Phenotypic spectrum of hereditary neuralgic amyotrophy caused by the SEPT9 R88W mutation.

BACKGROUND: Hereditary neuralgic amyotrophy (HNA), also known as hereditary brachial plexus neuropathy, has phenotypic and genetic heterogeneity. Mutations in the septin 9 (SEPT9) gene were recently identified in some HNA patients. The phenotypic spectrum of HNA caused by SEPT9 mutations is not well known. OBJECTIVE: To characterise the phenotype of a large family of HNA patients with the SEPT9 R88W mutation. METHODS: We report clinical, electrophysiological, neuroimaging and genetic findings of six HNA patients from a Japanese family. RESULTS: All 17 neuropathic episodes identified were selectively and asymmetrically distributed in the upper-limb nerves. Severe pain was an initial symptom in 16 episodes (94%). Motor weakness occurred in 15 (88%) and sensory signs in 10 (59%). A minor dysmorphism, hypotelorism, was seen in all. Nerve conduction studies revealed focal demyelination as well as prominent axonal degeneration changes. Needle electromyography revealed chronic neurogenic patterns only in the upper limbs. An MRI study showed a gadolinium-enhanced brachial plexus. The missense mutation c.262C>T; p.R88W was found in exon 2 of SEPT9 in all patients. CONCLUSIONS: The SEPT9 R88W mutation in this family causes selective involvement of the brachial plexus and upper-limb nerves. Wider and more universal recognition of clinical hallmarks and genetic counselling are of diagnostic importance for HNA caused by the SEPT9 mutation.

Reduced angiotensin II levels in the cerebrospinal fluid of patients with amyotrophic lateral sclerosis.

BACKGROUND: Recent studies suggest that angiotensin II, a major substrate in the renin-angiotensin system, protects neurons through stimulation of its type 2 receptors. However, quite a few clinical studies of angiotensin II levels have shown their relation to disease severity in neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS). AIMS OF THE STUDY: To clarify the significance of angiotensin II in ALS. METHODS: We assayed angiotensin II concentrations in cerebrospinal fluid (CSF) samples from 23 patients with ALS, nine patients with spinocerebellar degeneration (SCD) and 24 control individuals. We evaluated the disability levels of patients with ALS using the Revised ALS Functional Rating Scale (ALSFRS-R) and calculated the disease progression rate (DPR). RESULTS: CSF angiotensin II levels were significantly lower in the ALS group compared with that in the control group (P = 0.00864), and showed a significant positive correlation with scores on the ALSFRS-R, and a significant negative correlation with the DPR. CONCLUSIONS: In the present study, we reveal for the first time that angiotensin II levels in the CSF from patients with ALS are significantly reduced and significantly associated with disease severity and progression rate. These findings suggest that reduced levels of intrathecal angiotensin II may play a role in ALS.

The effect of CYP2C19 genotypes on the pharmacokinetics of warfarin enantiomers.

The aim of this study was to elucidate the pharmacokinetics and pharmacodynamics of warfarin enantiomers in relation to cytochrome P450 2C19 (CYP2C19) genotypes. Fourteen subjects, of whom seven were homozygous extensive metabolizers (hmEMs) and seven were poor metabolizers (PMs) for CYP2C19, were enrolled. After a single oral 10 mg dose of racemic warfarin, the plasma concentrations of the warfarin enantiomers and prothrombin time expressed as international normalized ratio (PT-INR) were measured over the course of 120 h. The mean plasma concentrations and elimination half-life of (R)-warfarin of all the subjects were about 2-fold greater than those of (S)-warfarin. Additionally, the area under the plasma concentration-time curve from zero to infinity (AUC(0-infinity)) and the elimination half-life of (R)-warfarin in PMs were significantly greater than those in hmEMs (P = 0.0005 and P = 0.0101 respectively). The S/R ratios of AUC of warfarin enantiomers were 0.51 in hmEMs and 0.37 in PMs (P = 0.0052). Whereas no difference was found in all pharmacokinetic parameters of (S)-warfarin in hmEMs compared with PMs. No significant difference in PT-INR, used as a measure of anticoagulant effect, was found between the hmEMs and PMs. These results show that CYP2C19 activity is important in the pharmacokinetics of (R)-warfarin. However, when warfarin is administered as a racemate, this difference is not translated into any significant effect in the pharmacodynamics of warfarin.

