effects of theophylline on caffeine biotransformation ...

Canadian Journal of Pure and Applied Sciences Vol. 12, No. 2, pp. 4475-4480, June 2018 Online ISSN: 1920-3853; Print ISSN: 1715-9997 Available online at www.cjpas.net

EFFECTS OF THEOPHYLLINE ON CAFFEINE BIOTRANSFORMATION BY CYP1A2 IN HUMAN LIVER MICROSOMES *Farhana Ali, Zeyad Alehaideb and Francis CP Law Department of Biological Sciences, Simon Fraser University, British Columbia V5A 1S6, Canada ABSTRACT The aim of this in vitro study was to determine theophylline (1,3-dimethylxanthine) inhibition of human cytochrome P450 1A2 isoenzyme and measure the kinetic parameters of IC50 and Ki. Caffeine (1,3,7-trimethylxanthine) was used as the substrate of CYP1A2. Paraxanthine (1,7-dimethylxanthine), a metabolite from 3-N-demethylation of caffeine, was measured to determine the kinetic parametersof KM, Vmax, IC50 and Ki. The measured KM and Vmax of caffeine 3-Ndemethylation was 0.66 ± 0.06 mM caffeine and 106.3 ± 3.4 ng paraxanthine/hour/mg protein, respectively. The measured IC50 and Ki of theophylline were 75.8 ± 5.2 µM and 0.41 ± 0.03 µM, respectively. The results of this study demonstrate that theophylline is a competitive inhibitor of caffeine 3-N-demethylation, and predicts potential in vivo drug-drug interaction of theophylline on P-450 CYP1A2 substrates. Keywords: Caffeine metabolism, Theophylline, Enzyme Kinetics, Cytochrome P450 inhibitors.

INTRODUCTION Caffeine (1,3,7-trimethylxanthine) is a xanthine alkaloid that is a Central Nervous System stimulant. It is the most widely used psychoactive substance used recreationally and medicinally (in cold and headache remedies) to restore mental alertness (Graham, 2001; Solinas et al., 2002). Caffeine has been found to possess neuroprotective properties (Kota and Daniel, 2008). Caffeine is principally biotransformed in the liver by the cytochrome P450 oxidase enzyme system (Fig. 1); especially by the 1A2 isoenzyme into three primary metabolic dimethylxanthines: paraxanthine (84%), theobromine (12%) and theophylline (4%) each with their own effects in the body (Krul and Hageman, 1998). Paraxanthine (1,7-dimethylxanthine) has the effect of increasing lipolysis leading to elevated glycerol and free fatty acid levels in the blood plasma. Theobromine (3,7dimethylxanthine) dilates blood vessels and increases urine volume. Theophylline (1,3-dimethylxanthine) relaxes smooth muscles of the bronchi and is used to treat asthma. Each of these metabolites are further metabolized and then excreted in the urine. Numerous studies (Ha et al., 1996; Peterson et al., 2006; Kota and Daniel, 2008) have shown that caffeine is primarily biotransformed by the CYP1A2 isoenzyme and 3-N-demethylation to paraxanthine. Moreover, the other oxidation pathways of caffeine may be mediated, at least partly, by P450 *Corresponding author e-mail: [email protected]

isoenzymes different from CYP1A2. Caffeine is an established marker substrate for testing CYP1A2 activity using 3-N-demethylation (Ha et al., 1996; Petersen et al., 2006) and 1-N-demethylation (Kota and Daniel, 2008) in humans. Theophylline, also known as dimethylxanthine, is a bronchodilator and respiratory stimulant that is effective in the treatment of acute and chronic asthma, CheyneStokes respirations, and apnea/bradycardia episodes in newborns. It is also used as an adjunct in the treatment of congestive heart failure and acute pulmonary edema (Hendeles and Weinberger, 1983). Theophylline is metabolized extensively in the liver, up to 90% in adults. It undergoes N-demethylation and 8-hydroxylation via cytochrome P450 1A2 into 1-methylxanthine, 3methylxanthine and 1,3-dimethyluric acid. 1,3dimethyluric acid is also catalyzed by CYP2E1 (Haley, 2008). The therapeutic range of theophylline is 10-20 mg/l; however, it is many times greater than the levels attained from caffeine metabolism (just ~4% biotransformed from caffeine) (Hendeles and Weinberger, 1983). The objective of our study was to determine the effects of theophylline on caffeine biotransformation pathways, specifically 3-N-demethylation, by CYP1A2 isoenzyme using human liver microsomes.

