Gene Polymorphisms and Drug-Drug Interactions Determine the Metabolic Profile of Blonanserin.

This study aimed to evaluate the effects of cytochrome P450 3A4 (CYP3A4) gene polymorphism and drug interaction on the metabolism of blonanserin. Human recombinant CYP3A4 was prepared using the Bac-to-Bac baculovirus expression system. A microsomal enzyme reaction system was established, and drug-drug interactions were evaluated using Sprague-Dawley rats. Ultra-performance liquid chromatography-tandem mass spectrometry was used to detect the concentrations of blonanserin and its metabolite. Compared with wild type CYP34A, the relative clearance of blonanserin by CYP3A4.29 significantly increased to 251.3%, while it decreased notably with CYP3A4.4, 5, 7, 8, 9, 10, 12, 13, 14, 16, 17, 18, 23, 24, 28, 31, 33, and 34, ranging from 6.09% to 63.34%. Among 153 tested drugs, nimodipine, felodipine, and amlodipine were found to potently inhibit the metabolism of blonanserin. Moreover, the inhibitory potency of nimodipine, felodipine, and amlodipine varied with different CYP3A4 variants. The half-maximal inhibitory concentration and enzymatic kinetics assay demonstrated that the metabolism of blonanserin was noncompetitively inhibited by nimodipine in rat liver microsomes and was inhibited in a mixed manner by felodipine and amlodipine in both rat liver microsomes and human liver microsomes. When nimodipine and felodipine were coadministered with blonanserin, the area under the blood concentration-time curve (AUC)(0-t), AUC(0-∞), and C max of blonanserin increased. When amlodipine and blonanserin were combined, the C max of blonanserin C increased remarkably. The vast majority of CYP3A4 variants have a low ability to catalyze blonanserin. With combined administration of nimodipine, felodipine, and amlodipine, the elimination of blonanserin was inhibited. This study provides the basis for individualized clinical use of blonanserin. SIGNIFICANCE STATEMENT: The enzyme kinetics of novel CYP3A4 enzymes for metabolizing blonanserin were investigated. Clearance of blonanserin by CYP3A4.4, 5, 7-10, 12-14, 16-18, 23-24, 28, 31, 33, and 34 decreased notably, but increased with CYP3A4.29. Additionally, we established a drug interaction spectrum for blonanserin, in which nimodipine, felodipine, and amlodipine kinetics exhibited mixed inhibition. Moreover, their inhibitory potencies decreased with CYP3A4.4 and 5 compared to CYP3A4.1. This study provides essential data for personalized clinical use of blonanserin.

Structural Basis of the Modulation of the Voltage-Gated Calcium Ion Channel Cav 1.1 by Dihydropyridine Compounds*.

1,4-Dihydropyridines (DHP), the most commonly used antihypertensives, function by inhibiting the L-type voltage-gated Ca2+ (Cav ) channels. DHP compounds exhibit chirality-specific antagonistic or agonistic effects. The structure of rabbit Cav 1.1 bound to an achiral drug nifedipine reveals the general binding mode for DHP drugs, but the molecular basis for chiral specificity remained elusive. Herein, we report five cryo-EM structures of nanodisc-embedded Cav 1.1 in the presence of the bestselling drug amlodipine, a DHP antagonist (R)-(+)-Bay K8644, and a titration of its agonistic enantiomer (S)-(-)-Bay K8644 at resolutions of 2.9-3.4 Å. The amlodipine-bound structure reveals the molecular basis for the high efficacy of the drug. All structures with the addition of the Bay K8644 enantiomers exhibit similar inactivated conformations, suggesting that (S)-(-)-Bay K8644, when acting as an agonist, is insufficient to lock the activated state of the channel for a prolonged duration.

Effects of atorvastatin on pharmacokinetics of amlodipine in rats and its potential mechanism.

