RESUMEN

Cytochrome P450 3A4 (CYP3A4) is involved in the metabolism of more drugs in clinical use than any other xenobiotic-metabolizing enzyme. CYP3A4-mediated drug metabolism is usually allosterically modulated by substrate concentration (homotropic allostery) and other drugs (heterotropic allostery), exhibiting unusual kinetic profiles and regiospecific metabolism. Recent studies suggest that residue Phe108 (F108) of CYP3A4 may have an important role in drug metabolism. In this work, residue mutations were coupled with well-tempered metadynamics simulations to assess the importance of F108 in the allosteric effects of midazolam metabolism. Comparing the simulation results of the wild-type and mutation systems, we identify that the π-π interaction and steric effect between the F108 side chain and midazolam is favorable for the stable binding of substrate in the active site. F108 also plays an important role in the transition of substrate binding mode, which mainly induces the transition of substrate binding mode by forming π-π interactions with multiple aromatic rings of the substrate. Moreover, the side chain of F108 is closely related to the radius and depth of the 2a and 2f channels, and F108 may further regulate drug metabolism by affecting the pathway, orientation, or time of substrate entry into the CYP3A4 active site or product egress from the active site. Altogether, we suggest that F108 affects drug metabolism and regulatory mechanisms by affecting substrate binding stability, binding mode transition, and channel characteristics of CYP3A4. Our findings could promote the understanding of complicated allosteric mechanisms in CYP3A4-mediated drug metabolism, and the knowledge could be used for drug development and disease treatment.

Asunto(s)

Citocromo P-450 CYP3A , Midazolam , Midazolam/química , Midazolam/metabolismo , Citocromo P-450 CYP3A/química , Citocromo P-450 CYP3A/genética , Citocromo P-450 CYP3A/metabolismo , Dominio Catalítico , Unión Proteica , Simulación por Computador

RESUMEN

CYP 3A4 and CYP 3A5 are two important members of the human cytochrome P450 family. Although their overall structures are similar, the local structures of the active site are different, which directly leads to obvious individual differences in drug metabolic efficacy and toxicity. In this work, midazolam (MDZ) was selected as the probe substrate, and its interaction with two proteins, CYP 3A4 and CYP 3A5, was studied by molecular dynamics simulation (MD) along with the calculation of the binding free energy. The results show that two protein-substrate complexes have some similarities in enzyme-substrate binding; that is, in both complexes, Ser119 forms a high occupancy hydrogen bond with MDZ, which plays a key role in the stability of the interaction between MDZ and the enzymes. However, the complex formed by CYP 3A4 and MDZ is more stable, which may be attributed to the sandwich structure formed by the fluorophenyl group of the substrate with Leu216 and Leu482. Our study interprets the binding differences between two isoform-substrate complexes and reveals a structure-function relationship from the atomic perspective, which is expected to provide a theoretical basis for accurately measuring the effectiveness and toxicity of drugs for individuals in the era of precision medicine.

Asunto(s)

Citocromo P-450 CYP3A , Midazolam , Humanos , Midazolam/química , Citocromo P-450 CYP3A/metabolismo , Sistema Enzimático del Citocromo P-450/metabolismo , Dominio Catalítico , Isoformas de Proteínas/metabolismo

RESUMEN

Cytochrome P450 3A4 (CYP3A4) is the main P450 enzyme for drug metabolism and drug-drug interactions (DDIs), as it is involved in the metabolic process of approximately 50% of drugs. A detailed mechanistic elucidation of DDIs mediated by CYP3A4 is commonly believed to be critical for drug optimization and rational use. Here, two typical probes, midazolam (MDZ, substrate) and testosterone (TST, allosteric effector), are used to investigate the molecular mechanism of CYP3A4-mediated heterotropic allosteric interactions, through conventional molecular dynamics (cMD) and well-tempered metadynamics (WT-MTD) simulations. Distance monitoring shows that TST can stably bind in two potential peripheral sites (Site 1 and Site 2) of CYP3A4. The binding of TST at these two sites can induce conformational changes in CYP3A4 flexible loops on the basis of conformational analysis, thereby promoting the transition of the MDZ binding mode and affecting the ratio of MDZ metabolites. According to the results of the residue interaction network, multiple allosteric communication pathways are identified that can provide vivid and applicable insights into the heterotropic allostery of TST on MDZ metabolism. Comparing the regulatory effects and the communication pathways, the allosteric effect caused by TST binding in Site 2 seems to be more pronounced than in Site 1. Our findings could provide a deeper understanding of CYP3A4-mediated heterotropic allostery at the atomic level and would be helpful for rational drug use as well as the design of new allosteric modulators.

