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. Author manuscript; available in PMC: 2020 Dec 1.
Published in final edited form as: Brain Res. 2019 Aug 27;1724:146410. doi: 10.1016/j.brainres.2019.146410

Clinically utilized kappa-opioid receptor agonist nalfurafine combined with low-dose naltrexone prevents alcohol relapse-like drinking in male and female mice

Yan Zhou 1, Mary Jeanne Kreek 1
PMCID: PMC6779526  NIHMSID: NIHMS1539729  PMID: 31469985

Abstract

Alcohol relapse is a treatment goal for alcohol dependence and the target for medications’ development. Clinically utilized nalfurafine (NFF) is a potent and selective kappa-opioid receptor (KOP-r) agonist, with fewer side effects (e.g., sedation or anhedonia) than classic KOP-r full agonists. We have recently found that NFF reduces excessive alcohol drinking in mice via a KOP-r-mediated mechanism. Here, we further investigated whether NFF alone (1-10 ug/kg) or in combination with naltrexone (NTX, mu-opioid receptor [MOP-r] antagonist) altered alcohol relapse-like drinking using a mouse alcohol deprivation effect (ADE) paradigm to mimic the relapse episodes in human alcoholics. Nalmefene (NMF, clinically utilized KOP-r partial agonist with MOP-r antagonism) was used as a reference compound for the effects on mouse ADE of new NFF+NTX combination. After exposed to 3-week intermittent-access alcohol drinking (two-bottle choice, 24-h access every other day), both male and female mice displayed excessive alcohol intake and then pronounced ADE after 1-week abstinence. NFF prevented the ADE in a dose-dependent manner in both male and female mice. A combination of NFF with NTX reduced the ADE without sex differences at doses lower than those individual effective ones, suggesting synergistic effects between the two compounds. NMF prevented the ADE in both sexes, while selective KOP-r antagonist nor-BNI had no effect. Our new study suggests that a combination of clinically-utilized, potent KOP-r agonist NFF with low-dose NTX has therapeutic potential in alcohol “relapse” treatment.

Keywords: combined therapy, KOP-r, nalfurafine, nalmefene, naltrexone, alcohol deprivation effect, relapse

1. INTRODUCTION

Nalfurafine (NFF), a potent kappa-opioid receptor (KOP-r) agonist, was well studied on its anti-pruritus effect in rodents during 1990’s [Shigeki 2015]. Since 2009, NFF has been used as the first clinically approved KOP-r agonist in Japan for anti-pruritus treatment [Kumagai et al 2012; Pongcharoen and Fleischer 2016; Kamimura et al 2017]. In a recent report on safety and efficacy of Remitch® (nalfurafine hydrochloride) after its 10-year post-marketing survey in more than 3700 patients, unlike other “classic” KOP-r agonists, NFF does not show strong side effects (e.g., sedation, depression or dysphoria) [Kozono et al 2018]. Recently, we examined the effect of NFF on voluntary alcohol consumption in mice and found that NFF dose-dependently reduces excessive alcohol drinking with few side effects [Zhou and Kreek 2019], suggesting that NFF may have potential for treating alcohol abuse. To date, however, there is no study investigating the effects of NFF on alcohol “relapse” in rodent models.

After certain time period of abstinence, there is a transient increase in alcohol intake observed in both humans and rodents, which is termed as the alcohol deprivation effect (ADE) [Burish et al 1981; Vengeliene et al 2014]. ADE has been demonstrated as an appropriate animal model for studying alcohol relapse and been widely used in rat and mouse studies [Holter et al 2000; Heyser et al 2003; Zhou et al 2017, 2018]. Specifically, in our mouse model, after chronic (1-week) abstinence from chronic (3-week) intermittent-access alcohol drinking with excessive alcohol consumption, both males and females show a significant increase in alcohol intake after 4 hours of alcohol access when alcohol is provided again [Hwa et al 2011; Zhou et al 2017, 2018]. Based on our recent new finding showing a reducing effect of NFF on excessive alcohol drinking, we proposed a new hypothesis that NFF could prevent alcohol relapse-like drinking. As alcohol relapse is an important target for medications development for alcoholism, we investigated the pharmacological effects of NFF using our mouse ADE model in both sexes, to ascertain its potential as an anti-relapse compound.

