Synthesis of a long acting nanoformulated emtricitabine ProTide

Abstract

While antiretroviral therapy (ART) has revolutionized treatment and prevention of human immunodeficiency virus type one (HIV-1) infection, regimen adherence, viral mutations, drug toxicities and access stigma and fatigue are treatment limitations. These have led to new opportunities for the development of long acting (LA) ART including implantable devices and chemical drug modifications. Herein, medicinal and formulation chemistry were used to develop LA prodrug nanoformulations of emtricitabine (FTC). A potent lipophilic FTC phosphoramidate prodrug (M2FTC) was synthesized then encapsulated into a poloxamer surfactant (NM2FTC). These modifications extended the biology, apparent drug half-life and antiretroviral activities of the formulations. NM2FTC demonstrated a >30-fold increase in macrophage and CD4+ T cell drug uptake with efficient conversion to triphosphates (FTC-TP). Intracellular FTC-TP protected macrophages against an HIV-1 challenge for 30 days. A single intramuscular injection of NM2FTC, at 45 mg/kg native drug equivalents, into Sprague Dawley rats resulted in sustained prodrug levels in blood, liver, spleen and lymph nodes and FTC-TP in lymph node and spleen cells at one month. In contrast, native FTC-TPs was present for one day. These results are an advance in the transformation of FTC into a LA agent.

Keywords: Emtricitabine; Formulation; Long-acting slow effective release anti-retroviral therapy (LASER) ART; Prodrug.

Fig. 1.. M2FTC physicochemical characterization.

(A) Synthesis of aryl amino phosphochloridate. N-Cbz-L-Phenylalanine was dissolved in DMF and CFM. Cooled on ice. HATU, docosanol, imidazole and Et₃N were added and reaction was stirred at RT for 48 h. Product was dissolved in CFM and MeOH. Palladium on carbon was added and cooled to 4 °C, Et₃SiH was added and stirred for 16 h. Product was dissolved in DCM at −78 °C, Phenyl dichlorophosphate was then added, followed by Et₃N/CFM addition, stirred at 48 h RT. (B) FTC was dissolved in THF at −78 °C, ^tBuMgCl was added and stirred at 48–90 h at RT. FTC was then reacted with product formed in previous step with a 61% yield (C) Solubility of FTC and M2FTC in 1-octanol and water was measured at 37°C. Data are expressed as mean ± SEM for n = 3 samples were evaluated. Statistical significance was measured using one way ANOVA at ****P < 0.0001. (D) Fourier transformed infra-red spectroscopic (FT-IR) analysis showed the presence of carbonyl and aromatic stretching vibrations in the region of 2900 – 3100 cm⁻¹ for FTC and M2FTC and phosphoramide (P-N-H) bending vibrations between 1600 – 1700 cm⁻¹ in M2FTC, confirming the presence of these functionalities. (E) XRD analysis of FTC and M2FTC supporting unique atomic arrangement in the compounds crystal lattice. F) Hydrolysis study in different species’ plasma demonstrated chemical stability of M2FTC over 90% in both normal and heat inactivated human plasma up to 24 h at 37 °C and gradual hydrolysis observed of up to 40% in mice and rats, dog, rabbit and in monkey plasma over 24 h.

Fig. 2.. NM2FTC characterizations.

A) Physical stability evaluation of NM2FTC at 25 °C in terms of particle size, zeta potential and polydispersity index over 1 month. (B) EC₅₀ was determined in MDM over a range of concentrations (0.001–1000 nM) by determining HIV-1 RT activity after FTC or NM2FTC treatments in cells infected with HIV-1_ADA. Results were analyzed by nonlinear regression least squares fit. Results are shown as the mean ± SEM of three replicates. C) Cell vitality was assessed in MDM by MTT assay 24 h after FTC, M2FTC or NM2FTC treatments over a range of concentrations (10 – 400 μM). Results were normalized to untreated control cells. Both M2FTC and NM2FTC were found to be non-cytotoxic at 100 μM or less. Data are represented as mean ± SEM for n = 3 samples per group. D) Transmission electron microscopy (TEM) of NM2FTC showed presence of nanoparticles predominantly in the size range of 100–250 nm. E) EC₅₀ was determined in CEM CD4+ T-cells by RT activity measurement in the supernatant after FTC or NM2FTC treatments over a range of concentrations (0.1–10,000 nM). Results were analyzed by nonlinear regression least squares fit. F) CEM-ss CD4+ T-cell vitality using LiveDead staining. All the treatments were non-toxic at 200 μM of drug or less. G) TEM morphological evaluation in MDM and CEM-ss CD4+ T-cells after NM2FTC (100 μM) treatment for 8 h, washing with PBS (2x) and re-suspension in TEM fixation buffer.

Fig. 3. NM2FTC uptake, retention and triphosphate (TP) conversion.

A) For uptake, MDM were treated with equal drug concentrations (100 μM) and uptake was determined over 8 h. B) For retention study, MDM were loaded with FTC or NM2FTC for 8 h followed by PBS washing and maintaining half media changes every other day for 30 days.. (C) Intracellular FTC TP levels during uptake studies was quantified at various time points over the 8h experiment time course. (D) Intracellular FTC TP levels were measured at different time points over 30-days. E) M2FTC, and F) FTC TP levels in parallel uptake studies in CEM-ss CD4+ T-cells were carried out as for MDMs.

Fig. 4.. Cell-based antiretroviral activities.

Antiretroviral activity was determined in MDM treated for 8 h with 100 μM FTC or NM2FTC and then infected with HIV-1_ADA at 1 to 30 days following drug loading. HIV-1 replication was determined 10 days after infection by A) Time course HIV-1 RT activity in culture supernatants. RT activity results were confirmed by B) HIV-1p24 antigen expression (brown stain) of adherent MDM. NM2FTC protected MDM from HIV-1 infection at all time points. Results were normalized to positive control cells. All results are shown as the mean ± SEM with n = 3. C) Comparative HIV-1 RT activity at day 30 showing statistical significance in the % RT activity between FTC and NM2FTC treatments using unpaired two tailed t-test at ****P < 0.0001.

Fig. 5.. PK and biodistribution of FTC and NM2FTC in rats.

Blood and tissue M2FTC and FTC concentrations were determined over 28 days. A) FTC and M2FTC levels in whole blood after a single IM injection of FTC or NM2FTC in Sprague Dawley rats (45 mg/kg FTC equivalents). M2FTC and FTC concentrations were determined in (B) lymph nodes, (C) spleen, and (D) liver. Data are expressed as mean ± SEM for n = 5 rats per group. Formation of FTC-TP in (E) lymph node and (F) spleen cells were determined. Drug concentrations were quantified by UPLC-MS/MS.