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Effects of Drugs on Conditioning in the Rat II. Synthesis of a Centrally Active Drug and the Effects of Nine Drugs on Operant Conditioning and Extinction
Effects of Drugs on Conditioning in the Rat I1 Synthesis of a Centrally Active Drug and the Effects of Nine Drugs on Operant Conditioning and Extinction By M. D. ACETOT, V. D. LYNCH$, and R. K. THOMS In this study, an operant conditioning and extinction technique is used to compare the effects of various so-called tranquilizing agents. In addition, a method is presented for the preparation of 2-ethyl-2-(3-methyl)-butylrnalondiamide. The statistical analysis of the data obtained is supported by observations which indicate that some of the effects of these drugs can be ascribed to secondary or side-effects.
HE TRANQUILIZING drugs comprise one of the Tareas of recent interest in the field of pharmacology. A considerable amount of experimentation and speculation has been undertaken in order to attempt to discover new compounds of this type and also to provide a basis for the understanding of the mode of action of these drugs. However, little progress has been reported towards developing a unique test to determine the value of these compounds as regards their ability to alter behavior. The reason may be that tranquilizing drugs, like most other drugs, have potential, actual, primary, and/or secondary effects manifested as ataxia, depression, anorexia, ptosis of the eyelids, flaccid paralysis, and the like. These latter points constitute an important aspect in the development of tranquilizers and the effects of these drugs on animal behavior. Equally important is the aspect of chemical structure-activity relationships. Indeed, a large number of tranquilizers used therapeutically were the result of investigation in this area. With these effects in mind, this problem was designed and undertaken in an effort to contrihute further to this area. It was felt that one approach in determining whether or not the actual, potential, primary, and/or secondary effects of tranquilizers and other centrally active drugs contributed to overall behavior was to compare the same drugs at the same dose levels using two different experimental techniques. Specifically, it was felt that the results obtained by the application of operant conditioning techniques would not only provide information based on its own merits Rewived November 5 . 1960. from the School of Pharmacv. ...~ .~ University of C o i n e i t i i i t , stoms. Accepted for publication March 27, 1961. Abstracted in part from dissertations submitted to the Graduate School of the University of Connecticut by Vincent D. Lynch and Mario D. Aceto in partial fulfillmentof therequirements for the degree of Doctor of Philosophy, June 1959. t Present address: School of Pharmacy, University of Pittsburgh Pittsburgh 13, Pa. Z Preseni address: College of Pharmacy, S t . John’s University, Jamaica 32, N. Y. ~
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but would contrast or complement the results obtained by using an avoidance-escape technique (1). The two techniques are based entirely on different psychological principles and might serve to emphasize towhat extent these actual, potential, primary, and/or secondary effects influence behavior and perhaps, within the limits of these two testing procedures, the type behavior. Pfeiffer, et al. (2), in a comparison of the effects of meprobamate with other classes of drugs active in the central nervous system concluded that this compound has a prolonged barbiturate-like action mixed with a moderate degree of trimethadione effect on the brain. Furthermore, they claimed that the pharmacological action resembled phenobarbital or trimethadione even more than it did its congener mephenesin. They felt that meprobamate is not an ataractic drug as is chlorpromazine, and that it exhibits few properties of mephenesin. It is interesting to note that Titus and Weiss (3) in an investigation of the metabolism of phenobarbital 2-CI4identified by means of paper chromatography nine metabolites, one of which was reported to be urea-C14 representing 2.5 per cent of the products. This metabolite strongly suggested that a certain amount of ring cleavage takes place in the metabolism of phenobarbital and possibly other barbiturates. An examination of the chemical structure of meprodibamate, 2-methyl-2-n-propyl-1,3-propanediol carbamate, showed various features in common with the barbiturates. The most obvious is the quaternary carbon atom. Both hydrogens attached to the carbon atom at position 5 in barbituric acid must be replaced by alkyl or aryl groups in order to manifest hypnotic properties. If the carbonyl group at position 2 in a barbiturate such as amobarbital (5-ethyl-5-isoamylbarbituric acid) is removed, the result will be amides not unlike the carbamide groups of meprobamate. Many compounds with quaternary carbon atoms have been shown to possess
823
Journal of Pharmaceutical Sciences
824 interesting central effects. Already mentioned were the sedative and hypnotic effects of the barbiturates. Meprobamate shows tranquilizing and spasmolytic efFects. Berger and Ludwig (4) examined various substituted propane-1,3diols and found tha,t diethyl propanediol (2,2diethylpropane-1,3-diol) has the same effect on the spinal cord as mephenesin. In view of the information available, it was proposed that various compounds closely related to both the barbiturates and meprobamate be investigated. Chosen for this study were 2ethyl-2-(3-methyl)-butylmalondiamide and 2ethyl-2- (3-methyl)-butylndonic acid diethylester. The ester contains no nitrogen and it was thought that its effects might provide an interesting contrast with 2-ethyl-2- (3-methyl)-butylmalondiamide when tested on animal behavior. Another compound, ethyl-2,4-toluene dicarbamate, was chosen for investigation because of the structural similarity between it and some of the otoloxy derivatives investigated by Berger (5) and also because it conta.ined the amide group which Toman (6) postulated should be present in order for a compound to have central depressant properties. Preliminary studies of several homologs of this compound demonstrated that this series of compounds possessed sedative and depressant properties worthy of further investigation.
