The effect of protein phosphatase 2A inhibitor LB100 on regulating methamphetamine induced conditioned place preference in mice.

Protein phosphatase 2A (PP2A) is an evolutionarily conserved serine/threonine phosphatase abundant in mammalian brains. Although recent research has revealed that PP2A plays important roles in cocaine and morphine addictions, the mechanism of action of PP2A in methamphetamine (METH) addiction is unclear. LB100 is a PP2A inhibitor able to penetrate the blood-brain barrier (BBB); the role of LB100 in METH-induced conditioned place preference (CPP) has not yet been reported. Here, we explored the roles of LB100 in distinct phases of METH-induced CPP. Our findings indicate that LB100 inhibits the acquisition and reinstatement of METH-induced CPP and promotes the extinction of METH-induced CPP. Moreover, LB100 alone did not affect the natural preference of mice. Intriguingly, repeated administration of LB100 in the extinction phase did not inhibit the reinstatement of METH-induced CPP, but LB100 injection prior to METH administration could significantly block it. Taken together, we found that LB100 has significant effects on different phases of METH-induced CPP, and is therefore, a potentially promising therapeutic for METH addiction.

The protein kinase Cß-selective inhibitor, enzastaurin, attenuates amphetamine-stimulated locomotor activity and self-administration behaviors in rats.

RATIONALE: Pathological amphetamine (AMPH) use is a serious public health concern with no pharmacological treatment options. Protein kinase Cß (PKCß) has been implicated in the mechanism of action of AMPH, such that inhibition of PKCß attenuates AMPH-stimulated dopamine efflux in vivo. With this in mind, inhibition of PKCß may be a viable therapeutic target for AMPH use disorder. OBJECTIVE: The purpose of this study is to demonstrate that selective pharmacological inhibition of PKCß alters AMPH-stimulated behaviors in rats. METHODS: Rats were administered intracerebroventricular (i.c.v.) injections of the PKCß-selective inhibitor enzastaurin 0.5, 3, 6, or 18 h before evaluating AMPH-stimulated locomotion (0.32-3.2 mg/kg). Rats were trained to make responses for different doses of AMPH infusions or sucrose under a fixed ratio 5 schedule of reinforcement, and the effects of enzastaurin pretreatment 3 or 18 h prior to a self-administration session were determined. Also, the effect of enzastaurin on AMPH-stimulated PKC activity in the ventral striatum was evaluated. RESULTS: A large dose of enzastaurin (1 nmol) decreased AMPH-stimulated locomotor activity 0.5 h following enzastaurin administration. Small doses of enzastaurin (10-30 pmol) attenuated AMPH-stimulated locomotor activity and shifted the AMPH dose-effect curve to the right following an 18-h pretreatment. Rats pretreated with enzastaurin 18 h, but not 3, prior to a self-administration session showed a decrease in the number of responses for AMPH, shifted the ascending limb of the amphetamine dose effect curve, and produced no change in responses for sucrose. AMPH-stimulated PKC activity was decreased following a 0.5- or 18-h pretreatment, but not a 3-h pretreatment of enzastaurin. CONCLUSIONS: These results demonstrate that inhibition of PKCß will decrease AMPH-stimulated behaviors and neurobiological changes and suggest that PKCß is potentially a viable target for AMPH use disorder.

Novel prodrug PRX-P4-003, selectively activated by gut enzymes, may reduce the risk of iatrogenic addiction and abuse.

