Estimation of endotoxin level in nanocrystal dispersion of DK-I-56-1 intended for parenteral administration

Abstract

Estimation of endotoxin level in nanocrystal dispersion of DK-I-56-1 intended for parenteral administration Jelena Mitrović1, Tanja Ilić1, Ivan Jančić2, Biljana Bufan2, Miroslav Savić3, Snežana Savić1 1 Department of Pharmaceutical Technology and Cosmetology, University of Belgrade – Faculty of Pharmacy, Vojvode Stepe 450, 11000 Belgrade, Serbia 2 Department of Microbiology and Immunology, University of Belgrade – Faculty of Pharmacy, Vojvode Stepe 450, 11000 Belgrade, Serbia 3 Department of Pharmacology, University of Belgrade – Faculty of Pharmacy, Vojvode Stepe 450, 11000 Belgrade, Serbia Estimation of endotoxin level in parenteral formulations is a prerequisite for numerous in vitro tests in preclinical studies and for future clinical development. However, the Limulus amoebocyte lysate (LAL) test in formulations containing nanoparticles could often lead to misinterpretation of results. Therefore, we tested if endotoxins could be detected in nanocrystal dispersions by the commercial gel clot assay kit. Nanocrystals of DK-I-56-1 (7‑methoxy‑2-(4‑methoxy‑d3-phenyl)-2,5-dihydro-3H-pyrazolo[4,3-c]quinolin-3-one) were prepared by wet-ball milling, lyophilized and reconstituted with water for injection prior experiment. Different dilutions of nanocrystal dispersion in LAL reagent water were prepared as well as positive and negative control. Despite difficulties to detect gel clots, they were visible in the sample at dilutions 1:75 and below. According to the protocol, the endotoxin limit was estimated to be 25.00 EU/ml, which corresponds to <12.50 EU/mg of DK-I-56-1. This value relates to the endotoxin limit for diazepam, with the similar dosing regimen as proposed for DK-I-56-1.