Substitution of temazepam and midazolam in pentobarbital-dependent rats

Physiology&Behavior,Vol. 46, pp. 55--60. ©Pergamon Press plc, 1989. Printed in the U.S.A.

0031-9384/89 $3.00 + .00

Substitution of Temazepam and Midazolam in Pentobarbital-Dependent Rats G . J. Y U T R Z E N K A , .1 G. A. P A T R I C K A N D W . R O S E N B E R G E R

*Department of Physiology and Pharmacology, University of South Dakota School of Medicine, Vermillion, SD and Department of Pharmacology and Toxicology, Medical College of Virginia Virginia Commonwealth University, Richmond, VA

YUTRZENKA, G. J., G. A. PATRICK AND W. ROSENBERGER. Substitution of temazepam and midazolara in pentobarbitaldependent rats. PHYSIOL BEHAV 46(1) 55-60, 1989.--The ability of temazepam and midazolam to substitute for pentobarbital and thus maintain the physical dependence state was used to assess the potential dependence liability of these two benzodiazepine compounds. Male Sprague-Dawley rats, weighing 175-200 g and having ad lib access to food and water, were determined to be dependent on pentobarbital following 12 days of continuous, intraperitoneal infusion of pentobarbital using an escalating drug infusion schedule. On day 13 (substitution phase) the pentobarbital was replaced with either temazepam, midazolam or vehicle and the rats were infused for an additional 24 hours. This was followed on Day 14 (withdrawal phase) by a 24 hr saline infusion period. Rats were observed for changes in overt behavior and alterations of body weight during both Day 13 and Day 14. Preliminary potency estimation studies had indicated that both drugs were more potent and longer acting than was pentobarbital. Temazepam, in doses of 32.5, 65 and 130 mg/kg/24 hr, was demonstrated to substitute for pentobarbital and provided dose-dependent suppression of overt behavioral signs indicative of withdrawal. Temazepam also suppressed the weight loss typically observed during withdrawal. Substitution of saline for temazepam resulted in an increased incidence of withdrawal signs and an approximate 10% decline in body weight. Midazolam, in doses of 60 and 120 mg/kg/24 hr, also substituted for pentobarbital and suppressed both overt behavior and weight loss. Following saline substitution on Day 14, a mild withdrawal syndrome was evident although body weight was noted to remain near control values. The data suggests that both temazepam and midazolam may be substituted for pentobarbital in dependent rats and, therefore, there is an indication that these drugs may possess a liability for the production of physical dependence. Substitution

Physical dependence

Benzodiazepines

Rats

THE benzodiazepines have proven to be of considerable value in the treatment of a variety of disorders including anxiety, insomnia, and muscle spasm. Along with their widespread use there has arisen concerns regarding the liability of these compounds towards the establishment of physical and/or psychological dependence. There is a growing sense of need to screen compounds for their abuse liability. One method by which to assess the dependence potential of a compound is by use of a cross-dependence paradigm (5,12). The usefulness of this method was first demonstrated as a result of studies utilizing a variety of opiate compounds (4). The crossdependence paradigm has subsequently been extended for use with sedative-hypnotic agents (10, 13, 14). The basic premise behind the cross dependence phenomena is that, if a drug can produce dependence and a second drug can be substituted for it and prevent or reverse the withdrawal syndrome associated with the first drug, then it is likely that the second drug will produce a dependence which is similar to that demonstrated for the first drug (4,12). A number of studies have provided evidence which establishes that cross-dependence can occur between barbiturates and benzo-

Temazepam

Midazolam

diazepines. Early investigators had described the ability of various benzodiazepines to ameliorate or reverse the withdrawal syndrome in barbital dependent dogs (3) and phenobarbital dependent mice (1) and rats (10). It has been determined that the reversal of the barbiturate withdrawal syndrome is not due simply to a nonspecific, sedative effect since compounds such as reserpine, clonidine, chlorpromazine and benzoctamine, while providing some sedation, were ineffective in suppressing the barbiturate withdrawal syndrome (10,13). It was also demonstrated, however, that not all barbiturates or benzodiazepines are equally effective in ameliorating all signs associated with the withdrawal syndrome (8,14). The current study was intended to further investigate the cross-dependence phenomena between benzodiazepines and barbiturates. The experiments investigated the ability of two short acting barbiturates, temazepam and midazolam to substitute for pentobarbital in dependent rats. These studies utilized the continuous infusion method for producing physical dependence on pentobarbital as well as for administering the substituted compounds.