Asunto(s)

Citocromo P-450 CYP3A , Midazolam , Midazolam/química , Midazolam/metabolismo , Citocromo P-450 CYP3A/química , Sistema Enzimático del Citocromo P-450/metabolismo , Sitios de Unión , Conformación Molecular

RESUMEN

Human cytochrome P450 CYP3A4 is involved in the processing of more than 35% of current pharmaceuticals and therefore is responsible for multiple drug-drug interactions (DDI). In order to develop a method for the detection and prediction of the possible involvement of new drug candidates in CYP3A4-mediated DDI, we evaluated the application of midazolam (MDZ) as a probe substrate. MDZ is hydroxylated by CYP3A4 in two positions: 1-hydroxy MDZ formed at lower substrate concentrations, and up to 35% of 4-hydroxy MDZ at high concentrations. The ratio of the formation rates of these two products (the site of metabolism ratio, SOM) was used as a measure of allosteric heterotropic interactions caused by effector molecules using CYP3A4 incorporated in lipid nanodiscs. The extent of the changes in the SOM in the presence of effectors is determined by chemical structure and is concentration-dependent. MD simulations of CYP3A4 in the lipid bilayer suggest that experimental results can be explained by the movement of the F-F' loop and concomitant changes in the shape and volume of the substrate-binding pocket. As a result of PGS binding at the allosteric site, several residues directly contacting MDZ move away from the substrate molecule, enabling the repositioning of the latter for minor product formation.

Asunto(s)

Citocromo P-450 CYP3A , Midazolam , Sitio Alostérico , Citocromo P-450 CYP3A/química , Interacciones Farmacológicas , Humanos , Membrana Dobles de Lípidos , Midazolam/química , Midazolam/metabolismo , Midazolam/farmacología

RESUMEN

Cytochrome P450 3A4 (CYP3A4) is the most important P450 enzyme for drug metabolism and drug-drug interaction, due to it being responsible for the biotransformation of approximately 50% of clinically used drugs. Advance knowledge of the molecular and mechanistic basis of CYP3A4 regioselective metabolism is beneficial for understanding the production of metabolites, and may allow personalized metabolic pathways or designing pathway-specific therapeutics. In this work, we focus on investigating the ligand-receptor interactions, substrate conformational transition, and key factors regulating the specificity of metabolic pathways using midazolam (MDZ) as a probe. Here, three types of substrate-binding conformations related to the diversity of MDZ metabolites are identified. The results also suggest that an allosteric site for MDZ is located near the F'-helix, A-anchor, and C-terminal loop of CYP3A4. The presence of an effector in the allosteric site can accelerate the conformational transition of the substrate via modulating a "sandwich" structure, and may affect the proportion of metabolites at high substrate concentration. We hope that the results can improve the understanding of the CYP3A4 structure and function, and provide a new perspective for drug development.

Asunto(s)

Citocromo P-450 CYP3A , Midazolam , Sitio Alostérico , Citocromo P-450 CYP3A/química , Citocromo P-450 CYP3A/metabolismo , Sistema Enzimático del Citocromo P-450/metabolismo , Interacciones Farmacológicas , Midazolam/química , Midazolam/metabolismo

RESUMEN

We developed an efficient and sensitive probe for drug-drug interactions mediated by human CYP3A4 by using midazolam (MDZ) as a probe substrate. Using global analysis of four parameters over several experimental data sets, we demonstrate that the first MDZ molecule (MDZ1) binds with high affinity at the productive site near the heme iron and gives only hydroxylation at the 1 position (1OH). The second midazolam molecule (MDZ2) binds at an allosteric site at the membrane surface and perturbs the position and mobility of MDZ1 such that the minor hydroxylation product at the 4 position (4OH) is formed in a 1:2 ratio (35%). No increase in catalytic rate is observed after the second MDZ binding. Hence, the site of the 1OH:4OH metabolism ratio is a sensitive probe for drugs, such as progesterone, that bind with high affinity to the allosteric site and serve as effectors. We observe similar changes in the MDZ 1OH:4OH ratio in the presence of progesterone (PGS), suggesting a direct communication between the active and allosteric sites. Mutations introduced into the F-F' loop indicate that residues F213 and D214 are directly involved in allosteric interactions leading to MDZ homotropic cooperativity, and these same residues, together with L211, are involved in heterotropic allosteric interactions in which PGS is the effector and MDZ the substrate. Molecular dynamics simulations provide a mechanistic picture of the origin of this cooperativity. These results show that the midazolam can be used as a sensitive probe for drug-drug interactions in human P450 CYP3A4.

