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Bisphosphonates are widely used in the treatment of osteoporosis and contribute to the reduction of bone fractures. Ibandronate (IBN) is a highly potent, nitrogen-containing bisphosphonate, which is administered orally or intravenously at extended dosing intervals. Vitamin D or active vitamin D3 derivatives are also used in the treatment of osteoporosis, and are often used in combination with other drugs. In this study, we investigated the effect of treatment with the combination of once-monthly s.c. dosing of IBN plus once-daily oral eldecalcitol (ELD), an active vitamin D3 derivative, using aged ovariectomized (OVX) rats. Treatment was started the day after OVX, and analyses were performed 4, 8, and 12weeks thereafter by determination of bone markers, bone mineral density, biomechanical properties, and histomorphometry. The combination treatment showed a synergistic effect in increasing both lumbar and femoral BMD, and resulted in a significant increase in bone ultimate load. The combination of IBN plus ELD acted synergistically to reduce bone resorption, whereas bone formation did not decrease any more than with monotherapy with either IBN or ELD. Bone formation independent of bone resorption (a process known as ‘minimodeling’) was not changed in vehicle treated OVX rats despite the increase in bone turnover. ELD upregulated minimodeling, which was however not diminished in the combination treatment. In conclusion, treatment with the combination of IBN plus ELD was beneficial in the treatment of osteoporosis in aged OVX rats. It exhibited a synergistic inhibitory effect on bone resorption and keeps bone formation at the level of sham controls. This uncoupling of bone resorption/bone formation was affected, to some extent, by minimodeling-based bone formation which is independent of bone resorption. This combination regimen which showed synergistic effect on BMD and bone ultimate load without inhibition of bone formation may be beneficial in long-term osteoporosis treatment to prevent bone fractures.

CUB-domain-containing-protein-1 (CDCP1) is an integral membrane protein whose expression is up-regulated in various cancer types. Although high CDCP1 expression has been correlated with poor prognosis in lung, breast, pancreas, and renal cancer, its functional role in tumor formation or progression is incompletely understood. So far it has remained unclear, whether CDCP1 is a useful target for antibody therapy of cancer and what could be a desired mode of action for a therapeutically useful antibody. To shed light on these questions, we have investigated the cellular effects of a therapeutic antibody candidate (RG7287). In focus formation assays, prolonged RG7287 treatment prevented the loss of contact inhibition caused by co-transformation of NIH3T3 cells with CDCP1 and Src. In a xenograft study, MCF7 cells stably overexpressing CDCP1 reached the predefined tumor volume faster than the parental MCF7 cells lacking endogenous CDCP1. This tumor growth advantage was abolished by RG7287 treatment. In vitro, RG7287 induced rapid tyrosine phosphorylation of CDCP1 by Src, which was accompanied by translocation of CDCP1 to a Triton X-100 insoluble fraction of the plasma membrane. Triggering these effects required bivalency of the antibody suggesting that it involves CDCP1 dimerization or clustering. However, this initial activation of CDCP1 was only transient and prolonged RG7287 treatment induced internalization and down-regulation of CDCP1 in different cancer cell lines. Antibody stimulated CDCP1 degradation required Src activity and was proteasome dependent. Also in three different xenograft models with endogenous CDCP1 expression RG7287 treatment resulted in significant tumor growth inhibition concomitant with substantially reduced CDCP1 levels as judged by immunohistochemistry and Western blotting. Thus, despite transiently activating CDCP1 signaling, the RG7287 antibody has a therapeutically useful mode of action.

Ibandronate is a highly potent, nitrogen-containing bisphosphonate. Unlike most other bisphosphonates, it is under clinical development for both oral and intravenous (i.v.) administration. Ibandronate can be used in convenient intermittent regimens that may optimize therapeutic outcome with enhanced compliance by patients. The preclinical pharmacokinetics (PK) and pharmacology of ibandronate have been extensively explored in a large preclinical development program involving various recommended animal models of human osteoporosis. These experimental studies of ibandronate indicate that the preclinical pharmacology and PK profile of ibandronate are broadly similar to those of other nitrogen-containing bisphosphonates. The efficacy of intermittent administration of subcutaneous (s.c.) and i.v. ibandronate has been demonstrated in four animal models (rat, dog, minipig, and monkey). Thus in rats, dogs, and monkeys with estrogen depletion, and in minipigs with glucocorticoid-induced bone loss, ibandronate administered s.c. or i.v. with extended intervals between doses reduces bone turnover, increases bone mineral density, and maintains bone quality in a dose-dependent manner. Furthermore, studies in rats and dogs comparing continuous and intermittent treatment schedules indicate similar efficacy when the same cumulative dose is applied over the duration of the study. These studies with ibandronate illustrate the concept that the total cumulative dose of bisphosphonate administered determines the response, independent of whether the dose is given daily or less frequently in a given time period. The efficacy of intermittent regimens has also been verified in models of secondary osteoporosis due to secondary hyperparathyroidism or immobilization (both in rats), or due to glucocorticoids in minipigs. Important factors for determining efficacy and the magnitude of response are the doses given, the length of the interval between doses, and the underlying bone turnover rate. The mechanisms underlying the remarkable efficacy of intermittent bisphosphonate dosing are not fully understood and further research is needed. Importantly, ibandronate is the only bisphosphonate so far proven to reduce the risk of vertebral fractures significantly with a between-dose interval >2 months, in a prospective clinical trial. Collectively, the preclinical studies on ibandronate have provided a sound basis for the design of the convenient regimens currently being examined in clinical trials.

Osteonecrosis of the jaw has been reported in patients receiving high doses of intravenous nitrogen-containing bisphosphonates (N-BPs) because of malignant disease. The exact pathomechanisms have been elusive and questions of paramount importance remain unanswered. Recent studies have indicated toxic effects of bisphosphonates on different cell types, apart from osteoclast inhibition. Multipotent stem cells play an important role in the processes of wound healing and bone regeneration, which seem to be especially impaired in the jaws of patients receiving high doses of N-BPs. Therefore, the aim of the present study was to investigate the effects of different bisphosphonate derivatives and dose levels combined with varying pH levels on the mesenchymal stem cells in vitro.The effect of 2 N-BPs (zoledronate and ibandronate) and 1 non-N-BP (clodronate) on immortalized mesenchymal stem cells was tested at different concentrations, reflecting 1, 3, and 6 months and 1, 3, 5, and 10 years of exposure to standard oncology doses of the 2 N-BPs and equimolar concentrations of clodronate at different pH values (7.4, 7.0, 6.7, and 6.3). Cell viability and activity were analyzed using a WST assay. Cell motility was investigated using scratch wound assays and visualized using time-lapse microscopy.Both types of bisphosphonates revealed remarkable differences. Zoledronate and ibandronate showed a dose- and pH-dependent cellular toxicity. Increasing concentrations of both N-BPs and an acidic milieu led to a significant decrease in cell viability and activity (P.01), with more pronounced effects for zoledronate. Equimolar concentrations of clodronate did not affect the cell survival or activity significantly, apart from the effect of pH reduction itself, which was also detectable in the patients in the control group who did not receive bisphosphonates.Our results have shown that high concentrations of N-BPs and a local acidic milieu, which is commonly present in infections of the jaw, might play a key role in the pathogenesis of osteonecrosis of the jaw in patients receiving high doses of N-BPs for malignant diseases. Also the potency of N-BPs might be different, suggesting a greater risk of osteonecrosis of the jaw with zoledronate.

Bisphosphonates are potent inhibitors of osteoclast-mediated bone resorption and play an important role in the treatment of osteoporosis, metastatic bone disease, and Paget disease. However, nephrotoxicity has been reported with some bisphosphonates. Nitrogen-containing bisphosphonates directly inhibit farnesyl diphosphate (FPP) synthase activity (mevalonate pathway) and reduce protein prenylation leading to osteoclast cell death. The aim here was to elucidate if this inhibition also occurs in kidney cells and may directly account for nephrotoxicity. In an exploratory study in rats receiving zoledronate or ibandronate an approximate 2-fold increase in FPP synthase mRNA levels was observed in the kidney. The involvement of the mevalonate pathway was confirmed in subsequent in vitro studies with zoledronate, ibandronate, and pamidronate, using the non-nitrogen containing bisphosphonate clodronate as a comparator. In vitro changes in FPP synthase mRNA expression, enzyme activity, and levels of prenylated proteins were assessed. Using two cell lines (a rat normal kidney cell line, NRK-52E, and a human kidney proximal tubule cell line, HK-2), ibandronate and zoledronate were identified as most cytotoxic (EC50: 23/>1000 microM and 16/82 microM, respectively) and as the most potent inhibitors of FPP synthase (IC50; 1.6/7.4 microM and 0.5/0.7 microM, respectively). In both cell lines, inhibition of FPP synthase activity occurred prior to a decrease in levels of prenylated proteins followed by cytotoxicity. This further supports that the mechanism responsible for osteoclast inhibition (therapeutic effect) might also underlie the mechanism of nephrotoxicity.