Metabolism of 5-methylchrysene and 6-methylchrysene by human hepatic and pulmonary cytochrome P450 enzymes.

The metabolism of environmentally occurring methylated polynuclear aromatic hydrocarbons by human cytochrome P450 (P450) enzymes has not been examined previously. We compared the metabolism of the tobacco smoke constituents 5-methylchrysene (5-MeC), a strong carcinogen, and 6-MeC, a weak carcinogen, in 18 hepatic and 11 pulmonary human microsomes. Major metabolites of 5-MeC were its proximate carcinogen trans-1,2-dihydroxy-1,2-dihydro-5-methylchrysene (5-MeC-1,2-diol), as well as 5-MeC-7,8-diol, bay region dihydrodiols, and phenols. 5-MeC-1,2-diol and 5-MeC-7,8-diol were formed stereoselectively, with the R,R enantiomers predominating. Major metabolites of 6-MeC were 6-MeC-1,2-diol, bay region dihydrodiols, phenols, and 6-(hydroxymethyl)chrysene. 6-MeC-1,2-diol was also formed stereoselectively in the 1R,2R configuration. All human liver samples formed the proximate carcinogenic 1,2-diols (0.2-2.3 pmol/mg protein/min for 5-MeC and 0.3-3.1 pmol/mg protein/min for 6-MeC). Comparable results were obtained in pulmonary microsomes, but the extent of metabolism was less than in the hepatic samples, and only 4 of 11 samples showed activity. Catalytic activities known to be associated with specific P450s were analyzed in each hepatic sample and correlated with levels of 5-MeC and 6-MeC metabolites in the same samples. The results of the correlation analysis indicated that P450s 1A1 and 1A2 were active in the formation of 5-MeC-1,2-diol and 6-MeC-1,2-diol, as well as several other metabolites resulting from ring oxidation. The formation of the hydroxymethyl metabolites was catalyzed by P450 3A4 (for 5-MeC) or P450s 3A4 and 1A2 (for 6-MeC). Experiments with chemical inhibitors and antibodies supported these results. The metabolism of 5-MeC and 6-MeC was also investigated using purified recombinant human P450s 1A1, 1A2, 2C10, 2D6, 2E1, 3A4, and 3A5. P450s 1A1, 1A2, and 2C10 had higher activities than the other enzymes for ring oxidation of 5-MeC and 6-MeC, whereas P450s 1A2 and 3A4 were more active than the other enzymes for methyl hydroxylation of 6-MeC. Only P450 3A4 showed substantial catalytic activity for methyl hydroxylation of 5-MeC. Collectively, the results of these studies demonstrate that P450s 1A2 and 2C10 are important catalysts of the metabolic activation of 5-MeC and 6-MeC in human liver, whereas P450 1A1 plays a major role in the metabolic activation of these compounds in human lung.

[Rapidly enlarging giant left ventricular pseudo-false aneurysm after myocardial infarction; report of a case].

A 71-year-old man was admitted to our hospital with acute myocardial infarction and cardiac tamponade. After pericardial drainage, his hemodynamics was improved. Because more than 3 days had been passed after the onset of myocardial infarction and he had severe renal dysfunction, emergent coronary angiography (CAG) was not performed. After improvement of his general status, coronary angiography and percutaneous catheter intervention was carried out, and his course was uneventful. But transthoracic echocardiography before discharge revealed a giant posterior psudoaneurysm. Patch closure and coronary artery bypass grafting was carried out under cardiopulmonary bypass, and postoperative course was uneventful. Postoperative left ventriculogram revealed disappearance of pseudoaneurysm, but relatively large akinetic area of posterior-inferior wall was left around a patch. Pseudo-false aneurysm was diagnosed by histological examination.