OBJECTIVES: Prescription stimulants are vulnerable to oral and parenteral abuse. Intravenous forms of abuse may be most detrimental due to an enhanced risk of dependence, overdose, and infectious diseases. Our objective was to discover an orally active prodrug of a stimulant that would not be easily converted to its parent when injected, thus hindering intravenous abuse. METHODS: Following an initial analysis of stimulant structures, the fencamfamine isomer [(-)-FCF; (N-ethyl-3-phenylbicyclo[2.2.1]heptan-2-amine)] was chosen as a parent drug due to its favorable biochemical properties. Subsequently, PRX-P4-003 {(-)-N-(Octadecanoyloxymethoxycarbonyl)-N-ethyl-3-phenylbicyclo[2.2.1]heptan-2-amine} qualified for further development. Experimental testing of PRX-P4-003 included radioligand binding assays, stability studies, and rodent pharmacokinetic and locomotor assays. RESULTS: Prodrug PRX-P4-003 is a pharmacologically inactive, hydrophobic compound, whereas its parent (-)-FCF is a dopamine reuptake inhibitor with weaker effects on norepinephrine reuptake (Ki = 0.07 and 0.80 µM, respectively). PRX-P4-003 is metabolized to (-)-FCF in simulated intestinal fluid (with pancreatin) but not in simulated gastric fluid (with pepsin). Finally, PRX-P4-003 shows a significant oral but no intravenous increase in locomotion, correlating with its pharmacokinetics by these different routes of administration. CONCLUSIONS: PRX-P4-003 is a novel prodrug stimulant enzymatically activated in the gut. Our data suggest a pancreatic, lipase-based mechanism of activation and as only 1% of this enzyme is found in the systemic circulation, PRX-P4-003 is unlikely to be bioactive if injected intravenously. Enzymatic release of (-)-FCF is needed prior to its systemic absorption, which may discourage oral abuse (e.g., by chewing). PRX-P4-003 is being developed for apathy in Alzheimer's disease and binge eating disorder.

Differential COMT expression and behavioral effects of COMT inhibition in male and female Wistar and alcohol preferring rats.

Polymorphisms of the catechol-O-methyl transferase (COMT) gene have been associated with alcoholism, suggesting that alterations in the metabolism of catecholamines may be a critical component of the neuropathology of alcoholism. In the current experiments, the COMT inhibitor tolcapone was utilized in an operant behavioral model of reinforcer-seeking and drinking to determine if this compound was capable of remediating the excessive seeking and drinking phenotype of the alcohol-preferring P rat. Tolcapone was administered to male and female alcohol-reinforced P and Wistar rats. Additionally, tolcapone was administered to male sucrose-reinforced P and Wistar rats to determine if its effects also extended to a natural reinforcer. Animals were trained to make an operant response that resulted in 20 min uninterrupted access to the reinforcer solutions. Tolcapone had no effect in female rats on either seeking or consumption of ethanol. However, reductions of both reinforcer seeking and consumption were observed in male P rats, but only of seeking in Wistars. In separate experiments, using reinforcer naïve male and female animals, COMT expression was assessed via Western Blot analysis. Sex differences in COMT expression were also observed, where male P rats exhibited a marked reduction in protein expression relative to females in the PFC. Sex differences were not observed for Wistars or in the striatum and hippocampus. These data complement our previous findings in which tolcapone reduced cue-evoked responses in P rats and further suggest clinical utility of COMT inhibitors in the treatment of addiction disorders, specifically in male high drinkers.

Short-term abstinence from cocaine self-administration, but not passive cocaine infusion, elevates αCaMKII autophosphorylation in the rat nucleus accumbens and medial prefrontal cortex.

Increases in alpha calcium/calmodulin-dependent protein kinase type II (αCaMKII) activity in the nucleus accumbens shell has been proposed as a core component in the motivation to self-administer cocaine and in priming-induced drug-seeking. Since cocaine withdrawal promotes drug-seeking, we hypothesized that abstinence from cocaine self-administration should enhance αCaMKII as well. We found that short-term abstinence from contingent, but not non-contingent, cocaine i.v. self-administration (2 h/d for 14 d; 0.25 mg/0.1 ml, 6 s infusion) elevates αCaMKII autophosphorylation, but not the kinase expression, in a dynamic, time- and brain region-dependent manner. Increased αCaMKII autophosphorylation in the nucleus accumbens (NAc) and medial prefrontal cortex (mPFC), but not dorsolateral striatum (dlS), was found 24 h, but not immediately, after the last cocaine self-administration session. Notably, in the mPFC, but not NAc and dlS, αCaMKII autophosphorylation was still enhanced 7 d later. The persistent enhancement in the mPFC of abstinent rats may represent a previously unappreciated contribution to initial incubation of cocaine-seeking.

