Monoolein-based nanoparticles containing indinavir: a taste-masked drug delivery system.

OBJECTIVE: This study developed a novel child-friendly drug delivery system for pediatric HIV treatment: a liquid, taste-masked, and solvent-free monoolein-based nanoparticles formulation containing indinavir (0.1%). SIGNIFICANCE: Adherence to antiretroviral therapy by pediatric patients is difficult because of the lack of dosage forms adequate for children. METHODS: Monoolein-based nanoparticles were developed. The particle size, zeta potential, pH, drug content, small angle X-ray scattering, stability, in vitro drug release profile, biocompatibility, toxicity, and taste-masking properties were evaluated. RESULTS: Monoolein-based formulations containing indinavir had nanosized particles with 155 ± 7 nm, unimodal particle size distribution, and polydispersity index of 0.16 ± 0.03. The zeta potential was negative (-31.3 ± 0.3 mV) and pH was neutral (7.78 ± 0.01). A 96% drug incorporation efficiency was achieved, and the indinavir concentration remained constant for 30 days. Polarized light microscopy revealed isotropic characteristics. Transmission electron microscopy images showed spherical shaped morphology. Small-angle X-ray scattering displayed a form factor broad peak. Indinavir had a sustained release from the nanoparticles. The system was nonirritant and was able to mask drug bitter taste. CONCLUSIONS: Monoolein-based nanoparticles represent a suitable therapeutic strategy for antiretroviral treatment with the potential to reduce the frequency of drug administration and promote pediatric adherence.

Tamizaje virtual de compuestosinhibidores de la SA P-2de Candida albicans / Virtual screening of inhibitory compounds SEP-2 Candida albicans

El incremento de la resistencia a antifúngicos en Candida spp. una levadurade carácter oportunista asociada a múltiples infecciones superficiales y sistémicas,plantea la necesidad de buscar diferentes estrategias para hallar nuevas opciones terapéuticas más selectivas y específicas. Una alternativa es el tamizaje basado en el acoplamiento virtual. En el presente trabajo, se analizaron 13418 compuestos con estructuras similares a compuestos reconocidos como inhibidores de la proteína SAP-2,seleccionada como diana antifúngica para el análisis virtual. Materiales y métodos: el estudio se realizó utilizando el programa SYBYL 8, al tiempo que se evaluó la afinidadde estas estructuras con los sitios activos de la enzima seleccionada mediante el programa FlexX integrado en SYBYL 8 y se realizó un consenso comparando los resultados con el programa Autodock Vina. Resultados: los resultados obtenidos mostraron una mayor afinidad por móleculas con estructura similar a los antirretrovirales,inhibidores de proteasas; amprenavir, saquinavir e indinavir. Conclusiones: los resultados obtenidos sugieren que estos lnhibidores de proteasas pueden ser propuestos como modelos para la búsqueda de nuevos antifúngicos que sean más selectivos contra Candida spp...

Novel protease inhibitor-loaded Nanoparticle-in-Microparticle Delivery System leads to a dramatic improvement of the oral pharmacokinetics in dogs.

With the advent of the Highly Active Antiretroviral Therapy, the morbidity and the mortality associated to HIV have been considerably reduced. However, 35-40 million people bear the infection worldwide. One of the main causes of therapeutic failure is the frequent administration of several antiretrovirals that results in low patient compliance and treatment cessation. In this work, we have developed an innovative Nanoparticle-in-Microparticle Delivery System (NiMDS) comprised of pure drug nanocrystals of the potent protease inhibitor indinavir free base (used as poorly water-soluble model protease inhibitor) produced by nanoprecipitation that were encapsulated within mucoadhesive polymeric microparticles. Pure drug nanoparticles and microparticles were thoroughly characterized by diverse complementary techniques. NiMDSs displayed an encapsulation efficiency of approximately 100% and a drug loading capacity of up to 43% w/w. In addition, mucoadhesiveness assays ex vivo conducted with bovine gut showed that film-coated microparticles were retained for more than 6 h. Finally, pharmacokinetics studies in mongrel dogs showed a dramatic 47- and 95-fold increase of the drug oral bioavailability and half-life, respectively, with respect to the free unprocessed drug. These results support the outstanding performance of this platform to reduce the dose and the frequency of administration of protease inhibitors, a crucial step to overcome the current patient-incompliant therapy.

