Reduction of alcohol drinking and upregulation of opioid receptors by oral naltrexone in AA rats

Alcohol 21 (2000) 215 ± 221

Reduction of alcohol drinking and upregulation of opioid receptors by oral naltrexone in AA rats J.H. Parkes, J.D. Sinclair* Department of Mental Health and Alcohol Research, National Public Health Institute, POB 719, FIN-00101 Helsinki, Finland Received 4 October 1999; received in revised form 24 February 2000; accepted 6 March 2000

Abstract Rats of the high-drinking AA line were given 1 mg/kg naltrexone (NTX) or vehicle orally with a stress-free procedure just before 1 h of access to 10% ethanol daily for 8 days and again, 8 h later on the first 7 days. Forebrain homogenate binding studies using 0.03 ± 6.00 nM [3H] naloxone were conducted from 1 to 4 days following treatment. NTX significantly suppressed alcohol intake, with the effect becoming progressively greater over days and continuing during the post-treatment period. Saturation binding studies in brain homogenate revealed that NTX had increased the Bmax for opioid receptors by 93%, 74%, 49%, and 28%, respectively, from post-treatment days 1 to 4 without altering Kd. Bmax was negatively correlated (r = ÿ 0.510, p = 0.008) with alcohol intake during the preceding hour, but in control rats, it was positively correlated with changes in alcohol intake over time (r = + 0.790, p = 0.020). These results are consistent with the hypothesis that opioid receptors mediate reinforcement from alcohol and that NTX reduces subsequent alcohol drinking by extinction. Opioid receptor upregulation can develop simultaneously with suppression of drinking and may partially counteract the clinical benefits from NTX in the treatment of alcoholism. D 2000 Elsevier Science Inc. All rights reserved. Keywords: Alcohol drinking; Opioid receptor; Extinction; Naltrexone; Upregulation; Supersensitivity

1. Introduction Opioid antagonists (naltrexone [NTX], naloxone, and nalmefene) have been shown repeatedly to reduce drinking and self-administration of ethanol by rats (Critcher et al., 1983; Sinclair, 1990, 1996; 1998a; HyytiaÈ & Sinclair, 1993). Double-blind, placebo-controlled clinical trials have confirmed that NTX and nalmefene administered with an appropriate protocol produce significant benefits in the treatment of alcoholism (O'Malley et al., 1992; Volpicelli et al., 1992; Balldin et al., 1997; HeinaÈlaÈ et al., 1999; Mansson et al., 1999; Mason et al., 1999). Another action of opioid antagonists is to cause an increase in opioid receptor binding density due to the upregulation of the number of opioid receptors (Pert et al., 1973; Lahti & Collins, 1978; Tang & Collins, 1978; Zukin et al., 1982; Yoburn et al., 1988, 1989). Such homeostatic compensation is a general phenomenon (Sinclair, 1981; Ruffolo, 1982). When receptors are not acti-

* Corresponding author. Tel.: +358-9-133-2857; fax: +358-9-133-2781. E-mail address: [email protected] (J.D. Sinclair).

vated for a prolonged period, either as a result of their agonist not being present or because they are blocked by an antagonist, the number of receptors is increased. The increase in opioid receptors is paralleled by an increase in the opioid agonist potency (functional supersensitivity) for both analgesia and toxic effects (Tang & Collins, 1978; Tempel et al., 1985; Yoburn et al., 1988; HyytiaÈ et al., 1999). The simultaneous increase in receptors and opioid agonist potency suggests that the new binding sites are functional. Both of the leading hypotheses for how NTX reduces alcohol drinking predict that the upregulation of opioid receptors would be clinically detrimental. The ``surfeit hypothesis'' postulates that ``an excess, or surfeit, of opioidergic activity is the critical event that enhances the frequency and extent of intake of alcohol beverages (or loss of control)'' (Reid & Hubbell, 1992, p. 129). Although there may be other ways of interpreting this idea, the simplest interpretation is that opioidergic activity is an ``event,'' which directly makes people and rats drink alcohol. NTX reduces the number of open opioidergic receptors and the potential for opioidergic activity, thus directly decreasing drinking and the risk of losing control. Upregulation of opioid receptors, in contrast, increases the

