Biomedical subjects

J K Coller

Publications and source records attributed to J K Coller.

7 recordsLinked to original sources

PubMed2004

A new simple diagnostic assay for the identification of the major CYP2D6 genotypes by DNA sequencing analysis.

AIM: To establish a method suitable for diagnostic genotyping of CYP2D6 alleles by DNA sequencing. METHODS: Initial PCR reactions were performed to specifically amplify exons 3, 4, 5 and 6 of the CYP2D6 gene using primers previously published. New primers were used to identify *2, *3, *4, *6, *7, *8, *9 and *41 in 2 sequencing reactions. Additional primers were designed for reverse sequencing in samples with 1 or 3 b.p. deletions. Previously published assays were used to detect *5, *10 and *16 alleles to complete genotype assignment. RESULTS: We reliably detected the nonfunctional alleles, *3, *4, *6, *7 and *8, which are associated with the poor metabolizer phenotype, and 2 important alleles associated with decreased enzyme activity, *9 and *41. Observed allele frequencies were comparable to those found previously in Caucasian populations. CONCLUSION: CYP2D6 genotype has been shown in previous clinical studies to be a good predictor of CYP2D6 phenotype and, therefore, related to therapeutic response and the risk of drug toxicity. This genotyping method is simple and reliable, and, therefore, can be routinely performed on an isolated patient sample, providing a relatively quick turnaround time needed for clinical practice. In addition, the simultaneous drawing of blood with the commencement of drug therapy will allow dosage adjustment on the basis of the CYP2D6 genotype to reduce the risk of adverse drug reactions.

Cytochrome P-450 CYP2D6↗

PubMed2003

Oxidative metabolism of tamoxifen to Z-4-hydroxy-tamoxifen by cytochrome P450 isoforms: an appraisal of in vitro studies.

1. Tamoxifen is used for the prevention and treatment of oestrogen receptor-positive breast cancer. 2. Tamoxifen is metabolized extensively and the formation of Z-4-hydroxy-tamoxifen (Z-4-OH-tam), a potent anti-oestrogen with high affinity for the oestrogen receptor, is believed to be strongly related to the therapeutic benefit achieved following tamoxifen treatment. 3. In vitro studies using human liver microsome preparations have shown considerable interindividual variability in the formation rates of Z-4-OH-tam. 4. Cytochrome P450 (CYP) isoform-specific chemical and monoclonal antibody inhibition studies have demonstrated that CYP2B6, CYP2C9, CYP2D6 and CYP3A4 all mediate the formation of Z-4-OH-tam. 5. Significant associations between the percentage inhibition of Z-4-OH-tam by CYP isoform-specific inhibitors and the rate of metabolism of CYP isoform-specific index reactions and between individual expression of CYP2B6, CYP2C9 and CYP2D6 and Z-4-OH-tam formation rates indicate predominant roles for these isoforms in this pathway. 6. Genotyping of patients with regards to CYP2B6, CYP2C9 and CYP2D6 may play a role in prediction of Z-4-OH-tam formation and, consequently, ultimate therapeutic benefit of tamoxifen treatment.

Animals↗

PubMed2001

Distribution of microsomal epoxide hydrolase in humans: an immunohistochemical study in normal tissues, and benign and malignant tumours.

Microsomal epoxide hydrolase is a biotransformation enzyme which is involved in the hydrolysis of various epoxides and epoxide intermediates. In the present study, its distribution was investigated in both normal human tissues and human tumours of different histogenetic origin using immunohistochemical techniques. In normal tissue, epithelial cells were more often and more intensely immunostained than mesenchymal cells. The main epithelial cell types expressing microsomal epoxide hydrolase were hepatocytes, acinus cells of the pancreas, and cells of salivary and adrenal glands. Immunostained cells of mesenchymal origin included monocytes, fibrocytes, fibroblasts, vessel endothelium, muscle cells, and cells of the reproductive system. Three patterns of expression were observed in tumour tissues: (1) moderate or strong in hepatocellular carcinomas, tumours of the adrenal gland, and theca-fibromas of the ovary; (2) inhomogeneous staining pattern of variable intensity in breast cancer, lung cancer, colorectal carcinomas, carcinoid tumours, and some tumours of mesenchymal origin; and (3) no expression in malignant melanomas, malignant lymphomas, and renal carcinomas. These data indicate that microsomal epoxide hydrolase expression is not restricted to tissue of any particular histogenetic origin. Nonetheless, immunohistochemical identification of microsomal epoxide hydrolase may be helpful in some well-defined histological settings, for example, confirmation of hepatocellular carcinoma.

Epithelial Cells↗

PubMed1999

Comparison of (S)-mephenytoin and proguanil oxidation in vitro: contribution of several CYP isoforms.

