Mark Pinches

Renal biomarker qualification submission: a dialog between the FDA-EMEA and Predictive Safety Testing Consortium

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 Consortiums (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.

Towards consensus practices to qualify safety biomarkers for use in early drug development

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.

Functional assessments in repeat-dose toxicity studies: the art of the possible

Clinical and nonclinical safety liabilities remain a major cause of adverse drug reactions, candidate drug attrition, delays during development, labelling restrictions, non-approval, and product withdrawal. Many of the toxicities are functional in nature and/or in origin. Whereas pharmacological responses tend to be fairly rapid in onset, and are therefore detectable after a single dose, some diminish on repeated dosing, whereas others increase in magnitude and therefore can be missed or underestimated in single-dose safety pharmacology studies. Functional measurements can be incorporated into repeat-dose toxicity studies, either routinely or on an ad hoc basis. Drivers for this are both scientific (see above), and regulatory (e.g., ICH S6, S7, S9). There are inherent challenges in achieving this: the availability of suitable technical and scientific expertise in the test facility; unsuitable laboratory conditions; use of simultaneous (as opposed to staggered) dosing; requirement for toxicokinetic sampling; unsuitability of certain techniques (e.g., use of anaesthesia; surgical implantation; food restriction); equipment availability at close proximity; sensitivity of the methods to detect small, clinically relevant, changes. Nonetheless, ‘fit-for-purpose’ data can still be acquired without requiring additional animals. Examples include assessment of behaviour, sensorimotor, visual, and autonomic functions, ambulatory ECG and blood pressure, echocardiography, respiratory, gastrointestinal, renal and hepatic functions. This is entirely achievable if functional measurements are relatively unobtrusive, both with respect to the animals and to the toxicology study itself. Careful pharmacological validation of any methods used, and establishing their detection sensitivity, is vital to ensure the credibility of generated data.

Characterization of renal biomarkers for use in clinical trials: biomarker evaluation in healthy volunteers.

Background Several preclinical urinary biomarkers have been qualified and accepted by the health authorities (US Food and Drug Administration, European Medicines Agency, and Pharmaceuticals and Medical Devices Agency) for detecting drug-induced kidney injury during preclinical toxicologic testing. Validated human assays for many of these biomarkers have become commercially available, and this study was designed to characterize some of the novel clinical renal biomarkers. The objective of this study was to evaluate clinical renal biomarkers in a typical Phase I healthy volunteer population to determine confidence intervals (pilot reference intervals), intersubject and intrasubject variability, effects of food intake, effect of sex, and vendor assay comparisons. Methods Spot urine samples from 20 male and 19 female healthy volunteers collected on multiple days were analyzed using single analyte and multiplex assays. The following analytes were measured: α-1-microglobulin, β-2-microglobulin, calbindin, clusterin, connective tissue growth factor, creatinine, cystatin C, glutathione S-transferase-α, kidney injury marker-1, microalbumin, N-acetyl-β-(D) glucosaminidase, neutrophil gelatinase-associated lipocalin, osteopontin, Tamm-Horsfall urinary glycoprotein, tissue inhibitor of metalloproteinase 1, trefoil factor 3, and vascular endothelial growth factor. Results Confidence intervals were determined from the single analyte and multiplex assays. Intersubject and intrasubject variability ranged from 38% to 299% and from 29% to 82% for biomarker concentration, and from 24% to 331% and from 10% to 67% for biomarker concentration normalized to creatinine, respectively. There was no major effect of food intake or sex. Single analyte and multiplex assays correlated with r2≥0.700 for five of six biomarkers when evaluating biomarker concentration, but for only two biomarkers when evaluating concentration normalized to creatinine. Conclusion Confidence intervals as well as intersubject and intrasubject variability were determined for novel clinical renal biomarkers/assays, which should be considered for evaluation in the next steps of the qualification process.

