The effects of ethanol alone and in combination with phenobarbital, chlorpromazine, or chlordiazepoxide

TOXICOLOOYANDAPPLIEDPHARMACOLOOY15,405-414(1969)

The

Effects

of Ethanol Alone bital, Chlorpromazine,

and in Combination with or Chlordiazepoxide132

Phenobar-

GERALD F. GEBHART, GABRIEL L. PLAA,~ AND C. L. MITCHELL Department of Pharmacology, College of Medicine, University of Iowa, Iowa City, Iowa 52240 Received November 21,1968

The Effects of Ethanol Alone and in Combination with Phenobarbital, Chlorpromazine, or Chlordiazepoxide. GEBHART, GERALD F., PLAA, GABRIEL L., and MITCHELL, C. L. (1969). Toxicol. Appl. Pharmacol. 15, 405414. The nature of the interaction of ethanol (E) in combinationwith phenobarbital(Pb), chlorpromazine(CPZ), or chlordiazepoxide(CDP) was investigatedin our laboratory. E, Pb, CPZ, or CDP wasadministeredip to male,albino miceand the dosecausing50% of the miceto fall from a 60” inclinedscreen(medianparalyzing dose,PD50)determinedfor eachagent. Sincethe dose-response curvesdid not deviatefrom parallelism(P > O.OS), one-halfthe PD50 of E was then given in combination with one-half the PD50 of Pb, CPZ, or CDP suchthat the times of peak effect coincided. Only CDP in combinationwith E produceda potentiation of effectsin this test method; Pb and CPZ in combination with E resultedin addition of effects.In a similarmanner,one-halfthePD50 of Pb, CPZ, or CDPwascombinedwith one-half the lossofrightingreflex ED50 of E. In this testingprocedure,all three agentsin combination with E demonstratedpotentiation. Blood levelsof ethanol (BLE) werefollowed in both of the abovestudies to determinethe possibilitythat increasedBLE might beresponsiblefor the potentiation seen.Blood sampletimeswere5, 15,30,60, and 120min post E administration.Combinationsof drug with E werecomparedto identical dosesof E administeredin the absenceof a drug. In no casewasa significant increasein BLE seenat the early collection periods,a time by which the behavioral effects would have already occurred. Therefore, elevationsin BLE cannot account for the behavioraleffectsseen.However, our data do suggestthat CPZ in combination with one-half the PD50 of E may retard the rate of disappearanceof E from the blood. The widespread use of psychotrophic drugs today greatly increasesthe possibility of, and concern about, combinations of these agents with other drugs, ethanol prominent among them. However, there exist in the literature conflicting reports concerning the nature of the interaction between ethanol and various psychotropic agents.Historically, studies addressingthe problem have utilized primarily barbiturates or chlorpromazine 1Thisinvestigationwassupportedby U.S. PublicHealthServicegrantsMH06564,GM12675,and 5TOlGMOO141. z Portionsof thisreportwerepresented at the 1968Fall Meetingof theAmericanSocietyfor PharmacologyandExperimentalTherapeutics in Minneapolis,Minnesota,August18-22. 3Presentaddress:Departmentof Pharmacology,Faculty of Medicine,University of Montreal, CasePostale6128,Montreal,Canada. 405