0741-8329/00/$ ± see front matter D 2000 Elsevier Science Inc. All rights reserved. PII: S 0 7 4 1 - 8 3 2 9 ( 0 0 ) 0 0 0 9 1 - 4

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potential for opioidergic activity once the receptors are no longer bound and thus increases the frequency and extent of alcohol drinking. The ``extinction hypothesis'' postulates that opioidergic activity reinforces alcohol drinking (Sinclair, 1990, 1996, 1997, 1998a,b). NTX blocks the reinforcement. Consequently, if alcohol is consumed while NTX is present, the alcohol-drinking behavior is extinguished. According to the extinction hypothesis, the number of opioid receptors does not directly affect craving, the chances of starting drinking, or loss of control, but rather the amount of reinforcement obtained after drinking and thus, the subsequent changes in the probability of drinking. The upregulation of opioid receptors during NTX treatment would be detrimental because it increases the rate of relearning if alcohol is consumed when NTX is no longer present. It is clear that NTX can under certain conditions reduce alcohol drinking and also that it can produce opioid upregulation. In the present study, however, we wanted to see if both effects could be produced at the same time. Furthermore, we wanted to see if this would happen in rats given NTX orally in a manner similar to that used clinically in humans. Only about a quarter of an oral NTX dose reaches the human brain (Kogan et al., 1977) because of high first pass metabolism. The study also provides an opportunity for testing opposing predictions from the two hypotheses for NTX actions. Both the surfeit and extinction hypotheses make similar predictions about the relation between the number of opioid receptors and alcohol consumption during the preliminary weeks of alcohol drinking and during NTX treatment of animals with simultaneous access to alcohol. They make contrasting predictions, however, about what should be expected during the first days after the end of NTX treatment. The surfeit hypothesis predicts a positive correlation between the number of opioid receptors and the amount of alcohol consumed under these conditions and all others. The extinction hypothesis, however, predicts a negative correlation between Bmax and alcohol consumption during the post-treatment period. 2. Method 2.1. Animals Male rats (n = 26) of the AA (Alko, Alcohol) line developed by selective breeding for high alcohol consumption were used. At the age of 3 months, the rats were placed in individual stainless steel cages (20  20  35 cm) in environmental rooms under conditions of constant temperature (222°C) and humidity (50 ‹ 5%) and on a 12:12 light/ dark cycle with lights off at 1800 h. Food (RM1; SDS, Witham, England) was always available. The Finnish regulations for the care and treatment of experimental animals were followed.

