Glucagon-Like Peptide-1 Receptor Signaling in the Ventral Tegmental Area Reduces Alcohol Self-Administration in Male Rats.

BACKGROUND: The misuse and abuse of alcohol is a major public health issue. However, available treatments are limited with variable efficacy. Recently, preclinical studies show that glucagon-like-peptide-1 (GLP-1) and its analogue Exendin-4 (Ex4) potently reduce a range of alcohol intake behaviors, thus highlighting its potential as a treatment for alcohol use disorders. However, the neural mechanisms and sites of action mediating the effects of Ex4 on alcohol intake behaviors remain to be characterized. This study examined the ventral tegmental area (VTA) as a site of action for the effects of GLP-1 on alcohol intake. METHODS: Male Long-Evans rats were given intermittent access to 20% alcohol and trained to nose poke for 20% alcohol. Rats received intra-VTA injections of Ex4 (vehicle, 0.01, 0.05 µg), and the effects of VTA Ex4 on alcohol self-administration, motivation, and relapse were assessed. RESULTS: When compared to vehicle treatment, intra-VTA Ex4 (0.01, 0.05 µg) delivery significantly reduced alcohol self-administration, an effect that was particularly prominent in high alcohol drinkers. However, VTA Ex4 did not reduce reacquisition of alcohol self-administration after extinction nor the motivation to obtain alcohol. Importantly, the lower dose of Ex4 (0.01 µg) used had no effect on food intake or locomotor activity, suggesting that the reduction in alcohol self-administration observed was not secondary to caloric intake or motor deficits. CONCLUSIONS: Together, these findings provide support for the VTA as a key site of action for GLP-1 on alcohol self-administration but not the reacquisition of alcohol self-administration or motivation to work for alcohol.

Interaction of disulfiram with antiretroviral medications: efavirenz increases while atazanavir decreases disulfiram effect on enzymes of alcohol metabolism.

BACKGROUND AND OBJECTIVES: Alcohol abuse complicates treatment of HIV disease and is linked to poor outcomes. Alcohol pharmacotherapies, including disulfiram (DIS), are infrequently utilized in co-occurring HIV and alcohol use disorders possibly related to concerns about drug interactions between antiretroviral (ARV) medications and DIS. METHOD: This pharmacokinetics study (n=40) examined the effect of DIS on efavirenz (EFV), ritonavir (RTV), or atazanavir (ATV) and the effect of these ARV medications on DIS metabolism and aldehyde dehydrogenase (ALDH) activity which mediates the DIS-alcohol reaction. RESULTS: EFV administration was associated with decreased S-Methyl-N-N-diethylthiocarbamate (DIS carbamate), a metabolite of DIS (p=.001) and a precursor to the metabolite responsible for ALDH inhibition, S-methyl-N,N-diethylthiolcarbamate sulfoxide (DETC-MeSO). EFV was associated with increased DIS inhibition of ALDH activity relative to DIS alone administration possibly as a result of EFV-associated induction of CYP 3A4 which metabolizes the carbamate to DETC-MeSO (which inhibits ALDH). Conversely, ATV co-administration reduced the effect of DIS on ALDH activity possibly as a result of ATV inhibition of CYP 3A4. DIS administration had no significant effect on any ARV studied. DISCUSSION/CONCLUSIONS: ATV may render DIS ineffective in treatment of alcoholism. FUTURE DIRECTIONS: DIS is infrequently utilized in HIV-infected individuals due to concerns about adverse interactions and side effects. Findings from this study indicate that, with ongoing clinical monitoring, DIS should be reconsidered given its potential efficacy for alcohol and potentially, cocaine use disorders, that may occur in this population.

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).

1H NMR of albumin in human blood plasma: drug binding and redox reactions at Cys34.

1H NMR methods are described which allow direct studies of the Cys34 binding site of albumin in intact human blood plasma in vitro. Antiarthritic gold drugs and the alcohol-aversive drug disulfiram induce a structural transition detectable via H epsilon 1 and H delta 2 resonances of His3 of albumin, and reactions of cystine, glutathione and captopril in plasma have also been investigated. Contrary to most assumptions, little of the albumin in normal plasma appears to be blocked at Cys34 as a cystine disulfide.

Cyanide is a product of the catalase-mediated oxidation of the alcohol deterrent agent, cyanamide.

Cyanide was detected as a product of cyanamide oxidation by bovine liver catalase in vitro under conditions that also produced an active aldehyde dehydrogenase (AlDH) inhibitor. Cyanide formation was directly related to both cyanamide and catalase concentrations and was also dependent on incubation time. The apparent Km for this reaction was 172 microM. Cyanide formation was blocked by ethanol, a known substrate for catalase Compound I. The toxic effects of cyanamide in the dog, a species with limited capacity to conjugate cyanamide by N-acetylation, may be causally related to enhancement of this catalase-mediated pathway for cyanamide metabolism.

