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Topical Tenofovir Disoproxil Fumarate Nanoparticles Prevent HIV-1 Vaginal Transmission in a Humanized Mouse Model
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"We focused on developing a TDF-loaded PLGA nanoparticle formulation in a TMS gel for improved delivery of TFV.....To achieve successful translation, nanoparticles should be able to achieve high encapsulation of TDF so that maximum drug is utilized. We therefore focused on an ion-pairing approach to increase the encapsulation efficiency of TDF in the nanoparticles (12)."
"TFV 1% vaginal gel and combinations of TDF-plus-emtricitabine tablets (Gilead Science) are currently the only prevention regimens that have shown efficacy in human trials. Additional prevention modalities are needed. Additionally, the prevention options should be controlled by the end-user. Each woman has different circumstances and needs. Therefore, vaginal and oral ingestion options are needed. We present a new preventive option that integrates a thermosensitive gel along with TDF nanoparticles that has shown sustained release properties in these humanized BLT mice when instilled locally. Further pharmacokinetic and efficacy studies in other animal models are needed to extend these results to humans."
"Our laboratory has been developing a nanotechnology-based gel delivery system (11-16). Our gel delivery system incorporates a thermosensitive (TMS) gel that is liquid at room temperature and a semisolid at body temperature. Tenofovir disoproxil fumarate (TDF) plus emtricitabine (Truvada; Gilead Sciences) is the only FDA-approved oral PrEP. TDF is a TFV prodrug with higher permeability and significantly lower 50% effective concentrations (EC50s) against HIV-1 than those of TFV (17). The TDF-loaded vaginal ring has shown significantly better vaginal delivery than the tenofovir ring (18). Incorporation of TDF into nanoparticles (TDF-NPs) was investigated for improved antiviral protection. The TMS gel allows for easy administration, and once in contact with vaginal tissue, it gelates instantaneously and becomes a pliable semisolid at body temperature. We now report the results of TDF-NPs in a TMS gel (TDF-NP-TMS)-based preventative strategy using TDF-NPs in a humanized BLT (hu-BLT) mouse model of HIV-1 vaginal transmission."
Tenofovir Intravaginal Ring.....
http://www.natap.org/2013/ICAAC/ICAAC_83.htm
Tenofovir Vaginal Ring Protects 5 of 6 Macaques From SHIV After High-Dose Depo-Provera /Intravaginal ring delivery of tenofovir disoproxil fumarate for prevention of HIV and herpes simplex virus infection.....
http://www.natap.org/2013/ICAAC/ICAAC_71.htm
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Topical Tenofovir Disoproxil Fumarate Nanoparticles Prevent HIV-1 Vaginal Transmission in a Humanized Mouse Model
Antimicrobial Agents and Chemotherapy
Christopher J. Destache,a Subhra Mandal,a Zhe Yuan,c Guobin Kang,c Abhijit A. Date,a* Wuxun Lu,c Annemarie Shibata,b Rachel Pham,b Patrick Bruck,b Michael Rezich,b You Zhou,c Renuga Vivekanandan,e Courtney V. Fletcher,d Qingsheng Lic
Creighton University School of Pharmacy & Health Professions, Omaha, Nebraska, USAa; Department of Biology, Creighton University, Omaha, Nebraska, USAb; Nebraska Center for Virology and School of Biological Sciences, University of Nebraska-Lincoln, Lincoln, Nebraska, USAc; University of Nebraska Medical Center College of Pharmacy, Omaha, Nebraska, USAd; Creighton University School of Medicine, Omaha Nebraska, USAe
ABSTRACT
Preexposure prophylaxis (PrEP) with 1% tenofovir (TFV) vaginal gel has failed in clinical trials. To improve TFV efficacy in vaginal gel, we formulated tenofovir disoproxil fumarate nanoparticles in a thermosensitive (TMS) gel (TDF-NP-TMS gel). TDF-NPs were fabricated using poly(lactic-co-glycolic acid) (PLGA) polymer and an ion-pairing agent by oil-in-water emulsification. The efficacy of TDF-NP-TMS gel was tested in humanized bone marrow-liver-thymus (hu-BLT) mice. Hu-BLT mice in the treatment group (Rx; n = 15) were administered TDF-NP-TMS gel intravaginally, having TDF at 0.1%, 0.5%, and 1% (wt/vol) concentrations, whereas the control (Ctr; n = 8) group received a blank TMS gel. All Rx mice (0.1% [n = 4], 0.5% [n = 6], and 1% [n = 5]) were vaginally