Alcohol increases mu-opioid receptor (MOP-r)/beta-endorphin -mediated transmission and binding of beta-endorphin to MOP-r is, in part, responsible for alcohol’s positive reinforcing and motivational properties, which is involved in excessive alcohol consumption and relapse episodes [Zhou and Kreek 2018]. Naltrexone (NTX, MOP-r antagonist) decreases alcohol craving and relapse in human alcoholics [O’Malley et al 1992, 2002], and relapse-like drinking in rat and mouse ADE models [Heyser et al 2003; Zhou et al 2018]. As neurobiological studies have demonstrated that both the KOP-r and MOP-r activities, among multiple actions of alcohol in the CNS, are profoundly altered by alcohol drinking [Vijay et al 2018; Zhou and Kreek 2018], we further hypothesized that by targeting on both the KOP-r and MOP-r pathways, the combination of the two clinically utilized compounds NFF and NTX would enhance efficacy over the single-compound approach on preventing relapse [Zhou and Kreek 2018]. Therefore, the present study tested whether the proper combination of these two compounds could be effective in preventing ADE at low doses of each one that had no effect on ADE. Nalmefene (NMF) is approved in Europe for reducing alcohol consumption in alcohol-dependent patients [Mason et al 1994; Mann et al 2016] and reduces alcohol self-administration in alcohol-dependent rats and mice [Walker and Koob 2008; Rose et al 2016]. As NMF is a mixed KOP-r partial agonist and MOP-r antagonist [Schluger et al 1998; Bart et al 2005], we used NMF as a reference compound and compared its effect with that of our new combination NFF+NTX.

During acute withdrawal from alcohol, KOP-r activation induces negative mood states and symptoms, which are involved in negative reinforcing aspects of alcohol addiction [Koob and Kreek 2007; Zhou and Kreek 2018]. Our particularly relevant question was whether there was an upregulation of KOP-r activity after prolonged abstinence and during alcohol relapse-like drinking, and if so, whether pharmacological blockade of KOP-r could reduce ADE or not. So, in addition, to further explore a possible involvement of KOP-r in alcohol relapse-like drinking, we purposely determined if there was any dose-dependent effect of selective KOP-r antagonist nor-binaltorphimine (nor-BNI) on mouse ADE in both sexes.

2. RESULTS

2.1. Effect of NFF on ADE.

In the 1st experiment, the effect of NFF at 1ug/kg dose was tested in both sexes, and there was no difference between saline control and NFF treatment in either males or females at 4, 8 or 24 hours (data not shown). In the 2nd experiment, both 3 and 10ug/kg doses were tested, and the results on alcohol intake at 4 hours are shown in Figure 1.

Figure 1.

Figure 1

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Effects of nalfurafine (NFF, 3 and 10 ug/kg) on alcohol intake in an alcohol deprivation effect (ADE) model at 4 hours in males (A) and females (B) after 1 week of abstinence from 3-week intermittent-access alcohol drinking. * p<0.05 vs. control Baseline, and + p<0.05 vs. control ADE.

In males (Figure 1A), two-way ANOVA revealed a significant effect of NFF [F(1,48)=4.6, p<0.05], Session [F(1,48)=5.6, p<0.05], and a significant interaction between Session and NFF [F(1,48)=6.4, p<0.01]. Post hoc analysis showed that: (1) males had more intake in the ADE session than that in the Baseline [p<0.05]; and (2) the 10 ug/kg NFF-treated males had less intake than the vehicle controls in the ADE session [p<0.01]. NFF at a low dose (3ug/kg), however, did not significantly reduce ADE at 4 hours in males.