AIMS: To compare the oxidative metabolism of (S)-mephenytoin and proguanil in vitro and to determine the involvement of various cytochrome P450 isoforms. METHODS: The kinetics of the formation of 4'-hydroxymephenytoin and cycloguanil in human liver microsomes from 10 liver samples were determined, and inhibition of formation was studied using specific chemical inhibitors and monoclonal antibodies directed towards specific CYP450 isoforms. Expressed CYP450 enzymes were used to characterize further CYP isoform contribution in vitro. Livers were genotyped for CYP2C19 using PCR amplification of genomic DNA followed by restriction endonuclease digestion. RESULTS: All livers were wildtype with respect to CYP2C19, except HLS#5 whose genotype was CYP2C19*1/CYP2C19*2. The Km, Vmax and CLint values for the formation of 4'-hydroxymephenytoin from (S)-mephenytoin and the formation of cycloguanil from proguanil ranged from 50.8 to 51.6 and 43-380 microm, 1.0-13.9 and 0.5-2.5 nmol mg-1 h-1, and 20.2-273.8 and 2.7-38.9 microl h-1 mg-1, respectively. There was a significant association between the Vmax values of cycloguanil and 4'-hydroxymephenytoin formation (rs=0.95, P=0.0004). Cycloguanil formation was inhibited significantly by omeprazole (CYP2C19/3A), troleandomycin (CYP3A), diethyldithiocarbamate (CYP2E1/3A), furafylline (CYP1A2), and (S)-mephenytoin. 4'-Hydroxymephenytoin formation was inhibited significantly by omeprazole, diethyldithiocarbamate, proguanil, furafylline, diazepam, troleandomycin, and sulphaphenazole (CYP2C9). Human CYP2E1 and CYP3A4 monoclonal antibodies did not inhibit the formation of cycloguanil or 4'-hydroxymephenytoin, and cycloguanil was formed by expressed CYP3A4 and CYP2C19 supersomes. However, only expressed CYP2C19 and CYP2C19 supersomes formed 4'-hydroxymephenytoin. CONCLUSIONS: The oxidative metabolism of (S)-mephenytoin and proguanil in vitro is catalysed by CYPs 2C19 and 1A2, with the significant association between Vmax values suggesting that the predominant enzymes involved in both reactions are similar. However the degree of selectively of both drugs for CYP isoforms needs further investigation, particularly the involvement of CYP3A4 in the metabolism of proguanil. We assert that proguanil may not be a suitable alternative to (S)-mephenytoin as a probe drug for the CYP2C19 genetic polymorphism.

Adult↗

PubMed1999

Flunitrazepam oxidative metabolism in human liver microsomes: involvement of CYP2C19 and CYP3A4.

The aims were to examine the kinetics of the oxidative metabolism of flunitrazepam in vitro when flunitrazepam was dissolved in dimethylformamide and acetonitrile, and to determine which cytochrome P450 isoform(s) are involved. The kinetics of the formations of 3'-hydroxyflunitrazepam and desmethyl-flunitrazepam were non-linear and best estimated using the Hill equation. Inhibition of their formation was studied using specific chemical inhibitors, expressed enzyme systems and specific antibodies. Ks, Vmax, Clmax and n (slope factor) for the formation of 3'-hydroxyflunitrazepam and desmethylflunitrazepam had ranges of 165-338 and 179 391 microM, 22-81 and 3-10 nmol x mg protein(-1) x h(-1), 6-17 and 0.9-1.9 microl x mg protein(-1) x h(-1), and 2.3-3.6 and 1.6-2.6 respectively when dimethylformamide was the organic solvent. When acetonitrile was the solvent, Ks, Vmax, Clmax and n (slope factor) for the formation of 3'-hydroxyflunitrazepam and desmethylflunitrazepam had ranges of 173-231 and 74-597 microM, 35-198 and 2.7-48 nmol.mg protein(-1) x h(-1), 1347 and 0.7-6.3 microl.mg protein(-1) x h(-1), and 1.5-3.6 and 1.1-2.7 respectively. CYP2C19, CYP3A4 and CYP1A2 mediated the formation of both 3'-hydroxyflunitrazepam and desmethylflunitrazepam. Investigators need carefully to consider the choice of organic solvent to avoid false CYP identification.

Acetonitriles↗

PubMed1998

Quantification of flunitrazepam's oxidative metabolites, 3-hydroxyflunitrazepam and desmethylflunitrazepam, in hepatic microsomal incubations by high-performance liquid chromatography.

A high-performance liquid chromatographic assay for the quantification of the oxidative metabolites of flunitrazepam, 3-hydroxyflunitrazepam and desmethylflunitrazepam, in human liver microsomal incubations was developed. Both metabolites were quantifiable in a single assay following a solvent extraction and reversed-phase high-performance liquid chromatography with UV detection. Standard curve concentrations for both metabolites ranged from 0.2 to 10 microM. Assay performance was determined using quality control samples and the intra- and inter-day accuracy and precision as determined by the coefficient of variations which were less than 15% (0.5-6 microM) for both metabolites. This method provides good precision and accuracy for use in kinetic studies of the oxidative metabolism of flunitrazepam in human liver microsomes.

Anti-Anxiety Agents↗

PubMed1997

Association between CYP2C19 genotype and proguanil oxidative polymorphism.

AIMS: To examine the relationship between proguanil metabolism and the number of mutations in CYP2C19 by comparing the CYP2C19 genotype and proguanil phenotype of 10 subjects. METHODS: Partial clearance and urinary metabolic ratio data were obtained from a previous study of 10 subjects [5]. Analysis of CYP2C19 genotypes was performed using PCR amplification followed by restriction endonuclease digestion of genomic DNA from a blood sample. RESULTS: The intrinsic partial clearance of PG to CG ranged from 0.41-10.11 h-1, and was related to the number of functional CYP2C19 alleles present. Genotypic PMs had metabolic ratios > 13, while genotypic heterozygote EMs had metabolic ratios < 9. CONCLUSIONS: Proguanil may be a suitable phenotyping probe for the CYP2C19 genetic polymorphism, however the exact antimode of the urinary metabolic ratio chosen to separate poor and extensive metabolisers needs further investigation.

Administration, Oral↗