Differential impacts of CYP2C19 gene polymorphisms on the antiplatelet effects of clopidogrel and ticlopidine.

We examined the influence of CYP2C19 polymorphisms on the antiplatelet effects of clopidogrel and ticlopidine. The platelet aggregation induced by 20 µmol/l adenosine diphosphate (ADP) and CYP2C19 single-nucleotide polymorphisms (*2 and *3) was determined in patients with coronary artery disease (CAD) who were taking aspirin alone (n = 21), aspirin plus clopidogrel (n = 97), or aspirin plus ticlopidine (n = 47). The degree of platelet aggregation in the clopidogrel group, although not in the ticlopidine group, depended on the CYP2C19 polymorphism, and the maximal platelet aggregation in poor metabolizers (PMs) taking clopidogrel was equivalent to that in the group taking aspirin alone. After being switched from clopidogrel to ticlopidine, all seven of the PMs showed markedly lower platelet aggregation. These results suggest that CYP2C19 polymorphisms have a profound impact on the antiplatelet effect of clopidogrel but not on that of ticlopidine. Ticlopidine may be an effective therapeutic option for CYP2C19 PMs.

Quantitative analysis of constitutive and inducible CYPs mRNA expression in the HepG2 cell line using reverse transcription-competitive PCR.

Drug interactions which affect drug metabolism are of clinical importance. It is, however, difficult to estimate drug interactions in human from results obtained in animal experiments. In our previous study, we demonstrated that a combination of the HepG2 cell line and semiquantitative reverse transcription-PCR (RT-PCR) could be used to evaluate the degree of CYP3A mRNA induction by various drugs. Using an RT-competitive PCR (RT-cPCR) with beta-actin as the standard in this study, the constitutive and rifampicin (RFP)-induced expression of CYP3A4, CYP2C9, CYP2E1, and CYP1A2 mRNA in the HepG2 cells could be quantitatively and reproducibly determined. 120 h-treatment of HepG2 cells with 50 micromol/l RFP induced maximally 8.4- and 6.0-fold the expression of CYP3A4 and CYP2C9 mRNA, respectively, in comparison with untreated cells. On the other hand, mRNA level in CYP2E1 and CYP1A2 was not significantly changed by 50 micromol/l RFP after 24 to 120 h. To our knowledge, we report for the first time quantitative profiles of CYPs mRNA in HepG2 cells. This study demonstrates the efficiency of a combination of HepG2 cells and RT-cPCR in the evaluation of CYPs mRNA-induction by drugs.

Relationship between mRNA levels quantified by reverse transcription-competitive PCR and metabolic activity of CYP3A4 and CYP2E1 in human liver.

Reverse transcription-competitive polymerase chain reaction is a powerful and sensitive tool for quantifying the absolute amount of mRNA. Using this method with beta-actin as the standard, we measured the mRNA level of CYP3A and CYP2E1 isoforms in human livers. We also determined the metabolic activities for both CYP isoforms. The absolute amounts of CYP3A4 and CYP2E1 mRNA in 15 liver tissues ranged from 15 to 5127 and 15163 to 69289 copies/10(4) copies of beta-actin (341- & 3.6-fold), respectively. The testosterone 6beta-hydroxylation for CYP3A4 and chlorzoxazone 6-hydroxylation activity for CYP2E1 ranged from 30 to 505 pmol/mg/min (16-fold) and from 0.59 to 2.73 nmol/mg/ml (3.6-fold), respectively. The correlation between the mRNA level and activity of CYP3A4 was significant (r = 0.94), while there was no significant correlation for CYP2E1 (r = 0.04). In conclusion, we observed a significant correlation between enzyme activity and mRNA expression for CYP3A4 but not for CYP2E1. This fact indicates that CYP2E1, in addition to being less variable between individuals than CYP3A4, differs in its regulation mechanism.

CYP2C19 genotypes and omeprazole metabolism after single and repeated dosing when combined with clarithromycin.

