A Concept Evaluation Study of a New Combination Bictegravir plus Tenofovir Alafenamide Nanoformulation with Prolonged Sustained-Drug-Release Potency for HIV-1 Preexposure Prophylaxis.

The antiretroviral treatment (ART) approach is the best-prescribed approach to date for preexposure prophylaxis (PrEP) for high-risk individuals. However, the daily combination antiretroviral (cARV) regimen has become cumbersome for healthy individuals, leading to nonadherence. Recent surveys showed high acceptance of parenteral sustained-release ART enhancing PrEP adherence. Our approach is to design a parenteral nanoparticle (NP)-based cARV sustained-release (cARV-SR) system as long-acting HIV PrEP. Here, we report a new combination of two potent ARVs (tenofovir alafenamide fumarate [TAF] and bictegravir [BIC]) loaded as a nanoformulation intended as a cARV-SR for PrEP. The BIC+TAF NPs were fabricated by using a standardized in-house methodology. In vitro intracellular kinetics, cytotoxicity, and HIV-1 protection studies demonstrated that BIC+TAF encapsulation prolonged drug retention, reduced drug-associated cytotoxicity, and enhanced HIV protection. In human peripheral blood mononuclear cells, nanoformulated BIC+TAF demonstrated significant (P < 0.05) improvement in the drug's selectivity index by 472 times compared to the BIC+TAF in solution. In vivo pharmacokinetic study of BIC, TAF, and respective drug metabolites in female BALB/c mice after single subcutaneous doses of BIC+TAF NPs demonstrated plasma drug concentrations of BIC and tenofovir above the intracellular 50% inhibitory concentration during the entire 30-day study period and prolonged persistence of both active drugs in the HIV target organs, including the vagina, colon, spleen, and lymph nodes. This report demonstrates that the encapsulation of BIC+TAF in a nanoformulation improved its therapeutic selectivity and the in vivo pharmacokinetics of free drugs. Based on these preliminary studies, we hypothesize that cARV-SR has potential as an innovative once-monthly delivery treatment for PrEP.

Correction to: Pharmacokinetic and Tissue Distribution Profile of Long Acting Tenofovir Alafenamide and Elvitegravir Loaded Nanoparticles in Humanized Mice Model.

There are errors in the published article (Volume 34, Number 12, pp. 2749-2755). The errors occurred in pharmacokinetic study design of materials and methods section on page 2750-51.

Development and validation of LC-MS/MS method for quantification of bictegravir in human plasma and its application to an intracellular uptake study.

A sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for quantification of bictegravir in human plasma. A small volume of only 50 µL aliquot of plasma was precipitated with acetonitrile containing an internal standard (IS). Chromatographic separation was performed on a Kinetex EVO C18 column, 50 × 3.0 mm, 5 µm using an isocratic mobile phase containing 80:20 acetonitrile-water with 0.1% formic acid. A mass spectrometer was operated in ESI positive multiple reaction monitoring mode using the m/z 450.1/289.1 for bictegravir and 420.1/277.1 for IS. The dynamic range of the method was 1-10,000 ng/mL with a correlation coefficient of ≥0.9991. The precision results of calibration standards were in the range of 0.05-4.57% and accuracies were 95.07-104.70%. The mean extraction recovery was 98.64% with a precision of 2.91%. The method was validated as per US Food and Drug Administration guidelines and was found to be accurate and precise. The method was successfully applied to in vitro cellular uptake study.

LC-MS/MS method for the simultaneous determination of tenofovir, emtricitabine, elvitegravir and rilpivirine in dried blood spots.

