**8. Preclinical considerations of targeting the DOP-R for the treatment of AUDs**

In view of the potential development of opioid-receptor selectively acting compounds for the treatment of AUDs, it is important to consider adverse effects associated with opioid receptor activity. A number of adverse effects have been reported with the use of the non-


Table 3. Table of DOP-R and MOP-R activity in brain regions following long-term ethanol consumption in rats.

The Role of Delta Opioid Receptors in Ethanol Consumption

effects using a number of these behavioral methods.

**8.1 Selective effects on ethanol and general consummatory behaviors** 

suggest that the MOP-R plays a more general role in ingestive behaviors.

Activation of the KOP-R has been suggested to affect taste responses due to a non-selective aversive action which could explain changes in levels of ethanol intake (Kovacs et al., 2005). KOP-R knockout mice have a reduced preference for saccharin solutions and a greater

A pharmacotherapeutic for the treatment of AUDs would ideally have selective activity on alcohol consumption and seeking over general consummatory behaviors such as food and fluid intake. Preclinical studies show that naltrexone reduces fat, sucrose and water consumption (Corwin & Wojnicki, 2009; Nielsen et al., 2008; Rao et al., 2008; Wong et al., 2009). Similarly, preclinical studies have shown reduced food intake and weight loss with the opioid antagonists, naloxone (Brown & Holtzman, 1979; Holtzman, 1979; Yuan et al., 2009a; Yuan et al., 2009b) and methylnaltrexone (Yuan et al., 2009a; Yuan et al., 2009b). Conversely, preclinical studies have shown that the MOP-R agonist, morphine, increases both ethanol and food intake (Barson et al., 2009) and the selective MOP-R agonist, DAMGO, increases intake of saccharin, salt, fat and ethanol (de Wet et al., 2001; Tatsuo et al., 1999). Clinical studies have reported that naltrexone treatment leads to reduced food intake and weight loss (Atkinson et al., 1985; Bertino et al., 1991; Spiegel et al., 1987; Sternbach et al., 1982; Yeomans & Gray, 1996; Yeomans & Gray, 1997), although the effects of naltrexone on the eating behavior of obese subjects have been less consistent with reports of either reductions (Spiegel et al., 1987) or no effects on food intake and body weight (Atkinson, 1987; Atkinson et al., 1985; Maggio et al., 1985; Malcolm et al., 1985). Conversely, studies in humans have found that treatment with an opioid agonist with highest affinity for the MOP-R, such as methadone and butorphanol, results in increased food intake and weight gain (Atkinson, 1987; Levine & Atkinson, 1987). Taken together, these studies

and Seeking: Implications for New Treatments for Alcohol Use Disorders 219

selective opioid antagonists (naloxone, naltrexone and methylnaltrexone) in both preclinical (Brown & Holtzman, 1979; Holtzman, 1979; Yuan et al., 2009a; Yuan et al., 2009b) and clinical studies (Bertino et al., 1991; Spiegel et al., 1987; Sternbach et al., 1982; Yeomans & Gray, 1996; Yeomans & Gray, 1997). In humans, naltrexone has neuropsychiatric side effects including anxiety, chills, dizziness, drowsiness, depression, headache, irritability, nervousness and insomnia (Oncken et al., 2001). Furthermore, gastrointestinal side effects associated with naltrexone treatment include appetite loss, constipation, diarrhea, nausea, vomiting and stomach pain/cramps (Oncken et al., 2001). High doses of naltrexone are also associated with hepatotoxicity (Mitchell et al., 1987). Preclinical testing of compounds utilize a number of animal behavioral models to test for adverse effects including effects on nonselective consummatory behavior (self-administration of sugar solutions, food, fat, water), anxiety (elevated plus maze), pain perception (tail-flick, hotplate, paw pressure, von Frey tests), abuse potential (conditioned place preference/aversion), depression (forced swim, learned helplessness), seizure thresholds, (scored observations of clonic movements and catalepsy-like behaviors), and sedation (rotarod and righting reflex tests). Using both opioid receptor knockout mice and opioid receptor subtypes-selective compounds, the roles of opioid receptors in the development of these adverse effects have been investigated in a number of studies. Compounds with affinity for the DOP-R have been tested for adverse