Atorvastatin combined with amlodipine (ALDP) can efficiently treat the hypertension with coronary heart disease. However, the drug-drug interaction between atorvastatin and ALDP are still unknown.This study investigates the effects of atorvastatin on the pharmacokinetics of ALDP in rats and clarifies its main mechanism.The pharmacokinetic profiles of oral administration of ALDP (1 mg/kg) in Sprague-Dawley rats, with or without pretreatment of atorvastatin (1.5 mg/kg/d for 7 d) were investigated. The effects of atorvastatin on the metabolism of ALDP were also investigated using rat liver microsomes.The results showed that atorvastatin could significantly increase the peak plasma concentration (from 18.28 ± 2.65 to 24.13 ± 1.96 ng/mL) and decrease the oral clearance (from 4.57 ± 1.15 to 1.79 ± 0.28 L/h/kg) of ALDP. In the rat liver microsome systems, the intrinsic clearance rate of ALDP was decreased by the pretreatment with atorvastatin (39.26 ± 2.1 vs. 33.24 ± 3.3 µL/min/mg protein).Those results indicated that atorvastatin could significantly affect the pharmacokinetic of ALDP, via inhibiting the metabolism of ALDP in rats.

UHPLC-MS/MS assay for simultaneous determination of amlodipine, metoprolol, pravastatin, rosuvastatin, atorvastatin with its active metabolites in human plasma, for population-scale drug-drug interactions studies in people living with HIV.

Thanks to highly active antiretroviral treatments, HIV infection is now considered as a chronic condition. Consequently, people living with HIV (PLWH) live longer and encounter more age-related chronic co-morbidities, notably cardiovascular diseases, leading to polypharmacy. As the management of drug-drug interactions (DDIs) constitutes a key aspect of the care of PLWH, the magnitude of pharmacokinetic DDIs between cardiovascular and anti-HIV drugs needs to be more thoroughly characterized. To that endeavour, an UHPLC-MS/MS bioanalytical method has been developed for the simultaneous determination in human plasma of amlodipine, metoprolol, pravastatin, rosuvastatin, atorvastatin and its active metabolites. Plasma samples were subjected to protein precipitation with methanol, followed by evaporation at room temperature under nitrogen of the supernatant, allowing to attain measurable plasma concentrations down to sub-nanogram per milliliter levels. Stable isotope-labelled analytes were used as internal standards. The five drugs and two metabolites were analyzed using a 6-min liquid chromatographic run coupled to electrospray triple quadrupole mass spectrometry detection. The method was validated over the clinically relevant concentrations ranging from 0.3 to 480 ng/mL for amlodipine, atorvastatin and p-OH-atorvastatin, and 0.4 to 480 ng/mL for pravastatin, 0.5 to 480 ng/mL for rosuvastatin and o-OH-atorvastatin, and 3 to 4800 ng/mL for metoprolol. Validation performances such as trueness (95.4-110.8%), repeatability (1.5-13.4%) and intermediate precision (3.6-14.5%) were in agreement with current international recommendations. Accuracy profiles (total error approach) were lying within the limits of ±30% accepted in bioanalysis. This rapid and robust UHPLC-MS/MS assay allows the simultaneous quantification in plasma of the major currently used cardiovascular drugs and offers an efficient analytical tool for clinical pharmacokinetics as well as DDIs studies.

DFT studies on global parameters, antioxidant mechanism and molecular docking of amlodipine besylate.

Amlodipine besylate (AMB) is a synthetic dihydropyridine calcium channel blocker with antihypertensive and anti-anginal effects. Quantum computational investigations on AMB were done using DFT/B3LYP/6-311++G (d, p) level of theory, to study the molecular structural properties, nonlinear properties and antioxidant properties of AMB. The electrophilic and nucleophilic sites along with complete NBO analysis helps to locate the intermolecular electronic interactions and their stabilization energies. Complete NBO analysis was additionally done to locate the intermolecular electronic interactions and their stabilization energies. Charge distributions of Mulliken population, NBO and MEP are correlated. Also, the antioxidant properties of AMB were assessed to check whether these antioxidant effects contribute to the effects of antioxidant therapy. Further, the molecular docking studies of these compounds demonstrated a good selectivity profile with Monoamine oxidase B with better binding affinity and confirms AMB is a potent antioxidant.

Vincristine combination with Ca+2 channel blocker increase antitumor effects.

