Development of Human Immunodeficiency Virus Type 1 Resistance to 4'-Ethynyl-2-Fluoro-2'-Deoxyadenosine Starting with Wild-Type or Nucleoside Reverse Transcriptase Inhibitor-Resistant Strains.

4'-Ethynyl-2-fluoro-2'-deoxyadenosine (EFdA, MK-8591, islatravir) is a nucleoside reverse transcriptase translocation inhibitor (NRTTI) with exceptional potency against wild-type (WT) and drug-resistant HIV-1 in phase III clinical trials. EFdA resistance is not well characterized. To study EFdA resistance patterns that may emerge in naive or tenofovir (TFV)-, emtricitabine/lamivudine (FTC/3TC)-, or zidovudine (AZT)-treated patients, we performed viral passaging experiments starting with WT, K65R, M184V, or D67N/K70R/T215F/K219Q HIV-1. Regardless of the starting viral sequence, all selected EFdA-resistant variants included the M184V reverse transcriptase (RT) mutation. Using recombinant viruses, we validated the role for M184V as the primary determinant of EFdA resistance; none of the observed connection subdomain (R358K and E399K) or RNase H domain (A502V) mutations significantly contributed to EFdA resistance. A novel EFdA resistance mutational pattern that included A114S was identified in the background of M184V. A114S/M184V exhibited higher EFdA resistance (∼24-fold) than either M184V (∼8-fold) or A114S alone (∼2-fold). Remarkably, A114S/M184V and A114S/M184V/A502V resistance mutations were up to 50-fold more sensitive to tenofovir than was WT HIV-1. These mutants also had significantly lower specific infectivities than did WT. Biochemical experiments confirmed decreases in the enzymatic efficiency (kcat/Km) of WT versus A114S (2.1-fold) and A114S/M184V/A502V (6.5-fold) RTs, with no effect of A502V on enzymatic efficiency or specific infectivity. The rather modest EFdA resistance of M184V or A114S/M184V (8- and 24-fold), their hypersusceptibility to tenofovir, and strong published in vitro and in vivo data suggest that EFdA is an excellent therapeutic candidate for naive, AZT-, FTC/3TC-, and especially tenofovir-treated patients.

Once-Weekly Oral Dosing of MK-8591 Protects Male Rhesus Macaques From Intrarectal Challenge With SHIV109CP3.

BACKGROUND: MK-8591 (4'-ethynyl-2-fluoro-2'-deoxyadenosine [EFdA]) is a novel reverse transcriptase-translocation inhibitor. METHODS: We assessed MK-8591 as preexposure prophylaxis in the rhesus macaque model of intrarectal challenge with simian/human immunodeficiency virus (SHIV). In study 1, 8 rhesus macaques received 3.9 mg/kg of MK-8591 orally on day 0 and once weekly for the next 14 weeks. Eight controls were treated with vehicle. All rhesus macaques were challenged with SHIV109CP3 on day 6 and weekly for up to 12 challenges or until infection was confirmed. The dose of MK-8591 was reduced to 1.3 and 0.43 mg/kg/week in study 2 and further to 0.1 and 0.025 mg/kg/week in study 3. In studies 2 and 3, each dose was given up to 6 times once weekly, and animals were challenged 4 times once weekly with SHIV109CP3. RESULTS: Control macaques were infected after a median of 1 challenge (range, 1-4 challenges). All treated animals in studies 1 and 2 were protected, consistent with a 41.5-fold lower risk of infection (P < .0001, by the log-rank test). In study 3, at a 0.1-mg/kg dose, 2 rhesus macaques became infected, consistent with a 7.2-fold lower risk of infection (P = .0003, by the log-rank test). The 0.025-mg/kg dose offered no protection. CONCLUSIONS: These data support MK-8591's potential as a preexposure prophylaxis agent.

Structural basis of HIV inhibition by translocation-defective RT inhibitor 4'-ethynyl-2-fluoro-2'-deoxyadenosine (EFdA).