In females (Figure 1B), two-way ANOVA showed a significant effect of Session [F(1,48)=4.1, p<0.05] and a marginally significant effect of NFF [F(1,48)=2.9, p=0.07]. To test our a priori hypothesis that NFF would reduce ADE, we included the Newman-Keuls post-hoc results: (1) females had more intake in the ADE session than that in the Baseline [p<0.05]; and (2) the 10 ug/kg NFF-treated females had less intake than the vehicle-treated controls in the ADE session [p<0.05], though 2-way ANOVA did not show a significant effect of NFF. Similar to males, NFF at 3ug/kg did not reduce ADE at 4 hours in females.

After 8 or 24 hours, there were no significant effects of NFF on alcohol intake at these 3 doses in either sex. The results at the highest dose 10ug/kg are shown in Table S3.

For alcohol preference, there was no significant effect of ADE or NFF on preference ratio in either sex at any doses tested. The results at 10ug/kg after 4 hours are presented in Table S4. NFF at these doses did not change water intake after 4, 8 or 24 hours, and Table S5 presents the data at 10ug/kg.

2.2. Effect of NTX on ADE.

In a pilot study, we tested the effect of 1mg/kg NTX, and found the NTX had no significant effect on ADE at 4 hours in either sex (data not shown).

In males with NTX at 3mg/kg (Table 1A), two-way ANOVA revealed significant effects of Session [F(1,24)=5.1, p<0.05], NTX [F(1,24)=4.6, p<0.05] and a significant interaction between Session and NTX [F(1,24)=4.5, p<0.05]. The post-hoc results showed that: (1) males had more intake in the ADE session than that in the Baseline [p<0.05]; and (2) the NTX-treated males had less intake than the vehicle controls in the ADE session [p<0.05]. After 8 or 24 hours, there were no significant effects of NTX on alcohol intake in males (data not shown).

Table 1.

Effects of NTX (3 mg/kg) on alcohol intake in an alcohol deprivation effect (ADE) model at 4 hours in males (A, n=6) and females (B, n=7-9) after 1 week of abstinence from 3-week intermittent-access alcohol drinking. * p<0.05 vs. control Baseline, and + p<0.05 vs. control ADE.

 A. Male
Vehicle 3 mg/kg NTX
Time point Baseline ADE Baseline ADE
Intake, g/kg 0–4 hour 5.3 ± 0.3 8.6 ± 1.1 * 5.5 ± 0.5 6.1 ± 0.9 * +
 B. Female
Vehicle 3 mg/kg NTX
Time point Baseline ADE Baseline ADE
Intake, g/kg 0-4 hour 6.5 ± 0.6 11.9 ± 1.2 * 6.4 ± 0.9 7.8 ± 0.9 * +
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In females with NTX at 3mg/kg (Table 1B), two-way ANOVA revealed significant effects of Session [F(1,26)=5.3, p<0.05], NTX [F(1,26)=5.4, p<0.05], and a significant interaction between Session and NTX [F(1,26)=4.8, p<0.05]. The Post-hoc analysis showed that: (1) females had more intake in the ADE session than that in the Baseline [p<0.05]; and (2) the NTX-treated females had less intake than the controls in the ADE session [p<0.05]. After 8 or 24 hours, there were no significant effects of NTX on alcohol intake in females (data not shown).

For alcohol preference, there was no significant effect of ADE or NTX in either sex at any time points (data not shown).

2.3. Effect of NFF combined with NTX on ADE.

In a pilot study, we tested the effect of 1ug/kg NFF combined with NTX at 0.3mg/kg, and found the combination had no significant effect on ADE at 4 hours in either sex, though a slight reduction in male only (Table S6). With a higher dose of NTX at 1mg/kg combined with 1ug/kg NFF, the ADE intake at 4 hour are shown in Figure 2.

Figure 2.

