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Reduction of alcohol intake by the positive allosteric modulator of the GABAB receptor, rac-BHFF, in alcohol-preferring rats
Alcohol 47 (2013) 69e73
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Reduction of alcohol intake by the positive allosteric modulator of the GABAB receptor, rac-BHFF, in alcohol-preferring rats Barbara Loi a, Paola Maccioni a, Carla Lobina a, Mauro A.M. Carai a, Gian Luigi Gessa a, Andrew W. Thomas b, Pari Malherbe b, Giancarlo Colombo a, * a b
Neuroscience Institute, National Research Council of Italy, Section of Cagliari, I-09042 Monserrato (CA), Italy Discovery Research CNS, F. Hoffmann-La Roche Ltd., Psychiatry Disease Area, Basel CH-4070, Switzerland
a r t i c l e i n f o
a b s t r a c t
Article history: Received 9 October 2012 Received in revised form 20 November 2012 Accepted 23 November 2012
Previous research has demonstrated that treatment with the positive allosteric modulator (PAM) of the GABAB receptor (GABAB PAM), rac-BHFF, suppressed lever-responding for alcohol and amount of selfadministered alcohol in Sardinian alcohol-preferring (sP) rats. The present study was designed to extend the investigation on the anti-alcohol effects of rac-BHFF to alcohol drinking behavior. To this end, sP rats were exposed to the homecage, 2-bottle “alcohol (10%, v/v) vs water” choice regimen, with unlimited access for 24 h/day. rac-BHFF was administered once daily and for 7 consecutive days at the doses of 0, 50, 100, and 200 mg/kg (i.g.). Treatment with rac-BHFF resulted in an immediate, stable, and dose-related reduction in daily alcohol intake; the overall magnitude of reduction in alcohol intake averaged approximately 25%, 40%, and 65% in 50, 100, and 200 mg/kg rac-BHFF-treated rat groups, respectively. An increase in daily water intake fully compensated the reduction in alcohol intake, so that daily total fluid intake was unaffected by treatment with rac-BHFF. Daily food intake tended to be reduced only by the highest dose of rac-BHFF. These results complement closely with previous data indicating that (a) rac-BHFF suppressed operant, oral alcohol self-administration in sP rats and (b) the prototypic GABAB PAMs, CGP7930 and GS39783, reduced alcohol drinking in sP rats. However, while the reducing effect of CGP7930 and GS39783 on the daily alcohol intake tended to vanish after the first 2e3 days of treatment, the reducing effect of racBHFF on daily alcohol intake remained unchanged over the entire 7-day treatment period. These data strengthen the hypothesis that GABAB PAMs may represent a step forward in the search for GABAB receptor ligands with therapeutic potential for alcoholism. Ó 2013 Elsevier Inc. All rights reserved.
Keywords: Positive allosteric modulation of the GABAB receptor rac-BHFF Alcohol drinking Sardinian alcohol-preferring rats
Introduction Positive allosteric modulators (PAMs) of the GABAB receptor (GABAB PAMs) represent a novel class of pharmacological agents with promising, therapeutic potential for alcoholism. Accumulating lines of experimental evidence indicate indeed that the currently available, in vivo effective GABAB PAMs invariably reduced different alcohol-related behaviors in rats and mice. Specifically, repeated treatment with the prototypic GABAB PAMs, CGP7930 and GS39783, reduced alcohol drinking in alcohol-preferring rats exposed to the 2-bottle “alcohol vs water” choice regimen (Orrù et al., 2005). Administration of CGP7930 and GS39783, as well as BHF177 and rac-BHFF (two additional GABAB PAMs synthesized more recently), reduced lever-responding for alcohol and amount of * Corresponding author. Neuroscience Institute, National Research Council of Italy, Section of Cagliari, S.S. 554, km. 4,500, I-09042 Monserrato (CA), Italy. Tel.: þ39 070 675 4342; fax: þ39 070 675 4320. E-mail address: [email protected] (G. Colombo). 0741-8329/$ e see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.alcohol.2012.11.002
self-administered alcohol in alcohol-preferring rats exposed to multiple procedures of operant, oral alcohol self-administration (Liang et al., 2006; Maccioni et al., 2007, 2008, 2009, 2012; Maccioni, Flore, et al., 2010; Maccioni, Thomas, et al., 2010). Treatment with GS39783 blocked alcohol-induced locomotor stimulation (although not alcohol-induced locomotor sensitization) in mice (Kruse, Linsenbardt, & Boehm, 2012). As predictable on the basis of their mode of action e i.e., absence of substantial intrinsic agonistic activity and activation of the GABAB receptor via synergistic enhancement of GABA effects only in those synapses in which and when endogenous GABA is present (see Froestl, 2010; Urwyler, 2011) e GABAB PAMs exert their antialcohol effects at doses far lower than those producing “undesired”, motor-incoordinating and sedative effects. For instance, CGP7930and GS39783-induced reduction in daily alcohol intake was associated to a fully compensatory increase in daily water intake (Orrù et al., 2005); acute treatment with GS39783, BHF177, and rac-BHFF reduced lever-responding for alcohol at doses that did not alter lever-responding for an alternative reinforcer such as a sucrose
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solution (with the sole exception of 200 mg/kg rac-BHFF, that reduced e although to a lower extent e lever-responding for the sucrose solution) (Maccioni et al., 2007, 2008, 2009, 2012; Maccioni, Thomas, et al., 2010). This selectivity for alcohol-related behaviors and the lack of sedative effects make GABAB PAMs clearly preferable over baclofen and other GABAB receptor agonists, the anti-alcohol effects of which in rats and mice often occur at doses not far from those producing behavioral toxicity (see Maccioni & Colombo, 2009; Vlachou & Markou, 2010). The present study was designed to provide additional clues to the preclinical, anti-alcohol profile of rac-BHFF [(R,S)-5, 7-di-tert-butyl-3-hydroxy-3-trifluoromethyl-3H-benzofuran-2-one (Malherbe et al., 2008)]. To this end, this study assessed the effect of the repeated treatment with rac-BHFF on alcohol drinking in selectively bred, Sardinian alcohol-preferring (sP) rats (see Bell et al., 2012; Colombo, Lobina, Carai, & Gessa, 2006). The procedure employed was the homecage, 2-bottle “alcohol vs water” choice regimen with unlimited access for 24 h per day, a proposed model for the “active drinking” phase of human alcoholism. Interest on racBHFF resides in the fact that it resulted to be, among the GABAB PAMs tested so far in sP rats, the most effective compound in reducing lever-responding for alcohol and amount of self-administered alcohol (Maccioni, Thomas, et al. 2010).
