RESUMO
OBJECTIVES: Free ritonavir, lopinavir and efavirenz injected intraperitoneally were compared with antiretroviral (AR) nanoparticles (NPs). METHODS: This is a prospective study in BALB/c mice comparing the pharmacokinetics of free drugs with AR NPs. All animals received free drugs or AR NPs (20 mg/kg) in PBS. In vitro replication assays were used for determination of the anti-HIV efficacy of NP formulations. At specific times (free drugs 0.08, 0.125, 0.25, 0.33, 1, 2 and 3 days; AR NPs 0.125, 0.33, 1, 2, 4, 7, 14, 21, 28, 35 and 42 days) mice were euthanized and serum and organs were harvested for determination of AR concentrations by HPLC. Single treatment of monocyte-derived macrophages (MDMs) infected with HIV-1(ada) compared AR NPs (0.005-0.05 mg/mL) with free efavirenz or lopinavir/ritonavir (0.01-0.1 mg/mL), blank NPs and controls. Results are presented as means ± SEM. RESULTS: Serum free AR drug concentrations peaked 4 h post-injection (ritonavir 3.9 ± 3.05, lopinavir 3.4 ± 2.5 and efavirenz 1.8 ± 0.63 µg/mL) and were eliminated by 72 h. Poly(dl-lactide-co-glycolide) NP animals had detectable ritonavir, lopinavir and efavirenz concentrations in all tissues for 28 days. Treatment of MDMs with AR NPs resulted in sustained inhibition of HIV-1(ada) replication. CONCLUSIONS: AR drug concentrations from NPs are sustained for 28 days in vivo and anti-HIV inhibition was comparable to that of free drugs in vitro and could be a sustained treatment for delivery of AR drugs.
Assuntos
Fármacos Anti-HIV/administração & dosagem , Fármacos Anti-HIV/farmacocinética , Sistemas de Liberação de Medicamentos , Nanopartículas/administração & dosagem , Poliésteres/administração & dosagem , Alcinos , Estruturas Animais/química , Animais , Benzoxazinas/administração & dosagem , Benzoxazinas/farmacocinética , Cromatografia Líquida de Alta Pressão , Ciclopropanos , Injeções Intraperitoneais , Lopinavir , Camundongos , Camundongos Endogâmicos BALB C , Monócitos/virologia , Pirimidinonas/administração & dosagem , Pirimidinonas/farmacocinética , Ritonavir/administração & dosagem , Ritonavir/farmacocinética , Soro/química , Fatores de TempoRESUMO
The use of combination antiretroviral nanoparticles (cART NPs) was investigated as a novel treatment approach for the inhibition of HIV-1 replication. We developed nanoparticles of biodegradable polymer, poly-(dl-lactide-co-glycolic acid; PLGA) containing efavirenz (EFV) and boosted lopinavir (lopinavir/ritonavir; LPV/r) by a high-pressure homogenization method. The method resulted in >79% drug entrapment efficiency for each of the three drugs. The average size of cART NPs was 138.3±55.4 nm as measured by dynamic light scanning, confirmed by scanning electron microscopy (SEM) with an average surface charge of -13.7±4.5. Lissamine-rhodamine-labeled fluorescent PLGA NPs exhibited efficient uptake in nonimmune (HeLa cells) and immune (H9 T cells) cells as measured by confocal microscopy. Cells treated with cART NPs resulted in minimal loss of cell viability over 28 days. Subcellular fractionation studies demonstrated that HIV-1-infected H9 monocytic cells treated with cART NPs contained significantly (p<0.05) higher nuclear, cytoskeleton, and membrane antiretroviral drug levels compared to cells treated with drug solutions alone. Finally, cART NPs efficiently inhibited HIV-1 infection and transduction. The IC50 for each of the three drugs in the cART NPs was <31 nM. These experiments demonstrate the efficacy of a novel PLGA NPs formulation for the delivery of cART to inhibit HIV-1 replication.
Assuntos
Fármacos Anti-HIV/administração & dosagem , Infecções por HIV/tratamento farmacológico , HIV-1/efeitos dos fármacos , Nanopartículas/uso terapêutico , Alcinos , Fármacos Anti-HIV/uso terapêutico , Benzoxazinas/administração & dosagem , Benzoxazinas/uso terapêutico , Linfócitos T CD4-Positivos/metabolismo , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Ciclopropanos , Quimioterapia Combinada , Células HeLa/metabolismo , Humanos , Ácido Láctico , Lopinavir/administração & dosagem , Lopinavir/uso terapêutico , Microscopia Confocal , Ácido Poliglicólico , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Frações Subcelulares/metabolismo , Células U937/metabolismoRESUMO
The objective of this investigation was to develop a thermosensitive vaginal gel containing raltegravir+efavirenz loaded PLGA nanoparticles (RAL+EFV-NPs) for pre-exposure prophylaxis of HIV. RAL+EFV-NPs were fabricated using a modified emulsion-solvent evaporation method and characterized for size and zeta potential. The average size and surface charge of RAL+EFV-NP were 81.8±6.4 nm and -23.18±7.18 mV respectively. The average encapsulation efficiency of raltegravir and efavirenz was 55.5% and 98.2% respectively. Thermosensitive vaginal gel containing RAL+EFV-NPs was successfully prepared using a combination of Pluronic F127 (20% w/v) and Pluronic F68 (1% w/v). Incorporation RAL+EFV-NPs in the gel did not result in nanoparticle aggregation and RAL+EFV-NPs containing gel showed thermogelation at 32.5°C. The RAL+EFV-NPs were evaluated for inhibition of HIV-1(NL4-3) using TZM-bl indicator cells. The EC(90) of RAL+EFV-NPs was lower than raltegravir+efavirenz (RAL+EFV) solution but did not reach significance. Compared to control HeLa cells without any treatment, RAL+EFV-NPs or blank gel were not cytotoxic for 14 days in vitro. The intracellular levels of efavirenz in RAL+EFV-NPs treated HeLa cells were above the EC(90) for 14 days whereas raltegravir intracellular concentrations were eliminated within 6 days. Transwell experiments of NPs-in-gel demonstrated rapid transfer of fluorescent nanoparticles from the gel and uptake in HeLa cells within 30 min. These data demonstrate the potential of antiretroviral NP-embedded vagina gels for long-term vaginal pre-exposure prophylaxis of heterosexual HIV-1 transmission.
Assuntos
Fármacos Anti-HIV/farmacologia , Benzoxazinas/farmacologia , Géis/farmacologia , HIV-1/efeitos dos fármacos , Nanopartículas/administração & dosagem , Pirrolidinonas/farmacologia , Alcinos , Fármacos Anti-HIV/química , Benzoxazinas/química , Cromatografia Líquida de Alta Pressão , Ciclopropanos , Portadores de Fármacos/administração & dosagem , Portadores de Fármacos/química , Avaliação Pré-Clínica de Medicamentos , Géis/química , Infecções por HIV/prevenção & controle , Infecções por HIV/virologia , Células HeLa , Humanos , Ácido Láctico/química , Testes de Sensibilidade Microbiana , Nanopartículas/química , Fagocitose , Poloxâmero/química , Ácido Poliglicólico/química , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Pirrolidinonas/química , Raltegravir Potássico , Temperatura , Fatores de TempoRESUMO
The antiretroviral nanoparticle formulation laboratory at Creighton University School of Pharmacy & Health Professions is lead by Chris Destache. Over the past 4 years, this laboratory has been investigating the use of antiretroviral nanoparticles as a sustained drug delivery method for HIV-1-infected patients.