Repurposing of rilpivirine for preventing platelet ß3 integrin-dependent thrombosis by targeting c-Src active autophosphorylation.

BACKGROUND: HIV-infected individuals are known to be at higher risk for thrombotic cardiovascular disease (CVD), which may also be differentially affected by components of anti-HIV drugs. To identify the effects of a series of FDA-approved anti-HIV drugs on platelet aggregation in humans, focusing on the novel pharmacological effects of rilpivirine (RPV), a reverse transcriptase inhibitor, on platelet function both in vitro and in vivo and the mechanisms involved. METHODS AND RESULTS: In vitro studies showed that RPV was the only anti-HIV reagent that consistently and efficiently inhibited aggregation elicited by different agonists, exocytosis, morphological extension on fibrinogen, and clot retraction. Treatment of mice with RPV significantly prevented thrombus formation in FeCl3-injured mesenteric vessels, postcava with stenosis surgery, and ADP -induced pulmonary embolism models without defects in platelet viability, tail bleeding, and coagulation activities. RPV also improved cardiac performance in mice with post-ischemic reperfusion. A mechanistic study revealed that RPV preferentially attenuated fibrinogen-stimulated Tyr773 phosphorylation of ß3-integrin by inhibiting Tyr419 autophosphorylation of c-Src. Molecular docking and surface plasmon resonance analyses showed that RPV can bind directly to c-Src. Further mutational analysis showed that the Phe427 residue of c-Src is critical for RPV interaction, suggesting a novel interaction site for targeting c-Src to block ß3-integrin outside-in signaling. CONCLUSION: These results demonstrated that RPV was able to prevent the progression of thrombotic CVDs by interrupting ß3-integrin-mediated outside-in signaling via inhibiting c-Src activation without hemorrhagic side effects, highlighting RPV as a promising reagent for the prevention and therapy of thrombotic CVDs.

Evolution of Antiretroviral Drug Rilpivirine and Approach to Oncology.

Rilpivirine is an antiretroviral drug used to treat AIDS worldwide. The drug is a non-nucleoside reverse transcriptase inhibitor that halts the cDNA elongation process and, thus, the capacity of the HIV-1 virus to replicate. With the new wave of drug repurposing in recent years, rilpivirine has been studied in this regard. This drug is useful in Zika virus treatment, with in vivo results indicating regression in neuronal effects often associated with this infection. Several cancer types have also been researched, from breast to leukemia and pancreatic cancer, and rilpivirine has proved to have inhibitory effects in various cell lines with low concentrations, causing cellular death, apoptosis, and cell cycle arrest. The pathways are not yet established, but some works have hypothesized and demonstrated that rilpivirine causes inhibition of Aurora A kinase and has effects on the Janus kinase-signal transducer and activator of transcription (JAK-STAT) signaling pathway and the vascular endothelial growth factors-receptors (VEGFs-VEGFRs) pathway, which are known to be altered in cancer and tumors and can be targeted for cancer treatment. Further testing and clinical trials are needed, but this review demonstrates the potential of rilpivirine's repurposing for cancer treatment.

Divergent effects of HIV reverse transcriptase inhibitors on pancreatic beta-cell function and survival: Potential role of oxidative stress and mitochondrial dysfunction.

Antiretroviral therapy (ART), a life-saving treatment strategy in HIV/AIDS, has been implicated in increasing the risk of type 2 diabetes mellitus (T2DM). Direct damaging effects on beta-cell function and survival by either non-nucleoside reverse transcriptase inhibitors (NNRTIs) or nucleoside/tide reverse transcriptase inhibitors (NRTIs) may predispose individuals to developing T2DM or if already type 2 diabetic, to insulin dependency. The aim of this study was to investigate the effects of the NNRTIs efavirenz, rilpivirine and doravirine, and the NRTIs tenofovir disoproxil fumarate and emtricitabine, on beta-cell function and survival while suggesting potential cellular and molecular mechanism(s). Our results show contrasting effects within the NNRTI class as doravirine did not cause damaging effects in the rat insulinoma INS-1E cells while efavirenz and rilpivirine reduced insulin release and cell viability, and induced apoptosis in INS-1E cells. Additionally, efavirenz and rilpivirine increased ROS generation, disrupted Δψm and upregulated the mRNA and protein expression of CHOP and GRP78, key markers of endoplasmic reticulum stress. In silico docking studies predict a possible inhibition of the mitochondrial ATP synthase by rilpivirine. On the contrary, both the NRTIs tenofovir disoproxil fumarate and emtricitabine did not affect GSIS, cell viability and apoptosis/necrosis levels in INS-1E cells. The deleterious effects observed in beta-cells exposed to efavirenz or rilpivirine may be, at least partially, mediated by oxidative stress and mitochondrial toxicity. These findings provide potential mechanism(s) by which efavirenz and rilpivirine may contribute to the pathogenesis of T2DM and the progression of T2DM to insulin dependency in HIV-infected type 2 diabetics.