EXPERIMENTAL. Preparation of 2-Ethyl-2-( 3-methyl)-butylmalondiamide.-Eighteen grams of 2-ethyl-2-(3-methyl)butylmalonic acid diethylester was hydrolyzed by refluxing on a steam bath for twenty-four hours with 30 ml. of a 50% hydroalcoholic (1:l) potassium hydroxide solution. The reaction mixture was diluted with 50 ml. of water and acidified to Congo red paper with concentrated hydrochloric acid. After cooling, the immiscible layers were separated and the aqueous portion was extracted with ether. The organic portions were combined and the ethereal solution was extracted with 5% aqueous sodium bicarbonate solution. The aqueous alkaline extract was acidified with hydrochloric acid and re-extracted with ether. Evaporation of the combined ether extracts yielded the free acid. After adding thionyl chloride dropwise to remove traces of water, an excess of the reagent was added, and the mixture was re3uxed on a steam bath for one hour. The residual thionyl chloride was removed by distillation and the remaining acyl chloride was dissolved in 100 ml. of dry hexane. Dry ammonia was bubbled through the solution until the resulting suspension of diamide became a. thick slurry. The solid was collected on a sintered-glass funnel and was washed with dry hexane, the filtrate being further treated with ammonia until no more precipitate formed. After being air dried, the crude diamide was extracted with hot ethanol and the filtered alcoholic solution was evaporated to dryness on a steam bath.
After washing the residue with water to remove ammonium chloride and other impurities, it was dried and recrystallized from ethanol. The yield was 42.1%, m. p. 190-191° (uncorr.). AnaZ.l-Calcd. for CIOHZONZOZ: N, 13.98; C, 59.97; H, 10.07. Found: N, 13.22, C, 59.62; H, 10.11. Drugs.-The drugs and dosage levels are listed in Table I. Except for the diamide and dicarbamate (7) which were synthesized in our laboratories, all other drugs were obtained commercially or supplied by the manufacturer.2 Distilled water was used as the solvent for the relatively water-soluble drugs. Methocel(O.5%) was used as the suspending agent for the poorly water-soluble drugs. All suspensions were shaken prior to use. In all cases, the drug was either dissolved or suspended sp that the desired dose per Kg. of body weight of the rat was contained in a 1-ml. h a 1 volume of water. When not in use, the solutions were stored in a refrigerator. Animals.-Descendants of a Wistar strain of rats were used in this study. As regards sex, the animals were chosen at random. These animals weighed at least 150 Gm. a t the beginning of the test period. However, because of weight gains during the test period, some of the same animals weighed up to 310 Gm. at the end of the period. Each animal was housed separately, with a food intake maintained at between 8-11 Gm. daily. Water was allowed ad libitum. Operant Conditioning and Extinction Procedure. -A bar-press apparatus was used for this procedure. This device permitted a food pellet to be dispensed each time the foot pedal inside the experimental chamber was depressed. Separate counters registered both the total number of bar presses and tablets dispensed. The food dispenser was arranged so that it could be disconnected during the extinction procedure. The experimental chamber was located in a windowless room adjacent to which was the room housing the control panel and recording devices. After determining the operant level for each rat, it was conditioned to press the bar to obtain food. All the animals were conditioned to respond at least 100 times during a fifteen-minute session. With the pellet dispenser disconnected, each animal was then extinguished to a rate of ten bar presses or less during a fifteen-minute session. When a sufficient number of animals had been conditioned and extinguished, they were assigned to groups of four animals each. Each group was then assigned a t random to one of the drugs and one of the dose levels being investigated. Drugs were administered intraperitoneally in all cases, in order to insure optimum absorption. With the exception of reserpine and meprobamate, animals were injected with drugs one hour before admission to the test apparatus. Reserpine was administered two hours before and meprobamate 1 The authors wish to thank Professor W. Williams who performed the nitrogen determination and Dr. Robert Gerraughty who performed the carbon and hydrogen determinations. Z The following drugs were supplied gratis from the firms listed: 2~ethyl-2-(3-methyl)-butylmalonicacid diethylester from Eli LiUy 8r Co.; meprobamate from Wyeth Laboratories; Thorazine hydrochloride from Smith Kline and French Laboratories; Atarax hydrochloride from Charles Pfizer and Co.; and Serpasil phosphate lyophilized crystals from Ciba Pharmaceutical Products, Inc.
Vol. 50, No. 10, October 1961 TABLE L-DRUGS
825 AND
DOSESUSED I N OPERANT CONDITIONING TECHNIQUE"
Drug
Lot and Control No.