Effects of L-cysteine on reinstatement of ethanol-seeking behavior and on reinstatement-elicited extracellular signal-regulated kinase phosphorylation in the rat nucleus accumbens shell.

BACKGROUND: Alcoholism is a neuroadaptive disorder, and the understanding of the mechanisms of the high rates of relapse, which characterize it, represents one of the most demanding challenges in alcoholism and addiction research. The extracellular signal-regulated kinase (ERK) is an intracellular kinase, critical for neuroplasticity in the adult brain that is suggested to play a fundamental role in the molecular mechanisms underlying drug addiction and relapse. We previously observed that a nonessential amino acid, L-cysteine, significantly decreases oral ethanol (EtOH) self-administration, reinstatement of EtOH-drinking behavior, and EtOH self-administration break point. METHODS: Here, we tested whether L-cysteine can affect the ability of EtOH priming to induce reinstatement of EtOH-seeking behavior. In addition, we determined the ability of EtOH priming to induce ERK phosphorylation as well as the ability of L-cysteine to affect reinstatement-elicited ERK activation. To these purposes, Wistar rats were trained to nose-poke for a 10% v/v EtOH solution. After stable drug-taking behavior was obtained, nose-poking for EtOH was extinguished, and reinstatement of drug seeking, as well as reinstatement-elicited pERK, was determined after an oral, noncontingent, priming of EtOH (0.08 g/kg). Rats were pretreated with either saline or L-cysteine (80 to 120 mg/kg) 30 minutes before testing for reinstatement. RESULTS: The findings of this study confirm that the noncontingent delivery of a nonpharmacologically active dose of EtOH to rats, whose previous self-administration behavior had been extinguished, results in significant reinstatement into EtOH-seeking behavior. In addition, the results indicate that reinstatement selectively activates ERK phosphorylation in the shell of the nucleus accumbens (Acb) and that pretreatment with L-cysteine reduces either reinstatement of EtOH seeking and reinstatement-elicited pERK in the AcbSh. CONCLUSIONS: Altogether, these results indicate that L-cysteine could be an effective pharmacological agent for the prevention of behavioral and molecular correlates of EtOH-primed reinstatement of EtOH seeking and that the shell of the Acb represents a critical neural substrate for priming-elicited reinstatement mechanisms involving ERK phosphorylation.

Robust escalation of nicotine intake with extended access to nicotine self-administration and intermittent periods of abstinence.

Although established smokers have a very regular pattern of smoking behavior, converging lines of evidence suggest that the escalation of smoking behavior is a critical factor in the development of dependence. However, the neurobiological mechanisms that underlie the escalation of smoking are unknown, because there is no animal model of the escalation of nicotine intake. On the basis of the pattern of smoking behavior in humans and presence of monoamine oxidase inhibitors in tobacco smoke, we hypothesized that the escalation of nicotine intake may only occur when animals are given extended-access (21 h per day) self-administration sessions after repeated periods of abstinence (24-48 h), and after chronic inhibition of monoamine oxidase using phenelzine sulfate. Intermittent access (every 24-48 h) to extended nicotine self-administration produced a robust escalation of nicotine intake, associated with increased responding under fixed- and progressive-ratio schedules of reinforcement, and increased somatic signs of withdrawal. The escalation of nicotine intake was not observed in rats with intermittent access to limited (1 h per day) nicotine self-administration or daily access to extended (21 h per day) nicotine self-administration. Moreover, inhibition of monoamine oxidase with daily administration of phenelzine increased nicotine intake by ≈ 50%. These results demonstrate that the escalation of nicotine intake only occurs in animals given intermittent periods of abstinence with extended access to nicotine, and that inhibition of monoamine oxidase may contribute to the escalation of smoking, thus validating both an animal model of the escalation of smoking behavior and the contribution of monoamine oxidase inhibition to compulsive nicotine-seeking.

