Preclinical Evaluation Of Efficacy Research Articles

Evaluation of helper-dependent canine adenovirus vectors in a 3D human CNS model.

Gene therapy is a promising approach with enormous potential for treatment of neurodegenerative disorders. Viral vectors derived from canine adenovirus type 2 (CAV-2) present attractive features for gene delivery strategies in the human brain, by preferentially transducing neurons, are capable of efficient axonal transport to afferent brain structures, have a 30-kb cloning capacity and have low innate and induced immunogenicity in preclinical tests. For clinical translation, in-depth preclinical evaluation of efficacy and safety in a human setting is primordial. Stem cell-derived human neural cells have a great potential as complementary tools by bridging the gap between animal models, which often diverge considerably from human phenotype, and clinical trials. Herein, we explore helper-dependent CAV-2 (hd-CAV-2) efficacy and safety for gene delivery in a human stem cell-derived 3D neural in vitro model. Assessment of hd-CAV-2 vector efficacy was performed at different multiplicities of infection, by evaluating transgene expression and impact on cell viability, ultrastructural cellular organization and neuronal gene expression. Under optimized conditions, hd-CAV-2 transduction led to stable long-term transgene expression with minimal toxicity. hd-CAV-2 preferentially transduced neurons, whereas human adenovirus type 5 (HAdV5) showed increased tropism toward glial cells. This work demonstrates, in a physiologically relevant 3D model, that hd-CAV-2 vectors are efficient tools for gene delivery to human neurons, with stable long-term transgene expression and minimal cytotoxicity.

Preclinical evaluation of efficacy and safety of an improved lentiviral vector for the treatment of β-thalassemia and sickle cell disease.

A previously published clinical trial demonstrated the benefit of autologous CD34+ cells transduced with a self-inactivating lentiviral vector (HPV569) containing an engineered β-globin gene (βA-T87Q-globin) in a subject with β-thalassemia major. This vector has been modified to increase transduction efficacy without compromising safety. In vitro analyses indicated that the changes resulted in both increased vector titers (3 to 4 fold) and increased transduction efficacy (2 to 3 fold). An in vivo study in which 58 β-thalassemic mice were transplanted with vector- or mock-transduced syngenic bone marrow cells indicated sustained therapeutic efficacy. Secondary transplantations involving 108 recipients were performed to evaluate long-term safety. The six month study showed no hematological or biochemical toxicity. Integration site (IS) profile revealed an oligo/polyclonal hematopoietic reconstitution in the primary transplants and reduced clonality in secondary transplants. Tumor cells were detected in the secondary transplant mice in all treatment groups (including the control group), without statistical differences in the tumor incidence. Immunohistochemistry and quantitative PCR demonstrated that tumor cells were not derived from transduced donor cells. This comprehensive efficacy and safety data provided the basis for initiating two clinical trials with this second generation vector (BB305) in Europe and in the USA in patients with β-thalassemia major and sickle cell disease.

Mouse models for therapeutic vaccination against hepatitis B virus.

A mouse model for persistent HBV infection is essential for the development of a therapeutic vaccine against HBV. Because HBV cannot infect mouse hepatocytes, even if the HBV receptor is introduced, surrogate models are used. A suitable model needs to establish persistent HBV replication and must allow the establishment of HBV-specific adaptive cellular and humoral immune responses. Therefore, an immunocompetent mouse model is needed in which one can break HBV-specific tolerance and ideally eliminate the HBV transcription template. The most widely used model for chronic HBV infection is the HBV transgenic mouse. Although HBV replicates from an integrated transgene, HBV-specific immune tolerance can be broken upon adequate immune stimulation because antigen expression only starts shortly before birth. Alternative mouse models of chronic HBV infection are generated by introducing HBV genomes either using viral vectors or using hydrodynamic injection. In these alternative models, the HBV transcription template is introduced into a proportion of hepatocytes and stays extra-chromosomal. It thus mimics the natural HBV transcription template, the HBV cccDNA in humans. Unlike an HBV transgene, however, it can be cleared upon appropriate treatment or immune stimulation. Human hepatocyte chimeric mice in which murine hepatocytes are widely replaced by human hepatocytes represent another important mouse model for persistent HBV infection. These mice are susceptible for HBV infection, but need to be severely immune deficient to accept human hepatocytes. In conclusion, a variety of mouse models for persistent HBV infection are available suitable for preclinical efficacy evaluations of therapeutic vaccination strategies against HBV.