1Requests for reprints should be addressed to G. J. Yutrzenka, Department of Physiology and Pharmacology, University of South Dakota School of Medicine, 414 East Clark Street, Vermillion, SD 57069.

55

56

YUTRZENKA, PATRICK AND ROSENBERGER

TABLE 1 RATING SCALE FOR ASSESSMENT OF WITHDRAWAL SIGNS

initially weighing 175-200 g were individually housed in stainless steel cages with food and water available ad lib. Male CF-1 mice (Dominion Labs, Dublin, VA) weighing 25-30 g were housed in plastic cages with food and water ad lib. All animals were acclimated to the animal facility for several days prior to use in any study. Rats were surgically prepared with an intraperitoneal cannula (PE 90) while under methoxyflurane anesthesia (15). All rats were allowed several days to recover from surgery prior to being placed into an infusion harness. Acclimation to the infusion system occurred for 3 days during which the rats were continuously infused with 0.9% saline. This was followed by the continuous infusion of either saline (control) or pentobarbital sodium for 12 consecutive days using an escalating drug dosage schedule (15). At the end of the infusion period rats were receiving pentobarbital at a dose of 950 mg/kg/24 hour. Body weight and water consumption were monitored daily during the drug infusion period. Following the final day of pentobarbital infusion a 24 hour substitution period commenced during which pentobarbital dependent rats were infused with either saline, vehicle, temazepam or midazolam. This was followed by a 24 hour drug withdrawal period during which all rats received saline. Every 2 hours for the first 12 hours and again, at 24 hours of each period, rats were assigned a withdrawal score based on the degree of expression of several behavioral responses (Table 1).

A) Response to air puff 0-- No response 1--Jumps (does not include flinches) 2--Jumps and vocalizes B) Response to prodding of flank 0--No response 1--Vocalizes C) Presence of "high" posture 0--Not present 1 --Present D)

Response to being grasped and held 0--No response 1--Struggles o r vocalizes 2--Struggles and vocalizes 3--Struggles, vocalizes, scratches and/or bites

METHOD Male Sprague-Dawley rats (Dominion Labs, Dublin, VA)

Day 1-12 Day 13

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Time(hr) FIG. 1. Mean withdrawal scores of control rats and pentobarbital-dependent rats during (A) temazepam or vehicle substitution (Day 13) or (B) saline substitution (Day 14). Temazepam was infused in doses of 32.5 mg/kg/24 hr or 65 mg/kg/24 hr. Each point represents the mean withdrawal score of 3 to 6 rats.

BENZODIAZEPINE SUBSTITUTION IN DEPENDENT RATS

57

WITHDRAWAL SCORE UPON SUBSTITUTION OF TEMAZEPAM--WITHDRAWAL PHASE

A

P

Day 1-12 Day 13

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Time (hr) FIG. 2. Mean withdrawal score of control rats and pentobarbital-dependentrats during (A) temazepam or vehicle substitution (Day 13) or (B) during saline substitution (Day 14). Temazepam was infused in a dose of 130 mg/kg/24 hr. Each point represents the mean withdrawal score of 3 to 6 rats. Scores were assigned by two observers who were blind to the drug treatment. In addition, body weight was determined at 0, 8 and 24 hours of each period. Investigators were blind to the identity of the compounds until all data was collected and analyzed. Preliminary studies to ascertain potency of these two benzodiazepine compounds, relative to pentobarbital, were conducted in mice. Drug-treated mice were assayed using the inverted screen test (2) and alteration of spontaneous locomotor activity. Three doses of each drug, with at least six mice per dose, were used to determine dose-response curves. Vehicle-treated mice served as controls and were assayed concurrently with drug-treated mice. The inverted screen test was conducted at 15, 30, 60 and 120 minutes following drug administration. The EDso dose, which was determined to be the dose at which one-half of the treated mice failed to right themselves within the 60 second time period, was computed for each time period. Spontaneous locomotor activity was determined using a single beam photocell which bisected a plastic cage containing 2 mice. Movement of the mice disrupted the beam and a "count" of activity was recorded. Following drug administration, activity was recorded at the following time intervals; 5-15 minutes, 35-50 minutes, 65-95 minutes, and 125-185 minutes. The EDso dose was determined to be that dose which reduced spontaneous locomotor activity to one-half that recorded for concurrently tested vehicle-treated control mice. Potency ratios of temazepam and midazolam, relative to pentobarbital, were determined at time of peak activity and when, in addition, the vehicle effect was no longer evident. Pentobarbital sodium was dissolved in 0.9% saline and mida-

