Francois Legay

Urinary clusterin, cystatin C, β2-microglobulin and total protein as markers to detect drug-induced kidney injury

Earlier and more reliable detection of drug-induced kidney injury would improve clinical care and help to streamline drug-development. As the current standards to monitor renal function, such as blood urea nitrogen (BUN) or serum creatinine (SCr), are late indicators of kidney injury, we conducted ten nonclinical studies to rigorously assess the potential of four previously described nephrotoxicity markers to detect drug-induced kidney and liver injury. Whereas urinary clusterin outperformed BUN and SCr for detecting proximal tubular injury, urinary total protein, cystatin C and β2-microglobulin showed a better diagnostic performance than BUN and SCr for detecting glomerular injury. Gene and protein expression analysis, in-situ hybridization and immunohistochemistry provide mechanistic evidence to support the use of these four markers for detecting kidney injury to guide regulatory decision making in drug development. The recognition of the qualification of these biomarkers by the EMEA and FDA will significantly enhance renal safety monitoring.

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.

Validation of immunoassay for protein biomarkers: Bioanalytical study plan implementation to support pre-clinical and clinical studies

Biomarkers have emerged as an important tool to optimize the benefit/risk ratio of therapeutics. The scientific impact of biomarker studies is directly related to the quality of the underlying data. It is therefore important that guidance be established for validation of assays used to support drug development. This paper specifically focuses on validation of immunoassay for protein biomarker to support pre-clinical and clinical studies. Therapeutics (small- and macro-molecules) and their respective target/ligand are out of scope. This paper describes the implementation of a bioanalytical study plan for the validation of immunoassays to support decision-making biomarkers and biomarker selection during preclinical and clinical studies. It establishes the complete operating procedure as well as the parameters and their respective acceptance criteria and defines milestones and decision points to be followed during the assay validation that should result in high quality bioanalytical data in a limited timeframe and with reduced costs. The bioanalytical study plan can be applied to the validation of a wild range of immunoassay technology such as monoplex ELISA, automated analyzer, multiplex assays or cutting edge technology. Before any validation, a feasibility study is performed to assess the performance of the immunoassay using biological samples which should mimic the clinical population. The feasibility study addresses the likelihood that an assay will be able to achieve its intended purpose with parallelism being the most critical element (milestone 1). At the end of the feasibility study, a decision is taken to either continue with the validation or change the assay (milestone 2). The milestone 3 consists of the establishment of the nominal value of quality control to be used during the validation. The quality controls used to validate an assay should preferentially be prepared using neat (non-spiked) biological matrix (ideally derived from the specific trial population). The last milestone (milestone 4), the formal validation, includes demonstration of the assay performance meeting accuracy and precision acceptance criteria within (intra-run) and between (inter-run) validation runs for each QC sample. Validation also includes the assessment of stability of the protein biomarker in the biological matrix. It is recognized that the extent of the validation should be correlated to the intended use of the data and the assay acceptance criteria should take into consideration the study objective(s), nature of the methodology and the biological variability of the biomarker.

Binding of artemether and lumefantrine to plasma proteins and erythrocytes.

The serum/plasma protein binding and blood distribution of artemether and lumefantrine was studied in vitro. The techniques used were the erythrocyte partitioning and ultrafiltration methods with 1499%, respectively. Under physiological protein concentrations, the distribution in blood showed that 33% of artemether was bound to alpha(1)-acid glycoprotein, 17% to albumin, 12% to high density lipoproteins (HDL), 9.3% to low density lipoproteins (LDL) and 12% to very low density lipoproteins (VLDL), with binding capacities (nKa) of 3.2 x 10(5), 6.2 x 10(3), 2.1 x 10(5), 1.7 x 10(6) and 2.0 x 10(7) lmol(-1), respectively. 77% of lumefantrine was bound to HDL, 7.3% to LDL and 6.6% to VLDL, with binding capacities of 2.7 x 10(7), 2. 6 x 10(7) and 2.4 x 10(8) lmol(-1), respectively. A negligible fraction of lumefantrine was bound to albumin and alpha(1)-acid glycoprotein. The fraction in erythrocytes was around 10% for both artemether and lumefantrine.