A simple, short, and rugged LC-MS/MS method for the simultaneous determination of tenofovir, emtricitabine, elvitegravir and rilpivirine was developed and validated. Dried blood spots were prepared with 25 µL of spiked whole blood. A 3 mm punch was extracted with methanol containing labeled internal standards. Ten microliters was injected into the LC-MS/MS using isocratic mobile phase composed of 0.1% formic acid in water and 0.1% formic acid in acetonitrile (45: 55 v/v) at a flow rate of 0.25 mL/min. The method was validated in the range of 10-2000 ng/mL for all four analytes. The intra-assay accuracy (RE) of the method was -4.73-4.78, 1.35-2.89, -8.89 to -0.49 and - 1.40-1.81 for tenofovir, emtricitabine, elvitegravir and rilpivirine, respectively. The inter-assay accuracy was within ±15% of nominal and precision (CV) was <15%. The hematocrit effect on quantification was nonsignificant at the tested hematocrit levels (35-70%). The dried blood spot method showed good agreement with the plasma method, and hence can be used as an alternative to plasma method.

Pharmacokinetic and Tissue Distribution Profile of Long Acting Tenofovir Alafenamide and Elvitegravir Loaded Nanoparticles in Humanized Mice Model.

PURPOSE: Non-adherence to the antiretroviral (ARV) regimen is a critical factor in determining efficacy of ARV drugs for pre-exposure prophylaxis (PrEP). A long-acting parenteral formulation may be an effective alternative to daily oral dosing. A pharmacokinetic and tissue distribution study of drug-loaded nanoparticle (NP) was performed in female humanized CD34+-NSG mice. METHODS: Mice received 200 mg/kg each of tenofovir alafenamide (TAF) and elvitegravir (EVG) as free drugs (TAF + EVG solution) or as drug loaded NP (TAF + EVG NP) formulation by subcutaneous (SubQ) administration. Plasma and tissue were collected to determine tenofovir (TFV) and EVG concentrations using LC-MS/MS. Non-compartmental analysis was performed using WinNonlin. RESULTS: SubQ administration of TAF + EVG NP formulation resulted in long residence time and exposure for both drugs. The AUC(0-72h) of TFV and EVG was 14.1 ± 2.0, 7.2 ± 1.8 µg × hr./mL from drugs in solution (free) and the AUC(0-14day) for the same drugs was 23.1 ± 4.4, 39.7 ± 6.7 µg × hr./mL from NPs. The observed elimination half-life (t1/2) for TFV of free and NPs were 14.2 h, 5.1 days and for EVG 10.8 h, 3.3 days, respectively. CONCLUSION: This study documents that a TAF + EVG NP provides sustained release, which can overcome patient non-adherence to dosing and may facilitate prediction of appropriate protective drug concentration for HIV prophylaxis.

Structural Determinants of Indole-2-carboxamides: Identification of Lead Acetamides with Pan Antimycobacterial Activity.

Tuberculosis (TB), caused by Mycobacterium tuberculosis (M.tb), is one of the leading causes of death in developing countries. Non-tuberculous mycobacteria (NTM) infections are rising and prey upon patients with structural lung diseases such as chronic obstructive pulmonary disease (COPD) and cystic fibrosis. All mycobacterial infections require lengthy treatment regimens with undesirable side effects. Therefore, new antimycobacterial compounds with novel mechanisms of action are urgently needed. Published indole-2-carboxamides (IC) with suggested inhibition of the essential transporter MmpL3 showed good potency against whole-cell M.tb, yet had poor aqueous solubility. This project focused on retaining the required MmpL3 inhibitory pharmacophore and increasing the molecular heteroatom percentage by reducing lipophilic atoms. We evaluated pyrrole, mandelic acid, imidazole, and acetamide functional groups coupled to lipophilic head groups, where lead acetamide-based compounds maintained high potency against mycobacterial pathogens, had improved in vitro ADME profiles over their indole-2-carboxamide analogs, were non-cytotoxic, and were determined to be MmpL3 inhibitors.

Targeted Immuno-Antiretroviral to Promote Dual Protection against HIV: A Proof-of-Concept Study.