Table 3. Table of DOP-R and MOP-R activity in brain regions following long-term ethanol

consumption in rats.

selective opioid antagonists (naloxone, naltrexone and methylnaltrexone) in both preclinical (Brown & Holtzman, 1979; Holtzman, 1979; Yuan et al., 2009a; Yuan et al., 2009b) and clinical studies (Bertino et al., 1991; Spiegel et al., 1987; Sternbach et al., 1982; Yeomans & Gray, 1996; Yeomans & Gray, 1997). In humans, naltrexone has neuropsychiatric side effects including anxiety, chills, dizziness, drowsiness, depression, headache, irritability, nervousness and insomnia (Oncken et al., 2001). Furthermore, gastrointestinal side effects associated with naltrexone treatment include appetite loss, constipation, diarrhea, nausea, vomiting and stomach pain/cramps (Oncken et al., 2001). High doses of naltrexone are also associated with hepatotoxicity (Mitchell et al., 1987). Preclinical testing of compounds utilize a number of animal behavioral models to test for adverse effects including effects on nonselective consummatory behavior (self-administration of sugar solutions, food, fat, water), anxiety (elevated plus maze), pain perception (tail-flick, hotplate, paw pressure, von Frey tests), abuse potential (conditioned place preference/aversion), depression (forced swim, learned helplessness), seizure thresholds, (scored observations of clonic movements and catalepsy-like behaviors), and sedation (rotarod and righting reflex tests). Using both opioid receptor knockout mice and opioid receptor subtypes-selective compounds, the roles of opioid receptors in the development of these adverse effects have been investigated in a number of studies. Compounds with affinity for the DOP-R have been tested for adverse effects using a number of these behavioral methods.

#### **8.1 Selective effects on ethanol and general consummatory behaviors**

A pharmacotherapeutic for the treatment of AUDs would ideally have selective activity on alcohol consumption and seeking over general consummatory behaviors such as food and fluid intake. Preclinical studies show that naltrexone reduces fat, sucrose and water consumption (Corwin & Wojnicki, 2009; Nielsen et al., 2008; Rao et al., 2008; Wong et al., 2009). Similarly, preclinical studies have shown reduced food intake and weight loss with the opioid antagonists, naloxone (Brown & Holtzman, 1979; Holtzman, 1979; Yuan et al., 2009a; Yuan et al., 2009b) and methylnaltrexone (Yuan et al., 2009a; Yuan et al., 2009b). Conversely, preclinical studies have shown that the MOP-R agonist, morphine, increases both ethanol and food intake (Barson et al., 2009) and the selective MOP-R agonist, DAMGO, increases intake of saccharin, salt, fat and ethanol (de Wet et al., 2001; Tatsuo et al., 1999). Clinical studies have reported that naltrexone treatment leads to reduced food intake and weight loss (Atkinson et al., 1985; Bertino et al., 1991; Spiegel et al., 1987; Sternbach et al., 1982; Yeomans & Gray, 1996; Yeomans & Gray, 1997), although the effects of naltrexone on the eating behavior of obese subjects have been less consistent with reports of either reductions (Spiegel et al., 1987) or no effects on food intake and body weight (Atkinson, 1987; Atkinson et al., 1985; Maggio et al., 1985; Malcolm et al., 1985). Conversely, studies in humans have found that treatment with an opioid agonist with highest affinity for the MOP-R, such as methadone and butorphanol, results in increased food intake and weight gain (Atkinson, 1987; Levine & Atkinson, 1987). Taken together, these studies suggest that the MOP-R plays a more general role in ingestive behaviors.

Activation of the KOP-R has been suggested to affect taste responses due to a non-selective aversive action which could explain changes in levels of ethanol intake (Kovacs et al., 2005). KOP-R knockout mice have a reduced preference for saccharin solutions and a greater

The Role of Delta Opioid Receptors in Ethanol Consumption

plasma corticosterone (Nielsen et al., 2011).