Abstract 3126: Renca RCC syngeneic model to evaluate efficacy of novel antisense oligonucleotides targeting TGF-β isoforms

Abstract Transforming growth factor beta (TGF-β represents a family of cytokines, which function as the primary mediators for TGF-β signaling via TGF-β receptor type II (TβIIR) and both non-canonical and canonical downstream signaling pathways. TGF-β is associated with a wide range of biological processes in oncology, including tumor cell invasion, migration, angiogenesis, immunosuppression, as well as regulation of tumor stem cell properties. Hence, optimal preclinical evaluation of efficacy of TGF-β antagonists is challenging. Isarna Therapeutics has designed and developed selective and potent LNA-modified antisense oligonucleotides targeting the various TGF-β isoforms. In order to adequately evaluate selected preclinical development candidates, Oncodesign has developed customized experimental Renca tumor models in syngeneic and/or immunodeficient mice. The Renca cell line was established from a murine transplantable renal adenocarcinoma of spontaneous origin, and has been used under various experimental conditions: (1) subcutaneous tumor model by inoculating cells into the flanks of the animals; (2) the pulmonary metastatic tumor model by an intravenous injection of cells into the tail vein; and (3) the orthotopic tumor model by injecting cells into the renal subcapsule (and subsequent pulmonary metastasis). Outcome of this development program and preliminary results for selected TGF-β antisense oligonucleotides will be presented and discussed. Citation Format: Hanna Korhonen, Julie-Orlane Redon, Damien France, Guillaume Serin, Francis Bichat, Frank Jaschinski, Katja Wosikowski, Michel Janicot. Renca RCC syngeneic model to evaluate efficacy of novel antisense oligonucleotides targeting TGF-β isoforms. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3126. doi:10.1158/1538-7445.AM2014-3126

Preclinical evaluation of efficacy and stability of docetaxel micelle-encapsulated by a tripodal cyclotriphosphazene amphiphile

Docetaxel formulated by micelle-encapsulation using a tripodal cyclotriphosphazene amphiphile [NP(MPEG750)(GlyPheLeu)2Et]3 (CP750) was named “Phostaxel” and compared in efficacy and stability with Taxotere® formulated using the surfactant polysorbate 80, which is currently in clinical use. Phostaxel has always shown better efficacy than Taxotere® in various xenograft trials at the same dosage and administration schedule against the tumor cell lines tested. The better efficacy of Phostaxel could be explained based on the difference in pharmacokinetic and biodistribution profiles of Phostaxel and Taxotere®. Phostaxel exhibited significantly slower clearance rate and larger AUClast value compared with Taxotere®. Phostaxel has also shown higher DTX distribution in tumor than Taxotere®. In addition, Phostaxel displayed better solution stability compared with Taxotere® both in distilled water and in saline solution at room and refrigerator temperatures.

A Three-dimensional Tissue Culture Model to Study Primary Human Bone Marrow and its Malignancies

Tissue culture has been an invaluable tool to study many aspects of cell function, from normal development to disease. Conventional cell culture methods rely on the ability of cells either to attach to a solid substratum of a tissue culture dish or to grow in suspension in liquid medium. Multiple immortal cell lines have been created and grown using such approaches, however, these methods frequently fail when primary cells need to be grown ex vivo. Such failure has been attributed to the absence of the appropriate extracellular matrix components of the tissue microenvironment from the standard systems where tissue culture plastic is used as a surface for cell growth. Extracellular matrix is an integral component of the tissue microenvironment and its presence is crucial for the maintenance of physiological functions such as cell polarization, survival, and proliferation. Here we present a 3-dimensional tissue culture method where primary bone marrow cells are grown in extracellular matrix formulated to recapitulate the microenvironment of the human bone (rBM system). Embedded in the extracellular matrix, cells are supplied with nutrients through the medium supplemented with human plasma, thus providing a comprehensive system where cell survival and proliferation can be sustained for up to 30 days while maintaining the cellular composition of the primary tissue. Using the rBM system we have successfully grown primary bone marrow cells from normal donors and patients with amyloidosis, and various hematological malignancies. The rBM system allows for direct, in-matrix real time visualization of the cell behavior and evaluation of preclinical efficacy of novel therapeutics. Moreover, cells can be isolated from the rBM and subsequently used for in vivo transplantation, cell sorting, flow cytometry, and nucleic acid and protein analysis. Taken together, the rBM method provides a reliable system for the growth of primary bone marrow cells under physiological conditions.

Development of an adenovirus-based respiratory syncytial virus vaccine: preclinical evaluation of efficacy, immunogenicity, and enhanced disease in a cotton rat model.