challenged with two transmitted/founder (T/F) HIV-1 strains (2.5 x 105 50% tissue culture infectious doses). Rx mice were challenged at 4 h (0.1%), 24 h (0.5%), and 7 days (1%) posttreatment (p.t.) and Ctr mice were challenged at 4 h p.t. Blood was drawn weekly for 4 weeks postinoculation (p.i.) for plasma viral load (pVL) using reverse transcription-quantitative PCR. Ctr mice had positive pVL within 2 weeks p.i. Rx mice challenged at 4 h and 24 h showed 100% protection and no detectable pVL throughout the 4 weeks of follow-up (P = 0.009; Mantel-Cox test). Mice challenged at 7 days were HIV-1 positive at 14 days p.i. Further, HIV-1 viral RNA (vRNA) in vaginal and spleen tissues of Rx group mice with negative pVL were examined using an in situ hybridization (ISH) technique. The detection of vRNA was negative in all Rx mice studied. The present studies elucidate TDF-NP-TMS gel as a long-acting, coitus-independent HIV-1 vaginal protection modality.
INTRODUCTION
Presently, a total of 36.9 million people worldwide are living with HIV-1 (1). Topical preexposure prophylaxis (PrEP) currently is a promising preventative strategy (2). The basic idea is to protect the vagina (and/or rectum) from HIV-1 infection by applying gel containing antiretroviral drug(s) around the time of sexual intercourse. This topical preparation is considered a microbicide, inhibiting infection by blocking viral transmission at the mucosal surface. To date, tenofovir (TFV) is the only drug administered locally as a 1% vaginal gel shown to be effective at preventing heterosexual contraction of HIV-1 (3). TFV tissue concentrations indicate a direct relationship between levels of TFV in genitals and protection (4-7). The minimum amount of TFV in cervicovaginal fluid levels when associated with gel that shows protection against HIV-1 infection has been reported to be >1,000 ng/ml (4). This level is greater than 10 times that seen in patients receiving oral TDF and emtricitabine (4). In female macaques given 1% TFV gel, the intracellular half-life for the active metabolite, tenofovir diphosphate, is significantly shorter (averaging 25 h) in vaginal lymphocytes than peripheral PBMCs (averaging 49 h) (7). A coitally independent strategy using 1% TFV gel has not shown efficacy in several clinical trials (8, 9). Based on the dramatic negative results of the Vaginal and Oral Interventions to Control the Epidemic (VOICE) trial, it is important to consider female attitudes and opinions for a vaginal gel-based prevention delivery system. A safe and effective female-controlled, discrete gel-based delivery system has the potential to prevent millions of HIV-1 infections worldwide annually.
When designing female-controlled preventative delivery systems, the gel-based system should have features important for the female user. Namely, the delivery system should be (i) easy to administer; (ii) adherent to the mucosal surface once applied vaginally; (iii) low seepage; and (iv) free of side effects or cytotoxicity to the mucosal surfaces of the female genital tract (10). All of these factors, if not optimized, could diminish gel effectiveness or lead to gel aversion. Finally, a long-acting preparation would be highly desirable if it offered long-term protection from HIV-1 (11).
Our laboratory has been developing a nanotechnology-based gel delivery system (11-16). Our gel delivery system incorporates a thermosensitive (TMS) gel that is liquid at room temperature and a semisolid at body temperature. Tenofovir disoproxil fumarate (TDF) plus emtricitabine (Truvada; Gilead Sciences) is the only FDA-approved oral PrEP. TDF is a TFV prodrug with higher permeability and significantly lower 50% effective concentrations (EC50s) against HIV-1 than those of TFV (17). The TDF-loaded vaginal ring has shown significantly better vaginal delivery than the tenofovir ring (18). Incorporation of TDF into nanoparticles (TDF-NPs) was investigated for improved antiviral protection. The TMS gel allows for easy administration, and once in contact with vaginal tissue, it gelates instantaneously and becomes a pliable semisolid at body temperature. We now report the results of TDF-NPs in a TMS gel (TDF-NP-TMS)-based preventative strategy using TDF-NPs in a humanized BLT (hu-BLT) mouse model of HIV-1 vaginal transmission.
