A computational evaluation of targeted oxidation strategy (TOS) for potential inhibition of SARS-CoV-2 by disulfiram and analogues.

In the new millennium, the outbreak of new coronavirus has happened three times: SARS-CoV, MERS-CoV, and SARS-CoV-2. Unfortunately, we still have no pharmaceutical weapons against the diseases caused by these viruses. The pandemic of SARS-CoV-2 reminds us the urgency to search new drugs with totally different mechanism that may target the weaknesses specific to coronaviruses. Herein, we disclose a computational evaluation of targeted oxidation strategy (TOS) for potential inhibition of SARS-CoV-2 by disulfiram, a 70-year-old anti-alcoholism drug, and predict a multiple-target mechanism. A preliminary list of promising TOS drug candidates targeting the two thiol proteases of SARS-CoV-2 are proposed upon virtual screening of 32,143 disulfides.

Development of new disulfiram analogues as ALDH1a1-selective inhibitors.

Disulfiram is an FDA-approved drug used to treat chronic alcoholism. This drug works by blocking the second step of ethanol metabolism by inhibiting aldehyde dehydrogenase-2 (ALDH2), the enzyme responsible for acetaldehyde oxidation into acetic acid. This leads to the accumulation of acetaldehyde in the blood following alcohol ingestion and to highly unpleasant symptoms known as acetaldehyde syndrome. Disulfiram also inhibits ALDH1a1, another member of the aldehyde dehydrogenases that catalyzes the oxidation of retinal into retinoic acid. ALDH1a1 represents a key therapeutic target for the treatment of important diseases such as cancer and obesity. The substrate tunnel is larger in ALDH1a1 than in ALDH2; therefore. Thus, replacing disulfiram ethyl groups with larger groups will yield selective ALDH1a1 inhibitors. In this work, we successfully synthesized derivative 2b, in which two ethyl groups were replaced by two para fluorobenzyl groups. The 2b derivative showed a comparable activity to disulfiram against ALDH1a1; however, it was completely devoid of inhibitory activity against ALDH2.

Disulfiram-loaded mixed nanoparticles with high drug-loading and plasma stability by reducing the core crystallinity for intravenous delivery.

To develop an injectable formulation and improve the stability of disulfiram (DSF), DSF was encapsulated into mixed nanoparticles (DSF-NPs) through a high-pressure homogenization method. The Flory-Huggins interaction parameters (χFH) were calculated to predict the miscibility between DSF and the hydrophobic core, resulting in PCL5000 selected as the hydrophobic block to encapsulate the DSF, as PCL5000 had a lower χFH 3.39 and the drug loading of the nanoparticles prepared by mPEG5000-PCL5000 was relatively higher. mPEG5000-PCL5000 and PCL5000 were blended to reduce the leakage of DSF during preparation, as well as increase the stability of the nanoparticles. The cargo-loading capacity of the nanoparticles was improved from 3.35% to 5.50% by reducing the crystallinity of the PCL nanoparticle core, and the crystallinity decreased from 51.13% to 25.15% after adding medium chain triglyceride (MCT). The DSF-NPs prepared by the above method had a small particle size of 98.1 ±â€¯10.54 nm, with a polydispersity index (PDI) of 0.036, as well as drug loading of 5.50%. Furthermore, DSF-NPs containing MCT showed higher stability than DSF-NPs without MCT and DSF-sol (DSF dissolved in Cremophor EL and ethanol) in water and 90% plasma-containing PBS. The pharmacokinetics proved that DSF-NPs containing MCT enhanced the DSF concentration in the blood. Finally, DSF-NPs effectively inhibited H22 xenograft tumor growth in vivo.

Pharmacological inhibition of Receptor Protein Tyrosine Phosphatase ß/ζ (PTPRZ1) modulates behavioral responses to ethanol.

