Potentiation of Yohimbine-Induced Lethality in Mice: Predictor of Antidepressant Potential

Drug Development Research 3:357-363 (1983)

Jeffrey B. Malick

Biomedical Research Department, Stuart Pharmaceuticals, Division of ICI Americas, Inc., Wilmington, Delaware

ABSTRACT Malick, J.B.: Potentiation of yohimbine-induced lethality in mice: Predictor of antidepressant potential. Drug Dev. Res. 3:357-363, 1983.

The ability of antidepressant agents to potentiate the lethal or toxic effects of yohimbine in mice was evaluated. With very few exceptions, the antidepressants were the only agents that significantly enhanced yohimbine-induced lethality. All of the clinically effective antidepressants, both typical (e.g., amine reuptake inhibitors, monoamine oxidase inhibitors) and atypical (e.g., mianserin, iprindole, bupropion, quipazine) drugs, produced a dose- related potentiation of yohimbine in mice. Representative anxiolytics and antipsychotics failed to potentiate yohimbine over a wide range of doses. Although several possible “false positives” (e.g., atropine, chlorpheniramine, and d-amphetamine) would be detected in this procedure, these same agents would be detected in other models (e.g., tetrabenazine antagonism, behavioral despair) considered predictive of antidepressant potential. Thus, the potentiation of yohimbine-induced lethality test in mice appears to represent a useful screening procedure for discovering potential antidepressant drugs.

Key words: yohimbine, antidepressants, potentiation

INTRODUCTION Antidepressants potentiate the actions of several types of drugs such as amphetamine [Miller et al., 1970; Vernier et al., 19621, serotonin [Sigg, 1959; Gogerty, 19751, L-dopa [Everett, 19671 and yohimbine [Quinton, 1963; Sanghvi et al., 19691; consequently, activity in these procedures has been considered to be predictive of antidepressant potential. However, many of these potentiation models exhibit significant drawbacks in that they detect “false positives” (i.e., compounds not considered to be clinically effective antidepres-

Received final version March 31, 1983; accepted April 22, 1983.

Address reprint requests to Jeffrey B. Malick, Biomedical Research Department, Stuart Pharmaceuticals, Division of ICI Americas, Inc., Wilmington, DE 19897.

0 1983 Alan R. Liss, Inc. 358 Malick sants) or they fail to detect all of the drugs which have been shown to be antidepressants in man. For example, amphetamine potentiation is particularly nonspecific in that numerous classes of drugs (i.e., anticholinergics [Carlton, 19611, antiserotonergics [Weiner ct al., 19731) are active in this procedure. Furthermore, a more serious problem with these procedures is that “atypical” antidepressants (i.e., drugs which are not potent amine reuptake inhibitors or monoamine oxidase [MAO] inhibitors), mianserin in particular, would not be detected in either the amphetamine or L-DOPA potentiation models [Brogden et al., 19781. Gershon and coworkers [Lang and Gershon, 1962; Sanghvi et al., 19691 have developed a yohimbine potentiation model in dogs which detects all of the clinically effective antidepressants that they have evaluated, including MA0 inhibitors, tricyclics, and the atypical iprindole; in this model, the antidepressants are potent enhancers of both the behavioral and cardiovas- cular responses elicited by yohimbine in the conscious dog. Although this procedure did not appear to detect false positives, the procedure is very time-consuming and expensive. In 1963, Quinton observed that imipramine and several other classes of drugs significantly enhanced the toxicity of yohirnbine in mice. In contrast to the yohimbine potcntiation model in dogs, the mouse procedure did not appear to be a specific test for antidepressants since representative agents from several other classes of drugs (most notably antipsychotics) also produced increased toxicity [Quinton, 19631. Thus, the mouse yohimbine potentiation model has not been as widely used to evaluate potential antidepressants as the dog procedure or other classical antidepressant models. The purpose of the present studies was to thoroughly reevaluate this model for the prediction of antidepressant potential.

