Jonathan L. Katz, Ph.D. - US grants

The primary focus of this research is to develop a better understanding of the pharmacological mechanisms underlying the behavioral effects of cocaine that lead to its abuse, and the consequences of that abuse. This better understanding will advance basic knowledge of the pharmacology of cocaine, and drug abuse. In addition, there is a large unmet medical need for cocaine addiction treatments. Recent (1999 to 2002) annual estimates of the number of individuals using cocaine range from 2 to 3.2 million in the United States alone. This research will ultimately lead to the discovery of new treatment modalities for cocaine abuse. Effective treatments for drug abuse will ultimately have a positive public health impact in curtailing drug abuse and the transmission of HIV infection. We examined the binding of atypical dopamine uptake inhibitors to dopamine (DA) transporter sites in vivo along with the behavioral effects of the drugs. Both cocaine and GBR 12909 increased locomotor activity in a dose-related manner, with the effects of GBR 12909 lasting longer than cocaine. In addition there were long lasting increases in locomotor activity produced by the atypical DA uptake inhibitors, however, maximal effects were generally lower than those produced by cocaine. Further, the stimulation produced by JHW 007 did not achieve statistical significance. The apparent rate of association for cocaine was 2.04% occupancy/min, whereas the rate of occupancy of JHW 007 was 10-fold lower. We further examined the relationship between locomotor-stimulant effects of cocaine, GBR 12909 and BZT analogues (AHN 1-055 and AHN 2-005) and their in vivo displacement of the DA transporter ligand [125I]RTI-121, by linear regression of dopamine transporter occupancy and the stimulation produced. For each of the drugs there was a significant positive correlation between occupancy and stimulation, as expected. However, for cocaine, the slope of the regression line was less than that for the other drugs. Contributing to this diminished strength of association between occupancy and effect were the effects of cocaine at early time points after injection. At these times the behavioral effect was a greater than predicted by the regression of displacement of [125I]RTI-121, and a trend for the stimulation to be less than predicted at times longer after injection. These data suggest that the on rate of occupancy of the dopamine transporter, in addition to percentage of sites occupied, contributes to the behavioral effects of cocaine. The present results suggest that among drugs that act at the DA transporter, the slower apparent rates of occupancy with the DA transporter by the BZT analogs may contribute in an important way to differences in their effectiveness. Studies described in Section III, Future Research Plans, address further tests of this hypothesis and the proposed further mechanistic studies that may account for differences in rates of association. The substantial occupancy of the dopamine transporter by JHW 007 obtained at 270 min after treatment was comparable to the occupancy obtained with cocaine at 30 min after treatment (Figure 7). Because this occupancy was sufficient to produce behavioral effects with cocaine but appeared to be inadequate for behavioral effects with JHW 007 we hypothesized that JHW 007 would function as an antagonist of cocaine. When we pretreated subjects with JHW 007 and later gave cocaine, the effects of cocaine were antagonized.