Mechanical strain effect on bone-resorbing activity and messenger RNA expressions of marker enzymes in isolated osteoclast culture.

Adaptive modeling and remodeling are controlled by the activities of osteoblasts and osteoclasts, which are capable of sensing their mechanical environments and regulating deposition or resorption of bone matrix. The effects of mechanical stimuli on isolated osteoclasts have been scarcely examined because it has proven to be difficult to prepare a number of pure osteoclasts and to cultivate them on mineralized substratum during mechanical stimulation. Recently, we developed an apparatus for applying mechanical stretching to the ivory slice/plastic plate component on which cells could be cultured. The loading frequency, strain rate, and generated strain over an ivory surface could be controlled by a personal computer. Using this apparatus, we examined the role of mechanical stretching on the bone-resorbing activity of the osteoclasts. Mature and highly enriched osteoclasts were cultured for 2, 12, and 24 h on the ivory/plate component while being subjected to intermittent tensile strain. The stretched osteoclasts showed enhanced messenger RNA (mRNA) expression levels of osteoclast marker enzymes, tartrate-resistant acid phosphatase (TRAP), and cathepsin K and increases of resorbed-pit formation, suggesting that the mechanical stretching up-regulated the bone-resorbing activity of the osteoclasts. A stretch-activated cation (SA-cat) channel blocker significantly inhibited the increases of the mRNA level and pit formation after 24 h of stretching. This study suggested the possibility that the mature osteoclasts responded to mechanical stretching through a mechanism involving a SA-cat channel in the absence of mesenchymal cells and, as a result, up-regulated their bone-resorbing activity.

Structural characterization of a new variant of the CYP2A6 gene (CYP2A6*1B) apparently diagnosed as heterozygotes of CYP2A6*1A and CYP2A6*4C.

During the course of investigating the frequency of a CYP2A6 whole deletion-type polymorphism (CYP2A6*4C) in Japanese, an unexpectedly large population of heterozygotes for CYP2A6*4C and the wild-type (CYP2A6*1A) was found. Cloning of a cDNA encoding CYP2A6 from the liver of individuals judged as heterozygotes for CYP2A6*4C and the CYP2A6*1A was carried out to identify the causal allele(s) responsible for a possible overestimation. A clone isolated from the liver cDNA library possessed 58 bp sequences in the 3'-untranslated region, which was replaced with the corresponding region of the CYP2A7 gene. The same gene conversion existed in the genomic DNA, indicating that the replacement was not a cloning artifact. Based on the gene structure of the allele (CYP2A6*1B), this variant was thought to be one of the causal alleles responsible for overestimation of heterozygotes for CYP2A6*4C and CYP2A6* A. To investigate this further, we developed a genotyping method which could distinguish the CYP2A6*A, CYP2A6*1B and CYP2A6*4C alleles from each other. The results clearly showed that CYP2A6*1B was the sole allele responsible for the overestimation. We conclude that the new genotyping method allows determination of six genotypes of the CYP2A6 gene, simultaneously and precisely, in both Oriental and Caucasian populations.

Animal model of depression. III. Mechanism of action of tetrabenazine.

A study was undertaken to gain an understanding of the biochemical mechanism whereby tetrabenazine (TBZ) produces a sedative effect on the locomotor activity of rats. Rats injected with L-5-hydroxytryptophan (L-5-HTP, 30 mg/kg), the immediate precursor of 5-hydroxytryptamine (5-HT), showed the characteristic bison appearance, pitosis, and catalepsy normally observed after injecting TBZ (30 mg/kg). The treatment of rats with low doses of L-5-HTP (9 mg/kg) plus TBZ (2 mg/kg) significantly decreased locomotor activity, whereas low doses of either one of these drugs given alone had no significant effect on locomotor activity. The level of 5-hydroxyindoleacetic acid (5-HIAA) was elevated in the brain of rats sacrificed 3 hr after treatment with low doses of either L-5-HTP or TBZ alone. Treatment of rats with p-chlorophenylalanine to inhibit the synthesis of 5-HT had an inhibitory effect on the duration of sedation following an injection of TBZ (30 mg/kg). The results of the biochemical and pharmacological studies as reflected by changes in locomotor activity were interpreted to indicate that the sedative action of TBZ was due to an excess of functional 5-HT.

