group). Groups were verified to be similar with respect to age, body weight, and lumbar spine BMD. Following an approximate 5-month monitoring period, animals were either ovariectomized (OVX) (control and ibandronate groups) or sham-operated (Sham control). At the end of the treatment period, animals were euthanized. Anesthesia The surgical procedures and the DXA and peripheral quantitative computed tomography (pQCT) measurements were performed under general anesthesia: a cocktail of glycopyrrolate, ketamine hydrochloric acid (HCl) injection, and xylazine, followed by anesthesia with isoflurane gas. Blood and urine samples were collected after an intramuscular injection of glycopyrrolate, ketamine HCl injection, and xylazine.

S.Y. Smith et al. / Bone 32 (2003) 45–55

Administration of drug/control materials Starting on the day of surgery, three groups of OVX monkeys were given iv bolus injections of ibandronate at doses of 10, 30, or 150 ␮g/kg (free acid equivalents) once every 30 days for a duration of 16 months. Sham and OVX control animals received placebo injection solution diluted with saline. Biochemical parameters Blood and urine samples were collected pretreatment and at 1, 3, 6, 12, and 16 months of the treatment period for evaluation of biochemical markers of bone turnover, hormones, serum and urinary calcium and phosphorus, and urinary creatinine. The following parameters were evaluated. Serum: osteocalcin (OC), DSL-6900 RIA kit; bonespecific alkaline phosphatase (sALP), Tandem-R Ostase IRMA kit; parathyroid hormone (PTH(1– 84)), Diagnostic Products Corp. Intact PTH IRMA kit; 1,25 dihydroxyvitamin D, Incstar 1,25(OH)2D RRA kit; estradiol, Diagnostics Products Corp. RIA kit; calcium, atomic absorption spectrophotometry; phosphorus, phosphomolybdate complex read at 340 nm. Urine: N-telopeptide (NTx), Osteomark ELISA kit; pyridinoline/deoxypyridinoline (PYD/DPD), Bio-Rad Crosslinks HPLC assay; calcium, atomic absorption spectrophotometry; creatinine, Jaffe kinetic; phosphorus, phosphomolybdate complex read at 340 nm.

47

vertebra (L4), rib, right proximal femur, and right distal and central radius and were retained in 70% alcohol. Fifteen and 5 days prior to necropsy sampling, each animal was given an iv injection of xylenol orange (90 mg/kg). Bones were dehydrated, defatted, and embedded in methylmethacrylate. Thick cross sections of approximately 80 ␮m were prepared from the rib, femoral neck, and central radius and thin sections were prepared from the iliac crest, proximal femur, fourth lumbar vertebra, and distal radius. Vertebral bodies were prepared in the parasagittal plane and the proximal femur and distal radius in the frontal plane. Histomorphometry was performed on unstained sections under ultraviolet light and stained sections under white light. The analysis followed standard laboratory procedures using BioQuant image analyzers (R and M Biometrics, Nashville, Tennessee, USA). Calculations and presentation of histomorphometry variables follow the guidelines of the report of the American Society for Bone and Mineral Research [27]. Biomechanical testing

Peripheral QCT (Norland/Stratec XCT 960A bone scanner, software version 5.20) was used to measure bone mineral content (BMC), volumetric BMD (vBMD), and geometric parameters of the right distal radius and right proximal tibia pretreatment and during months 4, 8, 12, and 16 of the treatment period. In vivo scans were acquired at a metaphyseal (cancellous bone) site at the tibia/fibula or radial/ulna junction and a diaphyseal (cortical bone) site at approximately 22% of the bone length measured from the ulna styloid to elbow or patella to lateral malleolus. In vivo scanning precision varied between CV 3 and 6%.

