RESUMEN
Cytochrome P450 (P450) 3A4 is the enzyme most involved in the metabolism of drugs and can also oxidize numerous steroids. This enzyme is also involved in one-half of pharmacokinetic drug-drug interactions, but details of the exact mechanisms of P450 3A4 inhibition are still unclear in many cases. Ketoconazole, clotrimazole, ritonavir, indinavir, and itraconazole are strong inhibitors; analysis of the kinetics of reversal of inhibition with the model substrate 7-benzoyl quinoline showed lag phases in several cases, consistent with multiple structures of P450 3A4 inhibitor complexes. Lags in the onset of inhibition were observed when inhibitors were added to P450 3A4 in 7-benzoyl quinoline O-debenzylation reactions, and similar patterns were observed for inhibition of testosterone 6ß-hydroxylation by ritonavir and indinavir. Upon mixing with inhibitors, P450 3A4 showed rapid binding as judged by a spectral shift with at least partial high-spin iron character, followed by a slower conversion to a low-spin iron-nitrogen complex. The changes were best described by two intermediate complexes, one being a partial high-spin form and the second another intermediate, with half-lives of seconds. The kinetics could be modeled in a system involving initial loose binding of inhibitor, followed by a slow step leading to a tighter complex on a multisecond time scale. Although some more complex possibilities cannot be dismissed, these results describe a system in which conformationally distinct forms of P450 3A4 bind inhibitors rapidly and two distinct P450-inhibitor complexes exist en route to the final enzyme-inhibitor complex with full inhibitory activity.
Asunto(s)
Clotrimazol/farmacología , Inhibidores del Citocromo P-450 CYP3A/farmacología , Citocromo P-450 CYP3A/química , Indinavir/farmacología , Itraconazol/farmacología , Cetoconazol/farmacología , Ritonavir/farmacología , Esteroide Hidroxilasas/antagonistas & inhibidores , Animales , Biocatálisis , Clonación Molecular , Clotrimazol/química , Citocromo P-450 CYP3A/genética , Citocromo P-450 CYP3A/metabolismo , Inhibidores del Citocromo P-450 CYP3A/química , Pruebas de Enzimas , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Humanos , Hidroxiquinolinas/síntesis química , Hidroxiquinolinas/metabolismo , Indinavir/química , Itraconazol/química , Cetoconazol/química , Cinética , Ratas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ritonavir/química , Esteroide Hidroxilasas/química , Esteroide Hidroxilasas/genética , Esteroide Hidroxilasas/metabolismoRESUMEN
Toxoplasma gondii is an obligate intracellular apicomplexan parasite causing lethal diseases in immunocompromised patients. UBL-UBA shuttle proteins (DDI1, RAD23, and DSK2) are important components of the ubiquitin-proteasome system. By degrading ubiquitinated proteins, UBL-UBA shuttle proteins regulate many cellular processes. However, the specific processes regulated by UBL-UBA shuttle proteins remain elusive. Here, we revealed that the deletion of shuttle proteins results in a selective accumulation of ubiquitinated proteins in the nucleus and aberrant DNA replication. ROP18 was mistargeted and accumulated in the shuttle protein mutant strain, resulting in the recruitment of immunity-related GTPases to the parasitophorous vacuole membrane (PVM). Furthermore, the mistargeting of ROP18 and the recruitment of Irgb6 to the PVM were also observed in the DDI1 mutant strain. DDI1 is a nonclassical UBL-UBA shuttle protein homologous to the HIV-1 protease. Molecular docking showed that DDI1 was a potential target of HIV-1 protease inhibitors. However, these inhibitors blocked the growth of T gondii in vitro but not in vivo. In conclusion, the Toxoplasma UBL-UBA shuttle protein regulates several important cellular processes and the mistargeting of ROP18 may be a representative of the abnormal homeostasis caused by shuttle protein mutation.
Asunto(s)
Indinavir/farmacología , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Protozoarias/metabolismo , Toxoplasma/metabolismo , Complejos de Ubiquitina-Proteína Ligasa/metabolismo , Ubiquitina/metabolismo , Animales , Replicación del ADN , Femenino , Inhibidores de la Proteasa del VIH/farmacología , Humanos , Ratones , Ratones Endogámicos BALB C , Proteínas Serina-Treonina Quinasas/genética , Proteínas Protozoarias/genética , Toxoplasma/efectos de los fármacos , Complejos de Ubiquitina-Proteína Ligasa/genética , UbiquitinaciónRESUMEN
A human immunodeficiency virus-1 (HIV-1) protease is a homodimeric aspartic protease essential for the replication of HIV. The HIV-1 protease is a target protein in drug discovery for antiretroviral therapy, and various inhibitor molecules of transition state analogues have been developed. However, serious drug-resistant mutants have emerged. For understanding the molecular mechanism of the drug resistance, an accurate examination of the impacts of the mutations on ligand binding and enzymatic activity is necessary. Here, we present a molecular simulation study on the ligand binding of indinavir, a potent transition state analogue inhibitor, to the wild-type protein and a V82T/I84V drug-resistant mutant of the HIV-1 protease. We employed a hybrid ab initio quantum mechanical/molecular mechanical (QM/MM) free-energy optimization technique which combines a highly accurate QM description of the ligand molecule and its interaction with statistically ample conformational sampling of the MM protein environment by long-time molecular dynamics simulations. Through the free-energy calculations of protonation states of catalytic groups at the binding pocket and of the ligand-binding affinity changes upon the mutations, we successfully reproduced the experimentally observed significant reduction of the binding affinity upon the drug-resistant mutations and elucidated the underlying molecular mechanism. The present study opens the way for understanding the molecular mechanism of drug resistance through the direct quantitative comparison of ligand binding and enzymatic reaction with the same accuracy.
