- Email: [email protected]
S05: Effects of PPAR agonists on proliferation and differentiation in the urothelium
ARTICLE IN PRESS 258
Abstracts / Experimental and Toxicologic Pathology 61 (2009) 257–295
genetic fingerprint. The use of this type of display may also have utility in examining and explaining differences in toxicity profiles among candidate compounds during preclinical development. doi: 10.1016/j.etp.2009.02.003
S03 PPAR-induced sarcoma: Cell proliferation and altered gene expression in tumour precursor cells Ulf S. Andersson AstraZeneca R&D, Safety Assessment So¨derta¨lje, Sweden The development of the dual PPAR a/g agonist tesaglitazar as an oral antidiabetic was recently discontinued. Here we present tumor data from a 2-year rat carcinogenicity study with focus on the findings of subcutaneous fibrosarcomas with a predominant spindle cell appearance that occurred at the highest dose level of 10 mmol/kg in both sexes. This dose level was at or above the maximum tolerated dose and caused considerable cardiovascular mortality. To investigate the mechanism for induction of fibrosarcomas, replicative DNA synthesis and expression of PPARg in subcutaneous adipose tissues was assessed in rats administered 1, 3 and 10 mmol/kg tesaglitazar for 2 weeks, 3 and 6 months. Tesaglitazar stimulated DNA synthesis mainly in subcutaneous interstitial mesenchymal cells. The percentage of BrdU-labeled interstitial cells was increased dose dependently after 2 weeks. The increase in DNA synthesis was still statistically significant at later time points but only at 10 mmol/kg, the dose producing fibrosarcoma. Immunohistochemical analyses showed no detectable PPARg protein in the majority of BrdUlabeled interstitial mesenchymal cells in white and brown fat. This indicates that stimulation of DNA synthesis is not mediated via direct activation of PPARg in these cells. Gene expression analysis of adipose tissue revealed that by six months the cells were less responsive to PPARg stimulation and that markers of adipose tissue differentiation were down regulated. Furthermore, increased gene expression of inflammatory cytokines was associated with increased occurrence of necrotic or fibrotic lesions in subcutaneous adipose tissues. In summary, the data suggest that high dose tesaglitazar increases cell turnover in mesenchymal interstitial cells that results in depletion of adipogenic precursor cells and preneoplastic lesions. doi: 10.1016/j.etp.2009.02.004
S04 Understanding the changes evoked by chronic peroxisome proliferators-activated receptor (PPAR) agonism Terrie-Anne Cock, David Hassall Investigative Pre-clinical Toxicology, Safety Assessment, GlaxoSmithKline, Ware, Herts SG120HU, UK Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear hormone receptor of ligandactivated transcription factors involved in regulating glucose and lipid metabolism. The PPAR subfamily consists of three members; PPARbeta/delta, PPARalpha, and PPARgamma. PPAR agonists from single subtype to multiple subtype or partial agonists have being developed for the treatment of type 2 diabetes, dyslipidemia and obesity. Even though PPAR agonists are not genotoxic, carcinogenicity studies in rodents with PPAR gamma and dual PPAR gamma/alpha agonists have found various tumours, especially mesenchymal sarcomas in rats, mice, and hamsters, and rat urinary bladder urothelial tumours (El-Hage, 2005). These mesenchymal sarcomas appear to be preferential to the subcutaneous adipose tissue rather than the visceral adipose tissue beds; however the mode of action for the development of these tumours has not been elucidated. To develop our understanding of why PPAR agonists have differences in tumour risk between adipose depots; we expanded our knowledge of the physiological and functional differences between these adipose depots, with a particular focus on factors that can impact and influence tumourigenesis, through the use of transcriptomics. doi: 10.1016/j.etp.2009.02.005
S05 Effects of PPAR agonists on proliferation and differentiation in the urothelium Claire L. Varley, Jennifer Southgate Jack Birch Unit of Molecular Carcinogenesis, Department of Biology, University of York, York YO10 5YW, UK Systemic treatment of rats with PPAR agonists (mainly of dual a/g activity) has indicated that they may invoke non-genotoxic carcinogenesis in the epithelial lining of the urinary tract (urothelium). Although there is evidence in the male rat to support an indirect effect via a crystaluria-induced urothelial damage response (Cohen SM, Toxicological Sciences 2005; 87 (2): 322–7), there is other evidence to indicate a direct signalling effect on the urothelium (Egerod FL, Nielsen