The first lumbar vertebra (L1), left femur, and left ulna were retained at necropsy, wrapped in saline-soaked gauze and plastic film, frozen at ⫺20 °C, and submitted for biomechanical testing. Testing of all specimens was performed using a servohydraulic material testing machine (Instron Model 8511, Instron Corp., Canton, Massachusetts, USA) with Labview data acquisition and analysis software (version 3.0.1, National Instruments, Austin, Texas, USA). The geometry, density, and structural properties of the specimens were assessed ex vivo by DXA (Hologic QDR 2000 plus) and/or pQCT (XCT 960A Norland/Stratec). The L1 vertebra was prepared for testing by removal of the spinal processes. Polymethylmethacrylate (PMMA) endcaps made with impressions of the superior and inferior ends of the L1 vertebra were used to apply the load to the vertebral bodies. Compression testing to failure at a strain rate of 0.5%/s was performed to determine ultimate load and stiffness from the resulting load vs displacement curve. Apparent strength and apparent modulus were calculated using the load, height, and midsection area. Ulnae were tested to failure in three-point bending with the upper support displacement at a rate of 1 mm/s until failure occurred. Ultimate load and stiffness were determined from the load vs displacement curve. Strength and modulus were calculated using load, stiffness, moment of inertia, and anterior–posterior diameter. The proximal portion of the left femur was embedded in PMMA up to the lesser trochanter and loaded to failure with a polyethylene cap attached to an actuator moving at a rate of 0.5 mm/s. Ultimate load and stiffness were determined from the load vs displacement curve.

Bone histomorphometry

Ibandronate concentration in bone

Bones were harvested at necropsy, 19 days following the last dose administration for the iliac crest, fourth lumbar

The left tibia and lumbar vertebra L6 were analyzed for ibandronate concentration. The tibiae were sawed in the

Bone densitometry by DXA BMD of the right femoral neck and lumbar spine (L1– L4) was measured using DXA (Hologic QDR 2000 plus) at baseline and during months 4, 8, 12, and 16 of the treatment period. The precision (CV%) of DXA scanning with repositioning was approximately 0.8% at the lumbar spine and approximately 4.5% at the femoral neck. Bone densitometry by pQCT

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S.Y. Smith et al. / Bone 32 (2003) 45–55

sagittal plane and the bone marrow rinsed out. Trabeculae were scraped from the distal and proximal parts for separate analyses. The remaining distal and proximal cortices and the midshaft diaphysis cortex were analyzed separately. Concentrations were combined to provide a total drug concentration for each individual tibia. The L6 lumbar vertebrae were analyzed for total drug concentration. Ibandronate was recovered from bones by dissolving in HCl, followed by precipitation and separation, with ibandronic acid measured as the ethyl ester by gas chromatography–mass spectrometry (GC-MS). Statistical analyses Statistical analyses were performed using SAS version 6.08 [32]. For data collected longitudinally (not histomorphometry and biomechanic parameters), the baseline value was subtracted from the posttreatment values of analyzed variables (adjusted means). Densitometry data were converted to percentages of the baseline value. Group variance homogeneity was assessed using Levene’s test (0.001 level of significance). Whenever group variances were found to be homogeneous, group means were compared within a one-factor analysis of variance. If a significant group effect was found, then the Sham and OVX control groups were compared and each dose group was compared against the sham control group as well as the OVX control group. The pairwise group comparisons were performed using protected t tests on the least square means and the comparisonwise error rate was set to 0.05√7, where 7 was the number of such pairwise comparisons. Dose–response relationships were evaluated when a significant group effect was found. The OVX control group and the treated groups were then used to assess the significance of a linear and/or quadratic trend across dose levels (0.05 level of significance). If group variance heterogeneity prevented the use of this parametric method, then the seven pairwise comparisons of interest were performed with the Welch test, provided that tested groups each had a nonzero variance [31]. The two-sample t test with the Satterhwaite’s approximation was used in all cases where one group had a zero variance. The comparisonwise error rate was set to 0.05√7. Pearson correlation coefficients were derived for bone density and biomechanical parameters.

