Quantities Of Cytochrome P-450 Research Articles

Effects of nifedipine on behavioral and biochemical parameters in rats after multiple morphine administration

Numerous studies indicate that opioid tolerance involves a disruption in Ca2+ homeostasis. In vivo studies have indicated the involvement of dihydropyridine-sensitive (L-type) voltage-gated channels in morphine abuse. In this study, the effect of multiple administration of the dihydropyridine calcium channel blocker nifedipine (5 mg/kg/twice daily), given in combination with morphine, on the signs of morphine withdrawal and some biochemical parameters were assessed. Multiple morphine administration in increasing doses (from 5 to 40 mg/kg for 7 days) and consequent withdrawal after 18 h, induced writhing, squealing, diarrhea, teeth chattering, eyelid ptosis and wet-dog type shaking. Coadministration of nifedipine prevented the squealing, diarrhea and teeth chattering. On a biochemical level, the activity of brain nitric oxide synthase (NOS) and the quantity of cytochrome P450 in rat brain and liver were measured. Nifedipine treatment decreased the brain nNOS activity, induced by multiple administration of morphine. The quantity of liver cytochrome P450, after multiple coadministration of morphine and nifedipine, was also increased. The quantity of brain cytochrome P450 was not significantly changed by morphine and nifedipine alone or in combination. The results of our study suggest that nifedipine influences the effects of morphine both at a pharmacokinetic and a pharmacodynamic level.

Mechanism-based inhibition of CYP activities in rat liver by fluoxetine and structurally similar alkylamines

1. The inhibition of cytochrome P450 (CYP)-mediated substrate oxidations by alkylamine-based drugs was investigated in rat hepatic microsomes. The effects of pre-incubation of the drugs with NADPH-fortified microsomes on inhibition potency was evaluated in relation to the formation of metabolite intermediate (MI) complexes with CYP in vitro.2. The selective serotonin-reuptake inhibitor fluoxetine (FLU) emerged as a potent and preferential inhibitor of CYP2C11 in rat liver microsomes. After FLU biotransformation in NADPH-supplemented microsomes, IC50 values of 2 and 1 μM were determined against CYP2C11-dependent testosterone 2α- and 16α-hydroxylation; in the absence of pre-incubation, the corresponding IC50 values were 47 and 39 μM. MI complexation of CYP appeared to contribute significantly to inhibition by FLU, as evidenced by the 21% decrease in apparent microsomal CYP content produced by 50 μM FLU in the presence of NADPH.3. The secondary amines nisoxetine (NIS), and especially, desipramine (DES) and nortriptyline (NOR), also inhibited CYP2C11 and generated MI complexes with microsomal CYP. In contrast, with the exception of SKF-525-A, tertiary alkylamines (10 compounds) inhibited specific CYP activities but did not form MI complexes. Pre-incubation of these agents with NADPH-supplemented microsomes did not enhance inhibition of CYP activities, thus suggesting that formation of inhibitory metabolites was minimal for these compounds.4. These findings implicate drug-mediated MI complexation of CYPs in the inhibition of hepatic biotransformation processes by secondary alkylamines. In contrast, tertiary amines did not generate significant quantities of CYP–MI complexes under the test conditions. Despite their diffusion from the CYP active site, inhibition produced by tertiary amines and stable metabolites of other drugs may be significant. However, such inhibition would be of shorter duration than that from MI complexation, which involves quasi-covalent binding to the haem and prevention of oxygen activation.