Pleiotrophin (PTN) and Midkine (MK) are neurotrophic factors that are upregulated in the prefrontal cortex after alcohol administration and have been shown to reduce ethanol drinking and reward. PTN and MK are the endogenous inhibitors of Receptor Protein Tyrosine Phosphatase (RPTP) ß/ζ (a.k.a. PTPRZ1, RPTPß, PTPζ), suggesting a potential role for this phosphatase in the regulation of alcohol effects. To determine if RPTPß/ζ regulates ethanol consumption, we treated mice with recently developed small-molecule inhibitors of RPTPß/ζ (MY10, MY33-3) before testing them for binge-like drinking using the drinking in the dark protocol. Mice treated with RPTPß/ζ inhibitors, particularly with MY10, drank less ethanol than controls. MY10 treatment blocked ethanol conditioned place preference, showed limited effects on ethanol-induced ataxia, and potentiated the sedative effects of ethanol. We also tested whether RPTPß/ζ is involved in ethanol signaling pathways. We found that ethanol treatment of neuroblastoma cells increased phosphorylation of anaplastic lymphoma kinase (ALK) and TrkA, known substrates of RPTPß/ζ. Treatment of neuroblastoma cells with MY10 or MY33-3 also increased levels of phosphorylated ALK and TrkA. However, concomitant treatment of neuroblastoma cells with ethanol and MY10 or MY33-3 prevented the increase in pTrkA and pALK. These results demonstrate for the first time that ethanol engages TrkA signaling and that RPTPß/ζ modulates signaling pathways activated by alcohol and behavioral responses to this drug. The data support the hypothesis that RPTPß/ζ might be a novel target of pharmacotherapy for reducing excessive alcohol consumption.

Disulfiram-loaded porous PLGA microparticle for inhibiting the proliferation and migration of non-small-cell lung cancer.

In this study, poly(lactic-co-glycolic acid) (PLGA) was used as a carrier to construct disulfiram-loaded porous microparticle through the emulsion solvent evaporation method, using ammonium bicarbonate as a porogen. The microparticle possessed highly porous surface, suitable aerodynamic diameter for inhalation (8.31±1.33 µm), favorable drug loading (4.09%±0.11%), and sustained release profile. The antiproliferation effect of release supernatant was detected through 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay using non-small-cell lung cancer A549 as a model, with only 13.3% of cell viability observed for the release supernatant at 7 days. The antiproliferation mechanism was elucidated to be associated with the enhanced induction of cell apoptosis and cell cycle arrest at S phase through flow cytometry and Western blotting analysis. Finally, wound healing and transwell migration assay showed that they could efficiently inhibit the cell migration. These results demonstrated that disulfiram-loaded porous PLGA microparticle could achieve favorable antitumor efficiency, implying the potential of treating non-small-cell lung cancer in a pulmonary administration.

Further characterization of the GlyT-1 inhibitor Org25935: anti-alcohol, neurobehavioral, and gene expression effects.

The glycine transporter-1 inhibitor Org25935 is a promising candidate in a treatment concept for alcohol use disorder targeting the glycine system. Org25935 inhibits ethanol-induced dopamine elevation in brain reward regions and reduces ethanol intake in Wistar rats. This study aimed to further characterise the compound and used ethanol consumption, behavioral measures, and gene expression as parameters to investigate the effects in Wistar rats and, as pharmacogenetic comparison, Alko-Alcohol (AA) rats. Animals were provided limited access to ethanol in a two-bottle free-choice paradigm with daily drug administration. Acute effects of Org25935 were estimated using locomotor activity and neurobehavioral status. Effects on gene expression in Wistar rats were measured with qPCR. The higher but not the lower dose of Org25935 reduced alcohol intake in Wistar rats. Unexpectedly, Org25935 reduced both ethanol and water intake and induced strong CNS-depressive effects in AA-rats (withdrawn from further studies). Neurobehavioral effects by Org25935 differed between the strains (AA-rats towards sedation). Org25935 did not affect gene expression at the mRNA level in the glycine system of Wistar rats. The data indicate a small therapeutic range for the anti-alcohol properties of Org25935, a finding that may guide further evaluations of the clinical utility of GlyT-1 inhibitors. The results point to the importance of pharmacogenetic considerations when developing drugs for alcohol-related medical concerns. Despite the lack of successful clinical outcomes, to date, the heterogeneity of drug action of Org25935 and similar agents and the unmet medical need justify further studies of glycinergic compounds in alcohol use disorder.

