RESUMEN
Many Proteobacteria use acyl-homoserine lactone-mediated quorum-sensing (QS) to activate specific sets of genes as a function of cell density. QS often controls the virulence of pathogenic species, and in fact a previous study indicated that QS was important for Burkholderia mallei mouse lung infections. To gain in-depth information on the role of QS in B. mallei virulence, we constructed and characterized a mutant of B. mallei strain GB8 that was unable to make acyl-homoserine lactones. The QS mutant showed virulence equal to that of its wild-type parent in an aerosol mouse infection model, and growth in macrophages was indistinguishable from that of the parent strain. Furthermore, we assessed the role of QS in B. mallei ATCC 23344 by constructing and characterizing a mutant strain producing AiiA, a lactonase enzyme that degrades acyl-homoserine lactones. Although acyl-homoserine lactone levels in cultures of this strain are very low, it showed full virulence. Contrary to the previous report, we conclude that QS is not required for acute B. mallei infections of mice. QS may be involved in some stage of chronic infections in the natural host of horses, or the QS genes may be remnants of the QS network in B. pseudomallei from which this host-adapted pathogen evolved.
Asunto(s)
Infecciones por Burkholderia/microbiología , Burkholderia mallei/patogenicidad , Percepción de Quorum/fisiología , Animales , Proteínas Bacterianas/genética , Proteínas Bacterianas/fisiología , Infecciones por Burkholderia/genética , Burkholderia mallei/genética , Burkholderia mallei/crecimiento & desarrollo , Células Cultivadas , Modelos Animales de Enfermedad , Femenino , Regulación Bacteriana de la Expresión Génica , Macrófagos/microbiología , Ratones , Ratones Endogámicos BALB C , Virulencia/genéticaRESUMEN
TLC-ART101 is a long-acting triple-HIV drug combination of lopinavir-ritonavir-tenofovir in one nanosuspension intended for subcutaneous injection. After a single TLC-ART 101 administration in nonhuman primates, drug concentrations in both plasma and HIV-target lymph node mononuclear cells were sustained for 2 weeks. Nevertheless, the mechanisms leading to the targeted long-acting pharmacokinetics remain elusive. Therefore, an intravenous study of TLC-ART 101 in nonhuman primates was conducted to elucidate the degree of association of drugs in vivo, estimate subcutaneous bioavailability, and refine a mechanism-based pharmacokinetic (MBPK2) model. The MBPK2 model considers TLC-ART 101 systemic drug clearances, nanoparticle-associated/dissociated species, more detailed mechanisms of lymphatic first-pass retention of associated-drugs after subcutaneous administrations, and the prediction of drug concentration time-courses in lymph node mononuclear cells. For all 3 drugs, we found a high association with the nanoparticles in plasma (>87% lopinavir-ritonavir, 97% tenofovir), and an incomplete subcutaneous bioavailability (<29% lopinavir-ritonavir, 85% tenofovir). As hypothesized by the MBPK2 model, the incomplete SC bioavailability observed is due to sequestration into a lymphatic node depot after subcutaneous absorption (unlike most intramuscular nanodrug products having near-to-injection depots), which contributes to long-acting profiles detected in plasma and target cells. This combined experimental and modeling approach may be applicable for the clinical development of other long-acting drug-combination injectables.