Decoding the anti-Trypanosoma cruzi action of HIV peptidase inhibitors using epimastigotes as a model.

BACKGROUND: Aspartic peptidase inhibitors have shown antimicrobial action against distinct microorganisms. Due to an increase in the occurrence of Chagas' disease/AIDS co-infection, we decided to explore the effects of HIV aspartic peptidase inhibitors (HIV-PIs) on Trypanosoma cruzi, the etiologic agent of Chagas' disease. METHODOLOGY AND PRINCIPAL FINDINGS: HIV-PIs presented an anti-proliferative action on epimastigotes of T. cruzi clone Dm28c, with IC50 values ranging from 0.6 to 14 µM. The most effective inhibitors, ritonavir, lopinavir and nelfinavir, also had an anti-proliferative effect against different phylogenetic T. cruzi strains. The HIV-PIs induced some morphological alterations in clone Dm28c epimastigotes, as reduced cell size and swollen of the cellular body. Transmission electron microscopy revealed that the flagellar membrane, mitochondrion and reservosomes are the main targets of HIV-PIs in T. cruzi epimastigotes. Curiously, an increase in the epimastigote-into-trypomastigote differentiation process of clone Dm28c was observed, with many of these parasites presenting morphological alterations including the detachment of flagellum from the cell body. The pre-treatment with the most effective HIV-PIs drastically reduced the interaction process between epimastigotes and the invertebrate vector Rhodnius prolixus. It was also noted that HIV-PIs induced an increase in the expression of gp63-like and calpain-related molecules, and decreased the cruzipain expression in epimastigotes as judged by flow cytometry and immunoblotting assays. The hydrolysis of a cathepsin D fluorogenic substrate was inhibited by all HIV-PIs in a dose-dependent manner, showing that the aspartic peptidase could be a possible target to these drugs. Additionally, we verified that ritonavir, lopinavir and nelfinavir reduced drastically the viability of clone Dm28c trypomastigotes, causing many morphological damages. CONCLUSIONS AND SIGNIFICANCE: The results contribute to understand the possible role of aspartic peptidases in T. cruzi physiology, adding new in vitro insights into the possibility of exploiting the use of HIV-PIs in the clinically relevant forms of the parasite.

In vivo assessment of antiretroviral therapy-associated side effects.

Antiretroviral therapy has been associated with side effects, either from the drug itself or in conjunction with the effects of human immunodeficiency virus infection. Here, we evaluated the side effects of the protease inhibitor (PI) indinavir in hamsters consuming a normal or high-fat diet. Indinavir treatment increased the hamster death rate and resulted in an increase in triglyceride, cholesterol and glucose serum levels and a reduction in anti-oxLDL auto-antibodies. The treatment led to histopathological alterations of the kidney and the heart. These results suggest that hamsters are an interesting model for the study of the side effects of antiretroviral drugs, such as PIs.

In vivo assessment of antiretroviral therapy-associated side effects

Antiretroviral therapy has been associated with side effects, either from the drug itself or in conjunction with the effects of human immunodeficiency virus infection. Here, we evaluated the side effects of the protease inhibitor (PI) indinavir in hamsters consuming a normal or high-fat diet. Indinavir treatment increased the hamster death rate and resulted in an increase in triglyceride, cholesterol and glucose serum levels and a reduction in anti-oxLDL auto-antibodies. The treatment led to histopathological alterations of the kidney and the heart. These results suggest that hamsters are an interesting model for the study of the side effects of antiretroviral drugs, such as PIs.

Production of pure indinavir free base nanoparticles by a supercritical anti-solvent (SAS) method.

