In vivo evaluation of coumarin and nicotine as probe drugs to predict the metabolic capacity of CYP2A6 due to genetic polymorphism in Thais.

The association between the distribution characteristics of CYP2A6 catalytic activities toward nicotine and coumarin, and the frequency distribution of CYP2A6 variant alleles reported was estimated in 120 healthy Thais. The distributions of the subjects as classified by the amounts of 7-hydroxycoumarin (7-OHC) excreted in the urine and by cotinine/nicotine ratio in the plasma were clearly bimodal. However, the numbers of apparently poor metabolizers for coumarin and nicotine were different. The inter-individual variability in the in vivo dispositions of coumarin and nicotine closely related to the CYP2A6 genetic polymorphism. There was a close correlation between the rate of 7-OHC excretion in the urine and cotinine/nicotine ratio in the plasma among subjects (R=0.92, p<0.001). The frequency of CYP2A6 allele found in the present study was: CYP2A6*1A=32% (95% CI, 22.1-39.4%), CYP2A6*1B=27% (95% CI, 19.4-33.5%), CYP2A6*9=20% (95% CI, 17.6-23.3%), CYP2A6*4=14% (95% CI, 9.6-17.8%), CYP2A6*7=5% (95% CI, 3.7-9.4%), CYP2A6*10=2% (95% CI, 0.8-5.1%). Subjects having CYP2A6*1A/*1B were found to have a higher rate of 7-OHC excretion, as well as a higher cotinine/nicotine ratio in the plasma compared with those of the other genotypes. In contrast, subjects with CYP2A6*4/*7 and CYP2A6*7/*7 almost lacked any cotinine formation, whereas urinary 7-OHC was still detectable. CYP2A6*9 allele clearly resulted in reduced enzyme activities. Despite the absence of the homozygote for CYP2A6*10 allele, the presence of CYP2A6*10 allele significantly decreased the enzyme activities. The results of the present study demonstrate that in vivo phenotyping of CYP2A6 using nicotine and coumarin are not metabolically equivalent. Nicotine is a better probe according to its specificity, while coumarin is still valuable to be used for a routine CYP2A6 phenotyping since the test employs a non-invasive method.

Interindividual variability of coumarin 7-hydroxylation in healthy volunteers.

A test designed to estimate the extent and rate of formation of 7-hydroxycoumarin by measuring the urinary excretion of the metabolite in humans after administering 5 mg coumarin was developed. Coumarin was rapidly metabolized after oral administration and more than 95% of the 7OHC formed was excreted in 4 h. The total amount of 7OHC formed was 64 +/- 15% (mean +/- SD, variation 20-100%) of the dose given. The percentage of 7OHC excreted in 2 h, as compared with the 7OHC excretion in 4 h, was found to be a constant and stable individual characteristic for the rate of the formation of 7OHC ('2 h coumarin test'). In 110 volunteers, there was a great interindividual variability in the extent and rate of 7OHC formation. Four individuals had relatively 'slow' coumarin test values (50-60%), but much larger populations would be needed for the demonstration of polymorphism.

Variation in coumarin 7-hydroxylase activity associated with genetic polymorphism of cytochrome P450 2A6 and the body status of iron stores in adult Thai males and females.

The relationships between catalytic activity of cytochrome P450 2A6 (CYP2A6), polymorphism of CYP2A6 gene, gender and levels of body iron stores were analysed in a sample group of 202 apparently healthy Thais, aged 19-47 years. Eleven individuals were found to have high activity of CYP2A6, judged by the relatively large amounts (11.2-14.6 mg) of 7-hydroyxcoumarin (7-OHC) excreted 3 h following administration of 15 mg of coumarin. Ten individuals, however, did not excrete any 7-OHC. Of these 10, four were found to have no CYP2A6 gene (whole gene deletion; CYP2A6*4 allele). The frequency of the CYP2A6 alleles; *1A, *1B and *4 in the whole sample group was 52, 40 and 8% while the frequency of the CYP2A6 gene types; *1A/*1A, *1A/*1B, *1B/*1B, *1A/*4, *1B/*4, *4/*4 was 29, 41, 16, 7, 5 and 2%. Subjects having CYP2A6*1A/*1B gene-type group were found to have higher rates of coumarin 7-hydroxylation compared with those of the CYP2A6*1B/*1B and CYP2A6*1A/*4 gene types. The inter-individual variability in CYP2A6 catalytic activity was therefore attributed in part to the CYP2A6 genetic polymorphism. Variation in CYP2A6 activity in this sample group was not associated with gender but, interestingly, it did show an inverse association with plasma ferritin; an indicator of body iron stores. Higher rates of coumarin 7-hydroxylation were found in individuals with low body iron stores (plasma ferritin < 20 microg/l) compared with subjects having normal body iron store status. Subjects (n = 16) with iron overload (plasma ferritin > 300 microg/l) also tended to have elevated rates of coumarin 7-hydroxylation. These results suggest an increased CYP2A6 expression in subjects who have excessive body iron stores. Further investigations into the underlying factors that may lead to increased expression of CYP2A6 in association with abnormal body iron stores are currently in progress in our laboratory.