4'-Ethynyl-2-fluoro-2'-deoxyadenosine (EFdA) is the most potent nucleoside analog inhibitor of HIV reverse transcriptase (RT). It retains a 3'-OH yet acts as a chain-terminating agent by diminishing translocation from the pretranslocation nucleotide-binding site (N site) to the posttranslocation primer-binding site (P site). Also, facile misincorporation of EFdA-monophosphate (MP) results in difficult-to-extend mismatched primers. To understand the high potency and unusual inhibition mechanism of EFdA, we solved RT crystal structures (resolutions from 2.4 to 2.9 Å) that include inhibition intermediates (i) before inhibitor incorporation (catalytic complex, RT/DNA/EFdA-triphosphate), (ii) after incorporation of EFdA-MP followed by dT-MP (RT/DNAEFdA-MP(P)• dT-MP(N) ), or (iii) after incorporation of two EFdA-MPs (RT/DNAEFdA-MP(P)• EFdA-MP(N) ); (iv) the latter was also solved with EFdA-MP mismatched at the N site (RT/DNAEFdA-MP(P)• EFdA-MP(*N) ). We report that the inhibition mechanism and potency of EFdA stem from interactions of its 4'-ethynyl at a previously unexploited conserved hydrophobic pocket in the polymerase active site. The high resolution of the catalytic complex structure revealed a network of ordered water molecules at the polymerase active site that stabilize enzyme interactions with nucleotide and DNA substrates. Finally, decreased translocation results from favorable interactions of primer-terminating EFdA-MP at the pretranslocation site and unfavorable posttranslocation interactions that lead to observed localized primer distortions.

MK-8591 (4'-Ethynyl-2-Fluoro-2'-Deoxyadenosine) Exhibits Potent Activity against HIV-2 Isolates and Drug-Resistant HIV-2 Mutants in Culture.

There is a pressing need to identify more effective antiretroviral drugs for HIV-2 treatment. Here, we show that the investigational compound MK-8591 (4'-ethynyl-2-fluoro-2'-deoxyadenosine [EFdA]) is highly active against group A and B isolates of HIV-2; 50% effective concentrations [EC50] for HIV-2 were, on average, 4.8-fold lower than those observed for HIV-1. MK-8591 also retains potent activity against multinucleoside-resistant HIV-2 mutants (EC50 ≤ 11 nM). These data suggest that MK-8591 may have antiviral activity in HIV-2-infected individuals.

4'-Ethynyl-2-fluoro-2'-deoxyadenosine (EFdA) inhibits HIV-1 reverse transcriptase with multiple mechanisms.

4'-Ethynyl-2-fluoro-2'-deoxyadenosine (EFdA) is a nucleoside analog that, unlike approved anti-human immunodeficiency virus type 1 (HIV-1) nucleoside reverse transcriptase inhibitors, has a 3'-OH and exhibits remarkable potency against wild-type and drug-resistant HIVs. EFdA triphosphate (EFdA-TP) is unique among nucleoside reverse transcriptase inhibitors because it inhibits HIV-1 reverse transcriptase (RT) with multiple mechanisms. (a) EFdA-TP can block RT as a translocation-defective RT inhibitor that dramatically slows DNA synthesis, acting as a de facto immediate chain terminator. Although non-translocated EFdA-MP-terminated primers can be unblocked, they can be efficiently converted back to the EFdA-MP-terminated form. (b) EFdA-TP can function as a delayed chain terminator, allowing incorporation of an additional dNTP before blocking DNA synthesis. In such cases, EFdA-MP-terminated primers are protected from excision. (c) EFdA-MP can be efficiently misincorporated by RT, leading to mismatched primers that are extremely hard to extend and are also protected from excision. The context of template sequence defines the relative contribution of each mechanism and affects the affinity of EFdA-MP for potential incorporation sites, explaining in part the lack of antagonism between EFdA and tenofovir. Changes in the type of nucleotide before EFdA-MP incorporation can alter its mechanism of inhibition from delayed chain terminator to immediate chain terminator. The versatility of EFdA in inhibiting HIV replication by multiple mechanisms may explain why resistance to EFdA is more difficult to emerge.

Preformulation studies of EFdA, a novel nucleoside reverse transcriptase inhibitor for HIV prevention.

