Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

Chlormethiazole is a sedative, hypnotic, neuroprotective, and anticonvulsant agent with the ability to potentiate -aminobutyric acid (GABA)ergic neurotransmission through an allosteric modulation of the GABA_A receptor complex (RC) (Smith and Jewkes, 1995). A specific binding site for chlormethiazole on the GABA_A RC has been conceptualized (Smith and Jewkes, 1995; Green, 1998). Consequently, chlormethiazole shows a distinctly different pharmacological and clinical profile from classic GABAergic drugs such as benzodiazepines, barbiturates, and neuroactive steroids (Smith and Jewkes, 1995; Green, 1998).

Chlormethiazole was developed by a structural modification of the thiazole ring of vitamin B₁ in the late 1950s (Fig. 1), and it was internationally introduced into clinical use (except the United States) some years later (Smith and Jewkes, 1995). Since then, the primary therapeutic indication of chlormethiazole has been the treatment of the ethanol withdrawal syndrome, including seizures, delirium tremens, and alcoholic dementia (Morgan, 1995). Chlormethiazole also is used to treat seizures and status epilepticus in epileptic patients unresponsive to barbiturates and benzodiazepines, and in the prevention and treatment of convulsive complications in pregnant women with pre-eclampsia and eclampsia (Smith and Jewkes, 1995). Hypnotic and sedative properties of chlormethiazole have been used in the pharmacological treatment of restlessness, agitation, and insomnia in both geriatric and psychiatric patients (Smith and Jewkes, 1995). Pharmacokinetic characteristics such as a rapid onset and a short, predictable duration of action (t_1/2  4 h), high lipid solubility, no intermediate metabolites, and lack of hepatic and systemic toxicity all make chlormethiazole very suitable for an acute, emergency treatment (Smith and Jewkes, 1995).

View larger version (14K): [in this window] [in a new window]   Fig. 1.   Structure of chlormethiazole and vitamin B1 (thiamine). The pyrimidine portion of vitamin B1 possesses convulsant properties, whereas the thiazole portion possesses anticonvulsant effects. This observation by Charronat led to the development of chlormethiazole by the structural modification of the thiazole ring in the 1950s (Smith and Jewkes, 1995).

There is a sparse clinical literature suggesting that chlormethiazole can be effective as an antidote against the toxic effects of other abused drugs such as heroin (Glatt et al., 1970) and 3,4-methylenedioxymethamphetamine ("ecstasy") (Bedford Russell et al., 1992). Experimental data provide additional impetus for the potential application of chlormethiazole in the treatment of toxicity from illicit psychomotor-stimulant drugs. Specifically, chlormethiazole attenuated the neurotoxic depletion of dopamine and 5-hydroxytryptamine content induced by methamphetamine and ecstasy in vitro (Green and Cross, 1994; Green, 1998). However, neither clinical nor experimental information is available on the efficacy of chlormethiazole against the toxic (seizures and lethality) effects of the prototypical dopaminergic psychomotor stimulant drug cocaine, the abuse of which is estimated to account for approximately one-third of all drug-related emergency department visits.

Cocaine-related emergency complications continue to be a major public health concern worldwide due to a high prevalence of cocaine abuse and high frequency of cocaine-related emergency department visits. In the United States in 1995 alone, there were an estimated 1.7 million regular users of cocaine; cocaine was implicated in approximately 150,000 emergency room visits and nearly 4,000 fatalities (National Institute on Drug Abuse, 1996; Substance Abuse and Mental Health Services Administration, 1997). Generalized clonic-tonic seizures and status epilepticus capable of producing progressive epileptogenic changes, long-term neurological and psychiatric impairment, and death are well-described sequelae of cocaine abuse (Kramer et al., 1990; Dhuna et al., 1991; Benowitz, 1993). Seizures can occur after the recreational use of relatively low doses of cocaine as well as after an overdose (Kramer et al., 1990; Dhuna et al., 1991). Regardless, seizures can be resistant to available anticonvulsant drugs (e.g., benzodiazepines, barbiturates) and are considered to be a major determinant of cocaine-related lethality (Dhuna et al., 1991; Benowitz, 1993). Up to 12% of patients admitted to emergency departments with cocaine intoxication require anticonvulsive therapy (Derlet and Albertson, 1989a; Dhuna et al., 1991).