A previous study from this laboratory showed that dopamine (DA) D2 receptors (D2Rs) are involved in the reinforcing effectiveness of different magnitudes of food reinforcement. We attempted to assess the contributions of the three D2-like receptor subtypes using knockout (KO) mice completely lacking DA receptors (D2R, D3R, or D4R KO mice) and their wild-type littermates. These subjects were exposed to a series of fixed-ratio (FR) food-reinforcement schedules in two contexts: an open economy with additional food provided outside the experimental setting and a closed economy with all food earned within the experimental setting. A behavioral-economic model was used to quantify reinforcer effectiveness with food pellets obtained as a function of price (FR schedule value) using an exponential model of demand with the equation logQ=logQ0+k(e-alphaQ0C-1), where alpha represents decline rate of the curve and is inversely related to reinforcer effectiveness, Q represents the number of reinforcers consumed, Q0 represents maximum consumption at zero price, C represents the cost of each reinforcer (i.e. FR value), and k is the y-axis range of the function. With both open and closed economies, as price increased, food consumption decreased, and did so more rapidly in DA D2R KO mice compared to WT littermates. Because an assessment of demand may be influenced by how rapidly responding extinguishes, we studied extinction of responding in two contexts: by eliminating food deliveries and by delivering food independently of responding. A hyperbolic model quantified rates of extinction with an equation of the form R=1/(1+kt), with R representing the response rate as a proportion of control, t representing session number, and k representing the decay parameter, which reflects rate of change (i.e., how quickly the behavior extinguishes). Extinction in DA D2R KO mice occurred less rapidly compared to WT mice in both contexts. Elasticity of food demand was higher in DA D4R KO than WT mice in the open, but not closed, economy. Extinction of responding in DA D4R KO mice was not different from that in WT littermates in either context. No differences in elasticity of food demand or extinction rate were obtained in D3R KO mice and WT littermates. These results indicate that the D2R is the primary DA D2-like receptor subtype mediating the reinforcing effectiveness of food. To identify novel ligands for the D2 dopamine receptor (D2R), we screened small molecule chemical libraries using high throughput screening. This identified a hit compound that was found to selectively activate the D2R in a functionally biased fashion. Chemical optimization resulted in a lead compound (VU207) that exhibits full agonist activity in three different D2R signaling assays: Ca2+ mobilization (Gqi5), inhibition of forskolin-stimulated cAMP accumulation (Gi/o), and β-arrestin recruitment. However, VU207 fails to activate D2R-Gβγ-mediated responses including GIRK channel activation and adenylyl cyclase potentiation. VU207 was also found to exhibit potent D3R antagonism with no functional activity at other dopamine receptors. Further, behavioral paradigms (hypothermia and yawning) indicated that VU207 is CNS-penetrant and acts as a D2R agonist and a D3R antagonist in vivo. Using ex vivo brain slices, we investigated the functional activity of VU207 at D2Rs located on dopaminergic neurons. In these cells, the D2Rs located on the cell bodies and dendrites (somatodendritic D2Rs) activate GIRK channels whereas identical D2Rs located on the nerve terminals inhibit dopamine release through a non-GIRK-mediated mechanism. We found that VU207 failed to activate GIRK currents through somatodendritic D2Rs and, in fact, acted as an antagonist of this D2R response. Notably, in contrast, VU207 acted as an agonist in inhibiting dopamine release via the D2Rs at the nerve terminals. This latter response was absent in tissue from autoD2R knockout mice that lack D2R expression in dopaminergic neurons. These findings suggest that VU207 exhibits a form of spatial or location bias acting as either an agonist or an antagonist depending on where in the neuron the D2R is located. It is expected that more cases of location/spatial bias will be observed upon further analyses of receptor-mediated signaling in cells with complex morphologies, especially neurons.

