[Safe administration of new drugs].
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Clinical Trials as Topic↗
Biomedical subjects
O Pelkonen
Publications and source records attributed to O Pelkonen.
Diagnosis of polymorphisms in carcinogen-activating and inactivating enzymes and cancer susceptibility--a review.
Up to 90% of all cancers are possibly caused by environmental factors, such as tobacco smoke, diet and occupational exposures. The majority of chemical carcinogens require metabolic activation before they interact with cellular macromolecules and can cause cancer initiation. The xenobiotic-metabolising machinery contains two main types of enzymes: the phase-I cytochromes P-450 (CYP) mediating oxidative metabolism, and phase-II conjugating enzymes. Several phase-I and phase-II genes have recently been cloned and identified in humans. Many of them show polymorphism and have been suggested to contribute to individual cancer susceptibility as genetic modifiers of cancer risk. Altered phenotypes and genotypes in the CYP subfamilies CYP1A1, CYP2D6 and CYP2E1 have been associated with tobacco smoke-induced lung cancer and other cancers. Defective glutathione S-transferase (GST) and N-acetyltransferase (NAT) enzymes have been associated with an increased risk of developing lung and bladder cancer. There are also several studies in each category in which no associations have been found. The risk of developing lung cancer is dramatically (up to 40-fold) elevated in subpopulations having simultaneously high-risk genotypes in CYP1A1 and GSTM1. There are several difficulties in this area of research. First, many of the observed restriction-fragment length polymorphisms (RFLPs) are due to mutations in introns or other silent areas of DNA, raising the possibility that any associations found between RFLPs and cancer occur only by chance. Second, biologically plausible mechanisms linking genotypes and cancer are lacking in most of the observed cases.(ABSTRACT TRUNCATED AT 250 WORDS)
Arylamine N-Acetyltransferase↗
Up-regulation of CYP2A5 expression by porphyrinogenic agents in mouse liver.
Coumarin 7-hydroxylase (COH) activity is catalyzed by the Cyp2a-5 gene product (CYP2A5 enzyme) in mice. Mouse hepatic CYP2A5 expression is often increased in conditions in which other P450 forms are repressed, e.g. after the administration of heavy metals and other toxic agents known to affect cellular heme balance. In this study, the effect of various porphyrinogenic chemicals on the expression CYP2A5 and the key enzymes in heme metabolism was studied. Administration of single doses of griseofulvin (1000 mg/kg), thioacetamide (10 mg/kg) and aminotriazole (1000 mg/kg) to DBA/2 and C57BL/6 mice produced up to 10-fold increases in hepatic COH catalytic activity. Dramatic, up to 130-fold increases in response to the inducers was observed in the amount of CYP2A5 steady-state mRNA. The mRNA contents of aminolevulinate synthase, ferrochelatase and heme oxygenase were also increased to a variable extent, possibly reflecting feed-back regulatory mechanisms. In D2 mice the CYP2A5 inducing effect of aminotriazole and thioacetamide, but not that of griseofulvin, pyrazole and phenobarbital, was abolished by exogenously administered heme arginate. In the B6 strain heme arginate treatment increased CYP2A5 expression but it did not affect the induction caused by porphyrinogenic agents. These results show that porphyrinogenic agents act as efficient inducers of CYP2A5, and suggest that regulation of the transcription of the Cyp2a-5 gene could in some instances involve heme-sensitive factors.
5-Aminolevulinate Synthetase↗
Serum sex hormone levels after replacing carbamazepine with oxcarbazepine.
The function of the hepatic P450 enzyme system was evaluated by measuring the kinetics of antipyrine and serum sex hormone levels were determined in 12 male patients with epilepsy during carbamazepine medication, and two and six months after changing their medication to oxcarbazepine. Antipyrine t1/2 increased and antipyrine CL decreased after the change reflecting normalisation of the liver P450 enzyme system function. Serum sex hormone binding globulin levels decreased, and serum dehydroepiandrosterone sulphate increased after the change. The results show that the carbamazepine-associated induction of the liver P450 enzyme system and changes in serum sex hormone balance can be avoided by replacing carbamazepine with oxcarbazepine.