The lack of a vaccine against respiratory syncytial virus (RSV) is a challenging and serious gap in preventive medicine. Herein, we characterize the immunogenicity of an adenovirus serotype 5-based RSV vaccine encoding the fusion (F) protein (Ad5.RSV-F) and the protection provided following immunization with Ad5.RSV-F and assess its potential for producing enhanced disease in a cotton rat (CR) model. Animals were immunized intranasally (i.n.) and/or intramuscularly (i.m.) and subsequently challenged with RSV/A/Tracy (i.n.) to assess protection. Robust immune responses were seen in CRs vaccinated with Ad5.RSV-F given i.m. or i.n., and these responses correlated with reduced replication of the virus in noses and lungs after challenge. Neutralizing antibody responses following immunization with a single dose of Ad5.RSV-F at 1 × 10(11) viral particles (v.p.) elicited antibody titers 64- to 256-fold greater than those seen after natural infection. CRs boosted with Ad5.RSV-F i.n. 28 days after an i.m. dose also had significant increases in neutralizing antibody titers. Antibody affinity for different F-protein antigenic sites revealed substantial differences between antibodies elicited by Ad5.RSV-F and those seen after RSV infection; differences in antibody profiles were also seen between CRs given Ad5.RSV-F i.m. and CRs given Ad5.RSV-F i.n. Ad5.RSV-F priming did not result in enhanced disease following live-virus challenge, in contrast to the histopathology seen in CRs given the formalin-inactivated RSV/A/Burnett vaccine. Respiratory syncytial virus (RSV) is the most common cause of acute lower respiratory infection in infants and young children and a serious health threat in the immunocompromised and the elderly. Infection severity increased in children in an immunization trial, hampering the over 4-decade-long quest for a successful RSV vaccine. In this study, we show that a genetically engineered RSV-F-encoding adenoviral vector provides protective immunity against RSV challenge without enhanced lung disease in cotton rats (CRs). CRs were vaccinated under a number of different regimens, and the immunity induced by the recombinant adenoviral RSV vaccine administered by use of an intramuscular prime-intranasal boost regimen may provide the best protection for young infants and children at risk of RSV infection, since this population is naive to adenoviral preformed immunity. Overall, this report describes a potential RSV vaccine candidate that merits further evaluation in a phase I clinical study in humans.

Sodium iodate induced retinal degeneration: new insights from an old model.

Studies on the selective retinal degeneration induced by sodium iodate (NaIO3) date back to 1941; Sorsby (1941) described the effect of intravenously injected NaIO3 solution on the rabbit retina. Since then, NaIO3-induced retinal degeneration has been described in different mammalian species including sheep, rabbit, rat and mouse with varying doses and routes of administration. At the present time, the murine NaIO3 model is the most widely used because it results in reproducible, patchy retinal degeneration (Figure 1) and can be studied in a wide variety of wild type and genetic knockout strains. Many studies reporting the pathologic changes in retinal structure at specific time points and electrophysiological changes in retinal function over time have followed (Franco et al., 2009; Redfern et al., 2011). More recently, the model has gained a resurgence of interest because of the ability to utilize high resolution in vivo imaging to study structural changes in the retina in live animals. Techniques such as spectral domain optical coherence tomography (OCT) and confocal scanning laser ophthalmoscopy (cSLO) allow longitudinal evaluation of retinal structure (Machalinska et al., 2014; Yang et al., 2014); thus making this model amenable to evaluation of novel therapeutic approaches to retinal degeneration such as cellular therapies.

Targeted Treatments: Pre-Clinical Evaluation Of Efficacy By Tracking Clonal Diversity In CLL Xenograft Models