2.2. Alcohol consumption Rats were offered a continual free choice between tap water and a 10% (v/v) ethanol solution (prepared from 96%, v/v, mixed with tap water) in graduated 100-ml Richter tubes, with the position of the two tubes reversed once a week. After the drinking reached a stable level, at about 1 month, the alcohol availability was restricted to 1 h of access daily (Sinclair et al., 1992), provided in 20-ml Richter tubes attached to the home cages. The availability of water and food was not restricted. The consumption of water was recorded every 24 h. 2.3. Drug treatments NTX treatment began after 12 days of limited-access to alcohol. Mean alcohol drinking (as g/kg) during the last week before treatment was used as the baseline measure. The experimental (n = 18) and control groups (n = 8) were matched for baseline ethanol consumption (experimental: 0.608 ‹ 0.035 g/kg; control: 0.626 ‹ 0.082 g/kg). NTX was administered with a novel stress-free oral technique (Sinclair & Stenberg, 1995). After the limitedaccess alcohol drinking had stabilized, the animals learned to eat a cocoa-flavored sucrose paste (98% powdered sugar, 2% cocoa powder, with water added to make a paste that can be extruded from a syringe without a needle) in a measured dose (1 ml/kg). Each day, all of the animals were weighed at 0800 h and returned to their home cage, at 0830 h, they were all given the paste to eat, and then at 0840 h, all were given their 1-h access to alcohol. After a few days of training, the rats immediately came up to the front of the cage and ate the sweet paste directly from the syringe placed through the wire mesh. Subsequently, NTX (Sigma, St. Louis, MO; N-3136) was added to the paste of the experimental group (n = 18), and a measured dose of 1 mg NTX per kg body weight was provided. A second dose of 1 mg/ kg was provided at 1630 h daily, except on the last treatment day when there was no second dose. The remaining eight rats constituted the control group and received the sucrose paste vehicle at these same times. The treatment continued for eight daily sessions. After the NTX series was completed, the rats were tested in daily sessions without NTX but with paste given at these same times over a final 4-day period to determine whether any effect of NTX on alcohol drinking would persist. After each of the four post-treatment alcohol drinking sessions, four or five experimental rats and two controls were randomly selected, decapitated, and their whole brains removed. 2.4. Membrane preparation Brains were removed and frozen at ÿ80°C until used. The forebrain was homogenized in 50 mM Tris ± HCL, pH 7.4, at 25°C, with a Polytron for 1 min at the medium setting. The homogenates (300 mg/5 ml) were centrifuged

J.H. Parkes, J.D. Sinclair / Alcohol 21 (2000) 215±221

at 40,000  g (20,000 rpm Sorvall SS-34) for 10 min at 4°C. The supernatant was decanted, and the pellets were resuspended in 20 volumes of 50 mM Tris ± HCL buffer containing 1 mg/ml of bovine serum albumin (BSA; Sigma, A-3059) and 100 mM NaCl and preincubated for 30 min at 25°C to facilitate dissociation of endogenous ligand and NTX occupying the receptors (Hawkins et al., 1989). It was then centrifuged for 10 min, the supernatant discarded, and the pellet resuspended in 20 volumes of buffer and centrifuged again. The pellets were finally resuspended in 50 mM Tris ±HCL with 1 mg/ml BSA, 100 mM phenylmethylsulfonylfluoride (PMSF; Sigma P7626), and 5 mM MgCl2 without sodium to prevent a decrease in binding affinity and homogenized again. 2.5. Binding assays [3H]Naloxone binding was measured by a modification of established procedures (Hawkins et al., 1989). Randomly chosen batches of one to four samples were analyzed together. Aliquots (approximately 320± 420 mg protein in a final volume of 100 ml) of this membrane preparation were assayed in triplicate in 1-ml incubation volume tubes containing six different concentrations of [3H] naloxone (DuPont NET-719, Boston, MA) ranging from 0.03 to 6.00 nM. Nonspecific binding was determined in the presence of 10 mM cold NTX (Sigma, N3136) at 25°C, 90 min (Misra et al., 1976). The incubation was terminated by the addition of 5-ml ice-cold 10 mM Tris ±HCl, pH 7.4. Bound and free [3H] naloxone were separated by filtration through Whatman GF/B filters (Whatman International, Maidstone, UK) using a Brandel Cell Harvester (Gaithersburg, MD), followed by rinsing three times in 5 ml of ice-cold Tris ±HCl buffer. The filters were presoaked for 2 h in 0.1% polyethylenimine (Sigma, P-3143) to reduce nonspecific filter binding. The filters were air dried, immersed in 4.5 ml of Optiphase HiSafe II (Walla, UK) scintillation cocktail, and counted for radioactivity with a Wallac model 1410 liquid scintillation counter (Pharmacia, Turku, Finland). Specific binding was determined as the difference between total binding in the absence of cold ligand and nonspecific binding in the presence of the cold ligand; it was expressed as fmole [3H] naloxone bound per mg protein. Protein was determined using a microassay technique based on the method of Bradford (Bradford, 1976), using reagent purchased from Bio-Rad (Richmond, CA). 2.6. Data analysis The saturation isotherms of [3H]naloxone binding were analyzed for the estimation of Kd, and Bmax by nonlinear regression with the Prism statistical program (GraphPad Software; San Diego, CA). Differences in Bmax on different post-treatment days were tested with a one-way ANOVA, and group differences in drinking with a two-way repeated