Asunto(s)
Fármacos Anti-VIH , Infecciones por VIH , Preparaciones Farmacéuticas , Animales , Fármacos Anti-VIH/uso terapéutico , Combinación de Medicamentos , Infecciones por VIH/tratamiento farmacológico , Lopinavir , RitonavirRESUMEN
Existing oral antiretroviral (ARV) agents have been shown in human studies to exhibit limited lymph node penetration and lymphatic drug insufficiency. As lymph nodes are a reservoir of HIV, it is critical to deliver and sustain effective levels of ARV combinations in these tissues. To overcome lymph node drug insufficiency of oral combination ARV therapy (cART), we developed and reported a long-acting and lymphocyte-targeting injectable that contains three ARVs-hydrophobic lopinavir (LPV) and ritonavir (RTV), and hydrophilic tenofovir (TFV)-stabilized by lipid excipients in a nanosuspension. A single subcutaneous (SC) injection of this first-generation formulation of drug combination nanoparticles (DcNPs), named TLC-ART101, provided persistent ARV levels in macaque lymph node mononuclear cells (LNMCs) for at least 1â¯week, and in peripheral blood mononuclear cells (PBMCs) and plasma for at least 2â¯weeks, demonstrating long-acting pharmacokinetics for all three drugs. In addition, the lymphocyte-targeting properties of this formulation were demonstrated by the consistently higher intracellular drug concentrations in LNMCs and PBMCs versus those in plasma. To provide insights into the complex mechanisms of absorption and disposition of TLC-ART101, we constructed novel mechanism-based pharmacokinetic (MBPK) models. Based upon plasma PK data obtained after single administration of TLC-ART101 (DcNPs) and a solution formulation of free triple-ARVs, the models feature uptake from the SC injection site that respectively routes free and nanoparticle-associated ARVs via the blood vasculature and lymphatics, and their eventual distribution into and clearance from the systemic circulation. The models provided simultaneous description of the complex long-acting plasma and lymphatic PK profiles for all three ARVs in TLC-ART101. The long-acting PK characteristics of the three drugs in TLC-ART101 were likely due to a combination of mechanisms including: (1) DcNPs undergoing preferential lymphatic uptake from the subcutaneous space, (2) retention in nodes during lymphatic first-pass, (3) subsequent slow release of ARVs into blood circulation, and (4) limited extravasation of DcNP-associated ARVs that resulted in longer persistence in the circulation.
Asunto(s)
Fármacos Anti-VIH/farmacocinética , Antirretrovirales/farmacocinética , Lopinavir/farmacocinética , Modelos Biológicos , Nanopartículas , Ritonavir/farmacocinética , Tenofovir/farmacocinética , Animales , Fármacos Anti-VIH/sangre , Antirretrovirales/sangre , Preparaciones de Acción Retardada/farmacocinética , Combinación de Medicamentos , Lopinavir/sangre , Macaca nemestrina , Masculino , Ritonavir/sangre , Tenofovir/sangreRESUMEN
Daily oral antiretroviral therapy regimens produce limited drug exposure in tissues where residual HIV persists and suffer from poor patient adherence and disparate drug kinetics, which all negatively impact outcomes. To address this, we developed a tissue- and cell-targeted long-acting 4-in-1 nanosuspension composed of lopinavir (LPV), ritonavir, tenofovir (TFV), and lamivudine (3TC). In 4 macaques dosed subcutaneously, drug levels over 5 weeks in plasma, lymph node mononuclear cells (LNMCs), and peripheral blood mononuclear cells (PBMCs) were analyzed by liquid chromatography-tandem mass spectrometry. Plasma and PBMC levels of the active drugs (LPV, TFV, and 3TC) were sustained for 5 weeks; PBMC exposures to LPV, ritonavir, and 3TC were 12-, 16-, 42-fold higher than those in plasma. Apparent T1/2z of LPV, TFV, and 3TC were 219.1, 63.1, and 136.3 h in plasma; 1045.7, 105.9, and 127.7 h in PBMCs. At day 8, LPV, TFV, and 3TC levels in LNMCs were 4.1-, 5.0-, and 1.9-fold higher than in those in PBMCs and much higher than in plasma. Therefore, 1 dose of a 4-drug nanosuspension exhibited persistent drug levels in LNMCs, PBMCs, and plasma for 5 weeks. With interspecies scaling and dose adjustment, this 4-in-1 HIV drug-combination could be a long-acting treatment with the potential to target residual virus in tissues and improve patient adherence.