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PLAA,

AND

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together with ethanol. Some investigators studying combinations of ethanol with barbiturates (Fearn and Hodges, 1953; Gruber, 1955; Archer, 1956; Aston and Cullumbine, 1959 ; Graham, 1960) or chlorpromazine (Eerola, 1963) report that the effect of such combinations is additive. Other workers, however, conclude that a potentiating effect occurs (Jetter and McLean, 1943; Brodie et al., 1955; Zirkle et al., 1959; Kopman and Hughes, 1959; Forney et al., 1962; Danechmand et al., 1966). Not only are the reported behavioral effects of the combined agents unclear, but the manner by which they are produced is likewise unknown. Tipton et al. (1961), using the rabbit, observed that chlorpromazine given in combination with ethanol greatly increased the blood level of ethanol over what would be expected after administration of a standardized dose of ethanol alone. These workers concluded from their study that an increased rate of absorption of ethanol was not responsible for the potentiated effects observed but suggested as a possible cause for the elevated blood level of ethanol an inhibition of metabolism of ethanol by chlorpromazine. While this hypothesis is attractive, it must be noted that other workers (Brodie et al., 1955; Kahn et al., 1964; Seidel and Soehring, 1965) have obtained no evidence to support the hypothesis. Indeed, Lind and Parkes (1967) report that in mice pretreatment with chlorpromazine will in fact shorten sleeping times induced by ethanol administration; they imply that this effect is due to an induction of the nonspecific oxidizing enzyme system of the liver microsomes by chlorpromazine. Clearly, the contradictions in the literature concerning addition versus potentiation of effects when ethanol is combined with barbiturates or chlorpromazine, and the discrepancies concerning the effect of chlorpromazine on the blood level of ethanol, are a source of confusion. The present study was undertaken, therefore, in an effort to clarify some aspects of this problem. In this study, we have investigated the effects of phenobarbital, chlorpromazine, and chlordiazepoxide in combination with ethanol on (a) alteration in gross behavior and (b) alteration in the blood level of ethanol. Chlordiazepoxide was included because it is an agent very commonly administered in humans and, second, because there are relatively few animal data available concerning the nature of its interaction with ethanol (Zbinden et al., 1961; Hughes, et al. 1963; Danechmand et al., 1966). Further, these animal data are not in agreement as to whether additive or potentiated effects are attained when chlordiazepoxide is given with ethanol. METHODS

Male, albino mice weighing 25-35 g and obtained from Sutter farms were used. The mice were allowed to acclimatize for not less than 4 days after arrival. The animals were permitted free accessto food and water up to the time they were used. All drug injections were given intraperitoneally. The drugs chosen for use in combination with ethanol (50 % v/v in normal saline), were phenobarbital sodium USP, chlorpromazine hydrochloride, and chlordiazepoxide hydrochloride. All solutions were prepared with normal saline in a manner such that 0.1 ml of drug solution was injected per 10 g of mouse weight. The initial testing procedure employed failure of mice to remain on a 60” inclined screen after drug administration hereafter referred to as paralysis for convenience. The 50% paralyzing dose (PD50) was determined for all four agents following the probit

