e Oral or topical daily administration of antiretroviral (ARV) drugs to HIV-1-negative individuals in vulnerable populations is a promising strategy for HIV-1 prevention. Adherence to the dosing regimen has emerged as a critical factor determining efficacy outcomes of clinical trials. Because adherence to therapy is inversely related to the dosing period, sustained release or long-acting ARV formulations hold significant promise for increasing the effectiveness of HIV-1 preexposure prophylaxis (PrEP) by reducing dosing frequency. A novel, subdermal implant delivering the potent prodrug tenofovir alafenamide (TAF) with controlled, sustained, zero-order (linear) release characteristics is described. A candidate device delivering TAF at 0.92 mg day ؊1 in vitro was evaluated in beagle dogs over 40 days for pharmacokinetics and preliminary safety. No adverse events related to treatment with the test article were noted during the course of the study, and no significant, unusual abnormalities were observed. The implant maintained a low systemic exposure to TAF (median, 0.85 ng ml ؊1 ; interquartile range [IQR], 0.60 to 1.50 ng ml ؊1 ) and tenofovir (TFV; median, 15.0 ng ml ؊1 ; IQR, 8.8 to 23.3 ng ml ؊1 ), the product of in vivo TAF hydrolysis. High concentrations (median, 512 fmol/10 6 cells over the first 35 days) of the pharmacologically active metabolite, TFV diphosphate, were observed in peripheral blood mononuclear cells at levels over 30 times higher than those associated with HIV-1 PrEP efficacy in humans. Our report on the first sustained-release nucleoside reverse transcriptase inhibitor (NRTI) for systemic delivery demonstrates a successful proof of principle and holds significant promise as a candidate for HIV-1 prophylaxis in vulnerable populations. O ral or topical daily administration of antiretroviral (ARV) drugs to HIV-1-negative individuals in vulnerable populations is a promising strategy for HIV-1 prevention, but clinical outcomes have varied widely (1-3). Adherence to frequent dosing is burdensome to the user and has emerged as a key factor in explaining the heterogeneous efficacy outcomes of HIV-1 preexposure prophylaxis (PrEP) clinical trials (4-7). It is well established across different delivery methods that adherence to therapy is inversely related with the dosing period (8-11). Sustained-release or long-acting ARV formulations hold significant promise as a means of reducing dosing frequency, thereby increasing the effectiveness of HIV-1 PrEP.Long-acting preexposure prophylaxis (LA-PrEP) is an alternative regimen to daily dosing designed to mitigate the abovedescribed adherence challenges (12, 13). LA-PrEP has been based primarily on ARV nanoparticles for parenteral administration as injections (12,14). Dosing intervals of 1 month or longer for injectable, long-acting, nanomilled formulations of the integrase strand-transfer inhibitor cabotegravir (GSK1265744) and the nonnucleoside reverse transcriptase inhibitor (NNRTI) rilpivirine are undergoing clinical evaluation as possible regimens for H...

Iron oxyhydroxide minerals, known to be chemically reactive and significant for elemental cycling, are thought to have been abundant in early-Earth seawater, sediments, and hydrothermal systems. In the anoxic Fe 2+ -rich early oceans, these minerals would have been only partially oxidized and thus redox-active, perhaps able to promote prebiotic chemical reactions. We show that pyruvate, a simple organic molecule that can form in hydrothermal systems, can undergo reductive amination in the presence of mixed-valence iron oxyhydroxides to form the amino acid alanine, as well as the reduced product lactate. Furthermore, geochemical gradients of pH, redox, and temperature in iron oxyhydroxide systems affect product selectivity. The maximum yield of alanine was observed when the iron oxyhydroxide mineral contained 1:1 Fe(II):Fe(III), under alkaline conditions, and at moderately warm temperatures. These represent conditions that may be found, for example, in iron-containing sediments near an alkaline hydrothermal vent system. The partially oxidized state of the precipitate was significant in promoting amino acid formation: Purely ferrous hydroxides did not drive reductive amination but instead promoted pyruvate reduction to lactate, and ferric hydroxides did not result in any reaction. Prebiotic chemistry driven by redoxactive iron hydroxide minerals on the early Earth would therefore be strongly affected by geochemical gradients of E h , pH, and temperature, and liquid-phase products would be able to diffuse to other conditions within the sediment column to participate in further reactions. life emergence | iron hydroxides | hydrothermal vents | early Earth | gradients T he synthesis of biomolecules, particularly amino acids and their condensation into peptides, from geochemical carbon and nitrogen sources is an important research topic for assessing the role of specific geochemical environments and mineral phases in the emergence of life. One mineral type of interest that would have been abundant in the mildly acidic, iron-rich oceans of the early Earth (1-3) is the iron oxyhydroxides, which can precipitate in a variety of stable or metastable redox states (4, 5). Iron oxides/oxyhydroxides are versatile reactive minerals that can drive redox reactions and concentrate phosphorus species, trace metals, organic molecules, and other anions (6-11). On the early Earth, iron oxyhydroxides and/or green rust would likely have been present in the water column as well as seafloor sediments, playing a fundamental role in elemental cycling and redox chemistry (4, 10). Iron oxyhydroxides would also have been a primary component in alkaline hydrothermal vent mounds and chimneys, which have been proposed as a possible environment for the emergence of metabolism due to their ambient pH, E h , ion/chemical, and temperature gradients (12-15).Seafloor hydrothermal sediments and chimneys are flowthrough gradient systems that combine reactive minerals with organic compounds in a variety of possible reaction conditions. Alkaline vents produ...

