Inhibition of In Vitro Metabolism of Opioids by Skeletal Muscle Relaxants

The purpose of this study was to test the hypothesis that skeletal muscle relaxants could inhibit the in vitro metabolism of common comedications opioids buprenorphine, methadone and oxycodone. The compounds [solubility‐limited concentration (μM) studied] were as follows: baclofen (1000), carisoprodol (200), its metabolite meprobamate (1000), chlorzoxazone (200), cyclobenzaprine (1000), metaxalone (50), methocarbamol (1000), orphenadrine (1000) and tizanidine (1000). Compounds were first incubated with human liver microsomes ± pre‐incubation, screened with pathway‐specific cDNA‐expressed cytochrome P450s (rCYP), and then IC₅₀ values determined using either 8‐concentration tests for those where the rCYP screen suggested an IC₅₀ was achievable, or a 3‐concentration test with downward extrapolation if screen suggested 50% inhibition was not achievable. These results were then extrapolated to determine an inhibitory potential. Six pathway inhibitor combinations were identified with a moderate inhibitory potential (≥2.0 < 5.0): five with chlorzoxazone, R‐EDDP, S‐EDDP and noroxycodone production by CYP3A4, and R‐ and S‐EDDP production by CYP2B6; and one for the meprobamate effect on noroxycodone production by CYP3A4. An additional eleven combinations were found with a weak inhibitory potential (≥1.25 < 2.0): five with carisoprodol, two each with methocarbamol and meprobamate, and one each with metaxalone and orphenadrine. This represents the first comprehensive study of the inhibitory effect of this class of drugs and suggests that some of them may produce significant drug–drug interactions with opioids that are frequent comedications with skeletal muscle relaxants.     

Inhibition of In Vitro Metabolism of Simvastatin by Itraconazole in Humans and Prediction of In Vivo Drug-Drug Interactions

Purpose. To evaluate an interaction between simvastatin and itraconazole in in vitro studies and to attempt a quantitative prediction of in vivo interaction in humans. Methods. The inhibitory effect of itraconazole on simvastatin metabolism was evaluated using human liver microsomes and the K_i values were calculated for the unbound drug in the reaction mixture. A physiologically-based pharmacokinetic model was used to predict the maximum in vivo drug-drug interaction. Results. Itraconazole competitively inhibited the metabolism of simvastatin to M-1 and M-2 with K_i values in the nM range. The area under the curve (AUC) of simvastatin after concomitant dosing with itraconazole was predicted to increase ca. 84-101-fold compared with that without administration of itraconazole. Taking into consideration the fact that this method predicts the maximum interaction, this agrees well with the clinical observation of a 19-fold increase. A similar prediction, based on the K_i value without taking into account the drug adsorption to microsomes, led to an underevaluation of the interaction. Conclusions. It was demonstrated that the competitive inhibition of CYP3A4-mediated simvastatin metabolism by itraconazole is the main cause of the drug interaction and that a K_i value corrected for drug adsorption to microsomes is the key factor in quantitatively predicting the maximum in vivo drug interactions.     

In Vitro Metabolism of p-Xylene by Rabbit Lung and Liver

1. Optimum conditions for metabolism in vitro of p-xylene by rabbit liver or lung microsomal enzymes have been studied. Reactions were linear with time for at least 30?min at a microsomal protein concentration of 1?mg/ml. Addition of cytosol fraction to incubation mixtures of microsomes, NADPH, and substrate increased enzyme activity but the increase was independent of amount of cytosol added (over range of 0.1 ml to 0.5 ml). The pH optimum for the lung and liver microsomal system was 7.3 and 7.8, respectively, using Hepes buffer. The apparent K_m and V_max for the liver and lung systems were determined. The NAD⁺ and NADP⁺ requirements were studied.

2. The major metabolite of p-xylene in vitro, as determined by t.l.c., of liver and lung microsomal incubation mixtures was p-toluic acid.