4'-Ethynyl-2-fluoro-2'-deoxyadenosine (EFdA) is a novel nucleoside analog of great interest because of its superior activity against wild-type and multidrug-resistant HIV-1 strains, and favorable safety profiles in vitro and in vivo. The aim of this work was to provide preformulation information of EFdA important for delivery system development. A simple, accurate and specific reverse-phase high performance liquid chromatographic (RP-HPLC) method with UV detection was developed for quantification of EFdA. In addition, physicochemical characterizations including pH solubility profile, octanol/water partition coefficient (Log Po/w), DSC analysis, field emission scanning electron microscopy, and stability studies under various conditions were conducted. EFdA existed in planar or flake shape, with a melting point of ∼130 °C, and had a pH dependent solubility. The log Po/w value of EFdA was -1.19. The compound was stable upon exposure to pH levels from 3 to 9 and showed good stability at elevated temperature (65 °C). In vitro cytotoxicity assessments were performed in two different epithelial cell lines. In cell-based studies, the EFdA selectivity index (50% cytotoxic concentration [CC50] values/50% effective concentration [EC50]) was found to be greater than 1 × 10(3). Permeability studies using cell- and tissue-based models showed that EFdA had an apparent permeability coefficient (Papp) <1 × 10(-6)cm/s and that the paracelluar pathway was the dominant transport route for EFdA. Overall, EFdA possesses favorable characteristics for further formulation development.

Intracellular Islatravir-Triphosphate in Dried Blood Spots and Peripheral Blood Mononuclear Cells from Pig-Tailed Macaques.

The purpose of this study was to evaluate the relationship between intracellular islatravir-triphosphate (ISL-TP) in paired peripheral blood mononuclear cells (PBMCs) and dried blood spots (DBS). Three pig-tailed macaques (PMs) were dosed with a single intravaginal extended-release ISL-etonogestrel film for a period of 31 days. After extraction and quantification, repeated measures correlation (rrm) was assessed between log-transformed DBS and PBMC ISL-TP concentrations. Twenty-six paired PBMC/DBS samples were included. Peak ISL-TP concentrations in DBS ranged from 262 to 913 fmol/punches, PBMC Cmax ranged from 427 to 857 fmol/106 cells. Repeated measures correlation yielded an rrm value of 0.96 (95% confidence interval 0.92-0.98; p < .0001). Importantly, ISL-TP was quantifiable in DBS and its pharmacokinetics were similar to PBMC in PMs. Human studies should evaluate DBS applications in clinical pharmacokinetic studies to help define ISL's place in the antiretroviral drug armamentarium.

HIV-1 Resistance to Islatravir/Tenofovir Combination Therapy in Wild-Type or NRTI-Resistant Strains of Diverse HIV-1 Subtypes.

Tenofovir disoproxil fumarate (TDF) and islatravir (ISL, 4'-ethynyl-2-fluoro-2'-deoxyadensine, or MK-8591) are highly potent nucleoside reverse transcriptase inhibitors. Resistance to TDF and ISL is conferred by K65R and M184V, respectively. Furthermore, K65R and M184V increase sensitivity to ISL and TDF, respectively. Therefore, these two nucleoside analogs have opposing resistance profiles and could present a high genetic barrier to resistance. To explore resistance to TDF and ISL in combination, we performed passaging experiments with HIV-1 WT, K65R, or M184V in the presence of ISL and TDF. We identified K65R, M184V, and S68G/N mutations. The mutant most resistant to ISL was S68N/M184V, yet it remained susceptible to TDF. To further confirm our cellular findings, we implemented an endogenous reverse transcriptase assay to verify in vitro potency. To better understand the impact of these resistance mutations in the context of global infection, we determined potency of ISL and TDF against HIV subtypes A, B, C, D, and circulating recombinant forms (CRF) 01_AE and 02_AG with and without resistance mutations. In all isolates studied, we found K65R imparted hypersensitivity to ISL whereas M184V conferred resistance. We demonstrated that the S68G polymorphism can enhance fitness of drug-resistant mutants in some genetic backgrounds. Collectively, the data suggest that the opposing resistance profiles of ISL and TDF suggest that a combination of the two drugs could be a promising drug regimen for the treatment of patients infected with any HIV-1 subtype, including those who have failed 3TC/FTC-based therapies.

EFdA efficiently suppresses HIV replication in the male genital tract and prevents penile HIV acquisition.