Virus-Mimicking Polymer Nanoparticles Targeting CD169+ Macrophages as Long-Acting Nanocarriers for Combination Antiretrovirals.

Monosialodihexosylganglioside (GM3)-presenting lipid-coated polymer nanoparticles (NPs) that recapitulate the sequestration of human immunodeficiency virus-1 (HIV-1) particles in CD169+ virus-containing compartments (VCCs) of macrophages were developed as carriers for delivery and sustained release of a combination of two antiretrovirals (ARVs), rilpivirine (RPV) and cabotegravir (CAB). RPV and CAB were co-loaded into GM3-presenting lipid-coated polylactic acid (PLA) and poly(lactic-co-glycolic acid) (PLGA) NPs without loss in potency of the drugs. GM3-presenting PLA NPs demonstrated the most favorable release properties and achieved inhibition of HIV-1 infection of primary human macrophages for up to 35 days. Intracellular localization of GM3-presenting PLA NPs in VCCs correlated with retention of intracellular ARV concentrations and sustained inhibition of HIV-1 infection. This work elucidates the design criteria of lipid-coated polymer NPs to utilize CD169+ macrophages as cellular drug depots for eradicating the viral reservoir sites or to achieve long-acting prophylaxis against HIV-1 infection.

Development of a novel in vitro assay to screen for neuroprotective drugs against iatrogenic neurite shortening.

This work tries to help overcome the lack of relevant translational screening assays, as a limitation for the identification of novel analgesics for neuropathic pain. Hyperexcitability and neurite shortening are common adverse effects of antiviral and antitumor drugs, leading to neuropathic pain. Now, as seen in the drug screening that we developed here, a high-content microscopy-based assay with immortalized dorsal root ganglia (DRG) neurons (differentiated F11 cells) allowed to identify drugs able to protect against the iatrogenic neurite shortening induced by the antitumor drug vincristine and the antiviral drug rilpivirine. We observed that vincristine and rilpivirine induced a significant reduction in the neurite length, which was reverted by α-lipoic acid. We had also evidenced protective effects of pregabalin and melatonin, acting through the α2δ-2 subunit of the voltage-dependent calcium channels and the MT1 receptor, respectively. Additionally, two hits originated from a previous primary screening aimed to detect inhibitors of hyperexcitability to inflammatory mediators in DRG neurons (nitrendipine and felodipine) also prevented neurite shortening in our model. In summary, in this work we developed a novel secondary assay for identifying hits with neuroprotective effect against iatrogenic neurite shortening, consistent with the anti-hyperexcitability action previously tested: highlighting nitrendipine and felodipine against iatrogenic damage in DRG neurons.

CARD8 is an inflammasome sensor for HIV-1 protease activity.

HIV-1 has high mutation rates and exists as mutant swarms within the host. Rapid evolution of HIV-1 allows the virus to outpace the host immune system, leading to viral persistence. Approaches to targeting immutable components are needed to clear HIV-1 infection. Here, we report that the caspase recruitment domain-containing protein 8 (CARD8) inflammasome senses HIV-1 protease activity. HIV-1 can evade CARD8 sensing because its protease remains inactive in infected cells before viral budding. Premature intracellular activation of the viral protease triggered CARD8 inflammasome-mediated pyroptosis of HIV-1-infected cells. This strategy led to the clearance of latent HIV-1 in patient CD4+ T cells after viral reactivation. Thus, our study identifies CARD8 as an inflammasome sensor of HIV-1, which holds promise as a strategy for the clearance of persistent HIV-1 infection.

