Marc R. Azar

CRF-CRF1 system activation mediates withdrawal-induced increases in nicotine self-administration in nicotine-dependent rats

Nicotine, the main psychoactive ingredient of tobacco, induces negative emotional symptoms during abstinence that contribute to a profound craving for nicotine. However, the neurobiological mechanisms underlying how nicotine produces dependence remains poorly understood. We demonstrate one mechanism for both the anxiety-like symptoms of withdrawal and excessive nicotine intake observed after abstinence, through recruitment of the extrahypothalamic stress peptide corticotropin-releasing factor (CRF) system and activation of CRF1 receptors. Overactivation of the CRF–CRF1 system may contribute to nicotine dependence and may represent a prominent target for investigating the vulnerability to tobacco addiction.

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.

CONTRIBUTION OF SEX AND GENETICS TO NEUROENDOCRINE ADAPTATION TO STRESS IN MICE

Male and female C57BL/6 (B6) and DBA/2 (D2) mice were subjected to either acute or 5 days of repeated restraint in ventilated, 50 ml centrifuge tubes. Control animals were not disturbed. The acute restraint animals were killed immediately following 15, 30 or 60 min of restraint and blood collected for corticosterone (CORT) analysis. The results of the acute restraint procedure revealed a strain difference in time to peak CORT in plasma with D2 animals showing an earlier peak. The males of both strains evinced similar maximum response and similar to B6 females; however, the D2 females showed a 2-fold greater CORT response than did the B6 females. Repeated restraint consisted of 5 days of 12 h in the tubes. At the end of 5 days, the animals were weighted and adrenalectomized in preparation for determination of brain corticosteroid receptors. Upon sacrifice, brains, thymus, adrenals and blood were harvested, the last for corticosteroid binding globulin (CBG). Five days of repeated restraint produced body weight loss in both strains, with B6s less affected than D2s. Repeated restraint reduced the mass of the adrenals in the B6s only. Restraint also reduced the mass of the thymus in both strains and sexes, but to a greater extent in the B6s. Plasma CBG densities were also sensitive to restraint, but only in females, showing a restraint-related decrease. Repeated restraint had no effect on hippocampal glucocorticoid or mineralocorticoid receptors; however for the latter, we observed significant strain and sex effects with D2 having higher Bmax than B6 and females having higher Bmax than males. In the pituitary, glucocorticoid receptors (GR) were reduced by repeated restraint in males, but increased in females, especially in the B6. These findings lend preliminary evidence for involvement of sex and genetics as sources of individual differences in bioadaptation to stress.

A critical evaluation of a nicotine vaccine within a self-administration behavioral model.

(S)-Nicotine is a psychostimulant legal drug responsible for causing addiction to tobacco smoking. Tobacco smoking has been irrevocably linked to a number of serious diseases and at present is considered the leading cause of preventable death in the United States. Despite well-documented adverse medical consequences, nicotine addiction has historically been one of the hardest to break. Current therapies have offered limited success and show high rates of relapse, emphasizing the need to engineer alternative therapies to aid nicotine cessation. The current study presents a protein-based immunopharmacotherapy approach for the treatment of nicotine addiction. Immunopharmacotherapy aims to use highly specific antibodies to blunt passage of drug into the brain thus minimizing reinforcing effects on the reward pathways of the central nervous system. Generation of a successful vaccine heavily relies on appropriate optimization of hapten design, immunogenic carrier and adjuvant. Modification of a classical nicotine hapten in conjugation with three distinct carrier proteins allowed for priming of a nicotine vaccine able to elicit significant amounts of nicotine-specific antibodies. Increased self-administration with use of a high drug dose (0.03 mg/kg/infusion; approximately 2 cigarettes in human) was observed in the vaccinated versus control animals suggesting a compensatory pattern and possibly reduced passage of nicotine to the brain. These results support the hypothesis that proper optimization of vaccine formulations could lead to successful nicotine vaccines for human use.