Results

low uterus weights [3.6, 3.1, 3.1, and 3.3 g (mean) in the vehicle, 10, 30, and 150 ␮g/kg/dose ibandronate (OVX) groups, respectively, compared with 11.3 g in the sham control group]. The presence of ectopic ovarian tissue in the uterus was identified for two OVX control animals that were subsequently excluded from analysis of all study data. Three animals died during the course of the study due to non-drugrelated reasons. Biochemical parameters OVX-induced increases in biochemical markers of bone formation (sALP and OC) and resorption [NTx, total DPD, and PYD (data not shown since a similar response was observed to the total DPD data)] were dose-dependently and significantly prevented by treatment with iv ibandronate bolus injections (Figs. 1A–1D). In general, for all doses of ibandronate, values for serum levels of calcium, phosphorus, PTH, and 1,25-hydroxyvitamin were within the normal range (data not shown). There was a trend throughout the treatment period for reduced serum calcium levels and increased PTH(1– 84) levels with 150 ␮g/kg of ibandronate, compared to OVX and sham control groups. Serum calcium levels were similar to those for sham and OVX controls for animals treated with 10 or 30 ␮g/kg of ibandronate. In animals receiving 150 ␮g/kg of ibandronate, serum phosphorus levels were decreased compared with those of OVX controls throughout most of the treatment period (months 1, 2, and 6). Throughout the study, serum phosphorus levels in animals receiving ibandronate were similar to levels observed with the sham controls. Bone densitometry by DXA Relative to sham controls, OVX controls showed decreases in bone density 16 months post-OVX of approximately 10% at the lumbar spine (Fig. 2A) and 11% at the femoral neck (Fig. 2B). Treatment with intermittent iv ibandronate bolus injections resulted in a significant dose-dependent prevention of the OVX-induced decrease in bone mass at these sites. Treatment with 30 and 150 ␮g/kg of ibandronate completely prevented bone loss at the spine and almost completely at the femoral neck. Partial prevention of bone loss at the spine was evident at 4 months post-OVX with 10 ␮g/kg of ibandronate, but was not significantly different from that of OVX controls at the end of the study.

Animal health Bone densitometry by pQCT Ibandronate was well tolerated by all animals. The mean body weights of animals in the various study groups were similar at baseline (3.0 –3.3 kg) compared with after 16 months (3.4 –3.8 kg). The status of ovarian function for OVX animals was confirmed by the reduction of serum estradiol to undetectable levels 1 week post-OVX, which persisted throughout the treatment period and was confirmed by the observation of uterine atrophy at necropsy and

Ovariectomy-induced changes at the proximal tibia and distal radius were characterized by a decrease in metaphyseal total slice BMC, trabecular vBMD, and diaphyseal slice cortical BMC and cortical vBMD. Data are shown only for the proximal tibia trabecular vBMD and diaphyseal cortical vBMD (Fig. 3). Bolus iv injections of ibandronate dose-dependently prevented the effects of OVX on metaphysis trabec-

S.Y. Smith et al. / Bone 32 (2003) 45–55

49

Fig. 1. Effect of ibandronate (Iban) on biochemical markers of bone turnover. Data are presented as means and 1 SD (n ⫽ 10 –15). Significances (relative to change from baseline) (P ⬍ 0.05): different from sham control, a; different from OVX control, b; different from sham and OVX controls, c.

ular vBMD. Animals treated with 30 and 150 ␮g/kg of ibandronate achieved vBMD levels similar to those of the sham control monkeys (nonsignificant difference) and achieved significantly greater vBMD values than OVX control animals (P ⬍ 0.05; Fig. 3A). The effect of OVX on cortical vBMD was also dose-dependently prevented by ibandronate (Fig. 3B). Data obtained from the distal radius showed a pattern of response to OVX and ibandronate treatment similar to that

of the tibia, but the changes were less marked (data not shown). Sham control data from the distal radius were noted to remain stable throughout the observation period. Bone histomorphometry of cancellous bone Histomorphometric results from cancellous bone sites obtained at necropsy are provided in Table 1. Ovariectomy

Fig. 2. Effect of OVX and treatment with ibandronate (Iban) on BMD measured by DXA at the lumbar spine L1–L4 (A) and femoral neck (B). Data are presented as mean percentages of baseline and 1 SEM (n ⫽ 10 –15). Significances (relative to change from baseline) (P ⬍ 0.05): different from sham control, a; different from OVX control, b; different from sham and OVX controls, c.