CONTEXT: This work investigated the production of pure indinavir free base nanoparticles by a supercritical anti-solvent method to improve the drug dissolution in intestine-like medium. OBJECTIVE: To increase the dissolution of the drug by means of a supercritical fluid processing method. MATERIALS AND METHODS: Acetone was used as solvent and supercritical CO2 as antisolvent. Products were characterized by dynamic light scattering (size, size distribution), scanning electron microscopy (morphology), differential scanning calorimetry (thermal behaviour) and X-rays diffraction (crystallinity). RESULTS AND DISCUSSION: Processed indinavir resulted in particles of significantly smaller size than the original drug. Particles showed at least one dimension at the nanometer scale with needle or rod-like morphology. Results of X-rays powder diffraction suggested the formation of a mixture of polymorphs. Differential scanning calorimetry analysis showed a main melting endotherm at 152 °C. Less prominent transitions due to the presence of small amounts of bound water (in the raw drug) or an unstable polymorph (in processed IDV) were also visible. Finally, drug particle size reduction significantly increased the dissolution rate with respect to the raw drug. Conversely, the slight increase of the intrinsic solubility of the nanoparticles was not significant. CONCLUSIONS: A supercritical anti-solvent method enabled the nanonization of indinavir free base in one single step with high yield. The processing led to faster dissolution that would improve the oral bioavailability of the drug.

Antirretrovirais estavudina (d4t) 30mg e indinavir (idv) 400mg / Antiretroviral stavudine (d4t) 30mg and indinavir (idv) 400mg

INTRODUÇÃO: A partir da Nota Técnica nº 90/2012, de março de 2012, o Ministério da Saúde recomendou a substituição de estavudina (d4T) 30mg e indinavir (IDV) 400mg por outros antirretrovirais (ARV) da mesma classe terapêutica para todos os adultos ainda em uso destes medicamentos, e reitera a não indicação de ambos na terapia antirretroviral inicial. Após sua publicação, houve redução no número de pessoas em uso de d4T e IDV, permanecendo, entretanto, um contingente de pessoas com esquemas antirretrovirais estruturados com estes ARV. Conforme as diretrizes nacionais de tratamento antirretroviral, estes medicamentos não devem ser utilizados para estruturar o esquema antirretroviral inicial, em razão da existência de outras opções terapêuticas com melhor perfil de toxicidade. Assim, toda nova prescrição de tratamento com estavudina (d4T) e/ou indinavir (IDV) como primeiro esquema de tratamento para adultos, terá sua dispensação bloqueada pelo Sistema de Controle Logístico de Medicamento (SICLOM). Toda prescrição que mantenha d4T e/ou IDV para pacientes que já vinham em uso destes medicamentos no passado, terá que ser avaliada em relação a existência de outras opções de tratamento e aprovada pelas Câmaras Técnicas dos Estados ou pelo Ministério da Saúde. Aqueles que estão em uso destes medicamentos seu fornecimento será mantido por no máximo 6 (seis) meses após a publicação deste Relatório. Após este período, a dispensação destes medicamentos será bloqueada no SICLOM. Para pacientes que apresentam supressão viral completa (carga viral indetectável), sugere-se: a) substituir d4T por zidovudina (AZT), tenofovir (TDF), didanosina (ddI) ou abacavir (ABC); b) substituir IDV por lopinavir/ritonavir (LPV/r), atazanavir/ritonavir (ATV/r) ou fosamprenavir/ritonavir (FPV/r). Esta avaliação deve ser individualizada, considerando o histórico de tratamento antirretroviral e genotipagens anteriores.Pacientes que já fazem uso de IDV ou d4T e que estão em falha terapêutica, devem realizar teste de genotipagem e ter seu esquema estruturado conforme as diretrizes expressas no documento "Recomendações para Terapia Antirretroviral em Adultos Infectados pelo HIV" vigente no País. RECOMENDAÇÃO DA CONITEC: Os membros da CONITEC presentes na 24ª reunião do plenário realizada nos dias 09/04/2014 e 10/04/2014 recomendaram a exclusão da estavudina (d4T) 30mg e indinavir (IDV) 400mg na terapia antirretroviral inicial. DECISÃO: PORTARIA Nº 36, de 26 de setembro de 2014 - Torna pública a decisão de excluir os antirretrovirais estavudina (d4t) 30mg e indinavir (idv) 400mg no âmbito do Sistema Único de Saúde - SUS.

Viral protease inhibitors affect the production of virulence factors in Cryptococcus neoformans.