Sigma receptors were initially proposed as opioid, and later phencyclidine receptors, and were finally demonstrated to represent unique binding sites in mammalian brain and peripheral tissues that are expressed throughout the CNS and have been implicated in a variety of physiological functions and disease states. Two subtypes of receptors have been distinguished molecularly and pharmacologically. Previous studies showed that sigma1 receptor (sigma1R) agonists (e.g. PRE-084, (+)-pentazocine) were reinforcing in rats with cocaine or d-methamphetamine self-administration experience, but not in experimentally nave rats. Further studies demonstrated that the induction of sigma1R agonist self administration specifically occurred with SA of drugs acting at the dopamine transporter (DAT), but not with other abused drugs (e.g. heroin, ketamine). In contrast to effects with sigma1R agonists, a recent report showed self administration of the non-selective sigma1/2R agonist DTG in nave rats. The present study assessed DTG self administration pharmacology, and whether self administration experience would induce PRE-084 self administration. Responding of rats trained with DTG self administration under an FR schedules was subsequently maintained with PRE-084. DTG self administration was insensitive to antagonism by dopamine (DA) receptor antagonists (SCH 39166, L-741626) which dose-dependently blocked cocaine self administration. Further, sigmaR antagonists (BD 1063, haloperidol) blocked DTG self administration, but only haloperidol, which is also a DA antagonist, blocked cocaine self administration. Pretreatments with PRE-084, the DAT inhibitor WIN 35,428, and DA D2R agonists (R(−)-NPA, (−)-quinpirole, which were themselves self administered) dose-dependently potentiated DTG and cocaine self administration. Treatment with DA D1R agonists (R(+)-SKF 81297, ()-SKF 82958, which were also self-administered) dose-dependently decreased maximal self administration of both cocaine and DTG. The results suggest that DA-uptake inhibition while sufficient is not necessary for induction of sigma1R agonist reinforcement, and that once induced, sigma1R-mediated reinforcement is independent of DA receptor antagonism, but is affected by DA agonists in a manner similar to that for cocaine. These results suggest that DA-uptake inhibition is not necessary for induction of reinforcement by sigma1R agonists. Further, positive modulation of DA systems and sigma1R agonism similarly enhance reinforcing effects of both cocaine and DTG. Thus, DA-uptake inhibition while sufficient is not necessary for induction of reinforcement by sigma1R agonists, and that once induced sigmaR-mediated reinforcement is insensitive to DAR antagonism.

DA D2 receptor activation stimulates AEA levels, thus, drugs that increase DA levels in the brain, like abused drugs, activate DA D2 receptors, and in turn, increase AEA levels. We have tested the effects of abused drugs in producing CB1-receptor mediated generalization in THC-discrimination tests. Cocaine, and amphetamine injected alone did not produce effects significantly different from vehicle, but potentiated the THC-like effects of THC. Nicotine and the D2/3 DA receptor agonist quinpirole alone did not generalize to the THC cue, but both drugs did so in animals pretreated with the inhibitor of FAAH that metabolizes AEA. Nicotine and quinpirole also potentiated the effects of THC. We have suggested that AEA is released by these drugs by a D2 receptor mediated mechanism. So, administered alone these drugs do not stimulate AEA levels sufficiently to provide CB1-mediated THC-like effects, but potentiate ineffective small doses of THC. However, when the same drugs are administered in combination with URB-597, AEA levels are magnified by blockade of its metabolism, and its concentration could thus activate CB1 receptors producing THC discriminative effects. Recently it has also been shown that effects of anandamide and blockers of its metabolism (e.g. URB-597) might be mediated not only by the endocannabinoid system, but also by PPAR-alpha receptors. It has also been demonstrated that blockade of anandamide metabolism through fatty acid amide hydrolase enzymes by URB-597 might lead to increased levels of oleoylamide (OEA) or palmytoilamide (PEA), as well as anandamide. While anadamide has both CB1 and PPAR-alpha receptor activities, OEA and PEA are selective ligands for PPAR receptors, with negligible activity at CB1 receptors. We showed that drugs acting specifically at brain PPAR-alpha receptors can block the addictive actions of nicotine in rats and monkeys. Psychostimulant sensitization might play a role in the path to abuse and addiction, and even a single exposure to psychostimulants could produce long-term sensitization by increasing strength of excitatory synapses in midbrain dopaminergic regions. Sensitization can be viewed as a type of synaptic plasticity, which is also related to alterations in the cannabinoid system. We hypothesized that development of psychostimulant sensitization might involve stimulation of brain endocannabinoid levels that can bind to and activate CB1 receptors. We started this project studying cocaine sensitization in mice, measured as increased stimulation of behavioral activities before and after sensitizing doses of cocaine. We are testing the hypothesis that the development of cocaine sensitization