Figure 2

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Effects of nalfurafine (NFF, 1ug/kg) combined with naltrexone (NTX, 1 mg/kg) on alcohol intake in an alcohol deprivation effect (ADE) model at 4 hours in males (A) and females (B) after 1 week of abstinence from 3-week intermittent-access alcohol drinking. * p<0.05 vs. control Baseline, and + p<0.05 vs. control ADE.

In males (Figure 2A), two-way ANOVA revealed significant effects of Session [F(1,36)=5.9, p<0.05], Combination [F(1,36)=4.7, p<0.05] and a marginally significant interaction between Session and Combination [F(1,36)=3.0, p=0.06]. To test our a priori hypothesis that there was an effect of the combination, we included the post-hoc results showing that: (1) males had more intake in the ADE session than that in the Baseline [p<0.05]; and (2) the NFF+NTX-treated males had less intake than the vehicle controls in the ADE session [p<0.05]. After 8 or 24 hours, there were no significant effects of the combination on alcohol intake in males (data not shown).

In females (Figure 2B), two-way ANOVA revealed significant effects of Session [F(1,32)=10.6, p<0.01] and Combination [F(1,32)=4.4, p<0.05], and a significant interaction between Session and Combination [F(1,32)=4.3, p<0.05]. Post-hoc analysis showed that: (1) females had more intake in the ADE session than that in the Baseline [p<0.05]; and (2) the NFF+NTX-treated females had less intake than the vehicle controls in the ADE session [p<0.05]. After 8 or 24 hours, there were no significant effects of the combination on alcohol intake in females (data not shown).

For alcohol preference, there was no significant effect of ADE or NFF+NTX in either sex at any time points (data not shown).

2.4. Effect of NMF on ADE.

In this experiment, the effect of NMF at 0.125, 0.25 or 0.5mg/kg on alcohol intake was tested and Figure 3 presents the data at 4 hours.

Figure 3.

Figure 3

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Effects of nalmefene (NMF, 0.125, 0.25 or 0.5mg/kg) on alcohol intake in an alcohol deprivation effect (ADE) model at 4 hours in males (A) and females (B) after 1 week of abstinence from 3-week chronic intermittent-access alcohol drinking. * p<0.05 vs. control Baseline, and ++ p<0.01 vs. control ADE.

In males (Figure 3A), two-way ANOVA showed a significant effect of NMF [F(1,72)=2.8, p<0.05], Session [F(1,72)=7.1, p<0.01] and a significant interaction between Session and NMF [F(1,72)=3.7, p<0.05]. Post hoc analysis showed that: (1) the males had more intake in the ADE session than that in the Baseline [p<0.05]; and (2) the 0.5 mg/kg NMF-treated males had less intake than the vehicle control in the ADE session [p<0.01]. NMF at low doses (0.125 or 0.25 mg/kg), however, did not significantly reduce ADE at 4 hours in males.

In females (Figure 3B), two-way ANOVA revealed a significant effect of NMF [F(1,84)=3.5, p<0.05], Session [F(1,84)=11, p<0.01] and a significant interaction between Session and NMF [F(1,84)=2.8, p<0.05]. Post hoc analysis showed that: (1) the females had more intake in the ADE session than that in the Baseline [p<0.05]; and (2) the 0.5mg/kg NMF-treated females had less intake than the vehicle-treated controls in the ADE session [p<0.01]. Similar to males, NMF at two lower doses (0.125 and 0.25 mg/kg) did not significantly reduce ADE in females.

After 8 or 24 hours, there were no significant effects of NMF on either alcohol intake in either sex (data not shown). For alcohol preference, there was no significant effect of ADE or NMF on preference ratio in either sex at any time points (data not shown).

2.5. Effect of nor-BNI on ADE.

At two low doses tested: 5 or 10 mg/kg, there were no significant effects of nor-BNI on alcohol intake in either sex (data not shown). The results at 20 mg/kg are shown in Table 2.

Table 2.

No effects of nor-BNI (20 mg/kg) on alcohol intake in an alcohol deprivation effect (ADE) model at 4 hours in males (A, n=9) and females (B, n=9) after 1 week of abstinence from 3-week intermittent-access alcohol drinking. * p<0.05 vs. control Baseline.