Differential inhibition of coumarin 7-hydroxylase activity in mouse and human liver microsomes.

Coumarin is 7-hydroxylated by the P450 isoform Cyp2a-5 in mice and CYP2A6 in humans. Various drugs, endogenous substances, plant substances and carcinogens, altogether about 90 chemicals, were evaluated as possible inhibitors of coumarin 7-hydroxylase (COH) activity in mouse microsomes. The effects of selected compounds on COH activity in human liver microsomes were also tested. The furanocoumarin derivatives methoxsalen (8-methoxypsoralen) and psoralen proved to be the most potent inhibitors of mouse COH activity (IC50 values 1.0 and 3.1 microM, respectively). The furanocoumarins bergapten (5-methoxypsoralen), isopimpinellin (5,8-dimethoxypsoralen), imperatorin and sphondin also effectively inhibited mouse COH activity (IC50 values 19-40 microM). Methoxsalen, isopimpinellin and metyrapone were also inhibitors in mice in vivo. Methoxsalen was a potent inhibitor of COH activity also in human liver microsomes, (IC50 value 5.4 microM), whereas bergapten, isopimpinellin and imperatorin had no effect. The imidazole antimycotic miconazole was a potent but non-specific inhibitor of COH activity. Several known substrates and inhibitors of members in the CYP1A, CYP2B, CYP2C, CYP2D and CYP3A subfamilies were poor inhibitors of COH activity. These results suggest that (i) the coumarin-type compounds in particular interact with the active sites of Cyp2a-5 and CYP2A6, and (ii) the active sites of Cyp2a-5 and CYP2A6 are structurally different, since a number of compounds inhibited mouse, but not human COH activity.

Antibody-based approaches to coumarin analysis.

7-Hydroxycoumarin (7-HC) was chemically conjugated by diazo coupling to carrier proteins such as bovine serum albumin (BSA), thyroglobulin and ovalbumin. These conjugates were characterised by sodium dodecyl sulphate/polyacrylamide gel electrophoresis (SDS-PAGE) and high-performance liquid chromatography (HPLC). Rabbits were immunised using the 7-HC-BSA conjugate. The highest antibody titre achieved was 1:10,000, as determined by competitive enzyme-linked immunosorbent assay (ELISA). The resulting antibodies were purified by ammonium sulphate precipitation, followed by protein A affinity chromatography. Their purity was assessed by SDS-PAGE and HPLC. These antibodies have been used in the development of a competitive ELISA, an amperometric biosensor and an electrochemical immunoassay. Both the ELISA and amperometric biosensor have been successfully applied to the analysis of 7-HC and its glucuronide conjugate in human urine samples. Each of these antibody-based methods provides a novel approach to the analysis of the main metabolites of coumarin.

Hepatitis A impairs the function of human hepatic CYP2A6 in vivo.

Hepatitis virus A (HVA) is a worldwide sporadic disease but its effects on pharmacokinetics and individual drug responses have not been studied. In this study, the 7-hydroxycoumarin (7OHC) excretion test used in vivo as a bioindex of hepatic CYP2A6 activity was performed in 20, previously healthy, acute jaundice HVA patients. Volunteers with an acute HVA were treated with one p.o. administration of 5 mg coumarin (Venalot). Among the patients, 11 were children (6-10 years; two girls and nine boys), the rest (15-40 years old) consisted of two men and seven women. Urinary excretion of 7OHC was measured after overnight fasting in four fractions: 0 h before any medication (to detect if any basal 7OHC excretion exits), and after a 5-mg coumarin capsule p.o., 0-2, 2-4 and 4-8 h fractions were collected and urine volumes were recorded. Urinary excretion of 7-hydroxycoumarin occurred to a similar extent in healthy adults and children. The first 2-h 7OHC excretion was decreased by 26% (P < 0.05) and total (0-8 h) 7OHC excretion was decreased by 37% (P<0.01) among HVA-positive adults (age range 15-40 years) compared with the values obtained from healthy volunteers. In 11 HVA-positive children (age 6-10 years), the first 2-h 7OHC excretion was only 20% (P < 0.0001) and the total 7OHC excretion 28% (P < 0.0001) of the value observed in healthy controls. These results suggest that (i) an acute HVA decreases the metabolic clearance of drugs such as coumarin which are rapidly metabolised by CYP2A6 and (ii) this decrease is even more prominent in children. Such metabolic responses may be of clinical importance and may also interfere with other drug therapy in these patients.