Asunto(s)

Citocromo P-450 CYP3A/metabolismo , Midazolam/química , Midazolam/farmacología , Regulación Alostérica/fisiología , Sitio Alostérico , Citocromo P-450 CYP3A/efectos de los fármacos , Citocromo P-450 CYP3A/fisiología , Interacciones Farmacológicas/fisiología , Humanos , Hidroxilación/efectos de los fármacos , Cinética , Simulación de Dinámica Molecular

RESUMEN

Hemoglobin-vesicles (Hb-V), hemoglobin encapsulated within a liposome, were developed as an artificial red blood cell (RBC). When Hb-V becomes clinically available in the future, patients would presumably be co-administered with one or more drugs. Since drug-drug interactions can cause serious adverse effects and impede overall curative effects, evidence regarding the risk associated with drug-drug interactions between Hb-V and such simultaneously administered drugs is needed. Therefore, we report on cytochrome P450 (CYP)-based drug interactions with Hb-V in healthy rats. At 1 day after the saline, Hb-V or packed RBC (PRBC) administration, the blood retention of CYP-metabolizing drugs (caffeine, chlorzoxazone, tolbutamide and midazolam) were moderately prolonged in the case of the Hb-V group, but not the PRBC group, compared to saline group. The results of a proteome analysis revealed that the Hb-V administration had only negligible effects on the protein expression of CYPs in the liver. Hb-V administration, however, clearly suppressed the CYP metabolic activity of the four target CYP isoforms compared with the saline and PRBC group. However, these alterations were nearly recovered at 7 day after the Hb-V administration. Taken together, these results suggest that the administration of Hb-V slightly and transiently affects the CYP-based metabolism of the above drugs.

Asunto(s)

Cafeína/farmacocinética , Clorzoxazona/farmacocinética , Sistema Enzimático del Citocromo P-450/metabolismo , Hemoglobinas/metabolismo , Midazolam/farmacocinética , Tolbutamida/farmacocinética , Animales , Cafeína/química , Clorzoxazona/química , Sistema Enzimático del Citocromo P-450/química , Interacciones Farmacológicas , Hemoglobinas/química , Liposomas/química , Liposomas/metabolismo , Masculino , Midazolam/química , Ratas , Ratas Sprague-Dawley , Tolbutamida/química

RESUMEN

Hemoglobin-vesicles (Hb-V) are being developed as red blood cell (RBC) substitutes. In this study, we report on quantitative and qualitative alterations of hepatic cytochrome P450 (CYPs) and the pharmacokinetics of CYP-metabolizing drugs, with a focus on four CYP isoforms (CYP1A2, CYP2C11, CYP2E1 and CYP3A2), after Hb-V resuscitation from a massive hemorrhage. The results of proteome analysis and western blot data indicate that resuscitation with both Hb-V and packed RBC (PRBC) resulted in a decrease in the protein levels of CYPs. Along with a decrease in the protein expression of CYPs, pharmacokinetic studies showed that the elimination of CYP-metabolizing drugs was prolonged in the Hb-V and PRBC resuscitation groups. It is also noteworthy that the CYP-metabolizing drugs in the Hb-V resuscitation group was retained for a longer period compared to the PRBC resuscitation group, and this is attributed to the CYP isoforms having a lower metabolic activity in the Hb-V resuscitation group than that for the PRBC resuscitation group. These findings suggest that resuscitation with Hb-V after a massive hemorrhage has a slight but not clinically significant effect on drug metabolism via CYPs in the liver due to decreased protein levels and the metabolic activity with respect to the CYPs.

Asunto(s)

Cafeína/metabolismo , Clorzoxazona/metabolismo , Sistema Enzimático del Citocromo P-450/metabolismo , Hemoglobinas/metabolismo , Midazolam/metabolismo , Choque Hemorrágico/metabolismo , Tolbutamida/metabolismo , Animales , Cafeína/química , Clorzoxazona/química , Sistema Enzimático del Citocromo P-450/química , Interacciones Farmacológicas , Hemoglobinas/química , Liposomas/química , Liposomas/metabolismo , Masculino , Midazolam/química , Ratas , Ratas Sprague-Dawley , Tolbutamida/química

