Biotransformation of caffeine, paraxanthine, theophylline, and theobromine by polycyclic aromatic hydrocarbon-inducible cytochrome(s) P-450 in human liver microsomes

The microsomal metabolism of caffeine and its primary dimethylxanthine metabolites, paraxanthine, theophylline, and theobromine, was investigated in 15 different human livers, including those from two known nonsmokers and one known smoker. At least two distinct enzymes with differing substrate affinities have the potential to catalyze most methylxanthine N-demethylations and C8-hydroxylations in vitro; however, at the low methylxanthine concentrations routinely encountered in vivo, participation by the high affinity site is expected to predominate. It appears that the high affinity enzyme is a polycyclic aromatic hydrocarbon-inducible isozyme of cytochrome P-450, based on competitive inhibition by 7-ethoxyresorufin and benzo[a]pyrene, and based on a significant (p less than 0.001) correlation between 7-ethoxyresorufin-O-deethylation and methylxanthine demethylation rates. alpha-Naphthoflavone inhibited all methylxanthine demethylations in excess of 80% in two high activity livers, whereas 8-hydroxylations were generally inhibited less. Kinetic analysis of paraxanthine 7-demethylation in four different liver preparations resulted in similar Km values of 1.2 +/- 0.5 mM (mean +/- SD), whereas Vmax values varied 8-fold, compatible with participation by the same high affinity isozyme. Notable was the high degree of inter-liver variation in metabolic rates, with the known smoker showing the second highest activity among a 20-fold range in paraxanthine demethylation rates, consistent with polycyclic aromatic hydrocarbon-related enzyme induction. Maximal inhibition of paraxanthine 8-hydroxylation by alpha-naphthoflavone left similar residual activities in the 15 liver preparations, indicating the presence of an enzyme activity that was not inducible. Furthermore, in low activity livers, more than 80% of paraxanthine 8-hydroxylation was mediated by an isozyme of cytochrome P-450 insensitive to inhibition by alpha-naphthoflavone. Our in vitro data show that the proportion of demethylation relative to hydroxylation products of paraxanthine correlate with 7-ethoxyresorufin O-deethylation rates. Taken together, the data provide a rationale for a potential in vivo marker of polycyclic aromatic hydrocarbon-inducible cytochrome P-450 activity based on a urinary metabolite ratio of paraxanthine 7-demethylation to 8-hydroxylation products after caffeine intake.     

Biotransformation of caffeine by cDNA-expressed human cytochromes P-450

Objectives: The biotransformation of caffeine has been studied in vitro using human cytochrome P-450 isoenzymes (CYPs) expressed in human B-lymphoblastoid cell lines, namely CYP1A1, 1A2, 2A6, 2B6, 2D6-Val, 2E1 and 3A4, and microsomal epoxide hydroxylase (EH). In addition, CYP 2D6-Met was also studied, in which a valine in the wild type (CYP2D6-Val) has been replaced by a methionine due to a G to A mutation in position 112. Results: At caffeine 3 mmol·l^-1, five CYPs (1A1, 1A2, 2D6-Met, 2E1 and 3A4) catalysed the biotransformation of caffeine. Among the enzymes studied, CYP1A2, which predominantly catalysed paraxanthine formation, had the highest intrinsic clearance (160 l h^-1·mmol^-1 CYP). Together with its high abundance in liver, it should be considered, therefore, to be the most important isoenzyme in caffeine metabolism. The affinity of caffeine for CYP1A1 was comparable to that of its homologue 1A2. CYP2D6-Met, which catalysed caffeine metabolism by demethylation and 8-hydroxylation, also had a relatively high intrinsic clearance (3.0 l·h^-1mmol^-1 CYP), in particular for theophylline and paraxanthine formation, with k_M values between 9–16 mmol·l^-1. In contrast, the wild type, CYP2D6-Val, had no detectable activity. In comparison, CYP2E1 played a less important role in in vitro caffeine metabolism. CYP3A4 predominantly catalysed 8-hydroxylation with a k_M value of 46 mmol·l^-1 and an intrinsic clearance of 0.60 l·h^-1·mmol^-1 CYP. Due to its high abundance in human liver, the latter CYP may contribute significantly to the in vivo formation of TMU. Conclusion: The findings of this study indicate that i) microsomes from transfected human B-lymphoblastoid cell lines give results close to those obtained with microsomes isolated from human liver, ii) at least four CYP isoforms are involved in caffeine metabolism, iii) at a substrate concentration <0.1 mmol·l^-1, CYP1A2 and 1A1 are the most important isoenzymes, iv) at higher concentrations the participation of other isoenzymes, in particular CYP3A4, 2E1 and possibly also CYP2D6-Met, are important in caffeine metabolism, and v) the nucleotide composition at position 1120 of CYP2D6 determines the activity of this isoenzyme in caffeine metabolism.Abbreviations AFMU 5-acetylamino-6-formylamino-3-methyluracil - CYP human cytochrome P-450 - PAH polycyclic aromatic hydrocarbon - 17X paraxanthine - 37X theobromine - 13X theophylline - 137U trimethyluric acid.     

