Effects of polyamines on ethylmorphine N-demethylation in rat liver microsomes

The time course linearity of ethylmorphine N-demethylation was improved by the addition of polyamines to the reaction mixture. The most remarkable effect on the time course linearity of ethylmorphine N-demethylation was observed when spermine was used. The apparent stimulatory effect of spermine was decreased remarkably by the simultaneous addition of EDTA to inhibit lipid peroxidation. Similar results were observed when an additional lipid peroxidation inhibitor such as Co2+, Mn2+ or 2,2'-bipyridine was used in place of EDTA. Hydrogen peroxide-dependent ethylmorphine N-demethylation activity in rat liver microsomes was not influenced by the addition of spermine. In addition, neither lipid peroxides formation nor the stimulatory effects of polyamines on ethylmorphine N-demethylation was observed in the reconstituted monooxygenase system. These results suggest that the inhibitory effects of polyamines on lipid peroxidation might be responsible for the stimulatory effects on drug oxidations.     

The effect of neuroleptics on imipramine demethylation in rat liver microsomes and imipramine and desipramine level in the rat brain

A study of the cytochrome P-450 level and imipramine (IMI) demethylase activity in liver microsomes of rats treated concurrently with IMI and chlorpromazine (CPZ) or IMI and chlorprothixene (CPX) for two weeks were carried out. Concomitant administration of IMI and CPZ or IMI and CPX elevated the cytochrome P-450 level and accelerated IMI demethylation in in vitro study. Kinetic study of IMI demethylation carried out in the absence or in the presence of CPZ or CPX revealed that those neuroleptics inhibited IMI demethylation via competitive mechanism. Simultaneously with the enzymatic study the brain level of IMI and its demethylated metabolite desipramine (DMI) was assessed. It was found that 1 hr after withdrawal of IMI and CPZ or IMI and CPX the brain level of IMI was elevated in comparison with that of IMI treated animals, and the ratio between DMI/IMI brain concentration was decreased. When the assessment of IMI and DMI brain level was performed 24 hr after withdrawal of IMI and CPZ or IMI and CPX, there was no difference between the concentration of IMI and DMI in both, experimental and control animals.     

Effect of pirenzepine on the N-demethylation of D(−)ephedrine and ethylmorphine by rabbit liver microsomes

The possible interaction of pirenzepine with the mixed-function oxidases obtained from phenobarbital-pretreated rabbit microsomes was examined in vitro. Under experimental conditions that did not lead to its own N-demethylation, the drug inhibited the microsomal oxidase systems responsible for the N-demethylation of D(-)ephedrine and ethylmorphine. Kinetic studies showed that pirenzepine inhibited the metabolism of both drugs in a competitive manner. The results indicated that the observed pirenzepine stability to the hepatic N-demethylating system is not a result of low affinity of the drug to the system.     

Comparison of the metabolism of the three antidepressants amitriptyline,imipramine, and chlorimipramine in vitro in rat liver microsomes

The metabolism of the tricyclic antidepressants amitriptyline (AMI), imipramine (IMI), chlorimipramine (CMI) and some of their metabolites was studied in vitro in isolated liver microsomes of female Spraque-Dawley rats. Nine metabolites of AMI, seven metabolites of IMI, and 11 metabolites of CMI were quantitatively determined with high-performance liquid chromatography. The main metabolic reactions, mediated by an NADPH generating system, were hydroxylation, demethylation, and N-oxidation. The ratio of these reactions was different for the three drugs. AMI was hydroxylated more than CMI and CMI more than IMI. The order for demethylation was CMI>AMI=IMI, the order for N-oxidation IMI>CMIAMI. The substrate dependence of metabolism was investigated. Demethylation and N-oxidation increased proportionally to increasing substrate concentrations, whereas formation of hydroxylated metabolites became saturated (in the concentration range of 10^–6–10^–5 M). The in vitro metabolism was compared with the in vivo metabolism in humans, reflected by the plasma concentrations of these drugs and their metabolites. A good agreement in metabolic pathways was found.     

O- and N-demethylation of venlafaxine in vitro by human liver microsomes and by microsomes from cDNA-transfected cells: effect of metabolic inhibitors and SSRI antidepressants.