Rod-shape theranostic nanoparticles facilitate antiretroviral drug biodistribution and activity in human immunodeficiency virus susceptible cells and tissues.

Human immunodeficiency virus theranostics facilitates the development of long acting (LA) antiretroviral drugs (ARVs) by defining drug-particle cell depots. Optimal drug formulations are made possible based on precise particle composition, structure, shape and size. Through the creation of rod-shaped particles of defined sizes reflective of native LA drugs, theranostic probes can be deployed to measure particle-cell and tissue biodistribution, antiretroviral activities and drug retention. Methods: Herein, we created multimodal rilpivirine (RPV) 177lutetium labeled bismuth sulfide nanorods (177LuBSNRs) then evaluated their structure, morphology, configuration, chemical composition, biological responses and adverse reactions. Particle biodistribution was analyzed by single photon emission computed tomography (SPECT/CT) and laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) imaging. Results: Nanoformulated RPV and BSNRs-RPV particles showed comparable physicochemical and cell biological properties. Drug-particle pharmacokinetics (PK) and biodistribution in lymphoid tissue macrophages proved equivalent, one with the other. Rapid particle uptake and tissue distribution were observed, without adverse reactions, in primary blood-derived and tissue macrophages. The latter was seen within the marginal zones of spleen. Conclusions: These data, taken together, support the use of 177LuBSNRs as theranostic probes as a rapid assessment tool for PK LA ARV measurements.

Molecular Docking Studies and Synthesis of Amino-oxy-diarylquinoline Derivatives as Potent Non-nucleoside HIV-1 Reverse Transcriptase Inhibitors.

In this study, amino-oxy-diarylquinolines were designed using structure-guided molecular hybridization strategy and fusing of the pharmacophore templates of nevirapine (NVP), efavirenz (EFV), etravirine (ETV, TMC125) and rilpivirine (RPV, TMC278). The anti-HIV-1 reverse transcriptase (RT) activity was evaluated using standard ELISA method, and the cytotoxic activity was performed using MTT and XTT assays. The primary bioassay results indicated that 2-amino-4-oxy-diarylquinolines possess moderate inhibitory properties against HIV-1 RT. Molecular docking results showed that 2-amino-4-oxy-diarylquinolines 8(A-D): interacted with the Lys101 and His235 residue though hydrogen bonding and interacted with Tyr318 residue though π-π stacking in HIV-1 RT. Furthermore, 8A: and 8D: were the most potent anti-HIV agents among the designed and synthesized compounds, and their inhibition rates were 34.0% and 39.7% at 1 µM concentration. Interestingly, 8A: was highly cytotoxicity against MOLT-3 (acute lymphoblastic leukemia), with an IC50 of 4.63±0.62 µg/mL, which was similar with that in EFV and TMC278 (IC50 7.76±0.37 and 1.57±0.20 µg/ml, respectively). Therefore, these analogs of the synthesized compounds can serve as excellent bases for the development of new anti-HIV-1 agents in the near future.

Effects of rilpivirine, 17ß-estradiol and ß-naphthoflavone on the inflammatory status of release of adipocytokines in 3T3-L1 adipocytes in vitro.