3. Phenobarbital pre-treatment of rabbits (multiple doses, intraperitoneal or intravenous) induced the liver microsomal xylene-metabolizing system approximately 3-fold, whereas there was no change in activity of the lung enzyme. Maximum induction of liver microsomal metabolism after a single intraperitoneal dose of phenobarbital (100?mg/kg) was reached in 2–3 days and declined slowly thereafter; during this same period lung xylene-metabolizing activity remained unchanged or increased only slightly. Chlorpromazine pre-treatment, intraperitoneal (but not intravenous), of rabbits stimulated hepatic microsomal xylene-metabolizing activity. Administration of 1,2,3,4-dibenzanthracene or 3-methylcholanthrene to rabbits resuited in decreased xylene metabolism in vitro by liver and lung microsomes.     

In Vitro Plasma Metabolism of RMP-7

Pharmaceutical Research -     

Study on In Vitro Metabolism and In Vivo Pharmacokinetics of Beauvericin

Beauvericin (BEA) is a well-known mycotoxin produced by many fungi, including Beaveria bassiana. The purpose of this study was to evaluate the in vitro distribution and metabolism characteristics as well as the in vivo pharmacokinetic (PK) profile of BEA. The in vitro metabolism studies of BEA were performed using rat, dog, mouse, monkey and human liver microsomes, cryopreserved hepatocytes and plasma under conditions of linear kinetics to estimate the respective elimination rates. Additionally, LC-UV-MSⁿ (n = 1~2) was used to identify metabolites in human, rat, mouse, dog and monkey liver microsomes. Furthermore, cytochrome P450 (CYP) reaction phenotyping was carried out. Finally, the absolute bioavailability of BEA was evaluated by intravenous and oral administration in rats. BEA was metabolically stable in the liver microsomes and hepatocytes of humans and rats; however, it was a strong inhibitor of midazolam 1′-hydroxylase (CYP3A4) and mephenytoin 4′-hydroxylase (CYP2C19) activities in human liver microsomes. The protein binding fraction values of BEA were >90% and the half-life (T_1/2) values of BEA were approximately 5 h in the plasma of the five species. The absolute bioavailability was calculated to be 29.5%. Altogether, these data indicate that BEA has great potential for further development as a drug candidate. Metabolic studies of different species can provide important reference values for further safety evaluation.     

In Vitro Inhibition of Thyroid Hormone Sulfation by Polychlorobiphenylols: Isozyme Specificity and Inhibition Kinetics

It was recently demonstrated by our laboratory that hydroxylatedmetabolites of polychlorinated biphenyls (PCB-OHs) are inhibitorsof thyroid hormone sulfation. In this study, a more detailedinvestigation on sulfotransferase isozyme specificity and thekinetics of inhibition was performed. Thyroid hormone sulfationwas determined using 3,3'-diiadothyronine (T2) as a substrate,and various sources of sulfotransferase (SULT) enzyme were used;e.g., female and male rat liver cytosol, male brain cytosoland cytclsolic preparations of V79 cells transfected with ratSULT1C1, and human SULT1A1 and human SULT1A3. The inhibitionpattern and IC50 values were very similar for male and femalerat liver and rSULT1C1 and hSULT1A1. PCB-OHs were not able toinhibit the T2 sulfotranferase activity using hSULT1A3. Metabolite3-hydroxy-2,3',4,4',5-pentachlorobiphenyl did not inhibit T2sulfotransferase activity in male brain cytosol, while it wasa very potent inhibitor in male and female rat liver cytosol.IC50 values for the tested PCB-OHs were not different with eitherT2 or 3,3',5-triiodothyronine (T3) as substrate, supportingthe hypothesis that T2 is the preferred iodothyronine substratefor the sulfotransferases catalyzing the sulfation of the activehormone T3. The Lineweaver-Burk plot obtained with rat livercytosol and T2 suggested that the nature of the T2 sulfationinhibition by 4-hydroxy-2',3,3',4',5-pentachlorobipheny is competitive.Finally, it was demonstrated that tested hydroxylated polychlorinateddibenzo-p-dioxins and biphenyls were, albeit poorly, sulfatedby sulfotransferases as measured by the production of ³⁵S-labeledmetabolites.     