Progressive CLL with DNA damage response defects caused by ATM or TP53 inactivation is still a therapeutic problem. There is currently a growing appreciation of the subclonal complexity in this type of leukaemia, mostly supported by whole genome analyses of CLL samples such as SNP arrays and next generation sequencing, both highlighting the role of subclonal variations in progression and therapeutic response. Although revealing, these analyses do not inform about the clonal make-up of individual proliferating cells and such information might be necessary to determine effectiveness of novel targeted treatments.In order to determine and follow the clonal evolutionary millieu in individual CLL cases we developed multi-colour fluorescence in situ hybridization (MCF) to analyse CLL samples at the single cell level. We screened 134 samples for the presence of 11q and 17p deletions of which 25 were identified with one of these cytogenetic defects and these were screened alongside 28 samples with normal 17p and 11q loci for 13q and 6q deletion as well as trisomy 12. We found that all but four of the samples with 11q or 17p deletions had at least one other genomic abnormality, whilst only two of the 28 samples with normal 17p and 11q loci harboured two genomic abnormalities. We subsequently performed MCF on the 27 samples with multiple genetic abnormalities and generated evolutionary trees for each of the samples. Two types of clonal evolution were identified: linear and branched, with the latter being the more common. We were able to analyse eleven of these samples post-treatment and found that whilst the clonality of some samples was largely unaffected by treatment, others showed treatment-induced differences in the subclonal make-up. Furthermore, some samples also exhibited signs of evolution with the generation of novel subclones upon treatment.Pre-clinical testing of novel therapeutic agents in xenograft models requires that the subclonal architecture of engrafted samples is representative of the donor sample. To this end, modifications were made to two primary CLL xenograft models. Firstly, samples from three different CLLs with a complex karyotype and multiple subclones were engrafted with autologous ex-vivo stimulated T-cells into NOG mice. All CLL subclones displayed a capacity to engraft and proliferate in this xenograft model. Furthermore, the MCF protocol was implemented to assess subclonality in vivo. Mice were randomised to Rituximab or control saline treatment three times over a period of five days. Analysis of engrafted cells in the spleen a week later displayed a good response to Rituximab with a significantly lower number of hCD45+ CD19+ CD5+cells. However, upon isolation of CLL cells by FACS and assessment of clonal architecture using MCF it was shown that Rituximab treatment affected all subclones with a differential subclonal response. This resulted in a greater proportion of the CLL subclones with the greatest genetic complexity harbouring both 11q and 6q deletions still remaining.Finally, to recapitulate patient response to therapy, T-cell depleted, pre-treatment PBMC from a patient poorly responsive to bendamustine + Rituximab were engrafted in humanised mice. As in the patient, bi-weekly therapy for 3 weeks resulted in a limited, poor response to therapy with a corresponding small reduction of CFSE-labelled CLL PBMC and hCD45+ CD19+ cells.We conclude that the majority of CLLs display a branched pattern of evolution and that the subclone dynamic is an important determinant of CLL proliferation and response to treatment. We suggest that novel targeted therapies should be tested in the context of their ability to eradicate CLL subclones with the highest proliferative capacity, as these subclones are most likely to evolve. The development of an MCF protocol, in combination with the xenograft model, provides a powerful tool to help predict overall and subclonal responses to therapy in the patient. Disclosures:No relevant conflicts of interest to declare.

Evaluation of Pre-Clinical Efficacy to HPV16 L2E6E7 Vaccine and HPV16 E6E7 Adenovirus-5 Vector Vaccine with Different Dosages and Prime- Booster Regiments in Mouse Model

Purpose: This study evaluates the dose-response and immunization procedure in mouse model and the efficacy of prime-booster regimens with HPV16 L2E6E7 vaccine and HPV16 E6E7 Ad5 vector vaccine. Methods: Experimental animals were C57 BL/6 mice. Each group included 10 or 20 C57 BL/6 mice. The tumor model used TC-1 tumor cells. The HPV16 L2E6E7 vaccine groups were treated using the following dosage: 15 μg/ml, 30 μg/ml, 60 μg/ml, 120 μg/ml, 240 μg/ml, and then 120 μg/ml was used for the following regimens: 0-7 days, 0-15 days, 0-7-15 days. The HPV16 E6E7 Adenovirus-5 vector vaccine groups were treated using the following dosage: 3.00×106 IU/ml, 3.00×107 IU/ml, 3.00×108 IU/ml, 3.00×109 IU/ml, and then 3.00×107 IU/ml was used for the following regimens: 0-7 days, 0-15 days, 0-7-15 days, and control group. Prime-booster combined regimens with HPV L2E6E7 vaccine (P, 120 μg/ml) and HPV16 E6E7 ad5 vector vaccine (V, 3.00×107 IU/ml) were set as follows: 0P-7P days, 0P-7V days, and 0P-7P-15V days, 0P-7V-15V days, and 0P-7P-15V-21V days. Results: Upon challenge with 104 TC-1 tumor cells, mice developed palpable, rapidly growing tumors within 7–14 days. These tumors became lethal to the mice within 21–28 days. HPV16 L2E6E7 vaccine (120 μg/ml, 0-7- 15 day’s procedure) protective efficacy was 85% and the HPV16 E6E7 Ad5 vector vaccine (3.00×107 IU/ml, 0 day procedure) that was 80%. Prime-booster regimens showed a protective efficacy of 80–90% for the 0P-7V days and 0P-7V-15V day’s schedules. Conclusion: HPV16 L2E6E7 vaccine and HPV16 E6E7 Ad5 vector vaccine are proved the candidate vaccine for therapeutic intervention against HPV16-induced tumor.

