Josef S. Ozer

Kidney injury molecule-1 outperforms traditional biomarkers of kidney injury in preclinical biomarker qualification studies

Kidney toxicity accounts both for the failure of many drug candidates as well as considerable patient morbidity. Whereas histopathology remains the gold standard for nephrotoxicity in animal systems, serum creatinine (SCr) and blood urea nitrogen (BUN) are the primary options for monitoring kidney dysfunction in humans. The transmembrane tubular protein kidney injury molecule-1 (Kim-1) was previously reported to be markedly induced in response to renal injury. Owing to the poor sensitivity and specificity of SCr and BUN, we used rat toxicology studies to compare the diagnostic performance of urinary Kim-1 to BUN, SCr and urinary N-acetyl-β-D-glucosaminidase (NAG) as predictors of kidney tubular damage scored by histopathology. Kim-1 outperforms SCr, BUN and urinary NAG in multiple rat models of kidney injury. Urinary Kim-1 measurements may facilitate sensitive, specific and accurate prediction of human nephrotoxicity in preclinical drug screens. This should enable early identification and elimination of compounds that are potentially nephrotoxic.

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.

A panel of urinary biomarkers to monitor reversibility of renal injury and a serum marker with improved potential to assess renal function

The Predictive Safety Testing Consortiums first regulatory submission to qualify kidney safety biomarkers revealed two deficiencies. To address the need for biomarkers that monitor recovery from agent-induced renal damage, we scored changes in the levels of urinary biomarkers in rats during recovery from renal injury induced by exposure to carbapenem A or gentamicin. All biomarkers responded to histologic tubular toxicities to varied degrees and with different kinetics. After a recovery period, all biomarkers returned to levels approaching those observed in uninjured animals. We next addressed the need for a serum biomarker that reflects general kidney function regardless of the exact site of renal injury. Our assay for serum cystatin C is more sensitive and specific than serum creatinine (SCr) or blood urea nitrogen (BUN) in monitoring generalized renal function after exposure of rats to eight nephrotoxicants and two hepatotoxicants. This sensitive serum biomarker will enable testing of renal function in animal studies that do not involve urine collection.

Urinary biomarkers trefoil factor 3 and albumin enable early detection of kidney tubular injury

The capacities of urinary trefoil factor 3 (TFF3) and urinary albumin to detect acute renal tubular injury have never been evaluated with sufficient statistical rigor to permit their use in regulated drug development instead of the current preclinical biomarkers serum creatinine (SCr) and blood urea nitrogen (BUN). Working with rats, we found that urinary TFF3 protein levels were markedly reduced, and urinary albumin were markedly increased in response to renal tubular injury. Urinary TFF3 levels did not respond to nonrenal toxicants, and urinary albumin faithfully reflected alterations in renal function. In situ hybridization localized TFF3 expression in tubules of the outer stripe of the outer medulla. Albumin outperformed either SCr or BUN for detecting kidney tubule injury and TFF3 augmented the potential of BUN and SCr to detect kidney damage. Use of urinary TFF3 and albumin will enable more sensitive and robust diagnosis of acute renal tubular injury than traditional biomarkers.

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.

Enhancing the utility of alanine aminotransferase as a reference standard biomarker for drug-induced liver injury

Drug-induced liver injury (DILI) is the most frequent cause of discontinuation of new chemical entities during development. DILI can either be intrinsic/predictable or an idiosyncratic type. These two forms of DILI are contrasted in their manifestation and diagnosis. Even with regulatory guidance (FDA, 2009), there is still a gap in our ability to identify predictable DILI, both specifically and sensitively. Alanine aminotransferase (ALT) is the principal reference standard biomarker to diagnose DILI, yet its current application in preclinical to clinical translation for decision-making purposes has imperfections: (1) analytical ALT assay uniformity across industry would be aided by common analytical processes; (2) assessment of ALT toxicological performance in a large preclinical analysis would help to establish a true threshold of elevation for predictable DILI and improve translational use across various stages of pharmaceutical development and finally, (3) clinical evaluation of ALT elevations prospectively and retrospectively is recommended to define and manage variations in clinical study subjects including rising body mass index (BMI) range and ALT upper limit of normal (ULN) in the broader population over time. The emergence of new hepatotoxicity biomarkers necessitates a parallel and equivalent assessment to the aminotransferases in a regulatory qualification model.