Topiramate as add-on therapy in non-respondent alcohol dependant patients: a 12 month follow-up study.

INTRODUCTION: Topiramate is a neuromodulator drug with different action mechanisms that could be implicated in alcohol dependence. It has been studied in open and double-blind studies. METHOD: In a group of patients (n = 64) undergoing standard treatment for alcohol dependence (according to ICD-10 criteria) with poor outcomes, a 12 month observational, prospective and multicenter study was conducted to assess the usefulness and tolerability of topiramate as addon therapy. Outcome measures were retention rate, alcohol consumption (days of drinking per month and number of Standard Drink Units [SDU] per day, and results of Alcohol Dependence Intensity Scale [ADIS]), craving and priming visual scales and serum transaminase levels. RESULTS: In these patients, adding topiramate leads to a significant decrease (p<0.001) in all the variables studied, including those derived from the craving and priming visual scales, the ADIS as well as the number of drinks/day and SDU/day consumed, the MCV and GGT values. Mean topiramate dose was almost 200 mg/day. Only three patients dropped out due to adverse reactions. CONCLUSIONS: Topiramate showed positive results for alcohol dependence in real clinical practice, with a significant decrease in craving-priming and dependence intensity scales, number of drinking days per month reported and transaminase levels. Topiramate seems to be a useful and well-tolerated pharmacological aid for patients with bad evolution in their alcohol dependence treatment.

Disulfiram generates a stable N,N-diethylcarbamoyl adduct on Cys-125 of rat hemoglobin beta-chains in vivo.

Disulfiram (DSF) is a drug used in aversion therapy to treat alcoholics and acts by inhibiting mitochondrial low-K(m) aldehyde dehydrogenase. Investigations into the mechanisms for in vivo inactivation suggest that the DSF metabolite S-methyl-N, N-diethylthiocarbamate sulfoxide reacts irreversibly with an active site Cys. This work aimed to determine if DSF generates monothiocarbamate adducts on cysteine residues in vivo by examining hemoglobin. Sprague-Dawley rats were treated with DSF po for 2, 4, and 6 weeks. Rats have four different globin beta-chains, of which three (beta-1-3) contain two cysteine residues each. MALDI-TOF MS analysis of two new globin species from DSF-treated rats collected by HPLC revealed increments of 99 Da above the mass of the unmodified chains (beta-2 and beta-3). In a separate experiment, the globin mixture was digested for 2 h with Glu-C and reanalyzed by MALDI-TOF MS. Results showed a peptide at m/z 2716.3 having a mass 99 Da higher than a known Cys-containing peptide. Subsequently, the Glu-C digest was analyzed using Q-TOF tandem MS, enabling observation of the +4 charge state of the peptide with m/z 2716.3. This peptide was fragmented to produce y-sequence ions that located the modification to Cys-125 (present on both beta-2 and beta-3). Cys-125 is the most reactive of two cysteine residues on these beta-chains. To confirm the structure of the modification, globin was hydrolyzed with 6 N HCl at 110 degrees C for 18 h. The adduct survived these conditions so that S-(N,N-diethylcarbamoyl)cysteine was detected in the hydrolysates of treated rats on the basis of comparison with the tandem MS spectrum of a standard. These results extend the findings of others obtained using glutathione conjugates and demonstrate the ability of DSF to covalently modify Cys residues of proteins in a manner consistent with the production of S-methyl-N, N-diethylthiocarbamate sulfoxide, or sulfone, intermediates.

Studies on absorption, distribution, metabolism and excretion of ceftazidime in Japan.

Ceftazidime achieved mean serum levels of 54.6 and 119.7 mg/l at 5 min and 0.9 and 1.9 mg/l at 8 h after an intravenous bolus injection of 0.5 and 1 g in healthy male adult volunteers, showing a dose-related increase in serum levels. Serum half-lives were in the range from 1.6 to 1.7 h. About 90% of the given dose was excreted in the urine during the first 24 h. Following a 1-h intravenous drip infusion of 1 and 2 g, ceftazidime attained peak serum levels of 69.5 and 150.8 mg/l at the end of the infusion. Eighty-three and 90%, respectively, of the given doses were excreted in the urine during 24 h after the administration of 1 and 2 g of ceftazidime. When serum levels were compared after intravenous bolus injections of 1 g of ceftazidime and 1 g of cefoperazone in a crossover fashion, results were very similar for both compounds, but the 24-h urinary recovery rate was about three times higher with ceftazidime than with cefoperazone. An intravenous injection of 1 g ceftazidime resulted in peak biliary levels about 4 h after administration, longer than that of ceftizoxime compared in a crossover fashion, and subsequently fairly high levels were maintained for a longer time. Ceftazidime penetrated gall bladder tissues at concentrations sufficient to inhibit most of the pathogenic bacteria. In the urine collected from healthy volunteers 1 h after an intravenous injection of 1 g ceftazidime, no active metabolite was detected.

