Tolerance and physical dependence to a short-acting benzodiazepine, midazolam.

Midazolam is a new ultra short-acting benzodiazepine whose physical dependence properties have not been well characterized. Our laboratory has demonstrated previously that physical dependence to the long-acting chlordiazepoxide in the rat is inducible by a single intoxicating dose, whereas maximal dependence required chronically equivalent maximally tolerable dosing b.i.d. for 5 weeks. Based on the methods developed in our laboratory to quantify benzodiazepine intoxication and withdrawal, Trs were designed to evaluate midazolam's capacity to induce dependence in the rat after definable acute (120 mg/kg p.o.), sub-acute (120 mg/kg q.i.d. x 3 days) and chronic (120-180 mg/kg bid. x 5 weeks) dosing that was near maximally tolerable. A single dose of midazolam failed to produce withdrawal signs. Tolerance and dependence increased as a function of midazolam dose and duration of Tr.

Pharmacologic characterization of acute chlordiazepoxide dependence in the rat.

A single intoxicating dose of chlordiazepoxide HCl (p.o.) in the rat can induce quantifiable manifestations of physical dependence. Dependence was revealed by antagonist precipitation (Ro 15-1788, CGS-8216) as well as spontaneous emergence of neurobehavioral signs of withdrawal observed by multiple raters blind to treatments. Ro 15-1788 was 45% more effective than CGS-8216 in both reversing chlordiazepoxide intoxication and expressing withdrawal signs. The severity of Ro 15-1788-precipitated withdrawal varied with chlordiazepoxide dose, Ro 15-1788 dose and the agonist-antagonist dose interval. Maximal precipitated dependence was evoked 3 days after chlordiazepoxide HCl (450 mg/kg) by Ro 15-1788 (25 mg/kg i.p.). The precipitated syndrome consisted of tail erection, reduced motor activity, high step, curled claw, arched back, muscle hypertonus and piloerection. Ro 15-1788-precipitated dependence emerged between 28 and 52 hr, peaked at 76 hr and disappeared by 124 hr. Spontaneous withdrawal had emerged from 100 to 124 hr and then faded gradually. The neurobehavioral expression of central nervous system depression and its reversal were necessary but not sufficient conditions for the induction and expression of acute chlordiazepoxide dependence. These results suggest caution in reviving acute benzodiazepine-overdosed patients to avoid iatrogenic withdrawal analogous to naloxone for opiates.

Benzodiazepine tolerance, physical dependence and withdrawal: electrophysiological study of spinal reflex function.

The in situ segmental spinal reflex system of the rat was used to determine changes in excitatory and inhibitory synaptic function associated with benzodiazepine tolerance, physical dependence and withdrawal. Rats were made physically dependent on chlordiazepoxide using a chronically equivalent dosing method. After spinalization, dorsal and ventral lumbar roots (L5 or L6) were isolated for extracellular stimulation and recording. Testing of spinal function was performed during peak withdrawal (8 days) and at peak effect (4 hr) after first ("acute") and last ("chronic") dose of chronically equivalent chlordiazepoxide. There were no quantitative or qualitative differences in the acute and chronic spinal actions of chlordiazepoxide. Polysynaptic discharges were markedly augmented during withdrawal (159% above control) and diminished during treatment (44% below control). Recovery of the 2 N reflex measured by twin pulse was shortened during withdrawal (23% above control) and lengthened during treatment (28% below control). Recovery of the 2 N reflex analyzed by low-frequency (10 Hz) stimulation was also elevated at peak withdrawal (40% above control) and depressed in treatment (41% below control). At peak withdrawal spinal inhibitions were reduced below control; presynaptic-dorsal root reflex (60%) and recurrent (62%). In contrast, drug treatment enhanced presynaptic (72%) and recurrent (48%) inhibitions above control. Only those synaptic parameters chronically altered by continuous chlordiazepoxide administration were oppositely affected during withdrawal. Consequently, benzodiazepine withdrawal is associated with rebound alterations of profound reductions in inhibitory synaptic transmission, increased net polysynaptic activity and shortened monosynaptic recovery times.

Phenobarbital tolerance and physical dependence: chronically equivalent dosing model.