OBJECTIVES: Omeprazole is metabolized mainly by CYP2C19 which has two major mutations (CYP2C19*2 in exon5 and CYP2C19*3 in exon4) associated with the poor metabolizer (PM) phenotype. The aim of this study was to examine the relationship between genetic polymorphism of CYP2C19 and metabolism of omeprazole administrated as a single dose or as repeated-doses, which were in both cases co-administered with clarithromycin. METHODS: Twelve healthy Japanese subjects were typed for CYP2C19 polymorphism. In the single-dose study, plasma levels of omeprazole and its metabolites were measured for 24 h after administration of 20 mg omeprazole and 400 mg clarithromycin to six healthy Japanese subjects. In the repeated-dose study, plasma levels of omeprazole and its metabolites were measured after repeated oral administration of 20 mg omeprazole and 400 mg clarithromycin twice daily for 6 days and then after 20 mg omeprazole and 400 mg clarithromycin once on the 7th day to the other 6 healthy Japanese subjects. RESULTS: In the single-dose study, the areas under the plasma concentration-versus-time curve (AUCs) of omeprazole of homozygotes for the wild-type allele (*1/*1 n = 2), heterozygotes (n = 3) for the CYP2C19*2 (*1/ *2) or for the CYP2C19*3 (*1/*3) and heterozygote (n = 1) for the two defects (*2/*3) were on average 450, 1007 and 6710 ng x h(-1) x ml(-1), respectively. The ratios of AUCs of omeprazole/5-hydroxyomeprazole for *1/*1, *1/*2 or *1/*3 and *2/*3 were 1, 2 and 30, respectively. In the repeated-dose study, the AUCs of omeprazole for * 1/ *1, *1/*2 or *1/*3 and *2/*3 were 4041 (n = 2), 3149 (n = 3) and 6684 (n = 1) ng x h(-1) x ml(-1), respectively. The ratios of AUCs of omeprazole/5-hydroxyomeprazole for *1/*1, * 1/*2 or * 1/*3 and *2/*3 were7, 11 and 30, respectively. In the repeated-dose study, the AUC of omeprazole of * 1/*1 genotypes was nine-fold higher, that of *1/*2 and *1/*3 genotypes was three-fold higher, and the Cmax value of omeprazole was three-fold higher compared with subjects with the same genotype in the single-dose study. However, there were few differences in the AUC and Cmax of omeprazole between the *2/*3 genotype in the single-dose study and the homozygote for the CYP2C19*2 (*2/*2) in the repeated-dose study. CONCLUSION: Subjects with *1/*1, *1/*2 and *1/*3 genotypes in the repeated-dose study had lower CYP2C19 activity than subjects of the same genotype in the single-dose study. The difference in omeprazole metabolism between subjects with different genotypes observed on day 1 seemed to disappear after 7 days of repeated-dose administration.

Evaluation of induction of CYP3A mRNA using the HepG2 cell line and reverse transcription-PCR.

Cytochrome P-450 3A (CYP3A) is a drug-metabolizing enzyme dominant in the human liver. We have designed a useful method for evaluation of induction of CYP3A mRNA by various drugs using HepG2 cells known to retain liver-cellular functions. Using semi-quantitative reverse transcription-PCR (RT-PCR), we demonstrated that cultured HepG2 cells constitutively expressed CYP3A mRNA. This mRNA was expressed at high levels in culture for several days and was further induced by several drugs (e.g. rifampicin (RFP), dexamethasone). Treatment of HepG2 cells with RFP induced CYP3A mRNA in a dose- and time-dependent manner. Cells in culture for 48 h with 1 and 50 micromol/l RFP increased 2.7- and 5.0-fold in CYP3A mRNA expression in comparison with untreated controls, respectively. In contrast, no change in the amount of CYP3A mRNA was observed when the cells were treated with cimetidine which has been shown to inhibit CYP3A activity. Our method using a combination of HepG2 cells and RT-PCR allowed evaluation of the degree of induction of CYP3A mRNA both easily and rapidly.