**8.3 Abuse potential** 

1999a).

and Seeking: Implications for New Treatments for Alcohol Use Disorders 221

receptors are not involved in the hormonal stress response. However, other studies have shown that rats housed in a stressful environment were more sensitive to the sedative effects of the DOP-R agonist, SNC80, compared to stimulant effects by SNC80 in rats that were not stressed (Pohorecky et al., 1999). In contrast, plasma corticosterone levels were increased in rats following acute intracerebral administration of the DOP-R agonists DPDPE and DADLE (Gonzalvez et al., 1991; Iyengar et al., 1987). Increased plasma corticosterone levels were found in rats administered naltrindole but not in rats co-administered naltrindole and SNC80 (Saitoh et al., 2005) or rats administered SoRI-9409 (Nielsen et al., 2008). Furthermore, pre-treatment with SoRI-9409 decreased yohimbine stress-induced reinstatement of ethanol-seeking in rats but did not affect yohimbine-induced increases in

An issue with the use of pharmacotherapeutics for the treatment of addiction is the incidence of potential abuse of the therapeutic itself. For example, the MOP-R agonist methadone, which is used to treat heroin addiction, has been reported to be widely abused (Li et al., 2011; Simonsen et al., 2011a; Simonsen et al., 2011b; Tormoehlen et al., 2011). Although the use of "substitution" therapy with opioid agonists has been effective for some patients, it has remained controversial (Gerra et al., 2009; Ling et al., 1994; Rhodes & Grossman, 1997). However, as naltrexone induces aversive side-effects in humans and conditioned place aversion in rats (Mitchell et al., 2009), it does not appear to be rewarding itself. Furthermore, naltrexone attenuates the expression of ethanol place conditioning in mice (Middaugh & Bandy, 2000). MOP-R agonists increase and MOP-R antagonists decrease, respectively, ethanol-induced CPP in rats (Matsuzawa et al., 1998; Matsuzawa et al., 1999a; Matsuzawa et al., 1999b). In comparison, activation of the KOP-R is associated with general aversive activity in rats (Shippenberg & Herz, 1986) and induces dysphoria in humans (Kumor et al., 1986; Pfeiffer et al., 1986; Rimoy et al., 1994). The KOP-R agonist, U50488, attenuates ethanol-induced CPP in rats (Matsuzawa et al., 1999a). KOP-R agonists, therefore, do not appear to be carry the risk of abuse potential. In comparison, KOP-R antagonists, such as nor-BNI do not produce CPP or CPA (Mitchell et al., 2005; Sante et al., 2000) although nor-BNI did increase ethanol-induced CPP in one study (Matsuzawa et al.,

As described earlier, DOP-R agonists increase and DOP-R antagonists attenuate, respectively, ethanol-induced CPP in rats (Bie et al., 2009; Matsuzawa et al., 1998; Matsuzawa et al., 1999a; Matsuzawa et al., 1999b) and DOP-R antagonists can make a nonaversive dose of alcohol aversive (Froehlich et al., 1998). DOP-R antagonists administered alone do not induce CPP or CPA (Nielsen et al., 2008) suggesting they are not rewarding themselves. The increase in the rewarding actions of ethanol by DOP-R agonists may contribute to the altered levels of ethanol consumption following treatment with DOP-R agonists, as described above (Barson et al., 2009; Barson et al., 2010; Margolis et al., 2008; van Rijn et al., 2010; van Rijn & Whistler, 2009). Furthermore, DOP-R (and MOP-R) agonists have been shown to be rewarding themselves such that DPDPE is self-administered into the VTA of rats (Devine & Wise, 1994; McBride et al., 1999). Activation of DOP-R (and MOP-R) leads to increased basal dopamine release in brain regions involved in the reward pathway

