Automated gas chromatographic assay for amlodipine in plasma and gingival crevicular fluid.

This paper describes an automated capillary gas chromatographic method for the determination of amlodipine in plasma, and in sub-microlitre volumes of gingival crevicular fluid (GCF), in order to assess if amlodipine is present in GCF under conditions of gingival overgrowth, as has been shown for nifedipine, another dihydropyridine drug. Liquid-liquid extraction followed by derivatisation was employed to isolate amlodipine and render it suitable for gas chromatography. Amlodipine was analysed in plasma and GCF of four patients undergoing amlodipine therapy for cardiovascular disorders, three of whom had significant gingival overgrowth. Amlodipine was detected in the plasma of all patients and in massive concentrations in the GCF of those patients with overgrowth, 23- to 290-fold greater than in their plasma. Like nifedipine, amlodipine sequestration into GCF appears to be linked with gingival overgrowth.

Identification of the major human hepatic cytochrome P450 involved in activation and N-dechloroethylation of ifosfamide.

Two NADPH-dependent metabolic routes for the anticancer drug ifosfamide, 4-hydroxylation (activation) and N-dechloroethylation (a detoxication pathway), were studied in human liver microsomes to identify the cytochrome P450 enzymes involved. Naringenin, a grapefruit aglycone and an inhibitor of cytochrome P450 3A4 (CYP3A4)-catalysed reactions, was found to inhibit ifosfamide activation and N-dechloroethylation by human liver microsomes. IC50 for both reactions was of the order of 70 microM. The CYP3A4-specific inhibitor triacetyloleandomycin inhibited ifosfamide N-dechloroethylation by human liver microsomes with an IC50 of approximately 10 microM. Furthermore, anti-human CYP3A4 antiserum inhibited by about 80% N-dechloroethylation of ifosfamide by human liver microsomes. The relative levels of cytochromes P450 1A, 2C, 2E and 3A4 in 12 human livers were determined by western blotting analysis. A strong correlation (P < 0.001) was observed between CYP3A4 expression and both activation and N-dechloroethylation of ifosfamide. A role for human CYP3A4 in both pathways of ifosfamide metabolism was thus demonstrated. This was substantiated by the observation that the nifedipine oxidase activities of the 12 samples of human liver microsomes correlated with ifosfamide activation (P < 0.009) and N-dechloroethylation (P < 0.001). These findings have important clinical implications. The involvement of the same key cytochrome P450 enzyme in both reactions prohibits selective inhibition of the N-dechloroethylation pathway, as might be desirable to reduce toxic side effects. They also demonstrate the need to consider interaction with co-administered drugs that are CYP3A4 substrates.

Determination of nifedipine in gingival crevicular fluid: a capillary gas chromatographic method for nifedipine in microlitre volumes of biological fluid.

This paper describes a sensitive capillary gas chromatographic (GC) method for the determination of nifedipine in sub-microliter samples of gingival crevicular fluid (GCF) in order to assess if nifedipine is present in the GCF and if so, whether the local tissue concentrations of this drug are an important determinant in the development of gingival overgrowth. Liquid-liquid and solid-phase extraction were combined to give adequate sample clean-up and concentration for measurement by automated capillary GC with electron capture detection. Nifedipine and its principal metabolite, M-I, were analysed in both plasma and GCF in 9 adult male patients who had been taking nifedipine for over six months. M-I could not be measured in GCF. Plasma nifedipine and M-I levels were normal, but the nifedipine levels found in the GCF of 7 patients (including all those with overgrowth) were remarkably elevated, 15 to 316-fold greater. This massive concentration of nifedipine into the GCF is therefore linked with gingival overgrowth. This is the first time that a GC method has been developed which permits determination of GCF pharmacokinetics of a drug which causes gingival overgrowth, and further investigation will lead to a better understanding of the tissue mechanisms involved.

Gingival sequestration of nifedipine in nifedipine-induced gingival overgrowth.

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.

A phase I study on the reversal of multidrug resistance (MDR) in vivo: nifedipine plus etoposide.

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.

Genetic and metabolic criteria for the assignment of debrisoquine 4-hydroxylation (cytochrome P4502D6) phenotypes.

A randomly selected population of 73 volunteers, together with 22 previously established poor metabolisers of debrisoquine, were phenotyped for their ability to 4-hydroxylate debrisoquine and were also analysed for a number of mutations in the CYP2D6 gene. Genotyping was performed using both restriction fragment length polymorphism with the restriction enzyme Xba I, together with two separate polymerase chain reaction assays. Together, these assays detected 98% of mutant alleles in the poor metaboliser group which corresponded to positive identification of 95% of this group. The most common mutant allele detected as the 29B which comprised 75% of total alleles in the poor metaboliser group, whereas the 29A had a frequency of 0.11. Two other allelic variants, which were detectable by restriction fragment length polymorphism analysis occurred at frequencies of 0.07 and 0.05. In the volunteer group, 2.7% of subjects were genotypically poor metabolisers, 35.6% heterozygous extensive metabolisers and 61.7% homozygous extensive metabolisers, on the basis of the genotyping assays used. A good correlation between debrisoquine metabolic ratio and genotype was obtained particularly for subjects genotyped as homozygous extensive metabolisers.

High performance liquid chromatographic measurement of lignocaine in tissue samples following transabdominal placental biopsy.

A method is described for the measurement of lignocaine in small samples of fetal and placental tissue. Tissue samples (ca 100 mg) are digested using a preteolytic enzyme. Lignocaine and an internal standard are extracted into methyl tert-butyl ether and analysed by high-performance liquid chromatography with electrochemical detection (+1.0 V vs Ag/AgCl). The limit of accurate measurement is better than 0.1 mg/kg wet weight for a 100 mg sample. This method has been used to assess fetal exposure to the drug when used as a local anaesthetic during transabdominal placental biopsy (chorionic villus sampling). The range of lignocaine concentrations found in the tissue samples was large (from less than 0.04 mg/kg wet weight to 15.4 mg/kg wet weight) although most samples contained less than 1.0 mg lignocaine/kg wet weight.