INTERACTIONS

OF DRUGS

WITH

ETHANOL

407

method described by Finney (1962). Subsequently, one-half of the PD50 of chlorpromazine, chlordiazepoxide, or phenobarbital was given in combination with one-half the PD50 of ethanol such that the times of peak effect of the two agents coincided. These times of peak effect were determined to be 60 min for chlorpromazine and phenobarbital, 30 min for chlordiazepoxide, and virtually instantly for ethanol; thus, chlorpromazine and phenobarbital were administered 1 hour, and chlordiazepoxide 30 min, prior to ethanol. The number of mice falling from the inclined screen following each combination was compared to that resulting from a PD50 of ethanol alone. In addition, the ability of the drugs in combination with ethanol to produce a loss of righting reflex was studied. Loss of the righting reflex was defined as a failure of mice to right themselves when placed on their backs and stimulated by a thumbnail tail pinch. The computations for fitting the probit-log dose regression line, the determination of the PD50, the 50% loss of righting reflex dose (LRRSO), and the 95% confidence intervals were done according to the methods described by Finney (1962). In determining each PD50 and the LRRSO, at least 3 dose levels, yielding between 10 % and 90 % effect, were used. At least 10 mice were utilized in determining each point. Blood levels of ethanol were determined from paired samples of blood by two methods (1) the microdiffusion method of Feldstein and Klendshoj (1954); and (2) a method employing a gas chromatographic technique modified from Davis (1966). In the microdiffusion method, ethanol in the blood was allowed to diffuse for not less than 3 hours at room temperature. The samples were diluted and the optical density was read on a.spectrophotometer at 450 rnp against distilled water set at zero density. The ethanol content in the blood was estimated from a previously prepared standard curve. In the gas chromatographic method, 1,4-dioxane (reagent grade) was employed as a protein precipitant to which isopropanol (reagent grade) had been added to serve as an internal standard. Aside from a difference in gas chromatographs and operating conditions thereof, the addition of isopropanol as an internal standard was the only modification of the method of Davis. The solutions were prepared to contain either 1.95 mg or 3.9 mg of isopropanol per 1.0 ml of 1,6dioxane. A Varian Aerograph model 1200-2 gas chromatograph equipped with a flame ionization detector was used for the analysis. The column used was l/8 inch x 10 feet containing 15 % Hallcomid M-18-OL on 70/80 Chromosorb W (obtainable from Varian Aerograph). Operating conditions were: column temperature, 90”; injector port temperature, 110”; detector temperature, 140”; hydrogen flow rate, 35 ml/min; and carrier gas (nitrogen) flow rate, 35 ml/min. In this procedure, the ethanol content in the blood was estimated by the ratio of peak heights of isopropanol to ethanol. Mouse blood for analysis was obtained via cardiac puncture and collected at 5, 15, 30, 60, and 120 min after ethanol administration. Blood (0.8 ml) from each of three mice was pooled; 1.0 ml of that pool was analyzed gas chromatographically and 0.8 ml was analyzed in the Conway microdiffusion cell. In the behavioral studies, statistical significance was determined using either the Exact probability method of Fisher or the Chi square (~2) test. In comparing the blood ethanol levels, the Student’s t was employed. P < 0.05 was considered as the level of statistical significance in all tests. There are available many definitions of addition and potentiation, and there exist undoubtedly more interpretations of the meanings of these terms than definitions. It is

408

GEBHART, PLAA, AND MITCHELL

therefore prudent before continuing further to provide operational definitions to prevent possible misinterpretations of the results to be reported by readers familiar with other definitions. For our purposes, potentiation as used herein will describe the effects of two drugs when given together producing a greater than algebraic summation of effects produced by either drug administered alone. Addition will be employed to describe the simple summation of the combined effects of two agents when given together. RESULTS

The dose-response curves for chlorpromazine, chlordiazepoxide, phenobarbital, and ethanol as determined in the inclined screen test are presented in Fig. 1. No deviation

CPZ

301

1 I I I IHI 5

Pb

600 OOSE,mg/kg (Log Scale)

Kx)O

2003

4000

9000

FIG. 1. Dose-response curves as determined on the 60” inclined screen. Chlorpromazine chlordiazepoxide (CDP), phenobarbital (Pb), and ethanol (A) (50% v/v).

(CPZ),

from parallelism (P > 0.05) was found for any of the curves. Table 1 indicates the 50 % paralyzing doses for the four agents and their respective relative potencies. The order of potency was found to be chlorpromazine > chlordiazepoxide > phenobarbital > ethanol. The results of combinations of one-half the PD50 of ethanol with one-half the PD50 of chlorpromazine, cblordiazepoxide, or phenobarbital are presented in Table 2. Only chlordiazepoxide and ethanol combined produced potentiation, as evidenced by a significantly greater number of animals falling from the screen when compared to mice given a 50% paralyzing dose of ethanol. Phenobarbital and chlorpromazine demonstrated only an addition of effects when given in combination with ethanol in this test procedure. An ED50 for ethanol producing a loss of the righting reflex (LRRSO) was determined and found to be 4.3 g/kg (95% confidence interval of 3.9-5.2 g/kg). One-half of this dose of ethanol was then given in combination with one-half the PD50 of chlorpromazine, chlordiazepoxide, phenobarbital, or ethanol (one-half the PD50 was used in combination with one-half the LRRSO of ethanol because an LRRSO could not be

