Short Communication In Vitro Interaction of Codeine and Diclofenac

There is very limited knowledge about possible pharmacokinetic interactions between opioid analgesics and nonsteroidal antiinflammatory drugs (NSAIDs), which are commonly used in combination for the treatment of chronic pain. The major metabolic pathway of the weak opioid codeine is glucuronidation to codeine6-glucuronide. Therefore we investigated the influence of the NSAID diclofenac on the formation of codeine-6-glucuronide in vitro, using human liver tissue homogenate. The formation of codeine-6-glucuronide exhibited single enzyme Michaelis-Menten kinetics with an average Vmax of 93.6 6 35.3 pmol/mg/min. A noncompetitive inhibition of codeine-6-glucuronidation by diclofenac was observed with an average Ki of 7.9 mM. These in vitro findings suggest that a pharmacokinetic interaction occurs in vivo, which has to be confirmed by an interaction study in human subjects. It can be speculated that in case of inhibition of glucuronidation, the amount of codeine available for other pathways especially O-demethylation to morphine is increased, resulting in higher morphine serum levels and therefore higher analgesic efficacy. The weak opioid codeine is widely used in the management of pain. The major metabolic pathway of codeine is the formation of codeine6-glucuronide, to a minor extent codeine is metabolized via O-demethylation to morphine and via N-demethylation to norcodeine (Yue et al., 1989; Chen et al., 1991; Vree and Verwey-van Wissen, 1992). There is good evidence that the analgesic effect of codeine is mediated by its O-demethylated metabolite morphine. This biotransformation step is catalyzed by cytochrome P450 2D6, which exhibits a genetic polymorphism. Five to 10% of the population who are designated as “poor metabolizers” do not express the functional enzyme. As a consequence after the administration of codeine no analgesia is observed since only trace amounts of morphine are formed (Poulsen et al., 1996; Eckhardt et al., 1998). The World Health Organization proposes a three-stage approach for the treatment of chronic pain, where on step two weak opioids are to be combined with nonsteroidal anti-inflammatory drugs (NSAIDs). There are several studies demonstrating the benefit of the combination of NSAIDs with opioids in comparison to opioids alone (Hodsman et al., 1987; Strobel, 1992; Björkman et al., 1993; Mercadante et al., 1997; Montgomery et al., 1996). The additive effect is thought to be caused by known different pharmacodynamic mechanisms as opioids displaying their analgesic activity in the central nervous system via opioid receptors, NSAIDs affecting the synthesis of inflammatory prostaglandins via inhibition of the enzyme cyclooxygenase. However, pharmacokinetic interactions cannot be excluded although until now only limited knowledge about pharmacokinetic interactions between opioids and NSAIDs is available. Recently, in vitro studies with human liver microsomes revealed that glucuronidation of the structural analog dihydrocodeine was inhibited by diclofenac and naproxen (Kirkwood et al., 1998). The aim of the present study was to investigate whether codeine glucuronidation can be inhibited by diclofenac in vitro and to assess the contribution of the gut wall and the liver to the presystemic glucuronidation of codeine. Because tissue samples of liver and gut wall were obtained from the same individual, interindividual variability in expression of the UGTs involved in codeine-6-glucuronidation could be excluded. Materials and Methods Chemicals and Reagents. Codeine phosphate-hemihydrate was purchased from Merck (Germany), codeine-6-glucuronide and codeine-6-glucuronide-d3 from Lipomed, via Inresa Arzneimittel GmbH, Freiburg, Germany; diclofenac sodium, Tris-HCl, Tris, and uridine 