In this study, it was aimed to determine the effects of Amlodipine, a calcium channel blocker and vincristine (VCR) an antineoplastic, on human neuroblastomas using different doses. The cytotoxicity assays of the study were performed using the MTT method depending on time and concentration. After obtaining the mixture (up to 85% for SH-SY5Y) and sufficient branches (cortex neurons), the cells were treated with amlodipine (10 µM) and vincristine (0.5, 1 and 2 µg) at different concentrations for 24 h. MTT assay was performed by the commercially available kit (Sigma Aldrich, USA). Cells were harvested, washed and stained with PI and Annexin V, respectively, according to the manufacturer's protocol (Biovision, USA). Than analyzes were carried out. The results were quite impressive. When amlodipine (10 µM) was administered alone there was little change compared to the control. However, all doses of amlodipine (10 µM) and vincristine (0.5, 1 and 2 µg) were greater than the deaths in the doses alone (0.5, 1 and 2 µg) of vincristine alone. (P < 0.05). As a result, the combination of vincristine and amlodipine is more effective than vincristine alone in reducing the viability of cancer cells.

Study of Interactions between Amlodipine and Quercetin on Human Serum Albumin: Spectroscopic and Modeling Approaches.

The aim of this study was to analyze the binding interactions between a common antihypertensive drug (amlodipine besylate-AML) and the widely distributed plant flavonoid quercetin (Q), in the presence of human serum albumin (HSA). Fluorescence analysis was implemented to investigate the effect of ligands on albumin intrinsic fluorescence and to define the binding and quenching properties. Further methods, such as circular dichroism and FT-IR, were used to obtain more details. The data show that both of these compounds bind to Sudlow's Site 1 on HSA and that there exists a competitive interaction between them. Q is able to displace AML from its binding site and the presence of AML makes it easier for Q to bind. AML binds with the lower affinity and if the binding site is already occupied by Q, it binds to the secondary binding site inside the same hydrophobic pocket of Sudlow's Site 1, with exactly the same affinity. Experimental data were complemented with molecular docking studies. The obtained results provide useful information about possible pharmacokinetic interactions upon simultaneous co-administration of the food/dietary supplement and the antihypertensive drug.

Effect Of epigallocatechin-3-gallate on the pharmacokinetics of amlodipine in rats.

This study investigates the effect of epigallocatechin-3-gallate (EGCG), a major ingredient of green tea, on the pharmacokinetics of amlodipine in rats. The pharmacokinetics of orally administered amlodipine (1 mg/kg) with or without EGCG pretreatment (30 mg/kg/day for 10 days) were investigated. Plasma concentrations of amlodipine were determined by using a sensitive and reliable liquid chromatography with tandem mass spectroscopy (LC-MS/MS) method. The effects of EGCG on the metabolic stability of amlodipine were investigated by using rat liver microsome incubation systems. The results indicated that when the rats were pretreated with EGCG, the Cmax of amlodipine increased from 16.32 ± 2.57 to 21.44 ± 3.56 ng/mL (p < 0.05), the Tmax decreased from 5.98 ± 1.25 to 4.01 ± 1.02 h (p < 0.05), and the AUC0-t increased from 258.12 ± 76.25 to 383.34 ± 86.95 µg h L-1 (p < 0.05), which suggested that the pharmacokinetic behavior of amlodipine was affected after oral co-administration of EGCG. Additionally, the metabolic half-life was prolonged from 31.3 ± 5.6 to 52.6 ± 7.9 min (p < 0.05) with the pretreatment of EGCG. It can be speculated that the drug-drug interaction between EGCG and amlodipine might occur, which might have resulted from the metabolism inhibition of amlodipine by EGCG when they were co-administered.

Evidence of human-like Ca2+ channels and effects of Ca2+ channel blockers in Acanthamoeba castellanii.

The evolution of voltage-gated calcium channel (Cav) in eukaryotes is an area of interest for biologists worldwide. The CLAN CL0030 and its family Ion_Trans 2 PF 07885 have been known to be present in prokaryotes, but the origin of these ion channels in Acanthamoeba spp. is yet to be determined. We inferred the origin of primitive forms of two-pore channels like proteins, human-like Cav 1.1 of L-type, and Cav subunit alpha-2/delta-1 in Acanthamoeba spp. early during evolution. By in-depth investigation into genomics, transcriptomics, use of bioinformatics tools and experimentations done with drugs like amlodipine and gabapentin on Acanthamoeba spp., we show the evidence of primitive forms of these channels in this protist pathogen. Genomics and transcriptomics of proteins ACA1_167020, 092610, and 270170 reflected their cellular expression in Acanthamoeba spp. We performed amino acid sequence homology, 3D structural modeling, ligand binding predictions, and dockings. Bioinformatics and 3D structural models show similarities between ACA1_167020, 092610, 270170, and different types of known human Cav. We show amoebicidal effects of amlodipine and gabapentin on Acanthamoeba spp., which can help design their structural analogs to target pathogenic genotypes of Acanthamoeba in diseases like Acanthamoeba keratitis and granulomatous amoebic encephalitis.

