Biomedical subjects
Publications and source records attributed to J R Idle.
A polymorphism that results in the loss of a CfoI site in the human cytochrome P450 CYP2D6 gene is described. This polymorphism does not appear to cause impaired enzyme activity.
It has been claimed that the amino acid derivative carbocisteine is predominantly metabolized by sulfoxidation and that this pathway exhibits a genetic polymorphism. Moreover, those subjects with a 'poor metabolizer' phenotype have been thought to have a genetic predisposition to developing certain diseases. We have confirmed the observations of others that this marker drug does not undergo significant S-oxidation. Furthermore, a novel urinary metabolite, S-(carboxymethylthio)-L-cysteine (CMTC) has recently been identified. To determine if a genetic polymorphism for this biotransformation pathway exists, metabolic ratios (% urinary excretion carbocisteine/% urinary excretion CMTC) for 120 healthy volunteers were assessed using high-performance thin-layer chromatography. Urinary excretion of the parent drug ranged from 6% of the dose administered to 56% (mean +/- SD, 23.4 +/- 0.8%). No cysteinyl sulfoxide metabolites were identified in the urine samples. The amount excreted as CMTC exhibited a 12-fold variation but only accounted for mean of 4.4% (1-12%) of the dose given. Two individuals initially had high metabolic ratios (> 30), however, on rechallenge both their MRs were less than 5. Therefore, carbocisteine is not an appropriate probe drug for sulfoxidation. The formation of the novel metabolite CMTC appears to exhibit polymorphism, although the considerable intra-subject variation for its formation does not allow assignment of a phenotype.
The present study investigates the relationship between the pharmacokinetic variables of nifedipine with the incidence and severity of gingival overgrowth in 9 adult male patients medicated with the drug for at least 6 months. Five of the patients had experienced significant gingival changes and were thus designated "responders". The remaining four patients exhibited no gingival overgrowth, and thus acted as a control. A baseline periodontal examination (plaque scores, bleeding index and gingival overgrowth assessment) was carried out on each patient, and confined to the upper and lower anterior teeth. Serial blood and gingival crevicular fluid samples were collected over an eight-hour investigation period. Samples were analyzed for nifedipine by gas chromatography. No significant difference (p > 0.05) was seen between responders and non-responders with regard to drug therapy, periodontal parameters or plasma pharmacokinetics of nifedipine. Nifedipine was detected in the gingival crevicular fluid of seven subjects (all responders, and two non-responders). The peak concentration of nifedipine in crevicular fluid was 15-90 fold greater than levels observed in plasma.
Genetic polymorphisms of drug-metabolizing enzymes, principally CYP2D6 (debrisoquine 4-hydroxylase), have long been considered influential on host responsiveness to environmental carcinogens. In several independent studies, lung cancer cases are more frequently associated with the extensive metabolizer phenotype of CYP2D6. However, assignment of phenotype has traditionally involved administration of debrisoquine and analysis of drug and metabolite concentrations in patient urine and is thus potentially confounded by concomitant drug therapy and the presence of the tumor itself. The development of molecular genotyping methods offers unique opportunities to obviate these problems and to ascertain the relationship between the presence of individual alleles and disease risk. Preliminary data are presented that indicate that the CYP2D6 wild-type allele may be a predisposing factor in lung cancer.
Primary biliary cirrhosis is a chronic cholestatic disease of unknown aetiology which predominantly affects middle-aged women. It is thought to be autoimmune in nature, but unlike many autoimmune diseases no clear HLA association has been described. Several studies have suggested conflicting associations with HLA class II, although a DR8 association is most frequently described. To test the hypothesis that primary biliary cirrhosis is associated with a certain HLA class II locus we genotyped 130 patients with the disease from the north-east region of England and 363 local healthy controls. HLA-DRB1 and confirmatory DQA and DQB genotypes were determined by TaqI restriction fragment DNA length polymorphism analysis. In addition, a polymerase chain reaction technique (double ARMS) was used to investigate the DRB3 locus (DR52) in 98 primary biliary cirrhosis patients and 107 local controls. We found an increased frequency of HLA-DR8 (18.5% vs 9.2%, p < 0.005, relative risk of 2.0 [1.3-3.1]) in the primary biliary cirrhosis group. HLA-DR8-positive primary biliary cirrhosis patients had a higher serum bilirubin level (p = 0.03) than DR8-negative patients. There was no difference in the DR52 frequencies and no association with markers of disease severity. These results support earlier serological findings, although the association between primary biliary cirrhosis and DR8 is weaker than previously described. In addition, DR8-positivity may identify a clinical subgroup with a worse prognosis.
What may appear to be a strategy for investigating pharmacogenetic variation must be seen in the context of an evolving understanding of the molecular biology of drug metabolism. New biochemical and analytical techniques permit the determination of mechanisms of drug action and the molecular biology of host and tumour factors influencing that action. However, the only example of the application of pharmacogenetics in cancer chemotherapy is that of 6MP in the treatment of ALL. This was based on a solid understanding of both the metabolism of the drug and therapeutic response and the ability to measure the polymorphic enzyme in erythrocytes. Identification of the gene responsible for this enzyme is thought to be near, with subsequent identification of mutant forms completing the pharmacogenetic story for this drug. However, pharmacogenetics appears to be an important component in the variability of both toxicity and tumour response for several chemotherapeutic agents. Determination of the complete molecular basis for this variation will no doubt make a major contribution to the increased understanding of host and tumour pharmacology, with the ultimate aim of optimization of therapy.
