Malliga R. Iyer

The sigma receptor antagonist BD-1063 decreases ethanol intake and reinforcement in animal models of excessive drinking

σ-Receptors (SigRs) have been implicated in behavioral and appetitive effects of psychostimulants and may also modulate the motivating properties of ethanol. This study tested the hypothesis that SigRs modulate ethanol reinforcement and contribute to excessive ethanol intake. The effects of subcutaneous treatment with the potent, selective Sig-1R antagonist BD-1063 on operant ethanol self-administration were studied in two models of excessive drinking—Sardinian alcohol-preferring (sP) rats and acutely withdrawn ethanol-dependent Wistar rats—and compared to ethanol self-administration in nondependent Wistar controls. To assess the specificity of action, the effects of BD-1063 on self-administration of an equally reinforcing saccharin solution were determined in Wistar and sP rats. Gene expression of Sig-1R in reward-related brain areas implicated in ethanol reinforcement was compared between ethanol-naive sP and Wistar rats and withdrawn ethanol-dependent Wistar rats. BD-1063 dose dependently reduced ethanol self-administration in sP rats (3.3–11 mg/kg) and withdrawn, dependent Wistar rats (4–11 mg/kg) at doses that did not modify mean ethanol self-administration in nondependent Wistar controls. BD-1063 did not reduce concurrent water self-administration and did not comparably suppress saccharin self-administration, suggesting selectivity of action. BD-1063 also reduced the breakpoints of sP rats to work for ethanol under a progressive-ratio reinforcement schedule. Ethanol-naive sP rats and 24-h withdrawn, dependent Wistar rats showed reduced Sig-1R mRNA expression in the nucleus accumbens. The results suggest that SigR systems may contribute to innate or ethanol-induced increases in susceptibility to self-administer high ethanol levels, identifying a potential neuroadaptive mechanism contributing to excessive drinking and a therapeutic target for alcohol abuse and dependence.

Antagonism of sigma-1 receptors blocks compulsive-like eating.

Binge eating disorder is an addiction-like disorder characterized by episodes of rapid and excessive food consumption within discrete periods of time which occur compulsively despite negative consequences. This study was aimed at determining whether antagonism of Sigma-1 receptors (Sig-1Rs) blocked compulsive-like binge eating. We trained male wistar rats to obtain a sugary, highly palatable diet (Palatable group) or a regular chow diet (Chow control group), for 1 h a day under fixed ratio 1 operant conditioning. Following intake stabilization, we evaluated the effects of the selective Sig-1R antagonist BD-1063 on food responding. Using a light/dark conflict test, we also tested whether BD-1063 could block the time spent and the food eaten in an aversive, open compartment, where the palatable diet was offered. Furthermore, we measured Sig-1R mRNA and protein expression in several brain areas of the two groups, 24 h after the last binge session. Palatable rats rapidly developed binge-like eating, escalating the 1 h intake by four times, and doubling the eating rate and the regularity of food responding, compared to Chow rats. BD-1063 dose-dependently reduced binge-like eating and the regularity of food responding, and blocked the increased eating rate in Palatable rats. In the light/dark conflict test, BD-1063 antagonized the increased time spent in the aversive compartment and the increased intake of the palatable diet, without affecting motor activity. Finally, Palatable rats showed reduced Sig-1R mRNA expression in prefrontal and anterior cingulate cortices, and a two-fold increase in Sig-1R protein expression in anterior cingulate cortex compared to control Chow rats. These findings suggest that the Sig-1R system may contribute to the neurobiological adaptations driving compulsive-like eating, opening new avenues of investigation towards pharmacologically treating binge eating disorder.