INTERACTIONS

OF DRUGS

WITH

TABLE

1

EFFECT OFDRUGSONTHEABILITV ON AN INCLINED

Drug

409

ETHANOL

OFMICETO REMAIN SCREEN”

50% paralyzing doseb Potency ratio= 6x&)

Ethanol

1820 (1436-2043)d

-

Chlorpromazine (lK2-205)

(9Y4) Chlordiazepoxide $8)

(2:834)

(82?02)

(1224)

Phenobarbital

(1Mice wereplacedon a 60” inclinedscreenat the following timesafter drugadministration:ethanol(50%v/v)-immediately ; chlordiazepoxide-30min;andchlorpromazineorphenobarbital60min. All druginjectionsweregivenintraperitoneally. bThe dosecausing50% of the miceto fall from the inclined screenasdetermined by the probit method. c The potencyratioswerecalculatedrelativeto ethanol. d95%confidence interval. determined for chlorpromazine, chlordiazepoxide, or phenobarbital which did not also produce varying degreesof lethality). The results of these combinations are presented in Table 3. In this testing procedure, all three agents in combination with ethanol exhibited a potentiation of effects when compared to one-half the LRRSO of ethanol in combination with one-half the PD50 of ethanol. This is indeed striking in view of the

fact that if one-half the LRRSO could have been employed for chlorpromazine, chlordiazepoxide, or phenobarbital, it would have been a much larger dose than the one-half PD50 which was used. TABLE EFFECT

OF DRUGS

2

IN COMBINATION WITH ETHANOL ON THE ABILITY TO REMAIN ON AN INCLINED SCREEN”

Drug-ethanol combination 50% paralyzing doseof ethanol + 50% paralyzing doses,ethanol + chlorpromazine 3 50% paralyzing doses,ethanol + chlordiazepoxide 3 50% paralyzing doses,ethanol + phenobarbital

OF

MICE

Number of Number of mice micetested responding 20

13

20 20

10 19 15

20

PC

> 0.30

< 0.05 > 0.70

’ Chlorpromazine andphenobarbitalweregiven60min prior to ethanol;chlordiazepoxide 30min prior to ethanol.Micewereplacedonthe60”inclinedscreen immediately afterethanoladministration. * Thenumberof micefallingfromthe60”inclinedscreen. c ProbabiIityof ~2(ethanol-drugcombinations YSethanolalone);P< 0.05isconsidered significant. 14

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GEBHART, PLAA, AND MITCHELL

TABLE 3 ABILITY OF DRUGS IN COMBINATION WITH ETHANOL TO PRODUCE A Loss OFRIGHTINGREFLEX (LRR)

3 LRRSO of ethanol plus : 3 PD50d of ethanol 3 PD50 of chlorpromazine ) PDSO of chlordiazepoxide f PD50 of phenobarbital

Number of mice tested

Number of mice responding

20 20 20 20

0 7 13 5

PC

i 0.004 -c 0.001

< 0.023

a Chlorpromazine and phenobarbital were given 60 min prior to ethanol; chlordiazepoxide 30 min prior to ethanol. The mice were tested immediately after ethanol administration. b The number of mice losing the righting reflex. c Fisher’s exact probability (f LRRSO ethanol + 3 PDSO of ethanol us 3 LRRSO of ethanol + 3 PD50 of drug); P < 0.05 is considered significant. d One-half the dose causing 50% of the mice to fall from a 60” inclined screen.