The effects of the protease inhibitors saquinavir, darunavir, ritonavir, and indinavir on growth inhibition, protease and phospholipase activities, as well as capsule thickness of Cryptococcus neoformans were investigated. Viral protease inhibitors did not reduce fungal growth when tested in concentrations ranging from 0.001 to 1.000 mg/L. A tendency toward increasing phospholipase activity was observed with the highest tested drug concentration in a strain-specific pattern. However, these drugs reduced protease activity as well as capsule production. Our results confirm a previous finding that antiretroviral drugs affect the production of important virulence factors of C. neoformans.

Effect of HIV protease inhibitors on New World Leishmania.

The incidence of HIV/Leishmania co-infection decreases after antiretroviral drug therapy; therefore, the in vitro and in vivo activity of three antiretroviral drugs against Leishmania (Viannia) braziliensis and L. (L.) amazonensis was evaluated. Different concentrations of indinavir (IDV), atazanavir (ATV), and ritonavir (RTV) were added to promastigote cultures, and the 50% inhibitory concentration (IC50) was determined. IDV and RTV were also evaluated against intracellular amastigotes, and the Infection Index determined. BALB/c mice, infected with L. (L.) amazonensis in the left footpad, were treated orally with IDV and RTV for 30 days, and monitored by measuring the footpad thickness and parasite load of regional lymph nodes and spleen. For promastigotes, IDV exhibited an IC50 value of 100 µM against L.(L.) amazonensis. The RTV IC50 for L. (L.) amazonensis and L. (V.) braziliensis were 40 and 2.3 µM, respectively, and the ATV IC50 for L. (V.) braziliensis was 266 µM. For intracellular amastigotes, IDV (25, 50, and 100 µM) significantly decreased the Infection Index of L. (L.) amazonensis (56.8%, 47.9%, and 65.0%) and L. (V.) braziliensis (37.8%, 48.7%, and 43.2%). RTV (12.5, 25, and 50 µM) decreased the infection index of L. (L.) amazonensis by 26.3%, 42.4%, and 44.0%, and that of L. (V.) braziliensis by 27.6%, 37.3%, and 39.2%. Antiretroviral-treated mice had a significant reduction in footpad thickness after the third week of IDV and after the fifth week of RTV treatment. However, there was no reduction in parasite load. These results suggest that IDV and RTV have anti-Leishmania activity, but only in higher concentrations.

Short communication: UGT1A1*28 variant allele is a predictor of severe hyperbilirubinemia in HIV-infected patients on HAART in southern Brazil.

Highly active antiretroviral therapy (HAART) has increased the survival of HIV-infected patients. However, adverse effects play a major role in adherence to HAART. Some protease inhibitors (mainly atazanavir and indinavir) act as inhibitors of uridine diphosphate-glucuronosyltransferase (UGT1A1), the enzyme responsible for hepatic conjugation of bilirubin. Variations in the promoter region of the UGT1A1 gene (UGT1A1*28, rs8175347) can influence bilirubin plasma levels, modulating the susceptibility to hyperbilirubinemia. Aiming to analyze the association between UGT1A1*28 allele and hyperbilirubinemia in individuals exposed to HAART, we evaluated 375 HIV-positive individuals on antiretroviral therapy. Individuals carrying the UGT1A1*28 allele had a higher risk of developing severe hyperbilirubinemia [prevalence ratio (PR)=2.43, 95% confidence interval (CI) 1.08-5.45, p=0.032] as well as atazanavir users (PR=7.72, 95% CI=3.14-18.98, p<0.001). This is the first description of such an association in Brazilian HIV patients, which shows that in African-American and Euroamerican HAART users, the UGT1A1*28 allele also predisposes to severe hyperbilirubinemia, especially in those exposed to atazanavir.

Chronic use of indinavir in albino rat pregnancy (Rattus norvegicus albinus, Rodentia, Mammalia): biological assay.