The effects of age and gender on the stereoselective pharmacokinetics of verapamil.

The effects of age and gender on the pharmacokinetics of verapamil stereoisomers were examined. Eighty milligrams of racemic verapamil was given orally to 12 young and 12 elderly healthy subjects, half of whom were women. The area under the plasma concentration-time curves (AUC) of (S)- and (R)-verapamil were greater in the elderly group than in the young group [(S)-/(R)-verapamil (mean +/- SD), 214.4 +/- 123.0/1582.2 +/- 763.0 and 50.4 +/- 36.5/584.9 +/- 252.4 ng.hr/ml for the elderly and young groups, respectively (p < 0.001/p < 0.001)]. Conversely, the apparent oral clearance values of (S)- and (R)-verapamil were significantly smaller in the elderly group than in the young group [(S)-/(R)-verapamil (mean +/- SD), 4.8 +/- 3.0/0.53 +/- 0.21 and 22.5 +/- 21.3/1.30 +/- 0.67 L/hr/kg for the elderly and young groups, respectively (p < 0.01/p < 0.001)]. The ratio of apparent oral clearance of (S)- to (R)-verapamil was significantly smaller in the elderly group than in the young group. As a consequence, the negative chronotropic and dromotropic effect of verapamil was observed in the elderly group. This study suggests that the effect of age on metabolism was greater for (S)-verapamil than for (R)-verapamil.

Interethnic difference in omeprazole's inhibition of diazepam metabolism.

OBJECTIVES: To compare the effect of omeprazole, a substrate and inhibitor of CYP2C19, on diazepam metabolism in white and Chinese subjects. SUBJECTS AND METHODS: The study, which took place at a clinical research center in a University Hospital, was designed as a double blind, crossover, two-stage study; each stage lasted 21 days and was separated by 4 weeks. Subjects were eight white and seven Chinese men who were extensive metabolizers of debrisoquin and mephenytoin. The subjects received, in a randomized order, omeprazole, 40 mg/day, and placebo for 21 days, followed by a 10 mg oral dose of diazepam. Diazepam and desmethyldiazepam plasma concentrations were determined by HPLC during a 26-day period after diazepam administration. RESULTS: In white subjects omeprazole treatment decreased diazepam clearance by 38% +/- 4.4% and increased desmethyldiazepam area under the plasma concentration-time curve (AUC) by 42.4% +/- 7.0%. In contrast, diazepam oral clearance decreased by only 20.7% +/- 7.3% and desmethyldiazepam AUC decreased by 25.4% +/- 4.6% in the Chinese group. The decrease in diazepam clearance and the prolongation in diazepam and desmethyldiazepam elimination half-lives after administration of omeprazole were significantly greater in the white group than in the Chinese group (p < 0.03, p < 0.001, and p < 0.004, respectively). In the absence of omeprazole, diazepam oral clearance was marginally greater (mean +/- SEM) (34.4 +/- 2.8 ml/min versus 25.2 +/- 3.5 ml/min, p = 0.057, respectively) and the AUC of desmethyldiazepam was significantly lower (8794 +/- 538 micrograms/L.hr versus 16,358 +/- 2985 mg/L.hr, p = 0.04, respectively) in the white subjects compared with the Chinese subjects. CONCLUSION: The extent of the inhibitory effect of omeprazole on diazepam metabolism is dependent on ethnicity. Further studies are needed to determine the mechanism responsible for this phenomenon.

Analysis of the CYP2D6 gene in relation to dextromethorphan O-demethylation capacity in a Japanese population.