Comparative pharmacokinetics of caffeine and its primary demethylated metabolites paraxanthine, theobromine and theophylline in man.

The pharmacokinetics of caffeine (CA), paraxanthine (PX), theobromine (TB) and theophylline (TP) were studied in six healthy male volunteers after oral administration of each compound on separate occasions. The total plasma clearances of CA and PX were similar in value (2.07 and 2.20 ml min-1 kg-1, respectively) as were those for TP and TB (0.93 and 1.20 ml min-1 kg-1, respectively). The unbound plasma clearances of CA and PX were also similar in magnitude (3.11 and 4.14 ml min-1 kg-1, respectively) as were those of TP and TB (1.61 and 1.39 ml min-1 kg-1, respectively). The half-lives of TP and TB (6.2 and 7.2 h, respectively) were significantly longer than those of CA and PX (4.1 and 3.1 h, respectively). The volume of distribution at steady state of TP (0.44 l kg-1) was lower than that of the other methylxanthines (0.63-0.72 l kg-1). The unbound volume of distribution of TP (0.77 l kg-1) was however the same as that of TB (0.79 l kg-1) whereas the unbound volume of distribution of PX (1.18 l kg-1) was similar to that of CA (1.06 l kg-1).     

Differences in caffeine and paraxanthine metabolism between human and murine CYP1A2

For the characterisation of murine models of CYP1A2 mediated metabolism in humans we compared the metabolism of caffeine and paraxanthine in human liver microsomes (LM) (two samples) and in LM from CYP1A2-null and wild-type mice. Inhibition experiments were carried out with the quinolones norfloxacin and pefloxacin and the substrate, caffeine. Additionally, in vivo pharmacokinetics of paraxanthine was determined in CYP1A2-null and wild-type mice. All LM produced the primary metabolites of caffeine and paraxanthine. In human LM, the main metabolite of caffeine was paraxanthine (K(M) 0.4 and 0.5 mmol L(-1)). In wild-type and CYP1A2-null mice LM, the main caffeine metabolite was 1,3,7-trimethylurate, but formation was not saturable. Apparent K(M) for paraxanthine formation from caffeine in wild-type and CYP1A2-null murine LM were 0.2 and 4.9 mmol L(-1), respectively. The main metabolite of paraxanthine was 1-methylxanthine in human (K(M) 0.13 and 0.2 mmol L(-1)) and in wild-type mice LM (K(M) 0.53 mmol L(-1)). In CYP1A2-null murine LM, the main paraxanthine metabolite was 7-methylxanthine. The quinolones competitively inhibited caffeine metabolism in human but not in wild-type or CYP1A2-null murine LM. No obvious differences were seen for blood pharmacokinetics and urinary metabolite excretion of paraxanthine between CYP1A2-null and wild-type mice. Thus, for paraxanthine, norfloxacin and pefloxacin interaction with CYP1A2 there were clear differences between mice and man. Our results suggest that an interspecies comparison is required for the metabolism of individual xenobiotics interacting with CYP1A2 prior to the use of mice models to predict its toxicity and/or pharmacological activity in man.     