The extent of drug-drug interaction between amlodipine and activated charcoal is attenuated by food intake in rats.

Activated charcoal decreases gastrointestinal absorption of concomitantly administered drugs. The absorption of amlodipine (AML) was reported as almost completely attenuated by 25 g of activated charcoal under a fasted condition, but not affected by 2 g of activated charcoal under a fed condition. However, it is not clear whether this difference resulted from the food intake or the dose of activated charcoal. The aim of this study was to quantitatively evaluate the effect of food intake on drug interactions caused by adsorption to activated charcoal in the gastrointestinal tract in rats. The rats were orally administered 0.08 mg/kg of AML, with or without 33 mg/kg of activated charcoal, under the fasted or fed condition and the plasma concentration profiles of AML were monitored. For the fed group, the standard breakfast used in clinical studies was smashed and administered at a dose of 11 g/kg. The AUC value of AML under the fasted condition was significantly decreased to 24.8% by coadministration of activated charcoal. On the other hand, activated charcoal moderately decreased the AUC value of AML to 74.8% under the fed condition. These results suggest that the extent of drug interactions caused by activated charcoal is attenuated by food intake.

Lactobacillus plantarum IS-10506 probiotic administration increases amlodipine absorption in a rabbit model.

OBJECTIVE: Probiotics are beneficial in human health. In this study, we investigated the effect of probiotics on absorption of amlodipine, a dihydropyridine calcium antagonist used in the treatment of angina and hypertension, in a rabbit model. METHODS: Lactobacillus plantarum IS-10506 probiotic was administered for 14 days to male New Zealand rabbits. Blood samples were collected before and after probiotic supplementation. Amlodipine (10 mg) was then administered to all groups. Blood samples from a marginal vein were withdrawn at 5, 15, 30, 60, and 120 minutes to determine amlodipine concentrations in rabbit plasma. RESULTS: Amlodipine concentrations in the L. plantarum IS-10506 group were 4.95 ± 1.22, 8.71 ± 0.69, and 12.48 ± 2.53 ng/ml, and those in the control group were 1.69 ± 0.31, 3.89 ± 1.23, and 7.17 ± 1.85 ng/ml at 30, 60, and 120 minutes, respectively after administration of amlodipine. Amlodipine concentrations in the L. plantarum IS-10506 group were significantly higher than those in the control group at 30, 60, and 120 minutes after amlodipine administration. CONCLUSION: Our results suggested that supplementation of L. plantarum IS-10506 significantly increases amlodipine plasma concentrations in rabbits.

Interaction of drugs amlodipine and paroxetine with the metabolizing enzyme CYP2B4: a molecular dynamics simulation study.

The interactions of the drugs amlodipine and paroxetine, which are prescribed respectively for treatment of hypertension and depression, with the metabolizing enzyme cytochrome CYP2B4 as the drug target, have been studied by molecular dynamics (MD) simulation. Poly ethylene glycol was used to control the drugs' interactions with each other and with the target CYP2B4. Thirteen simulation systems were carefully designed, and the results obtained from MD simulations indicated that amlodipine in the PEGylated form prescribed with paroxetine in the nonPEGylated form promotes higher cytochrome stability and causes fewer fluctuations as the drugs approach the target CYP2B4 and interact with it. The simulation results led us to hypothesize that the combination of the drugs with a specific drug ratio, as proposed in this work, manifests more effective diffusivity and less instability while metabolizing with enzyme CYP2B4. Also, the active residues in the CYP2B4 enzyme that interact with the drugs were determined by MD simulation, which were consistent with the reported experimental results. Graphical Abstract Efficient drug-enzyme interactions, as a result of PEGylation.

Validated LC-MS/MS Method for the Simultaneous Determination of Amlodipine and Its Major Metabolites in Human Plasma of Hypertensive Patients.