891 VITAMIN D AND BCG IMMUNOTHERAPY

You have accessJournal of UrologyBladder Cancer: Basic Research II1 Apr 2012891 VITAMIN D AND BCG IMMUNOTHERAPY Jong-Wei Hsu, Edward Messing, and Yi-Fen Lee Jong-Wei HsuJong-Wei Hsu Rochester, NY More articles by this author , Edward MessingEdward Messing Rochester, NY More articles by this author , and Yi-Fen LeeYi-Fen Lee Rochester, NY More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2012.02.986AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Bacillus Calmette-Guérin (BCG), a vaccine against tuberculosis (TB), has been used and proved to be one of the most effective treatments for superficial bladder cancer (BCa). However, the mechanisms of BCG action have not been completely understood, which limits the improvement of BCG therapy. Vitamin D deficiency has been associated with the high risk of TB infection, and the beneficial effect of UV exposure in TB patients was proven to be mediated via activation of vitamin D signals of innate immune cells. Thus, vitamin D signals might be involved in mediating BCG immunotherapy for BCa. METHODS We applied a transwell migration assay to examine vitamin D signal¡ߣs impact on the BCG-mediated innate immune cells¡ߣ migration. The corresponding pro-inflammatory cytokines induced by vitamin D/vitamin D receptor were screened by cytokine array and confirmed by ELISA. Preclinical evaluation of efficacy of intravesical BCG with 1£\,25-dihydroxyvitamin D3 (1,25-VD) therapy was performed using Nbutyl- N-(4-hydroxybutyl)-nitrosamine (BBN)-induced BCa mouse model. RESULTS ELISA assay revealed that treatment of BCa cells with 1,25-VD promotes BCG-induced interleukin 8 (IL-8) production, consequently inducing the migration of neutrophil-like cells, HL-60, and monocyte-like cells, THP-1. This 1,25-VD-mediated IL-8-induced innate immune cells migration was blocked by IL-8 neutralized antibody. These in vitro studies suggest that 1,25-VD could improve BCG responsiveness by enhancing recruitment of innate immune cells to promote cytotoxic killing of BCa cells. More importantly, our in vivo data revealed that intravesical treatment of 1,25-VD improves BCG efficacy where 1,25-VD+BCG treatment increased the survival rate of mice with BCa induced by BBN compared to BCG treatment alone. CONCLUSIONS The results of these studies indicate that 1,25-VD signaling is critical for success of BCG immunotherapy and co-treatment with 1,25-VD will enhance BCG's anti-tumor effects in BCa. © 2012 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 187Issue 4SApril 2012Page: e362-e363 Advertisement Copyright & Permissions© 2012 by American Urological Association Education and Research, Inc.MetricsAuthor Information Jong-Wei Hsu Rochester, NY More articles by this author Edward Messing Rochester, NY More articles by this author Yi-Fen Lee Rochester, NY More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

892 PRELIMINARY RESULTS OF PERIOPERATIVE OUTCOMES AND ONOCOLIGIC EFFICACY FROM A SINGLE INSTITUTION RANDOMIZED CONTROLLED TRIAL OF OPEN VERSUS ROBOTIC ASSISTED RADICAL CYSTECTOMY

cancer (BCa). However, the mechanisms of BCG action have not been completely understood, which limits the improvement of BCG therapy. Vitamin D deficiency has been associated with the high risk of TB infection, and the beneficial effect of UV exposure in TB patients was proven to be mediated via activation of vitamin D signals of innate immune cells. Thus, vitamin D signals might be involved in mediating BCG immunotherapy for BCa. METHODS: We applied a transwell migration assay to examine vitamin D signalis impact on the BCG-mediated innate immune cellsi migration. The corresponding pro-inflammatory cytokines induced by vitamin D/vitamin D receptor were screened by cytokine array and confirmed by ELISA. Preclinical evaluation of efficacy of intravesical BCG with 1£ ,25-dihydroxyvitamin D3 (1,25-VD) therapy was performed using NbutylN-(4-hydroxybutyl)-nitrosamine (BBN)-induced BCa mouse model. RESULTS: ELISA assay revealed that treatment of BCa cells with 1,25-VD promotes BCG-induced interleukin 8 (IL-8) production, consequently inducing the migration of neutrophil-like cells, HL-60, and monocyte-like cells, THP-1. This 1,25-VD-mediated IL-8-induced innate immune cells migration was blocked by IL-8 neutralized antibody. These in vitro studies suggest that 1,25-VD could improve BCG responsiveness by enhancing recruitment of innate immune cells to promote cytotoxic killing of BCa cells. More importantly, our in vivo data revealed that intravesical treatment of 1,25-VD improves BCG efficacy where 1,25-VD BCG treatment increased the survival rate of mice with BCa induced by BBN compared to BCG treatment alone. CONCLUSIONS: The results of these studies indicate that 1,25-VD signaling is critical for success of BCG immunotherapy and co-treatment with 1,25-VD will enhance BCG’s anti-tumor effects in BCa.