Rilpivirine is a non-nucleoside reverse transcriptase inhibitor, recently developed as a drug of choice for initial anti-retroviral (ARV) treatment of HIV-1 infection, whereas estradiol is a major component of hormonal contraceptives. Both drugs have effects on lipid metabolism, impairment of adipocyte differentiation and alteration of adipose tissue distribution and function.This study investigated the effects of different concentrations of either rilpivirine or estradiol either alone or in combination on adipocyte differentiation and adipocytokines status in vitro in the absence and presence of ß-naphthoflavone, (BNF),a potent agonist of the aryl hydrocarbon receptor. 3T3-L1 human pre-adipocytes were cultured and differentiated with different concentrations of treatment drugs. After 10 days of differentiation procedure, cells were examined for their morphology and viability. Glycerol,adiponectin, leptin, resistin and interleukin-8 (IL-8) were quantified using commercially available kits. The results show that either rilpivirine or estradiol individually or during their combination can evoke significant increases in glycerol release and a concomitant significant decrease of adiponectin from adipocytes. These effects were dose-dependent. The effects of combined treatments were much larger than individual concentration for each drug. Both drugs had little of no effect on leptin levels, except for a small decrease with 10 µM rilpivirine alone or when combined with estradiol. In addition, both drugs evoked small increases in the release of resistin and interleukin-8 with significant values at higher doses compared to untreated adipocytes.When adipocytes were pretreated with BNF, either rilpivirine or, estradiol or when combined evoked a much larger release in glycerol and a much larger decrease in adiponectin compared to the absence of BNF. In contrast, BNF pretreatment had little of no effect on either leptin, resistin or IL-8 metabolism compared to the results obtained in the presence of either rilpivirine or estradiol alone or in combination.These results show that rilpivirine and estradiol either alone or when combined or pretreated with BNF can evoke marked effects on glycerol and cytokines levels from adipocytes. However, their mechanism (s) in inducing adipogenesis warrants further investigation of different transcription factors at gene expression levels.

Frequent cross-resistance to rilpivirine among subtype C HIV-1 from first-line antiretroviral therapy failures in South Africa.

Background Rilpivirine (TMC278LA) is a promising drug for pre-exposure prophylaxis of HIV-1 because of its sub-nanomolar potency and long-acting formulation; however, increasing transmission of non-nucleoside reverse transcriptase inhibitor-resistant HIV-1 with potential cross-resistance to rilpivirine could reduce its preventive efficacy. This study investigated rilpivirine cross-resistance among recombinant subtype C HIV-1 derived from 100 individuals failing on first-line non-nucleoside reverse transcriptase inhibitor-containing antiretroviral therapy in South Africa whose samples were sent for routine HIV-1 drug resistance testing to Lancet Laboratories (Johannesburg, South Africa). Methods Plasma samples were selected from individuals with HIV-1 RNA > 10,000 copies/ml and ≥1 non-nucleoside reverse transcriptase inhibitor-resistance mutation in reverse transcriptase. Recombinant HIV-1LAI-containing bulk-cloned full-length reverse transcriptase sequences from plasma were assayed for susceptibility to nevirapine (NVP), efavirenz (EFV) and rilpivirine in TZM-bl cells. Fold-change (FC) decreases in drug susceptibility were calculated against a mean IC50 from 12 subtype C HIV-1 samples from treatment-naïve individuals in South Africa. Cross-resistance was evaluated based on biological cutoffs established for rilpivirine (2.5-FC) and the effect of mutation combinations on rilpivirine phenotype. Results Of the 100 samples from individuals on failing antiretroviral therapy, 69 had 2.5- to 75-fold decreased susceptibility to rilpivirine and 11 had >75-fold resistance. Rilpivirine resistance was strongly associated with K103N especially in combination with other rilpivirine-associated mutations. Conclusion The frequently observed cross-resistance of HIV-1 suggests that the preventive efficacy of TMC278LA pre-exposure prophylaxis could be compromised by transmission of HIV-1 from individuals with failure of first-line non-nucleoside reverse transcriptase inhibitor-containing antiretroviral therapy.

MDR1 and BCRP Transporter-Mediated Drug-Drug Interaction between Rilpivirine and Abacavir and Effect on Intestinal Absorption.

Rilpivirine (TMC278) is a highly potent nonnucleoside reverse transcriptase inhibitor (NNRTI) representing an effective component of combination antiretroviral therapy (cART) in the treatment of HIV-positive patients. Many antiretroviral drugs commonly used in cART are substrates of ATP-binding cassette (ABC) and/or solute carrier (SLC) drug transporters and, therefore, are prone to pharmacokinetic drug-drug interactions (DDIs). The aim of our study was to evaluate rilpivirine interactions with abacavir and lamivudine on selected ABC and SLC transporters in vitro and assess its importance for pharmacokinetics in vivo Using accumulation assays in MDCK cells overexpressing selected ABC or SLC drug transporters, we revealed rilpivirine as a potent inhibitor of MDR1 and BCRP, but not MRP2, OCT1, OCT2, or MATE1. Subsequent transport experiments across monolayers of MDCKII-MDR1, MDCKII-BCRP, and Caco-2 cells demonstrated that rilpivirine inhibits MDR1- and BCRP-mediated efflux of abacavir and increases its transmembrane transport. In vivo experiments in male Wistar rats confirmed inhibition of MDR1/BCRP in the small intestine, leading to a significant increase in oral bioavailability of abacavir. In conclusion, rilpivirine inhibits MDR1 and BCRP transporters and may affect pharmacokinetic behavior of concomitantly administered substrates of these transporters, such as abacavir.