BACKGROUND: No information on the pharmacokinetic characteristics of amlodipine (AML) metabolites is available. This study aimed to develop a method based on isocratic liquid chromatography coupled to tandem mass spectrometry for the simultaneous determination of AML and its 2 major metabolites, dehydroamlodipine (DH-AML) and O-des[2-aminoethyl]-O-carboxymethyl DH-AML (CM-DH-AML), and to use it for monitoring this drug in hypertensive patients. METHODS: Acetonitrile-deproteinized plasma specimens were separated using an octadecyl-silica column (3-µm particle size) with a mobile phase consisting of 50% methanol containing 0.15% of formic acid in water. The run time was 9 minutes. The mass spectrometer was run in the positive ion electrospray ionization mode. This method was applied for the determination of AML and its metabolites in plasma samples from patients treated with this drug. RESULTS: The calibration curves in human plasma of AML, DH-AML, and CM-DH-AML were linear over the concentration ranges of 0.5-64, 1-64, and 0.5-64 ng/mL, respectively, and their lower limits of quantification were 0.5, 1, and 0.5 ng/mL, respectively. Their extraction recovery rates and matrix factors in human plasma were 94.8%-109.0% and 97.0%-101.4%, respectively. The intra-assay and interassay imprecisions and accuracies were within 10.8% and 95.4%-111.2%, respectively. The plasma concentration ranges of AML, DH-AML, and CM-DH-AML were 6.5-20.9, 1.4-10.9, and 5.6-38.3 ng/mL, respectively. CONCLUSIONS: The present method with acceptable analytical performance can be helpful for monitoring the plasma concentration of AML, including the determination of its metabolites in patients with hypertension.

Construction and verification of CYP3A5 gene polymorphisms using a Saccharomyces cerevisiae expression system to predict drug metabolism.

The present study evaluated the ability of a Saccharomyces cerevisiae expression system to predict the pharmacokinetic (PK) activity of a calcium channel blocker in patients with distinct cytochrome P450 3A5 (CYP3A5) polymorphisms. The blood pressure lowering activity of amlodipine in 57 hypertensive patients with CYP3A5*1/*1, CYP3A5*1/*3, CYP3A5*4 and CYP3A5*6 polymorphisms was evaluated by the current study. Subsequently, a Saccharomyces cerevisiae expression system for CYP3A5 gene polymorphisms was constructed to examine the PK activity of CYP3A5*1/*1, CYP3A5*4 and CYP3A5*6 polymorphisms. This system was used to predict the PK of amlodipine and was compared with the in vivo data from different gene polymorphism groups. The blood pressure lowering activity of amlodipine in hypertensive patients varied among CYP3A5 polymorphisms. The in vivo results demonstrated that CYP3A5*6 exhibited the highest metabolic rate, followed by CYP3A5*1/*1, CYP3A5*4 and CYP3A5*1/*3. The difference between CYP3A5*6 and CYP3A5*1/*1 was not statistically significant (P=0.5). In accordance with in vivo data, CYP3A5*1/*1 exhibited the highest in vitro metabolic rate, followed by CYP3A5*6 and CYP3A5*4. With the exception of the comparison between CYP3A5*6 and CYP3A5*1/*1, polymorphisms exhibited statistically significant differences compared with CYP3A5*1/*1 (P<0.05). The Saccharomyces cerevisiae expression system may be a cost effective and potentially useful tool for assessing the PK activity of drugs that are metabolized by CYP3A5.

In Vitro Assessment of CYP-Mediated Drug Interactions for Kinsenoside, an Antihyperlipidemic Candidate.

Kinsenoside, the herb-derived medicine isolated from the plant Anoect chilus, has diverse pharmacological actions, and it is considered to be a promising antihyperlipidemic drug candidate. This study evaluates the effects of kinsenoside on CYP enzyme-mediated drug metabolism in order to predict the potential for kinsenoside-drug interactions. Kinsenoside was tested at different concentrations of 0.1, 0.3, 1, 3, 10, 30, and 100 µM in human liver microsomes. The c Cktail probe assay based on liquid chromatography-tandem mass spectrometry was conducted to measure the CYP inhibitory effect of kinsenoside. Subsequently, the metabolism profiles of amlodipine and lovastatin in human liver microsomes were analyzed following co-incubation with kinsenoside. The concentration levels of the parent drug and the major metabolites were compared with the kinsenoside-cotreated samples. The effect of kinsenoside was negligible on the enzyme activity of all the CYP isozymes tested even though CYP2A6 was slightly inhibited at higher concentrations. The drug-drug interaction assay also showed that the concomitant use of kinsenoside has a non-significant effect on the concentration of lovastatin or amlodipine, and their major metabolites. So, it was concluded that there is almost no risk of drug interaction between kinsenoside and CYP drug substrates via CYP inhibition.

