Cedo M. Bagi

Dual focal adhesion kinase/Pyk2 inhibitor has positive effects on bone tumors: implications for bone metastases.

Lytic bone metastases occur frequently in cancer patients and present major clinical issues including lack of effective therapies. The mechanism of lytic bone metastases involves interactions between tumor cells, bone matrix, and bone cells. Both focal adhesion kinase (FAK) and Pyk2 are implicated in the biology and physiology of bone and cancer.

Treatment of ovariectomized rats with the complex of rhIGF-I/IGFBP-3 Increases cortical and cancellous bone mass and improves structure in the femoral neck

Sixteen-week-old Sprague-Dawley rats were ovariectomized (Ovx) or sham-operated and housed for 8 weeks to develop osteopenia prior to systemic administration of rhIGF-I (0.9 and 2.6 mg/kg) alone or the rhIGF-I/IGFBP-3 (0.9, 2.6 and 7.5 mg/kg) complex. After 8 weeks of treatment, proximal femurs were fixed, embedded, and cut through the midneck region. Structural and dynamic histomorphometric analyses were performed using standard techniques. Ovx increased endocortical resorption and modeling-dependent periosteal formation which resulted in decreased cortical bone area. Despite increased bone formation, trabecular number, thickness, and area were all reduced due to increased resorption. Structural changes following Ovx included fewer struts and nodes, a higher percentage of the simpler strut forms, and reduced endocorticotrabecular cnnnectivity. Eight weeks of treatment with rhIGF-I or rhIGF-I/IGFBP-3 promoted periosteal and endocortical bone formation and reduced the endocortical resorption induced by Ovx. Both rhIGF-I formulations stimulated bone formation on existing trabecular surfaces which increased trabecular thickness and area but not trabecular number. These treatments prevented further deterioration of the trabecular network caused by Ovx and preserved endocortico-trabecular connectivity. In summary, changes in the femoral neck following Ovx appear to be similar in rats and humans. The highest dose of rhIGF-I/IGFBP-3 used in this study showed the best results in promoting cortical and cancellous bone formation, and appears to be promising therapy for human osteopenias.

Sunitinib and PF-562,271 (FAK/Pyk2 inhibitor) effectively block growth and recovery of human hepatocellular carcinoma in a rat xenograft model

Experimental Design: To investigate the anti-tumor effect of sunitinib and FAK/Pyk2 tyrosine kinase inhibitor (PF-562,271) combination therapy in vivo, utilizing human hepatocellular carcinoma (HCC) cells Huh7.5. Nude rats were inoculated subcutaneously with Huh7.5 hepatoma cells. Dosing for Phase 1 was initiated on day 5 post tumor inoculations with Vehicle (Group 1), sunitinib (25 mg/kg/day; Group 2) and sunitinib plus PF-562,271 combination (15 mg/kg/day; Group 3). Phase 2 of the study started on day 26, and each of the 3 original groups were divided in 2 subgroups; half of the rats remained on original therapy (Groups 1A and 2A) with the exception of Group 3A that was euthanized after Phase 1. The other half of the rats were switched to sunitinib and PF-562,271 combination (Group 1B) or vehicle (Groups 2B and 3B). Tumor volume and weight, serum alpha feto-protein (AFP), contrast-enhanced ultrasound imaging (CEUS) and tumor histology were used to evaluate effects of treatment on tumor growth. Results: The results from this study indicate that the combination of sunitinib and PF- 562,271 TKI has the potential to target different aspects of angiogenesis and tumor aggressiveness and may have significantly greater effect than relevant single agent, blocking not only tumor growth, but also impacting the ability of the tumor to recover upon withdrawal of the therapy.

Systemic administration of rhIGF-I or rhIGF-I/IGFBP-3 increases cortical bone and lean body mass in ovariectomized rats

The purpose of this study was to compare dose-related effects on cortical bone and lean body mass following subcutaneous administration of rhIGF-I alone, or bound to an equimolar amount of rhIGFBP-3 to adult Ovx rats. At the age of 16 weeks, rats were ovariectomized or sham-operated and were allowed 8 weeks to develop osteopenia. After being divided into control (saline treated) or treatment groups, rats were injected daily during an 8-week period with 0.9 and 2.6 mg/kg of rhIGF-I, or with 0.9, 2.6, and 7.5 mg/kg of rhIGF-I bound to rhIGFBP-3. Fluorescent bone markers were given 9 and 2 days prior to necropsy. Body weights and lean body mass were monitored throughout the experiment. Cortical bone histomorphometry was performed on tibial cross-sections at the tibiofibular junction, and endochondral bone growth was measured at the distal femoral metaphysis. All rats treated with rhIGF-I or the rhIGF-I/IGFBP-3 complex had increased body weights, corresponding to a dose-dependent increase in lean body mass. Endochondral growth was slightly increased in all experimental groups, but was not dose-dependent. A dramatic increase in periosteal, modeling-dependent formation, coupled with decreased or unchanged resorption on the endocortical envelope resulted in a dose-dependent increase in cortical thickness and cross-sectional area in groups treated with the complex of rhIGF-I/IGFBP-3. This complex appeared to be more effective in promoting positive musculoskeletal changes than rhIGF-I alone.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of systemically administered rhIGF-I/IGFBP-3 complex on cortical bone strength and structure in ovariectomized rats.