59-diphosphoglucuronic acid (UDPGA), pepstatin, leupeptin, and EDTA were purchased from Sigma (Taufkirchen, Germany). Pefa Bloc was purchased from Roth (Karlsruhe, Germany). All other reagents and solvents were purchased from commercial sources and were of analytical reagent grade. Human Liver and Small Intestine Tissue. Liver tissue was obtained from three male donors (age range 68–79 years) undergoing duodenopancreatectomy; in two of them, additional tissue of small intestine was obtained. The tissue sampling was approved by the local Ethics Committee and each donor gave written informed consent before study entry. The study was carried out in accordance to the Declaration of Helsinki. Organ Procurement. After resection, liver biopsies were directly stored on ice and frozen in liquid nitrogen within 10 min. Frozen liver tissue was ground in a ball mill (Braun Biotech, Melsungen, Germany) at 2500 rpm for 2 min. Ground tissue was suspended in protein storage buffer (100 mM Tris, pH 7.4, 1 mM Pefa Bloc, 1 mM EDTA, 1 mg/ml leupeptin, and 1 mg/ml pepstatin) and homogenized with a motor-driven glass Teflon homogenizer at 1000 rpm for 2 min. The homogenate was further sonicated at 12 W for 30 s (Sononplus HD 200; Bandelin, Berlin, Germany). Homogenates were stored at 280°C until The study was fully supported by the Robert Bosch Foundation, Stuttgart, Germany. 1 Abbreviations used are: NSAIDs, nonsteroidal anti-inflammatory drugs; UGT, UDP-glucuronosyltransferase; UDPGA, uridine 59-diphosphoglucuronic acid; CV, coefficient of variation; Cli, intrinsic clearance. Send reprint requests to: Susanne Ammon, M.D., Institute for Pharmacology and Toxicology, Otto-von-Guericke-University Magdeburg, Leipziger Str. 44, D-39120 Magdeburg, Germany. E-mail: [email protected] 0090-9556/00/2810-1149–1152$03.00/0 DRUG METABOLISM AND DISPOSITION Vol. 28, No. 10 Copyright © 2000 by The American Society for Pharmacology and Experimental Therapeutics 91/856963 DMD 28:1149–1152, 2000 Printed in U.S.A. 1149 at A PE T Jornals on July 5, 2017 dm d.aspurnals.org D ow nladed from further use. Enterocytes were isolated from human duodenal specimens as described (Fasco et al., 1993) and homogenized as described above. Protein concentrations were determined according to the method of Smith et al. (1985). Enzyme Kinetic Studies. Incubations were performed using a modification of the assay described by Yue et al. (1990). Incubations were carried out in a final volume of 250 ml containing 5 mM to 10 mM codeine phosphate, 50 mg of protein homogenate, 5 mM MgCl2, and 100 mM Tris, pH 7.4. Reactions were started by the addition of 10 mM UDPGA and was stopped after 60 min by adding 1.5 ml of ice-cold ethanol. The incubation tubes were vortexed and placed on ice, 100 pmol of internal standard (codeine-6-glucuronide-d3) was added. After centrifugation (10,000 g, 10 min), supernatant was taken and evaporated under nitrogen gas at 37°C. The residue was dissolved in mobile phase containing 94% distilled water, 5% acetonitrile, and 1% acetic acid. Formation of codeine-6-glucuronide in relation to time and protein concentration was linear under given conditions. Production of codeine-6-glucuronide was not observed in negative controls, which did not contain UDPGA. The formation of codeine-6-glucuronide was calculated as picomoles formed per minute per milligram of protein. Inhibition Studies with Diclofenac. Inhibition studies were performed on liver homogenates with a final concentration of 0.5, 5, 10, 50, and 100 mM diclofenac (methanolic solution) on three different concentrations of codeine phosphate (0.1, 1, and 10 mM). In the final concentration 1% methanol produced an average inhibition of glucuronidation of less than 5%. Analysis of Codeine-6-Glucuronide. Codeine-6-glucuronide was determined with HPLC electrospray mass