200 mg/kg. This dose range was identical to that used in the previous study testing rac-BHFF on operant, oral alcohol selfadministration in sP rats (Maccioni, Thomas, et al., 2010). Measured variables were: (a) daily alcohol intake (expressed in g/kg pure alcohol); (b) daily water intake (ml/kg); (c) daily total fluid intake (sum of alcohol solution and water; ml/kg); (d) daily preference ratio (percent ratio between daily intake of alcohol solution and daily total fluid intake); (e) daily food intake (g/kg). Recording was performed once daily (before the start of the dark phase) throughout the 7 consecutive days of the Treatment Phase as well as for an additional 5 consecutive days after treatment termination (Post-Treatment Phase).
Materials and methods
Alcohol intake
All experimental procedures employed in the present study were in accordance with the Italian Law on the “Protection of animals used for experimental and other scientific reasons”.
Treatment Phase e ANOVA indicated a significant effect of treatment [F(3,28) ¼ 15.51, p < 0.0001] and time [F(6,168) ¼ 5.69, p < 0.0001], and a significant interaction [F(18,168) ¼ 2.13, p < 0.01], on daily alcohol intake over the 7-day Treatment Phase. Treatment with rac-BHFF resulted in a dose-related reduction in daily alcohol intake (Fig. 1, panel A); the reducing effect of rac-BHFF on daily alcohol intake was evident from the first day of treatment and lasted for the entire Treatment Phase (Fig. 1, panel A). With respect to the vehicle-treated rat group, the overall magnitude of reduction in alcohol intake averaged approximately 25%, 40%, and 65% in 50, 100, and 200 mg/kg rac-BHFF-treated rat groups, respectively. Post-Treatment Phase e ANOVA indicated a significant effect of time [F(4,112) ¼ 4.03, p < 0.005], but not treatment [F(3,28) ¼ 0.94, p > 0.05], and no significant interaction [F(12,112) ¼ 0.87, p > 0.05], on daily alcohol intake over the 5-day Post-Treatment Phase. After treatment discontinuation, daily alcohol intake returned immediately to baseline levels, with no rebound increase and group difference on any day (Fig. 1, panel A).
Animals Male sP rats, from the 79th generation and 75-days-old at the start of the study, were used. Rats weighed approximately 450 g at the start of treatment with rac-BHFF. Rats were individually housed in standard plastic cages with wood chip bedding. The animal facility was under an inverted 12:12 h lightedark cycle (lights on at 9:30 p.m.), at a constant temperature of 22 2 C and relative humidity of approximately 60%. Rats were extensively habituated to handling and intragastric infusion (by means of a metal gavage). Standard rat chow (Mucedola, Settimo Milanese, Italy) was always available. Experimental procedure Alcohol was offered under the standard, homecage 2-bottle choice regimen between the alcohol solution (10% in tap water, v/v) and tap water, with unlimited access for 24 h/day. Bottles were refilled every day with fresh solution and their lefteright position interchanged at random to avoid development of position preference. Alcohol, water, and food intake was recorded once daily (before the start of the dark phase) by weighing the bottles and food pellets (0.1-g accuracy). Rats were exposed to the 2-bottle choice regimen for 4 consecutive weeks before the start of treatment with rac-BHFF, so that high and stable intakes of alcohol (averaging approximately 7 g/kg/day) were reached (“alcohol-experienced” rats at the time of treatment with rac-BHFF). Rats were then divided into 4 groups (n ¼ 8), matched for body weight as well as alcohol, water, and food intake during the last 3 days preceding the start of treatment with rac-BHFF. rac-BHFF was suspended in a mixture containing racBHFF, Cremophor EL, 1,2-propanediol, and distilled water (ratio of the mixture components: 1:7.6:1.9:9.5) and administered intragastrically (infusion volume: 2 ml/kg), once daily (1 h before lights off) and for 7 consecutive days, at the doses of 0, 50, 100, and
Statistical analyses Data on daily alcohol, water, total fluid, and food intake, and preference ratio during the 7-day Treatment Phase as well as the 5-day Post-Treatment Phase were evaluated by separate 2-way [treatment (rac-BHFF dose); time (day)] ANOVAs with repeated measures on the factor “time”, followed by the NewmaneKeuls test for post hoc comparisons. Results
Water intake Treatment Phase e ANOVA indicated a significant effect of treatment [F(3,28) ¼ 20.60, p < 0.0001] and time [F(6,168) ¼ 5.47, p < 0.0001], but not a significant interaction [F(18,168) ¼ 1.33, p > 0.05], on daily water intake over the 7-day Treatment Phase. Treatment with rac-BHFF resulted in a dose-related increase in daily water intake (Fig. 1, panel B). The overall intake of water was approximately 2.5-, 5-, and 8-fold higher in 50, 100, and 200 mg/kg rac-BHFF-treated rat groups, respectively, than in vehicle-treated rat group. Post-Treatment Phase e ANOVA indicated a significant effect of treatment [F(3,28) ¼ 7.59, p < 0.001] and time [F(4,112) ¼ 11.46, p < 0.0001], and a significant interaction [F(12,112) ¼ 5.89, p < 0.0001], on daily water intake over the 5-day Post-Treatment Phase. On the first 2 days after treatment discontinuation, daily water intake was significantly higher in the rat group treated with 200 mg/kg rac-BHFF than in vehicle-treated rat group; then, it returned progressively to baseline levels (Fig. 1, panel B).
