Individual Forms Of Cytochrome P450 Research Articles

Clinical utility of drug measurement and pharmacokinetics – therapeutic drug monitoring in psychiatry

Based on the assumption that a relationship between blood levels and clinical effects (therapeutic effects, adverse events and toxicity) can be defined and considering that after equal doses plasma concentrations vary markedly between individual patients, therapeutic drug monitoring (TDM) can assist to personalize dose adjustment. Taken together, drug levels and a knowledge of the pharmacological profile of the administered drugs can enable the optimal dosage to be tailored according to the need of the individual patient. Therapeutic drug monitoring has been established for a limited number of drugs. In psychiatry, it has a 40-year-long history, which started with nortriptyline. Evidence has accumulated which shows that TDM is a valid tool for the optimization of psychopharmacotherapy. When used adequately, TDM is helpful for many patients and in many situations. Combined with pharmacogenetic tests, the metabolic status of a patient can be well characterized. Several new observations have been made during routine TDM that have stimulated clinical pharmacological research, such as investigations on inherited differences in drug metabolism that are closely linked to TDM in psychiatry. The contributions of individual forms of cytochrome P450 (CYP) to the metabolism of drugs was elicited by clinical observations on pharmacokinetic drug interactions. Therapeutic drug monitoring requires a close collaboration between the prescribing physician, the laboratory specialist, the clinical pharmacologist and the patient. This complexity may result in errors which can be detected by analysing the appropriate use of TDM in clinical practice. More education has to be provided to the prescribing clinicians on the pharmacology of the drugs and the algorithm of TDM. Moreover, clinical trials should include measurements of blood concentrations during drug development to generate valid data on the relationships between drug concentrations and clinical outcomes under well-controlled conditions. This would merely increase the amount of work and costs, as high-throughput methods are now available in many laboratories. Any progress in TDM has direct benefits for the treatment of many individual patients.

RT-PCR-based cytochrome P450 expression profile of oral tissue samples1

To analyse the expression of individual forms of cytochrome P450 (CYP) at the mRNA level in five homogenized oral buccal tissue samples from four individuals with or without oral malignancy. Individual forms of CYPs were studied by reverse transcriptase-polymerase chain reaction (RT-PCR), using specific primers for CYPs 2B6, 2C, 2D6, 2E1, 3A3/4 and 3A5, and oral CYP expressions were compared with CYP expression in liver tissue. Consistent expression of CYPs 2C, 2E1 and 3A5 was observed in oral buccal tissue at mRNA level. These particular CYPs have possible roles in the protection of the body against orally ingested xenobiotics as well as influence the bioavailability of therapeutic compounds.

Forms of cytochrome P450 in the liver microsome of oxidized frying oil-fed guinea pigs.

The aim of this study is to investigate the influence of oxidized frying oil (OFO) on the induction of individual forms of cytochrome P450 (CYP450) in guinea pigs. The OFO samples were obtained by frying potato chips in soybean oil at 200+/-5 degrees C for 24 h. Sixteen male weaning guinea pigs were fed for 12 wk on a diet which included 15% of either OFO or fresh soybean oil supplemented with 300 ppm ascorbic acid. It was demonstrated that guinea pigs fed with the OFO diet (D300) exhibited inferior growth rates and lower feed efficiency than the control group (F300). The vitamin C contents of plasma, liver, and kidney in the D300 group were lower than those in the F300 group. Further, the thiobarbituric acid-reactive substance content in D300 liver and kidney was higher than that in the F300 equivalent. The liver UDP-glucuronyl-transferase activity in the D300 group was higher than that in the F300 group, and there was no difference in comparing the glutathione-S-transferase activity levels of the two groups. Notably, the total CYP450 content and the NADPH-cytochrome c reductase activity were significantly elevated in the D300 group. The ethoxyresorufin O-deethylase activity, which mainly represents CYP1A1 activity, for the detection of CYP450 isozyme characteristics was elevated more in the D300 than in the F300 group. There was no difference in the pentoxyresorufin O-dealkylase activity, which mainly reflects the CYP2B isozyme, between the two groups. However, the quantity of CYP1A1 isoform determined in the D300 group did not differ from that in the F300 group, as revealed by SDS-PAGE and Western blot testing. Our results demonstrate that the relative enzyme activity to CYP1A1-like activity of the guinea pig hepatic xenobiotic metabolizing enzyme system may be induced by OFO feeding, but more advanced research is needed to identify the predominant form of CYP450 isozyme induced as a result of this condition.