BACKGROUND: Carboxyphosphamide is an inactive metabolite of cyclophosphamide, which is a widely used antineoplastic drug. Deficiencies in the production of this metabolite have been reported. Such deficiencies would have important consequences for therapeutic and toxic effects of oxazaphosphorines like cyclophosphamide. PURPOSE: This study further investigates the variability in cyclophosphamide metabolism and carboxyphosphamide recovery in urine. METHODS: The 24-hour urinary metabolic profile of cyclophosphamide was investigated in 17 Turkish patients receiving doses of 100-1080 mg orally or by short intravenous infusion. Urine samples were assayed quantitatively for cyclophosphamide and its principal metabolites (phosphoramide mustard, 4-ketocyclophosphamide, carboxyphosphamide, and dechloroethylcyclophosphamide) with combined thin-layer chromatography-photography-densitometry. The amount of each metabolite excreted in 24 hours was expressed as a percentage of the dose. RESULTS: Recovery of drug and metabolites varied greatly among individuals (range, 0.01%-13.56% of dose). In particular, the amount of carboxyphosphamide varied over a thousandfold range and was undetectable in urine from four patients. The patients were classified by phenotype as demonstrating low or high carboxylation. Those with low carboxylation excreted less than 0.2% of the cyclophosphamide dose as carboxyphosphamide, while those with high carboxylation excreted 0.8%-13.6% (median, 1.81%). No association was observed between carboxylation phenotype and patient age, sex, disease, or concomitant therapy, although the three lifetime nonsmokers all showed poor carboxylation. No correlation was observed between the percent of dose excreted as any of the other metabolites and that excreted as carboxyphosphamide. There was a statistically significant inverse correlation between the combined recovery of carboxyphosphamide and phosphoramide mustard and the dose of prednisolone administered. CONCLUSIONS: These data confirm an earlier observation of a phenotypic deficiency of carboxyphosphamide excretion in British patients treated with cyclophosphamide. This deficiency may arise from a polymorphism in the enzyme aldehyde dehydrogenase. Carboxylation phenotype may have important implications for both the therapeutic effect and toxicity of cyclophosphamide.
The mechanism of gingival overgrowth associated with long-term use of nifedipine and of other drugs that affect calcium homoeostasis, such as cyclosporin and phenytoin, is unknown. With an ultrasensitive assay, we measured the pharmacokinetics of nifedipine in plasma and gingival crevicular fluid (GCF) of nine patients receiving this drug for angina and hypertension. In seven patients, the maximum nifedipine concentration was in the range 15-316 (mean 84 [SD 105]) times greater in GCF than in plasma. The two patients with low (undetectable) GCF nifedipine did not have overgrowth. We propose that gingival tissues sequester nifedipine and that the very high nifedipine concentrations predispose the tissues to overgrowth.
The frequency of fifteen genotypes of CYP2D6 (debrisoquine 4-hydroxylase) in 53 patients with Parkinson's disease was determined by the polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) analyses and compared with the findings in 72 healthy controls. The commonest mutant allele, CYP2D6B, was twice as frequent among patients as in controls, with an approximate relative risk ratio of 2.70 (95% confidence interval 1.14-6.41; p = 0.0063) for subjects homozygous or heterozygous for this allele.
A novel method for the determination of 7-hydroxycoumarin in human urine which combines thin-layer chromatography (TLC) with fluorescence detection (FD) has been devised. The limit of detection (1 ng/ml) enables determination of 7-hydroxycoumarin after both administration of coumarin and environmental exposure to this fragrance material. When compared to a spectrofluorometric method of analysis, the TLC-FD method proved to be more selective for the analysis of 7-hydroxycoumarin in human urine.
A method has been devised for the determination of the anticancer drug ifosfamide and its principal metabolites in urine, plasma and cerebrospinal fluid (CSF). The urine and CSF samples are absorbed onto Amberlite XAD-2 eluting the compounds of interest with methanol. Plasma is deproteinated using cold acetonitrile and centrifuged to yield a clear supernatant. The eluate and supernatant are analyzed by thin-layer chromatography, with spot visualization using 4-(4-nitrobenzyl)pyridine. The plates are photographed for subsequent densitometeric analysis. The intra-assay coefficient of variation for each compound in both urine and plasma was less than 10% and the lower limit of detection was 1 microgram/ml. The method provides a means of determining the full spectrum of metabolic products of ifosfamide in patients and will allow detailed investigation of variability in metabolism and pharmacokinetics of this drug.