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Fig. 1. Effect of the repeated administration of the positive allosteric modulator of the GABAB receptor, rac-BHFF, on alcohol drinking behavior in alcohol-experienced, Sardinian alcoholpreferring (sP) rats. rac-BHFF (0, 50, 100, and 200 mg/kg; i.g.) was administered once daily (1 h before lights off) for 7 consecutive days. Alcohol (10%, v/v) and water were offered under the homecage, 2-bottle choice regimen with unlimited access for 24 h/day. Food pellets were always available. Alcohol, water, total fluid (sum of alcohol solution and water), and food intake was recorded once a day (before the start of the dark phase) during the 7-day Treatment Phase as well as the subsequent, 5-day Post-Treatment Phase. Each point is the mean SEM for n ¼ 8 rats. *: p < 0.05, x: p < 0.01, þ: p < 0.005, U: p < 0.0005, and #: p < 0.0001 with respect to 0 mg/kg rac-BHFF-treated rat group (NewmaneKeuls test).
Total fluid intake Treatment Phase e ANOVA indicated no significant effect of treatment [F(3,28) ¼ 1.74, p > 0.05] and time [F(6,168) ¼ 1.74, p > 0.05], as well as no significant interaction [F(18,168) ¼ 1.18, p > 0.05], on daily total fluid intake over the 7-day Treatment Phase (Fig. 1, panel C).
Post-Treatment Phase e ANOVA indicated a significant effect of treatment [F(3,28) ¼ 5.18, p < 0.01], but not time [F(4,112) ¼ 1.39, p > 0.05], and no significant interaction [F(12,112) ¼ 0.32, p > 0.05], on daily total fluid intake over the 5-day Post-Treatment Phase. Daily total fluid intake tended to be higher in the rat group treated with 200 mg/kg rac-BHFF than in the other three rat groups (Fig. 1, panel C).
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Preference ratio Treatment Phase e ANOVA indicated a significant effect of treatment [F(3,28) ¼ 23.30, p < 0.0001] and time [F(6,168) ¼ 5.73, p < 0.0001], but no significant interaction [F(18,168) ¼ 1.49, p > 0.05], on preference ratio over the 7-day Treatment Phase. Treatment with rac-BHFF resulted in a dose-related reduction in preference ratio (Fig. 1, panel D); the reducing effect of rac-BHFF on preference ratio was already evident on the first day of treatment (Fig. 1, panel D). With respect to the vehicle-treated rat group, the overall magnitude of reduction in preference ratio averaged approximately 15%, 35%, and 60% in 50, 100, and 200 mg/kg racBHFF-treated rat groups, respectively. Post-Treatment Phase e ANOVA indicated a significant effect of treatment [F(3,28) ¼ 5.31, p < 0.01] and time [F(4,112) ¼ 10.63, p < 0.0001], and a significant interaction [F(12,112) ¼ 4.16, p < 0.0001], on preference ratio over the 5-day Post-Treatment Phase. After treatment discontinuation, preference ratio was still reduced, on Day 1, in the rat group treated with 200 mg/kg racBHFF; subsequently, it returned to baseline levels, with no group difference on any further day (Fig. 1, panel D). Food intake Treatment Phase e ANOVA indicated a significant effect of treatment [F(3,28) ¼ 12.39, p < 0.0001], but not time [F(6,168) ¼ 1.66, p > 0.05], and no significant interaction [F(18,168) ¼ 1.05, p > 0.05], on daily food intake over the 7-day Treatment Phase. Daily food intake tended to be reduced only in the rat group treated with 200 mg/kg rac-BHFF, reaching statistical significance at post hoc analysis on Days 3 and 7 (Fig. 1, panel E). Post-Treatment Phase e ANOVA indicated a significant effect of time [F(4,112) ¼ 7.58, p < 0.0001], but not treatment [F(3,28) ¼ 2.10, p > 0.05], and no significant interaction [F(12,112) ¼ 1.24, p > 0.05], on daily food intake over the 5-day Post-Treatment Phase. On the first 2 days after treatment discontinuation, daily food intake tended to be higher in the rat group treated with 200 mg/kg rac-BHFF (Fig. 1, panel E). Discussion The results of the present study indicate that repeated treatment with the GABAB PAM, rac-BHFF, reduced daily alcohol intake in selectively bred, alcohol-preferring sP rats exposed to the homecage, 2-bottle “alcohol vs water” choice regimen. Reduction in alcohol intake was dose-related, of remarkable magnitude, and stable over the 7-day treatment period (with no development of any degree of tolerance). Reduction in alcohol intake was associated to a fully compensatory increase in daily water intake; subsequently, daily total fluid intake (i.e., the sum of alcohol solution and water consumed over the 24 h) was unchanged by treatment with rac-BHFF. A further consequence of rac-BHFF-induced reduction in alcohol drinking and increase in water drinking was the marked reduction in preference ratio: in the rat group treated with 200 mg/ kg rac-BHFF, water tended to be preferred over the alcohol solution. Daily food intake (a parameter useful to assess the selectivity of the tested drug on alcohol intake) tended to be reduced solely by treatment with 200 mg/kg rac-BHFF. Together, these results depict a virtually ideal profile for the anti-alcohol properties of rac-BHFF: its repeated administration profoundly, steadily, and selectively (in comparison to water and food intake) reduced alcohol drinking in a rat model of excessive and chronic alcohol consumption. A previous experiment demonstrated that treatment with 50e200 mg/kg rac-BHFF did not alter spontaneous locomotor activity in male, adult, alcohol-experienced sP rats (Maccioni,