Asunto(s)
Inhibidores de la Proteasa del VIH , Indinavir , Sitios de Unión , Farmacorresistencia Viral , Proteasa del VIH/metabolismo , Inhibidores de la Proteasa del VIH/química , Humanos , Indinavir/química , Indinavir/metabolismo , Indinavir/farmacología , Simulación de Dinámica Molecular , MutaciónRESUMEN
In this work, we report the benchmark binding energies of the seven complexes within the L7 data set, six host-guest complexes from the S12L data set, a C60 dimer, the DNA-ellipticine intercalation complex, and the largest system of the study, the HIV-indinavir system, which contained 343 atoms or 139 heavy atoms. The high-quality values reported were obtained via a focal point method that relies on the canonical form of second-order Møller-Plesset theory and the domain-based local pair natural orbital scheme for the coupled cluster with single double and perturbative triple excitations [DLPNO-CCSD(T)] extrapolated to the complete basis set (CBS) limit. The results in this work not only corroborate but also improve upon some previous benchmark values for large noncovalent complexes albeit at a relatively steep cost. Although local CCSD(T) and the largely successful fixed-node diffusion Monte Carlo (FN-DMC) have been shown to generally agree for small- to medium-size systems, a discrepancy in their reported binding energy values arises for large complexes, where the magnitude of the disagreement is a definite cause for concern. For example, the largest deviation in the L7 data set was 2.8 kcal/mol (â¼10%) on the low end in C3GC. Such a deviation only grows worse in the S12L set, which showed a difference of up to 10.4 kcal/mol (â¼25%) by a conservative estimation in buckycatcher-C60. The DNA-ellipticine complex also generated a disagreement of 4.4 kcal/mol (â¼10%) between both state-of-the-art methods. The disagreement between local CCSD(T) and FN-DMC in large noncovalent complexes shows that it is urgently needed to have the canonical CCSD(T), the Monte Carlo CCSD(T), or the full configuration interaction quantum Monte Carlo approaches available to large systems on the hundred-atom scale to solve this dilemma. In addition, the performances of cheaper popular computational methods were assessed for the studied complexes with respect to DLPNO-CCSD(T)/CBS. r2SCAN-3c, B97M-V, and PBE0+D4 work well in large noncovalent complexes in this work, and GFN2-xTB performs well in π-π stacking complexes. B97M-V is the most reliable computationally efficient approach to predicting noncovalent interactions for large complexes, being the only one to have binding errors within the so-called 1 kcal/mol "chemical accuracy". The benchmark interaction energies of these host-guest complexes, molecular materials, and biological systems with electronic and medicinal implications provide crucial reference data for the improvement of current and future lower-cost methods.
Asunto(s)
Elipticinas , Infecciones por VIH , Benchmarking , ADN , Humanos , Indinavir , Teoría CuánticaRESUMEN
The human immunodeficiency virus type 1 (HIV-1) has continued to be a global concern. With the new HIV incidence, the emergence of multi-drug resistance and the untoward side effects of currently used anti-HIV drugs, there is an urgent need to discover more efficient anti-HIV drugs. Modern computational tools have played vital roles in facilitating the drug discovery process. This research focuses on a pharmacophore-based similarity search to screen 111,566,735 unique compounds in the PubChem database to discover novel HIV-1 protease inhibitors (PIs). We used an in silico approach involving a 3D-similarity search, physicochemical and ADMET evaluations, HIV protease-inhibitor prediction (IC50/percent inhibition), rigid receptor-molecular docking studies, binding free energy calculations and molecular dynamics (MD) simulations. The 10 FDA-approved HIV PIs (saquinavir, lopinavir, ritonavir, amprenavir, fosamprenavir, atazanavir, nelfinavir, darunavir, tipranavir and indinavir) were used as reference. The in silico analysis revealed that fourteen out of the twenty-eight selected optimized hit molecules were within the acceptable range of all the parameters investigated. The hit molecules demonstrated significant binding affinity to the HIV protease (PR) when compared to the reference drugs. The important amino acid residues involved in hydrogen bonding and п-п stacked interactions include ASP25, GLY27, ASP29, ASP30 and ILE50. These interactions help to stabilize the optimized hit molecules in the active binding site of the HIV-1 PR (PDB ID: 2Q5K). HPS/002 and HPS/004 have been found to be most promising in terms of IC50/percent inhibition (90.15%) of HIV-1 PR, in addition to their drug metabolism and safety profile. These hit candidates should be investigated further as possible HIV-1 PIs with improved efficacy and low toxicity through in vitro experiments and clinical trial investigations.