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The pharmacokinetics and metabolism of cyclophosphamide were studied in nine paediatric patients. Plasma samples were obtained from eight subjects and urine was collected from six children during a 24-h period after drug administration. Cyclophosphamide and its major metabolites phosphoramide mustard (PM), carboxyphosphamide (CX), dechloroethylcyclophosphamide (DCCP) and 4-ketocyclophosphamide (KETO) were determined in plasma and urine using high-performance thin-layer chromatography-photographic densitometry (HPTLC-PD). Cyclophosphamide (CP) was nearly, if not completely, cleared from plasma by 24 h after its administration. The plasma half-life of CP ranged from 2.15 to 8.15 h; it decreased following higher doses and was shorter than that previously reported for adult patients. Both the apparent volume of distribution (0.49 +/- 1.4 l/kg) and the total body clearance (2.14 +/- 1.4 l m-2 h-1) increased with increasing dose. Renal clearance ranged between 0.12 and 0.58 l/h (mean, 0.43 +/- 0.19 l/h). Between 5.4% and 86.1% of the total delivered dose was recovered as unchanged drug in the urine. The major metabolites identified in plasma and urine were PM and CX. One patient appeared to be deficient in CX formation. This study suggests that there is interpatient variability in the pharmacokinetics and metabolism of CP in paediatric patients. The shorter half-life and higher clearance as compared with adult values indicate faster CP metabolism in children.
Recent advances in the study of human cytochromes P450 by protein purification, molecular cloning techniques and analysis of polymorphisms has led to increased understanding of the role of the various forms in the metabolism of clinically important drugs. In particular, the substrate specificity of one form, CYP2D6, is well established. CYP2D6 shows polymorphism, with 5-10% of Caucasians (poor metabolizers) not expressing this enzyme. The molecular basis of this deficiency is now well understood and methods for the detection of poor metabolizers are discussed, as well as the effect of the polymorphism on drug metabolism. Substrate specificities and possible polymorphisms in other cytochromes P450 are also discussed.
Multidrug resistance (MDR) is one of the mechanisms of resistance to multiple cytotoxic drugs and is mediated by the expression of a membrane pump called the P-glycoprotein. Nifedipine is one of the calcium channel blocking agents which reverses MDR in vitro. Fifteen patients with various malignancies received nifedipine at three dose levels: 40 mg, 60 mg and 80 mg orally twice daily for 6 days. Etoposide was administered intravenously on day 2 in a dose of 150-250 mg m-2 and orally 150-300 mg twice daily on days 3 and 4. Cardiovascular effects of nifedipine were dose limiting and the maximum tolerated dose was 60 mg bid. Mean area under the plasma concentration curve (AUC0-00) and plasma half-life (beta) of nifedipine and its major metabolite MI at the highest dose level were 7.87 microM.h, 7.97 h and 4.97 microM.h, 14.0 h respectively. Nifedipine did not interfere with the pharmacokinetics of etoposide.
Previous studies have indicated that there is weak genetic linkage between the defective gene in cystic fibrosis (CFTR) and the gene encoding the nifedipine metabolizing enzyme P4503A4 which are both located on chromosome 7. To examine further this possible association, nifedipine metabolism was investigated in a group of 59 volunteers, and 17 adult cystic fibrosis patients and 37 of their relatives. In agreement with the majority of previous studies, the volunteer group showed a unimodal distribution of recoveries for the major metabolite M-II ranging from 33 to 78% excretion in 8 h. In the case of both the cystic fibrosis patients and their parents, the distribution of recoveries was shifted to the left with five out of 20 parents and three out of 11 unrelated cystic fibrosis patients showing recoveries below the range observed in the volunteer group. This poor metabolism appeared to be both reproducible and heritable and did not appear to be a consequence of mutations in the CFTR gene.
The human body is endowed with a large number of xenobiotic chemical metabolizing enzymes, a significant proportion of which are polymorphic and thus render one individual at greater or lesser risk than another of chemically-induced disease. All examples of genetic polymorphism of chemical metabolizing enzymes have been reviewed in relation to their potential to activate and detoxicate procarcinogens and promutagens. Many examples are cited whereby phenotype can act as a carcinogenic risk factor. With the availability of a large amount of DNA sequence data for chemical metabolizing enzymes there has emerged a number of polymerase chain reaction (PCR) strategies aimed at discerning one metabolic phenotype or another. This is seen as a very positive and democratic scientific development, widening the franchise for studies of disease risk. Nevertheless, it is argued that, at these early stages with many laboratory-based scientists scarcely familiar with epidemiological study design, a cautious approach should obtain when interpreting single studies.
The gingival health of 19 patients with cardiovascular problems who were medicated with nifedipine was compared with a similar cohort treated with atenolol and a control group of healthy patients. In the nifedipine and atenolol groups, patients had been taking their respective medication for a minimum period of 6 months. Plaque scores were similar for all three groups. However, patients medicated with nifedipine had a significantly higher gingival index (P less than 0.005), gingival overgrowth scores (P less than 0.02) and probing sites greater than 3 mm (P less than 0.005) when compared with the atenolol and control groups. 4 patients in the nifedipine group experienced clinically significant gingival overgrowth which required surgical excision. Gingival changes in the nifedipine patients were not related to drug dosage or plaque scores. It is concluded that nifedipine therapy results in significant gingival changes, an effect which may be mediated by the drug's action on calcium transport.