 A. Male
Vehicle 20 mg/kg Nor-BNI
Time point Baseline ADE Baseline ADE
Intake, g/kg 0-4 hour 5.2 ± 0.5 7.6 ± 0.9 * 5.3 ± 0.7 7.1 ± 1.0 *
 B. Female
Vehicle 20 mg/kg Nor-BNI
Time point Baseline ADE Baseline ADE
Intake, g/kg 0-4 hour 6.7 ± 0.7 12.3 ± 1.1 * 6.6 ± 0.5 12.0 ± 1.2 *
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In males at 4 hours (Table 2A), two-way ANOVA revealed a significant effect of Session [F(1,48)=8.5, p<0.01]. Post hoc analysis showed that males had more intake in the ADE session than that in the Baseline [p<0.05]. In females at 4 hours (Table 2B), two-way ANOVA revealed a significant effect of Session [F(1,48)=9.6, p<0.01]. Post hoc analysis showed that females had more intake in the ADE session than that in the Baseline [p<0.05]. After 4 hours, there were no significant effects of nor-BNI on alcohol intake in either sex at 20 mg/kg.

2.6. Effect of nor-BNI plus NTN on ADE.

After 4, 8 or 24 hours, there were no significant effects of nor-BNI+NTN on ADE in either sex (data not shown).

3. DISCUSSION

In our recent study using mouse excessive alcohol drinking model, we have found that NFF reduces alcohol intake in both male and female mice via a KOP-r-mediated mechanism [Zhou and Kreek 2019]. To further examine whether NFF altered alcohol relapse-like drinking, the present study used a mouse ADE paradigm to mimic the relapse episodes in human alcoholics, and investigated the potential of NFF in preventing ADE after chronic (1-week) abstinence from excessive alcohol drinking. ADE is considered as a rodent model of “relapse” drinking and craving behaviors with good predictive validity [Vengeliene et al 2014]. Therefore, we tested NFF with 1-10 ug/kg doses and found that NFF at 10ug/kg significantly reduced ADE intake after 4 hours in both males and females (Figure 1). The current study confirmed and extended our recent observation on the reducing effect of NFF on excessive alcohol drinking with no sex difference [Zhou and Kreek 2019]. The effect of NFF on the mouse ADE intake may not be due to its general inhibition of appetitive behavior (anhedonic effect) or consumption, as NFF at this dose range did not change sucrose or saccharin intake [Zhou and Kreek 2019]. Hence, our finding that NFF decreased alcohol relapse-like consumption would constitute additional information of the anti-addiction properties of NFF observed in cocaine, nicotine or opiate related behaviors [Tsuji et al 2000; Mori et al 2002]. Though “classic” KOP-r agonists (U50,488H and U69,593) reduce alcohol-induced reward and attenuate alcohol drinking in rodents [Lindholm et al 2007; Logrip et al 2009], their side effects (sedation, anxiety-like and depression-like behaviors) limit their clinical potential. Therefore, NFF, due to its few side effects observed in both humans and rodents, may provide potential treatments for alcohol relapse.

It has been found that “classic” KOP-r agonists induce alcohol-seeking behavior [Funk et al 2014] and promote alcohol relapse-like drinking [Holter et al 2000] (see recent reviews [Anderson and Becker 2017; Zhou and Kreek 2018]). Therefore, our new data showing that NFF, a potent KOP-r agonist, reduced rather than promoted relapse-like drinking, contradicts the findings of “classic” KOP-r agonists. “Classic” KOP-r agonists induce aversion, anxiety-, or depression-like behavior that may be responsible for alcohol seeking or relapse-like drinking. However, NFF does not produce the above side effects in rodents [Suzuki et al 2004; Inan et al 2009; Liu et al 2019; Zhou and Kreek 2019] or dysphoria in humans [Kamimura et al 2017; Kozono et al 2018], as NFF has possible different cellular signaling properties than “classic” KOP-r agonists [Schattauer et al 2017; Liu et al 2019]. As acute alcohol stimulates dopamine release in the striatum [Lindholm et al 2007], NFF may prevent the dopamine surge induced by alcohol. Alternatively, NFF may activate hypothalamic-pituitary-adrenal (HPA) axis, and thus the enhanced HPA activity may prevent alcohol relapse drinking and craving, as demonstrated in humans [O’Malley et al 2002]. With few side effects, such as sedation, dysphoria, anxiety and anhedonia, the NFF effects on dopamine and HPA axis could be responsible, at least in part, for the observed reduction of ADE in mice.