Asunto(s)
Fármacos Anti-VIH , Inhibidores de la Proteasa del VIH , VIH-1 , Humanos , Inhibidores de la Proteasa del VIH/química , Proteasa del VIH/química , Darunavir/farmacología , Indinavir/química , Indinavir/metabolismo , Indinavir/farmacología , Nelfinavir/química , Nelfinavir/metabolismo , Nelfinavir/farmacología , Ritonavir/química , Saquinavir/metabolismo , Saquinavir/farmacología , Lopinavir/farmacología , Sulfato de Atazanavir/farmacología , Simulación del Acoplamiento Molecular , Fármacos Anti-VIH/farmacología , Aminoácidos/farmacologíaRESUMEN
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.
Asunto(s)
Glicéridos/química , Indinavir , Nanopartículas , Niño , Sistemas de Liberación de Medicamentos , Liberación de Fármacos , Humanos , Tamaño de la Partícula , GustoRESUMEN
Orosomucoid polymorphisms influence plasma drug binding in humans; however, canine variants and their effect on drug plasma protein binding have not yet been reported. In this study, the orosomucoid gene (ORM1) was sequenced in 100 dogs to identify the most common variant and its allele frequency determined in 1,464 dogs (from 64 breeds and mixed-breed dogs). Plasma protein binding extent of amitriptyline, indinavir, verapamil, and lidocaine were evaluated by equilibrium dialysis using plasma from ORM1 genotyped dogs (n = 12). Free and total drug plasma concentrations were quantified by liquid chromatography-mass spectrometry. From the five polymorphisms identified in canine ORM1, two were nonsynonymous. The most common was c.70G>A (p.Ala24Thr) with an allele frequency of 11.2% (n = 1464). Variant allele frequencies varied by breed, reaching 74% in Shetland Sheepdogs (n = 21). Free drug fractions did not differ significantly (p > .05; Mann-Whitney U) between plasma collected from dogs with c.70AA (n = 4) and those with c.70GG (n = 8) genotypes. While c.70G>A did not affect the extent of plasma protein binding in our study, the potential biological and pharmacological implication of this newly discovered ORM1 variant in dogs should be further investigated.
Asunto(s)
Proteínas Sanguíneas/metabolismo , Perros/genética , Genotipo , Orosomucoide/metabolismo , Polimorfismo Genético , Amitriptilina/farmacocinética , Anestésicos Locales/farmacocinética , Animales , Antiarrítmicos/farmacocinética , Antidepresivos Tricíclicos/farmacocinética , Perros/sangre , Perros/metabolismo , Regulación de la Expresión Génica/fisiología , Inhibidores de la Proteasa del VIH/farmacocinética , Indinavir/farmacocinética , Lidocaína/farmacocinética , Orosomucoide/genética , Unión Proteica , Verapamilo/farmacocinéticaRESUMEN
BACKGROUND: Increased pericardial adipose tissue is associated with higher risk of cardiovascular disease. We aimed to determine whether human immunodeficiency virus (HIV) status was independently associated with larger pericardial adipose tissue volume and to explore possible HIV-specific risk factors. METHODS: Persons with HIV (PWH) were recruited from the Copenhagen Comorbidity in HIV Infection (COCOMO) Study and matched 1:1 on age and sex to uninfected controls. Pericardial adipose tissue volume was measured using cardiac computed tomography. RESULTS: A total of 587 PWH and 587 controls were included. Median age was 52 years, and 88% were male. Human immunodeficiency virus status was independently associated with 17 mL (95% confidence interval [CI], 10-23; Pâ <â .001) larger pericardial adipose tissue volume. Larger pericardial adipose tissue volume was associated with low CD4+ nadir and prior use of stavudine, didanosine, and indinavir. Among PWH without thymidine analogue or didanosine exposure, time since initiating combination antiretroviral treatment (per 5-year use) was associated with l6 mL (95% CI, -6 to -25; Pâ =â .002) lower pericardial adipose tissue volume. CONCLUSIONS: Human immunodeficiency virus status was independently associated with larger pericardial adipose tissue volume. Severe immunodeficiency, stavudine, didanosine, and indinavir were associated with larger pericardial adipose tissue volume. Persons with HIV with prior exposure to these drugs may constitute a distinct cardiovascular risk population.