RESUMEN

Drug-drug interactions (DDI) have been examined for various drugs for oral use, but less for non-oral applications. This study provides DDI prediction methods for non-orally administered CYP3A4 substrates based on clinical DDI data of oral dosages. Gut availability (Fg) and fraction contribution of CYP3A4 to hepatic intrinsic clearance (fmCYP3A4) were predicted by AUC ratio (AUCR) in oral DDI study with/without grapefruit juice, and alteration in intrinsic clearances with/without ketoconazole, respectively. AUCRs of non-orally administered CYP3A4 substrates with/without inhibitors or inducers were predicted with the estimated Fg, fmCYP3A4 and changes in liver CYP3A4 activities with inhibitors/inducers predicted using Simcyp library. DDIs of intravenously administered midazolam and alfentanil with CYP3A4 inhibitors/inducers could be predicted well by this method with predicted AUCRs within ±64% of observed values. Moreover, maximum DDIs with strong CYP3A4 inducers could be predicted by comparing hepatic clearance with hepatic blood flow, as hepatic blood flow indicates the possible maximum hepatic clearance after strong enzyme induction. Predicted AUCRs of midazolam, alfentanil and R- and S-verapamil were less than, but not far from observed ratios, suggesting good conservative prediction. These methods were applied to blonanserin transdermal patch, suggesting much smaller interaction with CYP3A4 inhibitors/inducers compared to oral dosage of blonanserin.

Asunto(s)

Alfentanilo/química , Citocromo P-450 CYP3A/metabolismo , Midazolam/química , Piperazinas/química , Piperidinas/química , Verapamilo/química , Administración Intravenosa , Administración Oral , Alfentanilo/administración & dosificación , Alfentanilo/metabolismo , Citocromo P-450 CYP3A/química , Interacciones Farmacológicas , Humanos , Midazolam/administración & dosificación , Midazolam/metabolismo , Piperazinas/administración & dosificación , Piperazinas/metabolismo , Piperidinas/administración & dosificación , Piperidinas/metabolismo , Especificidad por Sustrato , Parche Transdérmico , Verapamilo/administración & dosificación , Verapamilo/metabolismo

RESUMEN

BACKGROUND: Combination antiemetic therapy has become a common practice for the prevention of postoperative nausea and vomiting (PONV). The aim of the present study was to evaluate the stability and compatibility of ramosetron hydrochloride and midazolam in 0.9% sodium chloride injection when stored at 4°C and 25°C for up to 14 days. METHODS: Admixtures were assessed initially and for 14 days after preparation in polyolefin bags and glass bottles using 0.9% sodium chloride injection as the diluent and stored at 4°C or 25°C. The initial concentrations were 0.3 mg/100 mL ramosetron hydrochloride and 0.5 mg/100 mL midazolam hydrochloride. For all samples, the compatibility parameters (including precipitation, cloudiness, discoloration and pH values) were evaluated. Chemical stability was also determined using high-performance liquid chromatography (HPLC) analysis. RESULTS: After a 14-day period of storage at 4°C or 25°C, the percent of the initial concentration of ramosetron hydrochloride and midazolam hydrochloride in the various solutions were maintained at a minimum of 97%. All of the mixtures remained clear and colourless throughout the observation period, and no colour change or precipitation was observed. CONCLUSION: The results indicate that admixtures of 0.3 mg/100 mL ramosetron hydrochloride and 0.5 mg/100 mL midazolam hydrochloride in normal saline were stable for 14 days at 4°C or 25°C when packaged in polyolefin bags or glass bottles and protected from light.

Asunto(s)

Antieméticos/química , Bencimidazoles/administración & dosificación , Bencimidazoles/uso terapéutico , Midazolam/administración & dosificación , Midazolam/uso terapéutico , Náusea y Vómito Posoperatorios/tratamiento farmacológico , Solución Salina Hipertónica/administración & dosificación , Solución Salina Hipertónica/química , Antieméticos/administración & dosificación , Antieméticos/uso terapéutico , Bencimidazoles/química , Cromatografía Líquida de Alta Presión , Incompatibilidad de Medicamentos , Estabilidad de Medicamentos , Humanos , Midazolam/química , Estructura Molecular