The role of catechol-O-methyltransferase in reward processing and addiction.

Catechol-O-methyltransferase (COMT) catabolises dopamine and is important for regulating dopamine levels in the prefrontal cortex. Consistent with its regulation of prefrontal cortex dopamine, COMT modulates working memory and executive function; however, its significance for other cognitive domains, and in other brain regions, remains relatively unexplored. One such example is reward processing, for which dopamine is a critical mediator, and in which the striatum and corticostriatal circuitry are implicated. Here, we discuss emerging data which links COMT to reward processing, review what is known of the underlying neural substrates, and consider whether COMT is a good therapeutic target for treating addiction. Although a limited number of studies have investigated COMT and reward processing, common findings are beginning to emerge. COMT appears to modulate cortical and striatal activation during both reward anticipation and delivery, and to impact on reward-related learning and its underlying neural circuitry. COMT has been studied as a candidate gene for numerous reward-related phenotypes and there is some preliminary evidence linking it with certain aspects of addiction. However, additional studies are required before these associations can be considered robust. It is premature to consider COMT a good therapeutic target for addiction, but this hypothesis should be revisited as further information emerges. In particular, it will be critical to reveal the precise neurobiological mechanisms underlying links between COMT and reward processing, and the extent to which these relate to the putative associations with addiction.

Are genetic variants of COMT associated with addiction?

The human catechol-O-methyl transferase (COMT) gene contains multiple single-nucleotide polymorphisms, some of which are postulated to have clinical significance. This article reviews human studies that have explored the association between COMT polymorphisms and addiction to drugs, alcohol or tobacco. Most studies concentrate on the Val108/158Met polymorphism. Although there are reports indicating a positive association with COMT polymorphisms and addiction, the majority of the studies failed to detect such a link between them with one exception, smoking. It is unlikely that there would be any single gene that could be designated as 'the addiction gene'. Rather, there seems to be a great number of genes that are associated with addiction, among which COMT seems to have a minor role. Environmental factors and genetic milieu have a great impact on whether the small effects of COMT polymorphisms on risk of addiction can be detected in a given population. Sex differences complicate the gene-environment interplay even further.

Protein kinase C isozymes as regulators of sensitivity to and self-administration of drugs of abuse-studies with genetically modified mice.

Studies using targeted gene deletion in mice have revealed distinct roles for individual isozymes of the protein kinase C (PKC) family of enzymes in regulating sensitivity to various drugs of abuse. These changes in drug sensitivity are associated with altered patterns of drug self-administration. The purpose of this review is to summarize behavioral studies conducted on mice carrying targeted deletions of genes encoding specific PKC isozymes (namely the beta, gamma, delta, and epsilon isozymes), and to critically evaluate the possibility of using pharmacological inhibitors of specific PKC isozymes as modulators of the sensitivity to various drugs of abuse, as well as potential aids in the treatment of substance use disorders.

Biphasic modulation of cocaine-induced conditioned place preference through inhibition of histone acetyltransferase and histone deacetylase.

OBJECTIVE: To examine the causative relationship between aberrant histone acetylation changes and cocaine-induced reward. METHODS: Male Sprague-Dawley rats (n=160) were tested by conditioned place preference (CPP) procedure, to evaluate the effects of inhibitors of histone deacetylase (HDAC) and histone acetyltransferase (HAT) on the conditioned effects of cocaine. Conditioning sessions were conducted twice daily for 2-4 days. For each conditioning session, rats were injected with either HDAC (or HAT) inhibitors or saline in home cages, followed by cocaine (intraperitoneally [ip]) or saline (ip) 30 minutes later, and then immediately confined for 50 minutes in the cue-specific chamber. On the day following the last conditioning session, the rats were tested for place preference for 15 minutes. The present study was carried out at the Department of Pharmacology of Jiaxing University, Jiaxing, Zhejiang, and Pharmacology Research Center of Fudan University, Shanghai, China between October 2007 and January 2009. RESULTS: Our results showed that pretreatment with HDAC inhibitor (sodium butyrate), potentiated cocaine-induced CPP, but did not itself lead to conditioned preferences, or aversions. On the contrary, rats pretreated with curcumin (HAT inhibitor) markedly inhibited cocaine-induced CPP, but did not itself lead to conditioned preferences or aversions. CONCLUSION: Histone modifications may be an important mechanism that underlies conditioned effects of cocaine. Moreover, HAT may be a potential therapeutic target for cocaine addiction.

