Preclinical Evaluation of Long-Acting Emtricitabine Semi-Solid Prodrug Nanoparticle Formulations.

Long-acting injectable (LAI) formulations promise to deliver patient benefits by overcoming issues associated with non-adherence. A preclinical assessment of semi-solid prodrug nanoparticle (SSPN) LAI formulations of emtricitabine (FTC) is reported here. Pharmacokinetics over 28 days were assessed in Wistar rats, New Zealand white rabbits, and Balb/C mice following intramuscular injection. Two lead formulations were assessed for the prevention of an HIV infection in NSG-cmah-/- humanised mice to ensure antiviral activities were as anticipated according to the pharmacokinetics. Cmax was reached by 12, 48, and 24 h in rats, rabbits, and mice, respectively. Plasma concentrations were below the limit of detection (2 ng/mL) by 21 days in rats and rabbits, and 28 days in mice. Mice treated with SSPN formulations demonstrated undetectable viral loads (700 copies/mL detection limit), and HIV RNA remained undetectable 28 days post-infection in plasma, spleen, lung, and liver. The in vivo data presented here demonstrate that the combined prodrug/SSPN approach can provide a dramatically extended pharmacokinetic half-life across multiple preclinical species. Species differences in renal clearance of FTC mean that longer exposures are likely to be achievable in humans than in preclinical models

Development of Prodrug Approaches for Long-Acting Injectable Nanoformulations of Emtricitabine-Based Regimens

Most current antiretroviral (ARV) medications for HIV treatment and prevention necessitate lifelong daily dosing, and require high levels of patient adherence to be effective. Long-acting injectable (LAI) ARVs would allow less frequent administration, improving adherence. While nanomilled solid drug nanoparticles (SDNs) displaying long-acting delivery of ARVs following intramuscular injection are reported, technologies currently used to produce LAI-ARVs are incompatible with the nucleos(t)ide reverse transcriptase inhibitors (NRTIs) due to their high water solubility. It is critical to overcome this challenge to development of LAI NRTI strategies as most current ARV combination therapies include NRTI backbone therapies. Our long-term goal is to develop LAI-ARV approaches using lipophilic NRTI prodrugs. These pro-moieties will decrease NRTI water solubility and improve compatibility for SDN formation by the proprietary emulsion templated/freeze drying (ETFD) approach developed at the University of Liverpool. To achieve this goal, I have designed and synthesized a variety of different prodrug strategies for the NRTI emtricitabine (FTC), a cytosine analog. My thesis research has explored the use of amino-masking groups, including carbamates, imines, and self-immolative strategies (trimethyl lock amide, and 4-alkyloxybenzyloxy), as well as 5’-hydroxyl masking groups, including carbonates and esters to decrease NRTI aqueous solubility. Prodrug activation kinetics were assessed in vitro via HPLC in human muscle, plasma, and liver fractions. Prodrugs were then screened for SDN compatibility via ETFD at the University of Liverpool. The most promising hits to emerge from these initial screens were then carried forward for screens at higher drug loading. Successful formulations emerging from these advanced screens were then used for preclinical PK studies in animal models, guided by in silico physiologically-based pharmacokinetic (PBPK) modeling. Carbamate-based FTC prodrugs demonstrated the feasibility of our prodrug approach. Carbamates bearing longer alkyl chains (C7 and C8) were most efficiently activated in all conditions, with hydrolysis rates in liver higher than those in plasma or muscle. Carbonate moieties at the 5’-position were hydrolyzed more efficiently than the carbamate moiety under all conditions. Structure-activity relationship studies at the 5’-OH of the octyl carbamate FTC prodrug demonstrated a tolerance for branching in close proximity to the core of the molecule by esterases responsible for prodrug cleavage. However, increasing the distance between the site of cleavage and the site of branching decreased the efficiency of cleavage. Nanoparticle screening at 10 wt% prodrug demonstrated a correlation between calculated logP and the number of hits obtained. LogP also correlated with the ability to form SDNs with higher drug loading, with a hit for an octyl carbamate/carbonate FTC prodrug SDN achieved at 70 wt. %. In vivo studies demonstrated that higher-loaded prodrug SDNs can provide plasma exposure of FTC in rats for up to 14 days. Taken together, this work suggests that carbamate-based prodrug approaches offer a promising starting point for development of LAI FTC toward a complete LAI-ARV regimen to improve adherence

Development of Prodrug Approaches for Long-Acting Injectable Nanoformulations of Emtricitabine-Based Regimens