It was notable that nor-BNI alone did not affect the ADE in either sex. Our dose-response experiment tested 5-20 mg/kg of nor-BNI and failed to observe any effect of the KOP-r antagonist on mouse relapse-like drinking. As shown in Table 2, though the highest dose of nor-BNI at 20 mg/kg showed a slight reduction in males, the effect could not reach significance. Interestingly, there was an early study reporting that nor-BNI at 10 mg/kg did not affect the ADE in a 4-bottle choice drinking paradigm in male rats with ~1.5 years of alcohol drinking experience [Holter et al 2000]. When further studying the nor-BNI’s effects in a 23-h operant paradigm, they found nor-BNI had no effect on the rat ADE again. The behavioral measures explored in both the previous rat and current mouse studies using nor-BNI suggest that activity of KOP-r/dynorphin is not involved in relapse-like drinking, as pharmacological blockade of KOP-r had no effect on ADE at all.

In a short (1-week) drinking-in-the-dark model with limit drinking time (4 h/day) and intake (5-7 g/kg/day), our pilot study showed an increase in alcohol intake with nor-BNI treatment at a lowest dose (2 mg/kg) (Table S2), suggesting that endogenous KOP-r activity plays a tonic role in inhibiting the initial alcohol intake with low consumption. Our data are in line with early reports of nor-BNI’s effects on increasing dopamine release induced by alcohol [Doyon et al 2006] which could result in enhancing alcohol intake observed in rats [Mitchell et al 2005]. During acute and protracted alcohol withdrawal, however, pharmacological blockade of KOP-r attenuates alcohol seeking or drinking in rodents [Walker and Koob 2008; Kissler et al 2014; Domi et al 2018], as well as anxiety/depression-like behaviors [Anderson and Becker 2017]. Increased KOP-r activity might occur during the acute phase of alcohol withdrawal and KOP-r antagonists might be useful during acute withdrawal. But the increase may disappear later after a period of abstinence, as downregulation of KOP-r mRNA levels was observed in rat ventral tegmentum and nucleus accumbens [Rosin et al 1999]. Recent human PET study found that compared to healthy controls, alcohol-dependent subjects have a significantly lower KOP-r availability across multiple brain regions, including the frontal cortex, dorsal striatum and amygdala [Vijay et al 2018]. Due to conflicting results of KOP-r in the literature with alcohol-induced changes at different drinking periods and withdrawal phases, it is difficult, at this time, to provide a clear picture on how the KOP-r mechanism is involved in rodent ADE or human relapse. Nevertheless, the observed non-behavioral action of nor-BNI on the ADE in rats [Holter et al 2000] and mice (current experiment) suggests that endogenous KOP-r activity may not be involved in relapse-like drinking, though more study is needed.