The same dosing schedules were used in following the blood levels of ethanol as those previously described for the behavioral studies; namely, chlorpromazine, chlordiazepoxide, or phenobarbital in combination with either one-half the PD50 of ethanol or one-half the LRRSO of ethanol. The blood ethanol disappearance curves as estimated gas chromatographically are presented in Figs. 2 and 3. Only chlorpromazine in combination with one-half the PD50 of ethanol resulted in a significantly elevated blood level of ethanol at the 15,60-, and 120-min collection periods when compared to the blood levels of ethanol produced by one-half the PD50 of ethanol (Fig. 2). In

0-0 EtOH o--o CPZ t EtOH A--A CDP t EtOH A--b PbtEtOH

20o-

I 5

I 15

I 30 TIME (minutes

1 60

I 120

post ethanol administration)

FIG. 2. Blood levels of ethanol as determined by the gas chromatographic method. The EtOH, ethanol (50% v/v), curve was obtained using one-half the PDSO. The remaining curves were obtained using one-half the PDSO of chlorpromazine (CPZ), chlordiazepoxide (CDP), or phenobarbital (Pb) in combination with one-halfthe PD50 of ethanol. * indicates significantly different (P < 0.05; Student’s t) from ethanol alone. Each mean represents the mean of 3 pools of blood. The vertical lines represent * the standard error.

INTERACTIONS

OF DRUGS

WITH

o--o *-a

100

, 5

411

ETHANOL

EtOH CPZ t EtOH CDP + EtOH Pb + EtOH

I

I

I

I

15

30

60

120

TIME

(minutes

post

ethanol

administration)

FIG. 3. Blood levels of ethanol as determined by the gas chromatographic method. The EtOH, ethanol (50% v/v), curve was obtained using one-half the LRRSO. The remaining curves were obtained using one-half the PD50 of chlorpromazine (CPZ), chlordiazepoxide (CDP), or phenobarbital (Pb) in combination with one-half the LRRSO of ethanol. * indicates significantly different (P < 0.05; Student’s t) from ethanol alone. Each mean represents the mean of 3 pools of blood. The vertical lines represent i the standard error.

to the blood level of ethanol produced by one-half the LRRSO of ethanol, chlorpromazine-ethanol and phenobarbital-ethanol combinations at 60-min and both chlorpromazine and chlordiazepoxide in combination with ethanol at 120 min resulted in significant elevations in the blood ethanol level (Fig. 3). Figure 4 shows a comparison of the blood levels of ethanol as determined using the microdiffusion method versus those determined by the gas chromatographic method at one-half the PDSO level of ethanol. With the exception of the first collection period, those levels determined by the microdiffusion method are significantly greater (P Q 0.05) than those found by utilizing the gas chromatographic procedure.

comparison

FIG. 4. Comparison of the blood levels of ethanol determined spectrophotometrically and gas chromatographically from matched samples of pooled blood. The ethanol (50% v/v) dose employed was one-half the PD50 (910 mg/kg). Solid line = gas chromatographic method; dashed line = spectrophotometric method. Points at the same collection time are significantly different (P < 0.05 ; Student’s t ) except at the 5-min collection period. Each mean represents the mean of 3 pools of blood. The vertical lines represent f the standard error.