Intravaginal delivery of microbicide combinations is a promising approach for the prevention of sexually transmitted infections, but requires a method of providing simultaneous, independent release of multiple agents into the vaginal compartment. A novel intravaginal ring (IVR) platform has been developed for simultaneous delivery of the reverse-transcriptase inhibitor tenofovir (TFV) and the guanosine analogue antiviral acyclovir (ACV) with independent control of release rate for each drug. The IVR is based on a pod design, with up to 10 individual polymer-coated drug cores embedded in the ring releasing through preformed delivery channels. The release rate from each pod is controlled independently of the others by the drug properties, polymer coating, and size and number of delivery channels. Pseudo-zero-order in vitro release of TFV (144 ± 10 µg day) and ACV (120 ± 19 µg day−1) from an IVR containing both drugs was sustained for 28 days. The mechanical properties of the pod IVR were evaluated and compared with the commercially available Estring® (Pfizer, NY, NY). The pod-IVR design enables the vaginal delivery of multiple microbicides with differing physicochemical properties, and is an attractive approach for the sustained intravaginal delivery of relatively hydrophilic drugs that are difficult to deliver using conventional matrix IVR technology.

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Vaginal microbicides may play an important role in protecting women from HIV infection. A strong synergy between HSV and HIV has been observed, and epidemiological studies demonstrate that HSV infection increases the risk of HIV acquisition. Incorporation of the antiretroviral tenofovir (TFV) along with the antiherpetic acyclovir (ACV) into combination intravaginal rings (IVRs) for sustained mucosal delivery of both compounds could lead to increased microbicide product adherence and efficacy compared with conventional vaginal formulations. A novel, dual-protection "pod IVR" platform developed in-house and delivering ACV and TFV was evaluated in rabbit and sheep models. The devices were safe and exhibited sustained release of both drugs independently and at controlled rates over the 28-day studies. Daily release rates were estimated based on residual drug content of the used devices: rabbits, 343 ؎ 335 g day ؊1 (ACV) and 321 ؎ 207 g day ؊1 (TFV); sheep, 174 ؎ 14 g day ؊1 (ACV) and 185 ؎ 34 g day ؊1 (TFV). Mean drug levels in sheep vaginal samples were as follows: secretions, 5.25 ؎ 7.31 g ml ؊1 (ACV) and 20.6 ؎ 16.2 g ml ؊1 (TFV); cervicovaginal lavage fluid, 118 ؎ 113 ng ml ؊1 (ACV) and 191 ؎ 125 ng ml ؊1 (TFV); tissue, 173 ng g ؊1 (ACV) and 93 ng g ؊1 (TFV). An in vitro-in vivo correlation was established for both drugs and will allow the development of future formulations delivering target levels for prophylaxis and therapy. These data suggest that the IVR based on the pod design has potential in the prevention of transmission of HIV-1 and other sexually transmitted pathogens. Significant progress has been achieved in providing increased access to HIV/AIDS services in several low-and middleincome countries (44). Despite these encouraging findings, women in the developing world remain disproportionately at risk of HIV infection. Seventy-six percent of new HIV infections in sub-Saharan Africa occur in women aged 15 to 24 years (42). The proportion is as high as 90% in South Africa (38). In the absence of a preventative vaccine, effective prophylactic biomedical technologies are urgently required. Campaigns aimed at encouraging monogamy and condom use have had limited success in areas where marriage has been identified as the major risk factor for HIV acquisition in women (3,15,16,21,25).Human immunodeficiency virus type 1 (HIV-1) and herpes simplex virus type 2 (HSV-2), the serotype most commonly associated with genital herpes, are responsible for two intersecting epidemics, where the disease caused by one virus facilitates the transmission of and pathogenesis by the other. A strong synergy between HSV and HIV has been observed, and epidemiological studies demonstrate that HSV infection increases the risk of HIV-1 acquisition (33,34,43). A meta-analysis found that prevalent HSV-2 infection is associated with a threefold-increased risk of HIV acquisition among both men and women. These results suggest that, in areas of high HSV-2 prevalence, a substantial proportion of HIV infection is linked to HSV-2 infection (...