Thomas, et al., 2010). These data tend to exclude that the reducing effect of 200 mg/kg rac-BHFF on both alcohol and food intake observed in the present study was secondary to motorincapacitating and sedative effects, that would have disrupted the rats’ regular rates of drinking and feeding. The results of the present study complement those collected in a recent study testing the same dose range of rac-BHFF on operant, oral alcohol self-administration in sP rats (Maccioni, Thomas, et al., 2010). The magnitude of the reduction of lever-responding for alcohol and amount of self administered alcohol (approximately 30%, 65%, and 90% in 50, 100, and 200 mg/kg rac-BHFF-treated rats, respectively) resembles the pattern of reduction of daily alcohol intake recorded on most days of the present study. A further similarity between the results of the two studies is provided by the relative selectivity of rac-BHFF effect on alcohol-related behaviors: in the operant study, lever-responding for and self-administered amount of a sucrose solution in male, adult sP rats was reduced only by treatment with 200 mg/kg rac-BHFF (Maccioni, Thomas, et al., 2010); notably, the magnitude of this reduction (approximately 50%) was smaller than that of the reducing effect of 200 mg/ kg rac-BHFF on lever-responding for alcohol and amount of selfadministered alcohol (approximately 90%) (Maccioni, Thomas, et al., 2010). The results of the present study are also in agreement with those of a previous study testing CGP7930 and GS39798 e the two GABAB PAMs most widely used in behavioral pharmacological studies e on alcohol drinking in sP rats (Orrù et al., 2005). In the latter study, alcohol-experienced sP rats were exposed to the same 2-bottle “alcohol vs water” choice regimen employed in the present study. Repeated, daily administration of 50 and 100 mg/kg CGP7930 and GS39783 (i.g.) selectively reduced daily alcohol intake. With the caution needed when data from two different studies are compared, it should be noted however that the reducing effect of CGP7930 and GS39783 on daily alcohol intake was of smaller magnitude (30e40% at the highest dose tested) (Orrù et al., 2005) than that produced by the same doses of rac-BHFF (present study). Further, the reducing effect of CGP7930 and GS39783 on daily alcohol intake tended to vanish after the first 2e3 days of treatment (Orrù et al., 2005), while the efficacy of the reducing effect of racBHFF on daily alcohol intake remained unchanged over the entire 7day treatment period. The reasons for these differences in the anti-alcohol profile of CGP7930, GS39783, and rac-BHFF may reside in their pharmacokinetic properties: the half-life of rac-BHFF is indeed remarkably longer than that of CGP7930 and GS39783 (Malherbe et al., 2008), which is probably more suitable for treatments where the drug is administered once daily. It is interesting to note that a recent report comparing in vivo effects of CGP7930 and rac-BHFF found that CGP7930 produced hypothermia in mice (C57BL/6J) in a similar manner as baclofen when given alone, whereas rac-BHFF did not produce hypothermic effects (Koek, France, Cheng, & Rice, 2010). Because CGP7930-induced hypothermia was not attenuated by the GABAB orthosteric antagonist CGP35348, and CGP7930 could directly activate the 7TMD of GABAB2 subunit (Binet et al., 2004). Koek et al. (2010) suggested that CGP7930 acts as an “allosteric agonist” at the specific GABAB receptor populations mediating hypothermia. Since rac-BHFF, acting as a positive allosteric modulator, amplifies the gain of the GABAergic tonus only in synergy with orthosteric agonist (GABA), it is expected that it promotes less receptor desensitization upon sustained exposure compared to an allosteric agonist; this might explain the difference in tolerating effect to alcohol between CGP7930 and rac-BHFF. Finally, it can be hypothesized that e as seen for the mGLU receptors (see Urwyler, 2011) e multiple, distinct allosteric binding sites may exist in the GABAB receptor structure; accordingly, CGP7930, GS39783, and
B. Loi et al. / Alcohol 47 (2013) 69e73
rac-BHFF might bind to different sites, exerting differential effects on downstream receptor signaling and, subsequently, producing different in vivo effects. In this context, it has recently been discovered that native GABAB receptors are heteromultimers with a family of auxiliary subunits belonging to a subfamily of the KCTD (potassium channel tetramerization domain-containing) proteins. The KCTD proteins, which have distinct expression profiles in the brain, associate tightly with the C-terminus of GABAB2 as tetramers and markedly alter the G-protein signaling of the receptors in a KCTDsubtype-specific manner; hence they determine the pharmacology and kinetics of the GABAB receptor response (Metz, Gassmann, Fakler, Schaeren-Wiemers, & Bettler, 2011; Schwenk et al., 2010). It is conceivable that the functional diversity promoted by KCTD proteins in complex with GABAB receptor might underlie the functional differences observed among allosteric modulators. In conclusion, the results of the present study add an important element to the anti-alcohol profile of the GABAB PAM, rac-BHFF, i.e., its capacity to reduce alcohol intake and preference for alcohol in alcohol-preferring rats, with no change in efficacy on continuing treatment. Data collected to date, demonstrating that treatment with rac-BHFF effectively reduces alcohol drinking (present study) and alcohol reinforcing and motivational properties (Maccioni, Thomas, et al., 2010), make rac-BHFF an interesting compound with therapeutic potential for alcoholism.
Acknowledgments The authors are grateful to Dr. Maria Paola Castelli for helpful discussions, Mrs. Carla Acciaro for animal breeding and care, and Ms. Anne Farmer for language editing of the manuscript. This work has been supported by the following grants: (a) “EtOH-PAMs” from Compagnia di San Paolo e Programma Neuroscienze 2008/09, Turin, Italy; (b) no. CRP-17596 from Regione Autonoma della Sardegna (L.R. 7 agosto 2007, n. 7).
