Effects of pioglitazone and fenofibrate co-administration on bone biomechanics and histomorphometry in ovariectomized rats.

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.

Osteonecrosis of the jaw: effect of bisphosphonate type, local concentration, and acidic milieu on the pathomechanism.

PURPOSE: 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. MATERIALS AND METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Preclinical evidence for nitrogen-containing bisphosphonate inhibition of farnesyl diphosphate (FPP) synthase in the kidney: implications for renal safety.

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.

A rapid histological score for the semiquantitative assessment of bone metastases in experimental models of breast cancer.

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 microg 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.

Assessment of susceptible chemical modification sites of trastuzumab and endogenous human immunoglobulins at physiological conditions.

The quality control testing of chemical degradations in the bio-pharmaceutical industry is currently under controversial debate. Here we have systematically applied in vitro and in vivo stress conditions to investigate the influence of protein degradation on structure-function. Extensive purification and characterization enabled identification and functional assessment of the physiological degradation of chemical modification sites in the variable complementarity-determining regions (CDRs) and conserved region of trastuzumab. We demonstrate that the degradation of the solvent-accessible residues located in the CDR and the conserved fragment crystallizable region (Fc) occurs faster in vivo (within days) compared to the levels observed for bio-process and real-time storage conditions. These results hence question the rationality of extreme monitoring of low level alterations in such chemical modifications as critical patient safety parameters in product quality control testing, given that these modifications merely mirror the natural/physiological aging process of endogenous antibodies.

Retention, distribution, and effects of intraosseously administered ibandronate in the infarcted femoral head.

UNLABELLED: 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 (14)C-labeled or unlabeled ibandronate directly into the infarcted head. (14)C-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 (14)C-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 (14)C-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.

Sequence- and concentration-dependent effects of acute and long-term exposure to the bisphosphonate ibandronate in combination with single and multiple fractions of ionising radiation doses in human breast cancer cell lines.

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.

Local bioavailability and distribution of systemically (parenterally) administered ibandronate in the infarcted femoral head.

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, P<0.05). 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 (P

Characterization of a re-engineered, mesothelin-targeted Pseudomonas exotoxin fusion protein for lung cancer therapy.

Mesothelin overexpression in lung adenocarcinomas correlates with the presence of activating KRAS mutations and poor prognosis. Hence SS1P, a mesothelin-targeted immunotoxin, could offer valuable treatment options for these patients, but its use in solid tumor therapy is hampered by high immunogenicity and non-specific toxicity. To overcome both obstacles we developed RG7787, a de-immunized cytotoxic fusion protein comprising a humanized SS1 Fab fragment and a truncated, B-cell epitope silenced, 24 kD fragment of Pseudomonas exotoxin A (PE24). Reactivity of RG7787 with sera from immunotoxin-treated patients was >1000 fold reduced. In vitro RG7787 inhibited cell viability of lung cancer cell lines with picomolar potency. The pharmacokinetic properties of RG7787 in rodents were comparable to SS1P, yet it was tolerated up to 10 fold better without causing severe vascular leak syndrome or hepatotoxicity. A pharmacokinetic/pharmacodynamic model developed based on NCI-H596 xenograft studies showed that for RG7787 and SS1P, their in vitro and in vivo potencies closely correlate. At optimal doses of 2-3 mg/kg RG7787 is more efficacious than SS1P. Even large, well established tumors (600 mm(3)) underwent remission during three treatment cycles with RG7787. Also in two patient-derived lung cancer xenograft models, Lu7336 and Lu7187, RG7787 showed anti-tumor efficacy. In monotherapy two treatment cycles were moderately efficacious in the Lu7336 model but showed good anti-tumor activity in the KRAS mutant Lu7187 model (26% and 80% tumor growth inhibition, respectively). Combination of RG7787 with standard chemotherapies further enhanced efficacy in both models achieving near complete eradication of Lu7187 tumors.

Ibandronate for prevention of femoral head deformity after ischemic necrosis of the capital femoral epiphysis in immature pigs.

BACKGROUND: 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. METHODS: 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. RESULTS: 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 (p < 0.001) 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 (p < 0.01). 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

Analytical methods for the quantification of ibandronate in body fluids and bone.

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.

Acute renal effects of intravenous bisphosphonates in the rat.

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.

Treatment with the combination of ibandronate plus eldecalcitol has a synergistic effect on inhibition of bone resorption without suppressing bone formation in ovariectomized rats.

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 12 weeks 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.

Intermittent ibandronate preserves bone quality and bone strength in the lumbar spine after 16 months of treatment in the ovariectomized cynomolgus monkey.

UNLABELLED: The dose-dependent effect of ibandronate treatment on bone mass and architecture was assessed in a large animal study of OVX monkeys using microCT 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 microg/kg), medium- (30 microg/kg), and high- (150 microg/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 microCT 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.

Ibandronate: a clinical pharmacological and pharmacokinetic update.

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.

Effect of daily and intermittent use of ibandronate on bone mass and bone turnover in postmenopausal osteoporosis: a review of three phase II studies.

BACKGROUND: 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. OBJECTIVE: 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. METHODS: 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. RESULTS: 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 (P<0.001), oral ibandronate 20 mg QOD given for 12 doses at the start of each 3-monthly period (P<0.001), and injections of ibandronate 2 mg IV given every 3 months (P<0.01). All treatment regimens produced comparable significant increases in bone mineral density at the lumbar spine (P<0.01) and hip (P<0.05). Ibandronate was well tolerated when administered both orally and as an IV injection. CONCLUSIONS: 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.

New model for simulation of fracture repair in full-grown beagle dogs: model characterization and results from a long-term study with ibandronate.

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 microg/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.

The renal effects of minimally nephrotoxic doses of ibandronate and zoledronate following single and intermittent intravenous administration in rats.

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.

Antibody mediated CDCP1 degradation as mode of action for cancer targeted therapy.

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.

Combination treatment with pioglitazone and fenofibrate attenuates pioglitazone-mediated acceleration of bone loss in ovariectomized rats.

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.