preference for quinine solutions (Kovacs et al., 2005). Unlike ligands with highest activity for the MOP-R (such as naltrexone), KOP-R, NOP-R and SIG-R, the ligands which have selective for DOP-R appear to have greater selectivity for ethanol consumption with reduced activity on general consummatory behavior, such as food, sugar or water consumption (Barson et al., 2009; Barson et al., 2010; Froehlich et al., 1991; Krishnan-Sarin et al., 1995b). Central administration of naltrexone, nor-BNI and -funaltrexamine, but not naltrindole, reduces intake of sucrose solutions in rats (Beczkowska et al., 1992; Koch et al., 1995). Furthermore, central administration of naltrexone, naloxonazine and nor-BNI, but not naltrindole or the DOP-R agonis DALCE, reduces fat intake in food-deprived rats (Koch & Bodnar, 1994). The enkephalinase inhibitor, thiorphan, increased ethanol, but not water, intake in alcohol-preferring rats (Froehlich et al., 1991). Central administration of the DOP-R agonist, DALA, to rats selectively increased ethanol consumption over food and water in comparison to non-selective actions on ethanol intake by the MOP-R agonists, DAMGO and morphine (Barson et al., 2009; Barson et al., 2010). The naltrexone-derived DOP-R antagonist, SoRI-9409, was shown to be much more effective and selective in reducing ethanol consumption than naltrexone such that, unlike naltrexone, SoRI-9409 did not reduce water intake and did not reduce sucrose intake in doses that effectively reduced ethanol intake (Nielsen et al., 2008). Although one study found that the DOP-R antagonist, naltrindole, reduced ethanol and saccharin, but not water, consumption in rats (Krishnan-Sarin et al., 1995a), further studies by these same researchers showed that the DOP-R antagonist, naltriben, reduced the intake of solutions containing ethanol with saccharin and ethanol with quinine but no effects on the intake of either saccharin or quinine solutions alone (Krishnan-Sarin et al., 1995b). Taken together, these studies suggest that compounds with activity at the DOP-R selectively alter ethanol intake over general consummatory behavior.

#### **8.2 Anxiety and stress**

A major problem in treating AUDs is the high rate of relapse which is usually triggered by stress and anxiety (Sinha, 2007; Sinha & Li, 2007). Recent preclinical studies have suggested that potential new pharmacotherapies for AUDs act by reducing anxiety and cravings in alcohol-dependent subjects (George et al., 2008; Heilig et al., 2010). Treatment options to control stress and anxiety disorders include benzodiazepines, which carry the risk of abuse potential, and antidepressants, which demonstrate a relative large interindividual variability in terms of drug response (O'Brien, 2005; Tiwari et al., 2009). Studies investigating the roles of opioid receptors in anxiety and stress indicate that the DOP-R plays a significant role. DOP-R knockout mice have increased anxiety (Filliol et al., 2000). In comparison, rats administered the DOP-R agonist, SNC80 have increased anxiolytic activity, an effect that is reversed by naltrindole (Perrine et al., 2006; Saitoh et al., 2004). Naltrindole was found to have anxiogenic activity when given in higher, but not lower doses (Perrine et al., 2006; Saitoh et al., 2004) although the DOP-R antagonist, SoRI-9409 has neither anxiogenic nor anxiolytic activity (Nielsen et al., 2008). In contrast, -funaltrexamine and nor-BNI did not produce any anxiogenic or anxiolytic effects, suggesting the MOP-R and KOP-R do not play a role in anxiety states (Saitoh et al., 2004). Following the forced swim test, plasma levels of the stress hormone, corticosterone are the same, in triple opioid receptor knockout (MOP-R, DOP-R, KOP-R) knockout and wild-type mice (Contet et al., 2006). This suggests that opioid receptors are not involved in the hormonal stress response. However, other studies have shown that rats housed in a stressful environment were more sensitive to the sedative effects of the DOP-R agonist, SNC80, compared to stimulant effects by SNC80 in rats that were not stressed (Pohorecky et al., 1999). In contrast, plasma corticosterone levels were increased in rats following acute intracerebral administration of the DOP-R agonists DPDPE and DADLE (Gonzalvez et al., 1991; Iyengar et al., 1987). Increased plasma corticosterone levels were found in rats administered naltrindole but not in rats co-administered naltrindole and SNC80 (Saitoh et al., 2005) or rats administered SoRI-9409 (Nielsen et al., 2008). Furthermore, pre-treatment with SoRI-9409 decreased yohimbine stress-induced reinstatement of ethanol-seeking in rats but did not affect yohimbine-induced increases in plasma corticosterone (Nielsen et al., 2011).