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Fig. 3. Effect of OVX and treatment with ibandronate (Iban) on BMD measured by pQCT at the proximal tibia. (A) Metaphysis trabecular vBMD. (B) Diaphysis cortical vBMD. Data are presented as mean percentages of baseline and 1 SEM (n ⫽ 10 –15). Significances (relative to change from baseline) (P ⬍ 0.05): different from sham control, a; different from OVX control, b; different from sham and OVX controls, c.

resulted in minor changes in cancellous bone structural variables. There was a slight reduction in trabecular bone volume (BV/TV) relative to sham controls after 16 months at all sites evaluated; however, significance was only attained at the radius. There was a tendency for trabecular separation to be increased at all sites (nonsignificant, data not shown), although trabecular thickness was decreased only at the femur and radius (attaining significance only at the radius). The BV/TV is known to be quite variable, and thus the power to detect differences is relatively weak given the small sample sizes in this study. BV/TV appears in the calculation of Tb.Th, and thus the same lack of statistical power exists for this derived variable. Nevertheless, the magnitude of the mean differences between sham and OVX control, though not statistically significant in every case, are as expected for both BV/TV and Tb.Th. At the femur, there was a dose-dependent trend for iv ibandronate bolus injections to preserve trabecular bone volume, accompanied by a significant increase in trabecular thickness (with 150 ␮g/kg), compared with OVX controls. Activation frequency for controls was increased in the order of 2.3- to 3.7-fold at all sites evaluated 16 months following OVX compared with that of sham controls. Increases were also observed for the variables expressing bone formation (osteoid surface/bone surface and osteoid thickness, data not shown). Ibandronate dose-dependently prevented the OVX-induced increase in activation frequency, reducing levels to below those of the sham controls at the radius, L4, and iliac crest for the animals receiving 150 ␮g/kg/dose and at L4 for those treated with the 10 or 30 ␮g/kg/dose. The lack of effect of ibandronate at the femur was attributed to the variation noted in this parameter with 30 and 150 ␮g/kg of ibandronate (120 –140%) in comparison with the other groups (66 –90%). The extent of mineralizing surface/bone surface was significantly increased in response to OVX at all sites and this effect was generally dose-dependently reduced by iban-

dronate treatment. Generally, significant reductions were observed at the femur, L4, and iliac crest, even (on occasion) with the lowest dose of ibandronate (P ⬍ 0.05). Erosion surface/bone surface and osteoclast surface/bone surface were increased significantly at the radius in response to OVX. Erosion surfaces were significantly increased at the femur, L4, and iliac crest following treatment with 30 and 150 ␮g/kg of ibandronate, relative to sham and/or OVX controls (P ⬍ 0.05). Osteoclast surfaces were significantly increased at the iliac crest and L4 in animals given 10 and 30 ␮g/kg of ibandronate, compared with sham or OVX controls (P ⬍ 0.05), while animals receiving the highest dose of ibandronate (150 ␮g/kg) obtained similar values to sham or OVX controls. The eroded surface measurement is a static variable and does not bear a necessary relationship to dynamic bone resorption. Thus, variation in this measure may take place due to a prolonged reversal phase interposed between resorption and formation phases, which, in turn could be as a result of accelerated osteoclast apoptosis due to the effect of the drug. Also, this variable has considerable intrinsic variation that can confound its measurement. Bone histomorphometry of cortical bone At cortical bone sites (Table 2), increased activation frequency, indexed by the fraction of Haversian systems with fluorochrome labeling and the area of active Haversian canals (area of all Haversian canals that have labeling or are considered resorptive cavities), was noted in response to OVX with increases in endocortical mineralizing surfaces. Treatment with ibandronate dose-dependently reduced these parameters at the rib and radius. At the femoral neck, a decrease in endocortical mineralizing surface was observed, with no effect of ibandronate on the number of labeled Haversian systems or on the area of active Haversian canals. Slight increases in mineral apposition rates in response to OVX at all sites were reduced by ibandronate only at the radius.

S.Y. Smith et al. / Bone 32 (2003) 45–55

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Table 1 Histomorphometric parameters of cancellous bone after 16 months treatment with ibandronate Sham

OVX Ibandronate (␮g/kg/dose)

Vehicle

Bone volume/tissue volume (%) Iliac crest Proximal femur Distal radius L4 Trabecular thickness (␮m) Iliac crest Proximal femur Distal radius L4 Mineralizing surface/bone surface (%) Iliac crest Proximal femur Distal radius L4 Bone formation rate/bone volume (mm2/mm2/yr) Iliac crest Proximal femur Distal radius L4 Activation frequency (per year) Iliac crest Proximal femur Distal radius L4 Erosion surface/bone surface (%) Iliac crest Proximal femur Distal radius L4 Osteoclast surface/bone surface (%) Iliac crest Proximal femur Distal radius L4