Structure of daidzin, a naturally occurring anti-alcohol-addiction agent, in complex with human mitochondrial aldehyde dehydrogenase.

The ALDH2*2 gene encoding the inactive variant form of mitochondrial aldehyde dehydrogenase (ALDH2) protects nearly all carriers of this gene from alcoholism. Inhibition of ALDH2 has hence become a possible strategy to treat alcoholism. The natural product 7-O-glucosyl-4'-hydroxyisoflavone (daidzin), isolated from the kudzu vine ( Peruraria lobata), is a specific inhibitor of ALDH2 and suppresses ethanol consumption. Daidzin is the active principle in a herbal remedy for "alcohol addiction" and provides a lead for the design of improved ALDH2. The structure of daidzin/ALDH2 in complex at 2.4 A resolution shows the isoflavone moiety of daidzin binding close to the aldehyde substrate-binding site in a hydrophobic cleft and the glucosyl function binding to a hydrophobic patch immediately outside the isoflavone-binding pocket. These observations provide an explanation for both the specificity and affinity of daidzin (IC50 =80 nM) and the affinity of analogues with different substituents at the glucosyl position.

A cocaine hydrolase engineered from human butyrylcholinesterase selectively blocks cocaine toxicity and reinstatement of drug seeking in rats.

Successive rational mutations of human butyrylcholinesterase (BChE) followed by fusion to human serum albumin have yielded an efficient hydrolase that offers realistic options for therapy of cocaine overdose and abuse. This albumin-BChE prevented seizures in rats given a normally lethal cocaine injection (100 mg/kg, i.p.), lowered brain cocaine levels even when administered after the drug, and provided rescue after convulsions commenced. Moreover, it selectively blocked cocaine-induced reinstatement of drug seeking in rats that had previously self-administered cocaine. The enzyme treatment was well tolerated and may be worth exploring for clinical application in humans.

Monoamine oxidase inhibition dramatically increases the motivation to self-administer nicotine in rats.

Nicotine is the major neuroactive compound of tobacco, which has, by itself, weak reinforcing properties. It is known that levels of the enzymes monoamine oxidase A (MAO-A) and MAO-B are reduced in the platelets and brains of smokers and that substances, other than nicotine, present in tobacco smoke have MAO-inhibitory activities. Here, we report that inhibition of MAO dramatically and specifically increases the motivation to self-administer nicotine in rats. These effects were more prominent in rats selected for high responsiveness to novelty than in rats with low responsiveness to novelty. The results suggest that the inhibition of MAO activity by compounds present in tobacco smoke may combine with nicotine to produce the intense reinforcing properties of cigarette smoking that lead to addiction.

Protein kinase C isozymes and addiction.

Protein kinase C (PKC) has long been recognized an important family of enzymes that regulate numerous aspects of neuronal signal transduction, neurotransmitter synthesis, release and reuptake, receptor and ion channel function, neuronal excitability, development, and gene expression. Much evidence has implicated PKCs in the effects of several drugs of abuse, and in behavioral responses to these drugs. The present review summarizes the effects of both acute and chronic exposure to various drugs of abuse on individual PKC isozymes in the brain. In addition, we summarize recent studies utilizing mice with targeted deletions of the genes for PKCgamma and PKCepsilon. These studies suggest that individual PKC isozymes play a role in the development of drug dependence and addiction.

Deletion of the fyn-kinase gene alters behavioral sensitivity to ethanol.

