Prevention of HIV- 1 or HIV-2

About

Abstract An antiretroviral composition that gels upon heating and can be administered prophylactically prior to exposure to a retrovirus following sexual intercourse, and methods of using the same. Background [0002] Prevention of HIV- 1 infection to reduce the number of newly infected patients is an international priority. Various modalities such as male circumcision, prophylactic HIV vaccines, vaginal microbicides and oral pre-exposure prophylaxis have been explored to prevent sexual contraction of HIV. Prevention of HIV infection by using anti-viral agents as vaginal microbicides has received more attention in recent years. [0003] Worldwide, nearly half of all individuals living with HIV are now women, who acquire the virus largely by heterosexual exposure. Many women, because of limited economic options and gender inequality, cannot reliably negotiate sexual encounters, leaving them vulnerable to unwanted pregnancy and sexually transmitted infections (STIs), including HIV. In the absence of an effective vaccine, topical microbicide formulations, which are applied vaginally or rectally, represent an attractive solution to stop HIV transmission. However, clinical trials focusing on vaginal prophylaxis of HIV using topical microbicides have shown mixed results. Several topical microbicides such as BufferGel™, PRO 2000, and Carraguard™ have failed to show efficacy in clinical trials whereas coitally-dependent administration of 1% tenofovir gel has shown success. Conversely, the VOICE trial employing a coitus-independent, once daily administration of 1% tenofovir gel was halted due to lack of efficacy. The VOICE trial setback has prompted investigators to examine alternatives. Therefore, there is a need for new anti-viral gels that provide sustained delivery of anti-viral therapy for the prevention of sexual HIV transmission but can be used coitally-independent. BRIEF DESCRIPTION OF THE FIGURES [0004] FIG. 1 depicts a scanning electron micrograph (SEM) image of the raltegravir + efavirenz loaded PLGA nanoparticles. [0005] FIG. 2 graphically depicts the cytotoxicity of RAL-EFV-NP and RAL-EFV solution to H9 cells (A and B) and HeLa cells (C and D) in vitro. FIG. 2 A and C show cell viability at 24 h and FIG. 2 B and D show cell viability at 48 h. [0006] FIG. 3 plots the IC90 curves for RAL-EFV solution and RAL-EFV-NP.AL-EFV solution and RAL-EFV-NP were incubated with HIV-1 indicator TZM-bl cells at different concentrations starting for RAL + EFV concentration of 10 μg/ml. [0007] FIG. 4 graphically depicts the intracellular (HeLa cells) and extracellular(media) concentrations of RAL and EFV released from RAL-EFV-NP over a period of 14 days (n=6). [0008] FIG. 5 depicts the sol-gel transition profile of thermosensitive gel (20%Pluronic F127 + 1% Pluronic F68) demonstrating variations of the elastic (G'; left y-axis) and viscous (G"; right y-axis) moduli, as a function of temperature. The elastic modulus increases after 30°C indicating initiation of thermogelation process and increases further with increase in the temperature indicating formation of firm gel at 37°C. [0009] FIG. 6 graphically depicts the dynamic viscosity of the thermosensitive gel as a function of temperature. The viscosity of the thermosensitive gel starts increasing after 30°C indicating initiation of thermogelation process and increases further with increase in the temperature. [0010] FIG. 7 shows images of the thermosensitive gel containing RAL-EFV-NP at room temperature (A) and at 37°C (B). [001 1] FIG. 8 shows fluorescent images of HeLa cells after 15 min (A) and 30 min (B) incubation with the thermosensitive gel containing rhodamine 6G labeled PLGA nanoparticles in the transwell. [0012] FIG. 9 shows graphs depicting anti-HIV activity of a thermosensitive gel containing CAP-NP or CAP-EFV-NP. Briefly, TZM-bl cells were pretreated with either gel- control (gel-ctrl), gel alone (gel), gel containing CAP-NP (gel-cap (10-fold higher)), and gel containing CAP-EFV-NP (gel cap-efv), and then inoculated with HIV-lNL4-3- After incubating the cells for a defined period of time, cells were lysed, a luciferase substrate (Bright-Glo) was added, and luminescence was expressed as relative luminescence units. [0013] FIG. 10 shows graphs depicting anti-HIV activity of a thermosensitive gel containing CAP-NP or CAP-EFV-NP prepared from two different batches. Briefly, TZM-bl cells were pretreated with either gel containing CAP-NP from batch 4 (gel cap-4), gel containing CAP-EFV-NP from batch 4 (gel cap-efv 4), gel containing CAP-NP from batch 5 (gel cap-5), gel containing CAP-EFV-NP from batch 5 (gel cap-efv 5) and then inoculated with HIV-lNL4-3. After incubating the cells for a defined period of time, cells were lysed, a luciferase substrate was added, and luminescence was expressed as relative luminescence units. [0014] FIG. 11 shows a line graph depicting cytotoxicity of gel containing CAP- NP (CAP), gel containing CAP-EFV-NP (CAP EFV), gel containing RAL-EFV solution (Ral + EFV) and a gel control (Blank Gel without any nanoparticles) to HeLa cells over 96 hours in vitro. The control (CTRL) are control cells without anything added to the wells (e.g. no nanoparticles or gel). Relative luminescence is graphed on the y-axis, corresponding to HeLa cell viability. [0015] FIG. 12 shows a line graph depicting raltegravir (A) and efavirenz (B) levels in tissue sampled from different parts of the female mouse reproductive tract. Here, 30 uL of gel containing RAL+EFV NPs in gel were intravaginally instilled into female mice. At specific times (plotted on the X-axis), the mice were euthanized and the tissue harvested. Tissue was homogenized in acetonitrile and an aliquot of the supernatants were analyzed by HPLC for raltegravir or efavirenz. The Y-axis is depicted as ng/mg tissue weight. Additionally, at specific times, 50 uL of PBS was instilled into the vaginal tract and collected as cervicovaginal lavage to determine amounts of drugs from NPs were remaining in the vaginal tracts of the mice. The cervicovaginal lavage was measured in ug/mL (1000 fold higher than the tissue levels). [0016] FIG. 13 shows graphs depicting anti-HIV activity of CAP-EFV-NP from batch 4 and 5. Briefly, TZM-bl cells were pretreated with either CAP-NP but no drug from bath 4 (cap-4) or batch 5 (cap-5), or CAP-NP loaded with EFV from batch 4 (cap-efv 4) or batch 5 (cap-efv 5), and then inoculated with HIV-lNL4-3. After incubating the cells for a defined period of time, cells were lysed, a luciferase substrate was added, and luminescence was expressed as relative luminescence units. [0017] FIG. 14 shows a line graph depicting cytotoxicity of CAP-NP from batch 5 to HeLa cells over 96 hours in vitro. Relative luminescence is graphed on the y-axis, corresponding to HeLa cell viability. [0018] FIG. 15 shows fluorescent images of HeLa cells after 4 hr incubation with the thermosensitive gel containing rhodamine 6G labeled CAP nanoparticles in the transwell. Nuclei of HeLa cells are stained with DAPI (A); Rhodamine conjugated nanoparticles are imaged in (B); and (C) depicts a merged image (DAPI stained cells are blue, Rhodamine- conjugated nanoparticles are red). [0019] FIG. 16 shows fluorescent images of HeLa cells after 24 hr incubation with the thermosensitive gel containing rhodamine 6G labeled CAP nanoparticles in the transwell. Nuclei of HeLa cells are stained with DAPI (A); Rhodamine conjugated nanoparticles are imaged in (B); and (C) depicts a merged image (DAPI stained cells are blue, Rhodamine-conjugated nanoparticles are red).