Roles of FMO and CYP450 in the metabolism in human liver microsomes of S-methyl-N,N-diethyldithiocarbamate, a disulfiram metabolite.

BACKGROUND: The conversion of S-methyl-N,N-diethyldithiocarbamate (MeDDC) to MeDDC sulfine is the first step after methylation in the metabolic pathway of disulfiram, an alcohol deterrent, to its ultimate active metabolite. Various isoforms of CYP450 have recently been shown to catalyze this reaction, but the involvement of flavin monooxygenase (FMO) in this metabolism in humans has not been evaluated. In this study we examined the ability of recombinant human FMO3 in insect microsomes to metabolize MeDDC, and investigated the relative roles of FMO and CYP450 in the metabolism of MeDDC in human liver microsomes. METHODS: HPLC-mass spectrometry was used to identify the products of MeDDC formed by human liver microsomes and by recombinant human FMO3. MeDDC metabolism in human liver microsomes was studied by using either heat inactivation to inhibit FMO, or N-benzylimidazole (NBI) or antibodies to the CYP450 NADPH reductase to inhibit CYP450. RESULTS: We confirmed by HPLC-mass spectrometry that MeDDC sulfine was the major product of MeDDC formed by human liver microsomes and by FMO3. Recombinant FMO3 was an efficient catalyst for the formation of MeDDC sulfine (5.3+/-0.2 nmol/min/mg, mean+/-SEM, n = 6). Inhibition studies showed MeDDC was metabolized primarily by CYP450 in human liver microsomes at pH 7.4, with a 10% contribution from FMO (total microsomal activity 3.1+/-0.2, n = 17). In the course of this work, methyl p-tolyl sulfide (MTS), sulfoxidation of which is used by some investigators as a specific probe for FMO activity, was found to be a substrate for both FMO and CYP450 in human liver microsomes. CONCLUSIONS: Our results prove that MeDDC sulfine is the major product of MeDDC oxidation in human liver microsomes, MeDDC is a good substrate for human FMO3, and MeDDC is metabolized in human liver microsomes primarily by CYP450. We also showed that use of MTS sulfoxidation as an indicator of FMO activity in microsomes is valid only in the presence of a CYP450 inhibitor, such as NBI.

Identification of the protein-drug adduct formed between aldehyde dehydrogenase and S-methyl-N,N-diethylthiocarbamoyl sulfoxide by on-line proteolytic digestion high performance liquid chromatography electrospray ionization mass spectrometry.

Disulfiram has been used clinically as an aversion therapy treatment for recovering alcoholics. One of its metabolites, S-methyl-N, N-diethylthiocarbamoyl sulfoxide (MeDTC-SO), is currently believed by some to be the active metabolite in vivo. We demonstrate in this report that MeDTC-SO is a potent irreversible inhibitor of recombinant rat liver mitochondrial aldehyde dehydrogenase (rlmALDH), the enzyme responsible for oxidizing acetaldehyde formed during ethanol metabolism. Recombinant rlmALDH was inhibited by MeDTC-SO after in vitro incubation with an IC(50) = 4.62 microM. The inhibition of rlmALDH was found to be accompanied by a concomitant increase of approximately 100 Da to the molecular mass of the native enzyme as determined by on-line high performance liquid chromatography (HPLC) electrospray ionization mass spectrometry (LC/MS), indicating that a covalent modification has occurred. To determine the site and structure of this covalent adduct, we developed a novel approach to characterize specific protein-drug interactions by linking a proteolytic enzyme digestion cartridge on-line with LC/MS. The on-line pepsin digestion LC/MS of MeDTC-SO-inhibited rlmALDH revealed an ion at MH(2)(2+) = 500.9, which was not present in the pepsin digestion of the non-inhibited enzyme. This peptide was tentatively attributed to the putative active site peptide (FNQGQC(301)C(302)C(303)) plus the adduct. This peptide was subjected to analysis by LC/MS/MS, which allowed us to determine that the covalent modification was associated with a single carbamoyl adduct at Cys-302, which has been shown to be the active site nucleophile of the enzyme.

S-methyl-N,N-diethylthiocarbamate sulfoxide and S-methyl-N,N-diethylthiocarbamate sulfone, two candidates for the active metabolite of disulfiram.