MicroRNA-200b Impacts Breast Cancer Cell Migration and Invasion by Regulating Ezrin-Radixin-Moesin.

BACKGROUND Ezrin-radixin-moesin (ERM) plays an important role in multiple links of tumors. It also involved in breast cancer invasion and metastasis, and might be a potential biomarker of breast cancer. Another study suggested that ERM expression was regulated directly by miR-200c, and had a critical role in miR-200c suppressing cell migration. This study aimed to investigate the effect of miR-200b on ERM expression in a breast cancer cell line and its influence on invasion and metastasis ability in vitro. MATERIAL AND METHODS Breast cancer cell lines MCF-7 and MDA-MB-231 with different metastatic potentials were selected as a model. MiR-200b overexpression or inhibition was achieved by Lipofectamine™ 2000-mediated miRNA transfection. RT-PCR was used to test miR-200b level, while Western blot was selected to detect ERM protein expression. Wound healing assay and Transwell assay were performed to determine cell migration and invasion ability. RESULTS RT-PCR revealed that miR-200b level in MDA-MB-231 was obviously lower than that in MCF-7, while Western blot analysis showed that ERM expression was significantly higher. MiR-200b inhibition by transfection in MCF-7 markedly decreased miR-200b level, elevated ERM expression, and enhanced cell migration and invasion. MiR-200b overexpression in MDA-MB-231 obviously increased miR-200b level, reduced ERM expression, and weakened cell migration and invasion. CONCLUSIONS MiR-200b participates in breast cancer cell migration and invasion through regulating ERM in MCF-7 and MDA-MB-231.

Investigation of the Interaction Between Human Serum Albumin and Two Drugs as Binary and Ternary Systems.

BACKGROUND AND OBJECTIVES: Human serum albumin (HSA) is the most frequent protein in blood plasma. Albumin transports various compounds, preserves osmotic pressure, and buffers pH. A unique feature of albumin is its ability to bind drugs and other bioactive molecules. However, it is important to consider binary and ternary systems of two pharmaceuticals to estimate the effect of the first drug on the second one and physicochemical properties. METHODS: Different techniques including time-resolved, second-derivative and anisotropy fluorescence spectroscopy, resonance light scattering (RLS), critical induced aggregation concentration (C CIAC), particle size, zeta potential and stability analysis were employed in this assessment to elucidate the binding behavior of Amlodipine and Aspirin to HSA. Moreover, isothermal titration calorimetric techniques were performed and the QSAR properties were applied to analyze the hydration energy and log P. Multiple sequence alignments were also used to predict the structure and biological characteristics of the HSA binding site. RESULT: Time-resolved fluorescence spectroscopy showed interaction of both drugs to HSA based on a static quenching mechanism. Subsequently, second-derivative fluorescence spectroscopy presented different values of parameter H in binary and ternary systems, which were suggested that tryptophan was in a more polar environment in the ternary system than in a binary system. Moreover, the polydispersity index and results from mean number measurements revealed that the presence of the second drug caused a decrease in the stability of systems and increased the heterogeneity of complex. It is also, observed that the gradual addition of HSA has led to a marked increase in fluorescence anisotropy (r) of Amlodipine and Aspirin which can be suggested that the drugs were located in a restricted environment of the protein as confirmed by Red Edge Excitation Shift (REES) studies. The isothermal titration calorimetric technique demonstrated that the interaction of the drugs with HSA was an enthalpically-driven process. CONCLUSIONS: The present experiment showed that the binding of Amlodipine and Aspirin to HSA induced a conformational change of HSA. It was also identified that the protein binding of the first drug could be affected by the second drug. Such results can be of great use for understanding the pharmacokinetic and pharmacodynamic mechanisms of drugs.

Effects of orally administered antibiotics on the bioavailability of amlodipine: gut microbiota-mediated drug interaction.