Adult↗
The role of CYP enzymes in cocaine-induced liver damage.
Cocaine is hepatotoxic in several species, including man. A high dose of cocaine produces metabolism-dependent, mainly pericentral, liver damage. At 24 h after a single dose of cocaine, mouse hepatic P450 content decreases but CYP2A activities; coumarin 7-hydroxylase and testosterone 15 alpha-hydroxylase increase concomitant with prominent diffuse cell necrosis. Repeated administration of cocaine for up to 5 days decreases CYP1A1/2, 2A4/5, 2Cx, and 2E1 related enzymatic activities. However, after five doses of cocaine, CYP2B10 increases in conjunction with the healing process. In the acute phase, the increased CYP2A activities do not participate in cocaine bioactivation. CYP3A enzymes are principally responsible for the cocaine N-demethylation in human and mouse liver microsomes. The hepatic metabolic CYP enzyme profile will change during prolonged cocaine intake, this being accompanied by altered cell morphology. Possible connections to cocaine toxicity in man are discussed.
Animals↗
Antipyrine, coumarin and glipizide affect n-acetylation measured by caffeine test.
The effect of various treatments on acetylation status measured by caffeine metabolites was investigated in 17 subjects with non-insulin dependent diabetes mellitus (NIDDM). The test drugs, caffeine (200 mg), antipyrine (20 mg/kg) and coumarin (5 mg), were given simultaneously, and urinary 5-acetylamino-6-formyl-amino-3-methyluracil/1-methylxanthine (AFMU/1X) molar ratio was measured before and after 8 weeks of therapy. The urinary AFMU/1X molar ratio decreased (p < 0.05) after 8 weeks of therapy with glipizide (2.5 mg), but remained unaltered in those treated with placebo or those who self-monitored blood glucose (SMBG) by glucometer. Antipyrine and coumarin decreased (p < 0.05) the AFMU/1X molar ratio both in diabetics and healthy volunteers. Our data demonstrate that glipizide, antipyrine and coumarin may interfere with the classification of acetylator status measured by caffeine metabolites.
Acetylation↗
Individual expression of carcinogen-metabolizing enzymes: cytochrome P4502A.
Expression of enzymes metabolizing drugs, carcinogens, and other chemicals ("xenobiotics") is regulated by the interplay of genetic, host, and environmental factors, leading in human populations to a marked interindividual variability. On this basis it is speculated that individual differences in cancer susceptibility could be explained to a certain extent by interindividual differences in metabolic activation. CYP dependence of carcinogen activation is briefly reviewed; CYP2A6 as a more specific example and some consequences and corollaries are briefly discussed. At present, no consensus has been reached about the significance of interindividual differences (genetic or nongenetic) in carcinogen metabolism in cancer etiology. The likely reason is that chemically induced cancer is still a multifactorial, multistage disease involving numerous events before a clinically manifested disease develops. Molecular biological methods such as RFLP and PCR-based techniques as well as molecular dosimetry are making it possible to investigate the genetic background of individual subjects and environmental influences without biases caused by diseases, age, treatment, and other factors, which have plagued studies thus far.
Animals↗
CYP3A4 and CYP2A6 activities marked by the metabolism of lignocaine and coumarin in patients with liver and kidney diseases and epileptic patients.