Reserpine phosphate Ciba E 4961 SKF Cu 7664 Chlorpromazine hydrochloride Mep r ob a mate Wyeth L 3286-1 Hydroxyzine hydrochloride Pfuer 71407 d-Amphetamine sulfate Lack DA 675 Amobarbital Eli Lilly AX-9643-F Ethyl-Z,4-toluene dicarbamate ... 2-Ethyl-2-( 3-methyl)-butylmalondiamide 2-Ethyl-2-( 3-methyl)-butylmalonic acid diethylester Eli Lilly 11039 Control a
Low, mg./Kg.
Medium, mg./Kg.
HY,, mg. Kg
0.5 0.5 10.0 5.0 0.1 5.0 50.0 10.0 50.0
1.0 2.0 50.0 25.0 1.0 10.0 75.0 50.0 100.0
2.0 4.0 100.0 35.0 5.0 20.0 90.0 100.0 200.0
All drugs given by i. p. injection.
one-half hour before the animal was placed in the conditioning chamber. The test animals were reconditioned under the influence of drugs for two days. After the drug was stopped, the animal continued to recondition until a rate of a t least 100 bar presses was achieved during a fifteen-minute session. The animal was then given the same drug and extinguished for two days. After the drug was withdrawn, extinction was continued until a rate of 10 bar presses or less was achieved in a fifteen-minute session. I n those cases where extinction at the desired rate resulted on the first and/or second day in rats under the influence of a drug, at least one additional day was allowed for the animal t o extinguish to the desired rate. It was felt this would disclose any obviously drug-induced depression of the extinction rate. All animals were reconditioned and extinguished with drugs as many times as was feasible. A control group of four rats was used for each progressive reconditioning and extinction series.
RESULTS The data obtained was analyzed statistically by means of the t test (8). Before subjecting the data t o the t test, it was first determined that deviations from the means of the responses in both acquisition and extinction were normally distributed. Then, in order to compensate for the different learning abilities of the animals used, the total number of bar presses for each animal obtained during its first fifteen-minute session either under the influence of the drug or when serving as a control animal during acquisition (D)was subtracted from the total number of bar presses obtained during the first five 15-minute trials during which the animal first received reinforcement in response t o each bar press ( N ) . Negative numbers which resulted were then eliminated by adding t o each result a constant (C), the largest negative number obtained from all the results. These numbers were used in the calculation of the t tests. The data obtained during extinction was similarly treated, with the exception that the total number of bar presses for each animal on the first 15-minute extinction trial was used compared to the total number of bar presses obtained during the first five 15-minute sessions of acquisition. Tables I1 and I11 list the results of the t tests for the drugs and dose levels shown in Table I during acquisition and extinction, respectively. Tables IV and V present the original and
transformed data used in the calculation of the t tests for the different drugs which showed significant effects a t the 0.05 level of significance when compared to the controls. It should be noted that the measures N - D are inversely related to the energizing effects of the drugs. Thus, if drugged animals pressed the bar significantly more than the controls, the sums in the columns labeled C N - D for the drugged animals would be less than the sums in the column labeled C N - D for the control.
+
+
TABLE IL-TABLE OF t TESTSDERIVEDFROM THE COMPARISON OF RESPONSESMADE BY DRUGGED ANIMALSAND CONTROL ANIMALSDURINGACQUISITION OF OPERANT CONDITIONING~ Drug
?Dosage LevelLow Medium High
Reserpine PO, 1.68 Chlorpromazine HCl 1.56 Meprobamate 0.67 1.18 Hydroxyzine HC1 0.42 d-Amphetamine SO4 Amobarbital 3.19 Ethvl-2.4-toluene dicarbamate ' 0.14 2-Ethyl-2-( 3-methyl)-butylmalondiamide 0.82 2-Ethyl-2-( 3-methyl)-butylmalonic acid diethylester 4.03
0.40 1.09 0.08 1.11 2.29 0.78
0.31 1.67 3.44 1.33 0.43 2.14
0.44
0.38
0.03
0.66
0.28
0.31
Value of 2 at the 0.05 level of significance for 6 degrees of freedom is 2.447 (8). a
TABLE III.-TABLE OF t TESTSDERIVEDFROM THE COMPARISON OF RESPONSES MADEBY DRUGGED AND CONTROL ANIMALS DURINGOPERANTEXTINCTION" Drug
Reserpine POr Chlorpromazine HCI Meprobamate Hydroxyzine HCI d-Amphetamine SO4 Amobarbital Ethyl-2,4-toluene dicarbamate 2- Ethyl-2-( 3-methyl)-butylmalondiamide 2-Ethyl-2-( 3-methyl)-butylmalonic acid diethylester
Y D o s a g e Level--Low Medium High
1.10 1.87 0.65 1.56 0.29 1.17
1.05 1.99 2.21 2.37 3.10 0.18
2.37 1.10 1.55 1.13 3.29 0.53
0.01
2.03
0.21
2.14
1.09
0.59
1.42
1.33
2.17
Value of f at the 0.05 level of significance for 6 degrees of freedom is 2.447 (8). a
826
Journal of Pharmaceutical Sciences
TABLEIV.-ORIGINAL AND TRANSFORMED DATAOF THOSEDRUGSSHOWING SIGNIFICANT EFFECTSO N OPERANT CONDCTIONING AT THE 0.05 LEVEL OF SIGNIFICANCE WHENCOMPARED TO CONTROLS Group No.