217 POSTER Pre-clinical evaluation of efficacy and PK/PD biomarkers of GDC-0941, a potent class 1 PI3K inhibitor

The path of new compounds from discovery to bedside is long and costly and a large majority of compounds never make it to the market. To improve drug development, better tools are needed to assess efficacy and safety early in the process. One approach that has been suggested by the FDA to help streamlining the drug development process is pharmacokinetic/pharmacodynamic (PK/PD) modeling and simulation. In this approach, a model-based link between PK profiles and corresponding PD is established. Once a suitable model is identified and validated, predictions about efficacy and safety of different dosing regimens can be made.The goal of this review is to examine the current state of PK, PD, and PK/PD in antibiotic and antiviral research and development.

Oral sensitization with shrimp tropomyosin induces in mice allergen-specific IgE, T cell response and systemic anaphylactic reactions

Appropriate murine models of shrimp tropomyosin (ST) allergy would be useful in investigating the mechanisms underlying food allergy in human subjects, as well as for the pre-clinical evaluation of efficacy and safety of novel therapeutic approaches. These models should mimic immune and clinical features of human disease, including anaphylactic response. We sensitized C3H/HeJ mice by the oral route with purified ST using cholera toxin (CT) as adjuvant. ST-specific IgE, IgG1, IgG2a and IgA responses were evaluated by ELISA. Spleen cell proliferation and cytokine production by allergen-specific activation were assessed. Jejunum and colon fragments were collected to evaluate the local expression of cytokine genes by PCR. Local and systemic anaphylactic reactions induced by oral ST challenge were scored according to symptoms observed. Faecal samples were collected to assess local IgA production and histamine levels. Oral sensitization with ST plus CT induced in mice significant levels of serum IgE and IgG1 and faecal IgA. ST-specific cell proliferation and IL-4, IL-13 and IFN-gamma cytokine production were induced in the spleen. After oral challenge, 100% of mice had anaphylactic symptoms while no symptoms were observed in challenged naive mice. Faecal histamine content after ST challenge appeared significantly increased in sensitized mice when compared with that observed in pre-immune mice. Jejunum mRNA expression of T(h)2 cytokines was up-regulated by ST sensitization. These results support the importance of the oral way of sensitization and of the in-depth characterization of the anaphylactic response for the development of a suitable in vivo model of food allergy.

The SARS-CoV ferret model in an infection–challenge study

Phase I human clinical studies involving therapeutics for emerging and biodefense pathogens with low incidence, such as the severe acute respiratory syndrome coronavirus (SARS-CoV), requires at a minimum preclinical evaluation of efficacy in two well-characterized and robust animal models. Thus, a ferret SARS-CoV model was evaluated over a period of 58 days following extensive optimization and characterization of the model in order to validate clinical, histopathological, virological and immunological endpoints. Ferrets that were infected intranasally with 10 3 TCID 50 SARS-CoV showed higher body temperature (2–6 d.p.i.), sneezing (5–10 d.p.i.), lesions (5–7 d.p.i.) and decreased WBC/lymphocytes (2–5 d.p.i.). SARS-CoV was detected up to 7 d.p.i. in various tissues and excreta, while neutralizing antibody titers rose at 7 d.p.i. and peaked at 14 d.p.i. At 29 d.p.i., one group was challenged with 10 3 TCID 50 SARS-CoV, and an anamnestic response in neutralizing antibodies was evident with no detectable virus. This study supports the validity of the ferret model for use in evaluating efficacy of potential therapeutics to treat SARS.