Asunto(s)
Fármacos Anti-VIH/administración & dosificación , Sistemas de Liberación de Medicamentos , Lamivudine/administración & dosificación , Lopinavir/administración & dosificación , Ritonavir/administración & dosificación , Tenofovir/administración & dosificación , Animales , Fármacos Anti-VIH/sangre , Fármacos Anti-VIH/farmacocinética , Portadores de Fármacos/química , Quimioterapia Combinada , Infecciones por VIH/tratamiento farmacológico , Inyecciones Subcutáneas , Lamivudine/sangre , Lamivudine/farmacocinética , Leucocitos Mononucleares/metabolismo , Lopinavir/sangre , Lopinavir/farmacocinética , Ganglios Linfáticos/metabolismo , Macaca nemestrina , Masculino , Nanopartículas/química , Ritonavir/sangre , Ritonavir/farmacocinética , Tenofovir/sangre , Tenofovir/farmacocinéticaRESUMEN
The outer membranes of Gram-negative bacteria and mitochondria contain proteins with a distinct ß-barrel tertiary structure that could function as a molecular pattern recognized by the innate immune system. Here, we report that purified outer membrane proteins (OMPs) from different bacterial and mitochondrial sources triggered the induction of autophagy-related endosomal acidification, LC3B lipidation, and p62 degradation. Furthermore, OMPs reduced the phosphorylation and therefore activation of the multiprotein complex mTORC2 and its substrate Akt in macrophages and epithelial cells. The cell surface receptor SlamF8 and the DNA-protein kinase subunit XRCC6 were required for these OMP-specific responses in macrophages and epithelial cells, respectively. The addition of OMPs to mouse bone marrow-derived macrophages infected with Salmonella Typhimurium facilitated bacterial clearance. These data identify a specific cellular response mediated by bacterial and mitochondrial OMPs that can alter inflammatory responses and influence the killing of pathogens.
Asunto(s)
Autofagia , Proteínas de la Membrana Bacteriana Externa/metabolismo , Membrana Celular/patología , Diana Mecanicista del Complejo 2 de la Rapamicina/antagonistas & inhibidores , Membranas Mitocondriales/patología , Monocitos/patología , Infecciones por Salmonella/patología , Animales , Membrana Celular/metabolismo , Células Cultivadas , Humanos , Diana Mecanicista del Complejo 2 de la Rapamicina/metabolismo , Ratones , Membranas Mitocondriales/metabolismo , Monocitos/metabolismo , Infecciones por Salmonella/microbiología , Salmonella typhimurium/aislamiento & purificación , Familia de Moléculas Señalizadoras de la Activación Linfocitaria/metabolismoRESUMEN
OBJECTIVE: The aim of the present study was to determine whether a combination of anti-HIV drugs - tenofovir (TFV), lopinavir (LPV) and ritonavir (RTV) - in a lipid-stabilized nanosuspension (called TLC-ART101) could enhance and sustain intracellular drug levels and exposures in lymph node and blood cells above those in plasma. DESIGN: Four macaques were given a single dose of TLC-ART101 subcutaneously. Drug concentrations in plasma and mononuclear cells of the blood (PBMCs) and lymph nodes (LNMCs) were analysed using a validated combination LC-MS/MS assay. RESULTS: For the two active drugs (TFV, LPV), plasma and PBMC intracellular drug levels persisted for over 2 weeks; PBMC drug exposures were three- to four-fold higher than those in plasma. Apparent terminal half-lives (t1/2) of TFV and LPV were 65.3 and 476.9âh in plasma, and 169.1 and 151.2âh in PBMCs. At 24 and 192âh, TFV and LPV drug levels in LNMCs were up to 79-fold higher than those in PBMCs. Analysis of PBMC intracellular TFV and its active metabolite TFV-diphosphate (TFV-DP) indicated that intracellular exposures of total TFV and TFV-DP were markedly higher and persisted longer than in humans and macaques dosed with oral TFV prodrugs, tenofovir disoproxil fumarate (TDF) or tenofovir alafenamide (TAF). CONCLUSIONS: A simple, scalable three-drug combination, lipid-stabilized nanosuspension exhibited persistent drug levels in cells of lymph nodes and the blood (HIV host cells) and in plasma. With appropriate dose adjustment, TLC-ART101 may be a useful HIV treatment with a potential to impact residual virus in lymph nodes.