Asunto(s)
Tejido Adiposo/efectos de los fármacos , Fármacos Anti-VIH/efectos adversos , Fármacos Anti-VIH/uso terapéutico , Enfermedades Cardiovasculares/inducido químicamente , Infecciones por VIH/tratamiento farmacológico , Pericardio/fisiopatología , Carga Viral , Tejido Adiposo/fisiopatología , Adulto , Enfermedades Cardiovasculares/fisiopatología , Dinamarca , Didanosina/efectos adversos , Femenino , Infecciones por VIH/fisiopatología , Inhibidores de la Proteasa del VIH/uso terapéutico , Voluntarios Sanos , Humanos , Indinavir/efectos adversos , Masculino , Persona de Mediana Edad , Factores de Riesgo , Estavudina/efectos adversosRESUMEN
Infection by human immunodeficiency virus type 1 (HIV-1) not only destroys the immune system bringing about acquired immune deficiency syndrome (AIDS), but also induces serious neurological diseases including behavioral abnormalities, motor dysfunction, toxoplasmosis, and HIV-1 associated dementia. The emergence of HIV-1 multidrug-resistant mutants has become a major problem in the therapy of patients with HIV-1 infection. Focusing on the wild type (WT) and G48T/L89M mutated forms of HIV-1 protease (HIV-1 PR) in complex with amprenavir (APV), indinavir (IDV), ritonavir (RTV), and nelfinavir (NFV), we have investigated the conformational dynamics and the resistance mechanism due to the G48T/L89M mutations by conducting a series of molecular dynamics (MD) simulations and free energy (MM-PBSA and solvated interaction energy (SIE)) analyses. The simulation results indicate that alterations in the side-chains of G48T/L89M mutated residues cause the inner active site to increase in volume and induce more curling of the flap tips, which provide the main contributions to weaker binding of inhibitors to the HIV-1 PR. The results of energy analysis reveal that the decrease in van der Waals interactions of inhibitors with the mutated PR relative to the wild-type (WT) PR mostly drives the drug resistance of mutations toward these four inhibitors. The energy decomposition analysis further indicates that the drug resistance of mutations can be mainly attributed to the change in van der Waals and electrostatic energy of some key residues (around Ala28/Ala28' and Ile50/Ile50'). Our work can give significant guidance to design a new generation of anti-AIDS inhibitors targeting PR in the therapy of patients with HIV-1 infection.
Asunto(s)
Proteasa del VIH/metabolismo , Simulación de Dinámica Molecular , Fármacos Anti-VIH/química , Fármacos Anti-VIH/metabolismo , Carbamatos/química , Carbamatos/metabolismo , Resistencia a Medicamentos/efectos de los fármacos , Resistencia a Medicamentos/genética , Furanos , Proteasa del VIH/genética , Indinavir/química , Indinavir/metabolismo , Conformación Molecular , Mutación , Nelfinavir/química , Nelfinavir/metabolismo , Unión Proteica , Ritonavir/química , Ritonavir/metabolismo , Sulfonamidas/química , Sulfonamidas/metabolismoRESUMEN
Midazolam is a widely used index substrate for assessing effects of xenobiotics on CYP3A activity. A previous study involving human hepatocytes showed the primary route of midazolam metabolism, 1'-hydroxylation, shifted to N-glucuronidation in the presence of the CYP3A inhibitor ketoconazole, which may lead to an overprediction of the magnitude of a xenobiotic-midazolam interaction. Because ketoconazole is no longer recommended as a clinical CYP3A inhibitor, indinavir was selected as an alternate CYP3A inhibitor to evaluate the contribution of the N-glucuronidation pathway to midazolam metabolism. The effects of indinavir on midazolam 1'-hydroxylation and N-glucuronidation were first characterized in human-derived in vitro systems. Compared with vehicle, indinavir (10 µM) inhibited midazolam 1'-hydroxylation by recombinant CYP3A4, human liver microsomes, and high-CYP3A activity cryopreserved human hepatocytes by ≥70%; the IC50 obtained with hepatocytes (2.7 µM) was within reported human unbound indinavir Cmax (≤5 µM). Midazolam N-glucuronidation in hepatocytes increased in the presence of indinavir in both a concentration-dependent (1-33 µM) and time-dependent (0-4 hours) manner (by up to 2.5-fold), prompting assessment in human volunteers (n = 8). As predicted by these in vitro data, indinavir was a strong inhibitor of the 1'-hydroxylation pathway, decreasing the 1'-hydroxymidazolam/midazolam area under the plasma concentration versus time curve (AUC)0-12h ratio by 80%. Although not statistically significant, the midazolam N-glucuronide/midazolam AUC0-12h ratio increased by 40%, suggesting a shift to the N-glucuronidation pathway. The amount of midazolam N-glucuronide recovered in urine increased 4-fold but remained <10% of the oral midazolam dose (2.5 mg). A powered clinical study would clarify whether N-glucuronidation should be considered when assessing the magnitude of a xenobiotic-midazolam interaction.
