The first formal qualification of safety biomarkers for regulatory decision making marks a milestone in the application of biomarkers to drug development. Following submission of drug toxicity studies and analyses of biomarker performance to the Food and Drug Administration (FDA) and European Medicines Agency (EMEA) by the Predictive Safety Testing Consortium's (PSTC) Nephrotoxicity Working Group, seven renal safety biomarkers have been qualified for limited use in nonclinical and clinical drug development to help guide safety assessments. This was a pilot process, and the experience gained will both facilitate better understanding of how the qualification process will probably evolve and clarify the minimal requirements necessary to evaluate the performance of biomarkers of organ injury within specific contexts.

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The proactive approach we advocate has the potential to benefit patients and health care providers by providing more comprehensive safety information at the time of new product marketing and beyond.

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This study reports the evaluation of four urinary biomarkers of renal toxicity, α-glutathione-S-transferase (α-GST), μ-GST, clusterin, and renal papillary antigen-1 (RPA-1), in male Sprague-Dawley and Han-Wistar rats given cisplatin, gentamicin, or N-phenylanthranilic acid (NPAA). Kidney injury was diagnosed histopathologically, according to site/nature of renal injury, and graded for severity. The area under the receiver operating characteristic (ROC) curve was used to compare the diagnostic accuracy of each exploratory renal biomarker with traditional indicators of renal function and injury (blood urea nitrogen [BUN], serum creatinine [sCr] as well as urinary N-acetyl-β-D-glucosaminidase [NAG] and protein). These analyses showed that increased urinary α-GST was superior to BUN, sCr, and NAG for diagnosis of proximal tubular (PT) degeneration/necrosis. Paradoxically, urinary α-GST was decreased in the presence of collecting duct (CD) injury without PT injury (NPAA administration). RPA-1 demonstrated high specificity for CD injury, superior to all of the reference biomarkers. The clusterin response correlated well with tubular injury, whatever the location, particularly when regeneration was present (superior to all of the reference markers for cortical tubular regeneration). There was no conclusive evidence for the diagnostic utility of μ-GST. The data were submitted for qualification review by the European Medicines Agency and the US Food and Drug Administration. Both agencies concluded that the data justified the qualification of RPA-1 and increased the level of evidence for, and clarified the context of use of, the previously qualified clusterin for use in male rats. These biomarkers can be used in conjunction with traditional clinical chemistry markers and histopathology in Good Laboratory Practice rodent toxicology studies used to support renal safety studies in clinical trials. Qualification of α-GST must await further explanation of the differences in response to PT and CD injury.

Groups of 10 male and 10 female Sprague-Dawley rats were given a single intravenous injection of gadolinium chloride solution at dosages of 0 (saline vehicle), 0.07, 0.14, and 0.35 mmol/kg. Apart from 1 top-dose female, which died during dosing, 5 rats/sex/ group were necropsied 48 hr postdose, and the remaining 5 rats/sex/group were necropsied 14 days postdose. Macroscopic, hematological, and clinical chemistry analyses were undertaken on all animals that were necropsied. Histopathological examination was undertaken on all organs from high-dose and control animals necropsied 48 hr postdose and on tissues that showed treatment-related changes from all other rats necropsied either 48 hr or 14 days postdose. Major lesions related to gadolinium chloride administration consisted of mineral deposition in capillary beds (particularly lung and kidney), phagocytosis of mineral by the mononuclear phagocytic system, hepatocellular and splenic necrosis followed by dystrophic mineralization, mineralization of the fundic glandular mucosa in the absence of necrosis followed by mucous cell hyperplasia, decreased platelet numbers and increased prothrombin time, and activated partial thromboplastin time. Electron microscopy and x-ray microanalysis of the spleen and liver revealed electron-dense deposits in splenic macrophages, Kupffer cells, and hepatocytes composed of gadolinium, calcium, and phosphate.

Application of any new biomarker to support safety-related decisions during regulated phases of drug development requires provision of a substantial data set that critically assesses analytical and biological performance of that biomarker. Such an approach enables stakeholders from industry and regulatory bodies to objectively evaluate whether superior standards of performance have been met and whether specific claims of fit-for-purpose use are supported. It is therefore important during the biomarker evaluation process that stakeholders seek agreement on which critical experiments are needed to test that a biomarker meets specific performance claims, how new biomarker and traditional comparators will be measured and how the resulting data will be merged, analyzed and interpreted.

Cisplatin is an anticancer agent that induces renal proximal tubule lesions in many species. Studies were conducted in Sprague-Dawley and Han-Wistar rats to evaluate the utility of novel preclinical biomarkers of nephrotoxicity for renal lesions caused by this compound. Groups of 10 males of each strain were given a single intraperitoneal injection of 0.3, 1, or 3 mg/kg cisplatin and were sacrificed on days 2, 3, and 5. The novel biomarkers a-glutathione-S-transferase (a-GST) (for proximal tubular injury), m-glutathione-S-transferase (m-GST) (for distal tubular injury), clusterin (for general kidney injury), and renal papillary antigen-1 (RPA-1) (for collecting duct injury) were measured in urine by enzyme immunoassay. Histologically, degeneration and necrosis of the S3 segment of the renal proximal tubule were observed on day 2 (Han-Wistar) and days 3 and 5 (both strains) at 1 and 3 mg/kg. Results showed that in both strains of rats, urinary a-GST and clusterin can be detected in urine soon after injury, are more sensitive than BUN and serum creatinine, and therefore are usable as noninvasive biomarkers of proximal tubule injury. Changes in both m-GST or RPA-1 were considered to represent secondary minor effects of proximal tubular injury on distal segments of the nephron.