RESUMEN

PURPOSE: Midazolam is a benzodiazepine derivative commonly used in intensive care units to control sedation. Its use requires dilution of a 5-mg/mL commercial solution to a target concentration of 1 mg/mL. A study was conducted to evaluate the stability of diluted ready-to-use 1-mg/mL midazolam solutions over 365 days when stored in cyclic olefin copolymer vials or polypropylene syringes. METHODS: A specific stability-indicating high-performance liquid chromatography coupled with UV detection method was developed for midazolam hydrochloride and validated for selectivity, linearity, sensitivity, precision, and accuracy. Three storage conditions were tested: -20°C ± 5°C, 5°C ± 3°C, and 25°C ± 2°C at 60% ± 5% relative humidity. Half of the vials were stored upside down to test for the absence of interaction between midazolam and the stopper. Particle contamination, sterility, and pH were assessed. RESULTS: The limit of stability was set at 90% of the initial concentration. After 1 year's storage at -20°C and 5°C, concentrations remained superior to 90% under all storage conditions. At 25°C, stability was maintained up to day 90 in syringes (mean [SD], 92.71% [1.43%]) and to day 180 in upright and upside-down vials (92.12% [0.15%] and 91.57% [0.15%], respectively). No degradation products were apparent, no variations in pH values were detected, and containers retained their sterility and conformity with regard to any specific contamination during the study. CONCLUSION: The evaluated 1-mg/mL midazolam solution was stable over a 1-year period when stored at a refrigerated (5°C) or frozen (-20°C) temperature in both vials and syringes; with storage at 25°C, the stability duration was lower. The preparation of ready-to-use midazolam solutions by a hospital pharmacy is compatible with clinical practice and could help to decrease risks inherent in dilution in care units.

Asunto(s)

Hipnóticos y Sedantes/química , Midazolam/química , Cromatografía Líquida de Alta Presión , Embalaje de Medicamentos , Estabilidad de Medicamentos , Almacenaje de Medicamentos , Humanos , Servicio de Farmacia en Hospital , Polipropilenos , Jeringas

RESUMEN

PURPOSE: To examine the theoretical/practical utility of the liver-to-blood partition coefficient (Kpuu) for predicting drug-drug interactions (DDIs), and compare the Kpuu-approach to the extended clearance concept AUCR-approach. METHODS: The Kpuu relationship was derived from first principles. Theoretical simulations investigated the impact of changes in a single hepatic-disposition process on unbound systemic (AUCB,u) and hepatic exposure (AUCH,u) versus Kpuu. Practical aspects regarding Kpuu utilization were examined by predicting the magnitude of DDI between ketoconazole and midazolam employing published ketoconazole Kpuu values. RESULTS: The Kpuu hepatic-disposition relationship is based on the well-stirred model. Simulations emphasize that changes in influx/efflux intrinsic clearances result in Kpuu changes, however AUCH,u remains unchanged. Although incorporation of Kpuu is believed to improve DDI-predictions, utilizing published ketoconazole Kpuu values resulted in prediction errors for a midazolam DDI. CONCLUSIONS: There is limited benefit in using Kpuu for DDI-predictions as the AUCR-based approach can reasonably predict DDIs without measurement of intracellular drug concentrations, a difficult task hindered by experimental variability. Further, Kpuu changes can mislead as they may not correlate with changes in AUCB,u or AUCH,u. The well-stirred model basis of Kpuu when applied to hepatic-disposition implies that nuances of intracellular drug distribution are not considered by the Kpuu model.

Asunto(s)

Cetoconazol/farmacocinética , Midazolam/farmacocinética , Modelos Químicos , Sangre , Interacciones Farmacológicas , Humanos , Cetoconazol/química , Hígado/efectos de los fármacos , Midazolam/química

RESUMEN

Midazolam is a rapidly effective benzodiazepine drug that is widely used as a sedative worldwide. Due to its poor solubility in a neutral aqueous solution, the clinical use of midazolam is significantly limited. As one of the most promising formulations for poorly water-soluble drugs, nanocrystals have drawn worldwide attention. We prepared a stable nanosuspension system that causes little muscle irritation. The particle size of the midazolam nanocrystals (MDZ/NCs) was 286.6 ± 2.19 nm, and the crystalline state of midazolam did not change in the size reduction process. The dissolution velocity of midazolam was accelerated by the nanocrystals. The pharmacokinetics study showed that the AUC0-t of the MDZ/NCs was 2.72-fold (p < 0.05) higher than that of the midazolam solution (MDZ/S), demonstrating that the bioavailability of the MDZ/NC injection was greater than that of MDZ/S. When midazolam was given immediately after the onset of convulsions, the ED50 for MDZ/NCs was significantly more potent than that for MDZ/S and DZP/S. The MDZ/NCs significantly reduced the malondialdehyde content in the hippocampus of the seizures model rats and significantly increased the glutathione and superoxide dismutase levels. These results suggest that nanocrystals significantly influenced the dissolution behavior, pharmacokinetic properties, anticonvulsant effects, and neuroprotective effects of midazolam and ultimately enhanced their efficacy in vitro and in vivo.