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Alcohol journal homepage: http://www.alcoholjournal.org/
Reduction of alcohol intake by the positive allosteric modulator of the GABAB receptor, rac-BHFF, in alcohol-preferring rats Barbara Loi a, Paola Maccioni a, Carla Lobina a, Mauro A.M. Carai a, Gian Luigi Gessa a, Andrew W. Thomas b, Pari Malherbe b, Giancarlo Colombo a, * a b
Neuroscience Institute, National Research Council of Italy, Section of Cagliari, I-09042 Monserrato (CA), Italy Discovery Research CNS, F. Hoffmann-La Roche Ltd., Psychiatry Disease Area, Basel CH-4070, Switzerland
a r t i c l e i n f o
a b s t r a c t
Article history: Received 9 October 2012 Received in revised form 20 November 2012 Accepted 23 November 2012
Previous research has demonstrated that treatment with the positive allosteric modulator (PAM) of the GABAB receptor (GABAB PAM), rac-BHFF, suppressed lever-responding for alcohol and amount of selfadministered alcohol in Sardinian alcohol-preferring (sP) rats. The present study was designed to extend the investigation on the anti-alcohol effects of rac-BHFF to alcohol drinking behavior. To this end, sP rats were exposed to the homecage, 2-bottle “alcohol (10%, v/v) vs water” choice regimen, with unlimited access for 24 h/day. rac-BHFF was administered once daily and for 7 consecutive days at the doses of 0, 50, 100, and 200 mg/kg (i.g.). Treatment with rac-BHFF resulted in an immediate, stable, and dose-related reduction in daily alcohol intake; the overall magnitude of reduction in alcohol intake averaged approximately 25%, 40%, and 65% in 50, 100, and 200 mg/kg rac-BHFF-treated rat groups, respectively. An increase in daily water intake fully compensated the reduction in alcohol intake, so that daily total fluid intake was unaffected by treatment with rac-BHFF. Daily food intake tended to be reduced only by the highest dose of rac-BHFF. These results complement closely with previous data indicating that (a) rac-BHFF suppressed operant, oral alcohol self-administration in sP rats and (b) the prototypic GABAB PAMs, CGP7930 and GS39783, reduced alcohol drinking in sP rats. However, while the reducing effect of CGP7930 and GS39783 on the daily alcohol intake tended to vanish after the first 2e3 days of treatment, the reducing effect of racBHFF on daily alcohol intake remained unchanged over the entire 7-day treatment period. These data strengthen the hypothesis that GABAB PAMs may represent a step forward in the search for GABAB receptor ligands with therapeutic potential for alcoholism. Ó 2013 Elsevier Inc. All rights reserved.
Keywords: Positive allosteric modulation of the GABAB receptor rac-BHFF Alcohol drinking Sardinian alcohol-preferring rats
Introduction Positive allosteric modulators (PAMs) of the GABAB receptor (GABAB PAMs) represent a novel class of pharmacological agents with promising, therapeutic potential for alcoholism. Accumulating lines of experimental evidence indicate indeed that the currently available, in vivo effective GABAB PAMs invariably reduced different alcohol-related behaviors in rats and mice. Specifically, repeated treatment with the prototypic GABAB PAMs, CGP7930 and GS39783, reduced alcohol drinking in alcohol-preferring rats exposed to the 2-bottle “alcohol vs water” choice regimen (Orrù et al., 2005). Administration of CGP7930 and GS39783, as well as BHF177 and rac-BHFF (two additional GABAB PAMs synthesized more recently), reduced lever-responding for alcohol and amount of * Corresponding author. Neuroscience Institute, National Research Council of Italy, Section of Cagliari, S.S. 554, km. 4,500, I-09042 Monserrato (CA), Italy. Tel.: þ39 070 675 4342; fax: þ39 070 675 4320. E-mail address: [email protected] (G. Colombo). 0741-8329/$ e see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.alcohol.2012.11.002
self-administered alcohol in alcohol-preferring rats exposed to multiple procedures of operant, oral alcohol self-administration (Liang et al., 2006; Maccioni et al., 2007, 2008, 2009, 2012; Maccioni, Flore, et al., 2010; Maccioni, Thomas, et al., 2010). Treatment with GS39783 blocked alcohol-induced locomotor stimulation (although not alcohol-induced locomotor sensitization) in mice (Kruse, Linsenbardt, & Boehm, 2012). As predictable on the basis of their mode of action e i.e., absence of substantial intrinsic agonistic activity and activation of the GABAB receptor via synergistic enhancement of GABA effects only in those synapses in which and when endogenous GABA is present (see Froestl, 2010; Urwyler, 2011) e GABAB PAMs exert their antialcohol effects at doses far lower than those producing “undesired”, motor-incoordinating and sedative effects. For instance, CGP7930and GS39783-induced reduction in daily alcohol intake was associated to a fully compensatory increase in daily water intake (Orrù et al., 2005); acute treatment with GS39783, BHF177, and rac-BHFF reduced lever-responding for alcohol at doses that did not alter lever-responding for an alternative reinforcer such as a sucrose
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solution (with the sole exception of 200 mg/kg rac-BHFF, that reduced e although to a lower extent e lever-responding for the sucrose solution) (Maccioni et al., 2007, 2008, 2009, 2012; Maccioni, Thomas, et al., 2010). This selectivity for alcohol-related behaviors and the lack of sedative effects make GABAB PAMs clearly preferable over baclofen and other GABAB receptor agonists, the anti-alcohol effects of which in rats and mice often occur at doses not far from those producing behavioral toxicity (see Maccioni & Colombo, 2009; Vlachou & Markou, 2010). The present study was designed to provide additional clues to the preclinical, anti-alcohol profile of rac-BHFF [(R,S)-5, 7-di-tert-butyl-3-hydroxy-3-trifluoromethyl-3H-benzofuran-2-one (Malherbe et al., 2008)]. To this end, this study assessed the effect of the repeated treatment with rac-BHFF on alcohol drinking in selectively bred, Sardinian alcohol-preferring (sP) rats (see Bell et al., 2012; Colombo, Lobina, Carai, & Gessa, 2006). The procedure employed was the homecage, 2-bottle “alcohol vs water” choice regimen with unlimited access for 24 h per day, a proposed model for the “active drinking” phase of human alcoholism. Interest on racBHFF resides in the fact that it resulted to be, among the GABAB PAMs tested so far in sP rats, the most effective compound in reducing lever-responding for alcohol and amount of self-administered alcohol (Maccioni, Thomas, et al. 2010).