AIM: Assess possible adverse effects of the chronic use of indinavir during pregnancy in a rat model. METHODS: 40 pregnant EOM-1 albino rats were randomly allocated into four groups of 10 animals each: a control (Ctr) group (without any handling) and three experimental groups (Exp 1, Exp 2 e Exp 3) which received indinavir 9, 27 e 81 mg/kg, respectively). Rats were treated by gavage once daily. The treatment period extended from day 0 until the 20th day of pregnancy. Body weights were recorded on days 0, 7, 14 and 20. At term, the rats were sacrificed, and the implantation sites, number of live and dead fetuses and placentas, resorptions, fetal and placental weights were recorded. The fetuses were evaluated for external abnormalities under a stereomicroscope. RESULTS: Weight gain during pregnancy did not differ significantly between the groups. Average weight gains between the 7th and 20th day were 7.95-42.70 g; 7.22-45.27 g; 7.12-46.26 g and 8.05-42.29 g in groups Ctr, Exp 1, Exp 2 and Exp 3, respectively. All other parameters assessed did not differ significantly between groups. CONCLUSIONS: Chronic use of various dosages of indinavir during pregnancy was not associated significant adverse outcomes.

Evaluation of HIV protease and nucleoside reverse transcriptase inhibitors on proliferation, necrosis, apoptosis in intestinal epithelial cells and electrolyte and water transport and epithelial barrier function in mice.

BACKGROUND: Protease inhibitors (PI's) and reverse transcriptase drugs are important components of highly active antiretroviral therapy (HAART) for treating human acquired immunodeficiency syndrome (AIDS). Long-term clinical therapeutic efficacy and treatment compliance of these agents have been limited by undesirable side-effects, such as diarrhea. This study aims to investigate the effects of selected antiretroviral agents on intestinal histopathology and function in vivo and on cell proliferation and death in vitro. METHODS: Selected antiretroviral drugs were given orally over 7 days, to Swiss mice, as follows: 100 mg/kg of nelfinavir (NFV), indinavir (IDV), didanosine (DDI) or 50 mg/kg of zidovudine (AZT). Intestinal permeability measured by lactulose and mannitol assays; net water and electrolyte transport, in perfused intestinal segments; and small intestinal morphology and cell apoptosis were assessed in treated and control mice. In vitro cell proliferation was evaluated using the WST-1 reagent and apoptosis and necrosis by flow cytometry analysis. RESULTS: NFV, IDV, AZT and DDI caused significant reductions in duodenal and in jejunal villus length (p < 0.05). IDV and AZT increased crypt depth in the duodenum and AZT increased crypt depth in the jejunum. NFV, AZT and DDI significantly decreased ileal crypt depth. All selected antiretroviral drugs significantly increased net water secretion and electrolyte secretion, except for DDI, which did not alter water or chloride secretion. Additionally, only NFV significantly increased mannitol and lactulose absorption. NFV and IDV caused a significant reduction in cell proliferation in vitro at both 24 h and 48 h. DDI and AZT did not alter cell proliferation. There was a significant increase in apoptosis rates in IEC-6 cells after 24 h with 70 ug/mL of NFV (control: 4.7% vs NFV: 22%) while IDV, AZT and DDI did not show any significant changes in apoptosis compared to the control group. In jejunal sections, IDV and NFV significantly increased the number of TUNEL positive cells. CONCLUSION: The PI's, NFV and IDV, increased cell apoptosis in vivo, water and electrolyte secretion and intestinal permeability and decreased villus length and cell proliferation. NFV was the only drug tested that increased cell apoptosis in vitro. The nucleoside reverse transcriptase inhibitors, AZT and DDI, did not affect cell apoptosis or proliferation. These findings may partly explain the intestinal side-effects associated with PI's.

Causas de cambio de tratamiento en un grupo de pacientes VIH/SIDA cubanos / Causes of change in treatment in a group of HIV/AIDS Cuban patients

