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Journal Article

Bioware; SKOV3-luc-D3 cells

Peritoneal dissemination is a common feature of human ovarian carcinoma. While this can be mimicked in preclinical models by intraperitoneal injection of human ovarian tumor cells into immunocompromised mice, the resulting tumor burden is difficult to monitor and quantify. Intraperitoneal tumor growth is typically evaluated indirectly by measured changes in mouse abdominal girth and body weight or, directly, by macroscopic and histological examination at the endpoint of the study. In order to establish a model system that allows continuous and accurate assessment of ovarian cancer growth and spread over time we transfected SKOV-3 cells with the firefly luciferase gene. The resulting cell line, SKOV3-luc-D3, expresses stable levels of luciferase in vitro and emits a strong luminescent signal when exposed to luciferin. Xenograft tumors established with this cell line can be tracked and quantified non-invasively by bioluminescent imaging using a highly sensitive, cooled CCD camera (IVIS(R) Imaging System, Xenogen Corp). In addition to providing a direct measure of primary tumor burden and growth, the SKOV3-luc-D3 cell line also allows for real-time evaluation of tumor response to various therapeutic agents, as well as enhanced detection of distal metastases.

Journal Article

Animals; Bioware; Chromatography, Liquid; Cyclin-Dependent Kinase 4; Drug Stability; Female; Humans; MDA-MB-231-D3H1 cells; Mice; Mice, SCID; Neoplasm Transplantation; Neoplasms, Experimental; Piperazines; Protein kinase inhibitors; Pyridines; Sensitivity and Specificity; Tandem Mass Spectrometry; Transplantation, Heterologous

Phase II attrition of clinical candidates in the drug development cycle is currently a major issue facing the pharmaceutical industry. To decrease phase II attrition, there is an increased emphasis on validation of mechanism of action, development of efficacy models and measurement of drug levels at the site of action. PD 0332991, a highly specific inhibitor of cyclin-dependent kinase 4 (CDK-4) is currently in clinical development for the treatment of solid tumor. A clinical presurgical study will be required to better understand how PD 0332991 affects signaling pathways and how the intratumoral concentration of PD 0332991 correlates with plasma PK parameters and molecular alterations in breast cancer tissues after PD 0332991 treatment. Before conducting such a clinical study, it is important to evaluate PD 0332991 levels in tumor tissue samples from a xenograft mouse model for the determination of drug exposure at the site of action. Therefore, the objectives of this study were (1) to develop and validate a sensitive LC-MS/MS method to quantify PD 0332991 in mouse tumor tissues from MDA-MB-231-Luc human breast tumor xenografts in SCID-beige mice; (2) to quantify PD 0332991 levels in mouse tumor tissues after oral administration of PD 0332991 at 10 and 100mg/kg using the validated LC-MS/MS method. Both liquid-liquid extraction (LLE) and supported liquid extraction (SLE) in a 96-well format were developed and evaluated to achieve optimal extraction recovery with minimal matrix effects. The newly developed SLE method is more efficient (speed and ease) and demonstrates comparable recovery (93.1-100% at three different concentrations) compared to the traditional LLE method. The validated LC-MS/MS for PD 032291 in mouse tumor tissue homogenate method exhibited a linear dynamic range of 0.1-100 ng/mL with inter-day accuracy and precision within 15%. The validated method was successfully applied to measure PD 0332991 levels in tumor tissues in MDA-MB-231-Luc human breast tumor xenografts in SCID beige mice. The mean tumor concentrations at 6h post-oral PD 0332991 administration at 10 and 100mg/kg were 1793 (+/-1008) and 25,163 (+/-3959) ng/g, respectively.

Journal Article

Amino Acid Chloromethyl Ketones; Animals; Apoptosis; Bacterial Proteins; Caspases; Lipopolysaccharides; Macrophages; Mice; Mice, Inbred Strains; Nasopharynx; Pneumococcal Infections; Streptococcus pneumoniae; Streptolysins; Xen10