The novel non-imidazole histamine H3 receptor antagonist DL77 reduces voluntary alcohol intake and ethanol-induced conditioned place preference in mice.

It has become clear that histamine H3 receptors (H3R) have been implicated in modulating ethanol intake and preference in laboratory animals. The novel non-imidazole H3R antagonist DL77 with excellent selectivity profile shows high in-vivo potency as well as in-vitro antagonist affinity with ED50 of 2.1 ± 0.2 mg/kg and pKi=8.08, respectively. In the present study, and applying an unlimited access two-bottle choice procedure, the anti-alcohol effects of the H3R antagonist, DL77 (0, 3, 10 and 30 mg/kg; i.p.), were investigated in adult mice. In this C57BL/6 line, effects of DL77 on voluntary alcohol intake and preference, as well as on total fluid intake were evaluated. Results have shown that DL77, dose-dependently, reduced both ethanol intake and preference. These effects were very selective as both saccharin and quinine, used to control for taste sensitivity, and intakes were not affected following DL77 pre-application. More importantly, systemic administration of DL77 (10 mg/kg) during acquisition inhibited ethanol-induced conditioned-place preference (EtOH-CPP) as measured using an unbiased protocol. The anti-alcohol activity observed for DL77 was abrogated when mice were pretreated with the selective H3R agonist R-(α)-methyl-histamine (RAMH) (10 mg/kg), or with the CNS penetrant H1R antagonist pyrilamine (PYR) (10mg/kg). These results suggest that DL77 has a predominant role in two in vivo effects of ethanol. Therefore, signaling via H3R is essential for ethanol-related consumption and conditioned reward and may represent a novel therapeutic pharmacological target to tackle ethanol abuse and alcoholism.

Topiramate in Alcohol Use Disorders: Review and Update.

To date, a limited number of pharmacological agents exist to treat alcohol use disorders (AUDs), and there is growing interest in new therapeutic tools. In this framework, topiramate may represent a useful treatment option, although its use is not yet approved for AUDs. The main focus of this review is to discuss all the existing data supporting the use of topiramate in AUDs, with an emphasis on the most recent and relevant clinical implications. In addition, the profile of the alcoholic patient who may benefit more from the use of topiramate is outlined. In this regard, the authors conducted a PubMed search of clinical human studies published in English using the following key words: topiramate alcohol dependence, topiramate alcohol withdrawal and topiramate alcoholism. The evidence suggests that topiramate could be an effective treatment option for the management of AUDs, while there are limited results for its use to treat alcohol withdrawal syndrome. In particular, topiramate shows a greater beneficial effect in subjects with a typology of craving characterised by drinking obsessions and automaticity of drinking. Topiramate, within the dosage range of 75-300 mg/day, could be considered as a first-line treatment option for the management of AUDs. Its use appears to be safe and well-tolerated, especially in light of very recent findings.

Intra-nucleus accumbens shell injections of R(+)- and S(-)-baclofen bidirectionally alter binge-like ethanol, but not saccharin, intake in C57Bl/6J mice.

The GABAB agonist baclofen has been widely researched clinically and preclinically as a treatment of alcohol use disorders (AUDs). However, the efficacy of baclofen remains uncertain. The clinically used racemic compound can be separated into separate enantiomers. These enantiomers have produced different profiles in behavioral assays, with the S- compound often being ineffective compared to the R- compound, or the S- compound antagonizing the effects of the R- compound. We have previously demonstrated that the R(+)-baclofen enantiomer decreases binge-like ethanol intake in the Drinking-in-the-Dark (DID) paradigm, whereas the S(-)-baclofen enantiomer increases ethanol intake. One area implicated in drug abuse is the nucleus accumbens shell (NACsh).The current study sought to define the role of the NACsh in the enantioselective effects of baclofen on binge-like ethanol consumption by directly microinjecting each enantiomer into the structure. Following bilateral cannulation of the NACsh, C57Bl/6J mice were given 5 days of access to ethanol or saccharin for 2h, 3h into the dark cycle. On Day 5 mice were given an injection of aCSF, 0.02 R(+)-, 0.04R(+)-, 0.08 S(-)-, or 0.16 S(-)-baclofen (µg/side dissolved in 200nl of aCSF). It was found that the R(+)-baclofen dose-dependently decreased ethanol consumption, whereas the high S(-)-baclofen dose increased ethanol consumption, compared to the aCSF group. Saccharin consumption was not affected. These results further confirm that GABAB receptors and the NACsh shell are integral in mediating ethanol intake. They also demonstrate that baclofen displays bidirectional, enantioselective effects which are important when considering therapeutic uses of the drug.