The Göttingen minipig is one of the few large animal models that show glucocorticoid (GC)-induced bone loss. We investigated whether GC-induced loss of bone mineral density (BMD) and bone strength in minipigs can be recovered by treatment with the bisphosphonate ibandronate (IBN). 40 primiparous sows were allocated to 4 groups when they were 30 months old: GC treatment for 8 months (GC8), for 15 months (GC15), GC treatment for 15 months plus IBN treatment for months 8-15 (GCIBN), and a control group without GC treatment. Prednisolone was given at a daily oral dose of 1 mg/kg body weight for 8 weeks and thereafter 0.5 mg/kg body weight. IBN was administered intramuscularly and intermittently with an integral dose of 2.0 mg/kg body weight. BMD of the lumbar spine (L1-3) was assessed in vivo by Quantitative Computed Tomography (QCT) at months 0, 8, and 15. Blood and urine samples were obtained every 2-3 months. After sacrificing the animals lumbar vertebrae L4 were tested mechanically (Young's modulus and ultimate stress). Histomorphometry was performed on L2 and mineral content determined in ashed specimens of T12 and L4. In the GCIBN group, the GC associated losses in BMD of -10.5%+/-1.9% (mean+/-standard error of the mean, p0.001) during the first 8 months were more than recovered during the following 7 months of IBN treatment (+14.8%+/-1.2%, p0.0001). This increase was significantly larger (p0.0001) than the insignificant +2.1%+/-1.2% change in group GC15. At month 15, the difference between groups GCIBN and GC15 was 22% (p0.01) for BMD, 48% (p0.05) for Young's modulus, and 31% (p0.14) for ultimate stress; bone-specific alkaline phosphatase showed trends to lower values (p0.2) while deoxypyridinoline was comparable. This minipig study demonstrates that GC-induced impairment of bone strength can be effectively and consistently treated by IBN. GCIBN associated alterations in BMD and bone turnover markers can be monitored in vivo using QCT of the spine and by biochemical analyses, reflecting the changes in bone strength.

The local distribution, retention, and effects of intraosseous administration of ibandronate in the infarcted femoral heads were studied. Intraosseous administration effectively delivered and distributed ibandronate in the infarcted femoral heads and decreased the femoral head deformity in a large animal model of Legg-Calve-Perthes disease. Introduction: Bisphosphonate therapy has gained significant attention for the treatment of ischemic osteonecrosis of the femoral head (IOFH) because of its ability to inhibit osteoclastic bone resorption, which has been shown to contribute to the pathogenesis of femoral head deformity. Because IOFH is a localized condition, there is a need to explore the therapeutic potential of local, intraosseous administration of bisphosphonate to prevent the femoral head deformity. The purpose of this study was to investigate the distribution, retention, and effects of intraosseous administration of ibandronate in the infarcted head. Materials and Methods: IOFH was surgically induced in the right femoral head of 27 piglets. One week later, a second operation was performed to inject 14C-labeled or unlabeled ibandronate directly into the infarcted head. 14C-ibandronate injected heads were assessed after 48 h, 3 weeks, or 7 weeks later to determine the distribution and retention of the drug using autoradiography and liquid scintillation analysis. Femoral heads injected with unlabeled ibandronate were assessed at 7 weeks to determine the degree of deformity using radiography and histomorphometry. Results: Autoradiography showed that 14C-Ibandronate was widely distributed in three of the four heads examined at 48 h after the injection. Liquid scintillation analysis showed that most of the drug was retained in the injected head, and almost negligible amount of radioactivity was present in the bone and organs elsewhere at 48 h. At 3 and 7 weeks, 50% and 30% of the 14C-drug were found to be retained in the infarcted heads, respectively. Radiographic and histomorphometric assessments showed significantly better preservation of the infarcted heads treated with intraosseous administration of ibandronate compared with saline (p < 0.001). Conclusions: This study provides for the first time the evidence that local intraosseous administration is an effective route to deliver and distribute ibandronate in the infarcted femoral head to preserve the femoral head structure after ischemic osteonecrosis. In a localized ischemic condition such as IOFH, local administration of bisphosphonate may be preferable to oral or systemic administration because it minimizes the distribution of the drug to the rest of the skeleton and bypasses the need for having a restored blood flow to the infarcted head for the delivery of the drug.

AIM The effects of a systemic treatment with the bisphosphonate ibandronate on osseointegration of uncoated and hydroxyapatite-coated titanium implants and on periprosthetic bone volume have been evaluated and the dosage of medication had to be defined. METHOD We used an animal model of the rat, the animals were assigned to three treatment groups receiving 1 microg, 5 microg and 25 microg/kg body weight and one control group receiving NaCl 0.9%. An uncoated and a hydroxyapatite-coated titanium rod were inserted into the medullary canal of the femur. After 28 days the specimens were harvested and histomorphometric evaluation revealed extend of osseointegrated implant surface and changes of periprosthetic bone volume. RESULTS Treatment groups receiving 5 microg and 25 microg ibandronate showed significant improvement of osseointegrated implant surface compared to the control group. Enhancement of periprosthetic bone volume was revealed in all treatment groups but only application of 25 microg ibandronate was significantly improved compared to the control group. CONCLUSION A minor dose of 1 microg ibandronate is not effective to improve osseointegration. A high dosed bisphosphonate treatment with 5 microg or 25 microg ibandronate is potent to improve osseointegrated implant surface significantly compared to an untreated control in both uncoated and hydroxyapatite-coated titanium implants and to enhance periprosthetic bone volume. By that, improved secondary stability and prolonged survival time of cementless metal implants can be expected.

Osteoporosis is known to impair the process of implant osseointegration. Bisphosphonates are drugs that inhibit osteoclast-mediated bone resorption and normalize the high rate of bone turnover that characterizes this disease. Consequently, there is a rationale for using bisphosphonates to enhance the early stabilization of implants in subjects with low bone mass. In this study, 84 rats received titanium-only or hydroxyapatite (HA)-coated titanium femoral implants, 3 months after being ovariectomized (OVX) or sham operated. They were then treated for 4 weeks. The OVX rats were randomly assigned to daily subcutaneous injections of either saline or the bisphosphonate ibandronate (at a dose of 1 microg/kg or 25 microg/kg), while the sham-operated animals received saline throughout. The 1 microg/kg or 25 microg/kg ibandronate doses are considered translatable to doses used to treat osteoporosis and metastatic bone disease (MBD), respectively, in rats, and roughly reflect those used in humans. At the end of the treatment period, bone mineral density (BMD) at the lumbar spine increased in both of the ibandronate-treated groups when compared with the OVX control animals and to a level similar to that of the sham-operated control group. Osseointegration, determined by histomorphometric analysis and expressed as percentage of osseointegration implant surface (OIS), did not differ between groups for the titanium-only implants. For the HA-coated implants, however, OIS was 113.5% and 185% higher in the groups receiving 1 microg/kg or 25 microg/kg ibandronate, respectively, relative to the OVX controls. In turn, the OIS of the HA-coated implants was 56.5% lower in the OVX control group than in the sham control group. These findings clearly demonstrate that OVX-induced osteopenia impairs the osseointegration of HA-coated titanium implants and that ibandronate, administered at doses analogous to those used to clinically treat osteoporosis and MBD, counters this harmful effect. Ibandronate may, therefore, have a role in improving the osseointegration of implants in patients with osteoporosis and MBD.

Femoral head deformity is the most serious sequela of ischemic necrosis of the immature femoral head. The purpose of this study was to determine if a highly potent antiresorptive agent, ibandronate, can inhibit bone resorption during the repair of the infarcted femoral head and thus alter the repair process. We hypothesized that preservation of the trabecular framework by inhibiting osteoclastic bone resorption would minimize the development of deformity in a piglet model of ischemic necrosis. The effect of ibandronate on long-bone growth was also assessed.Ischemic necrosis of the right femoral head was produced in twenty-four piglets by placing a ligature tightly around the femoral neck. The animals were divided into three groups according to whether they received saline solution, prophylactic treatment, or post-ischemia treatment. The contralateral, untreated femoral heads from the animals that had received saline solution served as the normal control group. At eight weeks, the femoral heads were assessed for deformity with radiography and for trabecular bone indices with histomorphometry. Also, the length of femur from the untreated side was measured on the radiographs and compared among the groups.Radiographic assessment showed that the epiphyseal quotient, determined by dividing the maximum height of the osseous epiphysis by the maximum diameter, was better preserved in the prophylactic (p0.001) and post-ischemia (p = 0.02) treatment groups than in the group treated with saline solution. Histomorphometric assessment also showed that the trabecular bone indices were better preserved in the prophylactic and the post-ischemia treatment groups than in the group treated with saline solution (p0.01). The mean femoral length on the untreated side of the animals treated with ibandronate was reduced compared with the length on the untreated side of the animals that had received saline solution (p/= 0.01).Ibandronate preserves the trabecular structure of the osseous epiphysis and prevents femoral head deformity during the early phase of repair of ischemic necrosis in the piglet model.