Negative Regulation of Human Pregnane X Receptor by MicroRNA-18a-5p: Evidence for Suppression of MicroRNA-18a-5p Expression by Rifampin and Rilpivirine.

Small noncoding microRNAs act as post-transcriptional regulators of gene expression involved in diverse biologic functions. Pregnane X receptor (PXR, NR1I2), a member of the superfamily of nuclear receptors, is a transcription factor governing the transport and biotransformation of various drugs and other chemicals. In the present study, we identified a specific microRNA (miR) involved in regulating the expression and functionality of human PXR (hPXR). According to bioinformatics analysis employing three commonly used algorithms (TargetScan, miRanda, and DIANA-microT-CDS), miR-18a-5p was predicted to be the top candidate microRNA regulator of hPXR. Consequently, this microRNA was selected for detailed experimental investigation. As shown in cell-based dual-luciferase reporter gene assays, functional interaction occurred between miR-18a-5p and the microRNA recognition element of miR-18a-5p in the 3'-untranslated region of hPXR mRNA. Transfection of LS180 human colorectal adenocarcinoma cells with an miR-18a-5p mimic decreased hPXR mRNA and protein expression, whereas transfection of LS180 cells with an miR-18a-5p inhibitor increased hPXR mRNA and protein expression. The decrease in hPXR expression by the miR-18a-5p mimic was associated with a reduction in the extent of hPXR target gene (CYP3A4) induction by rifampin and rilpivirine. Treatment of untransfected LS180 cells with either of these hPXR agonists decreased endogenous expression of miR-18a-5p, and this preceded the onset of CYP3A4 induction. In conclusion, miR-18a-5p is a negative regulator of hPXR expression and the hPXR agonists rifampin and rilpivirine are chemical suppressors of miR-18a-5p expression.

Differential activation of human constitutive androstane receptor and its SV23 and SV24 splice variants by rilpivirine and etravirine.

BACKGROUND AND PURPOSE: Rilpivirine and etravirine are second-generation non-nucleoside reverse transcriptase inhibitors (NNRTIs) indicated for the treatment of HIV/AIDS. The constitutive androstane receptor (CAR) regulates the expression of genes involved in various biological processes, including the transport and biotransformation of drugs. We investigated the effect of rilpivirine and etravirine on the activity of the wild-type human CAR (hCAR-WT) and its hCAR-SV23 and hCAR-SV24 splice variants, and compared it with first-generation NNRTIs (efavirenz, nevirapine, and delavirdine). EXPERIMENTAL APPROACH: Receptor activation, ligand-binding domain (LBD) transactivation, and co-activator recruitment were investigated in transiently transfected, NNRTI-treated HepG2 cells. Nuclear translocation of green fluorescent protein-tagged hCAR-WT and CYP2B6 gene expression were assessed in NNRTI-treated human hepatocytes. KEY RESULTS: Rilpivirine and etravirine activated hCAR-WT, but not hCAR-SV23 or hCAR-SV24, and without transactivating the LBD or recruiting steroid receptor coactivators SRC-1, SRC-2, or SRC-3. Among the first-generation NNRTIs investigated, only efavirenz activated hCAR-WT, hCAR-SV23, and hCAR-SV24, but none of them transactivated the LBD of these receptors or substantively recruited SRC-1, SRC-2, or SRC-3. Rilpivirine, etravirine, and efavirenz triggered nuclear translocation of hCAR-WT and increased hCAR target gene (CYP2B6) expression. CONCLUSION AND IMPLICATIONS: NNRTIs activate hCAR-WT, hCAR-SV23, and hCAR-SV24 in a drug-specific and isoform-selective manner. The activation occurs by a mechanism that does not appear to involve binding to the LBD or recruitment of SRC-1, SRC-2, or SRC-3.