Cytochromes of the P450 2C subfamily are the major enzymes involved in the O-demethylation of verapamil in humans

The calcium channel blocker verapamil [2,8-bis-(3,4-dimethoxyphenyl)-6-methyl-2-isopropyl-6-azaoctanitrile] undergoes extensive biotransformation in man. We have previously demonstrated cytochrome P450 (CYP) 3A4 and 1A2 to be the enzymes responsible for verapamil N-dealkylation (formation of D-617 [2-(3,4-dimethoxyphenyl)-5-methylamino-2-isopropylvaleronitrile]), and verapamil N-demethylation (formation of norverapamil [2,8-bis(3,4-dimethoxyphenyl)-2-isopropyl-6-azaoctanitrile]), while there was no involvement of CYP3A4 and CYP1A2 in the third initial metabolic step of verapamil, which is verapamil O-demethylation. This pathway yields formation of D-703 [2-(4-hydroxy-3-methoxyphenyl)-8-(3,4-dimethoxyphenyl)-6-methyl-2-isopropyl-6-azaoctanitrile] and D-702 [2-(3,4-dimethoxyphenyl)-8-(4-hydroxy-3-methoxyphenyl)6-methyl-2-isopropyl-6-azaoctanitrile]. The enzymes catalyzing verapamil O-demethylation have not been characterized so far. We have therefore identified and characterized the enzymes involved in verapamil O-demethylation in humans by using the following in vitro approaches: (I) characterization of O-demethylation kinetics in the presence of the microsomal fraction of human liver, (II) inhibition of verapamil O-demethylation by specific antibodies and selective inhibitors and (111) investigation of metabolite formation in microsomes obtained from yeast strain Saccharomyces cerevisiae W(R), that was genetically engineered for stable expression of human CYP2C8, 2C9 and 2C18.In human liver microsomes (n=4), the intrinsic clearance (CL_int), as derived from the ratio of V _max/K_m, was significantly higher for O-demethylation to D-703 compared to formation of D-702 following incubation with racemic verapamil (13.9±1.0 vs 2.4±0.6 ml^*min^{-1 *}g^-1 mean±SD; p<0.05), S-Verapamil (16.8±3.3 vs 2.2±1.2 ml^* mini^*g^-1, p<0.05) and R-verapamil (12.1±2.9 vs 3.6 ±1.3 ml^*min^{-1 *} g^-1; p<0.05), thus indicating regioselectivity of verapamil O-demethylation process. The CL_int of D-703 formation in human liver microsomes showed a modest but significant degree of stereo selectivity (p<0.05) with a S/R-ratio of 1.41±0.17. Anti-LKM2 (anti-liver/kidney microsome) autoantibodies (which inhibit CYP2C9 and 2C19) and sulfaphenazole (a specific CYP2C9 inhibitor) reduced the maximum rate of formation of D-703 by 81.5±4.5% and 45%, that of D-702 by 52.7±7.5% and 72.5%, respectively. Both D-703 and D-702 were formed by stably expressed CYP2C9 and CYP2C18, whereas incubation with CYP2C8 selectively yielded D-703.In conclusion, our results show that enzymes of the CYP2C subfamily are mainly involved in verapamil O-demethylation. Verapamil therefore has the potential to interact with other drugs which inhibit or induce these enzymes.     

In vitro development and stability of tolerance to cloxacillin and vancomycin inStaphylococcus aureus

The stability of tolerance ofStaphylococcus aureus during subculturing at 37°C and development of this property after repeated exposure to cloxacillin or vancomycin were investigated in vitro. Four of five tolerant strains lost this property during repeated subculturing at 37°C for 50 days. Conversely, tolerance emerged in two of four nontolerant strains after repeated cycles of exposure to 25 µg of cloxacillin/ml or 10 µg of vancomycin/ml alternating with growth in antibiotic-free medium. Previous in vivo exposure to cloxacillin did not enhance the development of tolerance in vitro. MICs of both cloxacillin and vancomycin did not change significantly during this procedure. Whether the conversion of nontolerant strains to the tolerant state can also occur during antibiotic exposure in treatment of patients remains to be determined.     

Assessment of the reliability and validity of panoramic imaging for assessment of mandibular condyle morphology using both MRI and clinical examination as the gold standard.

OBJECTIVES: The purpose of this study was to evaluate both reliability and validity of the assessment of the shape of the mandibular condyle in panoramic images of the TMJ. STUDY DESIGN: Forty subjects were included and were examined according to the Research Diagnostic Criteria for Temporomandibular Disorders. Panoramic radiographs (PRs) and magnetic resonance images (MRIs) were completed for all subjects. Both MRIs and PRs were rated by raters blinded to the clinical diagnosis. Kappa statistics were used to compare the results of the raters of the PRs. Additionally, the specificity and the sensitivity of the PRs were calculated for 2 scenarios: one with MRI and the other with clinical findings as the gold standard. RESULTS: The sensitivity was 0.94 (specificity = 0.45) for the assumption that MRI is the gold standard and 0.86 (specificity = 0.49) for the assumption that the clinical examination is the gold standard. For reliability, the results for kappa ranged from 0.06 to 0.327. CONCLUSION: It can be concluded that PRs are not a reliable method of accurately judging the shape of the mandibular condyle.     

Regional Distribution of Neurofibrillary Tangles and Senile Plaques in the Cerebral Cortex of Elderly Patients: A Quantitative Evaluation of a One-Year Autopsy Population from a Geriatric Hospital

Detailed analyses of the neuropathologic changes in the cerebralcortex of elderly individuals and Alzheimer's disease patientshave demonstrated that certain components of the neocorticaland hippocampal circuits are likely to be selectively vulnerable.Based on the distribution of neurofibrillary tangles (NFTs)and senile plaques, it has been proposed that a global cortico-corticaldisconnection leads to the loss of integrated functions observedin Alzheimer's disease. In order to investigate the distributionof lesions associated with aging as well as with the earliestsymptoms of senile dementia, we performed a quantitative neuropathologicavaluation of a large series of elderly patients representingthe entire autopsy population for the year 1989 from a geriatrichospital. Among the 145 cases quantitatively assessed, therewere 102 nondemented patients, 33 patients presenting clinicallywith globally intact intellectual function but early signs ofimpairment of specific cognitive functions, and 10 cases withsenile dementia of the Alzheimer type. All of the cases hadNFTs in layer II of the entorhinal cortex, regardless of theirclinical diagnosis, and most cases had some NFTs in the CA1field of the hippocampus. Severe pathologic changes within theinferior temporal neocortex were observed only in the dementedcases. The extent of amyloid deposition was not correlated withthe clinical diagnosis and seemed to be present in the neocorticalareas earlier than in the hippocampal formation. Also, severalcases contained NFTs without amyloid deposition, but amyloidnever occurred without NFTs. These results suggests that involvementof certain structures within the hippocampal formation is aconsistent feature of aging. Thus, involvement of the hippocampalformation may be a necessary, but not sufficient, conditionfor the clinical expression of dementia, which is likely tobe more closely related to the progressive degeneration of selectneuronal populations in the neocortex.     

Problems and needs for improving primary care of osteoarthritis patients: the views of patients, general practitioners and practice nurses

Background  

Osteoarthritis (OA) is highly prevalent and has substantial impact on quality of life as well as on healthcare costs. The general practitioner (GP) often is the first care provider for patients with this chronic disease. The aim of this study was to identify health care needs of patients with OA and to reveal possible obstacles for improvements in primary care management of OA patients.