Because there is no specific treatment for cocaine-related convulsive states, the search for effective and safe treatments continues (Taylor and Slaby, 1992; Gasior et al., 1999; Witkin et al., 1999). Given the clinical and experimental findings with chlormethiazole and other abused drugs as mentioned above, we predicted that chlormethiazole also might be effective against the toxic effects of cocaine. We therefore performed a series of experiments to characterize the effectiveness of chlormethiazole against the seizure-generating (acute and chronic) and lethal effects of cocaine in mice. The effects of chlormethiazole were compared with the effects of two classic benzodiazepine antiepileptic drugs, diazepam and clonazepam.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

Subjects. Experimentally naive, male Swiss-Webster mice (Taconic Farms, Germantown, NY) between 10 and 12 weeks old and weighing 30 to 44 g were housed six per cage in an environmentally controlled vivarium (temperature, 24 ± 2°C; humidity, 45 ± 5%). All animals were acclimated to their home cages and to the light/dark cycle for at least 5 days before testing. Tap water and food pellets (NIH-07 diet; Zeigler Bros. Inc., Gardners, PA) were available ad libitum. Experiments were conducted between 9:00 AM and 3:00 PM during the light phase of a 12-h light/dark cycle (lights on between 7:00 AM and 7:00 PM) in an experimental room. At least eight mice per group were used, and all mice were experimentally naive.

Drugs. Chlormethiazole [also called clomethiazole, Heminevrin; 5-(2-chloroethyl)-4 methylthiazole, mol. wt. = 161.5) was obtained from AstraZeneca (Södertälje, Sweden). Diazepam (mol. wt. = 284.8) was obtained from Hoffmann-LaRoche (Nutley, NJ). Clonazepam (mol. wt. = 315.7) was obtained from Sigma Chemical Co. (St. Louis, MO). ()-Cocaine hydrochloride was obtained from the National Institute on Drug Abuse (Rockville, MD). Chlormethiazole and cocaine were dissolved in sterile 0.9% NaCl solution and administered i.p. Diazepam and clonazepam were suspended in 20% (v/v) propylene glycol (PEG) (Sigma Chemical Co.) with mild heat and administered s.c. Injection volume was 0.1 ml/10 g b.wt. Doses of drugs were expressed as milligrams of salt per kilogram of body weight. The pretreatment time of chlormethiazole was 45 min with the exception of the time course studies (see below) where pretreatment times ranged from 5 to 180 min. The pretreatment time for diazepam and clonazepam was 30 min. These pretreatment times were selected based on information on biological activity from the literature and confirmed in the present study.

Motor Toxicity. Immediately before administration of cocaine, mice were first tested on the inverted-screen test. The inverted-screen test was used to assess one form of behavioral toxicity induced by chlormethiazole. In this test, compounds with sedative and/or ataxic properties produce dose-dependent increases in screen-test failures, whereas other classes of drugs (e.g., psychomotor stimulants) do not. Mice were pretreated with either saline or chlormethiazole and returned to their home cage for the appropriate pretreatment interval. They were then individually placed on a 14 × 14-cm wire mesh screen (0.8-cm screen mesh) elevated 38 cm above the ground. After slowly inverting the screen, the mice were tested during a 2-min trial for their ability to climb to the top. Mice unable to climb to the top (all four paws on the upper surface) were counted as a failure. Immediately after completion of the screen test, mice were given cocaine whereupon toxicity tests were conducted as described below. Information about the effects of diazepam and clonazepam in this test can be found in Witkin et al. (1999).