zolam was dissolved in distilled water. Temazepam was dissolved in a vehicle consisting of distilled water:Polysorbate 80:dimethyl sulfoxide (60:20:20) at pH 2.1. Eight ml of each solution was infused over a 24 hour period. For this study, the benzodiazepines were provided in coded vials by the Committee on Problems of Drug Dependence (CPDD). Investigators were blind to the identity of the compounds until after the completion of the study. Withdrawal scores for each treatment group were compared to the control by use of the Mann-Whitney U-test (6). Alteration in body weight was tested for significance by use of t-test (6). EDso values and 95% confidence intervals were also determined (7). RESULTS

The continuous infusion of pentobarbital for twelve consecutive days resulted in the establishment of physical dependence. This was evidenced, upon substitution of saline for pentobarbital, by the observation of behavioral and physiological signs indicative of withdrawal from barbiturates (Figs. 1A and 4A). The behavioral signs tended to increase in severity and reached peak intensity at about 12 hours following discontinuation of pentobarbital administration. This was followed by a decline in the intensity of the withdrawal signs over the next several hours. Likewise, the rats demonstrated a steady decrease in body weight with the maximum decline observed at about 24-32 hours following the cessation of pentobarbital administration (Fig. 4A). In initial studies temazepam, a short acting benzodiazepine, was found to be between 15 and 20 times more potent than

58

YUTRZENKA, PATRICK AND ROSENBERGER

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Time (hr)

FIG. 3. Mean withdrawal score of control rats and pentobarbital-dependentrats during (A) midazolam substitution(Day 13) or (B) during saline substitution(Day 14). Midazolam was infused in doses of 60 mg/kg/24 hr and 120 mg/kg/24 hr. Each point represents the mean withdrawal score of 3-6 rats.

pentobarbital (Table 2). Temazepam provided dose-dependent substitution for pentobarbital (Figs. 1A, 2A) with the highest dose providing essentially complete suppression of the withdrawal syndrome. Upon substitution of saline for temazepam on Day 14 there was a marked increase in the severity of the withdrawal signs exhibited by these rats (Figs. 1B, 2B). There was also evidence that the vehicle may have provided some suppression of withdrawal signs as noted by a somewhat elevated withdrawal score in the vehicle-treated rats following substitution of saline on Day 14. Midazolam, another short acting benzodiazepine, was shown to be approximately 7-10 times more potent than pentobarbital (Table 2). When substituted into dependent rats on Day 13, midazolam suppressed the withdrawal syndrome (Fig. 3A). Upon substitution of saline on Day 14 there was an increase in severity of withdrawal signs in rats receiving the lower dose of midazolam (Fig. 3B). However, the higher dose of midazolam continued to suppress the withdrawal signs. It was also noted that the degree of intensity of withdrawal signs in this series of experiments was relatively low and thus interpretation of the results of the midazoiam administration must be approached with caution. Alteration of body weight of the rats during the substitution (Day 13) and withdrawal (Day 14) periods also adds to the general impression about the effectiveness of these benzodiazepines in suppressing withdrawal signs in pentobarbital-dependentrats (Fig. 4). It was noted that dependent rats infused with vehicle show a marked reduction in body weight which is most evident by 24-32

hours after the last dose of pentobarbital. The administration of either temazepam or midazolam tended to suppress the typical loss of body weight. DISCUSSION

These experiments serve to show the utility of the continuous infusion method for both the establishment of physical dependence on pentobarbital as well as for the administration of compounds during cross dependence studies. The advantages of the method include: a constant delivery of drug to the subject which, presumably, allows for a more consistent interaction of the drug with the site of action; elimination of the need for multiple drug dosing each day; and ease of adjustment of doses of drug as dictated by the behavioral and physiological signs from individual subjects. Both midazolam and temazepam were demonstrated to be able to substitute for pentobarbital in dependent rats. Both benzodiazepines provided dose-dependent suppression of behavioral signs of withdrawal. In addition, both compounds also prevented the loss of body weight which is characteristic for rats undergoing withdrawal from pentobarbital. A complicating feature of the model described above was the need to determine doses which may be adequate to substitute for pentobarbital. As was clearly shown for temazepam, there is a necessity to administer adequate doses of the drug being substituted in order to show effective suppression of typical withdrawal