Asunto(s)

Hipocampo/metabolismo , Midazolam , Músculo Esquelético/metabolismo , Nanopartículas , Convulsiones , Animales , Disponibilidad Biológica , Modelos Animales de Enfermedad , Hipocampo/patología , Masculino , Midazolam/química , Midazolam/farmacocinética , Midazolam/farmacología , Músculo Esquelético/patología , Nanopartículas/química , Nanopartículas/uso terapéutico , Nanotecnología , Tamaño de la Partícula , Ratas , Convulsiones/tratamiento farmacológico , Convulsiones/metabolismo , Convulsiones/patología , Solubilidad

RESUMEN

BACKGROUND: Calycosin (CAL), a type of O-methylated isoflavone extracted from the herb Astralagusmembranaceus (AM), is a bioactive chemical with antioxidative, antiphlogistic and antineoplastic activities commonly used in traditional alternative Chinese medicine. AM has been shown to confer health benefits as an adjuvant in the treatment of a variety of diseases. AIM: The main objective of this study was to determine whether CAL influences the cytochrome P450 (CYP450) system involved in drug metabolism. METHODS: Midazolam, tolbutamide, omeprazole, metoprolol and phenacetin were selected as probe drugs. Rats were randomly divided into three groups, specifically, 5% Carboxymethyl cellulose (CMC) for 8 days (Control), 5% CMC for 7 days + CAL for 1 day (single CAL) and CAL for 8 days (conc CAL), and metabolism of the five probe drugs evaluated using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). RESULTS: No significant differences were observed for omeprazole and midazolam, compared to the control group. T max and t1/2 values of only one probe drug, phenacetin, in the conc CAL group were significantly different from those of the control group (T max h: 0.50±0.00 vs 0.23±0.15; control vs conc CAL). C max of tolbutamide was decreased about two-fold in the conc CAL treatment group (conc vs control: 219.48 vs 429.56, P<0.001). CONCLUSION: Calycosin inhibits the catalytic activities of CYP1A2, CYP2D6 and CYP2C9. Accordingly, we recommend caution, particularly when combining CAL as a modality therapy with drugs metabolized by CYP1A2, CYP2D6 and CYP2C9, to reduce the potential risks of drug accumulation or ineffective treatment.

Asunto(s)

Inhibidores Enzimáticos del Citocromo P-450/metabolismo , Medicamentos Herbarios Chinos/metabolismo , Isoflavonas/metabolismo , Animales , Inhibidores Enzimáticos del Citocromo P-450/química , Inhibidores Enzimáticos del Citocromo P-450/farmacología , Sistema Enzimático del Citocromo P-450/metabolismo , Medicamentos Herbarios Chinos/química , Medicamentos Herbarios Chinos/farmacología , Isoflavonas/química , Isoflavonas/farmacología , Medicina Tradicional China , Metoprolol/química , Metoprolol/metabolismo , Midazolam/química , Midazolam/metabolismo , Omeprazol/química , Omeprazol/metabolismo , Fenacetina/química , Fenacetina/metabolismo , Ratas , Tolbutamida/química , Tolbutamida/metabolismo

RESUMEN

Promiscuous and allosteric drug interactions with cytochrome P450 3A4 (CYP3A4) are ubiquitous but incompletely understood at the molecular level. A classic allosteric CYP3A4 drug interaction includes the benzodiazepine midazolam (MDZ). MDZ exhibits homotropic and heterotropic allostery when metabolized to 1'-hydroxy and 4-hydroxy metabolites in varying ratios. The combination of hydrogen-deuterium exchange mass spectrometry (HDX-MS) and Gaussian accelerated molecular dynamics (GaMD) simulations of CYP3A4 in lipid nanodiscs and in a lipid bilayer, respectively, reveals MDZ-dependent changes in dynamics in a membrane environment. The F-, G-, and intervening helices, as well as the loop preceding the ß1-sheets, display the largest observed changes in HDX. The GaMD suggests a potential allosteric binding site for MDZ in the F'- and G'-regions, which undergo significant increases in HDX at near-saturating MDZ concentrations. The HDX-MS and GaMD results confirm that changes in dynamics are most significant near the developing consensus allosteric site, and these changes are distinct from those observed previously with the nonallosteric inhibitor ketoconazole. The results suggest that the allosteric MDZ remains mobile in its binding site at the Phe-cluster. The results further suggest that this binding site remains dynamic or changes the depth of insertion in the membrane.