Activation of σ-Receptors Induces Binge-like Drinking in Sardinian Alcohol-Preferring Rats

Sigma (σ) receptors have been implicated in the behavioral and motivational effects of alcohol and psychostimulants. Sigma receptor antagonists reduce the reinforcing effects of alcohol and excessive alcohol intake in both genetic (alcohol-preferring rats) and environmental (chronic alcohol-induced) models of alcoholism. The present study tested the hypothesis that pharmacological activation of σ-receptors facilitates ethanol reinforcement and induces excessive, binge-like ethanol intake. The effects of repeated subcutaneous treatment with the selective σ-receptor agonist 1,3-di-(2-tolyl)guanidine (DTG; 15 mg/kg, twice a day for 7 days) on operant ethanol (10%) self-administration were studied in Sardinian alcohol-preferring (sP) rats. To confirm that the effect of DTG was mediated by σ-receptors, the effects of pretreatment with the selective σ-receptor antagonist BD-1063 (7 mg/kg, subcutaneously) were determined. To assess the specificity of action, the effects of DTG on the self-administration of equally reinforcing solutions of saccharin or sucrose were also determined. Finally, gene expression of opioid receptors in brain areas implicated in ethanol reinforcement was analyzed in ethanol-naive sP rats treated acutely or repeatedly with DTG, because of the well-established role of the opioid system in alcohol reinforcement and addiction. Repeatedly administered DTG progressively and dramatically increased ethanol self-administration in sP rats and increased blood alcohol levels, which reached mean values close to 100 mg% in 1 h drinking sessions. Repeated DTG treatment also increased the rats’ motivation to work for alcohol under a progressive-ratio schedule of reinforcement. BD-1063 prevented the effects of DTG, confirming that σ-receptors mediate the effects of DTG. Repeated DTG treatment also increased the self-administration of the non-drug reinforcers saccharin and sucrose. Naive sP rats repeatedly treated with DTG showed increased mRNA expression of μ- and δ-opioid receptors in the ventral tegmental area. These results suggest a key facilitatory role for σ-receptors in the reinforcing effects of alcohol and identify a potential mechanism that contributes to binge-like and excessive drinking.

Liposomes containing monophosphoryl lipid A: a potent adjuvant system for inducing antibodies to heroin hapten analogs.

In order to create an effective immunization approach for a potential vaccine to heroin, liposomes containing monophosphoryl lipid A [L(MPLA)] were tested as an adjuvant system to induce antibodies to heroin hapten analogs. Four synthetic haptens and two immunization strategies were employed. In the first strategy, a hydrophobic 23 amino acid immunogenic peptide derived from the membrane proximal external region of gp41 from HIV-1 envelope protein was embedded as a carrier in the outer surface of L(MPLA), to which was conjugated a 15 amino acid universal T cell epitope and a terminal heroin hapten analog. In the second strategy, tetanus toxoid (TT) carrier protein was decorated with haptens by conjugation, and the hapten-conjugated protein was mixed with L(MPLA). After immunization of mice, each of the immunization strategies was effective for induction of IgG anti-hapten antibodies. The first immunization strategy induced a mean end-point IgG titer against one of two haptens tested of approximately 12,800; however, no detectable antibodies were induced against the liposome-associated HIV-1 carrier peptide. In the second immunization strategy, depending on the hapten used for decorating the TT, end-point IgG titers ranged from 100,000 to 6,500,000. In this strategy, in which hapten was conjugated to the TT, end-point IgG titers of 400,000 to the TT carrier were observed with each conjugate. However, upon mixing unconjugated TT with L(MPLA), anti-TT titers of 6,500,000 were observed. We conclude that L(MPLA) serves as a potent adjuvant for inducing antibodies to candidate heroin haptens. However, antibodies to the carrier peptide or protein were partly or completed inhibited by the presence of conjugated hapten.