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measures ANOVA. Specific comparisons were done with ttests. The relations between drinking and Bmax were determined with Pearson correlations. 3. Results 3.1. NTX effect on alcohol intake NTX significantly suppressed ethanol consumption during the 8-day treatment period relative to that by the control animals (F[1,22] = 34.69, p = 0.000006). As shown in Fig. 1, the suppression grew progressively stronger during the treatment period, producing a highly significant group  days interaction (F[7,735] = 9.267, p = 0.00000000005). The baseline levels of ethanol drinking during the week before treatment were 0.63 ‹ 0.08 (S.E.) g ethanol/kg body weight in 1 h for the eight controls and 0.61 ‹ 0.04 g/kg for the 18 experimental rats. Although the change in drinking from the baseline level was significant on each day, the level of significance increased with the duration of treatment. The

Fig. 1. Suppression of alcohol drinking during and after oral naltrexone treatment. The rats were given 1 mg/kg NTX orally twice daily for 7 days and in the morning of the 8th treatment day. The bars show the mean (‹ S.E.) decrease in alcohol consumption (g/kg body weight) during the 1-h access periods relative to their own mean intake during the week before treatment. Numbers on the bars represent the number of animals. Significant differences from baseline: *p < 0.05, **p < 0.01, ***p < 0.001; significant differences from vehicle control rats #p < 0.05; ##p < 0.01, ###p < 0.001.

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difference between NTX and control animals was not significant on the first treatment day but was highly significant ( p < 0.001) on the last 2 treatment days. During the 4-day post-treatment period as a whole, ethanol intake by the previously NTX-treated rats remained suppressed relative to both baseline (t[17] = 3.88, p = 0.001) and controls (t[24] = 3.04, p = 0.006). The reduction on individual days was significant on the first day without NTX (t[17] = 4.14, p = 0.0007, for the comparison with baseline; t[24] = 2.48, p = 0.02, for the comparison with controls). The reduction approached significance on the second day off of NTX (t[12] = 2.099, p = 0.058, for the comparison with baseline). Alcohol drinking on subsequent post-treatment days tended to return to the higher pre-treatment levels. There were no significant differences between groups in the their water intake (ml/kg) during treatment (35.68 ‹ 3.54 ml/kg for the controls, 36.99 ml/kg for the NTX treated rats; t[24] = 0.64, p > 0.05), after it (t[24] = 0.55, p > 0.05) or in the changes from baseline (t[24] = 1.33, p > 0.05). No measurable amounts of water were consumed during the ethanol sessions. There also were no group differences in body weight: the NTX-treated rats went from a baseline of 432 ‹ 10 (S.E.) g to 435 ‹ 9 g during treatment, and then to 439 ‹ 9 g in the post-treatment period. 3.2. NTX effect on opioid receptors Oral NTX for 8 days produced a dramatic upregulation of forebrain opioid receptors shown by a significant effect on

Fig. 3. Negative correlation between opioid Bmax and contiguous alcohol drinking after naltrexone treatment. The experimental rats (filled circles) had previously been treated with oral NTX for 8 days and then been off of it for 1 ± 4 days. Open circles represent the control rats. Alcohol drinking was measured during the hour just preceding sacrifice and binding measurement with [3H]naloxone. Although there was no clear relation between Bmax and contiguous alcohol drinking in the controls alone, NTX reduced drinking while increasing Bmax; during the days following NTX treatment, both values returned progressively toward control levels, thus producing the significant negative correlation.