Determination of aliphatic aldehydes as their thiazolidine derivatives in foods by gas chromatography with flame photometric detection.

A selective and sensitive gas chromatographic method for the determination of saturated and unsaturated aliphatic aldehydes in foods has been developed. After extraction of the sample with acetonitrile, aldehydes were converted into their thiazolidine derivatives by the reaction with cysteamine, and then measured by gas chromatography with flame photometric detection. The calibration curves for aliphatic aldehydes in the range 20-2500 ng were linear and the detection limits at a signal-to-noise ratio of 3 were ca. 4-100 pg injected. Aliphatic aldehydes in foods could be selectively determined by this method without any interference from coexisting substances. Overall recoveries of aldehydes added to food samples were 82-111%. Analytical results for the determination of aliphatic aldehydes in various food samples are presented.

Coordination of cortically induced rhythmic jaw and tongue movements in the rabbit.

1. Rhythmic movements of the jaw, tongue, and hyoid that were induced by stimulation of the cortical masticatory area (CMA) were recorded cineradiographically in the anesthetized rabbit. Jaw movements were also recorded by a laser position detector. 2. The evoked jaw movements were classified into four types: small circular (type A), large circular (type B), large vertical (type C), and crescent-shaped (type D). Among these, types B and D resembled the jaw movements of the food transport cycle and those of the chewing cycle in a masticatory sequence. 3. Each type of jaw movement was associated with a particular pattern of tongue and hyoid movements. In general, the tongue protruded during jaw opening and retracted during jaw closure. The hyoid generally moved upward and forward during jaw opening but downward and backward during jaw closure. 4. Electromyograms (EMGs) were recorded from jaw muscles [masseter (Ma) and digastric (Di) muscles], extrinsic tongue muscles [styloglossus (Sg) and genioglossus (Gg) muscles], and hyoid muscles [sternohyoid (Sh) and geniohyoid (Gh) muscles] during cortically induced rhythmic jaw and tongue movements (CRJTMs). These muscles were classified into two groups: group 1 was activated mainly in the jaw opening phase, and group 2 was activated mainly in the jaw closing and power phases. The Di, Gg, and Gh were included in the former, and the Ma, Sg, and Sh were included in the latter. 5. The timings of EMG activation to a jaw movement cycle were relatively constant for the muscles of group 1, irrespective of the types of CRJTMs, whereas those for the muscles of group 2 altered considerably with the different types of CRJTMs. 6. Relationships of the integrated muscle activity between the Di and Gg and between the Di and Gh were significant, whereas those between the Ma and Sg and between the Ma and Sh were not. 7. When a small strip of polyurethane form of various degrees of hardness was inserted between the opposing molars during CRJTMs, EMG activity of the muscles of group 2 increased with the hardness of the strip. On the other hand, EMG activities of the muscles of group 1 were less affected by the same intraoral stimuli. 8. Two conclusions were reached: first, physiological properties of the CRJTMs and cortically induced rhythmic movements of the hyoid were essentially similar to those observed in natural mastication. This fictive mastication might thus be regarded as a suitable model for simulating natural mastication.(ABSTRACT TRUNCATED AT 400 WORDS)

The integral dose in panoramic intraoral x-ray tube radiography.

A Monte Carlo computer program was developed to estimate the integral dose to the head and thyroid for panoramic intraoral x-ray tube radiography. The advantage of this computer simulation is that it is able to avoid many of the difficulties associated with low-energy and low-dose x-ray dosimetry. The calculations were made for maxillary and mandibular projections separately, using 10 kv. increments between 40 and 60 kv. The results obtained were presented in terms of the integral dose per milliampere second. Typical integral doses for a routine examination of the head are 2.1 mJ. and 8.5 microJ for the thyroid during mandibular radiography and 1.7 microJ for the thyroid during radiography of the maxilla using 55 kv. and 0.5 mAs.