Characterization and optimization of heroin hapten-BSA conjugates: method development for the synthesis of reproducible hapten-based vaccines

A potential new treatment for drug addiction is immunization with vaccines that induce antibodies that can abrogate the addictive effects of the drug of abuse. One of the challenges in the development of a vaccine against drugs of abuse is the availability of an optimum procedure that gives reproducible and high yielding hapten-protein conjugates. In this study, a heroin/morphine surrogate hapten (MorHap) was coupled to bovine serum albumin (BSA) using maleimide-thiol chemistry. MorHap-BSA conjugates with 3, 5, 10, 15, 22, 28, and 34 haptens were obtained using different linker and hapten ratios. Using this optimized procedure, MorHap-BSA conjugates were synthesized with highly reproducible results and in high yields. The number of haptens attached to BSA was compared by 2,4,6-trinitrobenzenesulfonic acid (TNBS) assay, modified Ellman’s test and matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Among the three methods, MALDI-TOF MS discriminated subtle differences in hapten density. The effect of hapten density on enzyme-linked immunosorbent assay (ELISA) performance was evaluated with seven MorHap-BSA conjugates of varying hapten densities, which were used as coating antigens. The highest antibody binding was obtained with MorHap-BSA conjugates containing 3–5 haptens. This is the first report that rigorously analyzes, optimizes and characterizes the conjugation of haptens to proteins that can be used for vaccines against drugs of abuse. The effect of hapten density on the ELISA detection of antibodies against haptens demonstrates the importance of careful characterization of the hapten density by the analytical techniques described.

Hybrid inhibitor of peripheral cannabinoid-1 receptors and inducible nitric oxide synthase mitigates liver fibrosis

Liver fibrosis, a consequence of chronic liver injury and a way station to cirrhosis and hepatocellular carcinoma, lacks effective treatment. Endocannabinoids acting via cannabinoid-1 receptors (CB1R) induce profibrotic gene expression and promote pathologies that predispose to liver fibrosis. CB1R antagonists produce opposite effects, but their therapeutic development was halted due to neuropsychiatric side effects. Inducible nitric oxide synthase (iNOS) also promotes liver fibrosis and its underlying pathologies, but iNOS inhibitors tested to date showed limited therapeutic efficacy in inflammatory diseases. Here, we introduce a peripherally restricted, orally bioavailable CB1R antagonist, which accumulates in liver to release an iNOS inhibitory leaving group. In mouse models of fibrosis induced by CCl4 or bile duct ligation, the hybrid CB1R/iNOS antagonist surpassed the antifibrotic efficacy of the CB1R antagonist rimonabant or the iNOS inhibitor 1400W, without inducing anxiety-like behaviors or CB1R occupancy in the CNS. The hybrid inhibitor also targeted CB1R-independent, iNOS-mediated profibrotic pathways, including increased PDGF, Nlrp3/Asc3, and integrin αvβ6 signaling, as judged by its ability to inhibit these pathways in cnr1-/- but not in nos2-/- mice. Additionally, it was able to slow fibrosis progression and to attenuate established fibrosis. Thus, dual-target peripheral CB1R/iNOS antagonists have therapeutic potential in liver fibrosis.

Design, Synthesis, and Biological Evaluation of Novel, Non-Brain-Penetrant, Hybrid Cannabinoid CB1R Inverse Agonist/Inducible Nitric Oxide Synthase (iNOS) Inhibitors for the Treatment of Liver Fibrosis

We report the design, synthesis, and structure-activity relationships of novel dual-target compounds with antagonist/inverse agonist activity at cannabinoid receptor type 1 (CB1R) and inhibitory effect on inducible nitric oxide synthase (iNOS). A series of 3,4-diarylpyrazolinecarboximidamides were synthesized and evaluated in CB1 receptor (CB1R) binding assays and iNOS activity assays. The novel compounds, designed to have limited brain penetrance, elicited potent in vitro CB1R antagonist activities and iNOS inhibitory activities. Some key compounds displayed high CB1R binding affinities. Compound 7 demonstrated potent in vivo pharmacological activities such as reduction of food intake mediated by the antagonism of the CB1Rs and antifibrotic effect in the animal models of fibrosis mediated by iNOS inhibition and CB1R antagonism.