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GEBHART,

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MITCHELL

DISCUSSION

It has already been pointed out (see results, Fig. 4) that the microdiffusion method of estimating the blood level of ethanol yields values significantly greater than those determined gas chromatographically. This inaccuracy by overestimation was discovered by virtue of the determinations of the blood levels of ethanol from paired samples of pooled blood. The microdiffusion method is a nonspecific oxidizing reaction, and hence substances other than ethanol, such as formaldehyde, acetaldehyde, and methanol, to mention the more common materials, will affect it. Our experience with both methods leads us to rely on the gas chromatographic technique for greater accuracy than on the method of Feldstein and Klendshoj (1954). However, regardless of the method, our data (although not included herein for the microdiffusion method) indicate that none of the three agents produce their effects by increasing the blood concentration of ethanol. The possibility exists, however, that the effects resulting from such combinations may be due to an elevation in blood-drug levels produced by ethanol. We can make no statement as to the validity of the possibility, but we can state that the effects resulting from these combinations do not arise from an elevated blood concentration of ethanol. This statement is made on the basis of the fact that the blood concentrations of ethanol were not shown to be elevated at a time when the behavioral effects had already occurred. Our data do suggest, however, that chlorpromazine may delay the rate of clearance of ethanol from the blood (Fig. 2). Although the same tendency was not apparent at one-half the LRRSO of ethanol, extended collection periods might indicate such a trend when one considers the difference in the blood levels of ethanol remaining at 120 min when this larger dose of ethanol was given as compared to one-half the PD50 of ethanol. Although statistical analysis indicates scattered differences from a dose of ethanol given alone as compared with that same dose given in combination with phenobarbital or chlordiazepoxide, no trend toward an increased blood level of ethanol is visible. We therefore conclude that, in fact, these two drugs in combination with ethanol do not alter the blood level of ethanol. With regard to the behavioral data, it should be pointed out that investigations into the nature of drug interactions require specific design. Without taking into consideration proper design, the experimental results cannot be properly evaluated either by themselves or when compared with results obtained by other workers. In his review, Gruber (1955) points out two of the difficulties in studies of this nature. First, it is necessary that the drugs when given in combination be administered apart in time such that the times of peak effect of the two agents coincide. Second, if the drugs appear to have a similar mode of action, it is impossible to distinguish between potentiated and additive effects without taking into consideration the nature of the dose-response curves for these agents and the inherent variability in biological material of the response of drugs. The doseresponse relationship for the agents must be established in order to provide a rational basis for the choosing of doses to be used in combination. If the dose-response curves are parallel (as was the case in this study) and if the drugs are acting in a similar manner, then one-half of the ED50 of one agent plus one-half of the ED50 of the other agent will yield results similar to an ED50 of either drug when given alone (provided, of course, that they are administered so that the times of peak effects coincide). On the other hand, ifpotentiation occurs, then the combination will yield results greater than those obtained

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413

with the ED50 alone. However, as is well known, an ED50 determined at one point in time will often produce different results when that identical dose is administered at some later time because of biological variation. Therefore, it is imperative that when combinations of drugs are given on the basis of some fraction of their respective EDSO’s, an ED50 of the agent against which the combination is to be compared must be given concurrently. Statistical analysis must be performed to assessthe significance of any difference observed among tests carried out at the same point in time. Finally, it is desirable to employ more than one index to measure the effects of ethanol in combination with psychotropic drugs. Stated differently, more than a single dose level of ethanol and/or drug(s) should be used in investigations of the problem. Unfortunately, the majority of investigations to date have not given adequate consideration to the design of their experiments. Hence, the literature is replete with contradicting reports. Gruber (1955) has provided adequate comment concerning the problems with earlier work. Since that time, however, problems remain with more recent work. Archer (1956) and Eerola (1963) for example, administered the agent to be tested in combination with ethanol concomitantly with no consideration given to times of peak effects. Danechmand et al. (1966) did not determine a dose-response relationship for the drugs (chlorpromazine and chlordiazepoxide included) given in combination with ethanol, nor a rationale for the times of administration of either ethanol or drugs relative to each other. Because of these inconsistencies one cannot compare our results with the results of others; there are simply too many factors that can influence the results obtained which are different between their work and ours. In our work, potentiated effects were observed in some cases while in others only additive effects obtained. It therefore cannot be unequivocally stated that ethanol in combination with chlorpromazine, chlordiazepoxide, or phenobarbital will always exhibit either potentiated or additive effects. Rather, the effects realized by such combinations depends, in part, upon the testing procedure employed (or more likely, upon the dose level of the drugs). With the doses of the other agents fixed, potentiated effects were observed with the higher dose level of ethanol whereas only additive effects were observed for phenobarbital and chlorpromazine when combined with the lower dose level of ethanol. That is, higher doses of ethanol (or perhaps also of the other drugs) yield different results than the lower dose level of ethanol in combination with identical dose levels of the other drugs. Thus, it is apparent that the initial test design pZays an influential role in the results obtained. REFERENCES

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424-429. DAVIS,

R. A. (1966). The determination of ethanol in blood or tissue by gas chromatography.

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