Pneumolysin, the cholesterol-dependent cytolysin of Streptococcus pneumoniae, induces inflammatory and apoptotic events in mammalian cells. Toll-like receptor 4 (TLR4) confers resistance to pneumococcal infection via its interaction with pneumolysin, but the underlying mechanisms remain to be identified. In the present study, we found that pneumolysin-induced apoptosis is also mediated by TLR4 and confers protection against invasive disease. The interaction between TLR4 and pneumolysin is direct and specific; ligand-binding studies demonstrated that pneumolysin binds to TLR4 but not to TLR2. Involvement of TLR4 in pneumolysin-induced apoptosis was demonstrated in several complementary experiments. First, macrophages from wild-type mice were significantly more prone to pneumolysin-induced apoptosis than cells from TLR4-defective mice. In gain-of-function experiments, we found that epithelial cells expressing TLR4 and stimulated with pneumolysin were more likely to undergo apoptosis than cells expressing TLR2. A specific TLR4 antagonist, B1287, reduced pneumolysin-mediated apoptosis in wild-type cells. This apoptotic response was also partially caspase dependent as preincubation of cells with the pan-caspase inhibitor zVAD-fmk reduced pneumolysin-induced apoptosis. Finally, in a mouse model of pneumococcal infection, pneumolysin-producing pneumococci elicited significantly more upper respiratory tract cell apoptosis in wild-type mice than in TLR4-defective mice, and blocking apoptosis by administration of zVAD-fmk to wild-type mice resulted in a significant increase in mortality following nasopharyngeal pneumococcal exposure. Overall, our results strongly suggest that protection against pneumococcal disease is dependent on the TLR4-mediated enhancement of pneumolysin-induced apoptosis.

Journal Article

Animals; Antineoplastic Agents; Bioware; Cell Line, Tumor; Drug Carriers; Drug Delivery Systems; Female; Hela Cells; HeLa-luc; Humans; Mice; Mice, SCID; Micelles; Neoplasms; Organoplatinum Compounds; Platinum; Polymers; Xenograft Model Antitumor Assays

Polymeric micelles are promising nanocarriers, which might enhance the efficacy of antitumor drugs. Herein, polymeric micelles incorporating dichloro(1,2-diamino-cyclohexane)platinum(II) (DACHPt), the oxaliplatin parent complex, were prepared through the polymer-metal complex formation of DACHPt with poly(ethylene glycol)-b-poly(glutamic acid) [PEG-b-P(Glu)] block copolymer having different lengths of the poly(glutamic acid) block [p(Glu): 20, 40, and 70 U]. The resulting micelles were studied with the aim of optimizing the system's biological performance. DACHPt-loaded micelles (DACHPt/m) were approximately 40 nm in diameter and had a narrow size distribution. In vivo biodistribution and antitumor activity experiments (CDF1 mice bearing the murine colon adenocarcinoma C-26 inoculated subcutaneously) showed 20-fold greater accumulation of DACHPt/m at the tumor site than free oxaliplatin to achieve substantially higher antitumor efficacy. Moreover, the micelles prepared from PEG-b-P(Glu) with 20 U of P(Glu) exhibited the lowest non-specific accumulation in the liver and spleen to critically reduce non-specific accumulation, resulting in higher specificity to solid tumors. The antitumor effect of DACHPt/m was also evaluated on multiple metastases generated from intraperitoneally injected bioluminescent HeLa (HeLa-Luc) cells. The in vivo bioluminescent data indicated that DACHPt/m decreased the signal 10-to 50-fold compared to the control indicating a very strong antitumor activity. These results suggest that DACHPt/m could be an outstanding drug delivery system for oxaliplatin in the treatment of solid tumors.

Journal Article

OsteoSense

Preclinical models for musculoskeletal disorders are critical for understanding the pathogenesis of bone and joint disorders in humans and the development of effective therapies. The assessment of these models primarily relies on morphological analysis which remains time consuming and costly, requiring large numbers of animals to be tested through different stages of the disease. The implementation of preclinical imaging represents a keystone in the refinement of animal models allowing longitudinal studies and enabling a powerful, non-invasive and clinically translatable way for monitoring disease progression in real time. Our aim is to highlight examples that demonstrate the advantages and limitations of different imaging modalities including magnetic resonance imaging (MRI), computed tomography (CT), positron emission tomography (PET), single-photon emission computed tomography (SPECT) and optical imaging. All of which are in current use in preclinical skeletal research. MRI can provide high resolution of soft tissue structures, but imaging requires comparatively long acquisition times; hence, animals require long-term anaesthesia. CT is extensively used in bone and joint disorders providing excellent spatial resolution and good contrast for bone imaging. Despite its excellent structural assessment of mineralized structures, CT does not provide in vivo functional information of ongoing biological processes. Nuclear medicine is a very promising tool for investigating functional and molecular processes in vivo with new tracers becoming available as biomarkers. The combined use of imaging modalities also holds significant potential for the assessment of disease pathogenesis in animal models of musculoskeletal disorders, minimising the use of conventional invasive methods and animal redundancy.