Arachidonate Metabolism is Changed by Dipyridamole in Guinea Pig Isolated Lungs

Abstract: Fifty nmol of ¹⁴C-arachidonic acid was injected into the pulmonary circulation of guinea pig isolated lungs and the metabolites were analysed from the non-recirculating perfusion effluent. The main metabolites in the effluent were thromboxane B₂ (TxB₂), 6-keto-PGF_1α, a metabolite group migrating on TLC plate near to 15-keto-PGE₂ and a group of two metabolites (probably 12L-hydroperoxy- and 12L-hydroxy-5,8,10,14-eicosatetraenoic acids). Pulmonary infusion of dipyridamole (20 μM) increased the amount of TxB₂ in the perfusion effluent. The amounts of other metabolites were unchanged. 2, 20 or 100 μM of dipyridamole had no effect in vitro on the activity of NAD⁺-dependent 15-hydroxyprostaglandin dehydrogenase in the 100,000Xg supernatant fraction of guinea pig lungs.     

Prenylated Phenols that Cause Contact Dermatitis from Trichomes of Phacelia ixodes.

The principal phenolic constituents were isolated from the exudate of the glandular trichomes of PHACELIA IXODES K ELLOGG (Hydrophyllaceae), a Mexican plant that causes allergic contact dermatitis. These are, in decreasing order of the amount in the trichomes: geranylhydroquinone (I), 3-geranyl-2,5-dihydroxyphenyl acetate (II), geranylbenzoquinone (III), 2-geranyl-6-hydroxy-4-methoxyphenyl acetate (IV), 2-geranyl-4-hydroxyphenyl acetate (V), 6-hydroxy-2-methyl-2-(4-methyl-3-pentenyl)-chromene (VI), and 6,7-dimethoxyapigenin. Compounds II, IV, and V are new natural products. Geranylbenzoquinone was the principal contact allergen in testing with guinea pigs.     

Pulmonary First-Pass and Steady-State Metabolism of Phenols

Purpose. To study (A) the effect of the administration route (i.v. and i.t.) on pre- and post-absorptive metabolism of phenol and 1-naphthol by the lung and (B) pulmonary extraction of phenol at steady-state. Methods. Phenols were administered intra-arterially, intravenously and intratracheally and the pre- and post-absorptive metabolism assessed by comparing the AUCs in arterial blood. Phenol was infused to steady-state and the pulmonary extraction assessed by measuring jugular vein and carotid artery blood concentrations. Results. In contrast to previously published data (e.g., Mistry and Houston, Drug Metab. Dispos. 13:740–745 (1985)) we could not detect pulmonary first-pass metabolism of the phenols; reasons for this disparity are discussed. An apparent negative pulmonary extraction of phenol at steady-state was observed, probably as a consequence of extraction by organs which are in series, and not parallel, with the lungs (e.g. liver, kidneys and GIT). Conclusions. (A) Phenol and 1-naphthol do not undergo pulmonary first-pass metabolism. (B) Traditional methods of assessing organ extraction and clearance at steady-state cannot be utilised when metabolising organs are in series.     

芩银注射液及相关单味药的体外抑菌试验

目的:测定芩银注射液的体外抗菌活性.方法:以液体培养基对倍稀释法测定芩银注射液及相关单味药对痢疾杆菌、大肠埃希菌的最低抑菌浓度(MIC)和最低杀菌浓度(MBC).结果:芩银注射液及相关单味药对痢疾杆菌和大肠杆菌均有不同程度的抑制和杀灭作用,芩银注射液对痢疾杆菌和大肠埃希菌的MIC、MBC均为1.56 mg&#8226;mL 1.结论:芩银注射液的抑菌和杀菌作用强于其相关单味药,处方合理,佐证了该药治疗痢疾杆菌和大肠埃希菌性腹泻的效果.     