Differential pulse voltammetric determination of 7-hydroxycoumarin in human urine.

The electrochemical behaviour of 7-OH-coumarin at the bare glassy carbon electrode has been studied using differential pulse voltammetry, and based on anodic detection of this metabolite at 0.66 V (vs SCE) using DC amperometry, a method has been developed for the determination of 7-OH-coumarin levels in urine samples, and a pharmacokinetic profile established.

Naringenin and interindividual variability in interaction of coumarin with grapefruit juice.

UNLABELLED: Grapefruit juice has been shown to enhance oral bioavailability of several drugs including coumarin. The degree of the interaction is highly variable among the individuals. OBJECTIVE: The aim of the study was to evaluate the interindividual variability in the pharmacokinetic profile of three components of grapefruit juice (naringin/naringenin, scopoletin, umbelliferone) and to compare it with the pattern of coumarin-grapefruit juice interaction. STUDY DESIGN: A two-set clinical study with the participation of 18 healthy volunteers was designed. In the first set of the experiment the total renal recovery of naringenin, scopoletin and umbelliferone within 13 hours after the intake of 1L grapefruit juice was estimated. Four individuals, who had demonstrated extremely high or extremely low excretion of the metabolites in the first set, were selected for the second set. The subjects took 10 mg coumarin with 1L grapefruit juice vs 10 mg coumarin with 1 L water in a cross-over manner. The interaction pattern was evaluated according to the time-course curves of 7-hydroxycoumarin (main metabolite of coumarin) excreted with urine. The detailed time-course excretory profiles of naringenin and scopoletin from grapefruit juice were also obtained. RESULTS: The screening demonstrated a significant interindividual variability in the renal excretion of naringenin (max/min > 15), scopoletin (max/min = 6.2), umbelliferone (max/min = 3.3). The interaction between coumarin and grapefruit juice has been observed by increase in the total recovery of 7-hydroxycoumarin up to 3 mg and by delay in time of its excretion by 2-3 hours. This interaction has been observed in 3 of 4 subjects and correlated with naringenin amounts in the urine. The mechanism and the sites of the interaction, as well as the causes for its wide interindividual variability are discussed in the paper.

Determination of coumarin metabolism in Turkish population.

Cytochrome P450 2A6 is an important human hepatic P450 which activates precarcinogens and oxidizes some drug constituents such as coumarin, halothane, and the major nicotine C-oxidase. Genetic polymorphism exists in the CYP2A6 gene. CYP2A6*1 (wild type) is responsible for the 7-hydroxylation of coumarin. The point mutation (T to A) in codon 160 leads to a single amino acid substitution (Leu to His) and the resulting protein, CYP2A*2 is unable to 7-hydroxylate coumarin. Gene conversion in exons 3, 6, and 8 between the CYP2A6 and the CYP2A7 genes creates another variant, CYP2A6*3. In this study, healthy male and female Turkish volunteers (n = 50) were administered 2 mg coumarin, and urine samples were analyzed for their content of the coumarin metabolite, 7-hydroxycoumarin (7OHC), by high-performance liquid chromatography (HPLC). Genetic polymorphism for CYP2A6 was detected by using two-step polymerase chain reaction (PCR) to identify CYP2A6*1, CYP2A6*2, and CYP2A6*3 in 13 of these subjects. The percentage of the dose excreted of total 7OHC in relation to CYP2A6 genotype and excretion of nicotine/cotinine was also evaluated to demonstrate the role of CYP2A6 in nicotine metabolism. The majority of Turkish subjects (68%) excreted less than 60% of the 2-mg dose as coumarin metabolite. The allelic frequencies were detected as 0.88 for CYP2A6*1 allele; 0.12 for CYP2A6*3 allele in 13 individuals. No heterozygous and homozygous individuals were identified for the CYP2A6*2 allelic variant. Phenotyping and genotyping for drug metabolizing enzymes are of great importance in studies correlating precarcinogen activation or drug metabolism to the CYP2A6 genotype in smoking behavior when populations are investigated.