10

30

150

27.31 (9.57) 26.03 (7.71) 19.44 (5.54) 27.01 (5.25)

20.70 (7.79) 18.84 (6.18) 12.48 (4.30)a 24.44 (4.34)

27.57 (11.11) 21.05 (6.86) 12.92 (5.48)a 23.86 (4.36)

25.61 (3.90) 22.21 (5.92) 11.61 (3.81)a 25.19 (3.79)

30.07 (8.80) 24.72 (9.33) 14.02 (5.41)a 26.81 (5.85)

173 (33) 196 (40) 153 (31) 127 (24)

171 (46) 166 (30) 121 (17)a 129 (19)

198 (62) 172 (39) 116 (25)a 119 (18)

170 (30) 189 (48) 109 (15)a 111 (13)

192 (32) 238 (95)b 126 (30)a 126 (21)

1.31 (1.44) 1.89 (2.68) 1.40 (1.75) 2.51 (2.55)

4.68 (3.65)a 6.13 (4.46)a 5.61 (4.02)a 6.77 (4.73)a

3.45 (3.92) 1.43 (1.55)b 5.53 (4.17)a 1.97 (1.20)b

2.31 (2.70) 3.00 (5.79) 2.57 (2.39)b 0.95 (0.87)c

0.23 (0.59)c 0.81 (2.52)b 0.75 (0.86)b 0.05 (0.12)c

0.10 (0.14) 0.10 (0.09) 0.11 (0.13) 0.20 (0.17)

0.21 (0.16)a 0.36 (0.25)a 0.38 (0.23)a 0.41 (0.24)a

0.10 (0.08)b 0.13 (0.16)b 0.28 (0.15)a 0.15 (0.10)b

0.06 (0.06)b 0.12 (0.20)b 0.19 (0.14)b 0.08 (0.07)c

0.01 (0.02)b 0.04 (0.11)b 0.05 (0.05)b 0.00 (0.01)c

0.176 (0.166) 0.237 (0.217) 0.131 (0.145) 0.294 (0.240)

0.407 (0.331) 0.874 (0.579) 0.377 (0.308)a 0.746 (0.499)a

0.308 (0.347) 0.229 (0.167) 0.304 (0.257) 0.206 (0.125)b

0.178 (0.187) 0.655 (0.917) 0.143 (0.121)b 0.152 (0.92)b

0.046 (0.071) 0.731 (0.903) 0.049 (0.042)b 0.023 (0.022)c

1.30 (0.71) 2.25 (1.74) 1.53 (0.98) 3.37 (1.65)

1.88 (0.99) 5.65 (3.84) 3.24 (1.57)a 4.09 (1.27)

2.38 (0.97)a 4.39 (4.73) 3.41 (1.87)a 7.38 (2.21)c

3.02 (1.02)c 4.18 (2.57) 2.28 (1.42) 7.78 (3.05)c

2.73 (0.93)a 10.03 (7.96)a 2.20 (1.61) 9.23 (3.83)c

0.79 (0.57) 0.17 (0.23) 0.48 (0.39) 0.98 (0.42)

1.00 (0.67) 0.48 (0.42) 1.40 (0.79)a 1.29 (0.65)

1.59 (0.69)a 0.26 (0.34) 1.45 (0.79)a 1.88 (0.67)a

2.20 (0.83)c 0.28 (0.30) 0.82 (0.77) 1.92 (0.75)c

1.28 (0.74) 0.36 (0.55) 0.60 (0.53)b 0.99 (0.60)

Data are means (SD). a Significantly different from sham controls. b Significantly different from OVX controls. c Significantly different from sham and OVX controls. P ⬍ 0.05.