Endogenous ethanolamide analysis in human plasma using HPLC tandem MS with electrospray ionization

A sensitive and selective liquid chromatography tandem mass spectrometry (LC\MS\MS) method has been developed for the simultaneous quantification in human plasma of the endocannabinoid anandamide (AEA) and three other related ethanolamides, linoleoyl ethanolamide (LEA), oleoyl ethanolamide (OEA), and palmitoyl ethanolamide (PEA). The analytical methodology requires 50 microL of human plasma which is processed via protein precipitation using a 96-well protein precipitation plate. Chromatographic separation of plasma extract was achieved with a Phenomenex Gemini C6-Phenyl HPLC column (2.1 mm x 50 mm, 5 microm) at a flow rate of 0.30 mL/min using gradient elution and a mobile phase consisting of acetonitrile and 5 mM ammonium formate. All four fatty acid ethanolamides were quantified by positive ion electrospray ionization tandem mass spectrometry, with the detection of ion current signal generated from the selected reaction monitoring (SRM) transition of [M+H](+)-->m/z 62. Deuterated anandamide (AEA-d8) was used as an internal standard for all four ethanolamides. The lower limit of quantitation was 0.05 ng/mL for AEA and LEA, 0.5 ng/mL for OEA and 1.0 ng/mL for PEA. Inter-assay precision and accuracy were typically within 12% for the four endogenous analytes and overall extraction recoveries ranged between 40% and 100%.

Liquid chromatography/tandem mass spectrometry based targeted proteomics quantification of P‐glycoprotein in various biological samples

P-Glycoprotein (P-gp/ABCB1) is expressed in membrane barriers to exclude pharmacological substrates from cells, and therefore influences the ADME/Tox properties and efficacy of therapeutics. In the present study, a liquid chromatography/tandem mass spectrometry (LC/MS/MS)-mediated targeted proteomics was developed to quantitate P-gp protein. With the aid of in silico predictive tools, a unique 9-mer tryptic peptide of P-gp protein was synthesized (with the stable isotope labeled (SIL) peptide as internal standard) and applied for quantitative LC/MS/MS method development. For LC/MS/MS quantification, the N-glycosylation of the peptide, polymorphism and transmembrane region was intended to be excluded during the peptide selection. The lower limit of quantification was established to be 0.025 nM with the linearity of the standard curve ranging to 20 nM of P-gp signature peptides in the matrix digested surrogate bovine serum albumin. The digestion efficiency, both the accuracy (relative error) and the precision (coefficient of variation) of the method, was verified by using the synthetic quantification peptide and the synthetic surrogate substrate peptide that mimics the sequence of tryptic peptide and associated flanking tryptic cleavage sites at the N- and C-terminals. By applying the method developed, the absolute amounts of human, dog and mouse P-gp (Mdr1a) were quantified in various biological samples. LC/MS/MS-mediated P-gp quantification was achieved as a highly sensitive, selective and reproducible assay and could be directly applicable to many current research needs related to P-gp.

Recommendations to qualify biomarker candidates of drug-induced liver injury

Certain compounds that induce liver injury clinically are not readily identified from earlier preclinical studies. Novel biomarkers are being sought to be applied across the pharmaceutical pipeline to fill this knowledge gap and to add increased specificity for detecting drug-induced liver injury in combination with aminotransferases (alanine and aspartate aminotransferase)--the current reference-standard biomarkers used in the clinic. The gaps in the qualification process for novel biomarkers of regulatory decision-making are assessed and compared with aminotransferase activities to guide the determination of safe compound margins for drug delivery to humans where monitoring for potential liver injury is a cause for concern. Histopathologic observations from preclinical studies are considered the principal reference standard to benchmark and assess subtle aminotransferase elevations. This approach correlates quite well for many developmental compounds, yet cases of discordance create dilemmas regarding which standard(s) indicates true injury. Concordance amongst a broader set of biomarker injury signals in a qualification paradigm will increase confidence, leading to accepted and integrated translational biomarker signals during safety assessment processes across the pharmaceutical industry, with academia, in government and in contractor laboratories.

A systematic approach to preclinical and clinical safety biomarker qualification incorporating Bradford Hill's principles of causality association.

The recently issued FDA/EMEA (US Food and Drug Administration/European Medicines Agency) guidelines pertaining to qualification of biomarkers are an important development in the regulatory approval process for new drug applications. 1, 2 A qualified biomarker approved by regulatory authorities allows sponsors across industry to use that marker in new drug applications on the basis of a single submission rather than requiring each sponsor to present a separate justification with each new drug application. Nevertheless, there are a number of unanswered questions regarding the specific criteria that such a biomarker should meet in order to be considered as adding value to historical clinical end points and preclinical pathology observations. There are also questions regarding the optimal methods of collecting and evaluating scientific evidence for the clinical qualification of a biomarker. We propose a novel application to assist and accelerate the drug development process by prioritizing biomarker candidates and evidence, an application based on Bradford Hills principles of causality association.