DISCUSSION
TFV is the most widely investigated antiretroviral drug used for HIV-1 prevention. However, clinical trial results employing 1% tenofovir vaginal gel have been discouraging (9, 26). Hydrophilicity and low cellular permeability can be reasons for the poor performance of TFV vaginal gel. Indeed, <5% of TFV permeated through HEC-1A cells using a transwell experimental design, corroborating low permeability (6). Therefore, it is essential to develop a strategy that will improve TFV cellular permeability when used for prevention of HIV-1. TDF has greater bioavailability and offers higher cellular permeability, as evidenced by a 100-fold lower 50% infectious dose (IC50) against HIV-1BaL than against TFV (7). TDF is an ester prodrug that hydrolyzes in water and/or in the presence of cellular esterases to TFV. We focused on developing a TDF-loaded PLGA nanoparticle formulation in a TMS gel for improved delivery of TFV. However, TDF has considerable water solubility (13.4 mg/ml at 25⋅C), and it is well known that hydrophilic drugs are not easy to encapsulate into nanoparticles. There have been few attempts to encapsulate TFV or TDF in PLGA nanoparticles (27, 28).
None of these investigations were able to achieve greater than 10% encapsulation of TFV or TDF in the nanoparticles. To achieve successful translation, nanoparticles should be able to achieve high encapsulation of TDF so that maximum drug is utilized. We therefore focused on an ion-pairing approach to increase the encapsulation efficiency of TDF in the nanoparticles (12).
The use of a thermosensitive vaginal gel incorporating TDF-NPs is a novel formulation for a female-controlled delivery system that has many advantages. As the gel is liquid at room temperature, it is easy to administer and forms a firm semisolid gel upon contact with body temperature surfaces; therefore, it is likely to be less prone to have vaginal seepage after application than the hydroxyethyl cellulose gel that is commonly used in vaginal preparations. Employing the thermosensitive properties of this gel will provide less vaginal seepage and increased compliance with use of the preparation. The TMS gel may also keep TDF nanoparticles in contact with the epithelial surface of the FRT over a longer time frame (15).
Rheology has been used to study the thermogelation properties of our gel in the presence of simulated cervicovaginal fluid (11-14). In the presence of simulated vaginal fluids, the optimized formulation of TMS gel was able to retain thermogelling behavior at approximately 32 to 34⋅C. We believe that this will increase acceptance and adherence for women. Additionally, this gel is a female-controlled, discrete prevention method not currently available. When TMS gel without TDF nanoformulation was used in the control mice, it offered no protection from HIV-1 challenge; thus, it is a delivery vehicle that allows for greater contact between the antiretroviral therapy and the cells on the epithelial surface (15).
The TDF-NPs with an ion-pairing agent, docusate sodium, required a cytotoxicity evaluation. Hence, we evaluated the cytotoxicity of TDF solution and TDF-NPs against various cell lines using a CellTiter-Glo assay (see Fig. S1 in the supplemental material). The TDF solution and TDF-NPs caused minimal toxicity to HeLa and H9 cells. There was no difference in cytotoxicity profile of TDF-NPs and TDF solution, indicating that docusate sodium did not adversely affect the cytotoxicity potential.
It is known that nucleoside reverse transcriptase inhibitors, notably stavudine, have been associated with mitochondrial toxicity (30). Other investigators that have fabricated nucleoside reverse transcriptase inhibitor-based nanoformulations containing zidovudine or didanosine triphosphate (at 15 μg/ml) have demonstrated reduced mitochondrial toxicity similar to these results (31). We carried out ex vivo cytotoxicity studies using three-dimensional human vaginal endocervical tissue (EpiVaginal; MatTek Corp). We evaluated the toxicity potential of TDF-NPs, TDF solution, and untreated and 1% Triton X-100-treated solutions (controls) for 1, 24, 48, and 96 h per the manufacturer's protocol (Fig. 2). As expected, the vaginal irritant Triton X-100 showed considerable cytotoxicity to vaginal endocervical tissues. TDF-NPs and solution demonstrated cytotoxicity at 48 h, which resolved at 96 h. These results could be due to the exchange of fresh media.