1. The in vitro hepatic metabolism of lignocaine to monoethylglycinexylide (MEGX) is mediated by CYP3A4 and that of coumarin to 7-hydroxycoumarin (7OHC) by CYP2A6. We investigated the usefulness of monitoring serum MEGX concentrations (after 1 mg kg-1 lignocaine i.v.) and urinary 7OHC excretion (after 5 mg coumarin p.o.) to reflect liver function in patients with liver (n = 36), kidney (n = 12) and epileptic (n = 12) disease and in control subjects (n = 20). The extent of liver disease was assessed using measurements of serum aminoterminal propeptide (PIIINP) and Child-Pugh grades. 2. Serum concentrations of MEGX were decreased in severe (4.6 +/- 3.0 s.d. ng ml-1), moderate (19.1 +/- 11.6 s.d. ng ml-1) and mild (32.8 +/- 14.2 s.d ng ml-1) liver disease as compared with controls (53.4 +/- 15.8 s.d ng ml-1). The excretion of 7OHC over 2 h was decreased in severe (18.0 +/- 10.3 s.d % of dose) and moderate (34.2 +/- 15.6 s.d %), but not in mild (49.7 +/- 19.0 s.d %) liver disease as compared with that in controls (56.2 +/- 11.6%). 3. In epileptic patients the urinary recovery of 7OHC was increased (2 h value 69.5 +/- 13.2 s.d %) suggesting enzyme induction. In contrast, serum MEGX concentration were low (40.0 +/- 14.1 s.d ng ml-1), possibly due to competition for CYP3A4 between lignocaine and antiepileptic drugs. 4. In patients with kidney disease serum MEGX concentration (56.5 +/- 26.1 s.d ng ml-1) was similar to that in controls.(ABSTRACT TRUNCATED AT 250 WORDS)
Administration, Oral↗
Selective serotonin reuptake inhibitors and theophylline metabolism in human liver microsomes: potent inhibition by fluvoxamine.
1. Fluvoxamine and seven other selective serotonin reuptake inhibitors (SRRI) were tested for their ability to inhibit a number of human cytochrome P450 isoforms (CYPs). 2. None of the drugs showed potent inhibition of CYP2A6 (coumarin 7-hydroxylase) or CYP2E1 (chlorzoxazone 6-hydroxylase), while norfluoxetine was the only potent inhibitor of CYP3A having IC50 values of 11 microM and 19 microM for testosterone 6 beta-hydroxylase and cortisol 6 beta-hydroxylase, respectively. 3. Norfluoxetine, sertraline and fluvoxamine inhibited CYP1A1 (7-ethoxyresorufin O-deethylase) in microsomes from human placenta (IC50 values 29 microM, 35 microM and 80 microM, respectively). Fluvoxamine was a potent inhibitor of CYP1A2-mediated 7-ethoxyresorufin O-deethylase activity (IC50 = 0.3 microM) in human liver. 4. In microsomes from three human livers fluvoxamine potently inhibited all pathways of theophylline biotransformation, the apparent inhibitor constant, Ki, was 0.07-0.13 microM, 0.05-0.10 microM and 0.16-0.29 microM for inhibition of 1-methylxanthine, 3-methylxanthine and 1,3-dimethyluric acid formation, respectively. Seven other SSRIs showed either weak or no inhibition of theophylline metabolism. 5. Ethanol inhibited the formation of 1,3-dimethyluric acid with K(i) value of 300 microM, a value which is consistent with inhibition of CYP2E1. Ethanol and fluvoxamine both inhibited 8-hydroxylation by about 45% and, in combination, the compounds decreased the formation of 1,3-dimethyluric acid by 90%, indicating that CYP1A2 and CYP2E1 are equally important isoforms for the 8-hydroxylation of theophylline. 6. It is concluded that pharmacokinetic interaction between fluvoxamine and theophylline is due to potent inhibition of CYP1A2.
Antidepressive Agents↗
Carbamazepine and its metabolites in human perfused placenta and in maternal and cord blood.