La infección por virus de inmunodeficiencia humana (VIH) es uno de los mayores problemas de salud actuales, se estima que más de 40 millones de personas están infectadas en el mundo. Se realizó un estudio descriptivo de corte transversal en 40 pacientes VIH/SIDA pertenecientes al servicio de Medicina del IPK con el propósito de caracterizar las causas del cambio de tratamiento antirretroviral y los tipos de reacciones adversas presentadas con este tratamiento en un grupo de pacientes VIH/SIDA..Los pacientes recibieron diferentes esquemas de antirretrovirales entre los que predominaron los siguientes: 3TC, d4T e Indinavir (57,5 por ciento), seguido de 3TC, AZT e Indinavir (22,5 por ciento). Las causas más frecuentes de cambio de tratamiento fueron las reacciones adversas y la mala adherencia al tratamiento. Entre las personas que están recibiendo terapia contra el VIH, existe una tendencia cada vez más frecuente de abandonar o cambiar la terapia. Las causas de estos cambios y del abandono de las terapias suelen estar relacionadas con los efectos secundarios, la fatiga del tratamiento, la fase de la infección por VIH en que se encuentra el paciente, y factores relativos a su estilo de vida. Las causas más frecuentes de cambio de tratamiento fueron las reacciones adversas, seguidas de la mala adherencia al tratamiento antirretroviral. Las reacciones adversas más frecuentes en el grupo de estudio fueron los vómitos y los trastornos digestivos respectivamente (AU)

The HIV virus infection is one of the major current problems of health estimating that more than 40 millions of persons are infected at world level. A cross-sectional and descriptive study was conducted in 40 HIV/AIDS patients from the Tropical Medicine Institute service to characterize the causes of the change in antiretroviruses treatment and the types of adverse reactions related to this treatment in a group of HIV/AIDS patients. Patients received different antiretroviruses schemes with predominance of 3TC, d4T and Indinarir (57,5 percent), followed by 3TC, AZT and Indinavir (22,5 percent). The more frequent causes of change of this treatment were the adverse reactions and a poor adherence to it. Among the persons with therapy HIV there is a more and more frequent trend to give up or to change of therapy. The causes of these changes and the leaving the therapy are related to side effects, treatment fatigue, and the HIV infection phase in patient and relative factors related to the lifestyle. The more frequent causes of the change of treatment were the adverse reactions followed by a poor adherence to antiretroviruses treatment. The more frequent adverse reactions were vomiting and digestive disorders, respectively (AU)

Causas de cambio de tratamiento en un grupo de pacientes VIH/SIDA cubanos / Causes of change in treatment in a group of HIV/AIDS Cuban patients

La infección por virus de inmunodeficiencia humana (VIH) es uno de los mayores problemas de salud actuales, se estima que más de 40 millones de personas están infectadas en el mundo. Se realizó un estudio descriptivo de corte transversal en 40 pacientes VIH/SIDA pertenecientes al servicio de Medicina del IPK con el propósito de caracterizar las causas del cambio de tratamiento antirretroviral y los tipos de reacciones adversas presentadas con este tratamiento en un grupo de pacientes VIH/SIDA..Los pacientes recibieron diferentes esquemas de antirretrovirales entre los que predominaron los siguientes: 3TC, d4T e Indinavir (57,5 por ciento), seguido de 3TC, AZT e Indinavir (22,5 por ciento). Las causas más frecuentes de cambio de tratamiento fueron las reacciones adversas y la mala adherencia al tratamiento. Entre las personas que están recibiendo terapia contra el VIH, existe una tendencia cada vez más frecuente de abandonar o cambiar la terapia. Las causas de estos cambios y del abandono de las terapias suelen estar relacionadas con los efectos secundarios, la fatiga del tratamiento, la fase de la infección por VIH en que se encuentra el paciente, y factores relativos a su estilo de vida. Las causas más frecuentes de cambio de tratamiento fueron las reacciones adversas, seguidas de la mala adherencia al tratamiento antirretroviral. Las reacciones adversas más frecuentes en el grupo de estudio fueron los vómitos y los trastornos digestivos respectivamente

The HIV virus infection is one of the major current problems of health estimating that more than 40 millions of persons are infected at world level. A cross-sectional and descriptive study was conducted in 40 HIV/AIDS patients from the Tropical Medicine Institute service to characterize the causes of the change in antiretroviruses treatment and the types of adverse reactions related to this treatment in a group of HIV/AIDS patients. Patients received different antiretroviruses schemes with predominance of 3TC, d4T and Indinarir (57,5 percent), followed by 3TC, AZT and Indinavir (22,5 percent). The more frequent causes of change of this treatment were the adverse reactions and a poor adherence to it. Among the persons with therapy HIV there is a more and more frequent trend to give up or to change of therapy. The causes of these changes and the leaving the therapy are related to side effects, treatment fatigue, and the HIV infection phase in patient and relative factors related to the lifestyle. The more frequent causes of the change of treatment were the adverse reactions followed by a poor adherence to antiretroviruses treatment. The more frequent adverse reactions were vomiting and digestive disorders, respectively

Mutation T74S in HIV-1 subtype B and C proteases resensitizes them to ritonavir and indinavir and confers fitness advantage.