Preexposure prophylaxis (PrEP) using FDA-approved antiretroviral (ARV) drugs is emerging as a promising strategy for the prevention of sexual HIV infection. There is growing consensus that a combination of ARV agents, analogous to highly active antiretroviral therapy (HAART), likely is essential for optimally effective PrEP (1, 2). Oral administration of tenofovir disoproxil fumarate (TDF) plus emtricitabine (FTC) (Truvada; Gilead Sciences, Inc.) is the first regimen approved by the FDA to reduce the risk of HIV infection in uninfected individuals (http://www.fda.gov/NewsEvents/Newsroom /PressAnnouncements/ucm312210.htm). Three recent clinical trials demonstrated that oral ARV regimens using the combination of TDF and FTC can be effective in susceptible men, women, and partners of HIV-infected individuals (3-5). However, the relative risk reduction in these trials varied widely (from 44 to 75%), and a study in which women used a daily oral TDF-FTC regimen was stopped early due to futility. A critical factor driving success in these trials appears to involve sustaining high levels of adherence to frequent dosing (6).It is well established across different delivery methods that adherence to therapy is inversely related to the dosing period (7-10). Controlled topical delivery of ARV drugs using intravaginal rings (IVRs) is thought to improve adherence (11) and to provide sustained mucosal levels independent of coitus and daily dosing (12). The delivery of two or more ARV drugs from conventional IVR designs involves significant technological and manufacturing hurdles. To meet these challenges, we have developed a novel IVR technology, the pod-IVR (13), that enables rapid development of devices capable of delivering multiple agents over a wide range of target delivery rates and aqueous solubilities (14-16). We recently published the design and 28-day pharmacokinetic (PK) evaluation in sheep of a five-drug pod-IVR as a proof-of-concept, advanced multipurpose prevention technology (MPT), combining three ARV drugs from different mechanistic classes (tenofovir [TFV], nevirapine, and saquinavir) with a proven estrogen-progestogen contraceptive for prevention of HIV infection and unintended pregnancy (17).Here we present the first report of an IVR delivering TDF and FTC, as well as the first report of a triple-combination IVR delivering TDF, FTC, and maraviroc (MVC) (an entry inhibitor/antagonist of chemokine receptor 5 [CCR5]). Preliminary local safety and pharmacokinetic (PK) findings for these devices were determined in pig-tailed macaques, which are considered by many to represent the most relevant animal model for HIV vaginal PrEP studies (15,18,19). Steady-state drug levels for all three ARV agents in vaginal fluids were sustained over the 28-day study period, with corresponding vaginal tissue concentrations suggesting putative efficacy in preventing HIV infection.

There is a pressing need for modeling of the symbiotic and at times dysbiotic relationship established between bacterial microbiomes and human mucosal surfaces. In particular clinical studies have indicated that the complex vaginal microbiome (VMB) contributes to the protection against sexually-transmitted pathogens including the life-threatening human immunodeficiency virus (HIV-1). The human microbiome project has substantially increased our understanding of the complex bacterial communities in the vagina however, as is the case for most microbiomes, very few of the community member species have been successfully cultivated in the laboratory limiting the types of studies that can be completed. A genetically controlled ex vivo model system is critically needed to study the complex interactions and associated molecular dialog. We present the first vaginal mucosal culture model that supports colonization by both healthy and dysbiotic VMB from vaginal swabs collected from routine gynecological patients. The immortalized vaginal epithelial cells used in the model and VMB cryopreservation methods provide the opportunity to reproducibly create replicates for lab-based evaluations of this important mucosal/bacterial community interface. The culture system also contains HIV-1 susceptible cells allowing us to study the impact of representative microbiomes on replication. Our results show that our culture system supports stable and reproducible colonization by VMB representing distinct community state types and that the selected representatives have significantly different effects on the replication of HIV-1. Further, we show the utility of the system to predict unwanted alterations in efficacy or bacterial community profiles following topical application of a front line antiretroviral.

Antiretroviral-based microbicides applied topically to the vagina may play an important role in protecting women from HIV infection. Incorporation of the nucleoside reverse transcriptase inhibitor tenofovir (TFV) into intravaginal rings (IVRs) for sustained mucosal delivery may lead to increased microbicide product adherence and efficacy compared with those of conventional vaginal formulations. Formulations of a novel "pod IVR" platform spanning a range of IVR drug loadings and daily release rates of TFV were evaluated in a pig-tailed macaque model. The rings were safe and exhibited sustained release at controlled rates over 28 days. Vaginal secretion TFV levels were independent of IVR drug loading and were able to be varied over 1.5 log units by changing the ring configuration. Mean TFV levels in vaginal secretions were 72.4 ؎ 109 g ml ؊1 (slow releasing) and 1.84 ؎ 1.97 mg ml ؊1 (fast releasing). The mean TFV vaginal tissue concentration from the slow-releasing IVRs was 76.4 ؎ 54.8 g g ؊1 and remained at steady state 7 days after IVR removal, consistent with the long intracellular half-life of TFV. Intracellular tenofovir diphosphate (TFV-DP), the active moiety in defining efficacy, was measured in vaginal lymphocytes collected in the study using the fast-releasing IVR formulation. Mean intracellular TFV-DP levels of 446 ؎ 150 fmol/10 6 cells fall within a range that may be protective of simian-human immunodeficiency virus strain SF162p3 (SHIV SF162p3 ) infection in nonhuman primates. These data suggest that TFV-releasing IVRs based on the pod design have potential for the prevention of transmission of human immunodeficiency virus type 1 (HIV-1) and merit further clinical investigation.