Asunto(s)
Fármacos Anti-VIH/farmacocinética , Terapia Antirretroviral Altamente Activa/métodos , Células Sanguíneas/química , Preparaciones de Acción Retardada/farmacocinética , Ganglios Linfáticos/química , Plasma/química , Suspensiones/farmacocinética , Animales , Fármacos Anti-VIH/administración & dosificación , Cromatografía Liquida , Preparaciones de Acción Retardada/administración & dosificación , Macaca , Suspensiones/administración & dosificación , Espectrometría de Masas en TándemRESUMEN
OBJECTIVE: To determine which route of inoculation produced consistent and frequent HIV infection in the central nervous system (CNS) and alterations in cognitive and motor development in infant macaques. METHODS: Infant macaques (Macaca nemestrina) were inoculated with the highly pathogenic strain HIV-2287 intravenously (n = 3) or intrathecally (n = 3). Uninfected infants were evaluated as controls. Disease progression was evaluated by virological assessment of blood and cerebral spinal fluid (CSF), CD4 T cell count in blood, and quinolinic acid levels in CSF (a surrogate marker of neuronal cell damage). The effect of HIV infection on cognitive and motor development in infants was monitored during the 6-month study. RESULTS: Either route of HIV-2287 inoculation produced detectable viral RNA in CSF and productive infection in blood. Detection of virus in CSF paralleled a rise in quinolinic acid levels. All HIV-infected infants experienced a severe and rapid decline in CD4 T cell counts by 10 weeks after viral infection. HIV-infected infants, particularly those infected by the intravenous route, exhibited delays in reaching cognitive and motor milestones, which paralleled neuropathological changes. CONCLUSIONS: The HIV-2287 infant model produced a high incidence of viral infection in the CNS regardless of the route of inoculation. Significant alteration in neurobehavioral development was observed in HIV-infected infants, and this measure was significantly impaired particularly in infants infected by the intravenous route. These data, coupled with the ability to detect viral RNA and changes in quinolinic acid levels in CSF, may allow quantitative evaluation of drug and immune candidates for treating neurological effects of AIDS.
Asunto(s)
Complejo SIDA Demencia/virología , VIH-2 , Complejo SIDA Demencia/líquido cefalorraquídeo , Complejo SIDA Demencia/patología , Análisis de Varianza , Animales , Conducta Animal , Cognición , Progresión de la Enfermedad , Macaca nemestrina , Modelos Animales , Destreza Motora , Ácido Quinolínico/líquido cefalorraquídeo , ARN Viral/líquido cefalorraquídeoRESUMEN
P-glycoprotein (P-gp) expression at the rodent blood-brain barrier (BBB) limits the central nervous system (CNS) distribution of anti-human immunodeficiency virus (HIV) protease inhibitors (PIs). However, it is not clear whether P-gp activity at the human BBB is as effective as that in rodents in preventing the distribution of PIs into the CNS. If it is, inhibition of P-gp at the human BBB could increase the distribution of the PIs into the CNS and, therefore, their efficacy against HIV-associated dementia. Because the distribution of the PIs into the human brain cannot be directly measured, we conducted studies in a more representative animal, the nonhuman primate. Specifically we investigated the distribution of nelfinavir (a PI and a P-gp substrate; 6 mg/kg i.v.) into the brain and cerebrospinal fluid (CSF) of nonhuman primates (cynomolgus monkeys, Macaca fascicularis) in the presence and absence of the potent and selective P-gp inhibitor, zosuquidar, and whether changes in brain nelfinavir concentration, after inhibition of P-gp, paralleled those in the CSF. Our data indicate that nelfinavir has poor penetration into the macaque's brain and CSF, and P-gp inhibition at the BBB by zosuquidar enhanced the distribution of nelfinavir into the brain by 146-fold. However, the concentration of nelfinavir in the CSF was unaffected by coadministration of zosuquidar (p > 0.05). In conclusion, P-gp inhibition at the nonhuman primate BBB significantly enhanced the distribution of nelfinavir into the brain, and this effect was not observed in the CSF. Therefore, as is common in human studies investigating P-gp inhibition at the BBB, CSF concentration of a drug should not be used as a surrogate marker for brain drug concentration.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/antagonistas & inhibidores , Barrera Hematoencefálica/fisiología , Encéfalo/metabolismo , Inhibidores de la Proteasa del VIH/farmacocinética , Nelfinavir/farmacocinética , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Animales , Área Bajo la Curva , Dibenzocicloheptenos/farmacología , Inhibidores de la Proteasa del VIH/líquido cefalorraquídeo , Inyecciones Intravenosas , Macaca fascicularis , Nelfinavir/líquido cefalorraquídeo , Quinolinas/farmacologíaRESUMEN