OBJECTIVE: To analyze the CYP2D6 allele frequencies in a Japanese population and to evaluate the effects of CYP2D6 variants on in vivo CYP2D6 activity as measured by the dextromethorphan metabolic ratio (MR). METHODS: Ninety-eight unrelated, healthy Japanese men were phenotyped with dextromethorphan and genotyped by the polymerase chain reaction amplification method for 7 CYP2D6 alleles. RESULTS: The CYP2D6*1, CYP2D6*10, CYP2D6*2, CYP2D6*5, CYP2D6*4, and CYP2D6*21 allele frequencies in our Japanese subjects were 0.423, 0.408, 0.092, 0.061, 0.020, and 0.010, respectively. Thirty-three subjects (33.7%) were heterozygous for *10/*1, and 18 (18.4%) and 17 (17.3%) subjects were homozygous for *1 and *10, respectively. Subjects who were homozygous for *10 showed the highest dextromethorphan MR among these 3 genotypes. Eighteen subjects (18.3%) were heterozygous for *2, but their dextromethorphan MR values were not greater than the MR values of subjects who were homozygous for *1. One subject was a poor metabolizer phenotypically, and he was homozygous for *5. CONCLUSIONS: The CYP2D6 allele frequencies in our Japanese subjects differed from those determined in previous studies of white subjects or mainland Chinese subjects. Individuals homozygous for *10 who have relatively low in vivo CYP2D6 activity represent almost 20% of the Japanese population. In addition, we did not identify any subjects with amplified *2 among our 98 Japanese men.

CYP3A activity in European American and Japanese men using midazolam as an in vivo probe.

OBJECTIVE: To investigate putative differences in CYP3A activity between European American and Japanese subjects using midazolam as an in vivo probe. METHODS: Midazolam was administered orally (2 mg) to 22 young healthy Japanese men and, on a separate occasion, to 19 of these by the intravenous route (1 mg). The disposition of the drug and its 1'-hydroxy metabolite were determined and compared with data collected in a similar fashion in 20 young healthy European American men. RESULTS: Plasma concentrations of midazolam, especially those attained soon after drug administration, were higher after intravenous injection in Japanese subjects than those in European American men. This observation was associated with smaller initial (2.5-fold) and steady-state (1.8-fold) volumes of distribution for the drug; normalization for body weight only modestly reduced these differences. The systemic clearance value of midazolam was 25% lower (P < .03) in Japanese subjects, but this difference was not apparent after accounting for the smaller body weights of that group. No statistical differences were noted in the elimination half-life (t 1/2) of midazolam between European American and Japanese subjects. Much greater interindividual variability was observed after oral administration compared with intravenous administration, but significant differences were not found between the 2 groups with respect to the maximum midazolam plasma level or its oral clearance. Absolute oral bioavailability and its associated gastrointestinal and hepatic extraction ratios also showed no statistically significant interracial differences. CONCLUSIONS: On average, hepatic CYP3A, as measured by the metabolism of midazolam, is lower in young healthy Japanese men compared with similar European Americans. However, there is considerable interindividual variability, and body size appears to be an important determinant. After oral administration, even greater variability in the plasma level-time profile of midazolam is present, and no statistically significant or clinically important interracial/ethnic difference is present. Possibly because of smaller body mass and differences in body composition, midazolam has a smaller distribution volume(s) in Japanese men than in European American men that might be an important factor when drugs are administered intravenously.

Osteopontin involvement in integrin-mediated cell signaling and regulation of expression of alkaline phosphatase during early differentiation of UMR cells.

To clarify the function of osteopontin in osteoblast differentiation, we have examined the signal transduction pathway in an osteoblastic cell line (UMR106-6) bound to osteopontin, fibronectin, vitronectin and collagen type I surfaces. This was done by investigating the production and autophosphorylation of focal adhesion kinase (FAK) and the expression of alkaline phosphatase (ALP) at the transcription level. Results suggest that osteopontin was not only responsible for the autophosphorylation of FAK but regulated the expression of ALP, which was strongly correlated with FAK activity. These results suggest that osteopontin might act as a trigger in the early differentiation of osteoblasts.

Tyrosine hydroxylase antisense gene therapy causes hypotensive effects in the spontaneously hypertensive rats.