Application of the PKCYP Test to Predict Caffeine Clearance Mediated by CYP1A2 in a Rat Acute Liver Injury Model

We previously established a method for assessing in vivo drug-metabolizing capacity by pharmacokinetic estimation of the quantity of cytochrome P450 (CYP) in vivo (PKCYP test), in which an apparent liver-to-blood free concentration gradient in vivo (qg) is introduced (Matsunaga et al., Jpn. J. Hosp. Pharm., 26: 492-504 (2000)). This method was applied to estimate the amount of CYP2C11 in rats treated with carbon tetrachloride (CCl(4)-treated rats). In this study, we estimated the amount of CYP1A2 in CCl(4)-treated rats by using acetanilide and caffeine as a probe and a model drug, respectively. In CCl(4)-treated rats, the total body clearance (CL(tot)) of acetanilide and caffeine was about one-fifth and one-eighth of that in control rats, respectively. In CCl(4)-treated rats, the amount of CYP1A2 was predicted as 0.60+/-0.06 nmol/kg from the clearance of acetanilide mediated by CYP1A2. Moreover, the clearance of caffeine mediated by CYP1A2 in CCl(4)-treated rats was estimated as 0.47+/-0.05 mL/min/kg by using the predicted amount of CYP1A2. The observed value was 0.44+/-0.03 mL/min/kg, and the predicted value was within the 95% confidence interval of the observed value. In conclusion, we have demonstrated that the PKCYP test can also be applied for estimating the amount of CYP1A2 in CCl(4)-treated rats.

Simultaneous Assessment of the In Vivo Amount of CYP1A2 and CYP3A2 by the PKCYP-test Using Theophylline in Rats

Recently, we developed a method for assessing in vivo drug metabolism capacity by pharmacokinetic estimation of the quantity of cytochrome P450 (CYP) in vivo (PKCYP-test), in which an apparent liver-to-blood free concentration gradient in vivo (qg) is introduced. The qg value can be alternatively defined as the ratio of the in vivo-in vitro clearance by a single CYP isoform. In this study, we examined the application of the PKCYP-test to drugs metabolized by multiple CYP isoforms in a rat model with fluctuating CYP1A2 levels using theophylline as a model drug. In control rats, the estimated qg values for each CYP1A2 and CYP3A2 based on the in vivo hepatic intrinsic clearance, in vitro Michaelis constant (K(m)) and maximal rate of metabolism (V(max)) values for liver slices agreed well. Moreover, the qg value for CYP1A2 determined by the K(m) and V(max) values for recombinant CYP1A2 was compatible with that based on liver slices. These qg values also agreed with that of rats pretreated with 3-methylcholanthrene. The time-course of theophylline concentrations in serum simulated by a physiologically-based pharmacokinetic model incorporating the hepatic clearance determined by the PKCYP-test agreed with the observed values. These results demonstrate that the qg value in the PKCYP-test is applicable to drugs metabolized by multiple CYP isoforms.

A comparative study of drug metabolizing enzymes in adrenal glands and livers of rats and chickens

1. 1. Drug metabolizing enzymes (cytochrome P450, glutathione- S-transferase, carboxylesterase) were compared in livers and adrenal glands from rats and chickens. 2. 2. Quantities of cytochrome P450 in chicken liver and adrenal glands were less than in rat liver and adrenals. 3. 3. Activities of carboxylesterase and of glutathione- S-epoxide transferase were similar in livers of rats and chickens. 4. 4. In the chicken, activities of carboxylesterase and of glutathione- S-epoxide transferase were less in adrenal glands than in livers. 5. 5. Carboxylesterase enzyme activities in adrenal glands of chickens were more sensitive to inhibition by antiesterase agents than were carboxylesterase enzyme activities in liver.

Inducibility of the hepatic microsomal monooxygenase system experimentally reduced to a minimum amount/activity: Effect of hypophysectomy, partial hepatectomy and phenobarbital/toluene treatment on the hepatic polysubstrate monooxygenase system in rats