ARTICLE IN PRESS Abstracts / Experimental and Toxicologic Pathology 61 (2009) 257–295
HS, Iversen L, et al., Biomarkers 2005; 10 (4): 295–309) and hence the full implications for using these drugs in man is unclear. Numerous reports have demonstrated that PPARs are expressed within the urothelium of different species, including man, and from an early developmental stage. We have developed methods to maintain normal human urothelial (NHU) cells in culture, where the cells retain PPAR expression and express a highly proliferative phenotype, mediated via autocrine stimulation of the epidermal growth factor receptor (EGFR). We have shown that specific activation of PPARg will result in a programme of gene expression changes associated with late/terminal cytodifferentiation, including induction of cytokeratins CK13 and CK20, tight junction-associated claudin 3 and uroplakins UPK1a and UPK2, but this is dependent upon inhibition of the signalling cascade downstream of EGFR. This indicates a subtle balance in the regulation of proliferation and differentiation in urothelium, with PPARg agonists promoting differentiation. Our data indicates that human urothelium is a target tissue for PPARg signalling, but it has yet to be determined whether dual agonists could have a modulatory effect on the proliferative/differentiation balance.
259
investigative new drug or new drug or biomarker applications. US FDA considers this approach to be a way to share information, realizing that most genomic data are currently used for exploration or research. The value to submission is to provide an opportunity for regulatory scientists to become familiar these data and to develop approaches for proper interpretation. The US Environmental Protection Agency has developed a draft guidance for submission of genomic data. Like the US FDA, these data are not required, but the opportunity to submit these data in an organized way allows both the regulated community and the agency to develop useful procedures for interpreting this new science. Currently, the value of this information has been in confirming biological or toxicity pathways and developing predictive models for prioritizing further testing. Establishing methods for application in safety and risk assessment are in the early stages of development. [This abstract of a proposed presentation does not reflect the policy or opinion of the US EPA] doi: 10.1016/j.etp.2009.02.007
doi: 10.1016/j.etp.2009.02.006
S07 EU Research Framework Programme 6 – Innovative medicines for Europe – InnoMed: Integrated project predictive toxicology – PredTox S06 US FDA and US EPA voluntary submission of genomic data guidance: Current and future use of genomics in decision making Douglas C. Wolf National Health and Environmental Effects Research Laboratory, Office of Research and Development, US Environmental Protection Agency; Research Triangle Park, NC, USA Appropriate utilization of data from toxicogenomic studies is an ongoing concern of the regulated industries and the agencies charged with assessing safety or risk. An area of current interest is the possibility of toxicogenomics to enhance our ability to develop higher or high-throughput models for predicting potential safety concerns or for rapidly identifying biomarkers of exposure or effect. These models could be developed to predict potential health concerns, prioritize testing strategies, or establish prognostic markers by helping to identify exposure to effect linkages. The US Food and Drug Administration has developed a guidance for voluntary submission of genomic data in support of
Friedlieb M. Pfannkuch F. Hoffmann-La Roche, Pharma Research-Global Non-Clinical Safety, CH-4070 Basel, Switzerland In order to increase early attrition in preclinical pharmaceutical drug development, the new omics technologies have been assessed by investigational toxicologists and technology experts. The InnoMed PredTox consortium with participation of 3 academic institutions, 2 technology vendors and 15 major pharmaceutical companies, has focused on a systematic and integrated -omics approach assessed against conventional toxicology assessment in the rat. EFPIA (European Federation of Pharmaceutical Industries and Associations) is the coordinator of the InnoMed project. As a unique feature, the consortium members have selected 14 proprietary compounds, troglitazone that had been withdrawn from the market, and a reference nephrotoxicant, gentamycin. The drug development program of the proprietary compounds was abandoned for hepato-/nephrotoxicity reasons at different development stages.