There was no evidence that iv ibandronate adversely affected mineralization at any bone sites as shown by no increase in osteoid volume, no increase in osteoid width, and no prolongation in mineralization lag time (data not shown). Furthermore, there were no abnormalities seen in the appearance of specimens by microscopic examination. Biomechanical testing Vertebral apparent bone strength and femoral neck strength (load) were decreased in response to OVX, an

effect that was prevented in the vertebrae by 30 and 150 ␮g/kg of ibandronate (Table 3). A significant positive correlation between the volumetric bone density (by pQCT) of prepared specimens and apparent strength was found for the L1 vertebra (r ⫽ 0.65, P ⬍ 0.0001). Prevention of the OVX-induced decreases in strength at the femoral neck was notably inconsistent. Although a meaningful correlation was established between ultimate stress and cortical density for the ulna (r ⫽ 0.51, P ⬍ 0.0001), no significant effects of either OVX or ibandronate on biomechanical variables were demonstrated. It is likely that larger changes in bone density are needed to have a detectable impact on cortical bone

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Table 2 Histomorphometric parameters of cortical bone after 16 months of treatment with ibandronate Sham

OVX Ibandronate (␮g/kg/dose)

Vehicle

10

30

150

2

Area of active Haversian canals (mm ) Rib Femoral neck Central radius Labeled Haversian systems (%) Rib Femoral neck Central radius Mineral apposition rate (␮m/day) Rib Femoral neck Central radius Endocortical mineralizing surface (%) Rib Femoral neck Central radius

0.026 (0.028) 0.130 (0.088) 0.023 (0.039)

0.058 (0.050)a 0.299 (0.174)a 0.272 (0.158)a

0.033 (0.028) 0.185 (0.094) 0.187 (0.083)a

0.035 (0.030) 0.236 (0.114) 0.161 (0.112)a

0.019 (0.018)b 0.214 (0.142) 0.099 (0.083)c

6.1 (4.5) 1.9 (1.8) 0.2 (0.6)

18.7 (9.1)a 3.6 (1.5) 4.8 (3.9)a

14.1 (12.9) 2.6 (1.7) 3.6 (1.9)a

10.0 (6.6)b 3.5 (3.5) 2.5 (1.9)a

3.7 (3.6)b 2.4 (1.9) 1.7 (3.6)c

0.85 (0.23) 0.76 (0.18) 0.78 (0.45)

0.93 (0.18) 0.83 (0.16) 1.00 (0.15)

0.87 (0.15) 0.73 (0.09) 0.80 (0.15)b

0.97 (0.24) 0.74 (0.21) 0.79 (0.23)b

0.91 (0.33) 0.78 (0.11) 0.76 (0.33)b

13.24 (13.83) 7.10 (8.95) 2.68 (4.90)

22.13 (11.90) 14.94 (10.10)a 13.91 (14.53)a

17.12 (20.71) 5.20 (6.99)b 17.87 (12.24)a

8.58 (6.92) 9.19 (8.37) 12.59 (10.92)a

14.87 (13.87) 3.71 (3.92)b 3.28 (6.26)b

Data are means (SD). a Significantly different from sham controls. b Significantly different from OVX controls. c Significantly different from sham and OVX controls. P ⬍ 0.05.

strength given the sample size. As a result of the small sample size, it is probable that this study was insufficiently powered to detect a significant effect on biomechanical variables in cortical bone.

Ibandronate concentration in bone The left tibia and lumbar vertebra L6 were analyzed for ibandronate concentrations (Fig. 4). The results of the anal-

Table 3 Biomechanical testing of lumbar vertebrae in compression, whole ulnae in three-point bending and femoral neck strength, after 16 months treatment with ibandronate Sham

OVX Vehicle

L1 whole vertebrae Ultimate load (N) Apparent strength (N/mm2) Stiffness (N/mm) Apparent modulus (N/mm2) pQCT BMD (mg/cm3) Ulna Ultimate load (N) Strength (N/mm2) Stiffness (N/mm) Modulus (N/mm2) Cortical pQCT BMD (mg/cm3) Femoral neck Ultimate load (N) Stiffness (N/mm) Proximal DXA BMD (g/cm2)

Ibandronate (␮g/kg/dose) 10

30

150

2116 (561) 22.80 (5.13) 1375 (395) 246.20 (68.25) 548.9 (81.1)

1586 (286)a 17.14 (4.15)a 1131 (257) 194.84 (49.21) 462.0 (98.6)a

1614 (163)a 17.61 (3.34)a 1345 (527) 220.94 (76.19) 468.9 (64.7)

1745 (299) 19.30 (3.26) 1266 (350) 233.64 (74.55) 477.0 (85.5)