In Vitro Assay of Translation Inhibition by Trichothecenes Using a Commercially Available System

Trichothecenes are a family of major secondary metabolites produced by some common filamentous fungi, including plant pathogenic and entomopathogenic fungi. It may be considered difficult to conduct a comparison between the toxicities of trichothecenes with consideration of different conditions and cell lines. In the current study, we developed an in vitro assay based on a commercially available system to estimate the translation inhibition, that is, the main toxicity, of trichothecenes. The assay was applied to estimate the inhibition of protein synthesis by trichothecenes. Initially, we examined the assay using trichothecene dissolved in water followed by an assessment of trichothecene solutions dissolved in acetonitrile. The obtained data showed that the assay tolerated the small amount of acetonitrile. The assay examined in this study has the advantages of a short operation time (one day), ease of use, and data stability, as it is a non-cell-based assay whose components are commercially available. It is expected that this assay will contribute to the evaluation of the toxicity of a vast number of trichothecenes.     

Transdermal Drug Transport and Metabolism. I. Comparison of In Vitro and In Vivo Results

Using excised human skin and tissue grafted to athymic mice, the in vitro and in vivo delivery and metabolism of a salicylate diester were compared. Concentration profiles of this drug and its metabolites were obtained for the outer several hundred microns of the skin. These results show significant differences in the extent of enzymatic cleavage and distribution of metabolites between in vitro and in vivo studies. Furthermore, these data suggest that in vitro results may overestimate metabolism because of increased enzymatic activity and/or decreased capillary removal.     

In Vitro Metabolism of Phenylspirodrimanes Derived from the Indoor Fungus Stachybotrys

Fungi belonging to the genus Stachybotrys are frequently detected in water-damaged indoor environments, and a potential correlation between emerging health problems of inhabitants of affected housing and the fungi is controversially discussed. Secondary metabolites (i.e., mycotoxins) produced by Stachybotrys, such as the highly toxic macrocyclic trichothecenes (MCTs), are of potential concern to human health. The present study, however, focused on the potential effects of the more broadly and abundantly formed group of phenylspirodrimanes (PSDs). The phase I and II metabolism of four structurally different PSDs were investigated in vitro using hepatic models in combination with high-performance liquid chromatography high-resolution mass spectrometry (HPLC-HRMS) analysis. In addition to metabolite detection by HRMS, isolation and structure elucidation by nuclear magnetic resonance spectroscopy (NMR) was part of the conducted study as well.     

In vivo and in vitro Inhibition of the Metabolism of N-Alkyl-substituted Amphetamines in Rat by Ferrocenylisopropylamine

Abstract

1. Ferrocenylisopropylamine (FIPA) inhibits the elimination of amphetamines in rat. The half-life of isopropylamphetamine was increased from approx. 30 to 85–100 min after administration of FIPA.

2. With isolated, perfused, rat liver, the half-lives of isopropylamphetamine, biamphetamine and benzylamphetamine were increased from 5–20 min to about 200 min by equimolar amounts of FIPA, indicating that the prolonging effect of FIPA is due to interference at the metabolic level.

3. Experiments with hepatic microsomal suspensions demonstrated that FIPA competitively inhibits the oxidative N-dealkylation of isopropylamphetamine; the K_i of FIPA is 4·1 × 10^?6 M.

4. Binding of isopropylamphetamine and FIPA to cytochrome P-450 was studied using hepatic microsomes of phenobarbital-treated rats. Isopropylamphetamine caused a type I, and FIPA a type II difference spectrum; FIPA showed a much higher binding affinity (K_s = 1·24 × 10^?2 M) than isopropylarnphetamine (K_s = 0·96 × 10^?3 M). FIPA acts as a modifier of the spectral changes induced by isopropylamphetamine.

