A comparison of tenofovir alafenamide (TAF) plus emtricitabine (FTC) in solution and as nanoparticles (NPs) for pre-exposure prophylaxis (PrEP) in humanized BLT (hu-BLT) mice Subhra Mandal1, Guobin Kang2, Pavan Kumar Prathipati1, Zhe Yuan2, You Zhou2, Wenjin Fan2, Qingsheng Li2, Christopher J Destache1 1Sch of Pharm & Health Professions, Creighton Univ, Omaha, NE  2Ctr for Virol, Sch of Bio Sci, Univ of Nebraska-Lincoln, Lincoln, NE 68583 9th IAS Conference on HIV Science (IAS 2017) July 23-26 2017 Paris Introduction Tenofovir and emtricitabine (TFV+FTC) has been approved for PrEP in a wide variety of high-risk people. TAF+FTC is currently under investigation for PrEP. My laboratory has been involved in fabricating nanomedicines for HIV-1 treatment and prevention since 2006. Results Figure 1. Formulation: Pharmacokinetic Disposition: 200 mg/kg SubQ NP: 100 mg/kg SubQ NP: ND=Not Detected Summary: TFV and FTC drug levels (> 50 ng/G) in vaginal tissues appears to correlate with PrEP efficacy for 200 mg/kg TAF+FTC NP. Lack of efficacy in 100 mg/kg TAF+FTC NP group correlates with low TFV and FTC vaginal levels. Active metabolite TFV or FTC peak or AUC in PBMCs did not correlate with PrEP efficacy for either 100 and 200 mg/kg dosages. Combined TFV and FTC drug levels on D14 were < 50 ng/G for 200 mg/kg TAF+FTC NP, potentially allowing HIV-1 virus to target CD4 within the vaginal tissue. PrEP experiments: Summary: Significant protection against HIV-1 was demonstrated for Hu-BLT mice receiving 200 mg/kg TAF+FTC NP and challenged with T/F virus at Day 4 and 7 with 60% efficacy compared to control mice. Hu-BLT mice challenged at Day 14, were moderately protected (40%). The 100 mg/kg TAF+FTC NP compared to drugs in solution experiment, did not appear efficacious regardless of the challenge date as all Hu-BLT mice became infected by D21. Delay in pVL positivity for D4 and D7 could be a result of declining vaginal tissue drug levels and once the drug levels were < 50 ng/G, allowed for a detectable pVL. Figure 4. Kaplan-Meier infectivity curve for Hu-BLT mice receiving (A) 200 mg/kg and (B) 100 mg/kg TAF+FTC NP or drugs in solution. Mice were challenged with T/F HIV-1 strains at 4,7, or 14 days after SubQ administration of PrEP. Size (nm) (mean ± SD) % Encapsulation efficiency TAF + FTC NP 233.2 ± 12.8 TAF: 69.2 ± 14.5 FTC: 65.9 ± 18.2 Purpose The present work was conducted to perform a comparative efficacy and pharmacokinetics of TAF+FTC loaded NPs to drug solution in a humanized mouse model for PrEP. Humanized CD34 mice (Hu-CD34) were used to characterize pharmacokinetic (PK) disposition study in the NP formulations versus solution. Humanized BLT mice (Hu-BLT) were used to test HIV-1 protection efficacy of TAF+FTC NP and solution over time. Figure 2. 200 mg/kg TAF+FTC NPs treated TFV+FTC PK analysis. Comparative study of TFV and FTC at (A) plasma, (B) active metabolites, (C) vaginal and (D) female reproductive tract (FRT) tissues. Concentration of (E) TFV and (F) FTC at all tissues obtained. Methods The TAF+FTC NPs were fabricated as previously described [1,2] with some modifications. Briefly, ARV drugs (TAF and FTC) loaded poly-(Lactic-co-Glycolic Acid) (PLGA) NPs were formulated along with the surfactant Pluronic F127 by water-in-oil-in-water (w-o-w) emulsion procedure (Figure 1). The size distribution were determined by dynamic light scattering (DLS) by using ZetaPlus instrument [1,2]. The percentage encapsulation efficiency (%EE) were evaluated by High Performance Liquid Chromatography (HPLC) analysis (Shimadzu, Kyoto, Japan) [1,2]. The morphology of the NPs were analyzed by scanning electron microscopy (SEM) imaging under a Hitachi S-4700 Field-emission SEM [1,2]. PK and HIV prevention studies, TAF+FTC NPs were reconstituted with 5% dextrose for subcutaneous (SubQ) administration. PK studies were performed as previously described [3]. Each time point, Hu-CD34 mice (n=3) received 100 or 200 mg/kg of each drug, TAF+FTC as NP encapsulated form (TAF+FTC NP) or in solution. 