Placental transfer and metabolism of carbamazepine (CBZ) was studied in a dual recirculating placental cotyledon perfusion system and was also evaluated in 16 pairs of maternal venous and cord blood samples. Among the parameters studied as possible indicators of a successful perfusion, volume changes in perfusate divided the perfusions into two groups, whereas no significant differences between perfusions were noted in blood gas analysis or in antipyrine transfer. CBZ added into the maternal circulation crosses the placenta in the beginning quicker than antipyrine which is in agreement with the different lipid solubilities of these compounds. Because the transfer rates of antipyrine and CBZ were about the same, the mechanism of transfer of CBZ is probably similar to that of antipyrine (passive diffusion). No metabolites of CBZ could be detected in the perfusate by high-performance liquid chromatography (HPLC) or gas chromatography/mass spectrometry. With the improved HPLC methodology for CBZ metabolites, six metabolites were detected in clinical samples, including 10-hydroxy-10,11-dihydro-CBZ (10-OH-CBZ), which has been described earlier in only 1 uremic patient. Relative levels of metabolites showed significant individual differences. CBZ crosses perfused placenta rapidly, but this does not contribute to CBZ metabolites detected in maternal and fetal circulation.
Antipyrine↗
Thyroid and myocardial function after replacement of carbamazepine by oxcarbazepine.
We determined changes in serum concentrations of thyroid hormones during carbamazepine (CBZ) therapy during a 5-year prospective follow-up study of 20 patients with newly diagnosed epilepsy. In addition, we evaluated the effects of replacing CBZ with oxcarbazepine (OCBZ) in 12 male patients with epilepsy in a 6-month prospective follow-up study. Circulating thyroxine and free thyroxine levels decreased after 2-month CBZ treatment and remained at a low level during the 5-year follow-up. There were no associated changes in serum thyrotropin (TSH) concentrations. When CBZ was replaced by OCBZ, the function of the liver's P450 enzyme system normalized, as shown by an increase in antipyrineT1/2, and a decrease in antipyrineCL. Serum total and free thyroxine levels increased, and thereafter serum TSH levels decreased. Indexes of diastolic heart function improved concomitantly, which may reflect subclinical hypothyroidism at the cellular level during CBZ treatment. We conclude that normal thyroid function can be restored in patients with epilepsy by replacing CBZ with OCBZ.
Adolescent↗
Transfer of lidocaine and bupivacaine across the isolated perfused human placenta.
Drug permeability and pharmacokinetics through the placenta are important factors determining foetal drug exposure. The purpose of the present study was to establish a perfused human placental cotyledon system to assess the placental transfer of lidocaine and bupivacaine, widely used local anaesthetics in obstetric anaesthesia. Term placentas were obtained immediately after delivery with maternal consent and a two-hour recycling perfusion of a single placental cotyledon was performed. Bupivacaine or lidocaine with antipyrine as a reference compound were added to the maternal reservoir and their disappearance from the maternal circulation and appearance to the foetal circulation were followed in five experiments for each drug. Drug concentrations were measured by gas chromatography. Bupivacaine disappeared more rapidly from the maternal circulation than lidocaine. At 2 hr, bupivacaine foetal:maternal concentration ratio was 0.56 +/- 0.12 and 14.6% +/- 2.99 of the total circulating amount was found in the foetal circulation. Lidocaine concentration increased more in the foetal circulation and the foetal:maternal concentration ratio at 2 hr was 0.90 +/- 0.09 (P < 0.01), and 22.1% +/- 2.21 (P < 0.01) was found in the foetal circulation. The maternal to foetal transfer of bupivacaine and lidocaine were 67.2% +/- 0.153 and 98.9% +/- 0.07 (P < 0.05) of that of freely diffusable antipyrine, respectively. Both amide local anaesthetics crossed the dually perfused human placenta rapidly. Bupivacaine disappeared faster than lidocaine from the maternal circulation but less was transferred to foetal circulation. This difference is probably explained by the greater lipophilicity of bupivacaine and hence higher placental binding. These results suggest less foetal drug exposure with bupivacaine than lidocaine.
Anesthetics, Local↗
A genetic polymorphism in coumarin 7-hydroxylation: sequence of the human CYP2A genes and identification of variant CYP2A6 alleles.