200 mg/kg. This dose range was identical to that used in the previous study testing rac-BHFF on operant, oral alcohol selfadministration in sP rats (Maccioni, Thomas, et al., 2010). Measured variables were: (a) daily alcohol intake (expressed in g/kg pure alcohol); (b) daily water intake (ml/kg); (c) daily total fluid intake (sum of alcohol solution and water; ml/kg); (d) daily preference ratio (percent ratio between daily intake of alcohol solution and daily total fluid intake); (e) daily food intake (g/kg). Recording was performed once daily (before the start of the dark phase) throughout the 7 consecutive days of the Treatment Phase as well as for an additional 5 consecutive days after treatment termination (Post-Treatment Phase).
Materials and methods
Alcohol intake
All experimental procedures employed in the present study were in accordance with the Italian Law on the “Protection of animals used for experimental and other scientific reasons”.
Treatment Phase e ANOVA indicated a significant effect of treatment [F(3,28) ¼ 15.51, p < 0.0001] and time [F(6,168) ¼ 5.69, p < 0.0001], and a significant interaction [F(18,168) ¼ 2.13, p < 0.01], on daily alcohol intake over the 7-day Treatment Phase. Treatment with rac-BHFF resulted in a dose-related reduction in daily alcohol intake (Fig. 1, panel A); the reducing effect of rac-BHFF on daily alcohol intake was evident from the first day of treatment and lasted for the entire Treatment Phase (Fig. 1, panel A). With respect to the vehicle-treated rat group, the overall magnitude of reduction in alcohol intake averaged approximately 25%, 40%, and 65% in 50, 100, and 200 mg/kg rac-BHFF-treated rat groups, respectively. Post-Treatment Phase e ANOVA indicated a significant effect of time [F(4,112) ¼ 4.03, p < 0.005], but not treatment [F(3,28) ¼ 0.94, p > 0.05], and no significant interaction [F(12,112) ¼ 0.87, p > 0.05], on daily alcohol intake over the 5-day Post-Treatment Phase. After treatment discontinuation, daily alcohol intake returned immediately to baseline levels, with no rebound increase and group difference on any day (Fig. 1, panel A).
Animals Male sP rats, from the 79th generation and 75-days-old at the start of the study, were used. Rats weighed approximately 450 g at the start of treatment with rac-BHFF. Rats were individually housed in standard plastic cages with wood chip bedding. The animal facility was under an inverted 12:12 h lightedark cycle (lights on at 9:30 p.m.), at a constant temperature of 22 2 C and relative humidity of approximately 60%. Rats were extensively habituated to handling and intragastric infusion (by means of a metal gavage). Standard rat chow (Mucedola, Settimo Milanese, Italy) was always available. Experimental procedure Alcohol was offered under the standard, homecage 2-bottle choice regimen between the alcohol solution (10% in tap water, v/v) and tap water, with unlimited access for 24 h/day. Bottles were refilled every day with fresh solution and their lefteright position interchanged at random to avoid development of position preference. Alcohol, water, and food intake was recorded once daily (before the start of the dark phase) by weighing the bottles and food pellets (0.1-g accuracy). Rats were exposed to the 2-bottle choice regimen for 4 consecutive weeks before the start of treatment with rac-BHFF, so that high and stable intakes of alcohol (averaging approximately 7 g/kg/day) were reached (“alcohol-experienced” rats at the time of treatment with rac-BHFF). Rats were then divided into 4 groups (n ¼ 8), matched for body weight as well as alcohol, water, and food intake during the last 3 days preceding the start of treatment with rac-BHFF. rac-BHFF was suspended in a mixture containing racBHFF, Cremophor EL, 1,2-propanediol, and distilled water (ratio of the mixture components: 1:7.6:1.9:9.5) and administered intragastrically (infusion volume: 2 ml/kg), once daily (1 h before lights off) and for 7 consecutive days, at the doses of 0, 50, 100, and
Statistical analyses Data on daily alcohol, water, total fluid, and food intake, and preference ratio during the 7-day Treatment Phase as well as the 5-day Post-Treatment Phase were evaluated by separate 2-way [treatment (rac-BHFF dose); time (day)] ANOVAs with repeated measures on the factor “time”, followed by the NewmaneKeuls test for post hoc comparisons. Results
Water intake Treatment Phase e ANOVA indicated a significant effect of treatment [F(3,28) ¼ 20.60, p < 0.0001] and time [F(6,168) ¼ 5.47, p < 0.0001], but not a significant interaction [F(18,168) ¼ 1.33, p > 0.05], on daily water intake over the 7-day Treatment Phase. Treatment with rac-BHFF resulted in a dose-related increase in daily water intake (Fig. 1, panel B). The overall intake of water was approximately 2.5-, 5-, and 8-fold higher in 50, 100, and 200 mg/kg rac-BHFF-treated rat groups, respectively, than in vehicle-treated rat group. Post-Treatment Phase e ANOVA indicated a significant effect of treatment [F(3,28) ¼ 7.59, p < 0.001] and time [F(4,112) ¼ 11.46, p < 0.0001], and a significant interaction [F(12,112) ¼ 5.89, p < 0.0001], on daily water intake over the 5-day Post-Treatment Phase. On the first 2 days after treatment discontinuation, daily water intake was significantly higher in the rat group treated with 200 mg/kg rac-BHFF than in vehicle-treated rat group; then, it returned progressively to baseline levels (Fig. 1, panel B).