Rat No.
N
D
N - D
C i - N - D
C4-N--I> 3rd Control
Amytal, 5 mg./Kg. (Third Reconditioning) 4 115 -111 29 116 Y-27 128 154 59 71 - 12 Y-29 53 159 4 91 87 Y-31 - 124 16 253 12 136 Y-33 Meprobamate, 100 mg./Kg. (First Reconditioning) 145" 4 48 - 44 96 11 Y-27 224a 59 61 - 2 138 Y-29 167a 4 82 - 78 62 Y-31 253a - 101 39 12 123 Y-33 2-Ethy1-2-(3-inethyl)-butylrnalonic Acid Diethylester, 50 mg./Kg. (First Reconditioning) 22 118 145" 62 84 13 Y-21 52 224a 4 88 - 84 Y-23 - 51 89 167a 7 58 Y-35 187 105 82 58 256" Y-40 11
-
-
LA
First control.
TABLEV.-ORIGINAL AND TRANSFORMED DATAOF T H O ~ DRUGS E SHOWING SIGNIFICANT EFFECTSO N OPERANT EXTINCTION AT THE 0.05 LEVEL OF SIGNIFICANCE WHENCOMPARED TO CONTROLS Group No.
3
11
Rat No.
N
D
N - D
Dextro-Amphetamine Sulfate, 1 mg./Kg. (Second Extinction) Y-35 79 55 15 84 - 4 65 Y-37 55 51 2NY-17 69 87 - 18 51 19 6 13 82 Y-28 Dextro-Amphetamine Sulfate, 5 mg./Kg. (Third Extinction) Y-27 128 0 128 197 133 0 133 202 Y-29 .. 183 Y-3i 114 0 114 285 354 Y-33 285 0 ~~
a
C4-N-D
C4-N-D 2nd Control
131 118 61 126 71a 101Q 122a 93Q
Third control.
SUMMARY A N D CONCLUSIONS Little has been reported previously concerning the acquisition of bar-pressing habit as a measure of the effects of drugs. As a rule, most of the experimentation of this nature has been concerned with the effects of drugs on already conditioned operant behavior, or on the extinction of such behavior. In the research presented here, it was proposed originally that an attempt would be made to determine the effects of drugs on the acquisition and extinction of operant behavior with the possibility in mind of comparing this technique to avoidance-escape conditioning relative t o the effects of drugs. It was determined, however, that the experimental design which was followed would allow only a gross measure of the effect of drugs on operant conditioning and ex tinction. This precluded any comparative evaluation of the two measures. The results of the r tests in both the operant conditioning and extisction procedures suggested statistically subliminal effects which probably were not detected because of the "noise" in the experimental design.
The results obtained through these procedures do allow some pertinent conclusions to be drawn, however. Thus, the effects of d-amphetamine sulfate indicated that 1 mg./Kg. appears to increase bar-press activity and 5 mg./Kg appears to decrease bar-press activity when compared to the controls. An examination d of the first day's effects of the different drugs tested at the dose ranges shown in Table I on both conditioning and extinction indicated that only those drugs which interfered with normal motor activity (e. g., amobarbital, meprobamate, and 2-ethyl-2-(3-methyl)-butylmalonic acid diethylester) and those drugs with anorexic properties (e. g., dextro-amphetamine sulfate) had significant effects when compared to the controls. While the t tests demonstrate that these several drugs do affect beFvior significantly, these results cannot be construed to mean that those drugs had any significant effect on the contingent learning process becaw of the associated effects of these compounds. On the other hand, it would be erroneous to suggest that these drugs had no effect on psycho-
Vol. SO, No. 10, Ortober 1961
827
logical processes. Rather i t is suggested that a
REFERENCES
refinement of technique is required to differentiate between the various effects. In the course of these experiments, it was further noted that the drugs used in this study had more pronounced pharmacological effects on the animals as compared to the animals used in avoidance-escape training which were allowed to feed ad libitum.
J o ( ~ ~ & ~ n $ $&5~i~&e'o~ M. D., and
R. K.,
( 2 ) Pfeiffer, C . C . , et a l . , A n n . N . Y . Acad. Sci., 67, 734 (1 957). E . , and Weiss, H . , J . B i d . Chem., 214, 807
($;)5,T't"",
(4) Berger, F. M., and Ludwig, B. J., J. Phaumacol. Ezptl.
Therap., 100, 27(1950).
( 5 ) Berger, F. M., ibid., 104, 229(1952). (6) Toman, J . E. P., el a l . , i b i d . , 106, 419(1952). (7) Summa, A. F . , and Jannke, P. J . , THISJOURNAL, 46,
363(1957). ( 8 ) Edwards, A. L., -Experimental ~~~i~~ in psycho. logical Research," Rinehart & Co., New York, N . Y., 1954, pp. 150, 151.