Bisphosphonates are widely used to prevent and treat skeletal complications of metastatic bone disease. There is increasing evidence that, besides inhibiting osteoclast activity and reducing bone resorption, bisphosphonates also have an anti-tumor effect. This paper reviews the preclinical data for ibandronate. Ibandronate increased the proportion of apoptotic tumor cells in vitro and in vivo, possibly following activation of caspase-like proteases. In vitro, ibandronate also prevented adhesion and spreading of tumor cells to bone, and tumor cell invasion. These inhibitory effects were additive when ibandronate was given with paclitaxel or docetaxel. In animal models of tumor-induced osteolysis, ibandronate significantly reduced the development of osteolytic lesions. Efficacy for the prevention and reduction of bone metastases was related to the timing of treatment; ibandronate treatment initiated prior to or shortly after tumor cell inoculation inhibited the growth of bone metastases and preserved skeletal integrity most effectively. As with other bisphosphonates, the influence of ibandronate on soft tissue metastases has been inconsistent. Overall, preclinical evidence supports the rationale for adjuvant treatment with ibandronate for patients at risk of metastatic bone disease. The renal safety profile of ibandronate supports its suitability for long-term adjuvant use, even with intermittent high dosing. Adjuvant clinical trials have been initiated. The ability of bisphosphonates to preserve skeletal integrity is also of benefit in other clinical settings. Recent studies in rat models demonstrate improved osseointegration of joint implants following ibandronate therapy, with potential application in patients with conditions such as degenerative arthritis or osteoporosis.

We examined the impact of urinary diversion using several types of intestinal segments on the bone metabolism of growing rats with renal insufficiency.A total of 110, 8-week-old Sprague-Dawley rats underwent 2-stage subtotal nephrectomy by removal of 5/6 of the renal mass or sham operation. Except for a uremic control group all uremic rats underwent enterocystoplasty using stomach, ileum or colon. An additional group with colic augmentation received the bisphosphonate ibandronate. Bone mineral density of the tibia and lumbar spine, serum analysis and urinary excretion of the bone resorption marker deoxypyridinoline were determined monthly for 12 weeks. At study termination bone ash weight, bone mineral analysis and serum osteotropic hormone levels were determined.All groups undergoing subtotal nephrectomy had a decreased endogenous creatinine clearance of approximately 30%. The lowest gains in tibial and lumbar spine bone mineral density were observed in animals undergoing ileocystoplasty. Femoral calcium content was significantly decreased in uremic ileocystoplasty rats compared with uremic controls. These changes were not induced by alterations in serum pH, nor were they associated with accelerated bone resorption as assessed by deoxypyridinoline. Ibandronate prevented changes related to bone resorption and increased bone mass.Our results suggest that cystoplasty using ileum segments can aggravate renal bone disease in growing rats with mild uremia. Since the acid-base state was unchanged, other properties of the interposed ileum segment must be responsible for the negative effect on bone metabolism.

Animal models are important tools to study the development of bone metastases and to evaluate strategies for their prevention and treatment. We here describe a new model in which tumour inoculation is achieved by injection of cancer cells into the femoral artery. This approach results in the development of multiple osteolytic lesions in the distal femora and proximal tibiae within 18 days after inoculation, with a success rate of 95–100% and no additional comorbidity. In untreated animals, osteolyses expanded continuously at a growth rate of 4.7–8.2 mm2/4 days, causing extensive destruction of resident bone structures by the tumour, significant loss of tibial bone density and a transient rise in urinary bone resorption markers. Continuous daily treatment with ibandronate (10 μg/kg) inhibited further growth of fully established metastases and reduced the mean osteolytic growth rate to 0.03 mm2/4 days. In lesions

Oral bisphosphonates are well established for the treatment and prevention of postmenopausal osteoporosis; however, they are poorly absorbed from the gastrointestinal (GI) tract and have been associated with GI adverse events. Thus, current dosing guidelines recommend that the patient not eat or lie down for at least 30 minutes after taking oral bisphosphonates, a requirement that is inconvenient and may be associated with reduced compliance. The drawbacks of these dosing requirements may be overcome either by reducing dosing frequency or by using alternative routes of administration.Ibandronate is a potent nitrogen-containing bisphosphonate that can be given orally or IV, daily or intermittently, with a between-dose interval of up to 3 months. This article presents the results of published Phase II trials of the efficacy and safety profile of oral and IV ibandronate administered daily or intermittently to postmenopausal women with low bone mass.MEDLINE was searched through January 2002 to identify all published Phase II clinical studies of oral and IV ibandronate in the treatment of post-menopausal osteoporosis.In the 3 Phase II studies identified, marked reductions in biochemical markers of bone resorption (50%-70%) and bone formation (40%-50%) were seen to a similar and statistically significant extent with oral ibandronate 2.5 mg/d (P0.001), oral ibandronate 20 mg QOD given for 12 doses at the start of each 3-monthly period (P0.001), and injections of ibandronate 2 mg IV given every 3 months (P0.01). All treatment regimens produced comparable significant increases in bone mineral density at the lumbar spine (P0.01) and hip (P0.05). Ibandronate was well tolerated when administered both orally and as an IV injection.In these Phase II studies, oral or IV ibandronate, administered continuously or intermittently, reduced markers of bone turnover, significantly increased bone mineral density, and was well tolerated in the treatment of osteoporosis in postmenopausal women. The data from these studies provided the rationale for further investigation of ibandronate in larger longer-term Phase III studies evaluating its potential as an efficacious and flexible alternative to existing bisphosphonate regimens.

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 microg/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 microg/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 microg/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 microg/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.

We determined the effects of the potent bisphosphonate ibandronate in a murine model of human myeloma bone disease. In this model, bone lesions typical of the human disease develop in mice following inoculation of myeloma cells via the tail vein. Treatment with ibandronate (4 μg per mouse per day) significantly reduced the occurrence of osteolytic bone lesions in myeloma-bearing mice. However, ibandronate did not prevent the mice from developing hindlimb paralysis and did not produce a detectable effect on survival. There was no significant effect of ibandronate on total myeloma cell burden, as assessed by morphometric measurements of myeloma cells in the bone marrow, liver, and spleen, or by measurement of serum IgG2b levels. These results support clinical findings that bisphosphonates may be useful for the treatment of myeloma-associated bone destruction, but suggest that other therapies are also required to reduce tumor growth.

We determined the effects of the potent bisphosphonate ibandronate in a murine model of human myeloma bone disease. In this model, bone lesions typical of the human disease develop in mice following inoculation of myeloma cells via the tail vein. Treatment with ibandronate (4 μg per mouse per day) significantly reduced the occurrence of osteolytic bone lesions in myeloma-bearing mice. However, ibandronate did not prevent the mice from developing hindlimb paralysis and did not produce a detectable effect on survival. There was no significant effect of ibandronate on total myeloma cell burden, as assessed by morphometric measurements of myeloma cells in the bone marrow, liver, and spleen, or by measurement of serum IgG2b levels. These results support clinical findings that bisphosphonates may be useful for the treatment of myeloma-associated bone destruction, but suggest that other therapies are also required to reduce tumor growth.

Bisphosphonates (BPs) are powerful inhibitors of bone resorption. They exert a strong affinity to bone mineral which allows the rapid and selective targeting to bone in vivo. We synthesized and evaluated two new classes of compounds: phosphono succinic acid (PSA) and phosphono glutaric acid (PGA) derivatives. In order to transfer the antiresorptive properties of bisphosphonates attempts have been made to elucidate the key elements of the putative BP pharmacophore. Whereas the new compounds revealed similar or better bone mineral affinity properties than BPs in vitro, the inhibition of endogenous bone resorption in vivo was less effective, but in contrast to BPs the effects were reversible after discontinuation of treatment, which suggests that these compounds are metabolically degradable.