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Fig. 1. Caffeine biotransformation by human P-450 cytochromes. Paraxanthine is the major metabolite of caffeine 3-Ndemethylation (Kot and Daniel, 2008). MATERIALS AND METHODS Chemicals and Biological Materials Caffeine, Theophylline, Adenine (6-aminopurine), 7-[βhydroxyethyl] theophylline and β-nicotinamide adenine dinucleotide phosphate (β-NADPH) were obtained from Sigma-Aldrich (St. Louis, MO). Sodium Phosphate monobasic/dibasic and Ammonium Sulphate were obtained from Anachemia (Norman Lachine, QC). All other chemicals and solvents used were of HPLC/analytical grade. Pooled human liver microsomes (HLM) were purchased from Becton Dickinson (NYC, NY). The protein concentration was 10mg/ml. HLMs were stored at -70°C until used for the experiment. Double distilled water was used with the resistivity of 18.2 MΩ. High Performance Liquid Chromatography Samples were analyzed using an Agilent 1090 HPLC (Palo Alto, CA) equipped with an Agilent ZORBAX

Eclipse Plus-C18 95 Å reverse-phase column (4.6 x 250 mm i.d., 5μm), a vacuum degasser, binary pump, autosampler, and an ultraviolent detector (UVD). The HPLC system was controlled by HP1090 series Chemstation® software. Caffeine and its metabolites were separated using a modified method by Bispo et al. (2002). The isocratic mobile phase consisted of methanol: water: acetic acid (75:20:5 v:v:v) with the flow rate of 0.7ml/min. The column was kept thermostated at the room temperature and xanthines were detected at 273 nm with a UVD (Fig. 2). Paraxanthine linearity was obtained using B.E.N. Software (DIN 32645) developed by the Institute of Legal Medicine and Traffic Medicine, Germany. The internal standard method, with at least five paraxanthine concentration points, was used to quantify paraxanthine amount produced in each incubation. Caffeine metabolites were quantified using internal standard of 7-[βhydroxyethyl] theophylline for caffeine biotransformation assay, and adenine (6-aminopurine) for theophylline inhibition study. The coefficients of paraxanthine linearity

Ali et al. was > 0.99. Results were reported as mean ± SD of three independent analyses. Caffeine Biotransformation Assay The assay’s method was adopted from Ha et al. (1996) with some modifications. Caffeine (0.128 - 32.78 mM), dissolved in water, was incubated in a sodium phosphate buffer of pH 7.4, in the presence of 1.12 mM NADPH in a final volume of 512.5 µl. After 2 min of pre-incubation at 37 °C, the reaction was started with the addition of 0.25

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mg HLM. After the 2 hr incubation at 37°C, the reaction was stopped by the addition of 50µl 2M HCl. Internal standard 7-[β-hydroxyethyl] theophylline, 0.5g ammonium sulfate and 7ml dichloromethane: isopropanol (80:20 v/v) were added after. The reaction mixture was shaken for 10 min and then centrifuged for 5 min at 3500 rpm. The organic phase was transferred into a test tube and evaporated to dryness. The residue was dissolved in 150 µl of the mobile phase and 120 µl of the solution was injected into the HPLC column for analysis.

Fig. 2. HPLC chromatogram showing separation of caffeine (4) and its metabolites: theobromine (1) and paraxanthine (2). 7-[β-hydroxyethyl] theophylline (3) was used as an internal standard in caffeine biotransformation assay. Theophylline Inhibition on Caffeine Metabolism Assay Methods for the determination of IC50 and Ki were also adopted from Ha et al. (1996) with the following modifications. Caffeine, Theophylline and NADPH (1.60 mM) were prepared in a 2% Ethanolic Sodium Phosphate buffer (pH 7.4) and same buffer was used in incubations to give a final volume of 500 µl. Caffeine concentration of 864 µM and theophylline concentrations of 977.60, 97.76 and 9.77 µM were used for the IC50 study. For the

Ki experiment, a 3x3 design method was adopted from Kakkar et al. (2000), with caffeine concentrations of 432, 554 and 864 µM and Theophylline concentrations of 9.7, 97.8 and 978 µM. After 2 min of pre-incubation at 37°C, the reactions were started with the addition of 1.0 mg HLM and stopped after 2 hr of incubation at 37°C with the addition of 50 µl of 2M HCl. Internal standard Adenin (6-aminopurine) and 0.5g ammonium sulfate were added. The reaction mixture was extracted twice with 4 ml of

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ethyl acetate. The rest of the procedure followed caffeine biotransformation assay. Data Analysis

CYP1A2 Activity Velocity (ng Paraxanthine/Hour/mg Protein)

GraphPad Prism version 5.0 software was used to calculate the KM, Vmax, IC50 and Ki from the incubation results. Michaelis-Menten kinetic model was used to determine KM and Vmax values for Caffeine metabolism. The IC50 value was obtained using a non-linear regression fit from the activity versus concentration curves from experimental analysis of % inhibition of caffeine metabolism in the presence of different theophylline concentrations. The Ki value for theophylline was

obtained by the competitive inhibition, non-linear regression model with a constraint on the Vmax to get a better fit. Results were reported as mean ± SD of three independent experiments. RESULTS AND DISCUSSION Figure 3 demonstrates the non-linear regression of caffeine concentrations (μM) versus enzyme activity rate (ng paraxanthine/hour/mg protein) with an R square > 0.99. The calculated KM and Vmax of caffeine 3-Ndemethylation were 0.66 ± 0.06 mM caffeine and 106.3 ± 3.4 ng paraxanthine/hour/mg protein, respectively.