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Fig. 1. Effect of the repeated administration of the positive allosteric modulator of the GABAB receptor, rac-BHFF, on alcohol drinking behavior in alcohol-experienced, Sardinian alcoholpreferring (sP) rats. rac-BHFF (0, 50, 100, and 200 mg/kg; i.g.) was administered once daily (1 h before lights off) for 7 consecutive days. Alcohol (10%, v/v) and water were offered under the homecage, 2-bottle choice regimen with unlimited access for 24 h/day. Food pellets were always available. Alcohol, water, total fluid (sum of alcohol solution and water), and food intake was recorded once a day (before the start of the dark phase) during the 7-day Treatment Phase as well as the subsequent, 5-day Post-Treatment Phase. Each point is the mean SEM for n ¼ 8 rats. *: p < 0.05, x: p < 0.01, þ: p < 0.005, U: p < 0.0005, and #: p < 0.0001 with respect to 0 mg/kg rac-BHFF-treated rat group (NewmaneKeuls test).
Total fluid intake Treatment Phase e ANOVA indicated no significant effect of treatment [F(3,28) ¼ 1.74, p > 0.05] and time [F(6,168) ¼ 1.74, p > 0.05], as well as no significant interaction [F(18,168) ¼ 1.18, p > 0.05], on daily total fluid intake over the 7-day Treatment Phase (Fig. 1, panel C).
Post-Treatment Phase e ANOVA indicated a significant effect of treatment [F(3,28) ¼ 5.18, p < 0.01], but not time [F(4,112) ¼ 1.39, p > 0.05], and no significant interaction [F(12,112) ¼ 0.32, p > 0.05], on daily total fluid intake over the 5-day Post-Treatment Phase. Daily total fluid intake tended to be higher in the rat group treated with 200 mg/kg rac-BHFF than in the other three rat groups (Fig. 1, panel C).
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Preference ratio Treatment Phase e ANOVA indicated a significant effect of treatment [F(3,28) ¼ 23.30, p < 0.0001] and time [F(6,168) ¼ 5.73, p < 0.0001], but no significant interaction [F(18,168) ¼ 1.49, p > 0.05], on preference ratio over the 7-day Treatment Phase. Treatment with rac-BHFF resulted in a dose-related reduction in preference ratio (Fig. 1, panel D); the reducing effect of rac-BHFF on preference ratio was already evident on the first day of treatment (Fig. 1, panel D). With respect to the vehicle-treated rat group, the overall magnitude of reduction in preference ratio averaged approximately 15%, 35%, and 60% in 50, 100, and 200 mg/kg racBHFF-treated rat groups, respectively. Post-Treatment Phase e ANOVA indicated a significant effect of treatment [F(3,28) ¼ 5.31, p < 0.01] and time [F(4,112) ¼ 10.63, p < 0.0001], and a significant interaction [F(12,112) ¼ 4.16, p < 0.0001], on preference ratio over the 5-day Post-Treatment Phase. After treatment discontinuation, preference ratio was still reduced, on Day 1, in the rat group treated with 200 mg/kg racBHFF; subsequently, it returned to baseline levels, with no group difference on any further day (Fig. 1, panel D). Food intake Treatment Phase e ANOVA indicated a significant effect of treatment [F(3,28) ¼ 12.39, p < 0.0001], but not time [F(6,168) ¼ 1.66, p > 0.05], and no significant interaction [F(18,168) ¼ 1.05, p > 0.05], on daily food intake over the 7-day Treatment Phase. Daily food intake tended to be reduced only in the rat group treated with 200 mg/kg rac-BHFF, reaching statistical significance at post hoc analysis on Days 3 and 7 (Fig. 1, panel E). Post-Treatment Phase e ANOVA indicated a significant effect of time [F(4,112) ¼ 7.58, p < 0.0001], but not treatment [F(3,28) ¼ 2.10, p > 0.05], and no significant interaction [F(12,112) ¼ 1.24, p > 0.05], on daily food intake over the 5-day Post-Treatment Phase. On the first 2 days after treatment discontinuation, daily food intake tended to be higher in the rat group treated with 200 mg/kg rac-BHFF (Fig. 1, panel E). Discussion The results of the present study indicate that repeated treatment with the GABAB PAM, rac-BHFF, reduced daily alcohol intake in selectively bred, alcohol-preferring sP rats exposed to the homecage, 2-bottle “alcohol vs water” choice regimen. Reduction in alcohol intake was dose-related, of remarkable magnitude, and stable over the 7-day treatment period (with no development of any degree of tolerance). Reduction in alcohol intake was associated to a fully compensatory increase in daily water intake; subsequently, daily total fluid intake (i.e., the sum of alcohol solution and water consumed over the 24 h) was unchanged by treatment with rac-BHFF. A further consequence of rac-BHFF-induced reduction in alcohol drinking and increase in water drinking was the marked reduction in preference ratio: in the rat group treated with 200 mg/ kg rac-BHFF, water tended to be preferred over the alcohol solution. Daily food intake (a parameter useful to assess the selectivity of the tested drug on alcohol intake) tended to be reduced solely by treatment with 200 mg/kg rac-BHFF. Together, these results depict a virtually ideal profile for the anti-alcohol properties of rac-BHFF: its repeated administration profoundly, steadily, and selectively (in comparison to water and food intake) reduced alcohol drinking in a rat model of excessive and chronic alcohol consumption. A previous experiment demonstrated that treatment with 50e200 mg/kg rac-BHFF did not alter spontaneous locomotor activity in male, adult, alcohol-experienced sP rats (Maccioni,