Sham-operated, hypophysectomized, partially hepatectomized and hypophysectomized + partially hepatectomized groups of CFY rats were treated with phenobarbital (40 mg/kg/day) per os or physiological saline once a day, or toluene-vapour (inhalation: 4,000 mg/m3, 8 h/day), for 3 days. The polysubstrate monooxygenase (PSMO) system of the liver was induced by phenobarbital after all kinds of surgical interventions. Inducibility of the enzyme system was the highest in the group with combined hypophysectomy + partial hepatectomy and decreased in order in the groups with partial hepatectomy, hypophysectomy and sham-operation. The relative cytochrome P-450 content (quantity of cytochrome P-450/100 g b.w.) was the lowest after combined operation, higher in partially hepatectomized and hypophysectomized animals and it was the highest in the sham-operated group. Hypophysectomy after partial hepatectomy seems to inhibit hepatic regeneration and to increase the inducibility of the enzyme by phenobarbital, at the same time. Phenobarbital treatment brought about SER proliferation in a part of liver cells, in each group. In case of repeated liver damage due to the consequences of hypophysectomy, partial hepatectomy, phenobarbital administration, a group of the liver cells responds to the first, another to the second, but even after the third injury a group of the liver cells maintained its regular structure. The hypothetical hepatotoxic effect of toluene, a widely used industrial solvent, has been tested. Hepatotoxicity has been excluded. The minimum non-specific hepatotoxic effect of the solvent was not augmented by either partial hepatectomy or hypophysectomy, or by the combination of the two. In all the experimental groups toluene increased the hepatic cytochrome P-450 level and induced a moderate increase in SER. Inducibility of the enzyme system after toluene inhalation was similar to that of the enzyme system after phenobarbital treatment in each group.

Ascorbic acid and drug metabolism

Studies in vitro with liver microsomes isolated from vitamin C deficient guinea pigs have indicated a significant decrease in over-all drug oxidation, as typified by aniline hydroxylation, aminopyrine N-demethylation and p-nitroanisole O-demethylation. Concommitant with decreased over-all drug oxidation, the quantity of cytochrome P-450 and cytochrome b 5, and the activity of NADPH cytochrome P-450 reductase and NADPH cytochrome c reductase also decreased significantly. It was not until the quantity of liver microsomal ascorbic acid had reached 30 per cent of normal values (11 μg/g wet weight compared to 3.5 μg/g wet weight) that a marked decrease in over-all drug oxidation activity, as well as the level and activity of individual electron transport components, occurred. In addition, the decrease in drug enzyme activities was not due to a calorie deficiency in the vitamin C deficient guinea pigs, since studies with fasted animals indicated normal or greater than normal drug enzyme activities. K m studies with microsomes isolated from normal and vitamin C deficient guinea pigs did not indicate a correlation in the apparent affinity of drug substrates such as aniline, aminopyrine, and p-nitroanisole with decreased microsomal enzyme activities in the vitamin C deficient guinea pigs. The K m value of aniline hydroxylase for aniline was approximately four times higher in normal guinea pig microsomes while the K m value of N-demethylase for aminopyrine and O-demethylase for p-nitroanisole was in the order of four times higher in the vitamin C deficient guinea pig microsomes. Studies concerned with aniline-cytochrome P-450 binding spectra indicated an atypical altered spectrum with microsomes isolated from vitamin C deficient animals. The usual trough of the spectrum shifted from 390 to 405 nm and the usual peak of the spectrum shifted from 430 to 440 nm. Also, the intensity of the trough and peak was at least 50 per cent lower than normal. Administration in vivo of ascorbic acid to vitamin C deficient animals was followed by reversal of over-all drug oxidation activities, quantity of cytochrome P-450, NADPH cytochrome P-450 reductase activity and altered aniline-cytochrome P-450 binding, but these changes required at least 6 days of treatment to return to normal even though normal levels of liver ascorbic acid were established within 3 days. Phenobarbital induction studies indicated that the microsomal protein-synthesizing system responds to such treatment in vitamin C deficient guinea pigs, and the increase in drug enzyme activities and the level and activity of electron transport components are equal to, if not greater than, those observed in normal animals. Alternative pathways involving an ascorbic acid dependent NADH oxidase system, which has been shown to be capable of metabolizing drugs in the absence of a microsomal NADPH P-450 reductase system, have been considered. The significance of vitamin C deficiency in human drug therapy has been discussed.