BENZODIAZEPINE SUBSTITUTION IN DEPENDENT RATS

59

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Time (hr) FIG. 4. Percent change in body weight of control rats and pentobarbital-dependent rats during both the drug substitution period (Day 13) and subsequent saline substitution period (Day 14). (A) Pentobarbital-dependent rats infused with either vehicle, or temazepam in doses of 32.5 mg/kg/24 hr or 65 mg/kg/24 hr. (B) Pentobarbital-dependent rats infused with temazepam at 130 mg/kg/24 hr. (C) Pentobarbital-dependent rats infused with midazolan at 60 mg/kg/24 hr or 120 mg/kg/24 hr. Each point is the mean percent change in body weight, as compared to time 0, for 3 to 6 rats.

signs. The use of the inverted screen test and the alteration of spontaneous locomotor activity have proven to be of some value in estimating potency of substituted compounds relative to pentobarbital. The need to use vehicles other than distilled water or saline

may further complicate estimation of potency and all potency estimation were calculated at a time of peak effect of the compound and when, in addition, the vehicle effect was no longer evident. In addition, a vehicle-treated control group was used to

60

YUTRZENKA, PATRICK AND ROSENBERGER

TABLE 2 ESTIMATION OF EDsoAND POTENCY RATIO OF TEMAZEPAMAND MIDAZOLAMRELATIVETO PENTOBARBITAL

Treatment

Time of Test After Treatment (Min) 30 60

15

Inverted Screen Test Pentobarbital EDso* Temazepam EDso PR~ Midazolam EDso PR

Treatment

17.8 (14.1-22.4)

24.5 (20.7-29.0)

0.79 (0.39-1.6) 22.5 and 31.0

1.43 (0.77-2.65) 12.4 and 17.1

2.09 (0.9--4.85) 8.5 and 11.7

---

2.6 (1.5-4.5) 6.8 and 9.4

7.3 (4.9-10.9) 2.4 and 3.4

correct for any activity intrinsic to the vehicle itself. Taken together, the data does support cross-dependence between pentobarbital and either temazepam or midazolam. The likelihood of cross dependence between barbiturates and benzodiazepines is further strengthened by recent findings that these two classes of CNS depressants may be acting through a common pathway involving regulation of GABAergic modulation of chloride ion flux in the neuronal membrane (9,11).

Time of Test After Treatment (Min) 5-15 35-50 Alteration of Locomotor Activity

Pentobarbital EDs0* Temazepam EDso PR:~ Midazolarn ED5o PR

24.7 (20.6-29.7)

--

1.34 (0.5-3.4) 18.4

---

1.38 (0.18-10.4) 17.9

3.49 (1.56-7.8) 7.1

*Indicates EDso values in mg/kg and 95% confidence intervals. tPR = potency ratio relative to pentobarbital at 15 and 30 minutes. SPotency ratio relative to pentobarbital at 5-15 minutes.

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tions. J. Neurochem. 37:1-13; 1981. 10. Tagashira, E.; Urano, T.; Hiramori, T.; Yanaura, S. Cross-physical dependence liability of psychotropic drugs in rats dependent on barbiturates. Jpn. J. Pharmacol. 33:659-669; 1983. 11. Ticku, M. K. Interaction of depressant, convulsant and anticonvulsant barbiturates with the (3H)-diazepam binding site of the benzodiazepine-GABA receptor-ionophore complex. Biochem. Pharmacol. 30: 1573-1579; 1981. 12. Woods, J. H.; Katz, J. L.; Winger, G. Abuse liability of benzodiazepines. Pharmacol. Rev. 39(4):251-413; 1987. 13. Yanagita, T.; Takahashi, S. Dependence liability of several sedative hypnotic agents evaluated in monkeys. J. Pharmacol. Exp. Ther. 185:303-316; 1973. 14. Yanagita, T. Dependence producing effects of anxiolytics: psychotropic agents. In: Hoffmeister, F., ed. Handbook of experimental pharmacology, vol. 55 part 2. New York: Springer-Verlag; 1981: 395--406. 15. Yutrzenka, G. J.; Patrick, G. A.; Rosenberger, W. Continuous intraperitoneal infusion of pentobarbital: A model of barbiturate dependence in the rat. J. Pharmacol. Exp. Ther. 232:111-118; 1985.