BACKGROUND: An earlier study showed that deletion of the fyn-kinase gene enhanced sensitivity to ethanol's sedative hypnotic effects and suggested that this was associated with diminished fyn-kinase phosphorylation of NMDA receptors. The authors of that study speculated that this resulted in an inability of the null mutants to develop acute tolerance to ethanol, leading to the longer ethanol-induced sleep times. However, in vivo acute tolerance to ethanol was not examined directly. METHODS: To address the role of fyn-kinase in mediating acute tolerance, as well as sensitivity to several other behavioral effects of ethanol, we studied an independently generated population of fyn null mutant and wild-type mice. RESULTS: Homozygous mutants exhibited longer ethanol sleep times that could not be attributed to differences in initial sensitivity, and impaired acute tolerance to the motor incoordinating effects of ethanol as measured by using the stationary dowel, but not the rotarod. Fyn-kinase null mutants were more sensitive to the anxiolytic effects of ethanol when tested using the elevated plus maze, and males displayed a lower preference for ethanol in a two-bottle choice paradigm. Finally, mutant and wild-type mice did not differ in sensitivity to the hypothermic effects of ethanol. The genotypes also did not differ in blood-ethanol clearance, eliminating a metabolic explanation for these behavioral differences. CONCLUSIONS: These results show that fyn-kinase modulates acute tolerance to ethanol and suggest a role for fyn in mediating ethanol's anxiolytic and reinforcing properties.

Gamma-vinyl GABA, an irreversible inhibitor of GABA transaminase, alters the acquisition and expression of cocaine-induced sensitization in male rats.

We examined the effect of (+/-)-gamma-vinyl GABA (GVG, Vigabatrin), an irreversible inhibitor of the enzyme GABA transaminase, on the acquisition and expression of cocaine-induced sensitization in albino male Sprague-Dawley rats. Animals received a single injection of 1 ml/kg i.p. of 0.9% saline or 15 mg/kg i.p. of (-)-cocaine and locomotor activity was assessed using automated locomotor cages and stereotyped behaviors were scored using a 4-point rating scale (Day 1). Subsequently, animals were given 15 mg/kg i.p. of cocaine every 48 h in their home cage for 1 week (Days 3, 5, and 7) and then given no treatment for 1 week. A challenge injection of 15 mg/kg i.p. of cocaine, but not vehicle, produced a significant increase in locomotor activity and stereotyped behaviors on Day 15 compared to animals that received cocaine on Day 1. Administration of 75 mg/kg i.p. of GVG 2.5 h before the cocaine injections did not significantly alter the acquisition of cocaine-induced locomotor sensitization. However, 150 mg/kg i.p. of GVG significantly attenuated the acquisition of cocaine-induced locomotor sensitization. Administration of 150 mg/kg i.p. of GVG 2.5 h before the cocaine challenge injection on Day 15 significantly attenuated the expression of cocaine-induced locomotor sensitization. Acquisition and expression of cocaine-induced sensitization of stereotypy was also significantly attenuated by 150 mg/kg i.p. of GVG. Since sensitization may be one of the factors involved in relapse to drug use, the present results, in combination with previous findings that GVG blocks the rewarding and incentive motivating effects of cocaine, suggest that GVG might prove useful in the treatment of cocaine addiction.

Circadian differences in behavioral sensitization to cocaine: putative role of arylalkylamine N-acetyltransferase.

Circadian rhythms might be involved in addictive behaviors. The pineal secretory product melatonin decreases cocaine sensitization in rats; mice mutant for the critical melatonin-synthesizing enzyme, arylalkylamine N-acetyltransferase (AANAT), exhibit altered behaviors. We hypothesized that AANAT/melatonin system, which is up-regulated at night, affects cocaine sensitization in mice. Intraperitoneal cocaine treatment (10 and 20 mg/kg) dose-dependently increased locomotor activity of both normal (C3H/HeJ) and AANAT mutant (C57BL/6J) mice; this effect was similar during the day and at night. Injections of cocaine during the day for three days resulted in behavioral sensitization in normal and AANAT mutant mice whereas treatment at night triggered sensitization in AANAT-deficient mice only. AANAT expression and synthesis of N-acetylserotonin/melatonin could play a role in addictive properties of cocaine.