METHODS Male Swiss-Webster mice (Hilltop Laboratories, Scottdale, PA), weighing 18-24 g, were used throughout these studies. Mice were housed ten per cage (15.5 X 30 X 9.5 cm) both prior to and after yohimbine administration, and studies were always performed in the afternoon (14OO-16OO hr). Mice were treated orally (5 ml/kg) with either HPMC vehicle (0.1% Tween@80, 0.5% hydroxypropylmethylcellulose in 0.9% NaCI) or test drug 60 min prior to yohimbine (30 mg/kg, s.c.); toxicity was assessed 18 hr postyohimbine. This dose of yohimbine very rarely killed any vehicle-treated control mice. A series of typical (e.g., tricyclics, MA0 inhibitors) and “atypical” (e.g., mianserin, iprindole) antidepressants as well as a wide range of psychotropic drugs were tested for their ability to potentiate yohimbine toxicity. Minimal effective doses (MED, i.e., the lowest dose of a drug which produced 50% or greater lethality when combined with yohimbine) for yohimbine potentiation were determined wherever possible.

RESULTS The effects of antidepressants which are amine reuptake inhibitors in the potentiation of yohimbine-induced lethality model are presented on Table 1. The three tricyclics that were evaluated, amitriptyline, imipramine, and clomipramine, all produced dose-related potentiation of yohimbine toxicity, and their MEDs were 6, 10, and 10 mg/kg, per 0s (P.o.), respectively. The selective amine reuptake inhibitors for serotonin (i.e., fluoxetine, zimelidine) and norepinephrine (i.e., nisoxetine. maprotiline) were also capable of potentiating yohimbine although they were relatively weak compared to the tricyclics (Table 1). Results with “atypical” and miscellaneous agents reported to be clinically effective antidepressants are summarized on Table 2. All of the antidepressant agents tested, including the atypicals (e.g. mianserin, iprindole), MA0 inhibitors (pargyline), and several nontricyclics (e.g., viloxazine, bupropion, quipazine, nomifensine), were found to significantly potentiate yohimbine in this procedure. Yohimbine Potentiation in Mice 359

TABLE 1. Effects of Antidepressants That are Amine Reuptake Inhibitors on Yohimbine Potentiation in Mice MED' (mgikg, p.0.) Dose for significant LDSOI-' Drug (mg/kg, p.0.) N" Lethality (%) potentiation (mgikg, pa.) Amitriptyline 1 10 0 6 289 3 10 30 6 30 60 10 20 60 15 10 90 Imipramine 3 10 0 10 400 6 10 20 10 10 60 15 10 70 30 20 75 Clomipramine 3 10 40 10 5 64 10 10 70 30 10 90 Fluoxetine 10 10 0 100 - 30 10 0 60 10 40 100 10 60 150 10 70 Zimelidine 10 10 0 1 50 > 300d 30 10 30 60 20 40 100 20 45 150 10 80 Nisoxetine 10 10 10 200 30 10 10 60 10 20 100 10 30 150 20 45 200 20 55 Maprotiline 10 10 0 200 > 400d 30 30 30 100 10 30 150 10 40 200 20 70 "No. of mice tested. %ED = minimal effective dose; i.e., the lowest dose producing 50% or greater lethality. 'LD,o = The dose that would be expected to produce lethality in 50% of the mice. Data from various literature sources for nonyohimbine-treated mice. dNone of the ten mice died at this dose; mice treated the same as the yohimbine potentiation groups except yohimbine was not administered.

Numerous other representatives of various classes of psychoactive drugs were also evaluated in this procedure (see Table 3). All of the antipsychotics (haloperidol, chlorpromazine, clozapine) that were tested were inactive (i.e., MED's could not be established) even up to high doses with the exception of fluotracen (MED = 3 mg/kg, P.o.), which is reported to possess both antidepressant and antipsychotic activity [Fowler et al., 1977; Itil et al., 19771. Likewise, the anxiolytics that were evaluated were all inactive with the exception of doxepin 360 Malick