Age and CYP3A4 and CYP2A6 activities marked by the metabolism of lignocaine and coumarin in man.

The effect of age on human liver drug-metabolizing ability was investigated by using probe drugs, metabolized by specific isozymes in liver, as an index. Formation of monoethylglycinexylide (MEGX) after i.v. infusion of lignocaine (1 mg/kg), metabolized by CYP3A4, and excretion of 7-hydroxycoumarin (7-OHC) after oral coumarin (5 mg) administration, hydroxylated by CYP2A6, were investigated in healthy young (< 25 years) and elderly (> 65 years) women and men (n = 10 in each group). MEGX content in young subjects (men 57.8 +/- 11.3 and women 52.9 +/- 13.1 ng/ml) did not diverge significantly but was reduced in elderly subjects (men 43.57 +/- 15.8 and women 29.2 +/- 13.6 ng/ml, p < 0.05 and 0.01, respectively). 7-OHC excretion at 2 h averaged 68.1 +/- 13.1 per cent (men) and 65.0 +/- 18.3 per cent (women) of the dose given in young subjects and was delayed in elderly persons (men 46.5 +/- 16.3 per cent and women 44.8 +/- 18.3 percent, p < 0.01 and 0.05, respectively). The change in probe drug metabolism was related to age (MEGX, r = -0.473 (men) and -0.682 (women) and 7-OHC; r = -0.690 (men) and -0.565 (women)). MEGX formation was reduced by 0.92 microgram/L per year and 7-OHC excretion by 0.85 per cent per year. The results indicate a decrease of CYP3A4 and CYP2A6 metabolic activities with age.

Relative bioavailability of coumarin from cinnamon and cinnamon-containing foods compared to isolated coumarin: a four-way crossover study in human volunteers.

SCOPE: Cassia cinnamon contains high levels (up to 1 %) of coumarin. Heavy consumption of this spice may result in a dose exceeding the tolerable daily intake (TDI). In this context, the question was raised whether coumarin in the plant matrix of cinnamon has the same bioavailability as isolated coumarin. METHODS AND RESULTS: A four-way crossover study was performed, in which the same dose of 12 mg coumarin was administered in different formulations to 24 healthy volunteers. The relative extent of absorption measured as urinary excretion of the main metabolite 7-hydroxycoumarin (7OHC) was found to be 62.8% for isolated coumarin in a capsule (reference), 56.0% for cinnamon in capsules, 66.1% for cinnamon tea, and 54.7% for cinnamon in rice pudding (means, n=23, observation period 8 hours). Additionally, 7OHC plasma levels were measured for 105 minutes after administration and revealed a fast absorption of coumarin from cinnamon tea leading to the highest peak concentrations. CONCLUSION: The relative extent of absorption of coumarin from powder of cassia cinnamon is only slightly lower than that of isolated coumarin. Therefore, the TDI of coumarin can be used for risk assessment of coumarin exposure from cinnamon-containing meals.

Evaluation of coumaphos exposure among tick eradication workers.

OBJECTIVE: To evaluate both the cholinesterase monitoring program and newer field methods of determining coumaphos exposure among tick eradication workers. METHODS: Measured blood cholinesterase by the Ellman and field testing methods and tested urine for chlorferon pre- and postshift; conducted personal air sampling, patch sampling of clothing, and wipe sampling of hands for coumaphos. RESULTS: Fifteen workers had normal plasma cholinesterase and acetylcholinesterase levels. No significant changes occurred pre- to postshift. High correlation was found between plasma cholinesterase and acetylcholinesterase levels by field testing and Ellman methods (r = 0.91, P < 0.01 and r = 0.63, P < 0.01, respectively). Chlorferon levels rose 4 to 6 hours after use (P < 0.01). Airborne coumaphos was detected in only one sample, in a trace amount. The majority of patch and hand wipe samples detected coumaphos. CONCLUSIONS: Dermal exposure to coumaphos resulted in significant increases in urinary metabolites of coumaphos.

Direct determination of 7-hydroxycoumarin and 7-hydroxycoumarin-glucuronide in urine by using capillary electrophoresis.

