Psychopharmacology (2005) 182: 58–64 DOI 10.1007/s00213-005-0030-7
ORIGINA L I NVEST IGATION
Letizia Antonilli . Emma Petecchia . Daniele Caprioli . Aldo Badiani . Paolo Nencini
Effect of repeated administrations of heroin, naltrexone, methadone, and alcohol on morphine glucuronidation in the rat Received: 14 December 2004 / Accepted: 11 April 2005 / Published online: 29 June 2005 # Springer-Verlag 2005
Abstract Rationale: Heroin is rapidly metabolized to morphine that in turn is transformed in morphine-3-glucuronide (M3G), an inactive metabolite, and morphine-6glucuronide (M6G), a potent mu-opioid receptor (MOR) agonist. We have found that heroin addicts exhibit higher M6G/M3G ratios relative to morphine-treated control subjects. We have also shown that heroin-treated rats exhibit measurable levels of M6G (which is usually undetectable in this species) and reduced levels of M3G. Objective: We investigated the role of MOR in these effects of heroin, by examining the effects of methadone, a MOR agonist, and of naltrexone, a MOR antagonist, on morphine glucuronidation. We also investigated the effects of alcohol, which is known to alter drug metabolism and is frequently coabused by heroin addicts. Methods: Morphine glucuronidation was studied in liver microsomes obtained from rats exposed daily for 10 days to saline, heroin (10 mg/kg, i.p.), naltrexone (20–40 mg/kg, i.p.), heroin + naltrexone (10 mg/kg+ 20–40 mg/kg, i.p.), methadone (5–20 mg/kg, i.p.), or 10% ethanol. Results: Heroin induced the synthesis of M6G and decreased the synthesis of M3G. Naltrexone exhibited intrinsic modulatory activity on morphine glucuronidation, increasing the synthesis of M3G via a low-affinity/ high-capacity reaction characterized by positive cooperativity. The rate of M3G synthesis in the heroin + naltrexone groups was not different from that of the naltrexone groups. Methadone and ethanol induced a modest increase in M3G synthesis and had no effect on M6G synthesis. Conclusion: The effects of heroin on morphine glucuronidation are not shared by methadone or alcohol (two drugs that figure prominently in the natural history of heroin addiction) and do not appear to depend on the activation of MOR. L. Antonilli . E. Petecchia . D. Caprioli . A. Badiani . P. Nencini (*) Department of Human Physiology and Pharmacology “Vittorio Erspamer”, University of Rome La Sapienza, Piazzale Aldo Moro 5, 00185 Rome, Italy e-mail:
[email protected] Tel.: +39-6-49912497 Fax: +39-6-49912499
Keywords Opiates . Morphine-3-glucuronide . Morphine-6-glucuronide . Liver microsomes
Introduction Morphine metabolism in mammals mainly consists of the mutually exclusive glucuronidation of the phenolic (position 3) and alcoholic (position 6) hydroxyl moieties (Milne et al. 1996). Being more hydrophilic than the parent compound, morphine-3-glucuronide (M3G) and morphine-6glucuronide (M6G) do not easily cross the blood–brain barrier and undergo renal clearance as rapidly as creatinine (Meineke et al. 2002). Nevertheless, glucuronidation does not terminate the effects of morphine, because M6G, unlike M3G, is a potent agonist to mu-opioid peptide receptors (MORs) (Ulens et al. 2001; Penson et al. 2000; Christrup 1997). Indeed, there is now substantial evidence that M6G contributes to both analgesic and toxic effects of morphine. M6G has been shown to produce analgesia when administered systemically (Romberg et al. 2004, Skarke et al. 2003) and is now under development as a therapeutic agent (Bayes et al. 2004). Furthermore, increasingly high blood levels of M6G due to impaired excretion might account for the central nervous system (CNS)-depressant effects produced by repeated morphine administrations in patients with renal failure (Pauli-Magnus et al. 1999; Peterson et al. 1990). M6G may also be implicated in the well-known individual differences in the responsiveness to morphine. For example, the unusual resistance to morphine overdosing exhibited by some nephropatic patients has been attributed to a single-nucleotide polymorphism of the MOR gene producing reduced responsiveness to M6G, but not to morphine (Lotsch et al. 2002). Moreover, Fugelstad et al. (2003) have hypothesized that individual variability in UGT activity may help explain cases of sudden death among heroin users. Not surprisingly, M3G/M6G ratio is now routinely assessed in clinical investigations of morphineinduced analgesia. We have recently reported that the M3G/M6G ratio in plasma and urine of heroin addicts is significantly lower