In Vitro Tests for Measuring Antibacterial Activity of Toilet Soap and Detergent Bars By L. J. VINSON, E. L. AMBYE, A. G. BENNETT, W. C. SCHNEIDER, and J. J. TRAVERS Two new simple in witro screening tests are described which have proved valuable in predicting the degerming effectiveness of germicidal toilet bars under regular use conditions. The tests, skin disk substantivity and finger imprint tests, measure the residual antibacterial activity of skin treated with soap solutions followed by a water rinse. Thus, a soap germicide is evaluated on the basis of its capacity to become adsorbed to skin and its ability, at low concentrations, to inhibit the growth of bacteria. In witro test data are described on 3,4',~-tribromosalicylanilide and other germicides incorporated into soap and detergent bars. Good correlation between the new in vitro test results and regular hand washing results (serial basin wash test) is achieved.
are being employed I more and more ingermicides toilet bars, contributing imN RECENT YEARS
portant properties not possessed by plain soaps. Although antiseptic agents have been used in soaps for many years it was only with the introduction of soap germicides like the bisphenolics (1, Z ) , tetramethylthiuramdisulfide (3, 4), and trichlorocarbanilide ( 5 ) that a substantial degerming of skin was achieved in regular washing. These germicides all possessed in common two properties to varying degrees: ( a ) antibacterial action a t low concentrations against Grampositive bacteria (micrococci), normal residents of skin which are not destroyed by exposure to plain soap washing; ( b ) an affinity for skin such that during washing with a soap, the germicide is adsorbed on the epidermis and is not washed out during the rinse operation. The presence of trace amounts of an effective germicide on the skin has the effect of suppressing the growth of the resident bacteria that feed on skin detritus. Received March 8, 1961, from Lever Brothers Co., Research and Development Division, Edgewater, N . J Accepted for publication March 17, 1SG1.
Thus, the typical "body odor" which arises from bacterial growth can be reduced. Standard bacteriological procedures like the phenol coefficient test or toxic dilution tests, employing seeded agar plates or broth tubes, are not good methods for predicting the effectiveness of germicides in toilet bars. A preparation may be effective at very low concentrations as demonstrated in such tests but may not be substantive to skin, or i t may prove incompatible with the vehicle. The practical usefulness of germicidal soaps is determined in the serial basin wash test which provides antibacterial data in terms of per cent reduction in hand bacterial counts after regular use of the test bar. This method, however, is tedious and time consuming and care must be taken in interpreting results with small panels (6). Another practical test for assessing germicidal bars is to determine the inhibitory effect on perspiration odor development of washed axillae (3). In the screening of a new germicide simple tests are necessary to determine not only the minimum effective concentration in various
HE TRANQUILIZING drugs comprise one of the Tareas of recent interest in the field of pharmacology. A considerable amount of experimentation and speculation has been undertaken in order to attempt to discover new compounds of this type and also to provide a basis for the understanding of the mode of action of these drugs. However, little progress has been reported towards developing a unique test to determine the value of these compounds as regards their ability to alter behavior. The reason may be that tranquilizing drugs, like most other drugs, have potential, actual, primary, and/or secondary effects manifested as ataxia, depression, anorexia, ptosis of the eyelids, flaccid paralysis, and the like. These latter points constitute an important aspect in the development of tranquilizers and the effects of these drugs on animal behavior. Equally important is the aspect of chemical structure-activity relationships. Indeed, a large number of tranquilizers used therapeutically were the result of investigation in this area. With these effects in mind, this problem was designed and undertaken in an effort to contrihute further to this area. It was felt that one approach in determining whether or not the actual, potential, primary, and/or secondary effects of tranquilizers and other centrally active drugs contributed to overall behavior was to compare the same drugs at the same dose levels using two different experimental techniques. Specifically, it was felt that the results obtained by the application of operant conditioning techniques would not only provide information based on its own merits Rewived November 5 . 1960. from the School of Pharmacv. ...~ .~ University of C o i n e i t i i i t , stoms. Accepted for publication March 27, 1961. Abstracted in part from dissertations submitted to the Graduate School of the University of Connecticut by Vincent D. Lynch and Mario D. Aceto in partial fulfillmentof therequirements for the degree of Doctor of Philosophy, June 1959. t Present address: School of Pharmacy, University of Pittsburgh Pittsburgh 13, Pa. Z Preseni address: College of Pharmacy, S t . John’s University, Jamaica 32, N. Y. ~