2089 (479)b 22.45 (3.64)b 1489 (417) 257.50 (51.14) 533.7 (93.3)

211 (70) 222.53 (29.85) 70 (29) 2.45 (0.34) 1357.0 (45.1)

160 (28)a 203.39 (31.28) 53 (11) 2.43 (0.40) 1296.7 (33.6)a

158 (37)a 214.89 (38.83) 51 (15) 2.51 (0.50) 1296.1 (43.8)a

172 (39) 206.50 (32.83) 57 (17) 2.38 (0.37) 1324.2 (36.5)

172 (43) 222.60 (33.93) 54 (16) 2.54 (0.41) 1344.6 (26.8)b

1624 (331) 1027 (245) 0.464 (0.037)

Data are means (SD). a Significantly different from sham controls. b Significantly different from OVX controls. P ⬍ 0.05.

1229 (203)a 862 (184) 0.403 (0.026)a

1246 (278)a 991 (242) 0.403 (0.044)a

1372 (285) 946 (279) 0.434 (0.063)

1362 (221) 881 (151) 0.470 (0.050)b

S.Y. Smith et al. / Bone 32 (2003) 45–55

53

Fig. 4. Concentration of ibandronic acid in the cortical bone of the diaphysis and proximal and distal metaphysis of the left tibia (left) and the total tibia and total lumbar vertebra L6 (right). Data represent the mean ⫾ SD (n ⫽ 10 –15).

yses in tibiae showed regional differences in concentration, with higher levels in the proximal metaphyseal cortex than the distal metaphyseal cortex. This relationship was also noted after separate analyses of both trabecular (data not shown) and cortical bone. Ibandronate concentration in the diaphyseal cortex was similar to that in the distal metaphyseal cortex. The concentration in lumbar vertebrae L6 was approximately three times that found in tibiae. The total mean concentration increased with the dose in a nearly linear manner, with an increase of approximately 7- to 10-fold when concentration in total bone was considered rather than that in separate regions.

Discussion In this study, estrogen depletion induced by surgical ovariectomy produced an osteopenia characterized by increased bone turnover and loss of BMD in the aged cynomolgus female monkey. The OVX-induced changes in biochemical markers and BMD suggest that the rapid phase of bone loss following estrogen depletion lasts approximately 8 –12 months following OVX. By 16 months post-OVX, a trend toward normalization of markers and stabilization of bone mass suggests a waning of this response. This is in agreement with the time course reported by Binckley et al [6] for rhesus monkeys. The use of older monkeys with stable bone mass in our study established a true osteopenia following OVX, consistent with recent studies [6,14], rather than a relative osteopenia as seen in earlier studies using wild caught or young, skeletally immature animals [1,15,21,38]. Intermittent iv bolus injections of ibandronate dose-dependently suppressed OVX-induced increases in biochemical markers of bone turnover to pre-OVX levels, an expected outcome observed following bisphosphonate treatment in humans and monkeys [1,6,11,34]. These data are consistent with those reported by Thie´ baud following

the administration of ibandronate to postmenopausal women as an iv bolus injection every 3 months [34]. In general, normalization of increased bone turnover as evidenced by decreased biochemical markers was accompanied by preservation of, or increase in, BMD in the ibandronate treatment groups relative to OVX controls. This has been consistently observed in bisphosphonate studies in animals and humans. It is the direct result of the ability of bisphosphonates to suppress increased activation frequency and accounts for the “antiresorptive” effect of these agents. Site-specific differences in BMD response and in tissue levels of bisphosphonate (see below) vary directly with site-specific differences in pretreatment remodeling rates. Results in human ibandronate trials parallel those seen here. Increases in lumbar spine BMD of up to 5.2% were observed in postmenopausal women following iv bolus injections of ibandronate every three months for 1 year [34]. In this female cynomolgus monkey prevention study, lumbar spine BMD was increased maximally by approximately 4%, relative to pretreatment values following eight doses of high-dose ibandronate (150 ␮g/kg/dose). Lumbar spine BMD was maintained at pretreatment levels for animals receiving middose ibandronate (30 ␮g/kg) consistent with complete prevention of the OVX response. Intermittent ibandronate dose-dependently prevented the OVX-induced decreases in proximal femur BMD at the femoral neck region, where a significant treatment effect was seen with the higher dose of ibandronate only at the end of the treatment period. The relatively lower BMD effect of ibandronate at the femoral neck region, compared with the spine, likely reflects the greater ratio of cortical bone to trabecular bone in the hip region and thus the lower remodeling rate and smaller remodeling space. These changes are consistent with those obtained following the administration of ibandronate to postmenopausal women for 1 year [34]. The ability of ibandronate treatment to preserve BMD to a greater degree at trabecular sites than cortical sites indicates that the dose responses for trabecular and Haversian

