Cryo-EM structures of wild-type and E138K/M184I mutant HIV-1 RT/DNA complexed with inhibitors doravirine and rilpivirine.

Structures trapping a variety of functional and conformational states of HIV-1 reverse transcriptase (RT) have been determined by X-ray crystallography. These structures have played important roles in explaining the mechanisms of catalysis, inhibition, and drug resistance and in driving drug design. However, structures of several desired complexes of RT could not be obtained even after many crystallization or crystal soaking experiments. The ternary complexes of doravirine and rilpivirine with RT/DNA are such examples. Structural study of HIV-1 RT by single-particle cryo-electron microscopy (cryo-EM) has been challenging due to the enzyme's relatively smaller size and higher flexibility. We optimized a protocol for rapid structure determination of RT complexes by cryo-EM and determined six structures of wild-type and E138K/M184I mutant RT/DNA in complexes with the nonnucleoside inhibitors rilpivirine, doravirine, and nevirapine. RT/DNA/rilpivirine and RT/DNA/doravirine complexes have structural differences between them and differ from the typical conformation of nonnucleoside RT inhibitor (NNRTI)-bound RT/double-stranded DNA (dsDNA), RT/RNA-DNA, and RT/dsRNA complexes; the primer grip in RT/DNA/doravirine and the YMDD motif in RT/DNA/rilpivirine have large shifts. The DNA primer 3'-end in the doravirine-bound structure is positioned at the active site, but the complex is in a nonproductive state. In the mutant RT/DNA/rilpivirine structure, I184 is stacked with the DNA such that their relative positioning can influence rilpivirine in the pocket. Simultaneously, E138K mutation opens the NNRTI-binding pocket entrance, potentially contributing to a faster rate of rilpivirine dissociation by E138K/M184I mutant RT, as reported by an earlier kinetic study. These structural differences have implications for understanding molecular mechanisms of drug resistance and for drug design.

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.

Europium sulfide nanoprobes predict antiretroviral drug delivery into HIV-1 cell and tissue reservoirs.

Background: Delivery of long-acting nanoformulated antiretroviral drugs (ARVs) to human immunodeficiency virus type one cell and tissue reservoirs underlies next generation antiretroviral therapeutics. Nanotheranostics, comprised of trackable nanoparticle adjuncts, can facilitate ARV delivery through real-time drug tracking made possible through bioimaging platforms. Methods: To model HIV-1 therapeutic delivery, europium sulfide (EuS) nanoprobes were developed, characterized and then deployed to cells, tissues, and rodents. Tests were performed with nanoformulated rilpivirine (NRPV), a non-nucleoside reverse transcriptase inhibitor (NNRTI) used clinically to suppress or prevent HIV-1 infection. First, CD4+ T cells and monocyte-derived macrophages were EuS-treated with and without endocytic blockers to identify nanoprobe uptake into cells. Second, Balb/c mice were co-dosed with NRPV and EuS or lutetium177-doped EuS (177LuEuS) theranostic nanoparticles to assess NRPV biodistribution via mass spectrometry. Third, single photon emission computed tomography (SPECT-CT) and magnetic resonance imaging (MRI) bioimaging were used to determine nanotheranostic and NRPV anatomic redistribution over time. Results: EuS nanoprobes and NRPV entered cells through dynamin-dependent pathways. SPECT-CT and MRI identified biodistribution patterns within the reticuloendothelial system for EuS that was coordinate with NRPV trafficking. Conclusions: EuS nanoprobes parallel the uptake and biodistribution of NRPV. These data support their use in modeling NRPV delivery to improve treatment strategies.

Cabotegravir Plus Rilpivirine: First Approval.

A regimen comprising extended release injectable suspensions of cabotegravir and rilpivirine for concurrent administration (CABENUVA™) is being developed by ViiV Healthcare and Janssen Pharmaceutica (Janssen) as a complete regimen for HIV infection. Based on the results of the ATLAS and FLAIR trials, the regimen was recently approved in Canada for the treatment of HIV-1 infection in adults to replace current antiretroviral therapy in patients who are virologically stable and suppressed. This article summarizes the milestones in the development of co-packaged cabotegravir and rilpivirine leading to this first approval.

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.

Investigation of Cyclodextrin-Based Nanosponges for Solubility and Bioavailability Enhancement of Rilpivirine.