In our second main objective, we provide clear experimental results showing that the NFF+NTX combination is more effective than either drug alone. In both male and female mice, the combination of NFF (1ug/kg) and NTX (1 mg/kg) together displayed a synergistic effect on preventing ADE (Figure 2), as each compound at this low dose alone had no effect. In our recent report, the same combination did not have any effect on sucrose or saccharin consumption, indicating an alcohol-specific effect [Zhou and Kreek 2019]. For comparison with NTX plus KOP-r agonist, we also purposely tested NTX combined with KOP-r antagonist nor-BNI in parallel and found no effect on the mouse ADE in either sex. In line with our observation with the new NFF+NTX combination, NMF (partial KOP-r agonist+MOP-r antagonist), as our reference compound, reduced relapse-like drinking in the ADE model at 0.5mg/kg (Figure 3), excessive consumption (intake and preference) in an intermittent-access drinking model at 0.5mg/kg (Figure S2), and even “binge” drinking in both sexes (Figure S1) at 1 mg/kg. The effect of NMF (at the dose range <1 mg/kg) was also specific to alcohol, as shown by the lack of the effect on sucrose or saccharin drinking (Table S7). Consistent with a previous study in alcohol-dependent rats [Walker and Koob 2008; Foo et al 2019], NMF (0.5mg/kg) was more effective in reducing ADE than NTX (3mg/kg) in alcohol-relapsing mice (Table 1), possible due to NMF targeting on both KOP-r and MOP-r receptors [Mason et al 1994; Brat et al 2005; Mann et al 2016]. Furthermore, the combination of NFF (1ug/kg) + NMF (0.125 mg/kg) showed more efficacious in reducing excessive drinking (Figure S3) and preventing alcohol relapse than each compound alone (Figure S4), as the two clinically utilized compounds in combination were designed and tested at low doses.

In conclusion, the single-receptor pharmacotherapies have been recently found to have modest therapeutic value, suggesting a need for better efficacy [Zhou and Kreek 2018]. Consistent with our recent study on excessive alcohol drinking in mice, our new finding here has provided further promising in vivo data demonstrating that the clinically utilized KOP-r agonist NFF, in combination with low-dose NTX, may offer a novel strategy to treat alcohol relapse.

4. Experimental Procedures

4.1. Animals.

C57BL/6J mice (8-week-old) in both sexes from The Jackson Laboratory (Bar Harbor, ME, USA) were purchased and housed in a temperature-controlled room (21 °C). After arrival, mice were maintained on a 12-hour reverse light-dark cycle (lights off at 7:00 am) and acclimated for at least one week prior to the experiments. Mice were individually housed and given ad libitum access to food and water. Animal care and experimental procedures were conducted according to Guide for Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources Commission on Life Sciences 1996) and were approved by the IACUC (Institutional Animal Care and Use Committee) of the Rockefeller University.

4.2. Materials.

Ethanol solution was prepared from 190 proof absolute ethyl alcohol (Pharmco-AAPER, Brookfield, CT, USA) and dissolved in tap water. Naltrexone hydrochloride was purchased from Sigma-Aldrich, nalmefene hydrochloride from Baker Norton Pharmaceuticals, and both dissolved in physiological saline. Nalfurafine and nor-BNI was obtained from the NIDA Division of Drug Supply and Analytical Services and dissolved in saline.

4.3. Procedures.

4.3.1. Alcohol deprivation effect (ADE) after chronic intermittent-access alcohol drinking (Table S1).

Chronic intermittent-access alcohol drinking model is a two-bottle choice paradigm with alcohol drinking every other day for 3 weeks. Three hours after lights off, both the water and alcohol (15%) solution sipper tubes were provided, and the sides (left or right one of the cage) of the bottles were randomly positioned on their home cages to avoid the development of side preference. The alcohol bottle was filled with fresh 15% alcohol and kept for 24 h before being replaced by the water bottle. We recorded both alcohol and water intake values after 4, 8 and 24 hours of alcohol access in the drinking days and calculated consumed alcohol intake (g/kg) and relative preference for alcohol (alcohol intake/total fluid intake).

At the end of the 3-week intermittent-access alcohol drinking, 30% alcohol and water bottles were provided on day 21 (week 3) and their intake values at 4, 8 and 24 hours were recorded in the Baseline session. Then, alcohol bottle was not provided for the following 7 days. On day 28 (week 5), after the 1-week abstinence, we provided alcohol (30%) bottles again 3 h after lights off and recorded the alcohol and water intakes at 4, 8 and 24 h in the ADE session.