Safety and efficacy of flumazenil for reversal of iatrogenic benzodiazepine-associated delirium toxicity during treatment of alcohol withdrawal, a retrospective review at one center.

Both alcohol withdrawal syndrome (AWS) and benzodiazepines can cause delirium. Benzodiazepine-associated delirium can complicate AWS and prolong hospitalization. Benzodiazepine delirium can be diagnosed with flumazenil, a GABA-A receptor antagonist. By reversing the effects of benzodiazepines, flumazenil is theorized to exacerbate symptoms of AWS and precludes its use. For patients being treated for alcohol withdrawal, flumazenil can diagnose and treat benzodiazepine delirium without precipitating serious or life-threatening adverse events. Hospital admission records were retrospectively reviewed for patients with the diagnosis of AWS who received both benzodiazepines and flumazenil from December 2006 to June 2012 at a university-affiliated inpatient toxicology center. The day of last alcohol consumption was estimated from available blood alcohol content or subjective history. Corresponding benzodiazepine, flumazenil, and adjunctive sedative pharmacy records were reviewed, as were demographic, clinical course, and outcome data. Eighty-five patients were identified (average age 50.3 years). Alcohol concentrations were detectable for 42 patients with average 261 mg/dL (10-530 mg/dL). Eighty patients were treated with adjunctive agents for alcohol withdrawal including antipsychotics (n = 57), opioids (n = 27), clonidine (n = 35), and phenobarbital (n = 23). Average time of flumazenil administration was 4.7 days (1-11 days) after abstinence, and average dose was 0.5 mg (0.2-1 mg). At the time of flumazenil administration, delirium was described as hypoactive (n = 21), hyperactive (n = 15), mixed (n = 41), or not specified (n = 8). Response was not documented in 11 cases. Sixty-two (72.9 %) patients had significant objective improvement after receiving flumazenil. Fifty-six patients required more than one dose (average 5.6 doses). There were no major adverse events and minor adverse effects included transiently increased anxiety in two patients: 1 patient who received 0.5 mg on abstinence day 2 and another patient who received 0.2 mg flumazenil on abstinence day 11. This is the largest series diagnosing benzodiazepine delirium after AWS in patients receiving flumazenil. During the treatment of AWS, if delirium is present on day 5, a test dose of flumazenil may be considered to establish benzodiazepine delirium. With the limited data set often accompanying patients with AWS, flumazenil diagnosed benzodiazepine delirium during the treatment of AWS and improved impairments in cognition and behavior without serious or life-threatening adverse events in our patients.

New pharmacological treatment strategies for relapse prevention.

Here we discuss treatment strategies that are based on pharmacological interventions to reduce craving and relapse in alcohol-dependent patients. We will first provide a historical overview about relapse prevention strategies. We will then review the development of disulfiram, naltrexone, acamprosate, and nalmefene and discuss their neurobiological modes of action. Then the concept of convergent genomic analysis will be introduced for the discovery of new molecular treatment targets. Finally, we will provide convincing evidence for the use of N-methyl-D-aspartate (NMDA) receptor channel blockers as substitution drugs. Important conclusions of this review are: (i) learning from other addictive substances is very helpful-e.g., substitution therapies as applied to opiate addiction for decades could also be translated to alcoholics, (ii) the glutamate theory of alcohol addiction provides a convincing framework for the use of NMDA receptor antagonists as substitution drugs for alcohol-dependent patients, (iii) a combination of behavioral and pharmacological therapies may be the optimal approach for future treatment strategies-one promising example concerns the pharmacological disruption of reconsolidation processes of alcohol cue memories, (iv) given that many neurotransmitter systems are affected by chronic alcohol consumption, numerous druggable targets have been identified; consequently, a "cocktail" of different compounds will further improve the treatment situation, (v) in silico psychopharmacology, such as drug repurposing will yield new medications, and finally, (vi) the whole organism has to be taken into consideration to provide the best therapy for our patients. In summary, there is no other field in psychiatric research that has, in recent years, yielded so many novel, druggable targets and innovative treatment strategies than for alcohol addiction. However, it will still be several years before the majority of the "treatment-seeking population" will benefit from those developments.