Asunto(s)
Inhibidores del Citocromo P-450 CYP3A/farmacología , Glucurónidos/metabolismo , Inhibidores de la Proteasa del VIH/farmacología , Indinavir/farmacología , Midazolam/farmacocinética , Estudios Cruzados , Interacciones Farmacológicas , Femenino , Hepatocitos/metabolismo , Humanos , Hidroxilación , Técnicas In Vitro , Masculino , Midazolam/sangre , Midazolam/orina , Estudios ProspectivosRESUMEN
The mechanism for differential effects of human immune deficiency virus protease inhibitors (HIVPIs), nelfinavir (NEL) and indinavir (IND) on collagen metabolism disturbances was studied in human skin fibroblasts. It has been considered that HIVPIs-dependent deregulation of collagen biosynthesis involves prolidase (an enzyme providing proline for collagen biosynthesis), glutamine (Gln) (a substrate for proline biosynthesis), nuclear factor-κB (NF-κB) (a transcription factor that inhibit expression of type I collagen genes), ß1 integrin receptor and Akt signalling. It was found that NEL impaired collagen biosynthesis and the process was more pronounced in the presence of Gln, while IND stimulated collagen biosynthesis. NEL-dependent inhibition of collagen biosynthesis was accompanied by massive intracellular accumulation of type I collagen, while IND slightly induced this process. This effect of NEL was reversed by ascorbic acid but not N-acetylcysteine. The mechanism for the NEL-dependent defect in collagen metabolism was found at the level of prolidase activity, ß1 integrin signalling and NF-κB. NEL inhibited expression of ß1 integrin receptor, Akt and ERK1/2 and increased expression of p65 NF-κB. However, inhibitors of p65 NF-κB did not prevent NEL-dependent inhibition of collagen biosynthesis suggesting that this transcription factor is not involved in studied mechanism. Using PI3K inhibitor wortmannin that prevent phosphorylation of Akt revealed that NEL-dependent inhibition of Akt results in inhibition of collagen biosynthesis. The data suggest that differential effect of NEL and IND on collagen metabolism involves NEL-dependent down-regulation of Akt signalling and proline availability for collagen biosynthesis.
Asunto(s)
Colágeno/metabolismo , Fibroblastos/efectos de los fármacos , Indinavir/farmacología , Nelfinavir/farmacología , Piel/efectos de los fármacos , Acetilcisteína/química , Ácido Ascórbico/química , Supervivencia Celular , ADN/análisis , Dipeptidasas/metabolismo , Regulación hacia Abajo , Fibroblastos/metabolismo , Glutamina/química , Inhibidores de la Proteasa del VIH/farmacología , Humanos , Subunidad p50 de NF-kappa B , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación , Prolina/química , Wortmanina/farmacologíaRESUMEN
BACKGROUND: Kidney injury is a serious comorbidity among HIV-infected patients. Intravenous drug use is listed as one of the risk factors for impaired renal function; however, this group is rarely assessed for specific renal-related risks. METHODS: Patients attending methadone program from 1994 to 2015 were included in the study. Data collected included demographic data, laboratory tests, antiretroviral treatment history, methadone dosing and drug abstinence. Patients' drug abstinence was checked monthly on personnel demand. We have evaluated two study outcomes: (1) having at least one or (2) three eGFR < 60 ml/min (MDRD formula). RESULTS: In total, 267 persons, with 2593 person-years of follow-up were included into analyses. At the time of analyses, 251 (94%) were on antiretroviral therapy (ARV). Fifty-two (19.5%) patients had 1eGFR and 20 (7.5%) 3eGFR < 60. In univariate analysis, factors significantly increasing the odds of impaired renal function were: female gender, detectable HIV RNA on ART, age at registration per 5 years older, atazanavir use and time on antiretroviral treatment per 1 year longer. In the multivariate model, only female gender (OR 4.7; p = 0.002), time on cART (OR 1.11; p = 0.01) and baseline eGFR (OR 0.71; p = 0.001) were statistically significant. CONCLUSIONS: We have demonstrated a high rate of kidney function impairment among HIV-1 positive patients in the methadone program. All risk factors for decreased eGFR in this subpopulation of patients were similar to those described for general HIV population with very high prevalence in women. These findings imply the need for more frequent kidney function monitoring in this subgroup of patients.
Asunto(s)
Infecciones por VIH/tratamiento farmacológico , Enfermedades Renales/epidemiología , Enfermedades Renales/fisiopatología , Metadona/uso terapéutico , Narcóticos/uso terapéutico , Trastornos Relacionados con Opioides/rehabilitación , Adulto , Fármacos Anti-VIH/uso terapéutico , Sulfato de Atazanavir/uso terapéutico , Quimioterapia Combinada , Femenino , Tasa de Filtración Glomerular , Infecciones por VIH/complicaciones , Humanos , Indinavir/uso terapéutico , Masculino , Tratamiento de Sustitución de Opiáceos , Trastornos Relacionados con Opioides/complicaciones , Polonia/epidemiología , Factores de Riesgo , Factores Sexuales , Tenofovir/uso terapéutico , Factores de TiempoRESUMEN
OBJECTIVE: Indinavir (IDV), an antiretroviral protease inhibitor used in treatment of HIV infection, has limited entry into brain due to efflux by the P-glycoprotein presented in blood-brain barrier. The aim of present study was to develop lactoferrin-treated nanoemulsion containing indinavir (Lf-IDV-NEs) for delivery to brain. METHODS: Indinavir-loaded nanoemulsions (IDV-NEs) were prepared by high-speed homogenization method, and then lactoferrin was coupled to IDV-NEs by water soluble EDC method. RESULTS: The hydrodynamic diameters, polydispersity index, and zeta potential of IDV-NEs were 112 ± 3.5 nm, 0.20 ± 0.02, and -33.2 ± 2.6 mV, respectively. From in vivo studies in animal model of rats, the AUC0-4 h of brain concentration-time profile of IDV-NEs and Lf-IDV-NEs were 1.6 and 4.1 times higher than free drug, respectively. The brain uptake clearance of IDV-NEs and Lf-IDV-NEs were, interestingly, 393- and 420-times higher than the free drug. CONCLUSIONS: It can be concluded that applying both lactoferrin-treated and non-treated nanoemulsions clearly leads to significant brain penetration enhancement of indinavir, an effect which is more pronounced in the case of Lf-IDV-NEs with the higher drug residence time in brain.