~~
_
I
but would contrast or complement the results obtained by using an avoidance-escape technique (1). The two techniques are based entirely on different psychological principles and might serve to emphasize towhat extent these actual, potential, primary, and/or secondary effects influence behavior and perhaps, within the limits of these two testing procedures, the type behavior. Pfeiffer, et al. (2), in a comparison of the effects of meprobamate with other classes of drugs active in the central nervous system concluded that this compound has a prolonged barbiturate-like action mixed with a moderate degree of trimethadione effect on the brain. Furthermore, they claimed that the pharmacological action resembled phenobarbital or trimethadione even more than it did its congener mephenesin. They felt that meprobamate is not an ataractic drug as is chlorpromazine, and that it exhibits few properties of mephenesin. It is interesting to note that Titus and Weiss (3) in an investigation of the metabolism of phenobarbital 2-CI4identified by means of paper chromatography nine metabolites, one of which was reported to be urea-C14 representing 2.5 per cent of the products. This metabolite strongly suggested that a certain amount of ring cleavage takes place in the metabolism of phenobarbital and possibly other barbiturates. An examination of the chemical structure of meprodibamate, 2-methyl-2-n-propyl-1,3-propanediol carbamate, showed various features in common with the barbiturates. The most obvious is the quaternary carbon atom. Both hydrogens attached to the carbon atom at position 5 in barbituric acid must be replaced by alkyl or aryl groups in order to manifest hypnotic properties. If the carbonyl group at position 2 in a barbiturate such as amobarbital (5-ethyl-5-isoamylbarbituric acid) is removed, the result will be amides not unlike the carbamide groups of meprobamate. Many compounds with quaternary carbon atoms have been shown to possess
823
Journal of Pharmaceutical Sciences
824 interesting central effects. Already mentioned were the sedative and hypnotic effects of the barbiturates. Meprobamate shows tranquilizing and spasmolytic efFects. Berger and Ludwig (4) examined various substituted propane-1,3diols and found tha,t diethyl propanediol (2,2diethylpropane-1,3-diol) has the same effect on the spinal cord as mephenesin. In view of the information available, it was proposed that various compounds closely related to both the barbiturates and meprobamate be investigated. Chosen for this study were 2ethyl-2-(3-methyl)-butylmalondiamide and 2ethyl-2- (3-methyl)-butylndonic acid diethylester. The ester contains no nitrogen and it was thought that its effects might provide an interesting contrast with 2-ethyl-2- (3-methyl)-butylmalondiamide when tested on animal behavior. Another compound, ethyl-2,4-toluene dicarbamate, was chosen for investigation because of the structural similarity between it and some of the otoloxy derivatives investigated by Berger (5) and also because it conta.ined the amide group which Toman (6) postulated should be present in order for a compound to have central depressant properties. Preliminary studies of several homologs of this compound demonstrated that this series of compounds possessed sedative and depressant properties worthy of further investigation.
EXPERIMENTAL. Preparation of 2-Ethyl-2-( 3-methyl)-butylmalondiamide.-Eighteen grams of 2-ethyl-2-(3-methyl)butylmalonic acid diethylester was hydrolyzed by refluxing on a steam bath for twenty-four hours with 30 ml. of a 50% hydroalcoholic (1:l) potassium hydroxide solution. The reaction mixture was diluted with 50 ml. of water and acidified to Congo red paper with concentrated hydrochloric acid. After cooling, the immiscible layers were separated and the aqueous portion was extracted with ether. The organic portions were combined and the ethereal solution was extracted with 5% aqueous sodium bicarbonate solution. The aqueous alkaline extract was acidified with hydrochloric acid and re-extracted with ether. Evaporation of the combined ether extracts yielded the free acid. After adding thionyl chloride dropwise to remove traces of water, an excess of the reagent was added, and the mixture was re3uxed on a steam bath for one hour. The residual thionyl chloride was removed by distillation and the remaining acyl chloride was dissolved in 100 ml. of dry hexane. Dry ammonia was bubbled through the solution until the resulting suspension of diamide became a. thick slurry. The solid was collected on a sintered-glass funnel and was washed with dry hexane, the filtrate being further treated with ammonia until no more precipitate formed. After being air dried, the crude diamide was extracted with hot ethanol and the filtered alcoholic solution was evaporated to dryness on a steam bath.