Preclinical efficacy evaluation of ixabepilone (BMS-247550) in combination with cetuximab or capecitabine in human colon and lung carcinoma xenografts

12017 Background: Ixabepilone belongs to a class of structurally novel, microtubule-stabilizing agents that exert their antimitotic action by binding to tubulin with a binding mode that is distinct from the taxanes. Preclinical findings that ixabepilone has antitumor activity in a broad spectrum of tumor types, including taxane-resistant tumors, is borne out by Phase II clinical trials where ixabepilone has demonstrated activities in multiple tumor types including breast, renal, pancreatic, prostate and lymphoma. The aim of this series of studies was to further characterize the therapeutic potential of ixabepilone in combination with currently approved chemotherapy agents. Methods: Antitumor activity was evaluated in the GEO human colon and L2987 human lung carcinoma xenografts. Therapeutic synergism of the combination was defined as the attainment of efficacy that was significantly better than the best response of the individual single agents administered at their maximum-tolerated dose (MTD) or optimal dose (OD). Results: In the GEO tumors, single-agent ixabepilone produced 1.1 log cell kill (LCK) at its MTD. Cetuximab at its OD yielded 0.8 LCK. The combination of ixabepilone and cetuximab produced 1.7 LCK which was significantly superior to ixabepilone alone (P=0.0173) and cetuximab alone (P=0.0002). Similar synergistic efficacy was observed in the L2987 tumors. The combined efficacy of capecitabine plus ixabepilone was evaluated in the GEO tumors. In this tumor, single-agent ixabepilone was modestly active (LCK = 0.8) at its MTD. Single-agent capecitabine was not effective (LCK = 0.4) at its MTD. However, the combination of the two agents produced therapeutic synergism, yielding antitumor efficacy (1.9 LCK) that was superior to either of the agents alone at their MTDs (P=0.035 and 0.0004, respectively). Conclusions: Ixabepilone demonstrates robust synergistic antitumor efficacy when used in combination with cetuximab or capecitabine in human xenografts providing a biologic rationale for these combinations in the treatment of cancer. [Table: see text]

Preclinical efficacy evaluations of XK-469: dose schedule, route and cross-resistance behavior in tumor bearing mice.

XK-469 is advancing to Phase I clinical trials. Preclinical studies were carried out to assist in clinical applications. DOSE-SCHEDULE ROUTE TESTING: Single dose i.v. treatment with XK-469 produced lethality (LD20 to LD100) above 142 mg/kg. Optimum treatment required total dosages of 350 to 600 mg/kg. Furthermore, high individual i.v. dosages (100 to 142 mg/kg) were poorly tolerated, producing substantial weight loss (8 to 18% of body weight), poor appearance, and slow recovery (8 to 12 days). A 1-hour infusion of dosages more than 140 mg/kg, or BID injections 6 hrs apart, did not reduce lethality. However, lower individual dosages of 40 to 50 mg/kg/injection i.v. were well tolerated and could be given daily to reach an optimum total dose with minimal toxicities. Likewise, 75 mg/kg/injection i.v. could be used every other day to reach optimal treatment. The necropsy profiles of deaths from toxic dosages were essentially identical regardless of schedule (deaths 4 to 7 days post treatment). The profiles were: paralytic ileus or gastroparesis; GI epithelial damage; and marrow toxicity. Interestingly, the key lethal events were rapidly reversible and simple to overcome with lower dosages given daily or every other day. Based on these results, the high dose, Q21 day schedule should be avoided in clinical applications. Instead, a split dose regimen is recommended (e.g., daily, every other day, or twice weekly). XK-469 was also well tolerated by the oral route, requiring 35% higher dosages p.o. to reach the same efficacy and toxicity as produced i.v.. CROSS-RESISTANCE STUDIES: XK-469 resistance was produced by optimum treatments of i.v. implanted L1210 leukemia over seven passage generations. This leukemia subline (L1210/XK469) had reduced sensitivity to VP-16 (with a 4.0 log kill in i.v. implanted L1210/XK469 compared to an 8.0 log kill against i.v. implanted L1210/0). It also had a reduction in the sensitivity to 5-FU (with a 2.0 log kill in the implanted L1210/XK469 compared to a 4.0 log kill against i.v. implanted L1210/0). Other agents were approximately as active against the resistant tumor, including: Ara-C, Gemzar, Cytoxan, BCNU, DTIC, and CisDDPT. No case of collateral sensitivity was observed; i.e., no agent was markedly more active against the resistant subline L1210/XK-469 than against the parent tumor in mice.

Corneal kindling in mice: behavioral and pharmacological differences to conventional kindling