A rat model of phenobarbital tolerance and physical dependence has been developed based on the 'maximally tolerable, chronically equivalent' dosing paradigm. Sodium phenobarbital was administered orally twice daily for 35 days in individually adjusted doses to achieve mean daily peak CNS depression culminating in severe ataxia. Dose to dose continuity of CNS depression was maintained throughout treatment. At the time of dosing rats were mildly ataxic. Following abrupt termination of chronic treatment, an abstinence syndrome characterized by CNS hyperexcitability was observed, which demonstrates physical dependence. Signs, which included motor, autonomic, and behavioral manifestations, appeared from 12-24 h and animals recovered by 12 days. Although tolerance development was nearly complete on day 10 of treatment, the abstinence syndrome observed was more severe following 35 than after 10 days of chronic treatment. Characteristics of tolerance and physical dependence are similar to those described for other species including humans.

Experimental induction of benzodiazepine tolerance and physical dependence.

To initiate studies of benzodiazepine tolerance and physical dependence, a reproducible animal model has been developed utilizing chlordiazepoxide in rats. Based on the "chronically equivalent" dosing principle, a regimen has been devised to maintain rats in a state of quantifiable intoxication for 5 weeks. Chlordiazepoxide was delivered intragastrically on a b.i.d. basis in doses individually adjusted day-to-day and animal-to-animal to produce an equivalent impairment of motor function evaluated by a gross neurological screen. Quantitative analysis of central nervous system depression ratings during the time of peak effect (4 hr postdose) confirmed that the criterion of chronic equivalence was indeed met. Over the 5-week period of repeated dosing, tolerance was reflected in a 5-fold increase in maintenance dose, from 163.3 mg/kg on day 2 to 839.3 mg/kg on day 35. Tolerance developed more rapidly during the first 9 to 10 days, but continued to develop thereafter more slowly without apparent ceiling. Upon abrupt withdrawal, a syndrome of hyperexcitation developed. Signs included twitches, tremors, muscle hypertonus, arched back, piloerection, myoclonic jerks, augmented struggle and vocalization upon handling, increased startle response, tail erection, teeth chatter, blanched ears and weight loss. No spontaneous convulsions occurred. Latency to onset of withdrawal ranged from 2 to 5 days, and signs peaked in intensity in 8 days and disappeared by 14 days posttreatment. This animal model appears to provide a useful tool for the study of specific mechanisms underlying benzodiazepine tolerance and physical dependence.

Physical dependence to barbital compared to pentobarbital. II. Tolerance characteristics.

This study describes the tolerance characteristics of barbital compared to pentobarbital, the standard drug, during "chronically equivalent" treatment. Barbiturate tolerance was assessed as the increase in dose from the beginning to the end of treatment required to achieve equieffective peak effect. Dispositional tolerance was assessed as a reduction in the elimination half-life of barbiturate from blood. Functional tolerance was assessed as the increase in blood concentration of barbiturate at the time of peak effect. Overall, greater tolerance was developed to pentobarbital than to barbital. For pentobarbital, tolerance was both dispositional and functional; the dispositional tolerance developed rapidly and was almost complete at 1 week. For barbital, tolerance was exclusively functional. A most interesting finding was that functional tolerance to barbital and pentobarbital developed at the same slow rate for chronically equivalent treatment. This finding suggests that functional tolerance development is independent of the particular barbiturate reflecting the adaptability of the central nervous system to chronic depression.

Physical dependence to barbital compared to pentobarbital. III. Withdrawal characteristics.

After "chronically equivalent" barbital and pentobarbital dosing for 5 weeks, treatments were abruptly stopped and the animals were carefully observed for signs of barbiturate withdrawal. The severity of withdrawal was assessed at preset times by counting the number of grand mal type convulsions and subjectively rating 20 additional motor, autonomic and behavioral signs including tremors, twitches, myoclonic jerks, postural disturbances and motor incoordination. Ratings achieved at peak intensity (raw scores) and their incidences were used to compute "total intensity scores" for each graded sign. For all quantitative measures, withdrawal signs were less severe for barbital than for pentobarbital, with strikingly lower (P less than .05) incidences of convulsions (6.3% vs. 100%), bizarre (hallucinatory) behavior (6.3% vs. 41.3), and death (0% vs. 100%). The withdrawal signs for barbital appeared later, developed more slowly and persisted longer than those for pentobarbital. That the onset and then peak of withdrawal signs occurred when the extents of decline from peak blood concentration of barbital and pentobarbital were similiar suggests that the time course of withdrawal might be inversely related to residual concentrations of drug, i.e., negative dose-response.

Physical dependence to barbital compared to pentobarbital. IV. Influence of elimination kinetics.