Improvement of drug tolerance in immunogenicity testing by acid treatment on Biacore

Detection of anti-drug antibodies (ADA) can be difficult, if not impossible, in the presence of drug in the sample. This is a particular concern with therapeutic monoclonal antibodies (mAbs), which have typically longer half-lives than other proteins. For detection of ADA in presence of high drug concentrations, assay choice is limited to ELISA-like methods, capable of incorporating acid dissociation procedures to separate drug-ADA immune complexes. To our knowledge, Biacore assays have not been shown to be directly compatible with acid dissociation procedures, until now. As a consequence, steps to ensure adequate clearance of the drug are prerequisite to enable sensitive detection of ADA. Here we describe the development of a novel, rapid and highly drug tolerant Biacore method that uses an acid dissociation step to detect ADA in the presence of excess drug in human serum. Removal of drug after acid treatment is not required.

Development and validation of a highly sensitive RIA for zoledronic acid, a new potent heterocyclic bisphosphonate, in human serum, plasma and urine.

Zoledronic acid is a new, highly potent bisphosphonate drug under clinical evaluation. A radioimmunoassay has been developed to determine zoledronic acid concentration in human serum, plasma, and urine. The assay utilizes rabbit polyclonal antisera against a zoledronic acid-BSA conjugate and a [125I]zoledronic acid derivative as tracer in a competitive format adapted to microtiter plates. The assay shows a LLOQ 0.4 ng/ml in serum or plasma (interassay%CV=17%, accuracy 97%), 5 ng/ml in urine (21%, 98%). In 23 patients receiving 4, 8 or 16 mg of zoledronic acid, drug concentrations in plasma were dose proportional and showed a multiphasic profile, followed by a prolonged gradual decline to concentrations near the LLOQ. Zoledronic acid disposition in plasma and the recovery of only 40-50% of the dose in urine are consistent with the rapid and extensive uptake by and slow release from bone in parallel with renal clearance, typically shown by bisphosphonates.

Development of protein microarray technology to monitor biomarkers of rheumatoid arthritis disease

Most biological processes are mediated by complex networks of molecular interactions involving proteins. The analysis of protein expression in biological samples is especially important in the identification and monitoring of biomarkers for disease progression and therapeutic endpoints. In this paper, the development of a protein microarray format for multiplexed quantitative analysis of several potential markers for rheumatoid arthritis (RA) is described. Development of a high-performance protein microarray system depends on several key parameters such as surface chemistry, capture agents, immobilization technology, and methods used for signal detection and quantification. Several technical possibilities were investigated and compared: poly-L-lysine versus self-assembled monolayer of octadecyl phosphoric acid ester for surface chemistries; noncontact piezoelectric versus contact printing technology for antibody deposition; CCD camera capture versus fluorescent scanning for image detection; and the concentration of coating antibody. On the basis of reproducibility, signal-to-noise ratio, and sensitivity we have selected self-assembled monolayer, noncontact piezoelectric printer, and high-read-out fluorescence scanning for our microarray format. This format was used to perform multiplexed quantitative analysis of several potential markers of disease progression of rheumatoid arthritis: IL-1β, IL-6, IL-8, MCP-1, and SAA. Some assays, such as MCP-1, provided a working range that covered physiologically relevant concentrations. Other assays, such as IL-6 and SAA, lacked sensitivity or were too sensitive for measuring biological concentrations, respectively. The results described demonstrate the applicability of protein microarrays to monitor RA markers; however, sandwich assay methodologies need to be further optimized to measure the appropriate biological ranges of these markers on one chip.