Acutely Induced Cocaine Seizures. A convulsant dose of cocaine (75 mg/kg) was administered, and the mice were immediately placed in individual Plexiglas containers (14 × 25 × 36 cm high) for observation. The dose of 75 mg/kg cocaine was selected to be close to the convulsive ED₉₀ value of cocaine as determined during pilot experiments and from the literature (Witkin and Tortella, 1991; Gasior et al., 1997, 1999). Cocaine-induced convulsions were defined as loss of the righting response lasting at least 5 s and the occurrence of clonic movements of all four limbs; tonic seizures were never observed. The presence or absence of convulsions was recorded for 30 min after cocaine injection; typically seizures occurred within 15-min postcocaine administration. Sudden locomotor activation with violent jumps and loss of the righting response often preceded clonic episodes in cocaine-challenged mice. Once seizures developed, the loss of the righting response often persisted over several minutes after cocaine injection; typically mice would then recover and show normal behavior by the end of the 30-min observation period.

Time Course Effects of Chlormethiazole. In addition to acute studies with a constant pretreatment time of 45 min, the behavioral and anticonvulsant effects of chlormethiazole were evaluated after a range of pretreatment times (5-180 min). Specifically, separate groups of mice were treated with 100 mg/kg chlormethiazole at different times before testing as described under Motor Toxicity and Acutely Induced Cocaine Seizures.

Chronically Induced Cocaine Seizures (Cocaine Kindling). Kindled seizures were produced by a total of five (or six) treatments with 60 mg/kg cocaine on days 1 to 5 (or 6). This treatment regime was reported to produce increases in the percentage of mice exhibiting clonic seizures after each successive injection with 60 mg/kg cocaine and long-term increases in sensitivity to the convulsive and lethal effects of cocaine without changes in spontaneous behavior (Miller et al., 2000). As with acutely induced cocaine seizures, mice were observed for the presence or absence of clonic convulsions for 30 min after cocaine injection. The behavioral manifestation of clonic seizures in cocaine-kindled mice did not differ qualitatively from that of the acutely induced clonic seizures as described above.

Cocaine-Induced Lethality. Acute lethality was produced by a single injection of cocaine (110 mg/kg). This dose of cocaine corresponds to a submaximal lethal value (LD_95-100) as determined by Miller et al. (2000) in the same strain of mice and confirmed in the present study. After cocaine administration, mice were placed in individual Plexiglas containers for 60 min. The protective effects of chlormethiazole were reflected by its ability to attenuate cocaine-induced lethality at 60-min postcocaine injection. Diazepam and clonazepam were evaluated for comparison.

Data Presentation and Statistical Calculations. Quantal dose(log)-effect functions were constructed for the acute behavioral and protective effects of chlormethiazole, clonazepam, and diazepam. At least three groups consisting of a minimum of eight mice each were used to construct one dose-effect function. Such dose-effect functions, where appropriate, were used to calculate drug potencies (for drug-induced motor toxicity a TD₅₀ was calculated, and for protective effects of antiepileptic drugs against cocaine-induced seizures and lethality ED₅₀ values were calculated). ED₅₀ and TD₅₀ values represent a dose of a drug predicted to produce an effect in 50% of the mice tested. ED₅₀ and TD₅₀ values with 95% CL were calculated and statistically analyzed according to the method described by Litchfield and Wilcoxon (1949). Relative potency estimates (with 95% CL) were derived as a conservative estimate of statistical differences in dose-effect functions. Relative potency estimates with 95% CL that did not encompass the value 1.00 (equivalent potencies) were considered statistically significant. Where appropriate, slopes (with 95% CL) of regression lines of dose (log)-effect functions were calculated and compared statistically for parallelism. Fisher's exact probability test was additionally used for specific comparisons between each dose-treatment and control group.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

Motor Toxicity and Anticonvulsant Effects of Chlormethiazole in Experimentally Naive Mice. Under control conditions, there were no screen failures on the inverted-screen test, and 87.5% of mice exhibited clonic seizures after administration of 75 mg/kg cocaine. Chlormethiazole (45 min before test) in a dose-dependent manner increased the percentage of mice falling off the screen, and the 300-mg/kg dose produced motor impairment in 100% of the mice tested (Fig. 2). The convulsive effects of cocaine were blocked dose dependently by chlormethiazole. Doses of 75 and 100 mg/kg chlormethiazole fully protected against cocaine-induced seizures. The anticonvulsant effects of 170 and 300 mg/kg chlormethiazole were not evaluated due to marked sedation produced by these doses of chlormethiazole (Fig. 2). Table 1 lists potencies of chlormethiazole to produce behavioral side effects (TD₅₀ value) and anticonvulsive effects (ED₅₀ value). There was a favorable separation between the TD₅₀ and ED₅₀ values that resulted in a PI of 22.3 (Table 1). Note, however, that the PI of chlormethiazole should be interpreted with caution due to a marked difference in slopes (Table 1) of the dose-effect functions to produce behavioral toxicity and protection against cocaine-induced seizures such that the separation between behavioral toxicity and anticonvulsive efficacy depends on the point of comparison along the dose-effect function (Fig. 2).

