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.

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.

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.

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.

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.

Targeted siRNA Delivery and mRNA Knockdown Mediated by Bispecific Digoxigenin-binding Antibodies.

Bispecific antibodies (bsAbs) that bind to cell surface antigens and to digoxigenin (Dig) were used for targeted small interfering RNA (siRNA) delivery. They are derivatives of immunoglobulins G (IgGs) that bind tumor antigens, such as Her2, IGF1-R, CD22, and LeY, with stabilized Dig-binding variable domains fused to the C-terminal ends of the heavy chains. siRNA that was digoxigeninylated at its 3'end was bound in a 2:1 ratio to the bsAbs. These bsAb-siRNA complexes delivered siRNAs specifically to cells that express the corresponding antigen as demonstrated by flow cytometry and confocal microscopy. The complexes internalized into endosomes and Dig-siRNAs separated from bsAbs, but Dig-siRNA was not released into the cytoplasm; bsAb-targeting alone was thus not sufficient for effective mRNA knockdown. This limitation was overcome by formulating the Dig-siRNA into nanoparticles consisting of dynamic polyconjugates (DPCs) or into lipid-based nanoparticles (LNPs). The resulting complexes enabled bsAb-targeted siRNA-specific messenger RNA (mRNA) knockdown with IC(50) siRNA values in the low nanomolar range for a variety of bsAbs, siRNAs, and target cells. Furthermore, pilot studies in mice bearing tumor xenografts indicated mRNA knockdown in endothelial cells following systemic co-administration of bsAbs and siRNA formulated in LNPs that were targeted to the tumor vasculature.Molecular Therapy - Nucleic Acids (2012) 1, e45; doi:10.1038/mtna.2012.39; published online 18 September 2012.

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.

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.

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.

Preclinical and clinical efficacy of the bisphosphonate ibandronate in cancer treatment.

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.

Ibandronate uptake in the jaw is similar to long bones and vertebrae in the rat.

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.

Intravenous ibandronate rapidly reduces pain, neurochemical indices of central sensitization, tumor burden, and skeletal destruction in a mouse model of bone cancer.

Over half of all chronic cancer pain arises from metastases to bone and bone cancer pain is one of the most difficult of all persistent pain states to fully control. Currently, bone pain is treated primarily by opioid-based therapies, which are frequently accompanied by significant unwanted side effects. In an effort to develop nonopioid-based therapies that could rapidly attenuate tumor-induced bone pain, we examined the effect of intravenous administration of the bisphosphonate, ibandronate, in a mouse model of bone cancer pain. Following injection and confinement of green fluorescent protein-transfected murine osteolytic 2472 sarcoma cells into the marrow space of the femur of male C3H/HeJ mice, ibandronate was administered either as a single dose (300 microg/kg), at Day 7 post-tumor injection, when tumor-induced bone destruction and pain were first evident, or in three consecutive doses (100 microg/kg/day) at Days 7, 8, and 9 post-tumor injection. Intravenous ibandronate administered once or in three consecutive doses reduced ongoing and movement-evoked bone cancer pain-related behaviors, neurochemical markers of central sensitization, tumor burden, and tumor-induced bone destruction. These results support limited clinical trials that suggest the potential of ibandronate to rapidly attenuate bone pain and illuminate the mechanisms that may be responsible for limiting pain and disease progression.

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.

Preventative ibandronate treatment has the most beneficial effect on the microstructure of bone in experimental tumor osteolysis.

We investigated the effect of ibandronate on three-dimensional (3-D) microstructure and bone mass in experimentally induced tumor osteolysis. Walker carcinosarcoma cells were implanted into the left femur of female rats that received 26-day ibandronate pretreatment followed by continued therapy or ibandronate posttreatment only. A tumor-only group received isotonic saline. At endpoint, excised femurs were scanned using microcomputed tomography (microCT) to assess bone volume density, bone mineral content, trabecular number/thickness, and separation for cortical plus trabecular bone or trabecular bone alone. Compared with the nonimplanted right femur, bone volume and surface density and trabecular number and thickness were reduced in the distal left femur following tumor cell implantation. microCT analysis revealed greater cortical and trabecular bone mineral content in the preventative and interventional (pre-post tumor) ibandronate group, and the interventional (post-tumor) ibandronate group, versus the tumor-only group. Bone volume density was significantly higher in pre-post and post-tumor groups compared to the tumor-only group. After preventative and interventional ibandronate, bone volume density and trabecular thickness were 13% and 60% greater, respectively, than in the post-tumor treatment group. 3-D microCT images confirmed microstructural changes. We conclude that combined interventional and preventative ibandronate preserves bone strength and integrity more than intervention alone.

The bisphosphonate ibandronate accelerates osseointegration of hydroxyapatite-coated cementless implants in an animal model.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

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

Ibandronate: the first once-monthly oral bisphosphonate for treatment of postmenopausal osteoporosis.

Osteoporosis is a major healthcare problem that continues growing as the population ages. Sufferers become increasingly susceptible to fractures, which compromise physical and emotional health and increase healthcare costs. Bisphosphonates are the most widely used medicines for the treatment and prevention of postmenopausal osteoporosis. However, therapeutic adherence is suboptimal, meaning that outcomes demonstrated in clinical trials are not realized in the real world. It is anticipated that reducing dosing frequency may facilitate medication intake and thereby improve adherence. Ibandronate is a potent nitrogen-containing bisphosphonate specifically developed for administration with long dose-free intervals. The comprehensive ibandronate preclinical development program has demonstrated dose-dependent improvements or preservation of bone quality and strength. The feasibility of intermittent dosing using the same total dose level as continuous dosing was also confirmed. In postmenopausal osteoporosis, once-monthly oral ibandronate has been shown to be therapeutically equivalent and even superior to daily oral ibandronate, which has demonstrated antifracture efficacy for vertebral and nonvertebral fractures, bone mineral density gains at the spine and hip, and reduction in bone resorption to premenopausal levels. Once-monthly oral ibandronate is also associated with excellent safety and tolerability, and promises to further improve therapeutic adherence to bisphosphonate treatment, thereby enhancing therapeutic outcomes.

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.