Development and validation of an IL-6 immuno-receptor assay based on surface plasmon resonance

Interleukin-6 (IL-6) is a pleiotropic cytokine which interacts with the specific IL-6 receptor at the surface of the T lymphocytes. A combined immuno- and receptor-assay has been developed and validated to characterize the biological activity of recombinant IL-6 (rIL-6). This assay is based on Surface Plasmon Resonance (SPR) technology. From each experiment two successive interactions were monitored: anti-IL-6 antibody/rIL-6 and rIL-6/IL-6 soluble Receptor (sIL-6R). Based on the first interaction an immuno-assay for rIL-6 was optimized and validated. Based on the second interaction a receptor-assay for rIL-6 biological activity was optimized and validated. The assays were validated by performing three different assays on three different days. The intra- and inter-day precisions (%CV) for the immuno-assay were respectively 0.9% and 1.7%. The overall recovery of the immuno-assay was 98.9 +/- 1.6. Intra- and inter-day precisions for the receptor-assay were respectively 1.1% and 1.4%. The overall recovery of the receptor-assay was 99.4% +/- 1.1. This immuno-receptor assay has allowed to compare the rIL-6 stability after storage at different temperatures. The results did not show significant difference between the three lower storage temperatures (-70, -20 and 5 degrees C). However, results obtained for the aliquot stored at 25 degrees C have shown a drastic denaturation of the rIL-6. These results illustrate the advantage of this method combining the evaluation of the immunological and biological integrity of the drug and high reproducibility and precision of the biosensor based technology.

Reduction of matrix interferences by the combination of chaotropic salt and DMSO in a broadly applicable target-based ELISA for pharmacokinetic studies of therapeutic monoclonal antibodies

Use of a synergistic effect of DMSO together with a chaotropic salt (NaSCN or MgCl2) allowed to drastically reduce matrix interferences in an ELISA for therapeutic monoclonal antibodies. Optimum combinations were found to be 0.4 M NaSCN together with 10.0% DMSO, and 1.0 M MgCl2 with 15.0% DMSO. At this optimum combination, quality controls spiked with mAb at 50.0 ng/ml in eighteen individual human sera and plasmas were quantified with an overall accuracy of 102.0%. All of these QCs fulfilled the acceptance criteria of 80.0-120.0% accuracy and precision below 20.0%. The assay was also successfully applied to the quantification of two other mAbs in human serum. Furthermore, the use of the assay was extended to pre-clinical species (cynomolgus monkey and rat serum). Here, the performed validation experiments confirmed the utility of the assay and demonstrated that the assay allowed quantification of mAb from 50.0 ng/ml to 100.0 microg/ml in cynomolgus monkey serum. The method has then been applied to a pharmacokinetic study in cynomolgus monkeys. In summary, this work demonstrates the efficacy of the combination of a chaotropic salt with DMSO to minimize matrix interferences in an ELISA. The robustness thus obtained allowed the successful establishment of a cost effective, target-based ELISA format for use in pharmacokinetic studies, that is easily applicable for the quantification of mAbs in various matrices such as human, cynomolgus monkey or rat serum and plasma.

Expression and Characterization of Recombinant, Tetrameric and Enzymatically Active Influenza Neuraminidase for the Setup of an Enzyme-Linked Lectin-Based Assay

Developing a universal influenza vaccine that induces broad spectrum and longer-term immunity has become an important potentially achievable target in influenza vaccine research and development. Hemagglutinin (HA) and neuraminidase (NA) are the two major influenza virus antigens. Although antibody responses against influenza virus are mainly directed toward HA, NA is reported to be more genetically stable; hence NA-based vaccines have the potential to be effective for longer time periods. NA-specific immunity has been shown to limit the spread of influenza virus, thus reducing disease symptoms and providing cross-protection against heterosubtypic viruses in mouse challenge experiments. The production of large quantities of highly pure and stable NA could be beneficial for the development of new antivirals, subunit-based vaccines, and novel diagnostic tools. In this study, recombinant NA (rNA) was produced in mammalian cells at high levels from both swine A/California/07/2009 (H1N1) and avian A/turkey/Turkey/01/2005 (H5N1) influenza viruses. Biochemical, structural, and immunological characterizations revealed that the soluble rNAs produced are tetrameric, enzymatically active and immunogenic, and finally they represent good alternatives to conventionally used sources of NA in the Enzyme-Linked Lectin Assay (ELLA).