The action of systematically administered recombinant human insulinlike growth factor-I (rhIGF-I) complexed to its natural binding protein-3 (rhIGFBP-3) on cortical bone dynamic, structural, and mechanical properties was tested in previously ovariectomized (Ovx) rats. Bilateral ovariectomy or sham surgery was performed on 16-week-old female Sprague-Dawley rats. Eight weeks after surgery basal Sham and Ovx rats were killed to establish baseline cortical bone values before the initiation of treatment with rhIGF-I/IGFBP-3 complex. At that time, Ovx rats had increased body weight and body fat mass with reduced femoral BMC and BMD relative to basal Shams. Bone formation rates in Ovx rats were increased on both cortical envelopes relative to time-matched controls. The thickness of the inner lamellar bone layer and average cortical width were reduced due to increased endocortical erosion. A similar ratio between Sham and Ovx rats in body mass and composition and femoral BMC and BMD continued throughout the experiment. Sixteen weeks after surgery bone formation rates at both cortical envelopes in Ovx rats were reduced relative to Shams, but endocortical erosion remained high causing a further decrease in thickness of the inner lamellar layer. As a result of periosteal bone modeling. Ovx rats exhibited a larger femoral cross-sectional area and periosteal perimeter, as well as a thicker outer lamellar layer. Newly deposited periosteal bone increased ultimate torque values in the Ovx rats relative to Shams at 16 weeks. Treatment of Ovx rats with the rhIGF-I/IGFBP-3 complex increased body weight, lean body mass, and femoral BMC and BMD.(ABSTRACT TRUNCATED AT 250 WORDS)

Age-related Changes in Marmoset Trabecular and Cortical Bone and Response to Alendronate Therapy Resemble Human Bone Physiology and Architecture

In older humans, bone elongation ceases, periosteal expansion continues, and bone remodeling remains a dominant metabolic process. An appropriate animal model of type I and type II osteoporosis would be a species with sealed growth plates and persistence of bone remodeling. The rat is commonly used as a primary model, but due to delayed epiphyseal closure with continuous modeling and lack of Haversian remodeling, Food and Drug Administration guidelines recommend assessment of bone quality in an additional, nonrodent, remodeling species. This study investigated the skeletal characteristics of senescent marmosets to evaluate their suitability as an osteoporosis model. Animals were randomized across three experimental groups; controls for both sexes and marmosets receiving alendronate for either 30 or 60 days (28 μg/kg, sc, twice per week). Outcome measures included serum chemistry and bone biomarkers, DEXA, histomorphometry, micro‐computed tomography, and histopathology. Results showed that the adult marmoset skeleton has similar anatomical characteristics to the adult human, including the absence of growth plates, presence of Haversian system, and true remodeling of cancellous and cortical bone. Structural analyses of senescent marmoset cancellous bone demonstrated loss of trabecular mass and architecture similar to skeletal changes described for elderly men and women. Treatment with alendronate improved trabecular volume and number by reducing bone resorption, although bone formation was also reduced through coupling of bone remodeling. The common marmoset may provide a valuable model for research paradigms targeting human bone pathology and osteoporosis due to skeletal features that are similar to age‐related changes and response to bisphosphonate therapy reported for humans. Anat Rec, 2007.

Models of Hepatocellular Carcinoma and Biomarker Strategy

The overwhelming need to improve preclinical models in oncology has stimulated research efforts to refine and validate robust orthotopic models that closely mimic the disease population and therefore have the potential to better predict clinical outcome with novel therapies. Sophisticated technologies including bioluminescence, contrast enhanced ultrasound imaging, positron emission tomography, computed tomography and magnetic resonance imaging have been added to existing serum- and histology-based biomarkers to assist with patient selection and the design of clinical trials. The rationale for the use of human hepatocellular carcinoma (HCC) cell lines, implementation of xenograft and orthotopic animal models and utilization of available biomarkers have been discussed, providing guidelines to facilitate preclinical research for the development of treatments for HCC patients.