OBJECTIVES: Several drug resistance and secondary mutations have been described in HIV-1 viruses from patients undergoing antiretroviral therapy. In this study, we assessed the impact of the protease substitution T74S on the phenotype and on the replicative fitness in HIV-1 subtypes B and C. METHODS: HIV-1 molecular clones carrying subtype B or C proteases had these coding regions subjected to site-directed mutagenesis to include T74S alone or in combination with four known protease inhibitor (PI) primary drug resistance mutations. All clones were used in a phenotypic assay to evaluate their susceptibility to most commercially available PIs. The impact of T74S on virus fitness was also assessed for all viruses through head-to-head competitions and oligonucleotide ligation assays to measure the proportion of each virus in culture. RESULTS: Viruses of both subtypes carrying T74S did not have their susceptibility altered to any tested PI. Viruses with the four resistance mutations showed strong resistance to most PIs with fold changes ranging from 5 to 300 times compared with their wild-type counterparts. Surprisingly, the addition of T74S to the multiresistant clones restored their susceptibilities to indinavir and ritonavir and partially to lopinavir, close to those of wild-type viruses. Most 74S-containing viruses were more fit than their 74T counterparts. CONCLUSIONS: Our results suggest that T74S is not a major drug resistance mutation, but it resensitizes multiresistant viruses to certain PIs. T74S is a bona fide accessory mutation, restoring fitness of multidrug-resistant viruses in both subtypes B and C. T74S should be further studied in clinical settings and considered in drug resistance interpretation algorithms.

Discordant genotypic interpretation and phenotypic role of protease mutations in HIV-1 subtypes B and G.

OBJECTIVES: HIV-1 group M is classified into nine different subtypes. Most antiretroviral (ARV) drugs have been developed for subtype B, and the response of non-B subtypes in terms of susceptibility and the acquisition of drug resistance when facing those drugs is largely unknown. In this study, we aimed to address differences in the impact of protease inhibitor (PI)-selected mutations on subtypes B and G. PATIENTS AND METHODS: ARV-treated, HIV-positive patients regularly monitored at the Hospital de Egas Moniz, in Lisbon, Portugal, were examined for the presence of PI-associated primary mutations (301 subtype B and 184 subtype G), and for the selection of those mutations over the time of PI exposure. Forty-three subtype G patients were phenotyped for susceptibility to all available PIs through VIRCO's Antivirogram, and compared with a similar dataset of subtype B patients. RESULTS: Mutation I54V/L was selected by nelfinavir in subtype G isolates, a mutation not previously described for this drug in subtype B. L90M was associated with a lower reduction in the susceptibility of subtype G to nelfinavir when compared with subtype B, and with no reduced susceptibility to saquinavir. This was compensated for by the acquisition of M89I in subtype G. L90M did not reduce the susceptibility of subtype G to saquinavir, in contrast to subtype B. Likewise, the pattern I54V/L-L90M did not reduce the susceptibility of subtype G to indinavir and saquinavir. Indinavir-associated mutations M46I/L, I84V and V82A/F/T developed earlier in subtype B across the time of exposure to that drug when compared with subtype G counterparts. CONCLUSIONS: Our results provide proof of principle and support the growing evidence that subtype-specific responses to ARVs exist. Data presented here highlight inconsistencies in current genotyping interpretation algorithms inadequately applied to all HIV-1 subtypes.

Indinavir-loaded pH-sensitive microparticles for taste masking: toward extemporaneous pediatric anti-HIV/AIDS liquid formulations with improved patient compliance.