Pioglitazone, the peroxisome proliferator-activated receptor-gamma (PPAR-γ) agonist is an effective therapy for type 2 diabetes, but has been associated with increased risk for bone fracture. Preclinical studies suggest that PPAR-α agonists (e.g., fenofibrate) increase bone mineral density/content, although clinical data on bone effects of fibrates are lacking. We investigated the effects of pioglitazone (10 mg/kg/day) and fenofibrate (25 mg/kg/day) on bone strength and bone histomorphometric parameters in osteopenic ovariectomized (OVX) rats. An additional group of rats received a combination of pioglitazone + fenofibrate to mimic the effects of a dual PPAR-α/γ agonist. The study consisted of a 13-week treatment phase followed by a 6-week treatment-free recovery period. Pioglitazone significantly reduced biomechanical strength at the lumbar spine and femoral neck compared with rats administered fenofibrate. Co-treatment with pioglitazone + fenofibrate had no significant effect on bone strength in comparison with OVX vehicle controls. Histomorphometric analysis of the proximal tibia revealed that pioglitazone suppressed bone formation and increased bone resorption at both cancellous and cortical bone sites relative to OVX vehicle controls. In contrast, fenofibrate did not affect bone resorption and only slightly suppressed bone formation. Discontinuation of pioglitazone treatment, both in the monotherapy and in the combination therapy arms, resulted in restoration of bone formation and resorption rates, demonstrating reversibility of effects. The above data support the concept that dual activation of PPAR-γ and PPAR-α attenuates the negative effects of PPAR-γ agonism on bone strength.

Peroxisome proliferator-activated receptor (PPAR) γ agonists, such as pioglitazone (Pio), improve glycemia and lipid profile but are associated with bone loss and fracture risk. Data regarding bone effects of PPARα agonists (including fenofibrate (Feno)) are limited, although animal studies suggest that Feno may increase bone mass. This study investigated the effects of a 13-week oral combination treatment with Pio (10 mg/kg per day)+Feno (25 mg/kg per day) on body composition and bone mass parameters compared with Pio or Feno alone in adult ovariectomized (OVX) rats, with a 4-week bone depletion period, followed by a 6-week treatment-free period. Treatment of OVX rats with Pio+Feno resulted in ∼50% lower fat mass gain compared with Pio treatment alone. Combination treatment with Pio+Feno partially prevented Pio-induced loss of bone mineral content (∼45%) and bone mineral density (BMD; ∼60%) at the lumbar spine. Similar effects of treatments were observed at the femur, most notably at sites rich in trabecular bone. At the proximal tibial metaphysis, concomitant treatment with Pio+Feno prevented Pio exacerbation of ovariectomy-induced loss of trabecular bone, resulting in BMD values in the Pio+Feno group comparable to OVX controls. Discontinuation of Pio or Feno treatment of OVX rats was associated with partial reversal of effects on bone loss or bone mass gain, respectively, while values in the Pio+Feno group remained comparable to OVX controls. These data suggest that concurrent/dual agonism of PPARγ and PPARα may reduce the negative effects of PPARγ agonism on bone mass.

Bisphosphonates, like ibandronate (Bondronat), represent the mainstay of treatment for metastatic bone disease. Ibandronate selectively binds to bone mineral and prevents osteoclast-mediated skeletal destruction. This review describes the preclinical and clinical profiles of ibandronate for treatment of cancer metastatic to bone. In preclinical studies ibandronate reduced metastatic processes and tumor growth, induced tumor cell apoptosis, decreased bone pain, and enhanced biomechanical indices. Skeletal destruction was completely prevented with ibandronate, and this directly correlated with histomorphometry and markers of bone turnover. Ibandronate efficacy in combination with anti-cancer therapies is discussed. Preclinical studies demonstrated that ibandronate does not compromise safety, including renal health. Intravenous and oral ibandronate had comparable efficacy in three Phase III clinical trials. Ibandronate achieved significant risk reductions in the incidence of skeletal-related events and bone pain. In additional clinical studies, ibandronate reduced markers of bone turnover. Furthermore, loading-dose ibandronate rapidly reduced bone pain in Phase II trials. Adjuvant trials are ongoing. The clinical safety profile (including a 4-year follow-up study) has demonstrated renal health is maintained with ibandronate. Overall, ibandronate preserves skeletal integrity, has a favorable safety profile, maintains renal function, and can rapidly reduce and maintain bone pain below baseline levels in patients with cancer metastatic to bone.

Background: Using a nude rat model of site-specific metastatic bone disease (MBD), we developed a semiquantitative histological score for rapid assessment of lytic lesions in bone. This provides additional information to conventional histological measurement by clarifying the extent and location of metastatic infiltration and the tumor growth pattern. The score can also be used to assess the action of bisphosphonates on bone metastases. Materials and Methods: Male nude rats (n = 12 per group) were inoculated with the human breast cancer cell line MDA-MB-231 via the femoral artery. Following appearance of radiographically visible osteolytic lesions on day 18, the animals received phosphate-buffered saline (PBS; controls) or ibandronate (IBN, 10 µg P/kg) daily until day 30. Whole body radiographs were obtained on days 18 and 30, and osteolytic areas (OA) were determined by radiographic computer-based analysis (CBA). On day 30, MBD was assessed in both tibias using conventional histological CBA and the new scoring system. Results: Metastatic tumor area correlated with the total sum of the new score in both PBS- (r = 0.762) and IBN-treated animals (r = 0.951; p < 0.001). OA correlated well with the total sum in both groups (r = 0.845 and 0.854, respectively; p < 0.001). Conclusion: Significant reduction of bone marrow and cortical infiltration of tumor cells with IBN suggested local control of metastases. Hintergrund: Mit Hilfe eines etablierten Tiermodells zur Erzeugung lokalisationsspezifischer Knochenmetastasen in der Nacktratte wurde ein semiquantitatives histologisches Graduierungssystem zur schnellen Bewertung osteolytischer Knochenmetastasen entwickelt. Das Graduierungssystem liefert hinsichtlich der Metastasenlokalisation, deren Ausmaß und Infiltrationsmuster wertvolle Zusatzinformationen zu den konventionellen histologischen Untersuchungsmethoden. Damit kann beispielsweise auch die pharmakologische Wirkung von Bisphosphonaten auf die Knochenmetastasierung beurteilt werden. Material und Methoden: Männlichen Nacktratten (n = 12 pro Gruppe) wurden Zellen der humanen Brustkrebszellinie MDA-MB-231 in die Oberschenkelarterie inokuliert. Ab dem Auftreten radiologisch erkennbarer Osteolysen 18 Tage nach Inokulation erhielten die Tiere bis zum Studienende (Tag 30) täglich entweder eine subkutane Applikation einer Phosphat-Puffer-Lösung (Kontrollgruppe) oder Ibandronat (IBN, 10 µg P/kg; Behandlungsgruppe). Konventionelle Röntgenaufnahmen wurden an den Tagen 18 und 30 nach Tumorinokulation angefertigt und die Osteolysenflächen mittels Computerauswertung bestimmt. Nach Studienende wurde der Metastasenbefall in beiden Tibiae sowohl konventionell histologisch als auch mittels des neuen Graduierungssystems ausgewertet. Ergebnisse: Die Metastasenfläche korrelierte mit der kummulativen Punktsumme des Graduierungssystems sowohl in der Kontrollgruppe (r = 0,762; p < 0,001) als auch in der Ibandronat- Gruppe (r = 0,951; p < 0,001). Ebenso war die Osteolysenfläche eng mit der Punktesumme in beiden Gruppen korreliert (r = 0,845 und 0,854; p < 0,001). Schlussfolgerung: Die signifikante Reduktion von Knochenmark- und Kortikalisbefall durch IBN deuten auf eine gute lokale Kontrolle des Metastasenwachstums hin. Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

Intravenous bisphosphonates may be involved in the development of osteonecrosis of the jaw (ONJ). However, a mechanistic causality has not been demonstrated. To evaluate whether there is higher drug uptake by the jaw versus other bones that might be involved in ONJ pathogenesis, we performed a pilot experiment comparing ibandronate uptake in rat mandible, femur, and lumbar vertebrae after repeated administration. Rats (n = 1/group) received daily subcutaneous injections of ibandronate in doses ranging from 0.003 to 0.3 mg/kg/day for 9 days. Five days after the last injection, the animals were killed and the right femur, lumbar vertebrae L3-L5, and the right mandible were removed. After cleaning and drying, bone dry weight was recorded, and ibandronate concentration was determined in whole-bone hydrolyzates by gas chromatography mass spectrometry. Concentrations of ibandronate increased dose-dependently in all bones with similar concentrations per bone at each dose level ranging from values below quantification limit (low dose) up to approximately 10 ng ibandronate/mg bone dry weight (high dose). In this rat study, there was a relatively similar bisphosphonate uptake between the femur and lumbar vertebrae bones whereas the uptake in the jaw was statistically smaller with regard to the absolute values (P < 0.05). Thus, there is no suggestion of preferential bisphosphonate uptake in the jaw.

