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

SANTIAGO, G; CABAÑA, E; MARELLI B; HEIN G; ORTEGA HH

Salta

Congreso; LV Annual SAIB Meeting and XIV PABMB; 2019

SAIB

The evaluation of new pharmaceutical productsrequires data about preclinical tests to ensure that the benefits will exceedany potential risk. Recently, clinical biomarkers are part of the diagnosis andmonitoring of different diseases in experimental models and concept proof. Inthis sense, a biomarker is defined as a substance made by a specific tissuethat can be detected in the circulation or tissues. To be useful from theclinical approach, it must be released in an amount proportional to theevolution of the specific pathological process and provide information on thepresence, severity and prognosis of the injury. Biochemical biomarkers havebeen used around for a long time to elucidate the physiologic, toxicologic,pharmacologic, or significance results to fill in gaps of uncertainty aboutdisease targets or variability in drug response. However, the few volumes ofbiological samples from small laboratory animals (rats and mice) limits thepossibility of carrying out commercial methods. Therefore, we aimed to developand validate an analytical platform for micro-volume assays (less than 50 µL oftotal serum) to design biomarker packages useful in preclinical trials. Allanalytical techniques were developed under an ISO 9001 certified QualityManagement System in accordance with the principles of Good LaboratoryPractices (BPL-OECD recognized facility). Mice, rats and rabbits were sampled andserum 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/Visspectrometers SPECTROstar Nano and CLARIOstar (BMG LABTECH GmbH, Germany) andcommercial kits (Wiener Labs, Argentina) designed for higher volumes of humansamples. The figures of merit were: limit of detection, limit ofquantification, quantifiable range, intra and inter-precision test, accuracy,inter-laboratories comparison, matrix effect, dilutional linearity andstability at -20°C and -80°C for 60 days. All these variables were analyzed andvalidated in accordance with EMA Guideline on bioanalytical method validationand FDA Guidance for Industry: Bioanalytical Method Validation. The resultsobtained with these validated micro-volume analytical techniques could contributeto the development of preclinical trials in small species, reducing the amountof necessary testing substance and adding complexity to the studies carriedout. This development also establishes a scientific framework that contribute toelucidate the pathophysiological changes in different animal models for thestudy 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´sprinciple and sustainability