Proc. Natl. Acad. Sci. USA Vol. 94, pp. 2719–2723, March 1997 Pharmacology Alprazolam dependence prevented by substituting with the b-carboline abecarnil (benzodiazepinesywithdrawalytoleranceyreplacement therapy) GRAZIANO PINNA*, RUGGERO GALICI*, HERBERT H. SCHNEIDER,DAVID N. STEPHENS†, AND LECHOSLAW TURSKI‡ Research Laboratories of Schering AG, Mu¨llerstrasse 178, D-13342 Berlin, Germany Communicated by Jan Bures, Czech Academy of Sciences, Prague, Czech Republic, December 26, 1996 (received for review December 2, 1996) ABSTRACT Abrupt termination of the treatment of hu- although this view is not adequately supported by controlled mans with benzodiazepines (BDZs) leads to a rapid onset of human data (5). There are also no adequately controlled discontinuation syndrome characterized by anxiety, muscle animal experimental data that allow comparison of the risk of spasms, and occasionally convulsions. For this reason, it is producing dependence between BDZs with short and long recommended in clinical practice to reduce the dose of the half-lives (7). Similarly, there is scanty experimental data BDZs gradually at the end of treatment. Nevertheless, many demonstrating how to discontinue safely treatment with BDZs clinicians report signs of dependence even during gradual with short half-lives. reduction of doses (tapering) of the BDZs in a large propor- To address this issue we have employed newly developed tion of patients. Thus, there is considerable interest in dis- methods for electroencephalographic (EEG) monitoring of covering means of weaning patients away from BDZs without seizures, for electromyographic (EMG) monitoring of muscle the risk of discontinuation syndrome. In the present study, tone, and for detecting anxiety-like behavioral changes after mice withdrawn from chronic treatment with alprazolam discontinuation of long-term treatment with sedative drugs in showed anxiety, muscle rigidity, and seizures between days 1 mice (8) for controlled and standardized assessment of de- and 28 after termination of the treatment. Replacement of pendence liability of alprazolam. Using these experimental alprazolam with the b-carboline abecarnil for 7 days pre- approaches, we describe the time course of the discontinuation vented the occurrence of the signs of dependence. In contrast, syndrome after termination of long-term treatment with al- substitution of the b-carboline antagonist ethyl-5-isopropoxy- prazolam in mice, and propose a therapeutic strategy for 4-methyl-b-carboline-3-carboxylate (ZK93426) for alprazo- preventing signs of withdrawal using substitution with the lam worsened the discontinuation syndrome. Replacement b-carboline abecarnil. therapy with abecarnil after long-term treatment with the BDZs offers a novel method for rapid tapering. MATERIALS AND METHODS Animals. Partial agonists at benzodiazepine (BDZ) receptors and com- Male NMRI mice (Schering), 20–24 g in weight, pounds selective for subtypes of BDZ receptors possess ad- were subjected to s.c. injections of 6 mgykg of alprazolam (8-chloro-1-methyl-6-phenyl-4H-[1,2,4]triazolo[4,3-a][1,4]- vantages over full agonists in that, following long-term treat- benzodiazepine; Upjohn) or vehicle (sesame oil) given twice ment, dependence is less (1, 2). The question arises whether daily (9:00 a.m. and 9:00 p.m.) for 12 consecutive days. such drugs may be useful in preventing the emergence of Alprazolam was dissolved in sesame oil and administered in a dependence on BDZs following long-term treatment, and volume of 0.04 ml 10 g body weight. Such treatment led to full whether subsequent termination of such treatment results in a y development of tolerance to the sedative action of alprazolam discontinuation syndrome. Therefore, the purpose of the (see Fig. 1). Monitoring of withdrawal signs started on the day present study was to examine the time course of dependence following the last administration of alprazolam or vehicle. This following termination of long-term treatment with the short day was designated withdrawal day 1 (W1). After withdrawal half-life BDZ alprazolam, and to study the ability of the all experimental animals received daily injections of vehicle. b-carboline BDZ receptor agonist abecarnil subtype selective Monitoring of Withdrawal Syndromes in Mice. Electroen- (3, 4) to prevent discontinuation syndrome in mice dependent cephalography. For long-term EEG monitoring [Grass poly- on alprazolam. graph (Grass Instruments, Quincy, MA); time constant, 0.03 s; Alprazolam, a BDZ with a short half-life, was chosen high cut-off filter, 15 Hz], mice were stereotaxically implanted because it recently has replaced diazepam as the most fre- with bipolar twisted electrodes (tip diameter, 100 mm; inter- quently prescribed anxiolytic drug (5). This shift in the pattern electrode distance, 500 mm) positioned in the dorsal hip- of drug prescription was justified by the low likelihood of pocampus (AP 2.5; L 2.0; V 3.5) (9) under sodium pentobar- alprazolam accumulation (compared with diazepam) and bital anesthesia [Nembutal; Ceva (Neuilly-sur-Seine, France), causing sedative effects on multiple doses (5). Nevertheless, a 50 mgykg i.p.]