After washing the residue with water to remove ammonium chloride and other impurities, it was dried and recrystallized from ethanol. The yield was 42.1%, m. p. 190-191° (uncorr.). AnaZ.l-Calcd. for CIOHZONZOZ: N, 13.98; C, 59.97; H, 10.07. Found: N, 13.22, C, 59.62; H, 10.11. Drugs.-The drugs and dosage levels are listed in Table I. Except for the diamide and dicarbamate (7) which were synthesized in our laboratories, all other drugs were obtained commercially or supplied by the manufacturer.2 Distilled water was used as the solvent for the relatively water-soluble drugs. Methocel(O.5%) was used as the suspending agent for the poorly water-soluble drugs. All suspensions were shaken prior to use. In all cases, the drug was either dissolved or suspended sp that the desired dose per Kg. of body weight of the rat was contained in a 1-ml. h a 1 volume of water. When not in use, the solutions were stored in a refrigerator. Animals.-Descendants of a Wistar strain of rats were used in this study. As regards sex, the animals were chosen at random. These animals weighed at least 150 Gm. a t the beginning of the test period. However, because of weight gains during the test period, some of the same animals weighed up to 310 Gm. at the end of the period. Each animal was housed separately, with a food intake maintained at between 8-11 Gm. daily. Water was allowed ad libitum. Operant Conditioning and Extinction Procedure. -A bar-press apparatus was used for this procedure. This device permitted a food pellet to be dispensed each time the foot pedal inside the experimental chamber was depressed. Separate counters registered both the total number of bar presses and tablets dispensed. The food dispenser was arranged so that it could be disconnected during the extinction procedure. The experimental chamber was located in a windowless room adjacent to which was the room housing the control panel and recording devices. After determining the operant level for each rat, it was conditioned to press the bar to obtain food. All the animals were conditioned to respond at least 100 times during a fifteen-minute session. With the pellet dispenser disconnected, each animal was then extinguished to a rate of ten bar presses or less during a fifteen-minute session. When a sufficient number of animals had been conditioned and extinguished, they were assigned to groups of four animals each. Each group was then assigned a t random to one of the drugs and one of the dose levels being investigated. Drugs were administered intraperitoneally in all cases, in order to insure optimum absorption. With the exception of reserpine and meprobamate, animals were injected with drugs one hour before admission to the test apparatus. Reserpine was administered two hours before and meprobamate 1 The authors wish to thank Professor W. Williams who performed the nitrogen determination and Dr. Robert Gerraughty who performed the carbon and hydrogen determinations. Z The following drugs were supplied gratis from the firms listed: 2~ethyl-2-(3-methyl)-butylmalonicacid diethylester from Eli LiUy 8r Co.; meprobamate from Wyeth Laboratories; Thorazine hydrochloride from Smith Kline and French Laboratories; Atarax hydrochloride from Charles Pfizer and Co.; and Serpasil phosphate lyophilized crystals from Ciba Pharmaceutical Products, Inc.
Vol. 50, No. 10, October 1961 TABLE L-DRUGS
825 AND
DOSESUSED I N OPERANT CONDITIONING TECHNIQUE"
Drug
Lot and Control No.
Reserpine phosphate Ciba E 4961 SKF Cu 7664 Chlorpromazine hydrochloride Mep r ob a mate Wyeth L 3286-1 Hydroxyzine hydrochloride Pfuer 71407 d-Amphetamine sulfate Lack DA 675 Amobarbital Eli Lilly AX-9643-F Ethyl-Z,4-toluene dicarbamate ... 2-Ethyl-2-( 3-methyl)-butylmalondiamide 2-Ethyl-2-( 3-methyl)-butylmalonic acid diethylester Eli Lilly 11039 Control a
Low, mg./Kg.
Medium, mg./Kg.
HY,, mg. Kg
0.5 0.5 10.0 5.0 0.1 5.0 50.0 10.0 50.0
1.0 2.0 50.0 25.0 1.0 10.0 75.0 50.0 100.0
2.0 4.0 100.0 35.0 5.0 20.0 90.0 100.0 200.0
All drugs given by i. p. injection.
one-half hour before the animal was placed in the conditioning chamber. The test animals were reconditioned under the influence of drugs for two days. After the drug was stopped, the animal continued to recondition until a rate of a t least 100 bar presses was achieved during a fifteen-minute session. The animal was then given the same drug and extinguished for two days. After the drug was withdrawn, extinction was continued until a rate of 10 bar presses or less was achieved in a fifteen-minute session. I n those cases where extinction at the desired rate resulted on the first and/or second day in rats under the influence of a drug, at least one additional day was allowed for the animal t o extinguish to the desired rate. It was felt this would disclose any obviously drug-induced depression of the extinction rate. All animals were reconditioned and extinguished with drugs as many times as was feasible. A control group of four rats was used for each progressive reconditioning and extinction series.
RESULTS The data obtained was analyzed statistically by means of the t test (8). Before subjecting the data t o the t test, it was first determined that deviations from the means of the responses in both acquisition and extinction were normally distributed. Then, in order to compensate for the different learning abilities of the animals used, the total number of bar presses for each animal obtained during its first fifteen-minute session either under the influence of the drug or when serving as a control animal during acquisition (D)was subtracted from the total number of bar presses obtained during the first five 15-minute trials during which the animal first received reinforcement in response t o each bar press ( N ) . Negative numbers which resulted were then eliminated by adding t o each result a constant (C), the largest negative number obtained from all the results. These numbers were used in the calculation of the t tests. The data obtained during extinction was similarly treated, with the exception that the total number of bar presses for each animal on the first 15-minute extinction trial was used compared to the total number of bar presses obtained during the first five 15-minute sessions of acquisition. Tables I1 and I11 list the results of the t tests for the drugs and dose levels shown in Table I during acquisition and extinction, respectively. Tables IV and V present the original and
transformed data used in the calculation of the t tests for the different drugs which showed significant effects a t the 0.05 level of significance when compared to the controls. It should be noted that the measures N - D are inversely related to the energizing effects of the drugs. Thus, if drugged animals pressed the bar significantly more than the controls, the sums in the columns labeled C N - D for the drugged animals would be less than the sums in the column labeled C N - D for the control.