Sexually transmitted HIV infections in heterosexual men are acquired through the penis. Low adherence to condom usage and the fact that 40% of circumcised men are not protected indicate the need for additional prevention strategies. Here, we describe a new approach to evaluate the prevention of penile HIV transmission. We demonstrated that the entire male genital tract (MGT) of bone marrow/liver/thymus (BLT) humanized mice is repopulated with human T and myeloid cells. The majority of the human T cells in the MGT express CD4 and CCR5. Direct penile exposure to HIV leads to systemic infection including all tissues of the MGT. HIV replication throughout the MGT was reduced 100-1,000-fold by treatment with 4'-ethynyl-2-fluoro-2'-deoxyadenosine (EFdA), resulting in the restoration of CD4+ T cell levels. Importantly, systemic preexposure prophylaxis with EFdA effectively protects from penile HIV acquisition. IMPORTANCE Over 84.2 million people have been infected by the human immunodeficiency virus type 1 (HIV-1) during the past 40 years, most through sexual transmission. Men comprise approximately half of the HIV-infected population worldwide. Sexually transmitted HIV infections in exclusively heterosexual men are acquired through the penis. However, direct evaluation of HIV infection throughout the human male genital tract (MGT) is not possible. Here, we developed a new in vivo model that permits, for the first time, the detail analysis of HIV infection. Using BLT humanized mice, we showed that productive HIV infection occurs throughout the entire MGT and induces a dramatic reduction in human CD4 T cells compromising immune responses in this organ. Antiretroviral treatment with novel drug EFdA suppresses HIV replication in all tissues of the MGT, restores normal levels of CD4 T cells and is highly efficient at preventing penile transmission.

Self-medication practice among pregnant women in Wolaita Zone, Southern Ethiopia: An institutionally based cross-sectional study.

Background: Self-medication is a treatment based on symptoms without prescription and medical consultation. Despite being one of the critical practices that impose a harmful effect on the fetus and the woman herself, evidence on its practice and associated factors are not well-documented. This study, therefore, assessed the self-medication practice and associated factors among pregnant women in Wolaita Zone, Southern Ethiopia. Methods: An institutionally based cross-sectional study was conducted at public health institutions in Wolaita Zone, Southern Ethiopia by recruiting a total of 408 pregnant women using a systematic random sampling technique between March 2019 and April 2019. We used the Antenatal care (ANC) registry as a sampling frame. A pre-tested, structured, interviewer-administered questionnaire used to depict Self-medication practice and associated factors. Data entered using Epi-data and analyzed by SPSS 23.0. Results: The overall prevalence of self-medication was 14.9% (95% CI:11-18). The odds of using self-medication may decreased by 75% for women who were in their third trimester (AOR = 0.25, 95% CI: 0.10, 0.64). However, the odds of practicing increased by 13-folds among pregnant women reported earlier (previous) self-medication experience (AOR = 13.62, 95% CI: 6.66-27.84). Conclusion: The prevalence of self-medication was high in the current study setting. Women's gestational period (third trimester) and earlier self-medication experience were associated with their current self-medication practice.

Drug Interactions in Lenacapavir-Based Long-Acting Antiviral Combinations.

Long-acting (LA) anti-HIV regimens show promise for increasing dosing intervals and consequently, improving the patients' quality of life. The first FDA-approved LA therapy is Cabenuva, which comprises rilpivirine (a non-nucleoside reverse transcriptase inhibitor) and cabotegravir (integrase strand transfer inhibitor). Novel promising LA anti-HIV agents such as lenacapavir (a capsid-targeting antiviral) and islatravir (EFdA, a nucleoside reverse transcriptase translocation inhibitor) need to be explored as combination therapies. Therefore, we sought to determine whether combination of lenacapavir with islatravir, rilpivirine, or cabotegravir displayed synergy, additivity, or antagonism. We performed dose-response matrices of these drug combinations in an HIV-1 reporter cell line and subsequently analyzed the data with SynergyFinder Plus, which employs four major drug interaction models: highest single agent, Bliss independence, Loewe additivity, and zero interaction potency. Most of these models predict additive inhibition by the studied drug combinations This work highlights the importance of effective drug combinations in LA-regimens.

Long-acting and extended-release implant and nanoformulations with a synergistic antiretroviral two-drug combination controls HIV-1 infection in a humanized mouse model.

The HIV pandemic has affected over 38 million people worldwide with close to 26 million currently accessing antiretroviral therapy (ART). A major challenge in the long-term treatment of HIV-1 infection is nonadherence to ART. Long-acting antiretroviral (LA-ARV) formulations, that reduce dosing frequency to less than once a day, are an urgent need that could tackle the adherence issue. Here, we have developed two LA-ART interventions, one an injectable nanoformulation, and the other, a removable implant, for the delivery of a synergistic two-drug ARV combination comprising a pre-clinical nonnucleoside reverse transcriptase inhibitor (NNRTI), Compound I, and the nucleoside reverse transcriptase inhibitor (NRTI), 4'-ethynyl-2-fluoro-2'-deoxyadenosine. The nanoformulation is poly(lactide-co-glycolide)-based and the implant is a copolymer of ω-pentadecalactone and p-dioxanone, poly(PDL-co-DO), a novel class of biocompatible, biodegradable materials. Both the interventions, packaged independently with each ARV, released sustained levels of the drugs, maintaining plasma therapeutic indices for over a month, and suppressed viremia in HIV-1-infected humanized mice for up to 42 days with maintenance of CD4+ T cells. These data suggest promise in the use of these new drugs as LA-ART formulations in subdermal implant and injectable mode.