Monoamine oxidase A rather than monoamine oxidase B inhibition increases nicotine reinforcement in rats

Although nicotine is considered to be responsible for the addictive properties of tobacco, growing evidence underlines the importance of non‐nicotine components in smoking reinforcement. It has been shown that tobacco smoke contains monoamine oxidase (MAO) A and B inhibitors and decreases MAO‐A and MAO‐B activity in smokers. Here, we investigated the effects of clorgyline hydrochloride (irreversible MAO‐A inhibitor; 2 mg/kg/day), selegiline (irreversible MAO‐B inhibitor; 4 mg/kg) and the beta‐carboline norharmane hydrochloride (reversible MAO‐B inhibitor; 5 mg/kg/day) treatments on nicotine self‐administration (30 µg/kg/infusion, free base) in rats. Independent of the responsiveness to novelty and locomotor activity stimulation, only clorgyline hydrochloride treatment increased the intake of nicotine in a fixed‐ratio schedule (FR5) of reinforcement. When a progressive‐ratio schedule was implemented, both clorgyline hydrochloride and norharmane hydrochloride treatments potentiated the reinforcing effects of nicotine, whereas selegiline had no effect. Taken together, these results indicate that MAO‐A inhibition interacts with nicotine to enhance its rewarding effects in rats and suggest that other compounds present in tobacco, such as beta‐carboline, may also play an important role in sustaining smoking behavior in humans.

Sex and Genetics are Important Cofactors in Assessing the Impact of Iron Deficiency on the Developing Mouse Brain.

The present study was designed to investigate the possible role of genetic background and sex in the behavioral and neurochemical responses to early iron deficiency. Male and female C57 and DBA mice were provided either an iron deficient (ID) or control (CN) diet during early growth periods. Dopamine D2 receptor densities, and dopamine and serotonin transporter densities were determined in specific brain regions. Iron deficient DBA mice had significantly lower D2 receptor densities in the frontal cortex (FC) and caudate putamen (CP) (19 vs. 35fmol/mg and 145 vs. 215 fmol/mg, respectively). Serotonin (5-HT) transporter densities in FC of iron deficient C57 mice tended to be lower than in control animals (40 vs. 60 fmol/mg) while in the nucleus accumbens (NA) the 5-HT transporter increased in density relative to controls (350 vs. 210 fmol/mg). Open field behaviors in naïve and cocaine treated mice were also affected by diet, suggesting that iron deficiency causes decreased dopamine output. These data indicate a substantial genetic-based variability in brain responses to iron deficiency anemia, some of which are in direct contrast to what other experimental data would have predicted. Future studies clearly need to consider genetic background and sex in their analytical approach.

Distribution and Clearance of Cocaine in Brain Is Influenced by Genetics

The purpose of this study was to examine the pharmacokinetics of cocaine in two inbred mouse strains, C57BL/6 (B6) and DBA/2 (D2). Male and female mice were administered 30 mg kg(-1) cocaine IP and killed after 5, 15, 30, or 60 minutes postinjection. Brains were removed quickly and assayed for total brain cocaine concentration. Quantification of cocaine was conducted using gas chromatography and mass spectrometry. The results of this study revealed a strain difference in total brain cocaine kinetics. Specifically, we observed that at 5 min onward, B6 mice cleared cocaine from the brain with a t1/2 estimated at 22.3 min, while distribution in D2 mice appeared to be incomplete until 15 min with a subsequent t1/2 estimated at 11.2 min. These results show that despite faster clearance by D2 mice, the prolonged time to distribution in this strain may help explain why D2 mice show initial greater locomotor activation by cocaine, compared to B6s.