Asunto(s)
Barrera Hematoencefálica/metabolismo , Portadores de Fármacos/química , Inhibidores de la Proteasa del VIH/farmacocinética , Indinavir/farmacocinética , Lactoferrina/química , Animales , Área Bajo la Curva , Liberación de Fármacos , Emulsiones , Infecciones por VIH/tratamiento farmacológico , Inhibidores de la Proteasa del VIH/administración & dosificación , Indinavir/administración & dosificación , Inyecciones Intravenosas , Masculino , Nanopartículas/química , Permeabilidad , Polisorbatos/química , Ratas , Ratas Sprague-DawleyRESUMEN
Cytochromes P450 (CYPs) oxidize alkylated amines commonly found in drugs and other biologically active molecules, cleaving them into an amine and an aldehyde. Metabolic studies usually neglect to report or investigate aldehydes, even though they can be toxic. It is assumed that they are efficiently detoxified into carboxylic acids and alcohols. Nevertheless, some aldehydes are reactive and escape detoxification pathways to cause adverse events by forming DNA and protein adducts. Herein, we modeled N-dealkylations that produce both amine and aldehyde metabolites and then predicted the reactivity of the aldehyde. This model used a deep learning approach previously developed by our group to predict other types of drug metabolism. In this study, we trained the model to predict N-dealkylation by human liver microsomes (HLM), finding that including isozyme-specific metabolism data alongside HLM data significantly improved results. The final HLM model accurately predicted the site of N-dealkylation within metabolized substrates (97% top-two and 94% area under the ROC curve). Next, we combined the metabolism, metabolite structure prediction, and previously published reactivity models into a bioactivation model. This combined model predicted the structure of the most likely reactive metabolite of a small validation set of drug-like molecules known to be bioactivated by N-dealkylation. Applying this model to approved and withdrawn medicines, we found that aldehyde metabolites produced from N-dealkylation may explain the hepatotoxicity of several drugs: indinavir, piperacillin, verapamil, and ziprasidone. Our results suggest that N-dealkylation may be an under-appreciated bioactivation pathway, especially in clinical contexts where aldehyde detoxification pathways are inhibited. Moreover, this is the first report of a bioactivation model constructed by combining a metabolism and reactivity model. These results raise hope that more comprehensive models of bioactivation are possible. The model developed in this study is available at http://swami.wustl.edu/xenosite/ .
Asunto(s)
Indinavir/metabolismo , Hígado/metabolismo , Microsomas Hepáticos/metabolismo , Piperacilina/metabolismo , Piperazinas/metabolismo , Tiazoles/metabolismo , Verapamilo/metabolismo , Aldehídos/química , Aldehídos/metabolismo , Aminas/química , Aminas/metabolismo , Remoción de Radical Alquila , Humanos , Indinavir/farmacología , Hígado/efectos de los fármacos , Microsomas Hepáticos/química , Microsomas Hepáticos/efectos de los fármacos , Modelos Moleculares , Estructura Molecular , Piperacilina/farmacología , Piperazinas/farmacología , Tiazoles/farmacología , Verapamilo/farmacologíaRESUMEN
PURPOSE: Lamivudine, a characterized substrate for human multidrug and toxin extrusion protein 1 (hMATE1) in vitro, was commonly used with indinavir as a therapy against human immunodeficiency virus (HIV). We aimed to investigate whether mouse MATE1 is involved in the disposition of lamivudine in vivo, and whether there is any transporter-mediated interaction between indinavir and lamivudine. METHODS: The role of MATE1 in the disposition of lamivudine was determined using Mate1 wild type (+/+) and knockout (-/-) mice. The inhibitory potencies of indinavir on lamivudine uptake mediated by OCT2 and MATE1 were determined in human embryonic kidney 293 (HEK 293) cells stably expressing these transporters. The role of MATE1 in the interaction between indinavir and lamivudine in vivo was determined using Mate1 (+/+) and Mate1 (-/-) mice. RESULTS: The plasma concentrations and tissue accumulation of lamivudine were markedly elevated in Mate1 (-/-) mice as compared to those in Mate1 (+/+) mice. Indinavir significantly increased the pharmacokinetic exposure of lamivudine in mice; however, the effect by indinavir was significantly less pronounced in Mate1 (-/-) mice as compared to Mate1(+/+) mice. CONCLUSION: MATE1 played an important role in lamivudine pharmacokinetics. Indinavir could cause drug-drug interaction with lamivudine in vivo via inhibition of MATE1 and additional mechanism.