There is evidence that bisphosphonates can improve fixation of cementless metal implants by enhancing the extent of osseointegration. The current preclinical study examined whether the nitrogen-containing bisphosphonate ibandronate can accelerate this process, resulting in early achievement of secondary stability and sealing of the bone-implant interface to prevent wear debris migration.The study was conducted on 88 female Sprague-Dawley rats in which uncoated titanium and hydroxyapatite-coated titanium implants were surgically inserted into the medullary canal of each femur. The animals were randomly assigned to receive subcutaneous treatment with 1.0, 2.5, or 5.0 microg/kg per day ibandronate or saline solution as a control. The extent of osseointegration expressed by the osseointegrated implant surface was quantified by histomorphometry at eleven time points during the study period. To determine the time course of osseointegration, the data were expressed using third-order polynomial regression analysis.For hydroxyapatite-coated implants, the highest dose of ibandronate (5 microg) reduced the time for a sufficient implant fixation of 60% osseointegrated implant surface to 18 days compared to 38 days in the control group. This reduction of 20 days (52.6%) represents a halving in the time required for sufficient osseointegration of the implant. For hydroxyapatite-coated implants and low-dose ibandronate application (1 microg, 2.5 microg) and for uncoated titanium implants, acceleration of osseointegration was not observed in any of the study arms.Continuous treatment with 5 microg/kg per day ibandronate is potent in accelerating osseointegration of hydroxyapatite-coated implants. As a result, improved early secondary stability and prevention of wear debris migration by the sealing of the implant-bone interface can be expected, therefore prolonging the long-term survival of the implant.

Bisphosphonates are potent osteoclast inhibitors that have been associated with renal toxicity following rapid intravenous administration of high doses, which was hypothesised to be due to precipitation of bisphosphonate aggregates or complexes in the kidney. Five studies were conducted in rats investigating the characteristics of bisphosphonate-related acute renal effects. These studies included single intravenous injections of the nitrogen-containing bisphosphonates (1) ibandronate (1-20 mg/kg), or (2) zoledronate (1-10 mg/kg); (3) a single nephrotoxic dose of the non-nitrogen-containing bisphosphonate, clodronate (2 x 200 mg/kg intraperitoneal injection); (4) a single low dose of ibandronate (1 mg/kg); (5) a single high dose of zoledronate (10 mg/kg). Clinical biochemistry and kidney histopathology were performed 1 and/or 4 days after bisphosphonate dosing. The proximal convoluted tubules were the primary target for renal injury. Tubular degeneration and single cell necrosis of the these tubules were observed with all three bisphosphonates on the fourth, but not the first day after dosing. Differences between the bisphosphonates in the type and/or localisation of the lesions were apparent. Granular deposits in the lumen of distal tubules were apparent with the highest dose of zoledronate (10 mg/kg). However, intraluminal debris was proteinaceous with no evidence of any precipitation of bisphosphonate, or formation of aggregates or complexes in the kidney. Generally, biochemical parameters of renal safety and urinary enzymes did not differ significantly from controls. In summary, bisphosphonate-related renal changes did not appear to be due to the precipitation, aggregation or complexation of bisphosphonate, and biochemical parameters of renal safety did not reliably detect this renal injury.

Both bisphosphonates and radiotherapy are highly effective for the management of bone metastases. Our in vitro study examined the cytotoxic effects resulting from combinations of ibandronate and ionising radiations (RX) in various sequences on breast cancer cells. Single radiation doses were given before, at halftime of, or after acute ibandronate incubation (48 h). Single or fractionated radiation doses were applied at the end of chronic ibandronate incubation (5 weeks). Combination of acute ibandronate exposure and single radiation doses led to synergistic cytotoxic effects in MDA-MB-231 cell line, but only with low ibandronate concentrations in MCF-7 cell line. In both cell lines, synergy was more marked when ibandronate followed RX. After long-term ibandronate exposure, only high single radiation doses induced synergistic effects in MDA-MB-231 cell line. Synergy was only detected with low ibandronate concentrations in MCF-7 cell line. In both cell lines, fractionated radiation doses exerted similar effects. The combination of ibandronate with radiation can exert synergistic effects on the inhibition of breast cancer cells growth, depending on cell line, drug sequence and dosage. Our data might provide a rationale for associating bisphosphonates and radiotherapy for the treatment of bone metastases from breast cancer.

Renal insufficiency is not rare in cancer patients who may receive nephrotoxic medications as antineo-plastic agents or for comorbid conditions. Thus, the choice of a particular bisphosphonate for patients with metastatic bone disease should be based not only on efficacy but also on the risk for renal deterioration. Some i.v. bisphosphonates have been associated with occasional renal toxicity in the clinical setting. Preclinical studies have also shown that there may be considerable differences among bisphosphonate renal safety profiles. Comparative studies show variations in the risk for histopathologic damage and the ability to cause cumulative toxicity during intermittent dosing. Reasons for the differences among bisphosphonates are not fully understood; however, research shows that they may be influenced by pharmacokinetic properties such as renal tissue half-life or protein binding and intracellular potency. Further preclinical analyses are needed to confirm and evaluate differences among bisphosphonates.

Recent studies show that bisphosphonates can decrease the development of femoral head deformity following ischemic osteonecrosis by inhibiting osteoclast-mediated bone resorption. Given the potential new indication, improved understanding of pharmacokinetics of bisphosphonates as it applies to the infarcted head would be beneficial. The purpose of this study was to investigate the local bioavailability and the distribution of ibandronate in the infarcted head at the avascular and vascular phases of the disease process. Ischemic osteonecrosis of the femoral head was surgically induced in 15 piglets. One, 3, and 6 weeks following the induction of ischemia, which represent various stages of revascularization and repair, 14C-labeled ibandronate was administered intravenously. Twenty-four hours following 14C-drug administration, the level of radioactivity and its distribution in the infarcted heads were determined using liquid scintillation analysis and autoradiography. A significant correlation was found between the extent of revascularization and the level of radioactivity measured in the infarcted heads (r=0.80, P0.05). The radioactivity level in the infarcted heads measured by liquid scintillation was similar to the negative controls at 1 week when revascularization was absent, but it increased significantly at 6 weeks when extensive revascularization was present (Por=0.00004). Autoradiographic assessment showed similar silver grain counts in the infarcted heads compared to the background at 1 week. At 3 weeks, a significant increase in the silver grain count was observed in the necrotic regions of the infarcted heads compared to the background (P=0.001) even though only a small area of the heads (2%) was found to be revascularized, suggesting diffusion of 14C-drug from the revascularized to the non-revascularized areas of the head. At 6 weeks, extensive 14C-drug binding was observed in the areas of revascularization with preferential binding of 14C-drug to the newly formed bone compared to the remaining necrotic bone (P=0.000001). These results indicate that revascularization and repair produce significant alteration of local bioavailability and distribution of ibandronate in the infarcted head. To our knowledge, this is the first study to examine the local bioavailability and distribution of bisphosphonate in the infarcted head. Current findings have important implications with regard to the timing and dosing of bisphosphonate after the onset of ischemic osteonecrosis.

Osteoporosis is a skeletal disorder characterized by low bone mass and deterioration of bone microarchitecture resulting in bone fragility, which increases the risk of fracture. The clinical efficacy of bisphosphonates is evaluated through improvements in bone mineral density (BMD) and reductions in the risk for fracture. However, as bisphosphonates are administered long term, there is increasing interest in their effects on bone quality, which includes bone mass, strength and architecture. Ibandronate is a potent, nitrogen-containing bisphosphonate with significant antifracture efficacy when administered daily and in regimens with extended between-dose intervals. Clinical studies with ibandronate are supported by an extensive preclinical program that investigated the efficacy and bone safety of ibandronate in various animal models of osteoporosis. In preclinical studies, treatment with ibandronate maintained, or improved the quality, strength and architecture of bone. Intermittent and daily ibandronate regimens provided similar benefits. During ibandronate treatment, the bone retains its capacity for repair and bone mineralization is not adversely affected. Notably, positive relationships among BMD, bone strength and bone architecture have been demonstrated. This review describes the preclinical evidence for the preservation of bone quality with ibandronate, irrespective of the dosing regimen and even when administered at doses higher than those used therapeutically.