Bmax (Fig. 2: F[4,21] = 162.84, p = 0.0000000000000017). Saturation binding tests in brain homogenate showed that opioid receptor density on the first day off of NTX was 93% higher than in vehicle-treated animals. The number of receptors decreased progressively with each additional day off of NTX, approaching the control level by the 4th day when there was only a 28% increase. There were no significant differences in mean Kd: 2.01 ‹ 0.55 for controls; 1.99 ‹ 0.22, 1.99 ‹ 0.37, 1.89 ‹ 0.46, and 1.95 ‹ 0.47 for the 1st, 2nd, 3rd, and 4th days off of NTX, respectively. 3.3. Correlations between Bmax and alcohol consumption Fig. 2. Recovery from NTX-induced opioid upregulation. The experimental rats were given 1 mg/kg NTX orally twice daily for 7 days and in the morning of the 8th day. Opioid binding was then measured after 1, 2, 3, or 4 days off of NTX and also in control rats. Shown are the mean ‹ S.E. values for Bmax measured as fmol/mg protein. The decrease back to control levels was highly significant (p < 0.001).

A significant negative correlation was found between each rat's Bmax value and its own alcohol intake during the last hour prior to decapitation: r = ÿ0.510, p = 0.008 (Fig. 3). The basis for the negative correlation was that NTX treatment decreased drinking but increased Bmax; then during the post-treatment period drinking increased pro-

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Fig. 4. Positive correlation between the opioid Bmax and the changes in alcohol consumption of control rats. The change shown is the one during the treatment week for the experimental animals, i.e., between the mean g/ kg consumed in the week before treatment (baseline) and that on the first 2 days after treatment (P1 and P2). The control animals here had had no specific treatment, continuing to get only the sweet paste vehicle prior to 1 h of access to alcohol. The [3H] naloxone bindings Bmax (mean, fmol/mg protein) values were obtained from the 1st to the 4th post-treatment days and are seen as representing the number of opioid receptors.

gressively toward control levels while Bmax decreased toward control levels. Among the eight control rats, Bmax correlated positively with the change in alcohol drinking during the treatment period, i.e., the difference between baseline drinking and that on the first 2 post-treatment days: r = + 0.790, p = 0.02 (Fig. 4). 4. Discussion The standard clinical procedure for treating alcoholism with NTX is to have the patients take a 50-mg pill of NTX once (O'Malley et al., 1992; Volpicelli et al., 1992) or twice (McCaul et al., 1997; Sinclair, 1997; Sinclair et al., 1998) daily. The preclinical procedure (Sinclair & Stenberg, 1995) in the present study was comparable: rats voluntarily ate a sweet paste containing 1 mg/kg NTX twice daily: first, shortly before drinking alcohol (as advised for patients in some clinical protocols (Sinclair, 1997, 1998b; Sinclair et al., 1998)) and, second, about 8 h later. The oral NTX significantly decreased the rats' alcohol intake. The magnitude of the suppression increased with each additional treatment session, and consumption remained reduced for days after the end of treatment. Similar results have been obtain previously using this oral procedure