OMEPRAZOLE HYDROXYLATION BY CYP2C19 AND CYP3A4: PREDICTION TOWARDS HUMAN LIVER ACTIVITIES USING THE DATA OF RECOMBINANT P450 ENZYMES

Omeprazole 5-hydroxylation and sulfoxidation activities were determined in liver microsomes of different humans whose levels of individual forms of cytochrome P450 (P450 or CYP) varied. Omeprazole 5-hydroxylation by liver microsomes of a human sample which contained relatively high levels of CYP3A4 and low levels of CYP2C19 were inhibited very significantly by ketoconazole and anti-CYP3A4 antibodies, while a human sample having high in CYP2C19 and low in CYP3A4 was found to be sensitive towards fluvoxamine and anti-CYP2C9 antibodies. Both recombinant human CYP2C19 and CYP3A4 enzymes had activities for omeprazole 5-hydroxylation, with low Km and high Vmax values for the former enzyme and high Km and low Vmax values for the CYP3A4. Omeprazole 5-hydroxylation activities of different human samples were found to be related to predicted values calculated from the kinetic parameters of recombinant enzymes and the levels of liver microsomal P450 enzymes. When recombinant human CYP2C19 and CYP3A4 were mixed at levels found in different human samples, relatively similar profiles of omeprazole oxidation by the recombinant and microsomal enzyme systems were determined. These results suggest that both CYP2C19 and CYP3A4 are involved in the 5-oxidation of omeprazole (at a substrate concentration of 10 μM) in human liver microsomes and that contributions of these P450 enzymes depend on the compositions of CYP2C19 and CYP3A4 in liver. Different contributions of CYP2C19 and CYP3A4 In the oxidation of testosterone and progesterone by human liver microsomes are also reported.

The in vitro effects of lipid peroxidation on the content of individual forms of cytochrome P-450 in liver microsomes of guinea-pigs

The effect of lipid peroxidation in vitro on the amounts of several forms of cytochrome P-450 in liver microsomes from guinea-pigs was investigated. Lipid peroxide formation in liver microsomes from ascorbic acid (VC)-deficient animals was much higher than that observed in control animals. The antibodies to rat P-450IA2 (P-448-H), P-450IIB 1 (P-450b) and human P-450IIIA4 (P-450NF) recognized one or two forms of cytochrome P-450 in liver microsomes of guinea-pigs. Neither cytochrome P-450 cross-reactive with anti-P-450IIB1 antibodies nor cytochrome P-450 cross-reactive with antibodies to P-450IIIA4 was virtually affected by microsomal lipid peroxidation induced by NADPH in vitro. In contrast, the forms of cytochrome P-450 immunochemically related to P-450IA2 were decreased with the increased level of lipid peroxide formation. The form-specific degradation of cytochrome P-450 due to lipid peroxidation was in agreement with our previous observation that the amounts of cytochrome P-450 cross-reactive with antibodies to P-450IA2 but not with antibodies to P-450IIIA (P-450PB-1) were predominantly decreased in VC-deficient guinea-pigs compared to control animals in vitro.

Cytochrome P-450 alterations in the BB/Wor spontaneously diabetic rat

The hepatic content of two individual cytochrome P-450 enzymes was analyzed in spontaneously diabetic (BB/Wor) male rats. The major male-specific form, RLM5, was found to be slightly decreased in livers of male rats shortly after the onset of diabetes. In contrast, the level of RLM6 was elevated in livers of diabetic rats that had not received insulin and had become ketotic. These results confirm that the changes in some individual forms of cytochrome P-450 after chemical (streptozotocin) induction of diabetes are also seen in the spontaneously diabetic animal. The earlier observed alterations in cytochrome P-450 are therefore due to the state of diabetes and not to inductive or depressive effects of streptozotocin.

Expression and function of three cytochrome P-450 isozymes in rat extrahepatic tissues