View larger version (14K): [in this window] [in a new window]   Fig. 2.   Effect of chlormethiazole on the inverted-screen test () and against 75 mg/kg cocaine-induced seizures () in experimentally naive mice. Mice pretreated with chlormethiazole or saline (control group) were first tested on the inverted-screen test and immediately after they received 75 mg/kg cocaine and were observed for the occurrence of clonic seizures. Each data point reflects the percentage of mice falling off the screen or protected against cocaine-induced clonic seizures (n = 8-16 mice/data point). All saline-treated mice correctly performed the inverted screen test (0% failures, n = 16), and 87.5% (n = 16) of mice developed clonic seizures after 75 mg/kg cocaine. Computed linear regressions (solid lines) were drawn for each dose-effect function. See Table 1 for statistical information on the dose-effect curves. Asterisks indicate statistically significant differences of the outcome of specific dose-treatments from the control performance on the inverted-screen test and in the seizure test (*P < .05, Fisher's exact test).

Time Course Effects of Chlormethiazole. During the above-described experiment, a marked sedation was observed within several minutes post administration of chlormethiazole (100 mg/kg) that was followed by recovery by the end of the 45-min pretreatment time. This unsystematic observation, suggestive of a temporal separation between behavior-disrupting and anticonvulsive effects, prompted evaluation of the time course of these two effects of chlormethiazole. As Fig. 3 shows, time courses of the behavior-disrupting and anticonvulsive effects of 100 mg/kg chlormethiazole did not overlap. The onset of the behavior-disrupting effect of chlormethiazole was rapid, evident within 5 to 15 min post injection, and was followed by complete recovery. In contrast, the onset of anticonvulsant efficacy was slower and longer lasting (Fig. 3). Chlormethiazole exerted a peak anticonvulsant effect 45 min post injection. From 45 to 90 min post chlormethiazole, anticonvulsant efficacy was observed without significant effect on behavior. The pretreatment time of 45 min was used in all subsequent experiments with chlormethiazole.

View larger version (13K): [in this window] [in a new window]   Fig. 3.   Time course for chlormethiazole's behavioral effects on the inverted-screen test () and anticonvulsant effects against 75 mg/kg cocaine-induced seizures (). Mice were pretreated with 100 mg/kg chlormethiazole at different times before testing. Each data point represents the percentage of mice falling off the screen or protected against cocaine-induced clonic seizures (n = 8-10 mice/data point). A separate group of mice was used for each pretreatment time. *P < .05, compared with the control performance on the inverted-screen test (0% failures, n = 24) and in the seizure test (91.7% clonic seizures, n = 24).

Effects of Chlormethiazole against Expression and Development of Cocaine-Kindled Seizures. Successive treatments with 60 mg/kg cocaine on days 1 to 6 resulted in the rapid development of kindled seizures (Fig. 4). Specifically, there was a 3.4-fold increase in the percentage of mice exhibiting clonic seizures after the second injection with cocaine relative to that after the first injection (P < .05). This trend continued after successive treatment with cocaine. Clonic seizures occurred in nearly 90% of mice by day 6. 

