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Normalization of cell associated antiretroviral drug concentrations with a novel RPP30 droplet digital PCR assay.
Dyavar, Shetty Ravi; Ye, Zhen; Byrareddy, Siddappa N; Scarsi, Kimberly K; Winchester, Lee C; Weinhold, Jonathan A; Fletcher, Courtney V; Podany, Anthony T.
Afiliación
  • Dyavar SR; Antiviral Pharmacology Laboratory, Department of Pharmacy Practice, College of Pharmacy (COP), University of Nebraska Medical Center (UNMC), Omaha, NE, 68198, USA. shettyravi.dyavar@unmc.edu.
  • Ye Z; Department of Pharmaceutical Sciences, COP, UNMC, Omaha, NE, 68198, USA.
  • Byrareddy SN; Department of Pharmacology and Experimental Neuroscience, College of Medicine, UNMC, Omaha, NE, 68198, USA.
  • Scarsi KK; Antiviral Pharmacology Laboratory, Department of Pharmacy Practice, College of Pharmacy (COP), University of Nebraska Medical Center (UNMC), Omaha, NE, 68198, USA.
  • Winchester LC; Antiviral Pharmacology Laboratory, Department of Pharmacy Practice, College of Pharmacy (COP), University of Nebraska Medical Center (UNMC), Omaha, NE, 68198, USA.
  • Weinhold JA; Antiviral Pharmacology Laboratory, Department of Pharmacy Practice, College of Pharmacy (COP), University of Nebraska Medical Center (UNMC), Omaha, NE, 68198, USA.
  • Fletcher CV; Antiviral Pharmacology Laboratory, Department of Pharmacy Practice, College of Pharmacy (COP), University of Nebraska Medical Center (UNMC), Omaha, NE, 68198, USA. cfletcher@unmc.edu.
  • Podany AT; Antiviral Pharmacology Laboratory, Department of Pharmacy Practice, College of Pharmacy (COP), University of Nebraska Medical Center (UNMC), Omaha, NE, 68198, USA. apodany@unmc.edu.
Sci Rep ; 8(1): 3626, 2018 02 26.
Article en En | MEDLINE | ID: mdl-29483619
Quantification of antiretroviral (ARV) drug concentrations in peripheral blood mononuclear cells (PBMCs) and tissue isolated mononuclear cells (TIMCs) from lymph node (LNMC) and rectum (RMC) is an important measure of bio-distribution. Normalization of drug concentrations is critical to represent tissue drug concentrations and to analyze both intra-individual and inter-individual variability in drug distribution. However, a molecular method to normalize intracellular drug concentrations in PBMCs and TIMCs methanol extracts is currently unavailable. In this study, a novel droplet digital PCR (ddPCR) assay was designed to amplify RPP30 gene sequence conserved in human and non-human primates (NHP). Genomic DNA (gDNA) isolated from 70 percent methanol embedded PBMCs and TIMCs was used as ddPCR template to quantitate precise RPP30 copies to derive cell counts. The novel molecular method quantitated RPP30 copies in human and rhesus macaque gDNA templates with greater accuracy and precision than qPCR. RPP30 ddPCR derived cell counts are strongly correlated with automated cytometer based cell counts in PBMC (R = 0.90, p = 0.001 and n = 20); LNMC (R = 0.85 p = 0.0001 and n = 22) and RMC (R = 0.92, p = 0.0001 and n = 20) and achieved comparable normalized drug concentrations. Therefore, the RPP30 ddPCR assay is an important normalization method in drug bio-distribution and pharmacokinetic studies in humans and NHPs.
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Leucocitos Mononucleares / Antirretrovirales / Reacción en Cadena en Tiempo Real de la Polimerasa Tipo de estudio: Risk_factors_studies Límite: Humans Idioma: En Revista: Sci Rep Año: 2018 Tipo del documento: Article País de afiliación: Estados Unidos Pais de publicación: Reino Unido
Texto completo
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Leucocitos Mononucleares / Antirretrovirales / Reacción en Cadena en Tiempo Real de la Polimerasa Tipo de estudio: Risk_factors_studies Límite: Humans Idioma: En Revista: Sci Rep Año: 2018 Tipo del documento: Article País de afiliación: Estados Unidos Pais de publicación: Reino Unido