requires release of endocannabinoids, and can be reversed by CB1 receptor blockade. Based on our original hypothesis, low doses of cocaine that do not induce behavioral sensitization might become effective when animals are pretreated with enhancers of endocannabinoid levels. DA transmission, believed to mediate behavioral and reinforcing effects of cocaine, will also be measured before and after cocaine sensitization. Our results have confirmed that a single exposure to cocaine induces behavioral sensitization in mice. Rimonabant, a CB1 antagonist, injected before the sensitizing dose of cocaine, reduced the sensitization produced by cocaine. In addition, cocaine-induced sensitization was paralleled by a sensitized, larger stimulation of DA levels, compared to saline treated animals in the nucleus accumbens core, but not in the NAC shell. Our results suggest also that blockade of endocannabinoid metabolism (obtained by pretreatment with URB-597 in mice) enhances the extracellular levels of endocannabinoids released by cocaine, and this enhancement could be related to the induction of behavioral sensitization by doses of cocaine otherwise not effective in inducing behavioral or neurochemical sensitization. We have also found that the enhancement of cocaine-induced anandamide levels in the brain will also result in a specific related neurochemical sensitization of DA stimulation in the core but not in the shell of the nucleus accumbens. It has been recently suggested that selected blockers of the dopamine transporter might possess the ability to negatively interact with an allosteric site of the cannabinoid CB1 receptors. It has also been suggested that this negative allosteric modulation of CB1 receptors might be important in these DAT blockers for expressing antagonism to the behavioral/reinforcing effects of cocaine. To test the validity of these suggestions, we tested antagonism of cocaine by JHW007, a DAT blocker that also show allosteric CB1 activity. Antagonism of cocaine was obtained in both CB1 knockout and wild-type mice suggesting that allosteric modulation of CB1 receptor actions has a minimal role in the cocaine-antagonist effects of JHW 007. The number of people seeking treatment for marijuana use in the United States per year (1,243,000) is higher than the number seeking treatment for cocaine or heroin use (787,000 or 507,000, respectively)1. THC, the main psychoactive ingredient in marijuana, activates brain pathways mediating its reinforcing effects by enhancing the firing of DA neurons in the ventral tegmental area (VTA), resulting in increased release of DA from nerve terminals in the shell of the nucleus accumbens (NAc). Developing medications that modulate these effects of THC as a reinforcer might provide a therapeutic approach for the treatment of marijuana dependence. For example, previously we found that reward-related behavioral and neurochemical effects of THC could be blocked by methyllycaconitine (MLA), a selective antagonist of α7nAChRs that are present in both the VTA and the NAc shell on glutamatergic nerve terminals. Their activation elicits glutamate release, which in turn acts at ionotropic glutamate receptors on dopaminergic terminals to stimulate dopamine release. Unfortunately, systemic use of direct antagonists of α7nAChRs is associated with side effects that limit their therapeutic utility. To avoid these unwanted effects, we tested a compound 3,4-dimethoxy-N-4-(3-nitrophenyl)thiazol-2-ylbenzenesulfonamide (Ro 61-8048), a potent, selective, peripherally acting kynurenine 3-monooxygenase (KMO) inhibitor, to indirectly increase brain KYNA, an endogenous negative allosteric modulators of α7nAChRs that might be better tolerated than directly acting cholinergic antagonists. Indeed, allosteric modulators change receptor conformations in the presence of orthosteric ligands and often have no effect on their own, acting only when physiological receptors are activated. Newly formed KYNA is promptly released into the extracellular compartment. Notably, no reuptake processes exist for KYNA, and extracellular KYNA is not degraded enzymatically but is slowly eliminated from the brain by a non-specific acid transporter. We found that administration of the kynurenine 3-monooxygenase (KMO) inhibitor Ro 61-8048 increases brain KYNA levels and attenuates THC-induced stimulation of extracellular levels of dopamine in reward-related brain areas. Administration of Ro 61-8048 also reduced the reinforcing effects of THC measured under self-administration behavioral procedures, also preventing relapse to drug-seeking induced by re-exposure to cannabinoids or cannabinoid-associated cues. Pharmacologic proof of involvement of α7nAChRs was obtained by administration of positive allosteric modulators of α7nAChRs. These results suggest a therapeutic strategy for treatment of marijuana dependence.