OBJECTIVE To investigate the effect of urinary diversion using several types of intestinal segments on cortical and cancellous bone structure of growing rats with renal insufficiency. MATERIAL AND METHODS In all, 110 female Sprague-Dawley rats (8 weeks old) had either a two-stage subtotal nephrectomy by removing five-sixths of the renal mass, or a sham operation. Except for a uraemic control group, all uraemic rats then had an enterocystoplasty using stomach, ileum or colon (20 animals per group). An additional group with colic augmentation received the bisphosphonate ibandronate. After 12 weeks, the left tibia was assessed using peripheral quantitative computed tomography and bone histomorphometry. RESULTS After subtotal nephrectomy all groups had ≈ 30% less endogenous creatinine clearance. Renal failure alone or in association with gastric or colic augmentation induced only negligible changes in the mass and structure of cortical and cancellous tibial bone. In contrast, rats after ileal augmentation and renal failure had a significant reduction in cancellous bone mineral density (P

The accurate determination of bisphosphonate levels in bone and biological fluids is important in both clinical and pharmacological/toxicological studies. Ibandronate is a potent nitrogen-containing bisphosphonate containing a tertiary amine group, which does not easily form chromophore derivatives that can be detected by UV light or fluorescence emissions. The current report describes the methodology and validation of a GC-MS assay for ibandronate in serum/plasma and urine, a similar, modified GC-MS method for measurement of bone ibandronate levels, and an ELISA for ibandronate determination in serum/plasma. The range of quantification for the GC-MS was 1-100 ng/ml and 2-7500 ng/ml in plasma or serum and urine, respectively, and 50-1600 pg/ml (potentially 10-320 pg/ml depending on sample size) for the ELISA in plasma or serum. These assays were comparable. The practical application of the assays in preclinical and clinical studies is briefly reviewed.

Ibandronate is a potent nitrogen-containing bisphosphonate. It has a strong affinity for bone mineral and potently inhibits osteoclast-mediated bone resorption. Ibandronate is effective for the treatment of hypercalcemia of malignancy, metastatic bone disease, postmenopausal osteoporosis, corticosteroid-induced osteoporosis, and Paget's disease. Oral ibandronate is rapidly absorbed (t(max)1 hour), with a low bioavailability (0.63%) that is further reduced (by up to 90%) in the presence of food. Ibandronate has a wide therapeutic index and is not metabolized and, therefore, has a low potential for drug interactions. Given its metabolic stability, ibandronate is eliminated from the blood by partitioning into bone (40%-50%) and through renal clearance (CL(R) approximately 60 mL/min). The CL(R) of ibandronate is linearly related to creatinine clearance. The sequestration of ibandronate in bone (V(D)90 L) results in a multiphasic elimination (t((1/2)) range approximately 10-60 hours), characterized by the slow release of ibandronate from the bone compartment. The potency of ibandronate and its sequestration into bone allow ibandronate to be developed as oral and intravenous injection formulations that can be administered with convenient extended between-dose intervals.

Using three well-designed experimental studies as illustration, we demonstrate that the biostatistical design and analysis of long-term animal studies simulating human osteoporosis should be analogous to the design and analysis of randomized clinical trials. This principal is in accordance with the recommendations from the International Conference on Harmonisation guidelines concerning statistical principles in clinical trials (1). An important element of biostatistical study design is sample size. The three studies that are described herein used an a-priori sample size estimation for the one-way layout that included controls and several treatment and dose groups. In these k-sample designs, with at least one control group, both the multiple comparison procedure and trend tests within procedures for identification of the minimal-effective dose are recommended. Although p-values in pharmacology are quite common, confidence intervals should be used according to their interpretation for both statistical significance and clinical relevance. The use of one-sided confidence intervals for both the difference and the ratio to control for proving either superiority or at least noninferiority is demonstrated by real data examples. Relevant and relatively straightforward software is available for biostatistical analysis and can also be used to aid design. In summary, referring to published, well-designed experimental studies can help to assist with ensuring the quality of future investigations. © 2004, Drug Information Association. All rights reserved.

The dose-dependent effect of ibandronate treatment on bone mass and architecture was assessed in a large animal study of OVX monkeys using μCT for quantitative bone morphometry and biomechanical testing for measures of bone strength. The study showed that intermittent ibandronate preserved lumbar spine bone quality and strength in these animals after 16 months of treatment. Introduction: Ibandronate is a bisphosphonate, which is a class of compounds that, in pharmacologically active doses, not only suppresses bone resorption and turnover but also prevents loss of bone mass and strength in the ovariectomized (OVX) rat. Materials and Methods: We evaluated the effects of ibandronate on bone mass and architecture in the OVX cynomolgus macaque. Sixty-one adult female macaques were divided into five groups (N = 11–15): sham control, OVX control, and OVX low- (10 μg/kg), medium- (30 μg/kg), and high- (150 μg/kg) dose ibandronate. Treatment was administered by intravenous bolus injection every 30 days for 16 months starting at ovariectomy. This dosing schedule is equivalent to a 3-monthly dosing regimen in human subjects over 4 years. Animals were killed at the conclusion of the study, and excised bone specimens of the first lumbar vertebra (L1) were evaluated for quantitative bone densitometry, morphometry, and mechanical properties. Architectural parameters were assessed by μCT including direct 3D bone morphometry. A measure of specimen strength was obtained using destructive compression testing. Results and Conclusions: A significant loss of bone mass and related changes in bone architecture after ovariectomy resulted in a reduction of whole bone strength as expressed by high correlations between architectural and mechanical properties. In this analysis, BMC was the best single predictor of whole bone strength (r2 = 67%). Nevertheless, including architectural indices in a multiple linear regression analysis increased that prediction to 88%. With respect to the treatment, the medium- and high-dose groups were not significantly different from the sham group for all bone mineral and structural parameters. Additionally, significant differences were seen for all measured parameters between the high-dose group and the OVX group, and for some parameters, between the medium-dose group and the OVX group. Intermittent ibandronate treatment effectively and dose-dependently prevented bone loss, architectural deterioration, and strength reduction in the lumbar spine of OVX monkeys.

Introduction: Given that bisphosphonates reduce bone turnover, it is important to establish that their long-term administration does not impair bone quality. This paper describes a new model for simulation of fracture repair to evaluate several aspects of bone quality following long-term administration (34 or 36 weeks) of ibandronate in full-grown beagle dogs. Methods: The treatment schedule consisted of continuous daily subcutaneous administration of a pharmacologically active dose (1 μg/kg/day) and two cyclical intermittent regimens providing a similar total dose per animal at the end of the experiment. Seven or 8 weeks before study end, 10 holes were drilled in the left tibia and bone marrow ablation was performed in the ipsilateral femur. Serial measurements for blood biochemistry (osteocalcin and iso-alkaline phosphatase) and bone mineral density (BMD; whole body and L1–L7) by dual-energy X-ray absorptiometry (DEXA) were performed during the experiment. Bone quality was determined at the end of the experiment by assessing early and late stage defect healing and structural, cellular, and dynamic histomorphometry (femur, tibia, and lumbar vertebrae L3 and L4). Results: Healing of the drill hole defects, which simulate the first stage of fracture healing, was neither qualitatively nor quantitatively influenced by ibandronate. The same was true for the activation of cortical remodeling that occurs in the later stage of fracture healing, which started in Week 4 after surgery and declined after Week 8 in all groups. Additionally, no difference was found between the various regimens and the controls with respect to DEXA analyses, trabecular bone volume, cancellous bone tissue area, cancellous bone perimeter, osteoclast count, serum osteocalcin, or bone-specific alkaline phosphatase. Discussion: In conclusion, the presence of the first and second steps of fracture healing and the fact that the histological features closely resemble those of fracture repair validate the development and characterization of a new model for simulation of fracture repair. A long-term study with a therapeutically active dose of ibandronate shows that ibandronate does not impair BMD, bone structure, bone repair, coupling, and serum parameters for bone formation and turnover after long-term administration.

Rapid, intravenous (i.v.) administration of high doses of bisphosphonates has been associated with acute renal toxicity. This controlled, preclinical study over 25 weeks investigated the potential for subclinical renal damage to accumulate to clinically relevant levels when minimally nephrotoxic doses of ibandronate (1 mg/kg) or zoledronate (1 or 3 mg/kg) were given intermittently, with a between-dose interval of 3 weeks, or as a single dose by i.v. injection. In rats, a single dose and intermittent dosing of ibandronate resulted in a similar incidence (one of six and two of six rats, respectively) and severity score (1.0 for both) of proximal tubular degeneration and single cell necrosis. No accumulation of histopathological renal damage occurred. However, intermittent dosing of zoledronate induced a higher incidence (six of six rats) and severity score (3.0) of renal damage compared with single dosing (four of six rats and 1.3, respectively). Accumulation of renal damage was also observed for a lower intermittent dose of zoledronate (1 mg/kg) that had not exhibited histopathological renal damage when given as a single 1 mg/kg dose. Biochemical parameters confirmed these histopathological findings. In summary, the results from this study indicate that administering ibandronate intermittently provides sufficient time for regeneration of potential subclinical renal damage.