A group of human cytochrome P450 genes encompassing the CYP2A, CYP2B, and CYP2F subfamilies were cloned and assembled into a 350-kb contig localized on the long arm of chromosome 19. Three complete CYP2A genes--CYP2A6, CYP2A7, and CYP2A13--plus two pseudogenes truncated after exon 5, were identified and sequenced. A variant CYP2A6 allele that differed from the corresponding CYP2A6 and CYP2A7 cDNAs previously sequenced was found and was designated CYP2A6v2. Sequence differences in the CYP2A6v2 gene are restricted to regions encompassing exons 3, 6, and 8, which bear sequence relatedness with the corresponding exons of the CYP2A7 gene, located downstream and centromeric of CYP2A6v2, suggesting recent gene-conversion events. The sequencing of all the CYP2A genes allowed the design of a PCR diagnostic test for the normal CYP2A6 allele, the CYP2A6v2 allele, and a variant--designated CYP2A6v1--that encodes an enzyme with a single inactivating amino acid change. These variant alleles were found in individuals who were deficient in their ability to metabolize the CYP2A6 probe drug coumarin. The allelic frequencies of CYP2A6v1 and CYP2A6v2 differed significantly between Caucasian, Asian, and African-American populations. These studies establish the existence of a new cytochrome P450 genetic polymorphism.
Alleles↗
Cytochrome P4502A5 expression and inducibility by phenobarbital is modulated by cAMP in mouse primary hepatocytes.
Factors involved in CYP2A5 expression were studied in mouse liver primary hepatocytes in culture. CYP2A5-mediated coumarin 7-hydroxylase (COH) activity was retained in simple culture conditions for at least 96 hours and the activity was inducible up to 33-fold by phenobarbital (PB). The constitutive activity and inducibility of COH was totally blocked by treatment of hepatocytes with actinomycin D, and short initial treatment with cycloheximide caused superinducibility when co-administered with PB. Treatment of hepatocytes with inhibitors of protein kinase C, tyrosine kinases and a generator of nitric oxide did not affect COH basal activity or inducibility. Administration of dibutyryl cAMP, forskolin, and 3-isobutyl-1-methylxanthine (IBMX) enhanced both basal and PB-induced COH activities and CYP2A5 mRNA levels, indicating that cAMP plays a major role in CYP2A5 expression.
1-Methyl-3-isobutylxanthine↗
Modification of hepatic cytochrome P450 profile by cocaine-induced hepatotoxicity in DBA/2 mouse.
Previous studies in our laboratory have shown that a hepatotoxic dose of cocaine increases coumarin 7-hydroxylase activity in male DBA/2 mouse liver. In the present study, the dose- and time-dependent responses of the hepatic CYP2A4/5 complex to cocaine-induced liver damage were studied. Cocaine increased CYP2A4/5 levels in a dose-dependent manner. The maximal increases in coumarin 7-hydroxylase activity (4-fold), microsomal CYP2A4/5 content (3-fold) and steady-state mRNA levels (10-fold) were observed at 24 h after administration of a single dose of 60 mg/kg cocaine coinciding with morphologically detectable diffuse liver damage, while the total P450 content was not changed. 3 and 5 days after the daily administration of cocaine severe, mainly pericentral (zone III of Rappaport), liver damage was apparent in parallel with a clear decline in CYP2A4/5 mRNA, protein content and coumarin 7-hydroxylase activity. After 5 days of treatment, CYP2A5 still remained at a very low level but an induction in CYP2B10 protein and related pentoxyresorufin O-dealkylase activity was observed. No marked changes in microsomal CYP2Cx and CYP1A1/2 contents or associated activities were observed. Dimethylnitrosamine N-demethylase activity, a marker for CYP2E1, decreased in parallel with increased cocaine dose and time and the severity of liver damage.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Metabolic interactions of methoxsalen and coumarin in humans and mice.