4.3.2. Administration of NFF, NTX or NFF+NTX in ADE (Table S1).

Mice in each sex were randomly assigned into the drug-treated and vehicle groups with similar alcohol consumption in the Baseline session on day 21. On day 28, the experimenter who was blinded to the treatment codes injected the vehicle and drugs before the ADE test. The mice in the vehicle control groups received one or two vehicle injections; and the mice in the drug groups received one drug (NFF, NTX, or NMF) or two drug (NFF+NTX) injections. Then, the alcohol bottles were presented, and alcohol and water intakes were recorded. [a] The range of NFF doses was based on our recent publication [Zhou and Kreek 2019]: mice received NFF (1, 3 or 10 ug/kg, i.p.) or vehicle (saline) 30 min before the ADE test; [b] The range of NTX doses was based on our recent study [Zhou et al 2018]: mice received NTX (0.3, 1 or 3 mg/kg, i.p.) or vehicle (saline) 10 min before the ADE test; and [c] The NFF+NTX dose chosen was also based on the above NFF and NTX experiments: mice received the first injection of NFF at low-dose 1ug/kg 30 min before the ADE test, followed by the second one of NTX (0.3 or 1 mg/kg) in saline vehicle 10 min before the ADE test.

4.3.3. Administration of NMF in ADE (Table S1).

The range of NMF doses (0.125, 0.25, 0.5 or 1 mg/kg, i.p.) was based on our pilot studies (Figure S1, Figure S2) and a previous publication [Walker and Koob 2008]: using the same paradigm for the above drugs, mice received one NMF injection or one saline 30 min before the ADE test.

4.3.4. Administration of nor-BNI in ADE (Table S1).

Selective KOP-r antagonist nor-BNI was tested on ADE, using the same paradigm for the above drugs with an exception: mice were pretreated with nor-BNI (5, 10 or 20 mg/kg, i.p.) or saline on day 27 (1 day before the ADE test). The nor-BNI dose was based on our pilot studies testing in a drinking-in-the-dark (DID) model and on our early publication in the intermittent-access drinking model [Zhou and Kreek 2019].

4.3.5. Administration of nor-BNI+NTX in ADE (Table S1).

The nor-BNI+NTX dose chosen was based on the above experiments. Using the same paradigm, mice were pretreated with nor-BNI (20 mg/kg, i.p.) or saline on day 27 (1 day before the ADE test), followed by the second one of NTX (1 mg/kg) in saline 10 min before the ADE test.

4.4. Data analysis.

Based on the levels of differences in our previous experiments [Zhou et al 2017, 2018], power analyses were performed to determine the number of animals required to provide statistical significances. We predicted that 8-12 mice per group in each sex were required in the present studies. If similar effects on the ADE with no significant sex differences were seen after each compound or their combination, data of each sex were analyzed and presented separately. Alcohol intake differences across the different groups were analyzed using two-way ANOVA for treatment (vehicle vs drug doses) and for sessions (Baseline vs ADE) in each sex, with testing our a priori hypothesis that there were effects of ADE or drug treatment, based on the published findings [Holter et al 2000; Heyser et al 2003; Walker and Koob, 2008; Zhou et al 2017, 2018] and our new hypothesis. Two-way ANOVA was followed by Newman-Keuls post-hoc tests, and the accepted level of significance was p<0.05. We performed all statistical analyses using Statistica (version 5.5, StatSoft Inc, Tulsa, OK).

Supplementary Material

1

Highlights:

  • Nalfurafine reduced alcohol “relapse” in male and female mice;

  • Nalmefene reduced alcohol “relapse” in male and female mice;

  • Synergistic blockade of “relapse” by a combination of nalfurafine and naltrexone;

Acknowledgement:

This work was supported by NIH AA021970 (YZ), and Miriam and Sheldon G. Adelson Medical Research Foundation (MJK), and NIDA Division of Drug Supply and Analytical Services.

Footnotes

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Conflict of interest: Authors declare that they have no conflicts of interest.

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