A method has been developed for the direct determination of 7-hydroxycoumarin (7HC) and 7-hydroxycoumarin-glucuronide (7HCG) in urine without sample clean-up. Separation was carried out in 90% 100 mmol l-1 phosphate buffer, 11 mmol l-1 deoxycholic acid (sodium salt) and 10% acetonitrile, on a 47 cm uncoated silica capillary at 20 kV with detection of the analytes at 320 nm. The linear detection range for concentration versus peak area for the assay is from 0 to 100 micrograms ml-1 for both analytes, with a limit of quantitation of 2 micrograms ml-1 for 7HC and 5 micrograms ml-1 for 7HCG in urine. Inter- and intra-assay results showed sr values in peak areas of between 0.5 and 13%. The method was applied to the direct determination of 7HC and the glucuronide conjugate in urine from two volunteers administered with 250 mg of coumarin. The samples were also analysed by another CE method and by using HPLC. There was no statical difference between the results determined by each of the methods. Up to 83% of the coumarin administered was excreted as 7HC or 7HCG. The majority of the 7HC excreted was in the glucuronide form (98%) with 2% occurring as free 7HC.

Comparison of a novel thin-layer chromatographic-fluorescence detection method with a spectrofluorometric method for the determination of 7-hydroxycoumarin in human urine.

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.

Spectrofluorimetric method for the quantification of 7-hydroxycoumarin in urine and plasma using both extracted and unextracted samples.

A sensitive spectrofluorimetric assay has been developed for the determination of 7-hydroxycoumarin (7-OHC) in urine and plasma. Assay systems for urine and plasma were developed and compared with regard to linearity, accuracy, precision, limit of quantification, percentage recovery and assay time. The amount of 7-OHC conjugated to glucuronide and excreted in the urine, was determined following treatment with beta-glucuronidase (5000 U ml(-1);1 U = 16.67 nkat) for 30 min at 37 degrees C. This allows the urinary concentration of free, total and conjugated 7-OHC to be determined. Samples were extracted with diethyl ether and a suitable aliquot of reconstituted extract diluted in 0.1 mol 1(-1) phosphate buffered saline (PBS), pH 10.0 and transferred to a 96 well microtitre plate. The 7-OHC concentration was also determined without prior solvent extraction. Aliquots (20 microl) of urine and plasma samples were transferred to a 96 well microtitre plate and dilutes to 200 microl with PBS, pH 10.0. The fluorescence intensity was determined at excitation and remission wavelengths of 370 and 450 nm, respectively, for both extracted and unextracted samples. Linear ranges of 7-OHC in urine and plasma, using either method, were found to be 0.5-10 and 10-100 micrograms ml(-1). Inter- and intra-day precision studies, using both methods, demonstrated relative standard deviations of below 10% across the linear range. The limit of quantification of 7-OHC in urine and plasma was 0.5 micrograms ml(-1) using both methods. Both methods have been used successfully to determine the concentration of 7-OHC excreted in the urine of patients. The percentage of the dose recovered as 7-OHC over a 24 h period was 92-98%, using unextracted and extracted samples, respectively, with 98% of this recovered as the glucuronide conjugate. The two methods are a significant improvement on a previously described method and provide an alternative to high-performance liquid chromatography.

The excretion of coumarin and hydroxycoumarins by the avian kidney in vivo.

The mechanics involved in the renal excretion of C14-labeled coumarin (C), 7-hydroxycoumarin (7HC) and 4-hydroxycoumarin (4HC) were studied in unanesthetized chickens by use of the Sperber technique. Analysis of urine collected during unilateral portal infusion of C, 4HC or 7HC indicated that the chicken excreted these coumarins almost entirely in conjugated form. Both 7HC and 4HC appeared in the urine only as the corresponding glucuronide. During C infusion, 84% of excreted radioactivity was identified as 7HC-glucuronide with only 8% appearing as a conjugate of open-ring metabolite o-hydroxyphenylacetic acid (HPAA). This demonstrated that, unlike other commonly-used laboratory animals, the chicken excretes C in a manner similar to man and suggests that this species is the most appropriate model for C pharmacodynamic studies applicable to humans. Comparative analysis of urines from the infused and control side kidneys indicated that neither C or 4HC undergoes net tubular secretion. In contrast 7HC was excreted by an active process which was significantly inhibited by simultaneous probenecid infusion. In addition it was demonstrated that the renal tubule cells conjugate 7HC as it crosses from blood to lumen suggesting that the kidney may contribute to the metabolism of C.