Western blots using a polyclonal and a monoclonal antibody raised against rat liver cytochrome P-450b indicate tissue-specific expression of low levels of cytochrome P-450's b and e. P-450b and P-450e were expressed very selectively in, respectively, lung and adrenal microsomes of untreated rats but neither isozyme was detected in the corresponding kidney or small intestine microsomes. The regioselectivity of microsomal metabolism of 7,12-dimethylbenz[ a]anthracene (DMBA) as well as the sensitivity to inhibition by anti P- 450 b e IgG established that low levels of “b-like” P-450's are functional in lung and adrenal microsomes from uninduced rats, but not in microsomes from the kidney or small intestine. Functional P-450c was also detected at low levels in liver, lung, kidney, and adrenals of untreated rats. Among the extrahepatic tissues examined, DMBA metabolism was the highest in rat adrenal microsomes. However, only 30% of this activity was due to P-450′s b, e, or c. Phenobarbital (PB) treatment of rats increased microsomal DMBA metabolism in all extrahepatic tissues examined. The selectivity of this increase for 12-methyl hydroxylation of DMBA and the near complete inhibition by anti- P- 450 b e are consistent with induction of P-450e even though P-450b was preferentially induced in each of the extrahepatic tissues examined. The levels of expression of P-450b were increased by PB in all sets of adrenal, lung, and intestinal microsomes and in three out of six sets of kidney microsomes. The levels of P-450e were also increased by PB in all sets of adrenal microsomes. Following PB treatment, P-450e became immunoquantifiable (>2 pmol/mg protein) in three of six sets of lung and kidney microsomes but remained below detection in all sets of intestinal microsomes. Based on the activity of purified P-450e, undetectable levels (<1 pmol/mg protein) could account for increased DMBA metabolism in this tissue. The high constitutive level of P-450b in the lung (~40 pmol/mg), was remarkably inactive in DMBA metabolism and was only slightly increased by PB treatment (50%). In contrast, PB treatment caused a 2.5- to 10-fold increase in 12-methyl hydroxylation of DMBA that was highly sensitive to anti- P- 450 b e . A protein comigrating with P-450e was well above detection (6–7 pmol/mg) in two of six preparations of lung microsomes that showed highest induction of this activity. PB treatment, therefore, increases both P-450's b and e in each of the extrahepatic tissues examined, consistent with hepatic induction although far less effectively. 3-Methylcholanthrene induced P-450c (>50-fold) in all tissues except the adrenal (only 3-fold) but with the exception of the latter, the associated activities were typically 10-fold lower than anticipated. These studies demonstrate that very small and even undetectable levels of individual forms of cytochrome P-450 play a major role in PAH metabolism in extrahepatic tissues.

Effect of suicide substrate on the metabolism of steroids and xenobiotics and on cytochrome P-450 apoproteins.

Xenobiotic administration can significantly alter forms of hepatic microsomal cytochrome P-450. Individual forms of cytochrome P-450 are known to be increased by agents, such as phenobarbital, 3-methylcholanthrene, β-naphthoflavone and pregnenolone-16α-carbonitrile1–3. Certain chemicals decrease the levels of these isoenzymes by modulating the heme biosynthetic pathway and, thereby, affect the availability of heme and the amount of hepatic cytochrome P-450, e.g. cobaltous chloride4,5. Some agents can act as suicide substrates (i.e., mechanism-based inhibitors) for one or more cytochrome P-450 forms and, thereby, promote catabolic destruction of these cytochrome P-450 species6,7. Derivatives of 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) interact rapidly with cytochrome P-450, promote the rapid destruction of hepatic microsomal cytochrome P-450 and lead to the generation in vivo of N-alkyl protoporphyrins which, in turn, inhibit the activity of mitochondrial ferrochelatase, the enzyme catalyzing the last step in heme biosynthesis8–11. The result is a decreased total hepatic microsomal cytochrome P-450 level and a marked hepatic porphyria12,13.

Altered distribution of constitutive forms of microsomal cytochrome P-450 in tumor-bearing mouse liver.

Changes of microsomal cytochrome P-450 species in the livers of mice were examined after the inoculation of Ehrlich ascites tumor cells i.p. into male ddY mice. The total content of hepatic cytochrome P-450 was observed to decrease around 6 days after the tumor-inoculation and reached the level of 40 to 50% of those of control mice (27.8 nmol P-450/g of control liver) after 10 days, while the liver weights of tumor-bearing mice slightly increased even at the end of the 2nd week. Microsomal proteins of tumor-bearing mice remained at the level of around 80% of control livers at the end of the 2nd week. The activity of 7-ethoxycoumarin deethylation decreased roughly in proportion to the content of microsomal cytochrome P-450. On the contrary, the specific activity of benzphetamine N-demethylase per nmol of P-450 in the tumor-bearing mouse liver increased to more than 2 fold of the value of normal liver (25 nmol HCHO/nmol P-450/min for control liver) during the last stage of the second week. These results suggest that the reduction of individual forms of cytochrome P-450 in the tumor-bearing mouse liver was not random and a new steady state of distribution of these hemeproteins was established under the pathological conditions.