Population pharmacokinetics of caffeine and its metabolites theobromine, paraxanthine and theophylline after inhalation in combination with diacetylmorphine

The stimulant effect of caffeine, as an additive in diacetylmorphine preparations for study purposes, may interfere with the pharmacodynamic effects of diacetylmorphine. In order to obtain insight into the pharmacology of caffeine after inhalation in heroin users, the pharmacokinetics of caffeine and its dimethylxanthine metabolites were studied. The objectives were to establish the population pharmacokinetics under these exceptional circumstances and to compare the results to published data regarding intravenous and oral administration in healthy volunteers. Diacetylmorphine preparations containing 100 mg of caffeine were used by 10 persons by inhalation. Plasma concentrations of caffeine, theobromine, paraxanthine and theophylline were measured by high performance liquid chromatography. Non-linear mixed effects modelling was used to estimate population pharmacokinetic parameters. The model was evaluated by the jack-knife procedure. Caffeine was rapidly and effectively absorbed after inhalation. Population pharmacokinetics of caffeine and its dimethylxanthine metabolites could adequately and simultaneously be described by a linear multi-compartment model. The volume of distribution for the central compartment was estimated to be 45.7 l and the apparent elimination rate constant of caffeine at 8 hr after inhalation was 0.150 hr(-1) for a typical individual. The bioavailability was approximately 60%. The presented model adequately describes the population pharmacokinetics of caffeine and its dimethylxanthine metabolites after inhalation of the caffeine sublimate of a 100 mg tablet. Validation proved the stability of the model. Pharmacokinetics of caffeine after inhalation and intravenous administration are to a large extent similar. The bioavailability of inhaled caffeine is approximately 60% in experienced smokers.     

CYP2A13 metabolizes the substrates of human CYP1A2, phenacetin, and theophylline.

Human cytochrome CYP2A13 shows overlapping substrate specificity with CYP2A6, catalyzing the metabolism of coumarin, nicotine, cotinine, and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone. Recently, it was found that CYP2A13 could catalyze the metabolic activations of 4-aminobiphenyl and aflatoxin B(1), which are known to be catalyzed by human CYP1A2. In the present study, we investigated the substrate specificity of CYP2A13. It was shown that CYP2A13 could catalyze ethoxyresorufin O-deethylation, methoxyresorufin O-demethylation, and phenacetin O-deethylation, which are used as marker activities for human CYP1A2. Although the intrinsic clearances (V(max)/K(m)) of the two former reactions by CYP2A13 were much lower than that of CYP1A2, the value of the last reaction by CYP2A13 was 2-fold higher than that of CYP1A2. Of particular interest was that CYP2A13 has higher affinity toward phenacetin than CYP1A2. In contrast, CYP2A6 hardly catalyzed these reactions, although the amino acid identity with CYP2A13 is as high as 93.5%. Furthermore, we found that CYP2A13 can catalyze theophylline 8-hydroxylation and 3-demethylation, which are known to be mainly catalyzed by human CYP1A2, although the intrinsic clearances were approximately one-tenth that of CYP1A2. CYP2A13 would not contribute to the systemic clearance of these drugs because CYP2A13 is hardly expressed in human liver. However, it may play a role in metabolism in local tissues such as lung or trachea. In conclusion, the results of the present study could extend our understanding of the substrate specificity of CYP2A13.     

HPC purity tests for caffeine, theophylline and theobromine]

After giving a survey of the literature of the HPLC methods for the determination of caffeine, theophylline and theobromine two methods are presented for their purity test, i.e. their detection in each other at the trace level. In the reversed-phase system the stationary phase was C18-silica and the 2:3 mixture of methanol and water was used as the eluent at a detection wavelength of 254 nm. The k' values for theobromine, theophylline and caffeine are 0,25, 0,62 and 1,12, respectively. This system enables the detection of 0,2% of theobromine and 0,1% of theophylline in caffeine and 0,2% of caffeine in theobromine or theophylline. In the normal phase system silica was used as the stationary phase while the eluent was 85:15:0,05 mixture of chloroform, dioxane and formic acid. The wavelength of the detection was 273 nm. The k' values of caffeine, theobromine and theophylline are 1,5 4,0 and 5,6, respectively. This system also enables the detection of 0,1% of theophylline and theobromine in caffeine or 0,2% of caffeine in the former. Theophyline and theobromine can be detected in each other at the 0,1% level.     