BACKGROUND: Amlodipine is a representative calcium channel blocker that is frequently prescribed for the treatment of hypertension. In this study, the possibility of drug-drug interactions between amlodipine and coadministered antibiotics (ampicillin) was investigated in rats; thus, changes in the metabolic activities of gut microflora and the consequent pharmacokinetic pattern of amlodipine following ampicillin treatment were characterized. METHODS AND RESULTS: In human and rat fecalase incubation samples, amlodipine was metabolized to yield a major pyridine metabolite. The remaining amlodipine decreased and the formation of pyridine metabolite increased with incubation time, indicating the involvement of gut microbiota in the metabolism of amlodipine. Pharmacokinetic analyses showed that systemic exposure of amlodipine was significantly elevated in antibiotic-treated rats compared with controls. CONCLUSION: These results showed that antibiotic intake might increase the bioavailability of amlodipine by suppressing gut microbial metabolic activities, which could be followed by changes in therapeutic potency. Therefore, coadministration of amlodipine with antibiotics requires caution and clinical monitoring.

Effect of Ginkgo Leaf Tablets on the Pharmacokinetics of Amlodipine in Rats.

BACKGROUND AND OBJECTIVE: Ginkgo leaf tablet (GLT) is an effective traditional Chinese multi-herbal formula, which is often combined with amlodipine for treating senile hypertension in clinic. The aim of this study was to study the pharmacokinetics of amlodipine after oral administration of amlodipine and GLT and to investigate the potential for pharmacokinetic herb-drug interactions between GLT and amlodipine in rats. METHODS: A liquid chromatography-tandem mass spectrometry (LC-MS/MS) analytical method was developed for quantification of amlodipine in rat plasma. The accuracy, precision, linearity, selectivity and recovery were all within an acceptable range. Male Sprague-Dawley rats were randomly assigned to two groups: amlodipine group and amlodipine + GLT group. Plasma concentrations of amlodipine were determined at the designated time points after oral administration by using the developed LC-MS/MS method, and the main pharmacokinetic parameters were calculated and compared. As ginkgolides A, ginkgolides B, bilobalide, quercetin and kaempferol were the main components of GLT, the effects of these ingredients in GLT on metabolism of amlodipine were further investigated in rat liver microsomes. RESULTS: The pharmacokinetic parameters, maximum plasma concentration (C max), time to reach C max (T max), area under the concentration-time curve (AUC), area under the first moment plasma concentration-time curve (AUMC) and elimination half-life (t 1/2), of amlodipine were significantly increased in amlodipine + GLT group, which suggested that GLT may influence the pharmacokinetic behavior after oral co-administration with amlodipine. Amlodipine is metabolized by cytochrome P450 (CYP) 3A4, so it was speculated that GLT may change the pharmacokinetic parameters of amlodipine through modulating the metabolism of CYP3A4 enzymes. When ginkgolides B, bilobalide, or quercetin and amlodipine were co-incubated in the rat liver microsomes, the metabolic rate of amlodipine was prolonged to 533.1, 216.1 and 407.6 min, respectively, from 73.7 min. CONCLUSIONS: These results suggested that these components in GLT inhibit the metabolism of amlodipine. So it can be speculated that the herb-drug interactions between GLT and amlodipine resulted from inhibiting the metabolism of amlodipine by GLT when they were co-administered.

The role of cytochrome P450 2B6 and 2B4 substrate access channel residues predicted based on crystal structures of the amlodipine complexes.

Recent X-ray crystal structures of human cytochrome P450 2B6 and rabbit cytochrome P450 2B4 in complex with amlodipine showed two bound ligand molecules, one in the active site and one in the substrate access channel. Based on the X-ray crystal structures, we investigated the interactions of P450 2B4 and 2B6 with amlodipine using absorbance spectroscopy, and determined the steady-state kinetics of 7-ethoxy-4-(trifluoromethyl)coumarin and 7-benzyloxyresorufin oxidation by some access channel mutants to evaluate the functional role of these residues in substrate turnover. The results of absorbance titrations are consistent with a simple mechanism with two parallel binding events that result in the formation of the enzyme complex with two molecules of amlodipine. Using this model we were able to resolve two separate ligand-binding events, which are characterized by two distinct KD values in each enzyme. The access channel mutants R73K in P450 2B6 and R73K, V216W, L219W, and F220W in P450 2B4 showed a significant decrease in kcat/KM with the both substrates. Overall, the results suggest that P450 2B4 and 2B6 form an enzyme complex with two molecules of amlodipine in solution, and R73, V216, L219 and F220 in P450 2B4 may play an important role in substrate metabolism.