The C-C motif chemokine receptor-5 (CCR5) expression on the T-cell surface is the prime barrier to HIV/AIDS eradication, as it promotes both active human immunodeficiency virus (HIV)-infection and latency; however, antiretrovirals (ARVs) suppress plasma viral loads to non-detectable levels. Keeping this in mind, we strategically designed a targeted ARVs-loaded nanoformulation that targets CCR5 expressing T-cells (e.g., CD4+ cells). Conceptually, CCR5-blocking and targeted ARV delivery would be a dual protection strategy to prevent HIV infection. For targeting CCR5+ T-cells, the nanoformulation was surface conjugated with anti-CCR5 monoclonal antibodies (CCR5 mAb) and loaded with dolutegravir+tenofovir alafenamide (D+T) ARVs to block HIV replication. The result demonstrated that the targeted-ARV nanoparticle's multimeric CCR5 binding property improved its antigen-binding affinity, prolonged receptor binding, and ARV intracellular retention. Further, nanoformulation demonstrated high binding affinity to CCR5 expressing CD4+ cells, monocytes, and other CCR5+ T-cells. Finally, the short-term pre-exposure prophylaxis study demonstrated that prolonged CCR5 blockage and ARV presence further induced a "protective immune phenotype" with a boosted T-helper (Th), temporary memory (TM), and effector (E) sub-population. The proof-of-concept study that the targeted-ARV nanoformulation dual-action mechanism could provide a multifactorial solution toward achieving HIV "functional cure."

A potential long-acting bictegravir loaded nano-drug delivery system for HIV-1 infection: A proof-of-concept study.

Bictegravir (BIC), a newly FDA-approved integrase strand transfer inhibitor (INSTI), as a single tablet regimen has proven efficacious in treating HIV-1 and SIV viruses, with reduced resistance. BIC clinical trials have not investigated its prophylaxis potency. This study investigates the HIV prevention potency of a novel long-acting BIC nano-formulation aimed to improve adherence. Poly (lactic-co-glycolic acid) loaded BIC nanoparticles (BIC NPs) were formulated using an oil-in-water emulsion methodology. BIC NPs were <200 nm in size, with 47.9 ±â€¯6.9% encapsulation efficiency. A novel, sensitive and high throughput LC-MS/MS method was used to estimate intracellular pharmacokinetics (PK) of BIC NPs and compared to BIC solution demonstrated prolonged intracellular BIC retention. BIC NPs safety was assessed based on cytotoxicity. Further, in-vitro prevention study of BIC NPs vs BIC solution was assessed against HIV-1NLX and HIV-1ADA on TZM-bl cell line and PBMCs, respectively. BIC nanoencapsulation demonstrated elevated cellular cytotoxicity concentration (CC50: 2.25 µM (BIC solution) to 820.4 µM (BIC NPs)] and lowers HIV-1 inhibitory concentration [EC50: 0.604 µM (BIC solution) to 0.0038 µM (BIC NPs)) thereby improving selectivity index (SI) from 3.7 (BIC solution) to 215,789 (BIC NP) for TZM-bl cells. Comparable results in PBMCs were obtained where BIC NPs improved SI from 0.29 (BIC solution) to 523.33 (BIC NPs). This demonstrates long-acting BIC nano-formulation with sustained drug-release potency, improved BIC cytotoxicity and enhanced HIV-1 protection compared to BIC in solution.

A review of CCR5 antibodies against HIV: current and future aspects.

HIV is one of the most devastating viral infections the world has ever encountered. Ever since HIV was first identified in the 1980s, it has claimed millions of lives worldwide. There has been tremendous research and development in the diagnosis, prevention and treatment of HIV. Small molecules have been shown to reduce the virus to nondetectable level in human plasma, however, there are reservoirs of latent virus that reemerge if antiretroviral therapy is stopped. There is no vaccine to prevent or cure HIV. A significant amount of research has been reported in the literature regarding antibodies for CCR5, a HIV entry host receptor. This report describes the role of CCR5 antibody in HIV prevention/treatment and how antibody-conjugated nanoparticles could be a future strategy with the potential to effectively eradicate the virus from the human system.

Nanoencapsulation introduces long-acting phenomenon to tenofovir alafenamide and emtricitabine drug combination: A comparative pre-exposure prophylaxis efficacy study against HIV-1 vaginal transmission.

