Tenofovir (TFV) is used in combination with other antiretroviral drugs for human immunodeficiency virus (HIV) treatment and prevention. TFV requires two phosphorylation steps to become pharmacologically active; however, the kinases that activate TFV in cells and tissues susceptible to HIV infection have yet to be identified. Peripheral blood mononuclear cells (PBMC), vaginal, and colorectal tissues were transfected with siRNA targeting nucleotide kinases, incubated with TFV, and TFV-monophosphate (TFV-MP) and TFV-diphosphate (TFV-DP) were measured using mass spectrometry–liquid chromatography. Adenylate kinase 2 (AK2) performed the first TFV phosphorylation step in PBMC, vaginal, and colorectal tissues. Interestingly, both pyruvate kinase isozymes, muscle (PKM) or liver and red blood cell (PKLR), were able to phosphorylate TFV-MP to TFV-DP in PBMC and vaginal tissue, while creatine kinase, muscle (CKM) catalyzed this conversion in colorectal tissue. In addition, next-generation sequencing of the Microbicide Trials Network MTN-001 clinical samples detected 71 previously unreported genetic variants in the genes encoding these kinases. In conclusion, our results demonstrate that TFV is activated in a compartment-specific manner. Further, genetic variants have been identified that could negatively impact TFV activation, thereby compromising TFV efficacy in HIV treatment and prevention.

Understanding small interfering RNA (siRNA) fraction unbound (f u) in relevant physiologic compartments is critical for establishing pharmacokinetic-pharmacodynamic relationships for this emerging modality. In our attempts to isolate the equilibrium free fraction of N-acetylgalactosamine-conjugated siRNA using classic smallmolecule in vitro techniques, we found that the hydrodynamic radius was critical in determining the size exclusion limit requirements for f u isolation, largely validating the siRNA "rigid rod" hypothesis. With this knowledge, we developed an orthogonally validated 50 kDa molecular-mass cutoff ultrafiltration assay to quantify f u in biologic matrices including human, nonhuman primate, rat, and mouse plasma, and human liver homogenate. To enhance understanding of the siRNA-plasma interaction landscape, we examined the effects of various common oligonucleotide therapeutic modifications to the ribose and helix backbone on siRNA f u in plasma (f u,plasma) and found that chemical modifications can alter plasma protein binding by at least 20%. Finally, to gain insight into which specific plasma proteins bind to siRNA, we developed a qualitative screen to identify binding "hits" across a panel of select purified human plasma proteins. All authors are employees and stock holders of Amgen, Inc.

Abnormal proliferation and migration of pulmonary arterial smooth muscle cells (PASMCs) are hallmark characteristics of vascular remodeling in pulmonary hypertension induced by chronic hypoxia. In this study, we investigated the role of the Na+/H+ exchanger (NHE) and alterations in intracellular pH (pHi) homeostasis in meditating increased proliferation and migration in PASMCs isolated from resistance‐sized pulmonary arteries from chronically hypoxic rats or from normoxic rats that were exposed to hypoxia ex vivo (1% or 4% O2, 24–96 h). We found that PASMCs exposed to either in vivo or ex vivo hypoxia exhibited greater proliferative and migratory capacity, elevated pHi, and enhanced NHE activity. The NHE inhibitor, ethyl isopropyl amiloride (EIPA), normalized pHi in hypoxic PASMCs and reduced migration by 73% and 45% in cells exposed to in vivo and in vitro hypoxia, respectively. Similarly, EIPA reduced proliferation by 97% and 78% in cells exposed to in vivo and in vitro hypoxia, respectively. We previously demonstrated that NHE isoform 1 (NHE1) is the predominant isoform expressed in PASMCs. The development of hypoxia‐induced pulmonary hypertension and alterations in PASMC pH i homeostasis were prevented in mice deficient for NHE1. We found that short‐term (24 h) ex vivo hypoxic exposure did not alter the expression of NHE1, so we tested the role of Rho kinase (ROCK) as a possible means of increasing NHE activity. In the presence of the ROCK inhibitor, Y‐27632, we found that pHi and NHE activity were normalized and migration and proliferation were reduced in PASMCs exposed to either in vivo (by 68% for migration and 22% for proliferation) or ex vivo (by 43% for migration and 17% for proliferation) hypoxia. From these results, we conclude that during hypoxia, activation of ROCK enhances NHE activity and promotes PASMC migration and proliferation.

Despite the exquisite potential of siRNA as a therapeutic, the mechanism(s) responsible for the robust indirect exposure-response relationships have not been fully elucidated. To understand the siRNA properties linked to potent activity, requires the disposition of siRNA to be characterized. A technical challenge in the characterization is the detection and quantitation of siRNA from biological samples. Described herein, a Locked Nucleic Acid (LNA) Hybridization-Ligation ECL ELISA was designed for ultra-sensitive quantification of both sense and antisense strands of siRNA independent of structural modifica-tions. This assay was applied to measure siRNA in serum and tissue homogenate in preclinical species. We observed rapid clearance of siRNA from the systemic circulation which contrasted the prolonged accumulation within the tissue. The assay was also able to distinguish and quantify free siRNA from RNA-induced silencing complex (RISC) and Argonaute 2 (Ago2) associated with therapeutic siRNA. We utilized an orthogonal method, LC-MS, to investigate 3′ exonuclease activity toward the antisense strand metabolism. Taken together, we have demonstrated that the LNA Hybridization-Ligation ECL ELISA is arobust analytical method with direct application to measuring the exposure of siRNA therapeutics seamlessly across biological matrices.