View larger version (39K): [in this window] [in a new window]   Fig. 4.   Anticonvulsant and antiepileptogenic effects of chlormethiazole, clonazepam, and diazepam against cocaine-kindled seizures. A control group () was treated with vehicle (saline or 20% PEG) before cocaine (60 mg/kg) injections on days 1 to 6. The anticonvulsant effects of drugs were evaluated in mice treated with each drug (doses in the legend) before cocaine injection on days 1 to 5 (open symbols). The antiepileptogenic effects were evaluated in drug-treated groups that on day 6 received saline instead of the antiepileptic drug before cocaine (, ,  and shaded region). Chlormethiazole and its vehicle, saline, were administered 45 min before cocaine injection. Clonazepam, diazepam, and their vehicle, 20% PEG, were administered 30 min before cocaine. Symbols with error bars represent the percentage (±S.E.M.) of mice exhibiting clonic seizures within 30 min post cocaine injection in three (chlormethiazole) or two (clonazepam and diazepam) independent appraisals, each consisting of 10 to 12 mice at the beginning of the experiment. Cumulative lethality in the control groups ranged from 17 to 25% by day 6. Symbols without error bars represent the percentage of mice exhibiting clonic seizures after cocaine injection in groups treated with an antiepileptic drug (n = 12-18 mice/group). *P < .05, compared with same-day outcome in saline-treated group (Fisher's exact test).

Motor Toxicity and Acute Anticonvulsant Effects of Chlormethiazole in Cocaine-Kindled Mice. Given the ability to suppress the expression and development of cocaine-kindled seizures, behavioral and protective effects of chlormethiazole in cocaine-kindled seizures were evaluated. As with the experimentally naive mice (Fig. 2), administration of chlormethiazole in cocaine-kindled mice resulted in a dose-dependent impairment of motor coordination on the inverted-screen test and protection against cocaine-induced seizures (Fig. 5). Potencies of chlormethiazole to produce behavioral side effects (TD₅₀ = 83.8 mg/kg) and protection against cocaine-induced seizures (ED₅₀ = 22.3 mg/kg) were favorably separated (PI = 3.76) (Table 1). The degree of separation between the TD₅₀ and ED₅₀ values of chlormethiazole in cocaine-kindled mice was substantially lower relative to that in naive mice, 3.76 versus 22.3, respectively (Table 1). The reduction was due to a decreased TD₅₀ value (increased potency) and increased ED₅₀ value (decreased potency) of chlormethiazole in cocaine-kindled mice relative to naive mice (Table 1). This resulted in statistically significant changes of the relative potencies of chlormethiazole to produce behavioral side effects (relative potency of 0.54) and protection against cocaine-induced seizures (relative potency of 3.18) in cocaine-kindled mice relative to naive mice (Table 1). The slopes of the dose-effect functions of chlormethiazole to produce behavioral side effects and protection did not differ significantly (P > .05) in cocaine-kindled mice relative to naive mice (Table 1). Of note however is that the slope of chlormethiazole's effect on the inverted screen test was 2.44-fold greater in cocaine-kindled seizures than in naive mice. In contrast, the slopes of the anticonvulsant dose-effect functions of chlormethiazole were comparable. Furthermore, like in naive mice, slopes (Table 1) of the dose-effect functions to produce behavioral toxicity and protect against cocaine-induced seizures (Fig. 5) were severalfold different.

View larger version (14K): [in this window] [in a new window]   Fig. 5.   Effect of chlormethiazole on the inverted-screen test () and against 60 mg/kg cocaine-induced seizures () in cocaine-kindled mice. Mice were kindled with cocaine on days 1 to 4. On day 5, mice (n = 8-10) pretreated with chlormethiazole or saline were tested on the inverted-screen test and then immediately observed for the occurrence of clonic seizures after 60 mg/kg cocaine. Cocaine-kindled mice correctly performed the inverted-screen test after saline administration (0% failures, n = 12), and 81.3% (n = 16) of mice developed clonic seizures after 60 mg/kg cocaine. Computed linear regressions (solid lines) were drawn for each dose-effect function. See Table 1 for statistical comparison of slopes of these regression lines and for TD50 value, ED50 value, and PI of chlormethiazole. Asterisks indicate statistically significant difference of the outcome of specific dose treatments from the control performance on the inverted-screen test and in the seizure test (*P < .05, Fisher's exact test).