Daily oral antiretroviral (ARV) drugs for pre-exposure prophylaxis (PrEP) has proven efficacy for diverse groups of high-risk individuals. However, daily dosing regimen has augmented non-adherence. These experiments comparatively investigated the long-acting (LA) PrEP potency of subcutaneous (SubQ) administrated tenofovir alafenamide (TAF) and emtricitabine (FTC) loaded nanoparticles (NPs) to solution in humanized (hu) mice. TAF + FTC NPs and TAF + FTC solution (each drug at 200 mg/kg) were administered to hu-CD34-NSG mice (n = 3/time point) for plasma and tissue pharmacokinetic parameter estimation using LC-MS/MS. NP enhanced tissue ARV assimilation compared to plasma. The same dose was administered for PrEP efficacy in HIV-1 challenged hu-BLT mice (n = 5/group). The hu-BLT mice were vaginally challenged with a transmission-founder (T/F) virus at 5 × 105 TCID50 inoculation, on day 4, 7 and 14 post-SubQ treatments (PT) and were compared to infected-untreated-control hu-BLT mice. By 21 days PT, 100% TAF + FTC solution-treated and control-untreated mice were infected. However, TAF + FTC NPs resulted in significant (p = .0002) protection from HIV-1 (day 4: 80%, day 7 and 14: 60%, respectively) compared to control mice. This proof-of-concept study demonstrated detectable TAF/FTC vaginal levels among TAF + FTC NP-treated hu-BLT mice correlating with prolonged PrEP efficacy, thus establishing long-acting TAF + FTC NPs as a potential PrEP modality.

Long-acting parenteral combination antiretroviral loaded nano-drug delivery system to treat chronic HIV-1 infection: A humanized mouse model study.

Human immunodeficiency virus (HIV) patients are often diagnosed in the chronic stage of HIV/AIDS. Combination antiretroviral therapy (cART) has improved quality of life for HIV-infected patients. Present study describes a novel long-acting parenteral formulation of combination antiretroviral (cARV) loaded nano-drugs for treating chronic HIV-1 (cHIV) in a humanized-BLT (hu-BLT) mice model. The cARV (elvitegravir+tenofovir alafenamide+emtricitabine; EVG+TAF+FTC) drugs (mimicking marketed Genvoya® one-pill for HIV-treatment) were encapsulated in poly (lactic-co-glycolic acid) nanoparticles (NPs). To establish cHIV, hu-BLT mice were intravaginally challenged with HIV-1 and maintained for 15 weeks. Plasma viral load (pVL) was monitored by RT-PCR to confirm cHIV. Baseline pVL (week 15) was comparable between treated (n = 10) and control (n = 5) mice groups. Subsequently, treatment hu-BLT mice received 3 subcutaneous doses of cARV NPs (417 mg/kg per dose; n = 10), biweekly, and a fourth/terminal dose a week later. Prior to each treatment and on sacrifice (week 24), pVL was determined. Within three subcutaneous doses of cARV NPs, a non-detectable pVL was established (week 19) and continued until week 22. After the establishment of a non-detectable pVL (week 19-22), 4 treated-mice were sacrificed for tissue drug concentration determination by LC-MS/MS analysis. A considerable amount of cARV was detected at the HIV-infection target and reservoir organs. Subsequently, pVL rebounded comparable to control group by week 24, (7 weeks post-terminal dosage). The present study demonstrated cARV NPs augments sustained ARV efficacy in the cHIV humanized-mouse model. Therefore, cARV NPs could be a novel delivery system to treat cHIV patients, by overcoming drawbacks of conventional cART.

Discovery of a non-nucleoside RNA polymerase inhibitor for blocking Zika virus replication through in silico screening.