Rilpivrine is BCS class II drug used for treatment of HIV infection. The drug has low aqueous solubility (0.0166 mg/ml) and dissolution rate leading to low bioavailability (32%). Aim of this work was to enhance solubility and dissolution of rilpivirine using beta-cyclodextrin-based nanosponges. These nanosponges are biocompatible nanoporous particles having high loading capacity to form supramolecular inclusion and non-inclusion complexes with hydrophilic and lipophilic drugs for solubility enhancement. Beta-cyclodextrin was crosslinked with carbonyl diimidazole and pyromellitic dianhydride to prepare nanosponges. The nanosponges were loaded with rilpivirine by solvent evaporation method. Binary and ternary complexes of drug with ß-CD, HP-ß-CD, nanosponges, and tocopherol polyethylene glycol succinate were prepared and characterized by phase solubility, saturation solubility in different media, in vitro dissolution, and in vivo pharmacokinetics. Spectral analysis by Fourier transform infrared spectroscopy, powder X-ray diffraction, and differential scanning calorimetry was performed. Results obtained from spectral characterization confirmed inclusion complexation. Phase solubility studies indicated stable complex formation. Saturation solubility was found to be 10-13-folds higher with ternary complexes in distilled water and 12-14-fold higher in 0.1 N HCl. Solubility enhancement was evident in biorelevant media. Molecular modeling studies revealed possible mode of entrapment of rilpivirine within ß-CD cavities. A 3-fold increase in dissolution with ternary complexes was observed. Animal studies revealed nearly 2-fold increase in oral bioavailability of rilpivirine. It was inferred that electronic interactions, hydrogen bonding, and van der Waals forces are involved in the supramolecular interactions.

Rilpivirine long-acting for the prevention and treatment of HIV infection.

PURPOSE OF REVIEW: Rilpivirine is a non-nucleoside reverse transcriptase inhibitor approved for the treatment of HIV in combination with other antiretrovirals (ARV). The development of the long-acting formulation of rilpivirine (RPV-LA) and its use in clinical settings is the subject of this review. RECENT FINDINGS: In 2017, the results of the phase IIb study LATTE2 have been published. This study demonstrated that the combination of RPV-LA with a second long-acting agent (cabotegravir) administered intramuscularly every 4--8 weeks was similar in virologic suppression rates to standard of care ARV treatment. SUMMARY: RPV-LA is an injectable nanoparticle suspension for intramuscular use. Phase I studies in healthy volunteers demonstrated that RPV-LA, administered at doses between 600 and 1200 mg, was well tolerated and effective in maintaining satisfactory drug concentrations in plasma, vaginal secretions and rectal tissue compartments for at least 4 weeks. RPV-LA efficacy was also shown in a phase II clinical trial in HIV infected patients and phase III studies are currently ongoing. Most study participants stated that they would use or continue using injectables, both in HIV treatment and prevention settings. Long-acting injectable ARVs have the potential to improve the convenience of HIV drug regimens.

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.

Simultaneous determination and validation of emtricitabine, rilpivirine and tenofovir from biological samples using LC and CE methods.

A combination of antiretroviral agents is frequently used in effective treatment of the human immunodeficiency virus infection. In this study, two different separation methods are presented for the simultaneous determination of emtricitabine, rilpivirine and tenofovir from raw materials and urine samples. Developed liquid chromatography and capillary electrophoresis methods were thoroughly optimized for high analytical performances. Optimization of multiple variables at the same time by performing a minimum number of experiments was achieved by the Box-Behnken design, which is an experimental design in response surface methodology, in capillary electrophoresis. The results of the experimental design ensure minimum analysis time with well-separated analytes. Separation conditions, such as different stationary phases, pH level, organic modifiers and temperatures in liquid chromatography method, were also optimized. In particular, among stationary phases, the core-shell column especially enhanced the effectiveness of separation in liquid chromatography. Both methods were fully validated and applied to real samples. The main advantage of the developed methods is the separation of the drug combination in a short time with high efficiency and without any time-consuming steps.

The HIV-1 p66 homodimeric RT exhibits different conformations in the binding-competent and -incompetent NNRTI site.

Non-nucleoside inhibitors of human immunodeficiency virus type 1 reverse transcriptase (RT), NNRTIs, which bind to the p66/p51 heterodimeric RT, also interact with the p66/p66 homodimer, whose structure is unknown. 19 F nuclear magnetic resonance of a single 4-trifluoromethylphenylalanine (tfmF) residue, incorporated into the NNRTI binding pocket of the p66/p66 homodimer at position 181, was used to investigate NNRTI binding. In the NNRTI-bound homodimer complex, two different 19 F signals are observed, with the resonance frequencies matching those of the NNRTI-bound p66/p51 heterodimer spectra, in which the individual p66-subunit or p51-subunit were labeled with tfmF at positions 181. These data suggest that the NNRTI-bound p66/p66 homodimer conformation, particularly around residue 181, is very similar to that in the p66/p51 heterodimer, explaining why NNRTI binding to p66/p66 enhances dimer formation.