We investigated the effect of antisense oligodeoxynucleotides (AS ODN) against tyrosine hydroxylase (TH) on hypertension and sympathetic nervous system activity in spontaneously hypertensive rats (SHR). Systolic blood pressure (SBP) in SHR treated with TH AS ODN (50, 200 microg/rat, i.v.) was significantly lower than that of control SHR. Epinephrine and norepinephrine levels, TH activity, and TH protein levels in the adrenal medulla of SHR were reduced concomitant with TH AS ODN treatment-induced changes in SBP. In contrast, TH AS ODN (200 microg/rat) had no effect on SBP in Wistar-Kyoto rats (WKY), despite significantly decreased catecholamine levels, TH activity, and TH protein levels. These findings suggest that peripheral systemic injection of TH AS ODN may be effective as hypotensive therapy in SHR.

The activity of dihydropyrimidine dehydrogenase from rat liver was not affected by any of five 5-hydroxytryptamine antagonists.

We estimate the influence of five 5-hydroxytryptamine receptor (5-HT3) antagonists on the activity of dihydropyrimidine dehydrogenase (DPDase), the rate-limiting enzyme in 5-fluorouracil (5FU) metabolism. The activity of DPDase from the rat liver was compared in the cytosol mixture of 5FU incubated with or without each of five 5-HT3 antagonists. DPDase activity was not altered in the presence of any 5-HT3 antagonist studied here. It may be inferred from these results that the any 5-HT3 receptor antagonist examined in this study has little or no effect on fluorouracil catabolism.

Multiple cytochrome P-450 subfamilies are co-induced with P-glycoprotein by both phenothiazine and 2-acetylaminofluorene in rats.

We studied the effects of two P-glycoprotein (P-gp) inducers, 2-acetylaminofluorene (2-AAF) and phenothiazine (PTZ), administered intraperitoneally, on the activities and content of hepatic cytochrome P-450 (CYP) subfamilies in hepatic microsomes of Sprague-Dawley rats. After 4-day administration of 2-AAF or PTZ, the P-gp content was increased. The total CYP content after PTZ treatment was significantly increased compared with that of controls. The CYP1A, CYP2B and CYP3A2 contents were induced, while the CYP2C6, CYP2C11 and CYP2E1 contents remained unaffected. A marked increase in CYP1A1 was found after administration of each compound. Ethoxyresorufin O-deethylase, pentoxyresorufin O-deethylase, and testosterone 6beta hydroxylation activities showed a significant increase after both 2-AAF and PTZ treatments. In particular, ethoxyresorufin O-deethylase exhibited more than ten times greater activity than that of the controls after the treatments. These results suggest that P-gp inducers affect several CYP subfamilies in addition to CYP3A, which is reported to be up-regulated coordinately with P-gp by a CYP3A inducer.

Serum lipid peroxide assayed by a new colorimetric method.

Serum lipid peroxide (LPO) in 72 healthy male subjects was studied using a newly developed assay method based on the reaction of LPO with methylene blue derivative (MCDP; 10-N-Methylcarbamoyl-3,7-dimethylamino-10 H-phenothiazine). This method is specific for lipid hydroperoxide and the relation is equimolar. The value obtained was 7.63 +/- 2.78 nmol/ml. Positive correlations were observed between LPO and height (r = 0.290), body weight (r = 0.244) and hemoglobin (r = 0.248). However, no significant correlation was observed between serum LPO level and other clinical data, including age. In addition, serum LPO value measured by this method did not show significant correlation with that measured by the thiobarbituric acid method in rat serum. This discrepancy might be due to the differences in substrates measured by each method.

Biotransformation of tritiated fentanyl in human liver microsomes. Monitoring metabolism using phenylacetic acid and 2-phenylethanol.