In HIV-infected persons on highly active antiretroviral therapy, residual virus is found in lymphoid tissues. Indinavir concentrations in lymph node mononuclear cells of patients on highly active antiretroviral therapy were approximately 25% to 35% of those in blood mononuclear cells, suggesting that drug insufficiency contributes to residual virus in lymphoid tissues. Therefore, we developed novel lipid-indinavir nanoparticles targeted to lymphoid tissues. Given subcutaneously, these nanoparticles provided indinavir concentrations 250% to 2270% higher than plasma indinavir concentrations in both peripheral and visceral lymph nodes. Improved indinavir delivery was reflected in reduced viral RNA and CD4(+) T-cell rebound. This study optimized lipid nanoparticle formulation with respect to indinavir in lymphoid tissues of HIV-infected macaques. Regardless of lipid characteristic tested (charge, fluidity, and steric modification), indinavir binds completely to lipid at pH 7.4 but is reversed at pH 5.5 or lower. Compared with previous formulations, nanoparticles composed of disteroyl phosphatidylcholine and methyl polyethylene glycol-disteroyl phosphatidylethanolamine (DSPC:mPEG-DSPE) provided 6-fold higher indinavir levels in lymph nodes and enhanced drug exposure in blood. Enhanced anti-HIV activity paralleled improved intracellular drug accumulation. Collectively, these data suggest that indinavir nanoparticles composed of DSPC:mPEG-DSPE provided the most effective lymphoid delivery and could maximally suppress the virus in lymphoid tissues.
Asunto(s)
Fármacos Anti-VIH/administración & dosificación , Fármacos Anti-VIH/farmacocinética , Infecciones por VIH/tratamiento farmacológico , VIH-2 , Indinavir/administración & dosificación , Indinavir/farmacocinética , Tejido Linfoide/química , Animales , Fármacos Anti-VIH/metabolismo , Fármacos Anti-VIH/farmacología , Recuento de Linfocito CD4 , Línea Celular , Modelos Animales de Enfermedad , Portadores de Fármacos , Infecciones por VIH/virología , Concentración de Iones de Hidrógeno , Indinavir/metabolismo , Indinavir/farmacología , Inyecciones Subcutáneas , Lípidos/administración & dosificación , Lípidos/farmacocinética , Ganglios Linfáticos/química , Macaca nemestrina , Nanoestructuras/química , Fosfatidilcolinas/farmacocinética , ARN Viral/sangre , Distribución TisularRESUMEN
Analysis of indinavir levels in HIV-positive patients indicated that drug concentrations in lymph node mononuclear cells (LNMCs) were about 25-35% of mononuclear cells in blood. To enhance lymphatic delivery of anti-HIV drugs, a novel drug delivery strategy was designed consisting of lipid-associated indinavir (50-80 nm in diameter) complexes in suspension for subcutaneous (SC) injection. Due to the pH-dependent lipophilicity of indinavir, practically all the drug molecules are incorporated into lipid phase when formulated at pH 7.4 and 5:1 lipid-to-drug (m/m) ratio. At pH 5.5, about 20% of drugs were found in lipid-drug complexes. Effects of lipid association on the time course of plasma indinavir concentrations were determined in macaques (Macaca nemestrina) administered with either soluble or lipid-associated formulation of indinavir (10 mg/kg, SC). Results yielded about a 10-fold reduction in peak plasma concentration and a 6-fold enhancement in terminal half-life (t1/2beta = 12 vs. 2 hours). In addition, indinavir concentrations in both peripheral and visceral lymph nodes were 250-2270% higher than plasma (compared with <35% with soluble lipid-free drug administration in humans). Administration of lipid-associated indinavir (20 mg/kg daily) to HIV-2287-infected macaques (at 30-33 weeks after infection) resulted in significantly reduced viral RNA load and increased CD4 T cell number concentrations. Collectively, these data indicate that lipid association greatly enhances delivery of the anti-HIV drug indinavir to lymph nodes at levels that cannot be achieved with soluble drug, provides significant virus load reduction, and could potentially reverse CD4 T cell depletion due to HIV infection.