Abstracts / Experimental and Toxicologic Pathology 61 (2009) 257–295
genetic fingerprint. The use of this type of display may also have utility in examining and explaining differences in toxicity profiles among candidate compounds during preclinical development. doi: 10.1016/j.etp.2009.02.003
S03 PPAR-induced sarcoma: Cell proliferation and altered gene expression in tumour precursor cells Ulf S. Andersson AstraZeneca R&D, Safety Assessment So¨derta¨lje, Sweden The development of the dual PPAR a/g agonist tesaglitazar as an oral antidiabetic was recently discontinued. Here we present tumor data from a 2-year rat carcinogenicity study with focus on the findings of subcutaneous fibrosarcomas with a predominant spindle cell appearance that occurred at the highest dose level of 10 mmol/kg in both sexes. This dose level was at or above the maximum tolerated dose and caused considerable cardiovascular mortality. To investigate the mechanism for induction of fibrosarcomas, replicative DNA synthesis and expression of PPARg in subcutaneous adipose tissues was assessed in rats administered 1, 3 and 10 mmol/kg tesaglitazar for 2 weeks, 3 and 6 months. Tesaglitazar stimulated DNA synthesis mainly in subcutaneous interstitial mesenchymal cells. The percentage of BrdU-labeled interstitial cells was increased dose dependently after 2 weeks. The increase in DNA synthesis was still statistically significant at later time points but only at 10 mmol/kg, the dose producing fibrosarcoma. Immunohistochemical analyses showed no detectable PPARg protein in the majority of BrdUlabeled interstitial mesenchymal cells in white and brown fat. This indicates that stimulation of DNA synthesis is not mediated via direct activation of PPARg in these cells. Gene expression analysis of adipose tissue revealed that by six months the cells were less responsive to PPARg stimulation and that markers of adipose tissue differentiation were down regulated. Furthermore, increased gene expression of inflammatory cytokines was associated with increased occurrence of necrotic or fibrotic lesions in subcutaneous adipose tissues. In summary, the data suggest that high dose tesaglitazar increases cell turnover in mesenchymal interstitial cells that results in depletion of adipogenic precursor cells and preneoplastic lesions. doi: 10.1016/j.etp.2009.02.004
S04 Understanding the changes evoked by chronic peroxisome proliferators-activated receptor (PPAR) agonism Terrie-Anne Cock, David Hassall Investigative Pre-clinical Toxicology, Safety Assessment, GlaxoSmithKline, Ware, Herts SG120HU, UK Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear hormone receptor of ligandactivated transcription factors involved in regulating glucose and lipid metabolism. The PPAR subfamily consists of three members; PPARbeta/delta, PPARalpha, and PPARgamma. PPAR agonists from single subtype to multiple subtype or partial agonists have being developed for the treatment of type 2 diabetes, dyslipidemia and obesity. Even though PPAR agonists are not genotoxic, carcinogenicity studies in rodents with PPAR gamma and dual PPAR gamma/alpha agonists have found various tumours, especially mesenchymal sarcomas in rats, mice, and hamsters, and rat urinary bladder urothelial tumours (El-Hage, 2005). These mesenchymal sarcomas appear to be preferential to the subcutaneous adipose tissue rather than the visceral adipose tissue beds; however the mode of action for the development of these tumours has not been elucidated. To develop our understanding of why PPAR agonists have differences in tumour risk between adipose depots; we expanded our knowledge of the physiological and functional differences between these adipose depots, with a particular focus on factors that can impact and influence tumourigenesis, through the use of transcriptomics. doi: 10.1016/j.etp.2009.02.005
S05 Effects of PPAR agonists on proliferation and differentiation in the urothelium Claire L. Varley, Jennifer Southgate Jack Birch Unit of Molecular Carcinogenesis, Department of Biology, University of York, York YO10 5YW, UK Systemic treatment of rats with PPAR agonists (mainly of dual a/g activity) has indicated that they may invoke non-genotoxic carcinogenesis in the epithelial lining of the urinary tract (urothelium). Although there is evidence in the male rat to support an indirect effect via a crystaluria-induced urothelial damage response (Cohen SM, Toxicological Sciences 2005; 87 (2): 322–7), there is other evidence to indicate a direct signalling effect on the urothelium (Egerod FL, Nielsen