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than in heroin-naive individuals treated with morphine for pain control (Antonilli et al. 2003a). Interestingly enough, low M3G/M6G ratios were observed even in abstinent heroin addicts receiving morphine for pain control. These findings are in agreement with the report by von Euler et al. (2003) of negligible urinary levels of M3G in some heroin addicts, despite the presence of 6-monoacetylmorphine (6-MAM). In an attempt to shed some light on the mechanisms responsible for the reduced M3G/M6G ratio in heroin addicts, we investigated the effects of repeated administrations of heroin on morphine glucuronidation in the rat (Antonilli et al. 2003b), a species that, under basal conditions, produces negligible levels of M6G. We found that rats that had received repeated heroin administration exhibited detectable plasma levels of M6G, which was absent in rats repeatedly treated with saline or morphine. Furthermore, microsomal preparations obtained from the livers of heroin-treated rats yielded, when incubated with morphine, measurable quantities of M6G (which was not detectable in microsomal preparations from rats treated with saline or morphine). Repeated heroin administration also affected the synthesis of M3G. Indeed, the microsomal preparations obtained from heroin-treated rats produced less M3G than those obtained from saline- or morphine-injected rats. Acute administration of heroin had no effect on the synthesis of either glucuronide. The fact that repeated administrations of medium to high doses of morphine (10–40 mg/kg per 10 days) failed to alter the synthesis of M6G may suggest that the ability of heroin to induce the synthesis of this active metabolite does not depend on its actions at MOR. The first goal of the present study was to investigate the effects of other pharmacological manipulations of the opioid system on morphine metabolism. In two independent experiments, we studied the effects on morphine metabolism of repeated administrations of naltrexone, a MOR antagonist, and of methadone, a nonphenanthrenic MOR agonist. Our interest in naltrexone and methadone was also related to the role these two drugs play in the therapy of heroin addiction. The second goal of our study was to investigate the effects on morphine glucuronidation produced by alcohol, which is known to alter the metabolism of various drugs (Klotz and Ammon 1998; Narayan et al. 1991) and is frequently coabused by heroin addicts.
Materials and methods
Treatments Experiment 1 The aim of experiment 1 was to examine the effects of repeated administrations of two different doses of the longlasting opiate antagonist naltrexone, alone or in combination with heroin on the formation of M3G and M6G. Twenty-five rats were separated into six groups. On days 1–10, the rats of the first three groups received daily i.p. injections of saline or of one of two doses of naltrexone (20 and 40 mg/kg, respectively). At the same time, the rats of the remaining three groups received ten injections of heroin (10 mg/kg), given alone or in combination with the two abovementioned doses of naltrexone. On day 10, at 14:00 h (i.e., 2 h after the last treatment), all rats were sacrificed to obtain blood samples for the quantification of heroin, 6monoacetylmorphine (6-MAM), morphine, M3G, and M6G (see below), and their livers were excised to obtain microsomal preparations (see below). Experiment 2 The aim of experiment 2 was to assess the effect of 10-day exposure to methadone or to ethanol on M3G and M6G formation in microsomal preparations. Twenty-five rats were separated into five groups. On days 1–10, the rats in the first four groups received a daily i.p. injection of 1 ml/ kg of saline or one of three doses of methadone (5, 10, and 20 mg/kg, respectively). At the same time, the rats in the fifth group were given round-the-clock access to a 10% ethanol solution as their only source of fluid. On day 10, at 14:00 h, all rats were sacrificed and their livers were excised to obtain microsomal preparations (see below). Microsomal preparations Liver microsomes were prepared as previously described (Antonilli et al. 2003b). Briefly, tissues were minced and rinsed in ice-cold 1.15% KCl and homogenized in 3 volumes of 100 mM phosphate buffer (pH 7.4) containing 0.25 M sucrose. The homogenate was centrifuged for 20 min at 9,000×g. The supernatant was further centrifuged for 60 min at 105,000×g. The resulting microsomal pellet was resuspended in 100 mM phosphate buffer containing 0.25 M sucrose to obtain a final protein concentration of 10 mg/ml.
Subjects A total of 59 male Sprague–Dawley rats (Harlan Italy, S. Pietro al Natisone, Italy), weighing 175–200 g upon arrival, was used in this study. The rats were individually housed in transparent plastic hanging cages in a temperature- and humidity-controlled colony room (lights on from 07:00 to 19:00 h) with ad libitum access to food and water.