5. Results suggest that the competitive inhibition of the N-dealkylation of N-alkylamphetamines, and thus the prolonging of their action, by FIPA is related to competition for binding to cytochrome P-450.     

细胞色素P450酶系体外药物代谢研究方法进展

目的 综述细胞色素P450 (CYP 450)酶影响药物代谢的体外研究方法.方法 参考国内外文献,对与药物代谢相关的CYP酶亚型、CYP酶种属差异、CYP酶体外反应体系、药物主要代谢酶的确认方法及体外CYP酶的抑制和诱导,进行分类、归纳和整理.结果与结论 CYP 450在药物代谢中具有重要作用,药物代谢研究是新药评价体系中重要的一部分.     

In Vitro andIn Vivo availability of some commercial prednisolone tablets

The in vitrodissolution rates of prednisolone from five commercially available 5- mg prednisolone tablets were determined in distilled water. The dissolution studies were repeated on the fastest- and slowest-dissolving brands using 0.01% polysorbate 80 in 0.1 NHCl as the dissolution medium. A two-way crossover bioavailability study was performed in 12 human male adult volunteers comparing the fastest- and slowest-dissolving brands. The plasma samples were assayed for prednisolone by a radioimmunoassay method. Statistical analysis of the data for the two brands showed no significant differences between average plasma levels of prednisolone at any sampling time. The results suggest that on the average the in vivorates of dissolution of the two brands were essentially the same, and in vitrodissolution in 0.01% polysorbate 80–0.1 NHCl medium was better correlated with these in vivoresults than dissolution in water.Supported by Contract CPF69-22, Food and Drug Administration, Washington, D.C., and in part by Public Health Service Grant 5-P11-GM15559.     

Kinetic Studies of Dose-Dependent Metabolism of Alprenolol: In Vitro and In Vivo Studies in Different Species

Abstract Kinetic studies of the metabolism of alprenolol were performed with isolated microsomes from the rat, guinea-pig, dog and man at an initial substrate concentration of 0.17-150 μM. In all species the rate of aromatic hydroxylation reached a plateu above 50 μM of alprenolol in contrast to the rate of desisopropylation, where consistent saturation level was not obtained. The Km-values for the aromatic hydroxylation in the guinea-pig and man, 2 ,7 μM and 1.3 μM respectively, showed no concentration dependency in contrast to the rat (Km₁ = 0.20 μM, Km₂ = 26 μM) and the dog (Km₁ = 0.78 μM, Km₂ = 66 μM). The apparent Km-value of 0.20 μM for aromatic hydroxylation in the rat seemed to be of the same order of magnitude as reported spectral dissociation constant (Ks = 0.34 μM). In vivo experiments in the rat by oral administration of 7-700 μmol/kg demonstrated a dose-dependent presystemic elimination of alprenolol. The urinary excretion of hydroxy-alprenolol was significantly lower after the highest dose. It is proposed, that the saturation of the aromatic hydroxylation, catalyzed by a high affinity site or subspecies of cytochrome P-450 with a low capacity. contributes to the dose-dependent kinetics in vivo.     

Inhibition of 1,2-Dimethylhydrazine Metabolism by Disulfiram

1. The effects of disulfiram on the metabolism of 1,2-dimethylhydrazine were studied in CDF rats.

2. Treatment with disulfiram causes enhanced elimination of azomethane in the expired air, inhibition of CO₂ production, and decreased levels of 1,2-dimethylhydrazine metabolites in the urine.

3. These results suggest that disulfiram inhibits the N-oxidation of azomethane to azoxymethane, thus preventing further metabolism to the ultimate carcinogenic species, and provide an explanation for the observations that disulfiram inhibits 1,2-dimethylhydrazine-induced neoplasia of the large intestine.