The biotransformation of venlafaxine (VF) into its two major metabolites, O-desmethylvenlafaxine (ODV) and N-desmethylvenlafaxine (NDV) was studied in vitro with human liver microsomes and with microsomes containing individual human cytochromes from cDNA-transfected human lymphoblastoid cells. VF was coincubated with selective cytochrome P450 (CYP) inhibitors and several selective serotonin reuptake inhibitors (SSRIs) to assess their inhibitory effect on VF metabolism. Formation rates for ODV incubated with human microsomes were consistent with Michaelis-Menten kinetics for a single-enzyme mediated reaction with substrate inhibition. Mean parameters determined by non-linear regression were: Vmax = 0.36 nmol/min/mg protein, K(m) = 41 microM, and Ks 22901 microM (Ks represents a constant which reflects the degree of substrate inhibition). Quinidine (QUI) was a potent inhibitor of ODV formation with a Ki of 0.04 microM, and paroxetine (PX) was the most potent SSRI at inhibiting ODV formation with a mean Ki value of 0.17 microM. Studies using expressed cytochromes showed that ODV was formed by CYP2C9, -2C19, and -2D6. CYP2D6 was dominant with the lowest K(m), 23.2 microM, and highest intrinsic clearance (Vmax/K(m) ratio). No unique model was applicable to the formation of NDV for all four livers tested. Parameters determined by applying a single-enzyme model were Vmax = 2.14 nmol/min/mg protein, and K(m) = 2504 microM. Ketoconazole was a potent inhibitor of NDV production, although its inhibitory activity was not as great as observed with pure 3A substrates. NDV formation was also reduced by 42% by a polyclonal rabbit antibody against rat liver CYP3A1. Studies using expressed cytochromes showed that NDV was formed by CYP2C9, -2C19, and -3A4. The highest intrinsic clearance was attributable to CYP2C19 and the lowest to CYP3A4. However the high in vivo abundance of 3A isoforms will magnify the importance of this cytochrome. Fluvoxamine (FX), at a concentration of 20 microM, decreased NDV production by 46% consistent with the capacity of FX to inhibit CYP3A, 2C9, and 2C19. These results are consistent with previous studies that show CYP2D6 and -3A4 play important roles in the formation of ODV and NDV, respectively. In addition we have shown that several other CYPs have important roles in the biotransformation of VF.     

氯米帕明在人肝微粒体中的N—去甲基代谢

目的：研究ＣＹＰ４５０选择性抑制剂对体外氯米帕明（Ｃｌｏ）Ｎ－去甲基代谢的影响。方法：应用米氏方程计算肝微粒体中Ｃｏｌ Ｎ－去甲基代谢的动力学参数，比较加入抑制剂前后这些参数的改变。结果：Ｋｍ１，Ｋｍ２，Ｖｍａｘ１，Ｖｍａｘ２，Ｖａｍｘ２／Ｖｍ１和Ｖａｍｘ２／Ｋｍ２分别为（０．１１±０．０６），（２４±１１）μｍｏｌ·Ｌ＾－１，（１１４±４７），（４２８±１８８）ｎｍｏｌ·ｇ＾－１·ｍｉｎ＾－１。     

Effects of alpha-carbon substituents on the N-demethylation of N-methyl-2-phenethylamines by rat liver microsomes

The effects of methyl, ethyl, isopropyl, isobutyl, and benzyl substituents at the alpha-carbon of N-methyl-2-phenethylamine on the kinetics of its N-demethylation in liver microsomes from both control and phenobarbital pretreated rats were studied. In control microsomes, the kinetic studies indicated that more than one enzyme was active for N-demethylation of N-methyl-alpha-methylphenethylamine (methamphetamine) while the other N-methyl-2-phenethylamines appeared to be demethylated by a single enzyme. In microsomes from phenobarbital pretreated animals, there appeared to be more than one enzyme system which was active for N-demethylation of all compounds except N-methyl-alpha-benzylphenethylamine. One of these had a much higher affinity for alpha-ethyl, isopropyl, and isobutyl N-methylphenethylamines while another exhibited affinities for substrates similar to the constitutive enzyme in control microsomes. A correlation was observed between the octanol-buffer or heptane-buffer distribution ratios of the compounds and the negative logarithm of the Michaelis constant (pKm) for the enzyme in control microsomes and for each of the enzyme systems in microsomes from phenobarbital-pretreated animals. Therefore, it is indicated that the concentration of a substrate at the active site of these microsomal enzymes is a function of its lipid solubility.     

The effect of age on sildenafil biotransformation in rat and mouse liver microsomes.