Glucuronidation assays The morphine glucuronidation assay was performed as described by Wielbo et al. (1993) under optimal conditions with respect to time and protein concentration. In detail, microsomal preparations were suspended in 100 mM phosphate buffer (pH 7.4) to a final protein concentration of 0.5 mg/ml. Microsomes were preincubated for 20 min in
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0.05% deoxycholic acid at 5°C to achieve full enzymatic activity. Morphine concentrations ranged from 0.1 to 4 mM for the calculation of M3G kinetics, and from 0.1 to 100 mM for the calculation of M6G kinetics. The incubation mixture consisted of 15 mM UDP–glucuronic acid (UDPGA), 100 mM phosphate buffer (pH 7.4), microsomes, and substrate (morphine) to a final volume of 1 ml. The reaction was started adding UDPGA. Samples and blanks (without UDPGA) were incubated in triplicates at 37°C for 30 min and the reaction was stopped with 0.5 ml of acetonitrile and centrifuged. Supernatants were then analyzed using high-performance liquid chromatography (HPLC). Plasma samples underwent solid phase extraction on LiChrolut TSC (200 mg) columns (Merck KGaA, Darmstadt, Germany) following the procedures described by Wielbo et al. (1993). Columns were conditioned with methanol (3 ml) followed by water (3 ml) and phosphate buffer (0.01 M, pH 3.0). After loading the sample, the column was washed with 0.01 mM phosphate buffer (pH 3.0) and methanol. The analytes were eluted with 3 ml of NH4OH 2% in methanol. The eluate was evaporated to dryness at 37°C under a gentle stream of nitrogen. The dry residue was dissolved in methanol and stored at 4°C until HPLC analysis was performed. Chromatographic analyses were carried out using a HPLC system equipped with automatic sampler (model L-7250), pump (model L-7100), diode array detector (model L-7455), and fluorescence detector (model L-7480), all purchased from Merck. Data were stored and processed using appropriate software (D-7000 HPLC System Manager Ver. 3.1, Hitachi).
activity. In the presence of data satisfying the normality test, group differences for Km, Vmax, and Hill coefficient were investigated using one-way ANOVAs. When appropriate, Fisher post hoc test was used for pairwise comparisons. The Km and Vmax values in experiment 2 (see Table 3) were analyzed using nonparametric statistics (Kruskal–Wallis ANOVA and Dunn’s pairwise multiple comparison procedure) because these data failed the normality test (P=0.004 and P=0.004, respectively). Significance level was set at P≤0.05.
Drugs Morphine hydrochloride, heroin hydrochloride, 6-MAM, M3G, and methadone were obtained from Salars (Como, Italy). Morphine-6-glucuronide, uridine diphosphoglucuronic acid (UDPGA), and alamethicin were obtained from Sigma-Aldrich (Milan, Italy). Naltrexone hydrochloride was kindly provided by Sirton SPA (Villaguardia, Como, Italy). Heroin, naltrexone, and methadone were all administered by i.p. injections at 12:00 h. All solvents were HPLC grade (Merck). Ethanol was administered as a 10% solution (v/v) in tap water. Statistical analyses Group differences for plasma levels (μg/ml) of heroin, 6MAM, morphine, M3G, and M6G were investigated using one-way ANOVAs. The saturation curves for the formation of M3G and M6G by liver microsomes leveled off at the highest morphine concentrations. Km (mM), Vmax (nmol/ min/mg protein), and Hill coefficient of M3G and M6G formation were determinated by nonlinear regression analysis. A Hill coefficient greater than 1 indicates that an enzymatic reaction does not follow Michaelis–Menten kinetics, but there is positive cooperation in the catalytic
Fig. 1 Mean (±SEM) rates of formation for M3G (a, b) and M6G (c) in microsomal preparations (0.5 mg/ml proteins) from livers excised 2 h after the last of ten daily administrations of saline, heroin, naltrexone, or heroin plus naltrexone. (same animals as in Table 2). Samples were incubated with morphine in triplicates at 37°C for 30 min. For the sake of clarity, data of saline and heroin groups are represented in both panels a and b
61 Table 1 Parameters of pharmacokinetics for the synthesis of M3G and M6G in microsomal preparations obtained from rats sacrificed after 10day exposure to saline, heroin, naltrexone, or heroin+naltrexone (same animals as in Fig. 1) M3G
Saline (1 ml/kg×10) Heroin (10 mg/kg×10) Naltrexone (20 mg/kg×10) Naltrexone (40 mg/kg×10) Heroin+naltrexone (10+20 mg/kg×10) Heroin+naltrexone (10+40 mg/kg×10)
M6G
Km (mM)
Vmax (nmol/min/mg)
Hill coefficient
Km (mM)
Vmax (pmol/min/mg)
Hill coefficient
0.25±0.04 0.22±0.04 0.57±0.12 0.55±0.05 0.42±0.06 0.65±0.10*
12.15±0.67 6.77±0.49* 15.99±0.67 20.14±1.51* 16.12±0.57 18.73±1.27*
0.98±0.04 0.92±0.06 1.60±0.15* 2.09±0.20* 1.27±0.13 1.36±0.10
n.d. 46.23±3.30 n.d. n.d. 45.22±4.59 55.07±2.89
n.d. 40.65±3.26 n.d. n.d. 22.59±3.75† 16.24±1.69†
n.d. 1.00±0.01 n.d. n.d. 0.96±0.01 0.95±0.01
Data are expressed as means±SEM n.d. Non detectable *P