Asunto(s)
VIH-1/efectos de los fármacos , Indinavir/química , Lamivudine/química , Lamivudine/farmacocinética , Proteínas de Transporte de Catión Orgánico/antagonistas & inhibidores , Animales , Fármacos Anti-VIH/química , Fármacos Anti-VIH/farmacocinética , Transporte Biológico/efectos de los fármacos , Técnicas de Cultivo de Célula , Interacciones Farmacológicas , Células HEK293 , Humanos , Riñón/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Distribución TisularRESUMEN
Hypoxis hemerocallidea (African potato) is a popular medicinal plant that has been used traditionally for the treatment of various disorders. Some HIV/AIDS patients use this traditional medicine together with their antiretroviral therapy. This study aimed to determine the impact of selected H. hemerocallidea materials (i.e., a commercial product, an aqueous extract, and biomass reference plant material) on the bidirectional permeability of indinavir across Caco-2 cell monolayers as well as the bioavailability of indinavir during an acute, single administration study in Sprague-Dawley rats. All of the selected H. hemerocallidea test materials demonstrated inhibition effects on indinavir efflux across Caco-2 cell monolayers, albeit to different extents. An increase in the bioavailability of indinavir was obtained in vivo when administered concomitantly with the H. hemerocallidea materials, albeit not statistically significantly. The change in bioavailability directly correlated with the in vitro permeability results. It can therefore be concluded that the change in permeability and bioavailability of indinavir was caused by efflux inhibition and this effect was dependent on the type of H. hemerocallidea material investigated, which was found to be in the following order: commercial product > aqueous extract > reference plant material. The clinical significance of the combined effect of efflux and metabolism inhibition by H. hemerocallidea should be determined in another in vivo model that expresses the cytochrome P450 3A4 enzyme.
Asunto(s)
Infecciones por VIH/tratamiento farmacológico , Inhibidores de la Proteasa del VIH/farmacocinética , Interacciones de Hierba-Droga , Hypoxis/química , Indinavir/farmacocinética , Extractos Vegetales/farmacología , Animales , Disponibilidad Biológica , Células CACO-2 , Cromatografía Liquida , Humanos , Masculino , Espectrometría de Masas , Medicina Tradicional , Extractos Vegetales/química , Extractos Vegetales/aislamiento & purificación , Ratas Sprague-DawleyRESUMEN
In the present day antiretroviral therapy, Ctrough is a key tool for efficacy assessment. The present work explored the feasibility of using Ctrough or Cmax in the area under the concentration-time curve (AUC) prediction of indinavir. A simple unweighted linear regression model was developed to describe the relationship between Cmax versus AUC (r = 0.8101, P < 0.001) and Ctrough versus AUC (r = 0.8127, P < 0.001) for indinavir. The regression lines were used to predict the AUC values from literature Cmax or Ctrough data of indinavir in HIV and healthy subjects. The fold difference, defined as the quotient of the observed and predicted AUC values, was evaluated along with statistical comparison, including root mean square error (RMSE) prediction for the 2 models. The correlation between Cmax versus AUC and Ctrough versus AUC was established. Majority of the predicted values for Cmax versus AUC were within 0.75- to 1.5-fold differences. However, the Ctrough versus AUC model showed larger variability with approximately one-third of the predictions within 0.75- to 1.5-fold differences. The r value and %RMSE for observed versus predicted AUC for Ctrough (r = 0.5925, n = 65, P < 0.001, and RMSE: 67%) were inferior to the Cmax (r = 0.8773, n = 86, P < 0.001, and RMSE: 46%). In conclusion, Cmax versus AUC and Ctrough versus AUC relationships were established for indinavir showing the utility of a single concentration time point for therapeutic drug monitoring purpose. The Cmax model for indinavir may be more relevant for AUC prediction as determined by the statistical criteria.