Structural Basis of Species-Dependent Differential Affinity of 6-Alkoxy-5-Aryl-3-Pyridinecarboxamide Cannabinoid-1 Receptor Antagonists

6-Alkoxy-5-aryl-3-pyridincarboxamides, including the brain-penetrant compound 14g [5-(4-chlorophenyl)-6-(cyclopropylmethoxy)-N-[(1R,2R)-2-hydroxy-cyclohexyl]-3-pyridinecarboxamide] and its peripherally restricted analog 14h [5-(4-chlorophenyl)-N-[(1R,2R)-2-hydroxycyclohexyl]-6-(2-methoxyethoxy)-3-pyridinecarboxamide], have been recently introduced as selective, high-affinity antagonists of the human cannabinoid-1 receptor (hCB1R). Binding analyses revealed two orders of magnitude lower affinity of these compounds for mouse and rat versus human CB1R, whereas the affinity of rimonabant is comparable for all three CB1Rs. Modeling of ligand binding to CB1R and binding assays with native and mutant (Ile105Met) hCB1Rs indicate that the Ile105 to Met mutation in rodent CB1Rs accounts for the species-dependent affinity of 14g and 14h. Our work identifies Ile105 as a new pharmacophore component for developing better hCB1R antagonists and invalidates rodent models for assessing the antiobesity efficacy of 14g and 14h.

Sigma-1 receptor mediates acquisition of alcohol drinking and seeking behavior in alcohol-preferring rats

Sigma-1 receptor (Sig-1R) has been proposed as a novel therapeutic target for drug and alcohol addiction. We have shown previously that Sig-1R agonists facilitate the reinforcing effects of ethanol and induce binge-like drinking, while Sig-1R antagonists on the other hand block excessive drinking in genetic and environmental models of alcoholism, without affecting intake in outbred non-dependent rats. Even though significant progress has been made in understanding the function of Sig-1R in alcohol reinforcement, its role in the early and late stage of alcohol addiction remains unclear. Administration of the selective Sig-1R antagonist BD-1063 dramatically reduced the acquisition of alcohol drinking behavior as well as the preference for alcohol in genetically selected TSRI Sardinian alcohol preferring (Scr:sP) rats; the treatment had instead no effect on total fluid intake, food intake or body weight gain, proving selectivity of action. Furthermore, BD-1063 dose-dependently decreased alcohol-seeking behavior in rats trained under a second-order schedule of reinforcement, in which responding is maintained by contingent presentation of a conditioned reinforcer. Finally, an innate elevation in Sig-1R protein levels was found in the nucleus accumbens of alcohol-preferring Scr:sP rats, compared to outbred Wistar rats, alteration which was normalized by chronic, voluntary alcohol drinking. Taken together these findings demonstrate that Sig-1R blockade reduces the propensity to both acquire alcohol drinking and to seek alcohol, and point to the nucleus accumbens as a potential key region for the effects observed. Our data suggest that Sig-1R antagonists may have therapeutic potential in multiple stages of alcohol addiction.

Probes for narcotic receptor mediated phenomena. 40. N-substituted cis-4a-ethyl-1,2,3,4,4a,9a-hexahydrobenzofuro[2,3-c]pyridin-8-ols.

A series of N-substituted rac-cis-4a-ethyl-1,2,3,4,4a,9a-hexahydrobenzofuro[2,3-c]pyridin-8-ols have been prepared using a simple synthetic route previously designed for synthesis of related cis-2-methyl-4a-alkyl-1,2,3,4,4a,9a-hexahydrobenzofuro[2,3-c]pyridin-6-ols. The new phenolic compounds, where the aromatic hydroxy moiety is situated ortho to the oxygen atom in the oxide-bridged ring, do not interact as well as the pyridin-6-ols with opioid receptors. The N-para-fluorophenethyl derivative had the highest mu-opioid receptor affinity of the examined compounds (K(i)=0.35 microM).