Asunto(s)

Sitio Alostérico/fisiología , Citocromo P-450 CYP3A/metabolismo , Membrana Dobles de Lípidos/metabolismo , Midazolam/metabolismo , Simulación de Dinámica Molecular , Nanopartículas/metabolismo , Ansiolíticos/química , Ansiolíticos/metabolismo , Citocromo P-450 CYP3A/química , Humanos , Membrana Dobles de Lípidos/química , Lípidos/química , Midazolam/química , Nanopartículas/química , Estructura Secundaria de Proteína

RESUMEN

Ketamine (Ket) and midazolam (MDZ) are commonly administered drugs in the intensive care setting for analgesia and sedation. Ket and MDZ are metabolized to dehydro-norketamine (DHNK), nor-ketamine (NK) and 1-hydroxy midazolam (1HMDZ). Limited studies evaluating their pharmacokinetics exists in patients on extracorporeal membrane oxygenation (ECMO) therapy. Therefore, we developed a quantitative, high-performance liquid chromatography-mass spectrometry (with single ion monitoring) method to simultaneously detect Ket, MDZ and their (DHNK, NK and 1HMDZ) metabolites in human plasma. Considerable sensitivity was obtained for the analytes using a C18 HILIC column operated by a high-performance liquid chromatography system coupled with a Thermo Exactive Orbitrap mass spectrometer. Calibration curves were developed for analyte molecules (n = 5) in the presence of carbamazepine (CBZ) as an internal standard. The lower limits of quantitation (LLOQ) for Ket and MDZ were 20 and 10 ng/mL, respectively with the LLOQ for DHNK, NK and 1HMDZ at 470, 320 and 150 ng/ml. Moreover, the percent coefficient of variance and precision for inter- and intra-day runs were within the standards set forth by the ICH and FDA guidelines. This method is sensitive and has been successfully applied to an ongoing pharmacokinetic study in patients on ECMO therapy.

Asunto(s)

Ketamina/química , Ketamina/metabolismo , Midazolam/química , Midazolam/metabolismo , Aminas/química , Calibración , Cromatografía Líquida de Alta Presión/métodos , Oxigenación por Membrana Extracorpórea/métodos , Humanos , Ketamina/análogos & derivados , Reproducibilidad de los Resultados , Espectrometría de Masas en Tándem/métodos

RESUMEN

Human CYP3A4 (Cyp3a11 in mice) is one of the most important enzymes for drug metabolism and detoxification. Here, we aimed to investigate a potential role for E4bp4 in regulation of Cyp3a11 expression and activity. The regulatory effects of E4bp4 on Cyp3a11 enzyme were assessed using E4bp4-/- mice and Hepa-1c1c7 cells. The mRNA and protein levels were quantified using qPCR and Western blotting, respectively. In vitro microsomal Cyp3a11 activity was probed using its specific substrates midazolam and testosterone. Pharmacokinetic studies were performed with wild-type and E4bp4-/- mice after midazolam administration. Global deletion of E4bp4 led to significant upregulation of Cyp3a11 mRNA and protein in major metabolic organs (i.e., the liver, kidney and small intestine). E4bp4 ablation also caused an increased microsomal Cyp3a11 activity consistent with the enzyme's expression change. Overexpression of E4bp4 in Hepa-1c1c7 cells resulted in reduced levels of Cyp3a11 mRNA and protein, whereas E4bp4 knockdown caused upregulation of Cyp3a11 expression. In addition, the systemic exposure of midazolam was lowered in E4bp4-/- mice compared with wild-type mice. This was accompanied by enhanced formation of its metabolite 1'-hydroxymidazolam. Furthermore, luciferase reporter and mobility shift assays revealed that E4bp4 repressed Cyp3a11 transcription via direct binding to C-site (-1539/-1529 bp) in the promoter region. In conclusion, E4bp4 negatively regulates Cyp3a11 expression, thereby impacting drug metabolism and pharmacokinetics.