Enhancement of oral bioavailability of anti-HIV drug rilpivirine HCl through nanosponge formulation.

OBJECTIVE: To synthesize ß cyclodextrin nanosponges using a novel and efficient microwave mediated method for enhancing bioavailability of Rilpivirine HCl (RLP). SIGNIFICANCE: Belonging to BCS class II RLP has pH dependent solubility and poor oral bioavailability. However, a fatty meal enhances its absorption hence the therapy indicates that the dosage form be consumed with a meal. But then it becomes tedious and inconvenient to continue the therapy for years with having to face the associated gastric side effects such as nausea. METHOD: Microwave synthesizer was used to mediate the poly-condensation reaction between ß-cyclodextrin and cross-linker diphenylcarbonate. Critical parameters selected were polymer to cross-linker ratio, Watt power, reaction time and solvent volume. Characterization studies were performed using FTIR, DSC, SEM, 1H-NMR and PXRD. Molecular modeling was applied to confirm the possibility of drug entrapment. In vitro drug dissolution followed by oral bioavailability studies was performed in Sprawley rats. Samples were analyzed using HPLC. RESULTS: Microwave synthesis yields para-crystalline, porous nanosponges (∼205 nm). Drug entrapment led to enhancement of solubility and a two-fold increase in drug dissolution (P < 0.001) following Higuchi release model. Enhanced oral bioavailability was observed in fasted Sprawley rats where Cmax and AUC0-∞ increases significantly (Cmax of NS∼ 586 ± 5.91 ng/mL; plain RLP ∼310 ± 5. 74 ng/mL). CONCLUSION: The approach offers a comfortable dosing zone for AIDs patients, negating the requirement of consuming the formulation in a fed state due to enhancement in drugs' oral bioavailability.

Drug-like property-driven optimization of 4-substituted 1,5-diarylanilines as potent HIV-1 non-nucleoside reverse transcriptase inhibitors against rilpivirine-resistant mutant virus.

On the basis of our prior structure-activity relationship (SAR) results, our current lead optimization of 1,5-diarylanilines (DAANs) focused on the 4-substituent (R1) on the central phenyl ring as a modifiable position related simultaneously to improved drug resistance profiles and drug-like properties. Newly synthesized p-cyanovinyl-DAANs (8a-8g) with different R1 side chains plus prior active p-cyanoethyl-DAANs (4a-4c) were evaluated not only for anti-HIV potency against both wild-type HIV virus and rilpivirine-resistant (E138K, E138K+M184I) viral replication, but also for multiple drug-like properties, including aqueous solubility, lipophilicity, and metabolic stability in human liver microsomes and human plasma. This study revealed that both ester and amide R1 substituents led to low nanomolar anti-HIV potency against wild-type and rilpivirine-resistant viral strains (E138K-resistance fold changes<3). The N-substituted amide-R1 side chains were superior to ester-R1 likely due to improved aqueous solubility, lipophilicity, and higher metabolic stability in vitro. Thus, three amide-DAANs 8e, 4a, and 4b were identified with high potency against wild-type and rilpivirine-resistant viral strains and multiple desirable drug-like properties.

UPLC-MS/MS method for the simultaneous quantification of three new antiretroviral drugs, dolutegravir, elvitegravir and rilpivirine, and other thirteen antiretroviral agents plus cobicistat and ritonavir boosters in human plasma.

Rilpivirine (RPV), dolutegravir (DTG) and elvitegravir (EVG) are the latest antiretroviral drugs approved for treatment of HIV infection. Currently, poor information is currently available concerning their pharmacokinetic and pharmacodynamic properties, thus making the use of therapeutic drug monitoring for these drugs not useful. This lack of information is partially due to the absence of an high-throughput method for their simultaneous quantification together with other antiretroviral drugs. In this work, we describe the development and validation of a new UPLC-MS/MS method to quantify these drugs, together with other fourteen antiretroviral agents, in human plasma. One hundred microliters of plasma samples were added with internal standard (6,7-Dimethyl- 2,3-di(2-pyridyl) quinoxaline), underwent a simple protein precipitation with methanol:acetonitrile (50:50v/v) followed by sample dilution with water. Chromatographic separation was performed on a Acquity® UPLC HSS T3 column (150mm x 2.1mm I.D) with a particle size of 1.8µm and compounds were detected with a tandem mass detector, monitoring two ion transitions for each drugs. The mean recovery of RPV, DTG and EVG were 101%, 87% and 112.3% respectively. Accuracy and precision inter/intra-day were below 15% for all drugs, in accordance to Food and Drug Administration guidelines requirements. The UPLC-MS/MS method reported here could be used routinely to monitor plasma concentrations of antiviral drugs, including RPV, DTG and EVG.