Effects of Chlormethiazole against Cocaine-Induced Lethality. Chlormethiazole produced dose-dependent protection against lethality induced by 110 mg/kg cocaine (Fig. 6). The protective effect of chlormethiazole was dose-dependent with a full protection conferred by 170 mg/kg. The protective potency of chlormethiazole was favorably separated (PI = 7.15) from the potency to produce behavioral side effects (Table 2). Both clonazepam and diazepam, like chlormethiazole, dose dependently protected against cocaine-induced lethality. The PI values were close to unity (Table 2). Unlike chlormethiazole, however, both clonazepam and diazepam failed to fully protect against cocaine-induced lethality. In fact, the highest doses of clonazepam (3.0 mg/kg) and diazepam (10 mg/kg) in combination with 110 mg/kg cocaine produced behavioral toxicity expressed as repetitive clonic jerks, severe sedation, and prolonged loss of the righting response. Slopes of the dose-effect functions (Fig. 6) of chlormethiazole (slope, 54.2; 95% CL, 30.2-78.2), clonazepam (slope, 41.0; 95% CL, 65.3-147.2), and diazepam (slope, 42.7; 95% CL, 17.1-102.5) did not differ significantly (P > .05).

View larger version (15K): [in this window] [in a new window]   Fig. 6.   Effects of chlormethiazole, clonazepam, and diazepam against lethality induced by cocaine (110 mg/kg). Mice (n = 8-10) were treated with a drug (doses on abscissa) 30 or 45 min before cocaine challenge. Lethality was recorded 60 min after cocaine challenge. Cocaine produced lethality in 19 of 20 (95%) of the control animals pretreated with vehicle instead of drugs. Solid lines represent computed linear regression lines of the dose-effect functions. See Table 2 for protective ED50 values of the drugs. *P < .05, compared with control group (Fisher's exact test).

View this table: [in this window] [in a new window]   TABLE 2 Motor toxicity (TD50), protective potency (ED50), and PI of drugs against cocaine-induced lethality TD50 values of clonazepam and diazepam are from Witkin et al. (1999). The remaining values (with 95% CL in parentheses) were calculated from the dose-effect functions as shown in Figs. 2 (TD50 value of chlormethiazole) and 6 (all ED50 values). ED50 and TD50 values represent a dose of a drug (mg/kg) predicted to produce an effect in 50% of the mice tested. PI values represent a ratio of the TD50 and ED50 values of a drug.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

The present study provides the first experimental evidence for the effectiveness of chlormethiazole against the toxic effects of cocaine. Chlormethiazole was effective against acute and kindled seizures induced by cocaine and against cocaine-induced lethality. Moreover, chlormethiazole attenuated the development of sensitization to the convulsive effects of cocaine, thus showing antiepileptogenic properties against cocaine-kindled seizures. The results of this study predict the potential utility of chlormethiazole for the treatment of life-threatening complications of cocaine use and abuse such as seizures and lethality for which no specific treatment has yet been identified. This prediction is further supported by the fact that chlormethiazole is a drug of choice in the emergency treatment of the ethanol withdrawal syndrome (Morgan, 1995) and that overlapping molecular substrates and neuronal pathways are associated with effects of cocaine and ethanol (Koob and Weiss, 1992; Ritz et al., 1992).

In the present study, chlormethiazole was fully efficacious against acute cocaine-induced seizures and showed an exceptionally favorable therapeutic index. In marked contrast to chlormethiazole, a number of classic (Witkin et al., 1999) and new (Gasior et al., 1999) antiepileptic drugs (e.g., phenytoin, carbamazepine, phenobarbital, clobazam, lamotrigine, topiramate, zonisamide) were without efficacy under the same experimental conditions. Furthermore, even in comparison to drugs demonstrating efficacy, chlormethiazole exhibited a higher PI (22) than classic (0.13-1.2) and new (1.3-7.7) antiepileptic drugs against cocaine-induced seizures (Gasior et al., 1999; Witkin et al., 1999). A high PI is predictive of a broad therapeutic window in patients and encourages clinical testing (Löscher and Nolting, 1991).

