Peripheral blood mononuclear cells isolation from mouse and rabbit blood to quantify active nucleotide and define brincidofovir dose for smallpox
Creators: name: Hart, Robert
name: Karnik, Shane
name: Van Sickle, Kathy
name: Mullin, Mark
name: Naderer, Odin
name: Tippin, Tim
name: Dunn, John
Publisher: name: Taylor & Francis
Year: 2024
Subjects: subject: Biochemistry
subject: Medicine
subject: Microbiology
subject: FOS: Biological sciences
subject: Cell Biology
subject: Genetics
subject: Molecular Biology
subject: Physiology
subject: Pharmacology
subject: Biotechnology
subject: Environmental Sciences not elsewhere classified
subject: Chemical Sciences not elsewhere classified
subject: Immunology
subject: FOS: Clinical medicine
subject: Hematology
subject: Infectious Diseases
subject: FOS: Health sciences
subject: Virology

description: <b>Aim:</b> Brincidofovir has been approved by the US FDA for the treatment of smallpox via the “Animal Rule”. The active moiety, cidofovir diphosphate (CDV-PP), was measurable in human peripheral blood mononuclear cells (PBMCs), but quantitation in animals was challenging given their limited blood volume. The aim of this study was to optimize PBMC isolation from rabbit and mouse blood to allow quantitation of CDV-PP. <b>Materials &amp; methods:</b> PBMC isolation methods using various separation media were evaluated using blood from naive and brincidofovir-dosed animals. CDV-PP was quantified using liquid chromatography tandem mass spectrometry. <b>Results &amp; conclusion:</b> PBMC isolation yields were increased by &gt;fourfold compared with yields obtained using human methods, allowing cross-species exposure comparisons. This work describes the development of novel methods to isolate animal peripheral blood mononuclear cells (PBMCs) from limited blood volumes that enabled key pharmacokinetic comparisons of nucleotide concentrations between animal models of smallpox and humans following administration of brincidofovir. Brincidofovir has been approved by the US FDA for the treatment of smallpox using the Animal Rule (21 CFR 314.600). The Animal Rule permits the use of well-characterized animal models and animal to human pharmacokinetic comparisons to demonstrate drug efficacy where human efficacy trials are not ethical nor feasible. For brincidofovir, intracellular concentrations of the active nucleotide metabolite, CDV-PP, were assessed in PBMCs, an established cellular matrix for other antivirals. PBMC isolation methods using various separation media were evaluated using blood from naive and brincidofovir-dosed rabbits and mice. An RBC lysis buffer and multiple rinse steps were key components of the optimized PBMC methods. PBMC isolation yields were increased by &gt;fourfold compared with yields obtained using human methods. High numbers of contaminating red blood cells in the PBMC isolate were minimized by the choice of separation media and inclusion of a red blood cell lysis buffer step in the isolation procedure. The yield and purity of animal PBMCs obtained with the developed methods enabled the FDA required animal to human exposure comparisons for the active metabolite, CDV-PP, thereby assuring the effectiveness of brincidofovir against human smallpox in the absence of human clinical trials.