Islatravir Is Not Expected to Be a Victim or Perpetrator of Drug-Drug Interactions via Major Drug-Metabolizing Enzymes or Transporters.

Islatravir (MK-8591) is a nucleoside reverse transcriptase translocation inhibitor in development for the treatment and prevention of HIV-1. The potential for islatravir to interact with commonly co-prescribed medications was studied in vitro. Elimination of islatravir is expected to be balanced between adenosine deaminase-mediated metabolism and renal excretion. Islatravir did not inhibit uridine diphosphate glucuronosyltransferase 1A1 or cytochrome p450 (CYP) enzymes CYP1A2, 2B6, 2C8, 2C9, 2C19, 2D6, or 3A4, nor did it induce CYP1A2, 2B6, or 3A4. Islatravir did not inhibit hepatic transporters organic anion transporting polypeptide (OATP) 1B1, OATP1B3, organic cation transporter (OCT) 1, bile salt export pump (BSEP), multidrug resistance-associated protein (MRP) 2, MRP3, or MRP4. Islatravir was neither a substrate nor a significant inhibitor of renal transporters organic anion transporter (OAT) 1, OAT3, OCT2, multidrug and toxin extrusion protein (MATE) 1, or MATE2K. Islatravir did not significantly inhibit P-glycoprotein and breast cancer resistance protein (BCRP); however, it was a substrate of BCRP, which is not expected to be of clinical significance. These findings suggest islatravir is unlikely to be the victim or perpetrator of drug-drug interactions with commonly co-prescribed medications, including statins, diuretics, anti-diabetic drugs, proton pump inhibitors, anticoagulants, benzodiazepines, and selective serotonin reuptake inhibitors.

Toward highly sensitive and reproducible LC-MS/MS analysis of MK-8591 phosphorylated anabolites in human peripheral blood mononuclear cells.

Aim: MK-8591 (EFdA), a novel anti-HIV nucleoside analog, is converted to mono-, di- and tri-phosphates (MK-8591-MP, MK-8591-DP and MK-8591-TP) intracellularly, among which MK-8591-TP is the active pharmacological form. An ultrasensitive LC-MS/MS assay was required to measure MK-8591-DP and MK-8591-TP levels in human peripheral blood mononuclear cells (PBMCs). Sensitivity and reproducibility were major bottlenecks in these analyses. Materials and methods: Human PBMCs were isolated from blood and lysed with 70/30 methanol/RPMI-1640. An LC-MS/MS method was developed to simultaneously quantify MK-8591-DP and MK-8581-TP in PBMC lysates. Results: Low flow LC and dimethyl sulfoxide mediated signal enhancement enabled an extreme sensitivity with limit of quantitation at 0.1 ng/ml. Assay accuracy was 92.5-106% and precision was 0.7-12.1% for a linear curve range of 0.1-40 ng/ml. Matrix variability and interference liability were comprehensively evaluated. Conclusion: Our study findings and steps taken in addressing clinical sample issues help understand and overcome the challenges facing intracellular nucleotide analog analysis.

The High Genetic Barrier of EFdA/MK-8591 Stems from Strong Interactions with the Active Site of Drug-Resistant HIV-1 Reverse Transcriptase.

4'-Ethynyl-2-fluoro-2'-deoxyadenosine (EFdA/MK-8591), a nucleoside reverse transcriptase inhibitor (NRTI) under clinical trials, is a potent and promising long-acting anti-HIV type 1 (HIV-1) agent. EFdA and its derivatives possess a modified 4'-moiety and potently inhibit the replication of a wide spectrum of HIV-1 strains resistant to existing NRTIs. Here, we report that EFdA and NRTIs with a 4'-ethynyl- or 4'-cyano-moiety exerted activity against HIV-1 with an M184V mutation and multiple NRTI-resistant HIV-1s, whereas NRTIs with other moieties (e.g., 4'-methyl) did not show this activity. Structural analysis indicated that EFdA and 4'-ethynyl-NRTIs (but not other 4'-modified NRTIs), formed strong van der Waals interactions with critical amino acid residues of reverse transcriptase. Such interactions were maintained even in the presence of a broad resistance-endowing M184V substitution, thus potently inhibiting drug-resistant HIV-1 strains. These findings also explain the mechanism for the potency of EFdA and provide insights for further design of anti-HIV-1 therapeutics.