This study was designed to investigate whether biochemical markers of bone resorption and formation could be determined in the serum and urine of marmosets (Callithrix jacchus), using standard laboratory chemistry methods and commercially available human kits. Consequently, the findings from this study will indicate whether the techniques and kits could serve as appropriate tools for assessing changes in bone turnover in this species. Two groups of animals (n = 12/group), consisting of a comparable number of young and old male and female marmosets, were given either isotonic saline or a single dose of the bisphosphonate ibandronate (0.1 mg/kg) s.c. in order to suppress bone turnover. Blood and urine were collected at baseline and 5 days after administration. Samples were analyzed for urinary (u) and serum (s) markers of bone formation (serum osteocalcin [sOC], serum N-terminal crosslinks of human pro-collagen type I [sP1NP]) and bone resorption (urinary pyridinoline [uPYD], urinary deoxypyridinoline [uDPD], serum C-terminal crosslinks of human collagen type I (C-telopeptide) [sCTX]), intact serum parathyroid hormone (iPTH) and urinary calcium and creatinine. Levels of all the markers of bone resorption and formation decreased during the study period. As expected, the bone formation markers decreased slightly less relative to the resorption markers. The most sensitive markers were sCTX (-33%; P < or =0.001) for bone resorption, and sP1NP (-3%; P < or =0.05) for bone formation. Serum PTH levels increased by 8% ( P < or =0.05), demonstrating a physiological reaction to prevent changes in serum calcium. Although not all variables reached statistical significance within the tested interval, the applied methods and kits were considered suitable for evaluating bone turnover changes in marmosets. Thus, these methods and kits can be utilized not only during the course of pharmacological investigations but also as additional tools to assess the overall bone health of this species.

To investigate the effects of treatment with ibandronate, a highly potent nitrogen-containing bisphosphonate, on bone loss, bone quality, biomechanical properties, and bone concentrations in aged ovariectomized rats.Eight-month-old female Wistar rats were ovariectomized (Ovx) or sham-operated. Treatment was started 10 weeks following Ovx with subcutaneous ibandronate in doses of 0.2, 1.0, 5.0, or 25 micro g/kg/day for 12 mo. Additional groups received 25 or 125 micro g/kg intermittently every 25 days, resulting in the same total dose as compared to 1.0 or 5.0 micro g/kg/day, respectively. Bone analyses by x-ray densitometry, peripheral quantitative computed tomography (pQCT), dual energy x-ray absorptiometry (DEXA), histomorphometry, 3-point bending, and compression tests were performed in femora, tibiae, and lumbar vertebrae in separate groups at the beginning and the end of treatment. Ibandronate concentration in tibiae and vertebrae was determined by gas chromatography mass spectroscopy at the end of the study.Ovariectomy resulted in a significant reduction in bone mass (por= 0.0001) and strength (p0.05) by 10 weeks after surgery in long bones, while only a trend was present in vertebrae. When compared to age matched Ovx controls, ibandronate resulted in a dose dependent increase in bone mineral density (BMD), trabecular bone volume and trabecular number, load to failure (Fmax), and yield load in long bones and vertebrae. The lowest significant dose, which was different from Ovx controls, ranged between 0.2 and 1.0 micro g/kg/day, with higher doses not differing from sham controls. Increased trabecular separation (por= 0.0001) was fully prevented by all doses. Vertebral BMD (pQCT and DEXA) positively correlated with Fmax by r = 0.88 (por= 0.0001) both; correlation of femoral Fmax versus cortical BMD was r = 0.61 (por= 0.0001).Bone concentrations of ibandronate were linear with the dose, suggesting linear kinetics in the applied dose range. In general, the same total cumulative ibandronate dose given provided equivalent results, independent of the administration schedule.

Ibandronate is a highly potent nitrogen-containing bisphosphonate that can prevent bone loss in various animal models as well as in clinical trials. We evaluated the effects of different doses and treatment schedules in ovariectomized aged rats, a model of human osteoporosis.Eight-month-old female Wistar rats were ovariectomized or sham operated (n = 15/group). Doses of 0.1 to 30 microg/kg/day ibandronate were administered subcutaneously over a period of 20 weeks with or without a 5 times higher single loading dose starting one day postsurgery. In a subsequent experiment, the optimal preventive dose, which is the lowest dose that prevented bone loss completely, or a suboptimal preventive dose were administered over the same period, either daily or by 3 cyclical intermittent regimens (on/off weeks = 1/2, 1/4, and 1/6), resulting in the same cumulative total dose.Ovariectomy induced significant bone loss in the following primary endpoints: femoral radiographic density, dry weight/tissue volume, and calcium content/tissue volume. Histomorphometry in the tibia resulted in reduced trabecular bone mass, thickness, and number, and increased separation. The optimal dose was 1.0 microg/kg/day, while 0.1 microg/kg/day was suboptimal. Higher doses resulted in a plateau. The loading dose had no effect on the results. Cyclical intermittent administration dose-dependently prevented bone loss, providing equivalent results per total dose, irrespective of the administration schedule.There were no differences between the various regimens, suggesting that it is the total dose of ibandronate rather than the treatment schedule that is important for efficacy, at least within the tested dosing intervals.

Bisphosphonate treatment is beneficial against symptoms of metastatic bone disease, although less is known about the effect of preventative treatment schedules. We investigated the effect of various treatment regimens of the bisphosphonate, ibandronate (IB), on the preservation of bone quality in a rat model of tumor-induced osteolysis. Osteolytic Walker 256 (W256) carcinosarcoma cells were implanted into the left femur of female Sprague-Dawley rats, resulting in a 10% reduction in bone mineral density (BMD), a 16% reduction in bone density (BD), and a 26% reduction in failure load compared with the right femur 28 days after implantation. IB was administered subcutaneously in five different treatment schedules: (1) IB PRE-POST received IB for 26 days, prior to implantation of W256 cells in the medullary canal of the femur, and for 28 additional days after surgery; (2) IB PRE-POST SHAM received the same IB administration, but with a sham operation; (3) IB PRE received IB injections before W256 cell insertion only; (4) IB PRE-0 received IB injections for 26 days and was then killed to serve as a time zero control; and (5) IB POST received sham injection with saline before W256 cell insertion, and then received IB injections for 28 days until killing. Controls (TUMOR ONLY) received sham injections with saline prior to W256 cell insertion, and then for 28 additional days until killing. We used dual-energy X-ray absorptiometry (DXA) to measure distal femur BMD and bone mineral content (BMC), peripheral quantitative computed tomography (pQCT) to measure distal femur BD, and torsion testing to obtain torsional failure load. Combined preventative and interventional IB treatment best preserved bone mass and strength, although all treatment schedules resulted in significant improvement compared with untreated controls (TUMOR ONLY). The possibility of reducing or even preventing skeletal morbidity in cancer patients with a high risk of developing metastatic spreading to bone is exciting, and warrants further exploration.

CUB domain-containing protein 1 (CDCP1) is a transmembrane glycoprotein and a substrate of Src family kinase (SFK). It has been shown to be upregulated and significantly contributing to a number of different cancers, including colon, lung, breast, kidney, and pancreas cancer. High expression of CDCP1 has been shown to correlate with poor prognosis. CDCP1 is involved in the regulation of anoikis resistance, cell migration and invasion. We have recently described RG7287, a humanized glycoengineered therapeutic anti-CDCP1 antibody with a dual mode of action. (1) downregulation of the receptor from the cell surface, and (2) engaging immune effector functions such as antibody-dependent cellular cytotoxicity (ADCC). Upon ligation of RG7287 to CDCP1 an initial phosphorylation of CDCP1 occurs, this transient activation results in the down-regulation of CDCP1. RG7287 prohibits the transformation potential of CDCP1 and Src in focus formation assays. In vivo, RG7287 increased the survival time of mice bearing tumors of MCF7 cells stably expressing CDCP1 compared to untreated CDCP1 overexpressing MCF7 tumors. Treatment of three different xenograft models with RG7287 inhibited tumor growth. In this study we looked at the possibility of eradicating the tumors by combining RG7287 with paclitaxel. Combining the cytostatic with RG7287 increases tumor growth inhibition compared to RG7287 alone in one xenograft model. In another model the combination of RG7287 with paclitaxel leads to tumor stasis. These results indicate a potential combination therapy for RG7287. We also tested whether RG7287 inhibits metastasis in vivo. Using a metastasis model we could show convincing inhibition of metastasis. Citation Format: Gwendlyn Kollmorgen, Alexander Lifke, Adam Nopora, Frieder Bauss, Gerhard Niederfellner, Birgit Bossenmaier. RG7287, a novel humanized anti-CDCP1 antibody with superior preclinical in vivo efficacy in combination with Paclitaxel. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2667. doi:10.1158/1538-7445.AM2014-2667