Metabolism of theophylline by cDNA-expressed human cytochromes P-450.

1. Theophylline metabolism was studied using seven human cytochrome P-450 isoforms (CYPs), namely CYP1A1, 1A2, 2A6, 2B6, 2D6, 2E1 and 3A4, and microsomal epoxide hydroxylase (EH), expressed in human B-lymphoblastoid cell lines. 2. At a high theophylline concentration of 10 mM four CYPs (1A1, 1A2, 2D6, 2E1) catalyzed the metabolism of theophylline. 3. Theophylline had the highest affinity (apparent Km range 0.2-1.0 mM) for the CYP1A subfamily and the kinetics of metabolic formation mediated by CYP1A2 indicated substrate-inhibition (Ki range 9-16 mM). 4. CYP1A2 catalyzed the demethylation of theophylline as well as its hydroxylation, and was associated with the highest intrinsic clearance (1995 l h-1 per mol CYP) to 1,3-dimethyluric acid (DMU). Therefore, this isoform can be considered to be the most important enzyme involved in theophylline metabolism in vitro. 5. CYP2E1 was responsible for a relatively high intrinsic clearance by 8-hydroxylation (289 l h-1 per mol CYP). The apparent Km value of this reaction was about 15 mM, suggesting that CYP2E1 may be the low-affinity high-capacity isoform involved in theophylline metabolism. 6. The affinity of theophylline for CYP1A1 was comparable with that of its homologue 1A2. When induced, the participation of CYP1A1 in theophylline metabolism may be important. 7. CYP2D6 played only a minor role and CYP3A4 was not active in the in vitro metabolism of theophylline. 8. Our findings confirm the major role of CYP1A2 in theophylline metabolism and explain why in vivo the elimination kinetics of theophylline are non-linear and in vitro theophylline metabolism by human liver microsomes does not obey monophasic kinetics. 9. The data suggest also that not only tobacco smoking but also chronic alcohol intake may influence theophylline elimination in man as ethanol induces CYP2E1.     

Effects of caffeine, theophylline and theobromine on scheduled controlled responding in rats.

1 Rats were trained to respond under a variable interval 30 s (VI 30) schedule of food reinforcement. Caffeine (0.32-32 mg/kg), theophylline (1.0-56 mg/kg) and theobromine (10-320 mg/kg) in general produced dose-related decreases in operant responding. At relatively low doses, caffeine (1.0 mg/kg) and theophylline (3.2 mg/kg) produced slight but nonsignificant increases in VI 30 responding. 3 The rank order of potency for producing decreases in responding was caffeine greater than theophylline greater than theobromine. 4 Daily caffeine injections (32 mg/kg, i.p.) resulted in the development of caffeine tolerance. This tolerance was characterized by a 6 fold shift to the right in the caffeine dose-effect curve. Saline substitution for the 32.0 mg/kg caffeine maintenance dose resulted in a substantial decrease in responding.     

Reactions of theophylline, theobromine and caffeine with Fenton's reagent--simulation of hepatic metabolism

Theophylline and caffeine undergo N-demethylation and hydroxylation by Fenton's reagent to give uric acid derivatives; theophylline is oxidized mainly to 1-methyluric acid, and 1,3-dimethyluric acid and 1-methyluric acid are the major products obtained from caffeine. Theobromine undergoes predominantly N-demethylation to give 7-methylxanthine. The nature of the products indicate that these reactions simulate hepatic drug metabolism.     

Reactions of theophylline,theobromine and caffeine with Fenton's reagent-simulation of hepatic metabolism

Abstract

1. Theophylline and caffeine undergo N-demethylation and hydroxylation by Fenton's reagent to give uric acid derivatives; theophylline is oxidized mainly to 1-methyluric acid. and 1,3-dimethyluric acid and 1-methyluric acid are the major products obtained from caffeine.