Synthesis and Biological Evaluation of α- and β-6-Amido Derivatives of 17-Cyclopropylmethyl-3, 14β-dihydroxy-4, 5α-epoxymorphinan: Potential Alcohol-Cessation Agents

Substituted aryl and aliphatic amide analogues of 6-naltrexamine were synthesized and used to characterize the binding to and functional activity of human mu-, delta-, and kappa-opioid receptors. Competition binding assays showed 11-25 and 27-31 bound to the mu (K(i) = 0.05-1.2 nM) and kappa (K(i) = 0.06-2.4 nM) opioid receptors. Compounds 11-18 possessed significant binding affinity for the delta receptor (K(i) = 0.8-12.4 nM). Functional assays showed several compounds acted as partial or full agonists of delta or kappa receptors while retaining an antagonist profile at the mu receptor. Structure-activity relationship for aryl amides showed that potent compounds possessed lipophilic groups or substituents capable of hydrogen bonding. Metabolic stability studies showed that 11, 12, and 14 possessed considerable stability in the presence of rat, mouse, or human liver preparations. The ED 50 of inhibition of 10% ethanol self-administration in trained rats, using operant techniques for 11, was 0.5 mg/kg.

Probing the protective effects of a conformationally constrained nicotine vaccine

Despite being consistently ranked as the leading cause of preventable death in the United States, about 20% of the population continues to smoke. Current smoking cessation therapies offer limited success, show high rates of relapse, and have potentially dangerous side effects, consequently emphasizing the need for alternative therapies. Immunopharmacotherapy aims to use highly specific antibodies to sequester nicotine in the bloodstream thus blunting passage into the brain and minimizing positive reinforcing effects. A successful vaccination strategy is dependent upon the appropriate hapten design, carrier protein and adjuvant which affect both the magnitude and affinity of the immune response elicited. Our laboratory previously demonstrated the use of molecular constraint as a means to increase the intrinsic immunogenicity and antigenicity of a nicotine vaccine. The present study expands upon those initial results and explores the protective effects of vaccination with both constrained hapten CNI and its unconstrained counterpart NIC. Our results demonstrate how immunization with CNI-KLH produces large amounts of moderate affinity anti-nicotine antibodies even when formulated with ALUM adjuvant, making it particularly relevant for human use. In contrast, vaccination with NIC-KLH produced moderate amounts of high affinity anti-nicotine antibodies. These differential responses proved critical in offering protecting effects. Vaccination with CNI, but not NIC, resulted in an increase of self-administration responding on a progressive ratio schedule using a high nicotine dose (0.03 mg/kg/infusion; ≈ 2 cigarettes in human) as compared to KLH-controls. Furthermore, vaccination with CNI was able to antagonize the analgesic effects of a heavy bolus dose of nicotine (0.35 mg/kg). These results support our hypothesis that molecular constraint can be advantageously utilized to increase the immunogenicity of a nicotine vaccine. Furthermore in correlating the behavioral effects with the differential responses elicited, we shed light on the distinct roles of antibody concentration and affinity.

Synthesis and pharmacological evaluation of 6-naltrexamine analogs for alcohol cessation

A series of substituted aryl amide derivatives of 6-naltrexamine, 3 designed to be metabolically stable were synthesized and used to characterize the structural requirements for their potency to binding and functional activity of human mu (mu), delta (delta) and kappa (kappa) opioid and nociceptin (NOP) receptors. Binding assays showed that 4-10 had subnanomolar K(i) values for mu and kappa opioid receptors. Functional assays for stimulation of [(35)S]GTPgammaS binding showed that several compounds acted as partial or inverse agonists and antagonists of the mu and delta, kappa opioid or NOP receptors. The compounds showed considerable stability in the presence of rat, mouse or human liver preparations and NADPH. The inhibitory activity on the functional activity of human cytochrome P450s was examined to determine any potential inhibition by 4-9. Only modest inhibition of CYP3A4, CYP2C9 and CYP2C19 was observed for a few of the analogs. As a representative example, radiolabeled 6 was examined in vivo and showed reasonable brain penetration. The inhibition of ethanol self-administration in rats trained to self-administer a 10% (w/v) ethanol solution, utilizing operant techniques showed 5-8 to have very potent efficacy (ED(50) values 19-50 microg/kg).