The mechanism of action of disulfiram involves inhibition of hepatic aldehyde dehydrogenase (ALDH). Although disulfiram inhibits ALDH in vitro, it is believed that the drug is too short-lived in vivo to inhibit the enzyme directly. The ultimate inhibitor is thought to be a metabolite of disulfiram. In this study, we examined the effects of S-methyl-N,N-diethylthiocarbamate (MeDTC) sulfoxide and S-methyl-N,N-diethylthiocarbamate sulfone (confirmed and proposed metabolites of disulfiram, respectively) on rat liver mitochondrial low K(m) ALDH. MeDTC sulfoxide and MeDTC sulfone, in 10-min incubations with detergent-solubilized mitochondria, inhibited ALDH activity with an IC50 (mean +/- SD) of 0.93 +/- 0.04 and 0.53 +/- 0.11 microM, respectively, compared with 7.4 +/- 1.0 microM for the parent drug disulfiram. Inhibition by MeDTC sulfone and MeDTC sulfoxide, both at 0.6 microM, was time-dependent, following apparent pseudo-first-order kinetics with a t1/2 of inactivation of 3.5 and 8.8 min, respectively. Dilution of ALDH inhibited by either sulfoxide or sulfone did not restore activity, an indication of irreversible inhibition. Addition of glutathione (50 to 1000 microM) to ALDH before the inhibitors did not alter the inhibition by MeDTC sulfoxide. In contrast, the inhibition by MeDTC sulfone was decreased > 10-fold (IC50 = 6.3 microM) by 50 microM of glutathione and almost completely abolished by 500 microM of glutathione. The cofactor NAD, in a concentration-dependent manner, protected ALDH from inhibition by MeDTC sulfoxide and MeDTC sulfone. In incubations with intact mitochondria, the potency of the two compounds was reversed (IC50 of 9.2 +/- 3.6 and 0.95 +/- 0.30 microM for the MeDTC sulfone and sulfoxide, respectively). Our results suggest that MeDTC sulfone is highly reactive with normal cellular constituents (e.g., glutathione), which may protect ALDH from inhibition, unless this inhibitor is formed very near the target enzyme. In contrast, MeDTC sulfoxide is a better candidate for the ultimate active metabolite of disulfiram, because it is more likely to be sufficiently stable to diffuse from a distant site of formation, such as the endoplasmic reticulum, penetrate the mitochondria, and react with ALDH located in the mitochondrial matrix.

Alcohol-histamine interactions.

Alcohol and histamine metabolic pathways in the body have the common enzymes aldehyde dehydrogenase and aldehyde oxidase. The metabolite of ethanol, acetaldehyde, can effectively compete with the metabolites of histamine, methylimidazole acetaldehyde, and imidazole acetaldehyde. At the periphery, alcohol and acetaldehyde liberate histamine from its store in mast cells and depress histamine elimination by inhibiting diamine oxidase, resulting in elevated histamine levels in tissues. Histamine mediates alcohol-induced gastric and intestinal damage and bronchial asthma as well as flushing in Orientals. On the other hand, alcohol provokes food-induced histaminosis and histamine intolerance, which is an epidemiological problem. There are many controversial reports concerning the effect of H2 receptor antagonists on ethanol metabolism and the activity of alcohol dehydrogenase in the stomach. In addition, alcohol affects histamine levels in the brain by modulating histamine synthesis, release, and turnover. Histamine receptor antagonists can affect ethanol metabolism and change the sensitivity of animals to the hypnotic effects of alcohol. In contrast to other neurotransmitters, the involvement of the brain histamine system in the mechanisms of the central actions of alcohol and in the pathogenesis of alcoholism is poorly studied and understood.

Interactions between taurine and ethanol in the central nervous system.

This purpose of this review will be to summarize the interactions between the endogenous amino acid taurine and ethyl alcohol (ethanol) in the central nervous system (CNS). Taurine is one of the most abundant amino acids in the CNS and plays an integral role in physiological processes such as osmoregulation, neuroprotection and neuromodulation. Both taurine and ethanol exert positive allosteric modulatory effects on neuronal ligand-gated chloride channels (i.e., GABA(A) and glycine receptors) as well as inhibitory effects on other ligand- and voltage-gated cation channels (i.e., NMDA and Ca(2+) channels). Behavioral evidence suggests that taurine can alter the locomotor stimulatory, sedating, and motivational effects of ethanol in a strongly dose-dependent manner. Microdialysis studies have revealed that ethanol elevates extracellular levels of taurine in numerous brain regions, although the functional consequences of this phenomenon are currently unknown. Finally, taurine and several related molecules including the homotaurine derivative acamprosate (calcium acetylhomotaurinate) can reduce ethanol self-administration and relapse to drinking in both animals and humans. Taken together, these data suggest that the endogenous taurine system may be an important modulator of effects of ethanol on the nervous system, and may represent a novel therapeutic avenue for the development of medications to treat alcohol abuse and alcoholism.