Although seizures often precede lethality, there is evidence to suggest cocaine-induced lethality is not always casually linked to seizures (cf. Witkin et al., 1989, 1993). The present experiments add to the list of drugs effective against both seizures and lethality induced by cocaine. Chlormethiazole, diazepam, and clonazepam each afforded a dose-dependent protection against seizures and lethality (present study; Derlet and Albertson, 1989b; Witkin et al., 1999). Parallel slopes of the dose-effect functions suggest the involvement of similar protective mechanisms. Of important note however is the fact that chlormethiazole produced protection at doses well below its TD₅₀ value (PI = 7.2), whereas protective potencies of diazepam and clonazepam were close to their TD₅₀ values (PI  1). Relative to anticonvulsant potencies, chlormethiazole was 3.1-fold less potent against cocaine-induced lethality, whereas diazepam and clonazepam were 8.8- and 1.4-fold more potent, respectively (Tables 1 and 2; Witkin et al., 1999). These differences provide additional support for the distinction between the convulsant and lethal effects of cocaine under acute conditions as well as to further differentiate the actions of chlormethiazole and benzodiazepine anticonvulsants.

The term "kindling" historically refers to a phenomenon where repeated exposure to an initially subconvulsant electrical stimulus results in intense and persistent limbic seizures. This term was later adopted to describe the development of sensitization to the convulsive effects of other stimuli, including cocaine (so called "pharmacological kindling") (Post and Rose, 1976). In the present study, both chlormethiazole and clonazepam suppressed the behavioral expression of cocaine-kindled seizures. Chlormethiazole demonstrated this anticonvulsant effect at doses below its TD₅₀ value. Clonazepam was effective at doses comparable with its TD₅₀ value. Diazepam was ineffective even when administered in severalfold higher doses than its TD₅₀ value.

It is largely accepted that the anticonvulsant efficacy of a drug does not necessarily reflect its antiepileptogenic efficacy in epilepsy models with electrical kindling of the central nervous system (Löscher and Schmidt, 1988; Silver et al., 1991). The present study is the first experimental demonstration of the dissociation between the chronic anticonvulsant and antiepileptogenic effects of drugs against cocaine-kindled seizures. Although both chlormethiazole and clonazepam produced quantitatively comparable suppression of clonic seizures during kindling acquisition, the development of sensitization to the convulsive effect of cocaine was attenuated by chlormethiazole but not clonazepam. Thus, the expression and development of cocaine-kindled seizures are two independent processes. Even the antiepileptogenic effectiveness of a drug in one model of kindled seizures may not generalize to cocaine kindling. For example, diazepam attenuated the development of amygdala- and pentylenetetrazole-kindled seizures (Löscher and Schmidt, 1988; Gasior et al., 2000) but was ineffective against cocaine-kindled seizures. Blockade of kindling development by chlormethiazole may have important clinical implications because the kindling phenomenon has been linked to the development of durable epileptogenic changes, increased seizure propensity (Dhuna et al., 1991), and an escalation of psychiatric symptoms in cocaine users (Davis, 1996).

Several pieces of evidence suggest that the blockade of the development of cocaine kindling by chlormethiazole was not due to existing brain levels of chlormethiazole or metabolites at the time of testing with cocaine alone 24 h after the last chlormethiazole injection. Chlormethiazole has a rapid onset and a short duration of action with no intermediate metabolites in clinical practice (Smith and Jewkes, 1995). In rats, there was no accumulation of chlormethiazole in plasma after daily treatment for 1 month in 100- and 175-mg/kg doses (Kalant et al., 1986); instead, there was a tendency for lower plasma levels of chlormethiazole after chronic treatment. In the present study, behavioral effects and protective efficacy were gone by 3 h post administration. Moreover, the convulsive threshold of cocaine was unchanged 24 h after repeated treatment with 100 mg/kg chlormethiazole.

Chlormethiazole was also effective against fully developed cocaine-kindled seizures. Relative to nonkindled mice, chlormethiazole was equieffective in attenuating seizures. However, there was a smaller separation between anticonvulsant and toxic effects in cocaine-kindled mice (PI = 3.76) relative to naive mice (PI = 22.3). The clinical implication of this finding is that chlormethiazole might show a smaller therapeutic window in cocaine abusers with a history of frequent and severe convulsive episodes. Increased sensitivity to the adverse effects of anticonvulsant drugs has been reported in amygdala-kindled rats (Honack and Löscher, 1995).