Simultaneous quantification of tenofovir, emtricitabine, rilpivirine, elvitegravir and dolutegravir in mouse biological matrices by LC-MS/MS and its application to a pharmacokinetic study.

Combination antiretroviral (cARV) treatment is more common in human immunodeficiency virus (HIV) infection. In many instances, treatment regimen includes two or more combination of drugs from six different classes. Some of the antiretroviral combination medications are under study at preclinical and clinical stages. A precise method is required to quantify the drug concentration in biological matrices to study pharmacokinetic behavior and tissue distribution profile in animals and/or humans. We have developed and validated a sensitive and precise liquid chromatography-tandem mass spectrometry method for simultaneous quantification of selected antiretroviral drugs, tenofovir (TNF), emtricitabine (FTC), rilpivirine (RPV), dolutegravir (DTG) and elvitegravir (EVG) in mouse biological matrices. This method involves a solid phase extraction, simple isocratic chromatographic separation using Restek Pinnacle DB BiPh column (50mm×2.1mm, 5µm) and mass spectrometric detection by an API 3200 Q Trap instrument. The total run time for each sample was 6min. The method was validated in the concentration range of 5-2000ng/mL for FTC, RPV, DTG, EVG and 10-4000ng/mL for TNF respectively with correlation coefficients (r(2)) higher than 0.9976. The results of intra and inter-run assay precision and accuracy were within acceptance limits for all the five analytes. This method was used to support the study of pharmacokinetics and tissue distribution profile of nanoformulated antiretroviral drugs in mice.

The impact of polymeric excipients on the particle size of poorly soluble drugs after pH-induced precipitation.

Active pharmaceutical ingredients (APIs) with strongly pH-dependent aqueous solubility can face the problem of precipitating from solution when the pH changes from acidic in the stomach to neutral in the intestine. The present work investigates the effect of two polymeric excipients - polyvinylpyrrolidone (PVP) and Soluplus - on the ability to either prevent precipitation, or to control the size distribution of precipitated particles when precipitation cannot be prevented. Two different APIs were compared, Dabigatran etexilate mesylate and Rilpivirine hydrochloride. The effect of excipient concentration on the precipitation behaviour during pH titration was systematically investigated and qualitatively different trends were observed: in case of Soluplus, which forms a micellar solution when critical micelle concentration is exceeded, precipitation was inhibited in the case of Dabigatran etexilate, which partitioned into the micelles. On the other hand, Rilpivirine precipitated independently of Soluplus concentration. In the case of PVP, which does not form micelles, precipitation could not be avoided. Increased polymer concentration, however prevented the aggregation of precipitated particles into larger cluster. The observed effect of PVP was especially pronounced for Rilpivirine. The main conclusion of this study is that a suitably chosen polymeric excipient can either prevent precipitation altogether or reduce the size of the resulting particles. The mechanism of action, however, seems-specific to a given molecule. It was also shown that the polymer-stabilised particles have a potential to redissolve.

Formulation and pharmacology of long-acting rilpivirine.

PURPOSE OF REVIEW: Rilpivirine (RPV), a nonnucleoside reverse transcriptase inhibitor, is a potent antiretroviral (ARV) effective for HIV treatment at 25 mg daily oral dose. Its physio-chemical and pharmacological properties enable formulation of RPV as a long-acting injectable nanosuspension. This review summarizes these properties supporting the potential of intermittent parenteral administration of rilpivirine long acting (RPV LA) in both treatment and prevention of HIV-1 infection. RECENT FINDINGS: RPV is unusual among ARVs in that its stability and solubility enable aqueous suspensions with high drug loading, so that injection volumes can be minimized. Such innovative nanosuspensions are well tolerated in animals and humans after intramuscular injection and provide sustained drug concentrations in systemic circulation. The pharmacological findings support further investigations of RPV LA injections every 4 or 8 weeks, both as a single agent for potential preexposure prophylaxis and as two-drug all-injectable maintenance therapy with cabotegravir long acting. SUMMARY: By building on expertise with long-acting injectable antipsychotic agents, RPV has been formulated as an agent for infrequent intramuscular dosing, in addition to its conventional oral tablet forms. The advantages of adherence to a regimen of intermittent injections may be significant.

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.