. Surface recordings were led from screws major drawback of long-term treatment with alprazolam is that positioned bilaterally over the occipital cortex. The surgery sustained effects could be achieved only on multiple daily was performed 3–5 days before the first administration of dosing regimens and that discontinuation of such therapy was alprazolam or vehicle. EEG recordings started on W1 at 9:00 most likely to induce signs of dependence (6). Indeed, it is commonly held that BDZs with short half-lives have greater dependence liability than compounds with longer half-lives, Abbreviations: BDZ, benzodiazepine; ZK93426, ethyl-5-isopropoxy- 4-methyl-b-carboline-3-carboxylate; EEG, electroencephalogramy electroencephalographic; EMG, electromyogramyelectromyographic; The publication costs of this article were defrayed in part by page charge W, withdrawal day(s). payment. This article must therefore be hereby marked ‘‘advertisement’’ in *G.P. and R.G. were on leave from the Department of Experimental accordance with 18 U.S.C. §1734 solely to indicate this fact. Biology, University of Cagliari, Cagliari, Italy, and have contributed equally to this work. Copyright q 1997 by THE NATIONAL ACADEMY OF SCIENCES OF THE USA †Present address: Laboratory of Experimental Psychology, University 0027-8424y97y942719-5$2.00y0 of Sussex, Brighton, U.K. PNAS is available online at http:yywww.pnas.org. ‡To whom reprint requests should be addressed. 2719 Downloaded by guest on September 28, 2021 2720 Pharmacology: Pinna et al. Proc. Natl. Acad. Sci. USA 94 (1997) a.m. and were carried out in circular Perspex cages (diameter treatment. The day following the last administration of alpra- 40 cm, height 40 cm) located in a sound-insulated room. Mice zolam was designated replacement day 1. The day following were chronically treated with alprazolam (6 mgykg s.c. twice the last administration of substituting agents (abecarnil, daily) or vehicle for 12 days. The monitoring was continued for ZK93426, alprazolam, or vehicle) was designated W1. During up to 28 days after the last alprazolam or vehicle administra- withdrawal all experimental animals received daily injections tion and was discontinued for periods of 10–15 minyday of vehicle. The treatment regimen chosen for abecarnil and (between 8:30–9:00 a.m.) for daily care. Video and EEG ZK93426 was selected in such a way that the doses used were signals were stored on computer discs and magnetic tape. the highest not inducing sedation or performance disturbances Videotaping during the nocturnal phase was made using of mice on the rota-rod as measured daily during the entire infrared cameras. Seizure recognition was performed on-line period of chronic administration. using an extended Compaq computer system (Biomedical Statistics. The experimental data were analyzed statistically Monitoring Systems, Campbell, CA) equipped with a detec- by means of two-way ANOVA, repeated measures ANOVA, tion program (monitor 5.1; Stellate Systems; Montreal) (8). and multivariate ANOVA. Computer-identified seizure patterns were reanalyzed off-line by an observer unaware of drug treatment, and artifacts were discarded. The correct location of the implanted deep elec- RESULTS trodes was histologically controlled in cresyl violet-stained Tolerance to Alprazolam. Long-term treatment with alpra- serial sections of the entire brain 5–7 days after the last zolam led to a rapid loss of its depressant action on exploratory observation session. activity in nonhabituated mice measured as the number of Electromyography. The spontaneous activity in the EMG was interruptions of horizontal sensors (Fig. 1). recorded from the gastrocnemius muscle of the mice using Withdrawal. Mice withdrawn from long-term (12 days) pairs of Teflon-insulated stainless-steel wire electrodes treatment with alprazolam showed electrographic seizures, (Cooner Wire, Chatsworth, CA; AS 632 SS) inserted percu- increase in EMG activity, and a pattern of exploratory activity taneously into the muscle (10). The mice were placed sepa- reminiscent of anxiety (Fig. 2). In animals withdrawn from rately in ventilated Perspex boxes and their hindlimbs, gently long-term treatment with vehicle, no seizures were detected in secured with adhesive tape, were extended through slots in the EEG, no changes in muscle tone were registered in EMG, and bottom of the boxes. The electrical signals