Zika virus (ZIKV), an emerging arbovirus, has become a major human health concern globally due to its association with congenital abnormalities and neurological diseases. Licensed vaccines or antivirals against ZIKV are currently unavailable. Here, by employing a structure-based approach targeting the ZIKV RNA-dependent RNA polymerase (RdRp), we conducted in silico screening of a library of 100,000 small molecules and tested the top ten lead compounds for their ability to inhibit the virus replication in cell-based in vitro assays. One compound, 3-chloro-N-[({4-[4-(2-thienylcarbonyl)-1-piperazinyl]phenyl}amino)carbonothioyl]-1-benzothiophene-2-carboxamide (TPB), potently inhibited ZIKV replication at sub-micromolar concentrations. Molecular docking analysis suggests that TPB binds to the catalytic active site of the RdRp and therefore likely blocks the viral RNA synthesis by an allosteric effect. The IC50 and the CC50 of TPB in Vero cells were 94 nM and 19.4 µM, respectively, yielding a high selective index of 206. In in vivo studies using immunocompetent mice, TPB reduced ZIKV viremia significantly, indicating TPB as a potential drug candidate for ZIKV infections.

Nucleotide Reverse Transcriptase Inhibitors: A Thorough Review, Present Status and Future Perspective as HIV Therapeutics.

BACKGROUND: Human immunodeficiency virus type-1 (HIV-1) infection leads to acquired immunodeficiency syndrome (AIDS), a severe viral infection that has claimed approximately 658,507 lives in the US between the years 2010-2014. Antiretroviral (ARV) therapy has proven to inhibit HIV-1, but unlike other viral illness, not cure the infection. OBJECTIVE: Among various Food and Drug Administration (FDA)-approved ARVs, nucleoside/ nucleotide reverse transcriptase inhibitors (NRTIs) are most effective in limiting HIV-1 infection. This review focuses on NRTIs mechanism of action and metabolism. METHODS: A search of PubMed (1982-2016) was performed to capture relevant articles regarding NRTI pharmacology. RESULTS: The current classical NRTIs pharmacology for HIV-1 prevention and treatment are presented. Finally, various novel strategies are proposed to improve the efficacy of NRTIs, which will increase therapeutic efficiency of present-day HIV-1 prevention/treatment regimen. CONCLUSION: Use of NRTIs will continue to be critical for successful treatment and prevention of HIV-1.

Cellulose Acetate Phthalate and Antiretroviral Nanoparticle Fabrications for HIV Pre-Exposure Prophylaxis.

To adequately reduce new HIV infections, development of highly effective pre-exposure prophylaxis (PrEP) against HIV infection in women is necessary. Cellulose acetate phthalate (CAP) is a pH sensitive polymer with HIV-1 entry inhibitory properties. Dolutegravir (DTG) is an integrase strand transfer inhibitor with potent antiretroviral activity. DTG delivered in combination with CAP may significantly improve current PrEP against HIV. In the present study the development of DTG-loaded CAP nanoparticles incorporated in thermosensitive (TMS) gel at vaginal pH 4.2 and seminal fluid pH 7.4 is presented as proof-of-concept for improved PrEP. Water-oil-in-water homogenization was used to fabricate DTG-loaded CAP nanoparticles (DTG-CAP-NPs). Size, polydispersity, and morphological analyses illustrate that DTG-CAP-NPs were smooth and spherical, ≤200 nm in size, and monodispersed with a polydispersity index PDI ≤ 0.2. The drug encapsulation (EE%) and release profile of DTG-CAP-NPs was determined by HPLC analysis. The EE% of DTG in DTG-CAP-NPs was evaluated to be ∼70%. The thermal sensitivity of the TMS gel was optimized and the pH dependency was evaluated by rheological analysis. DTG release studies in TMS gel revealed that DTG-CAP-NPs were stable in TMS gel at pH 4.2 while DTG-CAP-NPs in TMS gel at pH 7.4 rapidly release DTG (≥80% release within 1 h). Cytotoxicity studies using vaginal cell lines revealed that DTG-CAP-NPs were relatively non-cytotoxic at concentration <1 µg/mL. Confocal microscopic studies illustrate that ≥98% cells retained DTG-CAP-NPs intracellularly over seven days. Antiretroviral drug loaded nanocellulose fabrications in TMS gel delivered intravaginally may enhance both microbicidal and antiretroviral drug efficacy and may present a novel option for female PrEP against HIV.

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.