100 80 60 40 20 0 0

1

2

3

4

5

Caffeine (mM) Fig. 3. KM and Vmax obtained from non-linear regression of caffeine biotransformation by human P-450 CYP1A2 (3-Ndemethylation). Figure 4 shows the non-linear regression of log theophylline concentrations (µM) versus enzyme inhibition activity percentage (% inhibition) with an R square > 0.99. The measured IC50 of theophylline on caffeine 3-N-demethylation was 75.8 ± 5.2 µM.

Figure 5 shows the three non-linear regressions of caffeine concentration versus P-450 CYP1A2 enzyme activity rate in the presence of different theophylline concentrations with an average R square of 0.79. The measured Ki of theophylline was determined to be 0.41 ± 0.03 µM from the graph.

% Inhibition (CYP1A2 Activity)

Ali et al.

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80 60 40 20 0 -15

-10

-5

0

5

Log [Theophylline uM] Fig. 4. IC50 obtained from non-linear regression of log theophylline concentrations versus P-450 CYP1A2 enzyme inhibition activity percentage.

Fig. 5. Ki obtained using three non-linear regressions of caffeine concentrations versus P-450 CYP1A2 enzyme activity rate in the presence of different theophylline concentrations.

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Table 1 summarizes the results obtained from: the caffeine biotransformation assays (KM, Vmax), the % inhibition of CYP1A2 activity by theophylline (IC50) and

effects of theophylline biotransformation (Ki).

inhibition

on

caffeine

Table 1. Enzyme kinetic parameters obtained from paraxanthine formation rate. Parameters Values KM 0.66 ± 0.06 mM Caffeine Vmax 106.3 ± 3.4 ng paraxanthine/hour/mg protein IC50 75.8 ± 5.2 µM Theophylline Ki 0.41 ± 0.03 µM Theophylline CONCLUSION With higher concentrations of theophylline present in caffeine incubations, the higher inhibition of caffeine 3-Ndemethylation by CYP1A2 was observed. The results also demonstrate that theophylline acts as a competitive inhibitor in the metabolism of caffeine by the CYP1A2 enzyme in the human liver microsomes. With the results showing theophylline affecting caffeine metabolism, it is very likely that theophylline may also affect metabolism of other caffeine metabolites such as theobromine. CYP1A2 metabolizes many drugs and carcinogens (Landis et al., 2018) and is also important in caffeine and theophylline metabolism. Thus these results can be used to further study the drug-drug interaction containing these xanthines. REFERENCES Bispo, MS., Veloso, MCC., Pinheiro, HLC., De Oliveira, RFS., Reis, JON. and De Andrade, JB. 2002. Simultaneous determination of caffeine, theobromine and theophylline by high-performance liquid chromatography. Journal of Chromatographic Science. 40 (1):45-48. Graham, TE. 2001. Caffeine and Exercise. Sports Medicine. 31 (11):785-807. Ha, HR., Chen, J., Krähenbühl, S. and Follath, F. 1996. Biotransformation of caffeine by cDNAexpressed human cytochrome P-450. European Journal of Clinical Pharmacology. 49 (4):309-315. Haley, TJ. 2008. Metabolism and pharmacokinetics of Theophylline in human neonates, children, and adults. Journal of Drug Metabolism Reviews. 14 (2):295-335. Hendeles, L. and Weinberger, M. 1983. Theophylline: "A state of the art" review. Pharmacotherapy. 3 (1):2-44. Kakkar, T., Pak, Y. and Mayersohn, M. 2000. Evaluation of a minimal experimental design for determination of enzyme kinetic parameters and inhibition mechanism.

Journal of Pharmacology and Experimental Therapeutics. 293 (3):861-869. Kota, M. and Daniel, WA. 2008. The relative contribution of human cytochrome P450 isoforms to the four caffeine oxidation pathways: An in vitro comparative study with cDNA-expressed P450s including CYP2C isoforms. Biochemical Pharmacology. 76 (4):543-551. Krul, C. and Hageman, G. 1998. Analysis of urinary caffeine metabolites to assess biotransformation enzyme activities by reversed-phase high-performance liquid chromatography. Journal of Chromatography B. 709 (1):27-34. Landis, WG., Sofield, RM. and Yu, Ming-Ho. 2018. Introduction to Environmental Toxicology: Molecular Substructures to Ecological Landscapes. (5th edi.). CRC Press. Boca Raton, Florida, USA. 178-181. Petersen, M., Halling, J., Damkier, P., Nielsen, F., Grandjean, P., Weihe, P. and Brøsen, K. 2006. Caffeine 3-N-demethylation (CYP1A2) in a population with an increased exposure to polychlorinated biphenyls. European Journal of Clinical Pharmacology. 62 (12):1041-1048. Solinas, M., Ferré, S., You, ZB., Karcz-Kubicha, M., Popoli, P. and Goldberg, SR. 2002. Caffeine induces dopamine and glutamate release in the shell of the nucleus accumbens. Journal of Neuroscience. 22 (15):6321-6324. Accepted: June 1, 2018 Copyright©2017, This is an open access article distributed under the Creative Commons Attribution Non Commercial License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.