TABLE 2. Effects of Selected “Atypical” and Miscellaneous Agents Reported to be Clinically Effective Antidepressants on Yohimbine Potentiation in Mice MEDb (mg/kg, p.0.) Dose for significant LD50C Dw (rng/kg, p.0.) N” Lethality (%) potentiation (mgkpa.) Mianserin 3 10 10 30 365 10 10 10 17 10 30 30 10 60 60 10 70 Iprindole 10 10 0 60 775 17 10 10 30 20 35 60 10 70 Viloxazine 17 10 0 25 1,050 25 10 50 30 20 65 60 10 70 Qu i pdzi ne 3 10 10 17 296 10 20 45 17 20 70 30 10 80 Nomi fensine 3 10 0 60 400 10 10 10 30 30 25 60 30 50 100 20 60 Bupropion 100 10 10 200 544 200 10 50 300 10 70 Pargyline 100 (i.p.) 20 35 200 (i.p.) 370 (i.p.) 200 10 60 ”No. of mice tested. bMED = minimal effective dose; i.e., the lowest dose producing 50% or greater lethality. ‘LD5o = the dose that would be expected to produce lethality in 50% of the mice. Data from various literature sources for nonyohirnbine-treated mice.

(MED = 30 mg/kg, p.o.), a tricyclic which possesses both antidepressant and anxiolytic activity [Goldstein and Pinosky, 19691. Representative anticholingeric (atropine), antihistam- ink (chlorpheniramine) and stimulant (d-amphetamine) agents were evaluated and found to be significant enhancers of yohimbine toxicity (see Table 3).

DISCUSSION In some respects, the results of these studies are in agreement with those of Quinton [ 19631. He reported that the tricyclics, arnitriptyline and imipramine, were potent enhancers of yohimbine lethality exhibiting ED50 values of 4 and 6 mg/kg, p.o., respectively; the MED values for amitriptyline and imipramine (6 and 10 mg/kg, pa., respectively) in the present study are in close agreement. The present study extended this observation to a number of relatively selective reuptake inhibitors for serotonin (fluoxetine, zimelidine) and norepinephrine (nisoxetine, maprotiline); these agents also produced a dose-related enhancement of yohimbine toxicity, although they were much less potent than the tricyclics. Although only a Yohimbine Potentiation in Mice 361

TABLE 3. Effects of Selected Psychoactive Drugs on Yohimbine Potentiation in Mice MEDb(mg/kg, P.0) for significant LD50C Drug Na potentiation (mglkg, P.o.) Antipsychotics Chlorpromazine 30 Inactive (10- 60)d 319 Clozapine 30 Inactive (10- 30) 210 Haloperidol 40 Inactive ( 1- 30) 145 Fluotracen 70 3 353 Anxiolytics Diazepam 40 Inactive (10-100) 970 Buspirone 40 Inactive (10-100) > 3me Fenobam 60 Inactive ( 3-100) > 3mr Meprobamate 30 Inactive (30-300) 980 Doxepin 50 30 135 Miscellaneous Atropine SO4 30 10 400 Chlorpheniramine 40 17 142 d-Amphetamine SO4 80 6 37 ’No. of mice tested. %ED = minimal effective dose; i.e., the lowest dose producing 50% or greater lethality. ‘LDSO = the dose that would be expected to produce lethality in 50% of the mice. Data from various literature sources for nonyohimbine-treated mice. dFor inactive compounds, dose ranges evaluated are given in parentheses. %one of the ten mice died at this dose; mice treated the same as the yohimbine potentiation groups except that yohimbine was not administered.

limited number of compounds have been evaluated, it is interesting to note that the least potent enhancers of yohimbine toxicity among the compounds evaluated thus far are the selective norepinephrine uptake inhibitors, maprotiline and nisoxetine. Maprotiline, a quadricyclic [Carney, 19721, fluoxetine, and zimelidine [Benkert et al., 19771 have all been shown to be clinically effective antidepressants. Unfortunately, nisoxetine was dropped due to toxicity before its antidepressant potential could be assessed. Atypical antidepressants were also significant potentiators of yohimbine in this procedure. Mianserin and iprindole, agents which are not potent reuptake inhibitors or MA0 inhibitors but are effective antidepressants, exhibited dose-related activity. Bupropion, a clinically effective antidepressant that may be somewhat “atypical” in that it mainly acts as a dopamine reuptake inhibitor, was also weakly active. Another effective antidepressant, viloxazine (ICI 58,834), a bicyclic agent which is a relatively specific norepinephrine uptake inhibitor and which possesses less peripheral anticholinergic activity than tricyclics [Green- wood, 19751, was found to be a potent enhancer of yohimbine, although its dose-response curve was relatively flat. Other clinically effective agents that exhibited significant activity in this test were quipazine, a serotonin agonist [Rodriguez and Pardo, 19711; pargyline, an MA0 inhibitor; and nomifensine, a stimulantlike agent in rodents [Pechnold et al., 19751. Thus, all of the major classes of antidepressants that have been evaluated have exhibited activity in the yohimbine potentiation test in mice. In order to determine whether a true drug interaction occurred between the test agents and yohimbine, comparative toxicological data (LDSOs wherever available; a few agents were tested in our laboratories at twice their MEDs for yohimbine potentiation when data was not 362 Malick

