August 1, 2014 (Vol. 34, No. 14)
Robert Durham, Ph.D. Director of Field Application Scientists Gyros U.S.
Wei Zheng, Ph.D. EMD Millipore
Nanoliter-Scale Immunoassays Can Be Used to Measure KIM-1 and Clusterin
The top reason for drug candidate attrition is not poor efficacy as one might expect but rather unacceptable toxicity, highlighting the need to identify drug-induced toxicity in the early stages of drug development. The challenge is to identify and validate sensitive and specific biomarkers that can detect drug-induced organ damage early in exposure and also fulfill the requirements of regulatory authorities.
In the pharmaceutical and chemical industry, the kidney is routinely assessed during preclinical safety evaluations. The role of the kidney as a central detoxification organ leads to a high exposure of renal tissue to drugs, reactive metabolites, or environmental compounds. Traditional markers for assessing renal toxicity, such as blood urea nitrogen (BUN) and serum creatinine (SCr), have low sensitivity. Although both are direct measurements of renal function, serum concentrations of these biomarkers increase only after substantial renal injury, demonstrating a clear need for improved biomarker assays to detect damage earlier.
The Predictive Safety Testing Consortium (PSTC) was formed in 2006 to support the development of new biomarkers. PSTC encourages pharmaceutical companies to share and validate innovative safety testing methods that can be submitted for formal regulatory qualification by the FDA (Food and Drug Administration), the EMA (European Medicines Agency), and the PMDA (Japanese Pharmaceutical and Medical Devices Agency). This is a major step forward to establishing improved cross-industry methods that are applicable throughout the drug development spectrum and can be independently validated and accepted as proof of safety by regulatory authorities.
KIM-1 and Clusterin Biomarkers
The first project of the PSTC identified eight effective rat urinary biomarkers, including KIM-1 (Kidney Injury Molecule 1) and clusterin, that were endorsed by FDA and EMA for the detection of acute kidney toxicity during nonclinical drug development. Rat urinary KIM-1 and clusterin can be detected by ELISA, but this method has a number of disadvantages, including narrow dynamic range, large sample consumption (particularly problematic in preclinical work with small laboratory animals), and long assay times and hands-on time, especially when a large number of samples need to be analyzed.
EMD Millipore has overcome these weaknesses with new kits for rat urinary KIM-1 and clusterin based on a nanoliter-scale immunoassay platform developed by Gyros AB. This combination of fit-for-purpose assay kits and an automated high-throughput immunoassay platform using small sample volumes offers the potential for a sensitive robust method with significant advantages.
Gyrolab™ xP workstation performs automated immunoassays within nanoliter-scale microfluidic structures in a compact disk (CD) format. Each structure on the Bioaffy CD comprises a 15-nanoliter affinity column prepacked with Streptavidin-coated particles, supporting a variety of assay types including sandwich and indirect antibody assays.
Working at the nanoliter scale means that the consumption of sample and reagents is dramatically reduced compared with plate-based ELISA. Apart from obvious cost-saving advantages of reducing use of expensive reagents, the availability of sample, often taken from small laboratory animals, can become a limiting factor with ELISA, especially as regulatory demands drive the need to perform an increasing number of analyses on each sample.
The microfluidics ensure controlled flow of capture reagents, sample, and detection reagents, so that all samples within a single CD are processed in parallel. Parallel processing eliminates time-dependent artifacts that may arise in a typical plate-based ELISA and ensures that samples are processed under uniform conditions. Each individual microstructure equates to one data point, thus eliminating cross talk. The control and analysis software is 21 CFR part 11 compliant and gives flexibility in run setup and data analysis, ensuring that assays can be developed and transferred through to GMP and GLP environments.
Development of Fit-for-Purpose Assays
Using Gyrolab xP workstation, EMD Millipore developed and validated fit-for-purpose GyroMark™ HT Kits for rat urinary KIM-1 and clusterin that had the following properties:
• High linearity over a broad dynamic range (Figure 1)
• High sensitivity with LLOQ down to 0.02 ng/mL
• Target detection of spiked control in rat urine samples = 80–120%
• Minimal matrix effects, with minimum required dilutions as low as 1:2
• Dilutional linearity accuracy well within acceptance criteria over a broad dilution range
• Inter- and intra-assay precision in the 3.6–8.6% range
The results confirmed that the combination could indeed be suitable for studying drug-induced nephrotoxicity. The results from GyroMark HT kits also correlated very well with those from an alternative method—MILLIPLEX® MAP multiplex assays based on Luminex® technology.
Case Study: In Vivo Evaluation of Sub-acute Nephrotoxicity Biomarkers
Researchers at Merck Serono decided to determine if rat urinary KIM-1 and clusterin could be used to detect sub-acute drug-induced kidney damage. They measured the urinary protein biomarkers using GyroMark HT kits run on Gyrolab xP workstation and performed immunohistochemistry (IHC) in a 28-day rat study using vancomycin as the toxicant. The study design involved 120 animals and 120 samples, run in duplicate or triplicate, thus generating hundreds of data points. Using Gyrolab xP workstation they were able to analyze all samples in one day.
The baseline study involved histopathology studies and IHC staining for KIM-1 on all animals to correlate and confirm kidney damage. Levels of the traditional biomarkers BUN and SCr also increased significantly after eight days at the high vancomycin dose, including a dip in the recovery phase (Figure 2). No changes were seen in low-dose animals. The results from assays using GyroMark HT KIM-1 and clusterin kits correlated well with levels of the traditional biomarkers for both male and female (results not shown) animals, with significant changes being detected after only four days of exposure.
Receiver-operating characteristic (ROC) analysis of urinary proteins confirmed that KIM-1 and clusterin indeed have great diagnostic value. The area under the curve (AUC) was used as a measure of the overall ability of the biomarker to discriminate animals without histopathological findings in kidneys from those with signs of tubular damage. KIM-1 had an AUC of 0.90–0.95 and clusterin 0.94–0.98 for males and females, respectively.
The study confirmed the high accuracy and predictivity of rat urinary KIM-1 and clusterin as markers to detect sub-acute nephrotoxicity when treated with vancomycin. The combination of GyroMark HT kits and Gyrolab xP workstation generated reliable and sensitive measurements of the markers from a large number of 1 μL samples per day, thus promising to be a useful tool for drug development and basic research when studying kidney damage in rat models.
Robert Durham, Ph.D. ([email protected]), is director of field application scientists, Gyros U.S. Wei Zheng, Ph.D. ([email protected]), is head of R&D and licensing, research content and reagents, bioscience, EMD Millipore.