Correlation between μCT imaging, histology and functional capacity of the osteoarthritic knee in the rat model of osteoarthritis

BackgroundTo acquire the most meaningful understanding of human arthritis, it is essential to select the disease model and methodology translatable to human conditions. The primary objective of this study was to evaluate a number of analytic techniques and biomarkers for their ability to accurately gauge bone and cartilage morphology and metabolism in the medial meniscal tear (MMT) model of osteoarthritis (OA).MethodsMMT surgery was performed in rats to induce OA. A dynamic weight bearing system (DWB) system was deployed to evaluate the weight-bearing capacity of the front and hind legs in rats. At the end of a 10-week study cartilage pathology was evaluated by micro computed tomography (μCT), contrast enhanced μCT (EPIC μCT) imaging and traditional histology. Bone tissue was evaluated at the tibial metaphysis and epiphysis, including the subchondral bone. Histological techniques and dynamic histomorphometry were used to evaluate cartilage morphology and bone mineralization.ResultsThe study results showed a negative impact of MMT surgery on the weight-bearing capacity of the operated limb. Surgery caused severe and extensive deterioration of the articular cartilage at the medial tibial plateau, as evidenced by elevated CTX-II in serum, EPIC μCT and histology. Bone analysis by μCT showed thickening of the subchondral bone beneath the damaged cartilage, loss of cancellous bone at the metaphysis and active osteophyte formation.ConclusionsThe study emphasizes the need for using various methodologies that complement each other to provide a comprehensive understanding of the pathophysiology of OA at the organ, tissue and cellular levels. Results from this study suggest that use of histology, μCT and EPIC μCT, and functional DWB tests provide powerful combination to fully assess the key aspects of OA and enhance data interpretation.

Antitumor effect of vascular endothelial growth factor inhibitor sunitinib in preclinical models of hepatocellular carcinoma.

Objective Tumor recurrence and metastasis is the most common cause of mortality in hepatocellular carcinoma (HCC) patients. Despite positive results with vascular endothelial growth factor (VEGF) inhibitors in preclinical studies using HCC xenograft models, the clinical outcome in HCC patients has been disappointing. So far, only the multitargeted tyrosine kinase inhibitor sorafenib has been shown to significantly improve survival in HCC patients, suggesting that this class of agents could be effective against HCC. Recently, another VEGF inhibitor, sunitinib, showed survival benefits in HCC hepatitis B-positive patients, but failed to improve survival in HCC hepatitis C-positive patients. Obviously, concomitant liver disease, liver function in general, and the local liver environment have a huge impact on treatment outcomes. In this study, we aimed to examine the antiproliferative effect of sunitinib in different HCC cell lines in vitro, and then in xenograft and orthotopic models of HCC in order to assess the effect of the local liver vasculature on drug efficacy. Methods Human cancer cell lines Huh7.5, Hep3B, and SK-Hep-1 were used for in-vitro studies. In in-vivo studies, each mouse carried Huh7.5 cells in both the subcutaneous and the intrahepatic compartment; therefore, drug exposure and treatment regimen were identical in both tumors. Results Sunitinib has the potential to moderately inhibit proliferation in the Huh7.5 cell line, induce p53 in the p53-wild-type cell line SK-hep-1, and to increase the S-phase and the sub-G1 component of the cell cycle in the Hep3B cell line. Diverse responses to sunitinib in HCC cell lines emphasize the heterogeneity of HCC tumors and may further explain the discrepancy between preclinical and clinical results. The in-vivo results show that sunitinib treatment was far less effective against intrahepatic tumors compared with xenografts. Histological data indicate that large solid intrahepatic tumors are severely affected by sunitinib as shown by large areas of necrosis and diminished number of viable tumor cells. Conclusion The real problem when treating intrahepatic tumors with sunitanib and/or other VEGF inhibitors seems to arise from unopposed local growth of the small tumors and perhaps the development of distant micrometastases. Even though both xenograft and orthotopic models have limitations, these models add value to our understanding of tumor biology and help to better design treatment paradigms for patients with HCC. In comparison with xenograft models, the orthotopic HCC model allows for a more realistic assessment of drug efficacy in patients, in particular by enhancing our knowledge of the role that organ vasculature plays in the development of local metastasis and tumor resistance to antiangiogenic treatments.

Oral Administration of Soluble Guanylate Cyclase Agonists to Rats Results in Osteoclastic Bone Resorption and Remodeling with New Bone Formation in the Appendicular and Axial Skeleton

Orally administered small molecule agonists of soluble guanylate cyclase (sGC) induced increased numbers of osteoclasts, multifocal bone resorption, increased porosity, and new bone formation in the appendicular and axial skeleton of Sprague-Dawley rats. Similar histopathological bone changes were observed in both young (7- to 9-week-old) and aged (42- to 46-week-old) rats when dosed by oral gavage with 3 different heme-dependent sGC agonist (sGCa) compounds or 1 structurally distinct heme-independent sGCa compound. In a 7-day time course study in 7- to 9-week-old rats, bone changes were observed as early as 2 to 3 days following once daily compound administration. Bone changes were mostly reversed following a 14-day recovery period, with complete reversal after 35 days. The mechanism responsible for the bone changes was investigated in the thyroparathyroidectomized rat model that creates a low state of bone modeling and remodeling due to deprivation of thyroid hormone, calcitonin (CT), and parathyroid hormone (PTH). The sGCa compounds tested increased both bone resorption and formation, thereby increasing bone remodeling independent of calciotropic hormones PTH and CT. Based on these studies, we conclude that the bone changes in rats were likely caused by increased sGC activity.