[Disulfiram and addiction: reminders and new perspectives of use]. / Le disulfirame (Esperal(®)) dans le traitement des addictions : rappels et nouvelles perspectives d'utilisation.

Disulfiram is a relatively old molecule, which today remains marginal in the treatment of alcoholics diseases. Using this type of treatment is the subject of ethical debate. The prescription of this therapeutic requires clinical and biological rigorous evaluations before treatment. Its main action in treatment of alcoholism is related to the restraint of acetaldehyde dehydrogenase action causing the antabuse reaction. Prescription of disulfiram, supported by specialized programs of compartmental integrated care, brings significant benefit for alcoholic patients. Recently, following the discovery of its action on dopamine metabolism, disulfiram has been a renewed interest in the treatment of addictions to cocaine and pathological gambling. Although current data are insufficient to generalize its use in routine practice, they constitute a line of research interest for the future.

Acamprosate: a prototypic neuromodulator in the treatment of alcohol dependence.

Alcoholism is one of the most prevalent substance dependence disorders in the world. Advances in research in the neurobiological mechanisms underlying alcohol dependence have identified specific neurotransmitter targets for the development of pharmacological treatments. Acamprosate, marketed under the brand name Campral, is an orally administered drug available by prescription in the U.S. and throughout much of the world for treating alcohol dependence. Its safety and efficacy have been demonstrated in numerous clinical trials worldwide. Here we provide an overview of acamprosate in the context of the neurobiological underpinnings of alcohol dependence. We propose that unlike previously available pharmacotherapies, acamprosate represents a prototypical neuromodulatory approach in the treatment of alcohol dependence. A neuromodulatory approach seeks to restore the disrupted changes in neurobiology resulting from chronic alcohol intake. We believe that a neuromodulatory approach will provide a heuristic framework for developing more effective pharmacotherapies for alcohol dependence.

A selective ALDH-2 inhibitor reduces anxiety in rats.

CVT-10216 is a highly selective, reversible inhibitor of ALDH-2 that reduces excessive alcohol drinking. Anxiety plays a role in alcoholism. The present study asks whether CVT-10216 has anxiolytic properties, as reflected in social interaction behavior in four unrelated rodent models: endogenous anxiety-like behavior in naïve Fawn-Hooded rats, repeated alcohol-withdrawal-induced anxiety, restraint stress-induced anxiety and drug-induced anxiety. CVT-10216 counteracted anxiety in all models except that produced by the 5-HT(2C) agonist, mCPP. CVT-10216 exhibited both acute and prophylactic inhibitions of repeated alcohol-withdrawal-induced anxiety. Importantly, anxiogenic behavior induced by the benzodiazepine receptor inverse agonist, DMCM, was counteracted dose-dependently by CVT-10216. Thus, a non-addictive selective inhibitor of ALDH-2 has both anxiolytic and antidipsotropic properties, which may be dependent, in part on the involvement of the GABA-benzodiazepine system.

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).

Investigation of the relationship between in vitro and in vivo release behaviors of acamprosate from enteric-coated tablets.