The hu-BLT mouse model of HIV-1 infection has gained attention for evaluation of microbicides. It has already been demonstrated that 1% TFV gel applied 4 h before and after HIV-1 challenge (same design as that of the CAPRISA 004 trial) offered partial protection in hu-BLT mice, indicating a good correlation with CAPRISA 004 results (32). Additionally, mice rapidly metabolize TFV compared to humans; therefore, they may not completely mimic humans for microbicide discovery. We elected not to include a mouse cohort that received TFV solution in TMS gel, as the control mice became infected after gel administration. All humanized BLT mice that received 1% TDF-NPs and were challenged 7 days after gel administration became infected (Fig. 4). This may be related to the TFV vaginal tissue levels, the active metabolite in tissue, or the volume of administered TMS gel not remaining in the vaginal tract for this length of time. Nevertheless, the hu-BLT mouse model offers an economical means of screening microbicides before moving to macaques and humans.
Previous data from humans have demonstrated that 1% TFV gel produces a protective effect when CVL levels were >1,000 ng/ml (4). Donnell et al. investigated TFV plasma levels in serodiscordant couples receiving an oral tenofovir-emtricitabine combination (33). The majority of HIV-negative individuals in the serodiscordant relationship had TFV plasma concentrations of >40 ng/ml, consistent with daily drug dosing, whereas individuals who contracted HIV did not have that threshold level of TFV. In our experiments with NSG mice, the CVL TFV levels from the NP formulation were 1.75 times higher than those for mice receiving the 1% TFV in HEC gel at 24 h after administration. More study of the concentration-response relationship is necessary to determine the amount of TFV and/or active metabolite required for HIV prevention. Veselinovic et al. investigated mucosal tenofovir tissue levels in humanized mice (34). These investigators used oral TDF at 61.5 mg/kg daily by oral gavage for 5 days before harvesting plasma and tissue for tenofovir determination. The half-life of oral TDF in these mice averaged 9 h. Our NP formulation (0.5%) instilled into the female mouse vagina was 5 mg/ml, or 150 μg in 30 μl, which could be administered into the female mouse vagina. We did not investigate TFV drug levels or obtain the levels of the active metabolite, tenofovir diphosphate, in the FRT tissue but did get cervicovaginal lavage fluid from NSG mice in parallel. The diphosphate metabolite was below detectable limits, as in the previous humanized mouse study (32). Further study in macaques is necessary to develop this microbicide.
Our nanoparticles, fabricated using an FDA-approved PLGA polymer, are different from the particles made by wet-milling nanofabrication methods that others have published for treatment as well as prevention (35-38). Roy et al. used their wet-milled nanoparticles containing atazanavir and ritonavir for treatment of HIV-1 in NSG mice reconstituted with human peripheral blood leukocytes (PBLs) (35). Jackson et al. utilized Elan nanofabrication technology to fabricate rilpivirine nanoparticles (36). In this study, HIV-negative human volunteers received increasing single doses of rilpivirine-LA and plasma, and genital levels were measured in females and plasma and rectal levels were assessed in men. The results of these experiments showed that rilpivirine-LA injected intramuscularly would be able to prevent HIV-1 challenge in both men and women, but protective concentrations in plasma or reproductive tract tissues are not known. Andrews et al. investigated the protective effect of increasing single or multiple doses of cabotegravir (GSK1265744) in nonhuman primates against multiple low doses of HIV challenge (37). Results demonstrated correlation between protection and plasma drug levels, and a potential every-3-month dosing schedule in humans would be useful to reduce adherence difficulties that are problematic for preexposure prophylaxis.
TFV 1% vaginal gel and combinations of TDF-plus-emtricitabine tablets (Gilead Science) are currently the only prevention regimens that have shown efficacy in human trials. Additional prevention modalities are needed. Additionally, the prevention options should be controlled by the end-user. Each woman has different circumstances and needs. Therefore, vaginal and oral ingestion options are needed. We present a new preventive option that integrates a thermosensitive gel along with TDF nanoparticles that has shown sustained release properties in these humanized BLT mice when instilled locally. Further pharmacokinetic and efficacy studies in other animal models are needed to extend these results to humans.
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