The aim of this work was to develop indinavir pediatric anti-HIV/AIDS formulations enabling convenient dose adjustment, ease of oral administration, and improved organoleptic properties by means of the generation of drug-loaded microparticles made of a polymer that is insoluble under intake conditions and dissolves fast in the stomach in order to completely release the active agent. Indinavir-loaded microparticles made of a pH-dependent polymeric excipient soluble at pH < 5, Eudragit E100, were prepared using a double emulsion solvent diffusion technique and the in vitro release profiles characterized. Finally, taste masking properties were evaluated in blind randomized sensory experiments by ten healthy human volunteers. The use of a w/o/o emulsion system resulted in indinavir loads around 90%. Thermal analysis of the microparticles by differential scanning calorimetry revealed that indinavir appeared mainly dispersed at the molecular level. Concentrations of residual organic solvents as determined by gas chromatography were below the upper limits specified by the European Pharmacopeia for pharmaceutical oral formulations. Then, the behavior of drug-containing microparticles in aqueous media at different pH values was assessed. While they selectively dissolved in gastric-like medium, in tap water (intake conditions), the matrix remained almost unchanged and efficiently prevented drug dissolution. Finally, sensoring taste tests performed by volunteers indicated that systems with indinavir loads approximately 15% displayed acceptable taste. This work explored the production of indinavir-containing microparticles based on a common pharmaceutical excipient as a means for the improvement of medicines of drugs involved in the treatment of HIV/AIDS. For systems containing about 15% drug, taste studies confirmed the acceptability of the formulation. In pediatric regimes, this composition would require an acceptable amount of formulation (0.7-1.5 g).

Relationship between P-glycoprotein activity measured in peripheral blood mononuclear cells and indinavir bioavailability in healthy volunteers.

Indinavir, a HIV-1 protease inhibitor, showed large inter-individual pharmacokinetic variability. It has been proposed as a substrate of P-glycoprotein (P-gp), an efflux transporter, that may contribute to limit indinavir bioavailability. A liquid formulation of indinavir was developed from indinavir capsules in order to study indinavir pharmacokinetics in healthy volunteers. Compartmental and noncompartmental analysis of indinavir plasma concentrations showed high inter-individual variability in terms of area under the curve (AUC) and maximal plasma concentration (C(max)). A significant negative association between AUC normalized to body weight (AUC x weight) and lymphocyte P-gp activity, using Rh123 efflux assay, was observed (p = 0.008; r = -0.75). AUC normalized to elimination rate constant (AUC x beta) also showed a significant negative relationship with lymphocyte P-gp activity (p = 0.03, r = -0.64). Apparent clearance (CL/[F x weight]) and volume of distribution (VD/[F x weight]) showed a positive correlation with P-gp activity. Conversely, elimination rate constant did not correlate with P-gp activity. Although there is not enough evidence of a correlation between lymphocitary and intestinal function of P-gp, our results suggest a relationship between a P-gp phenotype marker, Rh123 efflux assay in lymphocytes, and indinavir bioavailability.

Pharmacokinetic study of the variability of indinavir drug levels when boosted with ritonavir in HIV-infected children.

The aim of this work is to: (1) assess therapeutic drug monitoring of indinavir (IDV) during clinical routine practice in HIV-infected children, whose antiretroviral treatment includes IDV boosted with ritonavir (RTV), and (2) describe a possible relationship between IDV pharmacokinetics and MDR1 genotypes. In 21 ambulatory pediatric patients receiving IDV plus RTV, IDV plasma levels and MDR1 genotypes on exon 26 (C3435T) were determined. Nine of the 21 patients initially receiving 250 mg/m(2) IDV yielded trough levels below 0.10 microg/ml (median: 0.21, range: 0.04-1.31 microg/ml). When the dosage was increased to 400 mg/m(2) IDV plus 100 mg/m(2) RTV b.i.d., all, except 1 patient, achieved levels above 0.10 microg/ml. Pharmacokinetic analysis showed higher volume of distribution median values related to the C/C genotype in comparison with C/T or T/T genotypes for exon 26 (4.57 vs. 1.20 and 1.50 l/kg, respectively; p = 0.002). Although a higher median value of clearance was observed with the C/C genotype, the difference was not statistically significant (1.43 vs. 0.27 and 0.42 l/h, respectively; p = 0.052). These results may be explained by a reduced absorption of the drug, related with lower plasma IDV levels in patients carrying the C/C genotype in exon 26.