100 mg/kg TAF+FTC NP treated mice, plasma and tissue (Figure 3) were harvested at 1, 2, 4, 7, 10, and 14 days after injection. Whereas, for 200 mg/kg TAF+FTC NP treated mice, plasma and tissue were harvested at 1, 4, and 12 h and 1, 2, 4, 7, 10, 14 days after injection (Figure 2). Further, peripheral blood mononuclear cells (PBMCs) were isolated to evaluate active metabolite concentration of TAF and FTC. PK samples were analyzed using an AB SciEx 5500, Qtrap LC-MS/MS. The LC-MS/MS method were used to analyze intracellular TFV active metabolite levels [4]. Quality control (QC) concentrations were calculated as ± 10% of the nominal value to be acceptable. Pharmacokinetic parameters were analyzed by Phoenix WinNonLin software. Area-under-the-vaginal-concentration-time data was determined using the trapezoid rule from the software (Table). These tissue concentrations were used to determine the amount of drugs at specific times at the site of infection For PrEP studies, Hu-BLT mice (n=5/experimental group) were treated with TAF+FTC NP or solution at 100 or 200 mg/kg each drug. On days 4, 7, 14 days post injection (PI) treatment (Rx) mice received 5 x 105 TCID50 in 20 mL from two HIV transmission/founder (T/F) viruses (WITO.c/2474 and SUMA.c/2821) from acutely infected patients intravaginally. Control (Ctr) mice (n=5) received blank NPs and were infected with the same T/F viruses on Day 4 PI. Mice receiving 100 mg/kg TAF+FTC solution were challenged with the T/F viruses on day 4 PI. Weekly blood was drawn for plasma viral load (pVL), starting 2 weeks after viral challenge & continued for 6 weeks. The pVL was determined by RT-PCR analysis as previously described (2). To evaluate HIV-1 viral infectivity function, Kaplan-Meier curve fitting was performed. A p-value of ≤ 0.05 was considered statistically significant. (Figure 4) Figure 3. 100 mg/kg TAF+FTC NPs treated TFV+FTC PK analysis. Comparative study of TFV and FTC at (A) plasma, (B) active metabolites, (C) vaginal and (D) female reproductive tract (FRT) tissues. Concentration of (E) TFV and (F) FTC at all tissues obtained Conclusions: TAF+FTC NP efficacy was significantly higher for 200 mg/kg at D4 and D7 [60% protection] compared to control Hu-BLT mice with 0% protection. Vaginal tissue TFV+FTC drug levels > 50 ng/G were indicative of HIV-1 protection. Further studies are needed to determine whether a concentration-response relationship exists between tissue drug levels and efficacy. Literature Cited: 1. Mandal S, Zhou Y, Shibata A, & Destache CJ (2015). AIP Advances 5(8):084803. 2. Mandal S, Prathipati PK, Kang G, et al. (2016). AIDS 2016 Dec DOI: 10.1097/AQD.0000000000001349. 3. Prathipati PK, Mandal S, Destache CJ (2016) J Pharm Biomed Analy 129:473-481. doi:http://dx.doi.org/10.1016/j.jpba.2016.07.040 4. Mandal S, Belshan M, Holec A, Zhou Y, Destache CJ (2017). Antimicrob Agents Chemother 61 (1). doi:10.1128/aac.01475-16 Pharmacokinetic parameter TFV from NP Formulation FTC from NP formulation Mean vaginal Cmax (mg/mL) 100: 0.434 200: 15.9 100: 0.02 200: 34.0 Mean vaginal Cmin (mg/mL) 4D 7D 14D 200 0.163 0.053 0.011 100 0.077 0.021 ND  200 0.029 0.090 0.004 Plasma AUCall (hr*mg/mL) 100: 65.3 200: 61.0 100: 2.6 200: 65.3 Acknowledgement: This research was supported by NIAID AI117740. Contact Information: Chris Destache, Pharm. D. Professor of Pharmacy Practice and Infectious Diseases Creighton University School of Pharmacy 2500 California Plaza Omaha, NE 68178 destache@creighton.edu