Most current antiretroviral (ARV) medications for HIV treatment and prevention necessitate lifelong daily dosing, and require high levels of patient adherence to be effective. Long-acting injectable (LAI) ARVs would allow less frequent administration, improving adherence. While nanomilled solid drug nanoparticles (SDNs) displaying long-acting delivery of ARVs following intramuscular injection are reported, technologies currently used to produce LAI-ARVs are incompatible with the nucleos(t)ide reverse transcriptase inhibitors (NRTIs) due to their high water solubility. It is critical to overcome this challenge to development of LAI NRTI strategies as most current ARV combination therapies include NRTI backbone therapies. Our long-term goal is to develop LAI-ARV approaches using lipophilic NRTI prodrugs. These pro-moieties will decrease NRTI water solubility and improve compatibility for SDN formation by the proprietary emulsion templated/freeze drying (ETFD) approach developed at the University of Liverpool. To achieve this goal, I have designed and synthesized a variety of different prodrug strategies for the NRTI emtricitabine (FTC), a cytosine analog. My thesis research has explored the use of amino-masking groups, including carbamates, imines, and self-immolative strategies (trimethyl lock amide, and 4-alkyloxybenzyloxy), as well as 5’-hydroxyl masking groups, including carbonates and esters to decrease NRTI aqueous solubility. Prodrug activation kinetics were assessed in vitro via HPLC in human muscle, plasma, and liver fractions. Prodrugs were then screened for SDN compatibility via ETFD at the University of Liverpool. The most promising hits to emerge from these initial screens were then carried forward for screens at higher drug loading. Successful formulations emerging from these advanced screens were then used for preclinical PK studies in animal models, guided by in silico physiologically-based pharmacokinetic (PBPK) modeling. Carbamate-based FTC prodrugs demonstrated the feasibility of our prodrug approach. Carbamates bearing longer alkyl chains (C7 and C8) were most efficiently activated in all conditions, with hydrolysis rates in liver higher than those in plasma or muscle. Carbonate moieties at the 5’-position were hydrolyzed more efficiently than the carbamate moiety under all conditions. Structure-activity relationship studies at the 5’-OH of the octyl carbamate FTC prodrug demonstrated a tolerance for branching in close proximity to the core of the molecule by esterases responsible for prodrug cleavage. However, increasing the distance between the site of cleavage and the site of branching decreased the efficiency of cleavage. Nanoparticle screening at 10 wt% prodrug demonstrated a correlation between calculated logP and the number of hits obtained. LogP also correlated with the ability to form SDNs with higher drug loading, with a hit for an octyl carbamate/carbonate FTC prodrug SDN achieved at 70 wt. %. In vivo studies demonstrated that higher-loaded prodrug SDNs can provide plasma exposure of FTC in rats for up to 14 days. Taken together, this work suggests that carbamate-based prodrug approaches offer a promising starting point for development of LAI FTC toward a complete LAI-ARV regimen to improve adherence

Semi-solid prodrug nanoparticles for long-acting delivery of water-soluble antiretroviral drugs within combination HIV therapies

The increasing global prevalence of human immunodeficiency virus (HIV) is estimated at 36.7 million people currently infected. Lifelong antiretroviral (ARV) drug combination dosing allows management as a chronic condition by suppressing circulating viral load to allow for a near-normal life; however, the daily burden of oral administration may lead to non-adherence and drug resistance development. Long-acting (LA) depot injections of nanomilled poorly water-soluble ARVs have shown highly promising clinical results with drug exposure largely maintained over months after a single injection. ARV oral combinations rely on water-soluble backbone drugs which are not compatible with nanomilling. Here, we evaluate a unique prodrug/nanoparticle formation strategy to facilitate semi-solid prodrug nanoparticles (SSPNs) of the highly water-soluble nucleoside reverse transcriptase inhibitor (NRTI) emtricitabine (FTC), and injectable aqueous nanodispersions; in vitro to in vivo extrapolation (IVIVE) modelling predicts sustained prodrug release, with activation in relevant biological environments, representing a first step towards complete injectable LA regimens containing NRTIs

Anhydrous nanoprecipitation for the preparation of nanodispersions of tenofovir disoproxil fumarate in oils as candidate long-acting injectable depot formulations

The facile formation of drug nanoparticles in injectable/ingestible oils, of water-soluble antiretroviral tenofovir disoproxil fumarate, using a novel nanoprecipitation is presented with studies showing drug release into relevant aqueous media