Attaching toxic payloads to antibodies has recently been established as a breakthrough in cancer therapy. Most of the currently developed antibody drug conjugate programs represent targeted chemotherapy with microtubule polymerization inhibiting drugs and hence share resistance mechanisms and side effects with classical chemotherapeutics like Taxol. Inhibition of protein synthesis by Pseudomonas Exotoxin (PE) is a more powerful mode of action that interferes with all hallmarks of cancer cells, not just with proliferation. However, its clinical use has been limited by immunogenicity as shown for SS1P, a mesothelin targeted immunotoxin. We have developed a novel de-immunized PE fusion protein (RG7787) for treatment of mesothelin-positive tumors. In order to de-immunize PE, point mutations have been introduced into domain III and the entire domain II has been deleted reducing the size of the effector moiety to approximately 24 kD. PE24 fusions with a disulfide-stabilized Fv fragment have previously been shown to be much better tolerated in rodents, but this could, at least in part, also be attributed to a much reduced serum half-life and exposure. In order to also de-immunize the targeting moiety of RG7787 and restore PK parameters similar to those of SS1P, we substituted the mouse dsFv moiety by a humanized Fab fragment. We show that RG7787 has indeed similar PK properties to SS1P in mouse and cyno. Cell viability assays show that RG7787 has similar cytotoxic potency as SS1P on different cell lines. In-vivo equipotency to SS1P in different xenograft models has been achieved at ∼3 fold higher doses indicating that tumor penetration of SS1P might be slightly better. However, RG7787 is up to tenfold better tolerated in rodents and cynomolgus monkeys, indicating that the therapeutic window is improved. RG7787 achieves potent tumor growth inhibition and even tumor regressions in several xenograft models. We also observed clearly synergistic efficacy with Taxol treatment in different tumor models making this a promising combination for clinical trials. Citation Format: Gerhard Niederfellner, Frieder Bauss, Sabine Imhof-Jung, Friederike Hesse, Sven Kronenberg, Roland Staak, Martin Lechmann, Ben Krippendorff, Wolfgang Richter, Rita Mateus, Gwendlyn Kollmorgen, Ulli Brinkmann, Masanori Onda, Ira Pastan, Klaus Bosslet. RG7787 - a novel de-immunized PE based fusion protein for therapy of mesothelin-positive solid tumors. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4510. doi:10.1158/1538-7445.AM2014-4510

The aim of this study was to evaluate the possible risk of impaired bone metabolism following augmentation cystoplasties with different gastrointestinal segments.60 young rats underwent augmentation cystoplasties using gastric, ileal or sigma segments, or sham operations. An additional group undergoing sigma-cystoplasty received the bisphosphonate ibandronate to inhibit osteoclast-mediated bone resorption. Bone mass in the lumbar spine and tibia was analyzed monthly by in vivo densitometry. Bone turnover was assessed monthly using current bone metabolism markers for a period of 16 weeks. Bone ashing and serum analyses of the osteotropic hormones parathyroid hormone (PTH), and 25-OH vitamin D3 were performed at study conclusion.Following ileocystoplasty, reduced bone mineral density (BMD) was seen throughout the observation period; this was pronounced in the trabecular bone. The decline in BMD was associated with decreased serum 25-OH vitamin D3 levels. Following sigmacystoplasty, bone calcium content was significantly decreased; this could be prevented by ibandronate. No skeletal changes occurred in the gastrocystoplasty group. Serum pH was not altered in any group, and markers of bone resorption indicated normal bone resorption rates.There is a significant correlation between impaired bone metabolism and the type of segment used for bladder augmentation. While the use of the ileum (and probably the colon too) causes osteopenia, gastrocystoplasties seem to have little influence on bone turnover.

Advanced tumor osteopathy is characterized by abnormal bone turnover. Using a rat model of parathyroid hormone-related peptide (PTHrP)-mediated tumor osteolysis, the aim of the present study was to define the sequential changes in, and the association between, biochemical and histomorphometric indices of bone metabolism during the early stages of developing tumor osteopathy. Eight-month-old Wistar rats (n = 48) were subcutaneously inoculated with either 2 x 10(6) cells of the Walker carcinosarcoma 256, or saline on day 0, and treated with either saline or the bisphosphonate ibandronate until killing on day 8. Serum calcium (sCa), alkaline phosphatase (sTAP), and osteocalcin (sOC) and urinary calcium (uCa), deoxypyridinoline (uDPD), and pyridinoline (uPYD) were measured daily. In a second semilongitudinal experiment (n = 70), the number of osteoclasts and osteoblasts (N.Oc, N.Ob), trabecular bone volume (BV/TV), and osteoid volume (O.Ar) were assessed by histomorphometry. In untreated tumor-bearing animals, osteoclast numbers increased by 74% on day 3 (5.4 +/- 2.4 vs. 3.1 +/- 1.5/mm(2), p0.05), and trabecular bone volume fell by 24% on day 4 (12.5 +/- 2.0 vs. 15.8 +/- 1.2%, p0.05). Both time course and magnitude of these changes were closely reflected by an increase in uDPD (0.46 +/- 0.14 vs. 0. 31 +/- 0.15 nmol/12 h, p0.05) and uPYD on day 4 (1.44 +/- 0.25 vs. 1.03 +/- 0.3 nmol/12 h, p0.05), sCa (3.8 +/- 0.52 vs. 3.0 +/- 0. 13 mmol/L, p0.01), and uCa (0.13 +/- 0.08 vs. 0.03 +/- 0.01 mmol/12 h, p0.001) on day 6, and sTAP (254 +/- 127 vs. 120 +/- 40 U/L, p0.001) on day 7 (mean +/- SD), whereas sOC remained unchanged until day 8. When combining the results of the two experiments, a high correlation was found between the number of osteoclasts and the urinary excretion of PYD (r = 0.91) and DPD (r = 0.89). Treatment with ibandronate delayed hypercalcemia, abolished hypercalciuria, and accelerated bone resorption. We conclude that osteoclast activation is an early event in PTHrP-mediated osteolysis, which is closely reflected by the renal excretion of pyridinium cross-links of type I collagen. Therefore, specific biochemical markers of collagen breakdown may be useful as early indicators of developing tumor osteopathy.

We previously found that bone loss occurs as soon as 1 month after ovariohysterectomy (OHX) in beagle dogs. Indirect evidence pointed to an early dramatic increase in bone resorption. To verify this hypothesis and evaluate the effects of a newly developed bisphosphonate, BM 21.0955 (Boehringer Mannheim), 36 beagle dogs were subjected to OHX and 12 dogs were sham operated (Sham). OHX dogs were divided into six groups (n = 6 each) and received subcutaneous injections of vehicle or BM 21.0955 at various doses (0.1, 0.3, 1, 10, and 100 micrograms/kg/day) for 1 month. Sham dogs were given vehicle (n = 6) or BM 21.0955 (1 microgram/kg/day, n = 6). Iliac crest biopsies and blood drawings were done at baseline and at month 1. OHX dogs given vehicle exhibited a decrease in cancellous bone volume associated with an increase in erosion depth and a decrease in serum levels of 1,25-dihydroxyvitamin D. BM 21.0955 prevented the bone loss at a dose > or = 1 microgram/kg and the increase in erosion depth and the decrease in serum levels of 1,25-(OH)2D at a dose > or = 0.3 microgram/kg. No osteomalacia was observed at any dose of BM 21.0955. Bone turnover was reduced only when BM 21.0955 was administered at doses of 10 or 100 micrograms/kg. There were no changes in body weight or serum levels of calcium, phosphorus, creatinine, parathyroid hormone, or osteocalcin in all groups. The increase in erosion depth in OHX dogs given vehicle proves that the early rapid bone loss after cessation of ovarian function is related to an increase in osteoclastic activity. The antiosteoclastic activity of BM 21.0955 at a dose > or = 1 microgram/kg prevents this increase and preserves bone volume. The absence of any signs of osteomalacia at any dose confers a relatively wide therapeutic margin to BM 21.0955. BM 21.0955 at a dose > or = 10 micrograms/kg also acts as an inhibitor of bone turnover. This is not observed at a dose of 1 microgram/kg, at least after 1 month of administration.