The positive allosteric modulation of the GABA_A RC is now a well-documented mechanism of action of chlormethiazole (Smith and Jewkes, 1995). The GABA_A RC is traditionally linked to experimental seizures, human epilepsy, and the anticonvulsant effects of drugs (Rogawski and Porter, 1990; Bradford, 1995). There is also evidence for the involvement of the GABA_A RC in the effects of repeatedly administered cocaine. Under these conditions receptor numbers are down-regulated in a region-specific manner (Goeders, 1991; Pecins-Thompson and Peris, 1993; Peris, 1996). A direct inhibitory effect of high doses of cocaine on the GABA_A receptor-mediated Cl current in hippocampal neurons has been demonstrated (Ye et al., 1997). Enhanced dopaminergic neurotransmission as with cocaine administration also can decrease the release of endogenous GABA (Melis and Gale, 1983; Lindefors, 1993). On the other hand, activation of the GABA_A RC has been shown to decrease baseline and cocaine-induced release of dopamine in the striatum and nucleus accumbens (Dewey et al., 1998; Morgan and Dewey, 1998). Accordingly, chlormethiazole was shown to decrease methamphetamine-induced dopamine release (Green and Cross, 1994; Green, 1998).

The differential effects of chlormethiazole and other GABAergic agents uncovered herein suggest that only specific molecular modifications of the GABA_A RC are relevant to this toxicity. The favorable anticonvulsant/antiepileptogenic profile of chlormethiazole might be attributed to its specific action on the GABA_A RC. First, chlormethiazole is thought to interact with the GABA_A RC at a site distinct from those ascribed to the benzodiazepines and barbiturates (Smith and Jewkes, 1995). Neuroactive steroids that also modulate the GABA_A RC in a manner distinct from that of benzodiazepines and barbiturates were recently reported also to be efficacious against acute convulsant effects of cocaine (Gasior et al., 1997). Second, chlormethiazole increases the duration of open state of the GABA_A Cl channel, whereas benzodiazepines increase the frequency of channel openings (Hales and Lambert, 1992; Macdonald and Olsen, 1994). Chlormethiazole also may affect glutamatergic excitatory mechanisms. Although chlormethiazole has no direct effect on the N-methyl-D-aspartate RC, it protects against N-methyl-D-aspartate-induced seizures (Green and Cross, 1994), whereas diazepam and phenobarbital show limited efficacy (Gasior et al., 1997). A prominent role of the N-methyl-D-aspartate RC in acute (Witkin et al., 1999) and kindled (Itzhak and Stein, 1992) seizures induced by cocaine has been documented. Finally, a GABA-dopamine interaction has been implicated as a molecular mechanism of the protective effect of chlormethiazole (Green and Cross, 1994; Green, 1998).

In conclusion, the results of the present study encourage clinical testing of chlormethiazole against the toxic effects of cocaine. The fact that chlormethiazole has already been approved for human use would make a clinical trial easier to launch than with compounds that are in early stages of preclinical development. Based on its mode of action in psychiatric patients (Smith and Jewkes, 1995), chlormethiazole also might be expected to offset cocaine-related psychiatric complications such as violent behaviors, hyperactivity, paranoid psychosis, anxiety, and panic attacks. The differentiation of chlormethiazole from other GABAergic compounds also provides potential clues for rational medication development as well as to the underlying neurological changes associated with the toxic effects of cocaine, especially those resulting from repeated exposure. There is need for compounds to treat a variety of symptoms in the progression of cocaine dependence therapy (cf. Witkin, 1994). Chlormethiazole has demonstrated efficacy for ethanol dependence treatment, symptoms that overlap those of cocaine-dependent individuals (Taylor and Slaby, 1992). Moreover, frequent concurrent use of cocaine and ethanol is associated with heightened morbidity and mortality risk (Taylor and Slaby, 1992; McCance-Katz et al., 1998). For these reasons, chlormethiazole would seem an ideal drug candidate for some phases of cocaine dependence and/or toxicity treatment. This suggestion is given added emphasis because chlormethiazole already has a long history of clinical use.