Electrical kindling via unilateral implanted depth electrodes in rats is currently the most commonly used model for temporal lobe epilepsy, but the use of this model in drug screening for the identification of novel anticonvulsants is markedly hampered by the laborious and time-consuming preparation and the size of the animals. Kindling of male mice via transcorneal electrical stimulation has recently proposed as a cost-effective screening model that may improve the preclinical evaluation of efficacy and adverse effect potential of drug candidates for treatment of partial epilepsy. In the present study, corneal kindling was characterized and compared in male and female mice. In fully kindled mice, the anticonvulsant efficacy of the standard antiepileptic drug phenytoin was determined. Large groups of kindled mice were used to examine whether phenytoin non-responders can be selected in the corneal kindling model as reported previously for amygdala kindling. Furthermore, in view of the enhanced adverse effect potential of NMDA antagonists in amygdala kindled rats, it was evaluated whether corneally kindled mice also differ in this respect from non-kindled animals. Mice of both genders could be kindled by twice daily transcorneal stimulation within 10–12 days. However, in contrast to traditional kindling, corneal kindling was associated with a high frequency of mortality, and persistence of the fully kindled state after 4 weeks without stimulation was not pronounced. Phenytoin proved highly potent and efficacious to block corneally kindled seizures. Only one non-responder could be selected out of 75 fully kindled mice repeatedly tested with phenytoin. At 6 days after the last kindled seizure, kindled mice were more sensitive than non-kindled mice to phencyclidine-like behavioral adverse effects of the competitive NMDA antagonist D-CPPene, but this altered sensitivity was not long-lasting, having almost disappeared 27 days after the last seizure, indicating that, in contrast to traditional kindling, brain alterations after corneal kindling are not permanent. In summary, although corneal kindling may have advantages for the identification of new drugs during initial screening of large numbers of compounds, it cannot replace traditional electrical kindling during later phases of drug development. Furthermore, the high mortality and insufficient persistence of corneal kindling in mice detract from the use of this model for repeated drug testing in the same group of animals.

Development of Breast Cancer Chemopreventive Drugs

Abstract: Chemoprevention, or intervention with chemical agents at the precancer stage to avoid or slow the development of the carcinogenic process, is one strategy to reduce both the incidence of breast cancer and its mortality. Systematic development of cancer chemopreventive drugs calls for evaluation of pre-clinical efficacy in well-characterized in vitro screens and animal cancer models. Highly promising agents are also included in traditional pre-clinical toxicity tests performed in two species. The most promising and least toxic agents enter clinical trials, including both phase I safety and pharmacokinetics evaluations and phase II and III efficacy studies. The use of populations with defined, measurable biologic alterations in tissue occurring prior to cancer development, i.e. intermediate biomarkers, is important to successful phase II chemoprevention trials. The intermediate biomarkers may be of several types, such as histological/premalignant lesions, proliferative, genetic/regulatory, differentiation-related, or biochemical. For example, ductal and lobular carcinoma in situ are premalignant lesions in the human breast. These lesions are tissue at very high risk of malignant progression and also have the potential to be modulated by chemopreventive agents. In addition, other types of biomarkers may be identified within the lesions. These biomarkers could then serve as surrogate trial end-points, instead of cancer incidence, for use in shorter, less costly trials.

Preclinical efficacy evaluation of potential chemopreventive agents in animal carcinogenesis models: methods and results from the NCI Chemoprevention Drug Development Program.

In the NCI, Chemoprevention Branch drug development program, potential chemopreventive agents are evaluated for efficacy against chemical carcinogen-induced tumors in animal models. This paper summarizes the results of 144 agents in 352 tests using various animal efficacy models. Of these results, 146 were positive, representing 85 different agents. The target organs selected for the animals model are representative of high-incidence human cancers. The assays include inhibition of tumors induced by MNU in hamster trachea, DEN in hamster lung, AOM in rat colon (including inhibition of AOM-induced aberrant crypts), MAM in mouse colon, DMBA and MNU in rat mammary glands, DMBA promoted by TPA in mouse skin, and OH-BBN in mouse bladder. The agents tested may be classified into various pharmacological and chemical structural categories that are relevant to their chemopreventive potential. These categories include antiestrogens, antiinflammatories (e.g., NSAIDs), antioxidants, arachidonic acid metabolism inhibitors, GST and GSH enhancers, ODC inhibitors, protein kinase C inhibitors, retinoids and carotenoids, organosulfur compounds, calcium compounds, vitamin D3 and analogs, and phenolic compounds (e.g., flavonoids). The various categories of compounds have different spectra of efficacy in animal models. In hamster lung, GSH-enhancing agents and antioxidants appear to have high potential for inhibiting carcinogenesis. In the colon, NSAIDs and other antiinflammatory agents appear particularly promising. Likewise, NSAIDs are very active in mouse bladder. In rat mammary glands, retinoids and antiestrogens (as would be expected) are efficacious. Several of the chemicals evaluated also appear to be promising chemopreventive agents based on their activity in several of the animal models. Particularly, the ODC inhibitor DFMO was active in the colon, mammary glands, and bladder models, while the dithiolthione, oltipraz, was efficacious in all the models listed above (i.e., lung, colon, mammary glands, skin, and bladder).