Sildenafil [SIL (Viagra); Pfizer, New York, NY] is a widely prescribed agent for erectile dysfunction in men older than 65 years. The present study evaluated experimental models to assess age-dependent changes in SIL biotransformation using hepatic microsomes from male rats and mice ranging from 6 weeks to 26 months of age. The role of specific isoforms in the conversion of SIL to its primary circulating metabolite, UK-103,320 (piperazine N-desmethyl sildenafil) in the mouse was also investigated using immunoinhibitory antibodies. Although CYP2C11 largely mediated UK-103,320 formation in the rat, UK-103,320 formation was principally inhibited by a CYP3A antibody in the mouse. An age-related decrement in metabolite formation rate was observed for both species, although this effect was more pronounced in the old rats (reduced to 7% of young) than in the old mice (reduced to 51% of young). CYP2C expression was assessed by Western blot analysis in rat and mouse livers. Age-related differences in hepatic CYP3A expression in the mouse were also compared with metabolite formation rates in the mouse model. Decrements with age in CYP2C and -3A expression in the aging rodents paralleled the decrements in SIL biotransformation, suggesting that age-related differences in SIL metabolic rate may, in part, reflect differences in expression. Although the role of specific CYP enzymes and the clearance values for this reaction may differ among species, age-related changes in these rodent models are consistent with the reduced clearance of SIL observed in human studies.     

Biphasic kinetics of imipramine N-oxidation in rat brain microsomes

Imipramine (IMI) N-oxidase activity in brain microsomes from rats of both sexes was determined by high performance liquid chromatography, and compared with the results in rat liver microsomes. Brain and liver microsomal IMI N-oxidation was sensitive to thermal inactivation and had an optimal pH at around 9.0. IMI N-oxidase activity (15.54 pmol/min/mg protein) in brain microsomes was about one-hundredth that of liver microsomes (2.08 nmol/min/mg protein) at a substrate concentration of 5 mM. IMI N-oxidase activities in both brain and liver microsomes displayed biphasic kinetics that associated a low Km-low Vmax element with a high Km-high Vmax component. Furthermore, a significant sex difference was observed in Vmax values (male>female) in both phases, but Km values were similar between male and female rats, resulting in a significant sex difference (male>female) in intrinsic clearance values (Vmax/Km) of the low-Km and the high-Km phases.     

Inhibitors of imipramine metabolism by human liver microsomes.

1. The aromatic 2-hydroxylation of imipramine was studied in microsomes from three human livers. The kinetics were best described by a biphasic enzyme model. The estimated values of Vmax and Km for the high affinity site ranged from 3.2 to 5.7 nmol mg-1 h-1 and from 25 to 31 microM, respectively. 2. Quinidine was a potent inhibitor of the high affinity site for the 2-hydroxylation of imipramine in microsomes from all three human livers, with apparent Ki-values ranging from 9 to 92 nM. This finding strongly suggests that the high affinity enzyme is CYP2D6, the source of the sparteine/debrisoquine oxidation polymorphism. 3. The selective serotonin reuptake inhibitors (SSRI), paroxetine, fluoxetine and norfluoxetine were potent inhibitors of the high affinity site having apparent Ki-values of 0.36, 0.92 and 0.33 microM, respectively. Three other SSRIs, citalopram, desmethylcitalopram and fluvoxamine, were less potent inhibitors of CYP2D6, with apparent Ki-values of 19, 1.3 and 3.9 microM, respectively. 4. Among 20 drugs screened, fluvoxamine was the only potent inhibitor of the N-demethylation of imipramine, with a Ki-value of 0.14 microM. 5. Neither mephenytoin, citalopram, diazepam, omeprazole or proguanil showed any inhibition of the N-demethylation of imipramine and the role of the S-mephenytoin hydroxylase for this oxidative pathway could not be confirmed.     

Acute effect of bromodichloromethane on lipid metabolism in rat liver microsomes

Acute effects of CHBrCl2 on microsomal lipid metabolism were studied in the liver of male Wistar rats administered a single oral dose (215 mg/kg). 3 h after administration, the modulation of phospholipid metabolism and the accumulation of triacylglycerol (TG) were noted in the microsomal membranes. A marked decrease in the synthetic rate of phospatidylcholine (PC) could be explained by a fall in the microsomal activity of CTP:phosphocholine cytidylyltransferase. A decrease in glycerophosphate acyltransferase activity and an enhancement in phosphatidate phosphatase activity seemed to offset each other, and consequently no significant change was observed in the synthetic rate of microsomal TG. These alterations in microsomal lipid metabolism can be regarded as one of the manifestations of hepatotoxicity of CHBrCl2.