available in the literature) in nonyohimbine-treated mice are also presented. All of the antidepressants clearly enhanced the toxicity of yohimbine since lethality occurred with the drug combinations at doses that were considerably lower than the LDSOs for the drugs by them- selves. This was also true for doxepin (anxiolytic/antidepressant): and fluotracen (antipsychotic/antidepressant) as well as for the false positives (i.e., atropine, chlorpheniramine, amphetamine) that were detected in this procedure. In order to determine the specificity of the yohimbine potentiation model in mice, representative agents from other classes of psychoactive agents were evaluated. All of the antipsychotics, both typicals (e.g.,halopcridol, chlorpromazine) and atypicals (clozapine) were inactive; fluotracen, an agent which appears to be both an antidepressant and an antipsychotic [Fowler et al., 1977; Itil et a]., 19771 was a potent enhancer of yohimbine. Thus, since the antipsychotics were inactive, the activity of fluotracen must be attributed to its antidepressant component of activity. The present data on the antipsychotics is in marked contrast to that of Quinton [ 19631, who reported that chlorpromazine, promazine, and perphenazine were active with EDSOs of 7,9, and SO mg/kg, p.o., respectively. This difference is difficult to reconcile; the most obvious difference between the studies being different strains of mice. Several representative anxiolytic (e.g., diazepam, meprobamate, fenobam, buspirone) agents have been evaluated, and with the exception of doxepin, all were inactive. Doxepin is a tricyclic which exhibits both anxiolytic and antidepressant activity in man [Goldstein and Pinosky, 19691; thus, its potent activity in this procedure must be attributable to its antidepressant component since all of the other anxiolytics were totally inactive. Several additional compounds have been found to be potent enhancers of yohimbine- induced toxicity in mice. Atropine, an anticholinergic drug, exhibited dose-related activity; this is in agreement with the findings of Quinton 119631, who reported an EDSO of 40 mg/kg, P.o., in his procedure. The antihistamine chlorpheniramine was also effective as a potentiator of yohimbine toxicity; however, Quinton [ 19631 reported that the antihistamines mepyramine and promethazine were essentially inactive in his procedure. In addition, the CNS stimulant d-amphetamine was found to be a potent enhancer of yohimbine lethality; this finding is in agreement with that of Quinton [ 19631. If the procedure were to be considered predictive of antidepressant potential, these agents might be categorized as false positives in this test; however, this is not a serious drawback to the use of this procedure for discovering antidepressant potential because these same agents would show up as false positives in several other procedures (e.g., antagonism of muricidal behavior [Vogel, 19751, tetrabenazine antagonism [Niemegeers, 197.51, and behavioral despair [Porsolt et al., 19781) that are commonly used to predict antidepressant activity. Thus, this model appears to represent a useful screen for potential antidepressant drugs. All of the clinically effective antidepressants that were evaluated in the yohimbine potentiation model in mice exhibited activity; i.e., all of the representative agents from tricyclic (e.g., amitriptyline, imipramine), MA0 inhibitor (e.g., pargyline), and atypical (e.g., iprindole, mianserin) categories of antidepressants as well as agents that combine antidepressant activity with either anxiolytic (e.g., doxepin) or antipsychotic (e.g., fluotracen) activities significantly potentiated yohimbine-induced toxicity in mice. Although the yohimbine potentiation test in dogs may be more selective than the mouse test in that it apparently will detect the antidepressants while not picking up stimulants as false positives, it is more costly and time-consuming. Thus, this yohimbine potentiation model in mice appears to be a useful primary screen, whereas the dog model may represent a useful secondary evaluation procedure for potential antidepressant activity.

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