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with nalmefene (Sinclair, 1998a; Sinclair & Stenberg, 1995) and with subcutaneous injections of naloxone (Sinclair, 1990; 1996), but this is the first report for the stress-free oral procedure (Sinclair & Stenberg, 1995) with NTX (the antagonist currently approved for treating alcoholism). The clinical results with oral NTX have been similar to those found in rats: alcohol drinking and alcohol craving decrease significantly and (if started without prior detoxification) progressively (Bohn et al., 1994; Sinclair, 1997, 1998b; Sinclair et al., 1998), and remain significantly suppressed for months after the end of treatment (Bohn et al., 1994; Mansson et al., 1999). The present study showed that such results from oral NTX could also be accompanied by significant upregulation of opioid receptors. The upregulation itself is consistent with recent findings by HyytiaÈ et al. (1999). They gave AA rats a very high dose of naloxone (72 mg/kg daily) administered continually with minipumps. They found a dramatic upregulation demonstrated, not only by an increase in opioid binding, but also functionally in a shift in the morphine ± analgesia curve and an increase in G-protein activation by opioid agonists. They found only a small reduction in alcohol drinking, the effect diminished in magnitude with time, and no continued suppression was found after the end of treatment. Results rather similar to theirs were obtained in another study with continual naloxone where the suppression of drinking diminished so much over time that it no longer was significant after the first 4 days of treatment (Crews et al., 1998; Overstreet et al., 1999). It seems likely that our procedure promoted the suppressive effect from NTX treatment and thus found increasing benefits that persisted after treatment; the other two studies (HyytiaÈ & Sinclair, 1993; Crews et al., 1998; Overstreet et al., 1999) used continual administration that is known to favor upregulation and, therefore, found only a limited reduction in drinking. In accord with the idea that opioidergic activation reinforces alcohol drinking, a highly significant positive correlation was now found between the opioid binding and the changes in alcohol consumption over time in the control rats. In contrast, a significant negative correlation was found between Bmax and the immediately preceding intake of alcohol. This finding is apparently contrary to the simple version of the surfeit hypothesis stipulating that opioidergic activation is ``event that enhances the frequency and extent of intake of alcohol beverages'' (Reid & Hubbell, 1992). The observed negative correlation is consistent with the extinction hypothesis (Sinclair, 1990). According to it, the number of opioid receptors has no direct control over the craving and drinking at that timeÐand thus, no positive correlation. The correlation is predicted to be negative because of the divergent actions of a third factor, NTX, with two separate actions: (1) extinguishing drinking and (2) upregulating opioid receptors. Extinction produces a change in the organism that persists after

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the end of NTX treatment so that alcohol drinking remains reduced. At the same time, Bmax remains increased. During the post-treatment period, alcohol drinking is relearned while Bmax goes back down to normal, therefore, resulting in a negative correlation. This interpretation might not be valid if the NTX remained in the rats' brains for several days after the end of treatment. There is evidence that the t1/2 for NTX in the human brain may be from 72 to 108 h (Lee et al., 1988). The initial t1/2 for NTX in the brains of rats, however, has been found by Misra et al. (Reid & Hubbell, 1992) to be only 18 min. This lasts for 2 h. It is followed by a terminal phase with a t1/2 of 14 h. Based on these values, the brain NTX levels in our rats at 24 h after oral intake of 1 mg/kg should have been only about 1.5 ng/g, i.e., lower than what Misra et al. described as ``barely detectable''. They also reported that very little of the active metabolite, naltrexol, is present in rat brain and none in the plasma. It should be noted that although the functional half-lives of NTX and naltrexol for their effects on alcohol drinking are not known, recent work suggests they may be shorter than what would be expected from brain or plasma levels and from morphine analgesia studies (McCaul et al., 1997). On the basis of these results, it is unlikely that persisting NTX could have been responsible the suppression of drinking 24 h after the last administration. Consistent with this conclusion, a previous study showed that drinking was not suppressed 24 h after a still larger dose of oral NTX given during abstinence to preclude extinction (Sinclair, 1990). Our finding that opioid receptor upregulation can coexist with a suppression of alcohol drinking suggests that a similar upregulation might be occurring clinically even in alcoholics whose drinking was reduced by NTX. An increase in opioidergic receptors is probably detrimental toward the clinical efficacy of NTX. It has been proposed that upregulation could eventually cause there to be more receptors than could be blocked with a standard dose of antagonist, producing tolerance to the beneficial effects on alcohol drinking (Crews et al., 1998; Overstreet et al., 1999). Even if a complete blockade were maintained when NTX is being taken, upregulation would increase reinforcement from alcohol if it were drunk without NTX, thus accelerating the relearning of the drinking behavior. Consequently, more attention might be directed towards factors in different clinical NTX protocols that affect the probability that opioid upregulation develops and then to testing whether these factors are indeed related to efficacy.

Acknowledgments The authors wish to thank Pirkko Johansson for her skillful technical assistance and Prof. H. Vapaatalo for his excellent advice and detailed comments on the manuscript.

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