Methoxsalen (8-methoxypsoralen) is a very potent inhibitor of human cytochrome P450 2A6 (CYP2A6) and mouse Cyp2a-5-mediated coumarin 7-hydroxylation in vitro. To determine the effect of methoxsalen on coumarin 7-hydroxylation in humans in vivo, five subjects were given 45 mg of methoxsalen and 5 mg of coumarin. Methoxsalen inhibited in vivo coumarin metabolism by 47 +/- 9.2% (mean +/- SEM). Methoxsalen was metabolized in human liver microsomes at the rate of 50-100 pmol/mg protein/min (approx. 30% of the activity in mouse liver microsomes). Metabolism was not inhibited by the anti-Cyp2a-5 antibody in human liver microsomes. NIH 3T3 cells stably expressing catalytically active CYP2A6 enzyme did not metabolize methoxsalen, indicating that CYP2A6 does not accept methoxsalen as a substrate. In pyrazole-induced mouse liver microsomes, methoxsalen metabolism was inhibited by the anti-Cyp2a-5 antibody. Cyp2a-5 protein expressed in the yeast Saccharomyces cerevisiae was capable of metabolizing methoxsalen, indicating that methoxsalen is a substrate of Cyp2a-5. Although kinetic studies indicated that the inhibition of coumarin 7-hydroxylation by methoxsalen is competitive in human liver microsomes, methoxsalen does not appear to be a substrate for CYP2A6. Methoxsalen and coumarin have the potential of strong metabolic interactions in man.
Animals↗
Expression of xenobiotic-metabolizing cytochrome P450 forms in human adult and fetal liver.
Expression of human cytochrome P450 (CYP) genes in human adult and fetal liver were studied using the reverse transcriptase-polymerase chain reaction (RT-PCR) method. In adult liver mRNA of CYPs 1A1, 1A2, 2A6/2A7, 2B6/2B7, 2C8-19, 2D6, 2E1, 3A3/3A4 and 3A7 were detected while CYPs 2F1 and 4B1 were absent. In fetal liver mRNA of CYPs 2C8, 2D6, 3A3/3A4 and 3A7 were found but all other forms studied were undetectable. The results provide a comprehensive qualitative picture of the expression of CYP genes in families CYP1 through CYP4 in human adult and fetal liver.
Adult↗
Cocaine N-demethylation and the metabolism-related hepatotoxicity can be prevented by cytochrome P450 3A inhibitors.
Cocaine is eliminated and detoxified principally through the metabolism of nonspecific plasma and tissue esterases. Microsomal oxidative metabolism is of importance in cocaine N-demethylation, this being a principal pathway of cocaine bioactivation and hepatotoxicity. The contribution of different cytochrome P450 (CYP) enzymes to cocaine N-demethylase activity was studied in vitro with DBA/2 mouse and human liver microsomes, and cocaine hepatotoxicity was examined in vivo in DBA/2 male mice. Species dependent enzyme kinetics was observed. Cocaine N-demethylase displayed two Km values in murine liver (40-60 microM and 2-3 mM), whereas only one Km value was observed in human liver microsomes (2.3-2.7 mM). We suggest that CYP3A plays a prominent role in the N-demethylation of cocaine for the following reasons: (i) pregnenolone-16 alpha-carbonitrile, an inducer of CYP3As increases cocaine N-demethylase in parallel with testosterone 6 beta-hydroxylase activity and immunoreactive 3A protein in mouse liver; (ii) human and mouse cocaine N-demethylase and testosterone 6 beta-hydroxylase activities can be inhibited by triacetyloleandomycin, cannabidiol, or gestodene, all selective inhibitors of CYP3A P450s; (iii) antibodies directed against P450s within subfamilies 1A, 2A, 2B, 2C, or 2E inhibited cocaine N-demethylase activity only marginally, and finally, (iv) treatment of mice with triacetyloleandomycin or cannabidiol in vivo significantly attenuated the cocaine-elicited hepatotoxicity as assessed by the serum alanine aminotransferase activity and liver histology in parallel with decreased cocaine N-demethylase activity.(ABSTRACT TRUNCATED AT 250 WORDS)
Adolescent↗