Thomas, et al., 2010). These data tend to exclude that the reducing effect of 200 mg/kg rac-BHFF on both alcohol and food intake observed in the present study was secondary to motorincapacitating and sedative effects, that would have disrupted the rats’ regular rates of drinking and feeding. The results of the present study complement those collected in a recent study testing the same dose range of rac-BHFF on operant, oral alcohol self-administration in sP rats (Maccioni, Thomas, et al., 2010). The magnitude of the reduction of lever-responding for alcohol and amount of self administered alcohol (approximately 30%, 65%, and 90% in 50, 100, and 200 mg/kg rac-BHFF-treated rats, respectively) resembles the pattern of reduction of daily alcohol intake recorded on most days of the present study. A further similarity between the results of the two studies is provided by the relative selectivity of rac-BHFF effect on alcohol-related behaviors: in the operant study, lever-responding for and self-administered amount of a sucrose solution in male, adult sP rats was reduced only by treatment with 200 mg/kg rac-BHFF (Maccioni, Thomas, et al., 2010); notably, the magnitude of this reduction (approximately 50%) was smaller than that of the reducing effect of 200 mg/ kg rac-BHFF on lever-responding for alcohol and amount of selfadministered alcohol (approximately 90%) (Maccioni, Thomas, et al., 2010). The results of the present study are also in agreement with those of a previous study testing CGP7930 and GS39798 e the two GABAB PAMs most widely used in behavioral pharmacological studies e on alcohol drinking in sP rats (Orrù et al., 2005). In the latter study, alcohol-experienced sP rats were exposed to the same 2-bottle “alcohol vs water” choice regimen employed in the present study. Repeated, daily administration of 50 and 100 mg/kg CGP7930 and GS39783 (i.g.) selectively reduced daily alcohol intake. With the caution needed when data from two different studies are compared, it should be noted however that the reducing effect of CGP7930 and GS39783 on daily alcohol intake was of smaller magnitude (30e40% at the highest dose tested) (Orrù et al., 2005) than that produced by the same doses of rac-BHFF (present study). Further, the reducing effect of CGP7930 and GS39783 on daily alcohol intake tended to vanish after the first 2e3 days of treatment (Orrù et al., 2005), while the efficacy of the reducing effect of racBHFF on daily alcohol intake remained unchanged over the entire 7day treatment period. The reasons for these differences in the anti-alcohol profile of CGP7930, GS39783, and rac-BHFF may reside in their pharmacokinetic properties: the half-life of rac-BHFF is indeed remarkably longer than that of CGP7930 and GS39783 (Malherbe et al., 2008), which is probably more suitable for treatments where the drug is administered once daily. It is interesting to note that a recent report comparing in vivo effects of CGP7930 and rac-BHFF found that CGP7930 produced hypothermia in mice (C57BL/6J) in a similar manner as baclofen when given alone, whereas rac-BHFF did not produce hypothermic effects (Koek, France, Cheng, & Rice, 2010). Because CGP7930-induced hypothermia was not attenuated by the GABAB orthosteric antagonist CGP35348, and CGP7930 could directly activate the 7TMD of GABAB2 subunit (Binet et al., 2004). Koek et al. (2010) suggested that CGP7930 acts as an “allosteric agonist” at the specific GABAB receptor populations mediating hypothermia. Since rac-BHFF, acting as a positive allosteric modulator, amplifies the gain of the GABAergic tonus only in synergy with orthosteric agonist (GABA), it is expected that it promotes less receptor desensitization upon sustained exposure compared to an allosteric agonist; this might explain the difference in tolerating effect to alcohol between CGP7930 and rac-BHFF. Finally, it can be hypothesized that e as seen for the mGLU receptors (see Urwyler, 2011) e multiple, distinct allosteric binding sites may exist in the GABAB receptor structure; accordingly, CGP7930, GS39783, and
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rac-BHFF might bind to different sites, exerting differential effects on downstream receptor signaling and, subsequently, producing different in vivo effects. In this context, it has recently been discovered that native GABAB receptors are heteromultimers with a family of auxiliary subunits belonging to a subfamily of the KCTD (potassium channel tetramerization domain-containing) proteins. The KCTD proteins, which have distinct expression profiles in the brain, associate tightly with the C-terminus of GABAB2 as tetramers and markedly alter the G-protein signaling of the receptors in a KCTDsubtype-specific manner; hence they determine the pharmacology and kinetics of the GABAB receptor response (Metz, Gassmann, Fakler, Schaeren-Wiemers, & Bettler, 2011; Schwenk et al., 2010). It is conceivable that the functional diversity promoted by KCTD proteins in complex with GABAB receptor might underlie the functional differences observed among allosteric modulators. In conclusion, the results of the present study add an important element to the anti-alcohol profile of the GABAB PAM, rac-BHFF, i.e., its capacity to reduce alcohol intake and preference for alcohol in alcohol-preferring rats, with no change in efficacy on continuing treatment. Data collected to date, demonstrating that treatment with rac-BHFF effectively reduces alcohol drinking (present study) and alcohol reinforcing and motivational properties (Maccioni, Thomas, et al., 2010), make rac-BHFF an interesting compound with therapeutic potential for alcoholism.
Acknowledgments The authors are grateful to Dr. Maria Paola Castelli for helpful discussions, Mrs. Carla Acciaro for animal breeding and care, and Ms. Anne Farmer for language editing of the manuscript. This work has been supported by the following grants: (a) “EtOH-PAMs” from Compagnia di San Paolo e Programma Neuroscienze 2008/09, Turin, Italy; (b) no. CRP-17596 from Regione Autonoma della Sardegna (L.R. 7 agosto 2007, n. 7).
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