Asunto(s)
Área Bajo la Curva , Monitoreo de Drogas/métodos , Inhibidores de la Proteasa del VIH/farmacocinética , Indinavir/farmacocinética , Infecciones por Retroviridae/tratamiento farmacológico , Estudios de Factibilidad , Inhibidores de la Proteasa del VIH/uso terapéutico , Humanos , Indinavir/uso terapéutico , Modelos LinealesRESUMEN
Pharmacologic HIV protease inhibitors (PIs) and structurally related oligopeptides are known to reversibly bind and inactivate the insulin-responsive facilitative glucose transporter 4 (GLUT4). Several PIs exhibit isoform selectivity with little effect on GLUT1. The ability to target individual GLUT isoforms in an acute and reversible manner provides novel means both to investigate the contribution of individual GLUTs to health and disease and to develop targeted treatment of glucose-dependent diseases. To determine the molecular basis of transport inhibition, a series of chimeric proteins containing transmembrane and cytosolic domains from GLUT1 and GLUT4 and/or point mutations were generated and expressed in HEK293 cells. Structural integrity was confirmed via measurement of N-[2-[2-[2-[(N-biotinylcaproylamino)ethoxy)ethoxyl]-4-[2-(trifluoromethyl)-3H-diazirin-3-yl]benzoyl]-1,3-bis(mannopyranosyl-4-yloxy)-2-propylamine (ATB-BMPA) labeling of the chimeric proteins in low density microsome fractions isolated from stably transfected 293 cells. Functional integrity was assessed via measurement of zero-trans 2-deoxyglucose (2-DOG) uptake. ATB-BMPA labeling studies and 2-DOG uptake revealed that transmembrane helices 1 and 5 contain amino acid residues that influence inhibitor access to the transporter binding domain. Substitution of Thr-30 and His-160 in GLUT1 to the corresponding positions in GLUT4 is sufficient to completely transform GLUT1 into GLUT4 with respect to indinavir inhibition of 2-DOG uptake and ATB-BMPA binding. These data provide a structural basis for the selectivity of PIs toward GLUT4 over GLUT1 that can be used in ongoing novel drug design.
Asunto(s)
Proteínas Facilitadoras del Transporte de la Glucosa/antagonistas & inhibidores , Inhibidores de la Proteasa del VIH/farmacología , Isoformas de Proteínas/antagonistas & inhibidores , Secuencia de Aminoácidos , Línea Celular , Clonación Molecular , Proteínas Facilitadoras del Transporte de la Glucosa/genética , Proteínas Facilitadoras del Transporte de la Glucosa/metabolismo , Inhibidores de la Proteasa del VIH/metabolismo , Humanos , Indinavir/metabolismo , Indinavir/farmacología , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
Human immunodeficiency virus (HIV)-1 protease is one of the most promising drug target commonly utilized to combat Acquired Immune Deficiency Syndrome (AIDS). However, with the emergence of drug resistance arising from mutations, the efficiency of protease inhibitors (PIs) as a viable treatment for AIDS has been greatly reduced. I50V mutation as one of the most significant mutations occurring in HIV-1 protease will be investigated in this study. Molecular dynamics (MD) simulation was utilized to examine the effect of I50V mutation on the binding of two PIs namely indinavir and amprenavir to HIV-1 protease. Prior to the simulations conducted, the electron density distributions of the PI and each residue in HIV-1 protease are derived by combining quantum fragmentation approach molecular fractionation with conjugate caps and Poisson-Boltzmann solvation model based on polarized protein-specific charge scheme. The atomic charges of the binding complex are subsequently fitted using delta restrained electrostatic potential (delta-RESP) method to overcome the poor charge determination of buried atom. This way, both intraprotease polarization and the polarization between protease and the PI are incorporated into partial atomic charges. Through this study, the mutation-induced affinity variations were calculated and significant agreement between experiments and MD simulations conducted was observed for both HIV-1 protease-drug complexes. In addition, the mechanism governing the decrease in the binding affinity of PI in the presence of I50V mutation was also explored to provide insights pertaining to the design of the next generation of anti-HIV drugs.
Asunto(s)
Carbamatos/farmacología , Infecciones por VIH/virología , Inhibidores de la Proteasa del VIH/farmacología , Proteasa del VIH/genética , VIH-1/enzimología , Indinavir/farmacología , Mutación Puntual , Sulfonamidas/farmacología , Furanos , Infecciones por VIH/tratamiento farmacológico , Proteasa del VIH/química , Proteasa del VIH/metabolismo , VIH-1/química , VIH-1/genética , Humanos , Simulación de Dinámica Molecular , Unión ProteicaRESUMEN
Despite the success of highly active antiretroviral therapy (HAART) in the management of human immunodeficiency virus (HIV)-1 infection, virological failure due to drug resistance development remains a major challenge. Resistant mutants display reduced drug susceptibilities, but in the absence of drug, they generally have a lower fitness than the wild type, owing to a mutation-incurred cost. The interaction between these fitness costs and drug resistance dictates the appearance of mutants and influences viral suppression and therapeutic success. Assessing in vivo viral fitness is a challenging task and yet one that has significant clinical relevance. Here, we present a new computational modelling approach for estimating viral fitness that relies on common sparse cross-sectional clinical data by combining statistical approaches to learn drug-specific mutational pathways and resistance factors with viral dynamics models to represent the host-virus interaction and actions of drug mechanistically. We estimate in vivo fitness characteristics of mutant genotypes for two antiretroviral drugs, the reverse transcriptase inhibitor zidovudine (ZDV) and the protease inhibitor indinavir (IDV). Well-known features of HIV-1 fitness landscapes are recovered, both in the absence and presence of drugs. We quantify the complex interplay between fitness costs and resistance by computing selective advantages for different mutants. Our approach extends naturally to multiple drugs and we illustrate this by simulating a dual therapy with ZDV and IDV to assess therapy failure. The combined statistical and dynamical modelling approach may help in dissecting the effects of fitness costs and resistance with the ultimate aim of assisting the choice of salvage therapies after treatment failure.