spectrometry analogous to a previously published method (Schänzle et al., 1999) with minor modifications. The mobile phases used for HPLC were: A) 1% acetic acid in water and B) 1% acetic acid in acetonitrile. HPLC separation was achieved on a LiChrospher 100 RP-18 end-capped analytical column (125 3 3-mm i.d., 5 mm particle size) at a flow rate of 0.5 ml/min using a linear gradient from 8% B to 40% B in 8 min. The mass spectrometer (HP 1100 MSD, Hewlett-Packard, Waldbronn, Germany) was operated in the selected ion monitoring mode using the respective MH ions, m/z 476 for codeine-6-glucuronide and m/z 479 for codeine-6-glucuronide-d3. Calibration points ranged from 1 to 1000 pmol of codeine-6-glucuronide. Coefficient of variation (CV) of the interassay variability (quality controls containing 10, 100, and 1000 pmol of codeine-6glucuronide) ranged between 4.7 and 9.2% (1 pmol: CV 12%), CV of the intraassay variability ranged between 0.7 and 3.2%. Data Analysis. The Michaelis-Menten constant Km and the maximum velocity Vmax for codeine glucuronidation and the inhibition constant Ki for diclofenac were calculated using the GRAPHIT 4.04 software (Erithacus Software Ltd., Surrey, UK, 1998). The untransformed kinetic data (mean of duplicates) were fitted to a one-enzyme Michaelis-Menten equation. The intrinsic clearance (Cli) was calculated as Vmax/Km. To determine diclofenacKi, the model of competitive inhibition and noncompetitive inhibition, using the equations V 5 VmaxS/(Km(1 1 I/Ki)) 1 S and V 5 VmaxS/(Km(1 1 I/Ki)) 1 (S(1 1 I/Ki)), were investigated. Goodness of fit was assessed using the x 2 test. Results and Discussion The formation of codeine-6-glucuronide in homogenate of human liver and enterocyte homogenate of exhibited Michaelis-Menten kinetics are shown in Table 1 and Fig. 1. Vmax of codeine-6-glucuronidation in liver homogenate was on average 93.6 6 35.3 pmol/ mg/min; in enterocyte homogenate, Vmax was less than 10% compared to liver (8.5 6 3.8 pmol/mg/min). The Km values were in the millimolar range (Table 1) and not different between liver (1488 6 253 mM) and enterocytes (18576 413 mM). The Km values were similar to those in human liver microsomes (Km 5 2.1 mM) reported by Yue et al. (1990). The calculated average intrinsic clearance of codeine6-glucuronidation in liver homogenate was 0.062 ml/min/mg. The intrinsic clearance in enterocyte homogenates was only 7.3% of the intrinsic clearance in corresponding liver homogenate. We therefore conclude that the small intestine contributes only to a minor extent to total codeine glucuronidation. TABLE 1 Michaelis-Menten constant (Km), maximum velocity (Vmax), inhibition constant for diclofenac (Ki), and intrinsic clearance (Cli) of codeine-6-glucuronide formation in homogenates of liver and small intestinal enterocytes Mean Km and Vmax of codeine-6-glucuronidation in liver homogenate: 1488 mM, 93.6 pmol/min/mg; mean Km and Vmax of codeine-6-glucuronidation in enterocyte homogenate (small intestine): 1857 mM, 8.5 pmol/min/mg; mean Ki of diclofenac on codeine-6-glucuronidation in liver homogenate: 7.9 mM. Homogenate Km S.E. Vmax S.E. Ki x 2 Cli mM pmol/min/mg mM ml/min/mg Liver donor no. 1 1547 55.8 89.0 1.23 9.1 1.91 0.058 Liver donor no. 2 1707 64.6 131 1.86 0.077 Liver donor no. 3 1211 40.8 60.8 0.68 6.8 2.12 0.050 Small intestine donor no. 2 2149 311 11.1 0.51 0.005 Small intestine donor no. 3 1565 219 5.80 0.28 0.004 FIG. 1. Codeine-6-glucuronide formation A, codeine-6-glucuronide formation from codeine in liver homogenate from three different donors (mean of duplicates 6S.D.). B, codeine-6-glucuronide formation from codeine in small intestine homogenate from two different donors (donors 2 and 3) (mean of duplicates 6S.D.). 1150 AMMON ET AL. at A PE T Jornals on July 5, 2017 dm d.aspurnals.org D ow nladed from