In vitro transport characteristics of EFdA, a novel nucleoside reverse transcriptase inhibitor using Caco-2 and MDCKII cell monolayers.

4'-Ethynyl-2-fluoro-2'-deoxyadenosine (EFdA) is a novel nucleoside reverse transcriptase inhibitor with a unique mechanism of action and highly potent activity against both wild-type and clinically relevant drug resistant HIV-1 variants. Furthermore, in vivo efficacy and safety evaluations have shown EFdA to be a promising therapeutic candidate for use in the treatment of HIV infection. However, little is known about the pharmacokinetic and biopharmaceutical properties of EFdA. In this study, we evaluated cellular EFdA transport using Caco-2 and Madin-Darby Canine Kidney II (MDCKII) in vitro cell models. Studies using Caco-2 cell monolayers showed that EFdA efflux ratios were >2.0, suggesting that active drug transport mechanisms may play a role in EFdA flux. ABCB1 transporter (PGP1) inhibition was assessed using the acetomethoxy derivate of calcein (calcein-AM) as a fluorescent probe in both wild-type MDCKII and PGP1 overexpressing MDCKII cells. Nonetheless, our data showed that EFdA is not a substrate of PGP1. Additionally, comparative bidirectional flux of EFdA and Lucifer yellow (LY, a well-known paracellular marker) was studied over a range of EFdA concentrations. In MDCKII monolayers, EFdA had an apparent permeability coefficient (Papp) (a-b) of <1×10(-6)cm/s. The Papp values significantly increased in the presence of the paracellular permeability enhancer, indicating that EFdA primarily permeates via the paracellular route.

Probing the molecular mechanism of action of the HIV-1 reverse transcriptase inhibitor 4'-ethynyl-2-fluoro-2'-deoxyadenosine (EFdA) using pre-steady-state kinetics.

The novel antiretroviral 4'-ethynyl-2-fluoro-2'-deoxyadenosine (EFdA) is a potent nucleoside HIV-1 reverse transcriptase (RT) inhibitor (NRTI). Unlike other FDA-approved NRTIs, EFdA contains a 3'-hydroxyl. Pre-steady-state kinetics showed RT preferred incorporating EFdA-TP over native dATP. Moreover, RT slowly inserted nucleotides past an EFdA-terminated primer, resulting in delayed chain termination with unaffected fidelity. This is distinct from KP1212, another 3'-hydroxyl-containing RT inhibitor considered to promote viral lethal mutagenesis. New mechanistic features of RT inhibition by EFdA are revealed.

Development of a vaginal delivery film containing EFdA, a novel anti-HIV nucleoside reverse transcriptase inhibitor.

The aim of this work was to develop a fast-dissolving film formulation containing EFdA for potential use as a topical vaginal microbicide for prevention of HIV sexual transmission. Solid state compatibility approaches were used to screen commonly used polymers for formulation development. Factorial design and desirability function were used to investigate the effect of two variables, the ratio of the polymers and the concentration of selected plasticizer on four mechanical responses including tensile strength, elongation at break, toughness and elastic modulus for optimization of the film formulation. Assessments of EFdA-loaded films included physicochemical characteristics, in vitro cytotoxicity, epithelia integrity, ex vivo permeability and bioactivity test. The optimal placebo film was composed of PVA, HPMC E5 and propylene glycol (7:3:3, w/w), and its mechanical characteristics were comparable to those of VCF(®) film (a commercial vaginal film product). Permeability studies using human ectocervical explants showed that there was no significant difference in cumulative permeated amount of EFdA between EFdA film and free EFdA. The results of in vitro cytotoxicity and bioactivity testing showed that 50% cytotoxic concentration (CC50) was several orders of magnitude higher than 50% effective concentration (EC50) of EFdA. Furthermore, epithelial integrity study showed that EFdA-loaded film had a much lower toxicity to HEC-1A cell monolayers as compared to VCF(®). Therefore, EFdA-loaded vaginal film may be considered as a promising vaginal microbicide for HIV prevention.