Norfentanyl has been identified previously as a urinary metabolite of fentanyl. However, at clinically relevant concentrations, norfentanyl are below the limits of detection. The use of labeled drug in metabolic studies is a standard approach to overcome the limitations imposed by metabolite concentrations that are below detection limits. Unfortunately, the available tritium-labeled fentanyl yields unlabeled norfentanyl following N-dealkylation. Thus, we have developed a technique to monitor the N-dealkylation of fentanyl using the other products of N-dealkylation. The biotransformation of fentanyl was studied in human liver microsomes. After incubation with human liver microsomes for 20 min, almost 50% of a 0.03 microM concentration of [3H]-fentanyl was metabolized to the [3H]N-dealkylated metabolite phenylacetaldehyde, which was then converted principally to [3H]2-phenylethanol and to a smaller extent to [3H]phenylacetic acid in microsomal incubates. The apparent Km,app and Vmax,app for norfentanyl formation were 82 +/- 21 microM and 4.7 +/- 0.4 nmol product formed/min/nmol cytochrome P450, respectively. Thus, this study defined methodology that can be used to evaluate the metabolism of fentanyl, both in vivo and in vitro, at clinically relevant concentrations.

Colchicine biotransformation by human liver microsomes. Identification of CYP3A4 as the major isoform responsible for colchicine demethylation.

Colchicine disposition involves both active biliary and renal excretion of parent drug, and at least in mammals a substantial fraction undergoes hepatic demethylation prior to excretion. We investigated the biotransformation of [3H]colchicine in a panel of microsomal preparations obtained from sixteen human liver samples. The production rate of the main metabolites of colchicine's 3-demethylcolchicine (3DMC) and 2-demethylcolchicine (2DMC), was linear in relation to incubation time, cytochrome (P450) content, and substrate concentration. Following the incubation of colchicine (5 nM) with microsomes in the presence of an NADPH-generating system for 60 min, 9.8% and 5.5% of the substrate were metabolized to 3DMC and 2DMC, respectively. The formation rate of colchicine metabolites exhibited a marked variation between the different microsomal preparations. The formation rates of both colchicine metabolites were correlated significantly with nifedipine oxidase activity, a marker of CYP3A4 activity (r = 0.96, P < 0.001), but not with the metabolic markers of CYP2A6, CYP2C19, CYP2C9, CYP2D6, and CYP2E1 activities. Chemical inhibition of CYP3A4 by preincubation with gestodene (40 microM) or troleandomycin (40 microM) reduced the formation of 3DMC and 2DMC by 70 and 80%, respectively, whereas quinidine, diethyldithiocarbamate, and sulfaphenazole had no inhibitory effect. Similarly, antibodies raised against CYP3A4 almost completely abolished colchicine demethylation and nifedipine oxidase activity, but preimmune IgG had no effect. In conclusion, colchicine was metabolized to 3DMC and 2DMC by human liver microsomes. The production of colchicine metabolites was mediated by CYP3A4, and its rate varied greatly between microsomal preparations obtained from different liver samples. The coadministration of colchicine with known inhibitors or substrates of CYP3A4 may inhibit colchicine metabolism, resulting in concentration-related toxicity.

No ethnic difference between Caucasian and Japanese hepatic samples in the expression frequency of CYP3A5 and CYP3A7 proteins.

Ethnic differences in the pharmacokinetics of nifedipine, a substrate of CYP3A, and in CYP3A7 expression have been reported. The aim of the present study was to measure the protein levels of CYP3A4, CYP3A5, and CYP3A7 and nifedipine oxidation activity in hepatic microsomes from 15 Caucasian and 15 Japanese patients for comparison between the two ethnic groups. Nifedipine oxidation activity and CYP3A4 protein level were well correlated. No significant difference between Caucasian and Japanese microsomal samples was found in nifedipine oxidation activity or in the CYP3A4 protein level. CYP3A5 was detected in 6 of 15 Caucasian samples and in 5 of 15 Japanese samples, but no ethnic difference was found in either the frequency of expression or its protein level. CYP3A7 was found in 10 of 15 Caucasian samples and in 14 of 15 Japanese samples. Although the estimated CYP3A7 protein level was higher in the Japanese than in the Caucasian samples, its protein level was much lower than that of CYP3A4. These results imply that the contribution of CYP3A5 or CYP3A7 to the purported Caucasian-Japanese ethnic difference in the overall CYP3A activity seems to be small.