The withdrawal characteristics of barbital and pentobarbital after "chronically equivalent" treatment suggested that the longer acting barbital was less liable to produce physical dependence. Therefore, to distinguish this potential pharmacodynamic difference from the known pharmacokinetic differences between the two drugs, the rate of elimination of each was adjusted to mimic that of the other. The rate of barbiturate elimination after chronically equivalent pentobarbital dosing was reduced by barbital substitution or by first-order pentobarbital dose reduction, with the result that withdrawal signs became mild and appeared later (3 days postdrug). The rate of barbiturate elimination after chronically equivalent barbital dosing was increased by pentobarbital substitution or by peritoneal dialysis of barbital, with the result that withdrawal signs became severe and appeared sooner (within 1 day). These findings conclusively support the key role of the rate of barbiturate elimination to expose underlying physical dependence to barbiturates. Furthermore, "physical dependence" and its expression in "withdrawal" must be regarded separately to evaluate and compare critically the dependence capability of different drugs.

Evaluation of anticonvulsants in barbiturate withdrawal.

Four prototypic anticonvulsants were tested for their effectiveness against barbiturate withdrawal in cats. The effects were evaluated on a total of over 20 motor, autonomic and behavioral withdrawal signs. The animals were made physically dependent by 5 weeks of twice daily "maximally tolerable" sodium pentobarbital dosing intragastrically. Anticonvulsants were administered by intravenous infusion 25 hours after the final dose of chronic pentobarbital treatment when all withdrawal signs had become severe and grand mal type withdrawal convulsions were observed. Phenobarbital blocked withdrawal signs quite effectively at doses that caused no significant acute central nervous system depression. Trimethadione also reversed most withdrawal signs, but some signs persisted even at doses causing overt acute toxicity. Dimethadione was less effective than the parent compound, trimethadione, in reversing withdrawal but caused greater acute toxicity. Phenytoin was in effective for most withdrawal signs and some signs were made worse. The clonic phase of withdrawal convulsions was accentuated and the overall condition of the animals worsened. During withdrawal, the animals were less sensitive (tolerant) to phenobarbital but were more sensitive to acute toxicity from the other drugs tested.

Withdrawal characteristics following chronic pentobarbital dosing.

Experiments were performed to provide a quantitative description of the barbiturate withdrawal syndrome. Physical dependence was produced in 63 cats by 'maximally tolerable' dosing with sodium pentobarbital. After 5 weeks of chronic treatment each animal was placed in an activity monitoring cage and observed closely for signs of barbiturate abstinence. Electroencephalographic monitoring of sleep--wake cycles was performed in 5 of these cats. Most withdrawal signs appeared in 12-18 h and rapidly intensified. These included signs of neural hyper-excitability that involved motor, autoncidence, severity and time course of many withdrawal signs. 26 animals (41%) died during abstinence, usually during or immediately following grand mal type convulsions. The importance of quantitating withdrawal phenomena is discussed with respect to investigation of the requirements for physical dependency production, comparison of different drug dependencies, and pre-clinical evaluation of potential treatments of sedative-hypnotic dependence.

Effect of chronic pentobarbital treatment on blood-cerebrospinal fluid kinetics.

Cats were made tolerant and physically dependent by twice daily equieffective anesthetic dosing with sodium pentobarbital intragastrically for 5 weeks. Serial blood and CSF samples were simultaneously collected on day 1 and day 35 and analyzed for pentobarbital by a gas chromatographic method. Pentobarbital penetration into CSF was rapid and unchanged by chronic treatment. The concentration in blood and CSF increased significantly with chronic treatment; however, the relative CSF/blood concentration ratios were unchanged.

Tolerance characteristics produced during the maximally tolerable chronic pentobarbital dosing in the cat.

The method of "maximally tolerable" dosing technique for establishing a reproducible state of barbiturate dependence in cats was used for the study. The development of tolerance in animals treated by this method has been quantitatively assessed. Sodium pentobarbital was administered morning and evening for 35 days via a plastic tube implanted into the stomach through the abdominal wall. A range of neurological signs of intoxication was scored before and after each dose and during the day at certain preset intervals. Based on the scoring of neurological impairment, each cat was given the maximally tolerable anesthetic dose of sodium pentobarbital. All of the animals treated by this method exhibited severe withdrawal signs upon abrupt withdrawal of the drug. For each cat, blood pentobarbital concentrations were determined every day before and 1 to 11/4 hours after the morning dose. Also, a complete blood pentobarbital elimination study was made on days 1, 7, 14, 21, 28 and 35 of the regimen. These results distinguished between dispositional and functional tolerance. The dispositional tolerance developed maximally within a week and was maintained at that level as long as the treatment was continued. The functional tolerance, on the other hand, developed more gradually and progressed with continued treatment.