54

S.Y. Smith et al. / Bone 32 (2003) 45–55

bone may be different. Whereas 30 ␮g/kg/dose of ibandronate appeared to normalize the rate of trabecular remodeling, even the 150 ␮g/kg/dose of ibandronate generally did not suppress the activation of remodeling Haversian systems to sham control levels and often not significantly below OVX control levels. The lower effect of ibandronate at predominantly cortical sites relative to cancellous sites is supported by the results of drug analysis in bone, which showed a higher incorporation of ibandronate in the predominantly trabecular bone of L6 compared with the tibia. Concentration differences observed between various regions can be explained by the relative proportion of bone surface available, i.e., there is a higher surface area available in regions of trabecular bone than in areas of cortical bone. Thus, this effect is most probably attributable to the higher surface area available for drug binding in the vertebrae. The higher concentration of ibandronate in vertebrae, as well as the nonhomogeneous distribution within the tibiae, is therefore considered a consequence of normal bone physiology. The concentration of ibandronate in total tibiae and lumbar vertebrae (L6) was dose-dependent in a weak nonlinear manner. This is consistent with the inhibitory effects of ibandronate on bone turnover and consequent reduction of remodeling sites. Activation frequency and remodeling surface were shown to decrease with increasing doses of ibandronate at most sites evaluated, thereby providing a relatively smaller surface area that could be targeted by the higher dose levels. However, this weak nonlinearity might also be a chance finding given that only three doses in a narrow dose range were assessed and that the ibandronate concentration in bone is strictly linear with the dose in aged rats when a dose range of 2 magnitudes was applied over 1 year [4]. In this study, we observed a dose-dependent preservation of bone strength at the spine and a less pronounced, slight, nonsignificant effect at the femur following treatment with intermittent iv ibandronate bolus injections. Vertebral trabecular bone mass was positively and significantly correlated with increases in apparent strength. Similar findings were obtained following lifelong administration of ibandronate to rats [17], where doses of ibandronate far in excess of any intended therapeutic dose increased apparent density and maintained bone mechanical properties of lumbar vertebrae. This implied that microfracture accumulation and thus a reduction in compressive strength did not occur [17]. In contrast, accumulation of microdamage and subsequent reduction in some mechanical properties have been reported in dogs treated with alendronate [22,23,39]. In summary, this is the first study to investigate the effect of a bisphosphonate in ovariectomized monkeys using a clinically relevant regimen. This preclinical study demonstrates that iv bolus injections of 30 ␮g/kg of ibandronate given every 30 days for 16 months (equivalent to 4 human years), which corresponds to an interval of 3 months in humans, prevents estrogen depletion-induced osteopenia and, in a noninferior manner, maintained bone strength.

Treatment with ibandronate at five times this dose level (150 ␮g/kg/dose) for 16 months shows no deleterious effects on bone and, in addition, provides a greater prevention of the OVX-induced changes in cortical bone. Bolus iv injections of ibandronate every three months are currently being extensively investigated clinically for the treatment and prevention of PMO. This study supports the clinical findings that intermittent ibandronate is effective for the prevention of osteoporosis and further verifies its potential for administration by iv bolus injection.

Acknowledgments We acknowledge the exceptional technical expertise of Martine Gagnon, the assistance of Christine Chevrier, Eric Perras, and Mathieu Sabourin, who performed the DXA and pQCT scanning, Toni Howard and Susan Bare, who assisted in the histomorphometry measurements, Jon Conta for performing the biomechanical testing, Dr. Richard Endele for measuring the ibandronate concentration in bones, and Linda Blunte, who assisted in the preparation of the manuscript.

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