Micro-volume assays for development and application of biochemical biomarker clusters in preclinical trials.

SANTIAGO, G.M. ; CABAÑA, E.R.; MARELLI, B.E.; HEIN, G.J.; ORTEGA, H.H.

Salta

Workshop; 1ST WORKSHOP ON DRUG DISCOVERY; 2019

LV Annual SAIB Meeting and XIV PABMB Congress.

The evaluation of new pharmaceutical products requires data about preclinical tests to ensure that the benefits will exceed any potential risk. Recently, clinical biomarkers are part of the diagnosis and monitoring of different diseases in experimental models and concept proof. In this sense, a biomarker is defined as a substance made by a specific tissue that can be detected in the circulation or tissues. To be useful from the clinical approach, it must be released in an amount proportional to the evolution of the specific pathological process and provide information on the presence, severity and prognosis of the injury. Biochemical biomarkers have been used around for a long time to elucidate the physiologic, toxicologic, pharmacologic, or significance results to fill in gaps of uncertainty about disease targets or variability in drug response. However, the few volumes of biological samples from small laboratory animals (rats and mice) limit the possibility of carrying out commercial methods. Therefore, we aimed to develop and validate an analytical platform for micro-volume assays (less than 50 μL of total serum) to design biomarker packages useful in preclinical trials. All analytical techniques were developed under an ISO 9001 certified Quality Management System in accordance with the principles of Good Laboratory Practices (BPL-OECD recognized facility). Mice, rats and rabbits were sampled and serum concentrations of glucose (Glc), triglycerides (Tg), cholesterol (Col), high (HDL) and low density lipoprotein (LDL), total protein (TP), albumin (Alb), uric acid (UA), and urea (U) were quantified using the ultra-fast UV/Vis spectrometers SPECTROstar Nano and CLARIOstar (BMG LABTECH GmbH, Germany) and commercial kits (Wiener Labs, Argentina) designed for higher volumes of human samples. The figures of merit were: limit of detection, limit of quantification, quantifiable range, intra and inter-precision test, accuracy, inter-laboratories comparison, matrix effect, dilutional linearity and stability at -20°C and -80°C for 60 days. All these variables were analyzed and validated in accordance with EMA Guideline on bioanalytical method validation and FDA Guidance for Industry: Bioanalytical Method Validation. The results obtained with these validated micro-volume analytical techniques could contribute to the development of preclinical trials in small species, reducing the amount of necessary testing substance and adding complexity to the studies carried out. This development also establishes a scientific framework that contribute to elucidate the pathophysiological changes in different animal models for the study of subclinical changes occurred in certain organs, such as liver, pancreas, kidney and heart not detectable by other techniques such as pathology. Furthermore, the reductions of sample and reagent volumes agree with the 3R´s principle and sustainability.