Intramuscular and subcutaneous administration of antiretroviral drugs, compared with oral, enhances delivery to lymphoid tissues in BALB/c mice.

BACKGROUND: Multiple tissue reservoirs are established soon after HIV infection, and some tissues may also be pharmacological sanctuaries. Parenteral administration of antiretroviral (ARV) drugs for treatment and prevention of HIV infection is an active area of drug development. The influence of route of administration on ARV tissue pharmacokinetics is not known. OBJECTIVES: To investigate ARV pharmacokinetics in lymphatic and select non-lymphatic tissues (e.g. brain and testes) after intramuscular and subcutaneous administration compared with oral in BALB/c mice. METHODS: Tissue concentrations of cobicistat, efavirenz, elvitegravir, maraviroc, rilpivirine, tenofovir alafenamide and tenofovir disoproxil fumarate were determined. The tissue penetration ratio (TPR) was the primary measure for comparison; a change in TPR arises from factors affecting tissue distribution controlling for changes in systemic bioavailability. RESULTS: Intramuscular and subcutaneous delivery increased TPRs in the lymph node and spleen for 27 of 28 (96%) drug administration events. Decreased TPRs, however, were found in some tissues such as the brain and testes. CONCLUSIONS: These results demonstrate a change in route of drug administration from oral to intramuscular or subcutaneous can change tissue uptake. This has implications for HIV pharmacotherapy. For example, HIV persists in lymphoid tissues despite long-term oral ARV therapy, and low ARV concentrations have been found in lymphoid tissues. The improved ARV lymphatic tissue bioavailability with intramuscular and subcutaneous administration allows future studies to investigate these routes of drug administration as a therapeutic manoeuvre to limit viral persistence and eliminate viral sanctuaries in the lymphatic tissues, which is a prerequisite for eradication of HIV.

Assessing the lymphoid tissue bioavailability of antiretrovirals in human primary lymphoid endothelial cells and in mice.

BACKGROUND: The secondary lymphoid tissues (LTs), lymph nodes (LNs) and gut-associated lymphoid tissue (GALT) are considered reservoirs for HIV. Antiretrovirals (ARVs) have lower penetration into LT. In vitro models predictive of ARV LT penetration have not been established. OBJECTIVES: To develop an in vitro model of LT bioavailability using human lymphoid endothelial cells (HLECs) and investigate its predictability with in vivo pharmacokinetic (PK) studies in mice. METHODS: ARV bioavailability in HLECs was evaluated at the maximum plasma concentration (Cmax) observed in HIV-infected patients. ARVs were: abacavir, atazanavir, darunavir, dolutegravir, efavirenz, elvitegravir, emtricitabine, maraviroc, raltegravir, rilpivirine, ritonavir, tenofovir disoproxil fumarate and the PK booster cobicistat. The LT PK of representative drugs showing high (efavirenz), intermediate (dolutegravir) and low (emtricitabine) HLEC bioavailability was investigated in BALB/c mice given 50/10/30 mg/kg efavirenz/dolutegravir/emtricitabine orally, daily for 3 days. The concordance of in vitro and in vivo ARV bioavailability was examined. RESULTS: ARVs showed high (>67th percentile; rilpivirine, efavirenz, elvitegravir and cobicistat), intermediate (67th-33rd percentile; ritonavir, tenofovir disoproxil fumarate, dolutegravir and maraviroc) and low (<33rd percentile; atazanavir, darunavir, raltegravir, emtricitabine and abacavir) HLEC bioavailability. The hierarchy of efavirenz, dolutegravir and emtricitabine bioavailability in LN, gut and brain tissues of mice was: efavirenz>dolutegravir>emtricitabine. CONCLUSIONS: ARVs displayed distinct HLEC penetration patterns. PK studies of representative ARVs in LT of mice were concordant with HLEC bioavailability. These findings support further development of this approach and its translational predictability in humans.

Normalization of cell associated antiretroviral drug concentrations with a novel RPP30 droplet digital PCR assay.

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.