2. Theobromine undergoes predominantly N-demethylation to give 7-methyl-xanthine.

3. The nature of the products indicate that these reactions simulate hepatic drug metabolism.     

Easy preparative scale syntheses of labelled xanthines: caffeine,theophylline and theobromine

Several easy preparative scale (0.5–1.5 g) syntheses of deuterium labelled caffeine, theophylline and theobromine are described. Some new selective syntheses of theophylline and theobromine have been developed. Labelled xanthines are of great interest in qualitative or quantitative isotope dilution‐mass spectrometry, coupled with gas or liquid chromatography, currently performed in anti‐doping and forensic laboratories. Copyright © 2007 John Wiley & Sons, Ltd.     

Effect of diethyldithiocarbamate (DDC) and ticlopidine on CYP1A2 activity and caffeine metabolism: an in vitro comparative study with human cDNA-expressed CYP1A2 and liver microsomes

The aim of the present study was to test the effect of diethyldithiocarbamate (DDC), which is regarded as a cytochrome P450 (CYP) CYP2A6 and CYP2E1 inhibitor, and ticlopidine, an efficient CYP2B6, CYP2C19 and CYP2D6 inhibitor, on the activity of human CYP1A2 and the metabolism of caffeine (1-N-, 3-N- and 7-N-demethylation, and C-8-hydroxylation). The experiment was carried out in vitro using human cDNA-expressed CYP1A2 (Supersomes) and human pooled liver microsomes. The effects of DDC and ticlopidine were compared to those of furafylline (a strong CYP1A2 inhibitor). A comparative in vitro study provides clear evidence that ticlopidine and DDC, applied at concentrations that inhibit the above-mentioned CYP isoforms, potently (as compared to furafylline) inhibit human CYP1A2 and caffeine metabolism, in particular 1-N- and 3-N-demethylation.     

Metabolic activation by human arylacetamide deacetylase,CYP2E1, and CYP1A2 causes phenacetin-induced methemoglobinemia

Phenacetin has been used as an analgesic antipyretic but has now been withdrawn from the market due to adverse effects such as methemoglobinemia and renal failure. It has been suggested that metabolic activation causes these adverse effects; yet, the precise mechanisms remain unknown. We previously demonstrated that human arylacetamide deacetylase (AADAC) was the principal enzyme catalyzing the hydrolysis of phenacetin. In this study, we assessed whether AADAC was involved in phenacetin-induced methemoglobinemia. A high methemoglobin (Met-Hb) level in the blood was detected 1 h after administration of phenacetin (250 mg/kg, p.o.) to male C57BL/6 mice. Pre-administration of tri-o-tolylphosphate, a general esterase inhibitor, was found to decrease the levels of Met-Hb and the plasma concentration of p-phenetidine, a hydrolyzed metabolite of phenacetin. An in vitro study using red blood cells revealed that incubation of phenacetin or p-phenetidine with human liver microsomes (HLM) increased the formation of Met-Hb. To identify the enzymes involved in the formation of Met-Hb, we used recombinant enzymes and HLM treated with inhibitors in the measurement of the formation of Met-Hb. High levels of Met-Hb were observed following incubation of human AADAC with either cytochrome P450 (CYP) 1A2 or CYP2E1. Furthermore, the increased Met-Hb formation by the incubation of HLM with phenacetin was significantly inhibited to 25.1 ± 0.7% of control by eserine, a potent AADAC inhibitor. In conclusion, we found that the hydrolysis by AADAC and subsequent metabolism by CYP1A2 and CYP2E1 play predominant roles in phenacetin-induced methemoglobinemia.     

HPLC法测定复方茶碱麻黄碱片中茶碱、咖啡因和可可碱的溶出度

目的:建立高效液相色谱法测定复方茶碱麻黄碱片中可可碱、茶碱和咖啡因的溶出度的方法.方法:采用溶出度测定法(桨法),以水为溶出介质,转速为50 r · min-1,45 min时取样.采用Apollo C18色谱柱(4.6 mm ×250 mm,5 μm),以醋酸盐缓冲液-乙腈(93:7)为流动相,流速0.8 mL · ...     

Determination of CYP1A2 and NAT2 phenotypes in human populations by analysis of caffeine urinary metabolites.