ARTICLE IN PRESS Abstracts / Experimental and Toxicologic Pathology 61 (2009) 257–295
HS, Iversen L, et al., Biomarkers 2005; 10 (4): 295–309) and hence the full implications for using these drugs in man is unclear. Numerous reports have demonstrated that PPARs are expressed within the urothelium of different species, including man, and from an early developmental stage. We have developed methods to maintain normal human urothelial (NHU) cells in culture, where the cells retain PPAR expression and express a highly proliferative phenotype, mediated via autocrine stimulation of the epidermal growth factor receptor (EGFR). We have shown that specific activation of PPARg will result in a programme of gene expression changes associated with late/terminal cytodifferentiation, including induction of cytokeratins CK13 and CK20, tight junction-associated claudin 3 and uroplakins UPK1a and UPK2, but this is dependent upon inhibition of the signalling cascade downstream of EGFR. This indicates a subtle balance in the regulation of proliferation and differentiation in urothelium, with PPARg agonists promoting differentiation. Our data indicates that human urothelium is a target tissue for PPARg signalling, but it has yet to be determined whether dual agonists could have a modulatory effect on the proliferative/differentiation balance.
259
investigative new drug or new drug or biomarker applications. US FDA considers this approach to be a way to share information, realizing that most genomic data are currently used for exploration or research. The value to submission is to provide an opportunity for regulatory scientists to become familiar these data and to develop approaches for proper interpretation. The US Environmental Protection Agency has developed a draft guidance for submission of genomic data. Like the US FDA, these data are not required, but the opportunity to submit these data in an organized way allows both the regulated community and the agency to develop useful procedures for interpreting this new science. Currently, the value of this information has been in confirming biological or toxicity pathways and developing predictive models for prioritizing further testing. Establishing methods for application in safety and risk assessment are in the early stages of development. [This abstract of a proposed presentation does not reflect the policy or opinion of the US EPA] doi: 10.1016/j.etp.2009.02.007
doi: 10.1016/j.etp.2009.02.006
S07 EU Research Framework Programme 6 – Innovative medicines for Europe – InnoMed: Integrated project predictive toxicology – PredTox S06 US FDA and US EPA voluntary submission of genomic data guidance: Current and future use of genomics in decision making Douglas C. Wolf National Health and Environmental Effects Research Laboratory, Office of Research and Development, US Environmental Protection Agency; Research Triangle Park, NC, USA Appropriate utilization of data from toxicogenomic studies is an ongoing concern of the regulated industries and the agencies charged with assessing safety or risk. An area of current interest is the possibility of toxicogenomics to enhance our ability to develop higher or high-throughput models for predicting potential safety concerns or for rapidly identifying biomarkers of exposure or effect. These models could be developed to predict potential health concerns, prioritize testing strategies, or establish prognostic markers by helping to identify exposure to effect linkages. The US Food and Drug Administration has developed a guidance for voluntary submission of genomic data in support of
Friedlieb M. Pfannkuch F. Hoffmann-La Roche, Pharma Research-Global Non-Clinical Safety, CH-4070 Basel, Switzerland In order to increase early attrition in preclinical pharmaceutical drug development, the new omics technologies have been assessed by investigational toxicologists and technology experts. The InnoMed PredTox consortium with participation of 3 academic institutions, 2 technology vendors and 15 major pharmaceutical companies, has focused on a systematic and integrated -omics approach assessed against conventional toxicology assessment in the rat. EFPIA (European Federation of Pharmaceutical Industries and Associations) is the coordinator of the InnoMed project. As a unique feature, the consortium members have selected 14 proprietary compounds, troglitazone that had been withdrawn from the market, and a reference nephrotoxicant, gentamycin. The drug development program of the proprietary compounds was abandoned for hepato-/nephrotoxicity reasons at different development stages.