Acamprosate calcium is a highly soluble drug with low permeability that is used to maintain abstinence in alcohol-dependent patients. The aim of this study was to investigate the relationship between in vitro and in vivo behaviors of acamprosate from enteric-coated tablets. The in vitro release behavior of acamprosate tablets in pH 6.8 buffer solution was determined in three dissolution conditions, 50 and 150 rpm (paddle method) and 180 rpm (basket method). The results of this in vitro experiment indicated that acamprosate tablets hardly disintegrated, and drug dissolution was retarded despite the extremely hydrophilic nature of the drug. A single dose (333 mgx2 tablets) of each formulation was orally administered to four beagle dogs under fasting conditions, and the pharmacokinetic parameters were calculated. The mean AUC0-48, Cmax, Tlag and Tmax for the two types of tablets ranged from 41.5-53.6 microg.h/mL, 4.3-4.5 microg/mL, 2.0-2.5 h and 3.8-4.0 h, respectively. In conclusion, it is suggested that retarded drug release from the tablets and the low drug permeability may result in poor absorption and erratic bioavailability of this drug in humans.

Synthesis and hydrolytic behavior of two novel tripartate codrugs of naltrexone and 6beta-naltrexol with hydroxybupropion as potential alcohol abuse and smoking cessation agents.

A codrug approach for simultaneous treatment of alcohol abuse and tobacco dependence is considered as very desirable because of substantial evidence that smoking is increased significantly during drinking, and that smoking is regarded as a behavioral 'cue' for the urge to consume alcohol. The purpose of this study was to design and synthesize codrugs for simultaneous treatment of alcohol abuse and tobacco dependence. Two novel tripartate codrugs of naltrexone (NTX) and naltrexol (NTXOL) covalently linked to hydroxybupropion (BUPOH) were synthesized (25 and 26, respectively), and their hydrolytic cleavage to the parent drugs was determined. These codrugs were generally less crystalline when compared to NTX, or NTXOL, as indicated by their lower melting points, and were expected to be more lipid-soluble. Also, the calculated clogP values were found to be higher for the codrugs compared to those for NTX and NTXOL. The studies on the hydrolysis of the codrugs provided good evidence that they could be efficiently converted to the parent drugs in buffer at physiological pH. Thus, these codrugs are likely to be cleaved enzymatically in vivo to generate the parent drugs, and are considered to be potential candidates for simultaneous treatment of alcohol abuse and tobacco dependence.

A new polyamine derivative, a structural analog of spermine, with in vivo activity as an inhibitor of ethanol appetite.

This report describes the design and synthesis of the synthetic polyamine DCD (N,N'-bis-(3-aminopropyl)cyclohexane-1,4-diamine, tetramethanesulfonate), a structural analog of spermine, and its in vivo activity as an inhibitor of alcohol consumption in a free-paradigm carried out on genetically high-ethanol-consuming UChB rats. After acute treatment with DCD (daily single dose, 20 mg/kg, p.o., 3 days), a 19% decrease in ethanol intake was obtained, without affecting the levels of food and water intake. After chronic treatment (daily single dose, 20mg/kg, p.o., 60 days) a decrease of up to 60% in ethanol intake with respect to the basal period was provoked; this effect was significantly maintained during the post-treatment period and, according to the data obtained from the determination of acetaldehyde levels in blood, was not related to a possible disulfiram-like effect. The design of this new compound was carried out using molecular modeling techniques, with the structures of natural polyamines (putrescine, spermidine, and spermine) and biosynthetically related diamines (1,3-diaminopropane; DAP) as templates. These polyamines have shown activity as inhibitors of ethanol appetite in the same experimental model.

Metadoxine in the treatment of acute and chronic alcoholism: a review.

Alcohol abuse and alcoholism are responsible for a wide variety of medical problems. The pharmaco-therapeutic aspect of alcoholism includes the use of drugs, with different actions and objectives. Among them, metadoxine seems to be of interest. Metadoxine is able to accelerate the elimination of alcohol from the blood and tissues, to help restore the functional structure of the liver and to relieve neuro-psychological disorders associated with alcohol intoxication. Metadoxine also seems to be safe; in more than 15 years of post-marketing surveillance only minor aspecific and reversible events were monitored in patients exposed to the treatment. In this review the preclinical and clinical results obtained using metadoxine in acute and chronic alcohol intoxication are reported.