Tissue Expression and Correlation of a Panel of Urinary Biomarkers Following Cisplatin-induced Kidney Injury

In recent years, there has been considerable activity to identify urinary biomarkers of nephrotoxicity as noninvasive measurements with greater sensitivity and specificity than traditional biomarkers, such as serum creatinine and blood urea nitrogen. Our study aimed to use cisplatin-treated rats to evaluate the use of immunohistochemistry directed at multiple urinary biomarkers in kidney tissue. Tissue levels were compared to urinary levels of these biomarkers to demonstrate tissue specificity and sensitivity. These techniques could also be used in studies where urine samples are not available, such as retrospective studies in drug safety testing, to demonstrate the potential utility of using these biomarkers in future preclinical or clinical studies. All of the biomarkers investigated showed either an increase (kidney injury molecule [KIM-1], osteopontin [OPN], and, clusterin) or a decrease (alpha-glutathione S-transferase and trefoil factor 3) except beta 2 microglobulin (β2MG) that showed no significant changes 5 days after 1.0 mg/kg or 2.5 mg/kg cisplatin treatment. All of the biomarkers except β2MG showed utility as tissue biomarkers, but KIM-1 and OPN expression correlated closely with urinary biomarker measurements and reflect tissue damage. Future studies are needed to determine the wider application of these two markers for detecting renal toxicity following administration of other nephrotoxicants.

Characterization of renal biomarkers for use in clinical trials: effect of preanalytical processing and qualification using samples from subjects with diabetes

Background Identifying the potential for drug-induced kidney injury is essential for the successful research and development of new drugs. Newer and more sensitive preclinical drug-induced kidney injury biomarkers are now qualified for use in rat toxicology studies, but biomarkers for clinical studies are still undergoing qualification. The current studies investigated biomarkers in healthy volunteer (HV) urine samples with and without the addition of stabilizer as well as in urine from patients with normoalbuminuric diabetes mellitus (P-DM). Methods Urine samples from 20 male HV with stabilizer, 69 male HV without stabilizer, and 95 male DM without stabilizer (39 type 1 and 56 type 2) were analyzed for the following bio-markers using multiplex assays: α-1-microglobulin (A1M), β-2-microglobulin, calbindin, clus-terin, connective tissue growth factor (CTGF), creatinine, cystatin-C, glutathione S-transferase α (GSTα), kidney injury marker-1 (KIM-1), microalbumin, neutrophil gelatinase-associated lipocalin, osteopontin, Tamm–Horsfall urinary glycoprotein (THP), tissue inhibitor of metalloproteinase 1, trefoil factor 3 (TFF3), and vascular endothelial growth factor. Results CTGF and GSTα assays on nonstabilized urine were deemed nonoptimal (>50% of values below assay lower limits of quantification). “Expected values” were determined for HV with stabilizer, HV without stabilizer, and P-DM without stabilizer. There was a statistically significant difference between HV with stabilizer compared to HV without stabilizer for A1M, CTGF, GSTα, and THP. DM urine samples differed from HV (without stabilizer) for A1M CTGF, GSTα, KIM-1, microalbumin, osteopontin, and TFF3. A1M also correctly identified HV and DM with an accuracy of 89.0%. Summary These studies: 1) determined that nonstabilized urine can be used for assays under qualification; and 2) documented that A1M, CTGF, GSTα, KIM-1, microalbumin, osteopontin, and TFF3 were significantly increased in urine from P-DM. In addition, the 89.0% accuracy of A1M in distinguishing P-DM from HV may allow this biomarker to be used to monitor efficacy of potential renal protective agents.

Letter on Behalf of the European College of Veterinary Clinical Pathology in Response to the Article “International Recommendations for Training Future Toxicologic Pathologists Participating in Regulatory-Type, Nonclinical Toxicity Studies”

We are a small but dedicated group of certified veterinary clinical pathologists. We are familiar with the practice of toxicologic clinical pathology in the industry in North America and Europe. We have read and discussed the manuscript titled ‘‘International Recommendations for Training Future Toxicologic Pathologists Participating in Regulatory-Type, Nonclinical Toxicity Studies.’’ On behalf of the European College of Veterinary Clinical Pathology, a European Board for Veterinary Specialization (EBVS)–recognized specialty college for clinical pathology in Europe, we would like to present our unanimous considered opinion on the two main issues that are of concern to us in your article.