Asunto(s)

Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Citocromo P-450 CYP3A/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Proteínas de la Membrana/metabolismo , Midazolam/farmacología , Testosterona/farmacología , Andrógenos/química , Andrógenos/metabolismo , Andrógenos/farmacología , Animales , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Línea Celular , Citocromo P-450 CYP3A/genética , Moduladores del GABA/metabolismo , Moduladores del GABA/farmacología , Regulación de la Expresión Génica/fisiología , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Masculino , Proteínas de la Membrana/genética , Ratones , Ratones Noqueados , Midazolam/química , Midazolam/metabolismo , Estructura Molecular , Testosterona/química , Testosterona/metabolismo , Regulación hacia Arriba

RESUMEN

This study provides whole-body physiologically-based pharmacokinetic models of the strong index cytochrome P450 (CYP)1A2 inhibitor and moderate CYP3A4 inhibitor fluvoxamine and of the sensitive CYP1A2 substrate theophylline. Both models were built and thoroughly evaluated for their application in drug-drug interaction (DDI) prediction in a network of perpetrator and victim drugs, combining them with previously developed models of caffeine (sensitive index CYP1A2 substrate), rifampicin (moderate CYP1A2 inducer), and midazolam (sensitive index CYP3A4 substrate). Simulation of all reported clinical DDI studies for combinations of these five drugs shows that the presented models reliably predict the observed drug concentrations, resulting in seven of eight of the predicted DDI area under the plasma curve (AUC) ratios (AUC during DDI/AUC control) and seven of seven of the predicted DDI peak plasma concentration (Cmax ) ratios (Cmax during DDI/Cmax control) within twofold of the observed values. Therefore, the models are considered qualified for DDI prediction. All models are comprehensively documented and publicly available, as tools to support the drug development and clinical research community.

Asunto(s)

Cafeína/farmacocinética , Citocromo P-450 CYP1A2/metabolismo , Fluvoxamina/farmacocinética , Midazolam/farmacocinética , Rifampin/farmacocinética , Teofilina/farmacocinética , Administración Oral , Algoritmos , Área Bajo la Curva , Cafeína/administración & dosificación , Cafeína/química , Citocromo P-450 CYP1A2/química , Citocromo P-450 CYP3A/química , Citocromo P-450 CYP3A/metabolismo , Interacciones Farmacológicas , Fluvoxamina/administración & dosificación , Fluvoxamina/química , Humanos , Midazolam/administración & dosificación , Midazolam/química , Modelos Biológicos , Modelos Moleculares , Rifampin/administración & dosificación , Rifampin/química , Teofilina/administración & dosificación , Teofilina/química

RESUMEN

Drug delivery to the neonatal and premature pediatric populations is very challenging. This research assessed the potential of delivering midazolam by transdermal iontophoresis as an alternative strategy in pediatric therapy. In vitro experiments used intact and tape-stripped porcine skin as models for the skin barrier function of full-term and premature newborns, respectively. Midazolam transdermal transport was significantly enhanced by applying higher currents, increasing the formulation pH, and optimizing the drug's mole fraction in the vehicle. When the skin barrier was decreased to half of its baseline competence, the passive permeation of midazolam increased by approximately 60-fold; and complete stratum corneum removal led to an additional 20-fold enhancement in permeation. Iontophoresis retained control of the drug transport trough partially compromised skin. However, a very high passive contribution undermined the iontophoretic control when the barrier was fully compromised. Overall, midazolam delivery could be rate-controlled by iontophoresis in most circumstances, and therapeutically useful fluxes could be achieved.

Asunto(s)

Hipnóticos y Sedantes/administración & dosificación , Iontoforesis , Midazolam/administración & dosificación , Administración Cutánea , Sistemas de Liberación de Medicamentos , Humanos , Hipnóticos y Sedantes/química , Recién Nacido , Midazolam/química , Nacimiento Prematuro , Factores de Tiempo

RESUMEN

The results of research on selected drugs used in palliative care are presented, including fentanyl, tramadol, metoclopramide, hyoscine butylbromide, midazolam, haloperidol, levomepromazine and clonazepam. Interpretation of their ESI mass spectra obtained by the use of a triple quadrupole linear ion trap mass spectrometer is given. As a result, fragmentation pathways described in the literature are complemented and presented with more details. On their basis, transitions for quantitative analysis are selected and chromatographic conditions for the determination of the palliative care drugs are proposed as well. These results enable future studies on palliative care drugs in elderly patients including both their quantitation in body fluids and easier identification of their metabolites.

Asunto(s)

Preparaciones Farmacéuticas/química , Espectrometría de Masa por Ionización de Electrospray/métodos , Adyuvantes Anestésicos/química , Analgésicos Opioides/química , Anticonvulsivantes/química , Antieméticos/química , Antipsicóticos/química , Clonazepam/química , Fentanilo/química , Haloperidol/química , Humanos , Metotrimeprazina/química , Metoclopramida/química , Midazolam/química , Cuidados Paliativos , Espectrometría de Masas en Tándem/métodos , Tramadol/química