The wide variations in urinary bladder and colo-rectal cancer incidence in humans have been attributed in part to metabolic factors associated with exposure to carcinogenic aromatic and heterocyclic amines. Cytochrome P-4501A2 (CYP1A2), which catalyses N-oxidation, and acetyltransferase (NAT2) which catalyses N- and O-acetylation, both appear to be polymorphically distributed in human populations; and slow and rapid NAT2 phenotypes have been implicated as risk factors for these cancers. Caffeine has also been shown to undergo 3-demethylation by CYP1A2, and it is further acetylated to 5-acetylamino-6-formylamino-3-methyluracil (AFMU) by the polymorphic NAT2. In this report, we describe a metabolic phenotyping procedure that can be used to determine concomitantly the hepatic CYP1A2 and NAT2 phenotypes. For the NAT2 phenotype, we confirm the valid use of the urinary molar ratio of AFMU/1-methylxanthine, even in alkaline urines. For the CYP1A2 phenotype, the urinary molar ratio of [1,7-dimethylxanthine + 1,7-dimethyluric acid]/caffeine, taken at 4-5 h after caffeine ingestion, was identified from pharmacokinetic analyses of 12 subjects as being better correlated (r = 0.73; p = 0.007) with the rate constant for caffeine 3-demethylation than other previously suggested ratios. This procedure was then used to determine the CYP1A2 phenotype in subjects from Arkansas (n = 101), Italy (n = 95), and China (n = 78). Statistical and probit analyses of nonsmokers indicated that the CYP1A2 activity was not normally distributed and appeared trimodal. This trimodality allowed arbitrary designation of slow, intermediate, and rapid phenotypes, which ranged from 12-13% slow, 51-67% intermediate, and 20-37% rapid, in the different populations. A reproducibility study of 13 subjects over a 5 day or 5 week period showed that, with one exception, intraindividual variability did not alter this CYP1A2 phenotypic classification. Induction of CYP1A2 by cigarette smoking was also confirmed by the increased caffeine metabolite ratios observed in the Arkansas and Italian smokers (blonde tobacco). However, Italian smokers of black tobacco and Chinese smokers did not appear to be induced. Furthermore, probit analyses of Arkansas and Italian blonde tobacco smokers could not discriminate between phenotypes, apparently as a consequence of enzyme induction.     

Oxidation of the flavonoids galangin and kaempferide by human liver microsomes and CYP1A1, CYP1A2, and CYP2C9.

There is very limited information on cytochrome P450 (P450)-mediated oxidative metabolism of dietary flavonoids in humans. In this study, we used human liver microsomes and recombinant P450 isoforms to examine the metabolism of two flavonols, galangin and kaempferide, and one flavone, chrysin. Both galangin and kaempferide, but not chrysin, were oxidized by human liver microsomes to kaempferol, with K(m) values of 9.5 and 17.8 microM, respectively. These oxidations were catalyzed mainly by CYP1A2 but also by CYP2C9. Consistent with these observations, the human liver microsomal metabolism of galangin and kaempferide were inhibited by the P450 inhibitors furafylline and sulfaphenazole. In addition, CYP1A1, although less efficient, was also able to oxidize the two flavonols. Thus, dietary flavonols are likely to undergo oxidative metabolism mainly in the liver but also extrahepatically.     

Differential inhibition of human CYP1A1 and CYP1A2 by quinidine and quinine.

1. The inhibition of recombinant CYP1A1 and CYP1A2 activity by quinidine and quinine was evluated using ethoxyresorutin O-deethylation, phenacetin O-deethylation and propranolol desisopropylation as probe catalytic pathways. 2. With substrate concentrations near the Km of catalysis, both quinidine and quinine potently inhibited CYP1A1 activity with [I](0.5) approximately 1-3 microM, whereas in contrast, there was little inhibition of CYP1A2 activity. The Lineweaver-Burk plots with varying inhibitor concentrations suggested that inhibition by quinidine and quinine was competitive. 3. There was only trace metabolism of quinidine by recombinant CYP1A1, whereas rat liver microsomes as a control showed extensive consumption of quinidine and metabolite production. 4. This work suggests that quinidine is a non-classical inhibitor of CYP1A1 and that it is not as highly specific at inhibiting CYP2D6 as previously thought.