Peroxisome proliferator-actuated receptors (PPARs) are nuclear hormone receptors targeted for therapeutic modulation in diabetes. Specifically, PPARa agonism will control dyslipidemia, while PPARy agonism affords improved glucose homeostasis. Nonclinical and clinical safety issues have prevented PPARay agonists from becoming drugs (Balakumar et al., 2007, Rubenstrunk et al., 2007). The results of two-year rodent carcinogenicity studies, including hemangiosarcoma, liposarcoma, and urinary bladder transitional cell carcinoma, have generally clouded a clear human risk assessment. With widespread distribution of PPARa and PPARy receptors in tissues, including those transformed in carcinogenesis, a clear separation of the potentially beneficial role of receptor agonism from the potentially adverse contribution to tumor development is complex to research and understand.
The investigative approaches directed toward establishing a cogent human risk assessment for dual PPAR agonist-tnduced urinary bladder transitional cell carcinoma in rodents are described here for the PPARa/y agonist mura-
glitazar (Dominick et al., 2006; Achanzar et al., 2007; Tannehill-Gregg et al., 2007; Waites et al., 2007). An increased incidence of ventral bladder wall transitional cell papillomas and carcinomas of the urinary bladder were noted in rats at doses as low as eight-times the projected human exposure at 5 mg/kg (Tannehill-Gregg et al., 2007). Histopathology and scanning electron microscopy revealed early microscopic injury associated with the presence of calcium phosphate crystals. Crystalluria was confirmed in studies designed to document the fragile and sometimes transient crystals in male rats dosed with muraglitazar. The crystal-induced epithelial injury was hypothesized as initiating the increased turnover as confirmed in BrdU-labeling experiments of the ventral bladder urothelium, a proliferative response strongly suspected in the genesis of tumor development. To determine the potential role of crystalluria in injury, and carcinogenesis, crystals were solubilized in rats through urinary acidification with 1% dietary ammonium chloride. Urinary acidification of male rats dosed with muraglitazar abrogated crystalluria, early urothelial injury, and cell proliferation (urothelial hyperplasia), and ultimately, urinary bladder carcinogenesis. This mode of action is recognized as a nongenotoxic mechanism of urinary bladder carcinogenesis in rats (Cohen, 1999).
To evaluate a potential role for pharmacology, the regulation of genes downstream of PPARa and PPARy in the rat bladder urothelium were evaluated in the presence of PPARay agonist-treated crystalluric and acidified diet, noncrystalluric rats. No changes in gene expression or traditional endpoints were observed, suggesting that PPAR-mediated changes were not directly causative in urothelial profileration or carcinogenesis (Achanzar et al., 2007).
To investigate further the mechanism of muraglitazar-induced crystalluria, urine samples were collected from treated rats for metabonomic analysis. NMR spectroscopic evaluation of urine from treated compared to control rats revealed a striking reduction in divalent acids, including citrate and 2-oxoglutarate. Subsequent analytical-grade analyses of urinary citrate to cre-atinine concentrations confirmed and extended these metabonomic findings. It was hypothesized that male-rat-specific decreased urinary excretion of divalent acids, and in particular citrate, contributed to a milieu highly permissive of calcium phosphate crystal formation.
Based on the results of studies conducted in rats, a final set of experiments examined the absolute excretion of citrate in urine from humans treated with muraglitazar. No reductions in citrate concentrations were observed across many patients compared to placebo and pretest populations. Therefore, a research strategy based on determining the role of urinary crystallogenesis in rats suggested that muraglitazar was unlikely to pose any risk to humans in inducing the early procarcinogenic change observed in rats.
The muraglitazar example demonstrates how preclinical metabonomics evaluations may identify biomarkers with potential clinical impact; however, the potential for this technology to yield specific and sensitive individual or multiple-entity biomarkers is also largely unrealized (Lindon et al., 2004; Robertson, 2005 ; Robertson et al., 2007 ).
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