+
+
TABLE IL-TABLE OF t TESTSDERIVEDFROM THE COMPARISON OF RESPONSESMADE BY DRUGGED ANIMALSAND CONTROL ANIMALSDURINGACQUISITION OF OPERANT CONDITIONING~ Drug
?Dosage LevelLow Medium High
Reserpine PO, 1.68 Chlorpromazine HCl 1.56 Meprobamate 0.67 1.18 Hydroxyzine HC1 0.42 d-Amphetamine SO4 Amobarbital 3.19 Ethvl-2.4-toluene dicarbamate ' 0.14 2-Ethyl-2-( 3-methyl)-butylmalondiamide 0.82 2-Ethyl-2-( 3-methyl)-butylmalonic acid diethylester 4.03
0.40 1.09 0.08 1.11 2.29 0.78
0.31 1.67 3.44 1.33 0.43 2.14
0.44
0.38
0.03
0.66
0.28
0.31
Value of 2 at the 0.05 level of significance for 6 degrees of freedom is 2.447 (8). a
TABLE III.-TABLE OF t TESTSDERIVEDFROM THE COMPARISON OF RESPONSES MADEBY DRUGGED AND CONTROL ANIMALS DURINGOPERANTEXTINCTION" Drug
Reserpine POr Chlorpromazine HCI Meprobamate Hydroxyzine HCI d-Amphetamine SO4 Amobarbital Ethyl-2,4-toluene dicarbamate 2- Ethyl-2-( 3-methyl)-butylmalondiamide 2-Ethyl-2-( 3-methyl)-butylmalonic acid diethylester
Y D o s a g e Level--Low Medium High
1.10 1.87 0.65 1.56 0.29 1.17
1.05 1.99 2.21 2.37 3.10 0.18
2.37 1.10 1.55 1.13 3.29 0.53
0.01
2.03
0.21
2.14
1.09
0.59
1.42
1.33
2.17
Value of f at the 0.05 level of significance for 6 degrees of freedom is 2.447 (8). a
826
Journal of Pharmaceutical Sciences
TABLEIV.-ORIGINAL AND TRANSFORMED DATAOF THOSEDRUGSSHOWING SIGNIFICANT EFFECTSO N OPERANT CONDCTIONING AT THE 0.05 LEVEL OF SIGNIFICANCE WHENCOMPARED TO CONTROLS Group No.
Rat No.
N
D
N - D
C i - N - D
C4-N--I> 3rd Control
Amytal, 5 mg./Kg. (Third Reconditioning) 4 115 -111 29 116 Y-27 128 154 59 71 - 12 Y-29 53 159 4 91 87 Y-31 - 124 16 253 12 136 Y-33 Meprobamate, 100 mg./Kg. (First Reconditioning) 145" 4 48 - 44 96 11 Y-27 224a 59 61 - 2 138 Y-29 167a 4 82 - 78 62 Y-31 253a - 101 39 12 123 Y-33 2-Ethy1-2-(3-inethyl)-butylrnalonic Acid Diethylester, 50 mg./Kg. (First Reconditioning) 22 118 145" 62 84 13 Y-21 52 224a 4 88 - 84 Y-23 - 51 89 167a 7 58 Y-35 187 105 82 58 256" Y-40 11
-
-
LA
First control.
TABLEV.-ORIGINAL AND TRANSFORMED DATAOF T H O ~ DRUGS E SHOWING SIGNIFICANT EFFECTSO N OPERANT EXTINCTION AT THE 0.05 LEVEL OF SIGNIFICANCE WHENCOMPARED TO CONTROLS Group No.
3
11
Rat No.
N
D
N - D
Dextro-Amphetamine Sulfate, 1 mg./Kg. (Second Extinction) Y-35 79 55 15 84 - 4 65 Y-37 55 51 2NY-17 69 87 - 18 51 19 6 13 82 Y-28 Dextro-Amphetamine Sulfate, 5 mg./Kg. (Third Extinction) Y-27 128 0 128 197 133 0 133 202 Y-29 .. 183 Y-3i 114 0 114 285 354 Y-33 285 0 ~~
a
C4-N-D
C4-N-D 2nd Control
131 118 61 126 71a 101Q 122a 93Q
Third control.
SUMMARY A N D CONCLUSIONS Little has been reported previously concerning the acquisition of bar-pressing habit as a measure of the effects of drugs. As a rule, most of the experimentation of this nature has been concerned with the effects of drugs on already conditioned operant behavior, or on the extinction of such behavior. In the research presented here, it was proposed originally that an attempt would be made to determine the effects of drugs on the acquisition and extinction of operant behavior with the possibility in mind of comparing this technique to avoidance-escape conditioning relative t o the effects of drugs. It was determined, however, that the experimental design which was followed would allow only a gross measure of the effect of drugs on operant conditioning and ex tinction. This precluded any comparative evaluation of the two measures. The results of the r tests in both the operant conditioning and extisction procedures suggested statistically subliminal effects which probably were not detected because of the "noise" in the experimental design.
The results obtained through these procedures do allow some pertinent conclusions to be drawn, however. Thus, the effects of d-amphetamine sulfate indicated that 1 mg./Kg. appears to increase bar-press activity and 5 mg./Kg appears to decrease bar-press activity when compared to the controls. An examination d of the first day's effects of the different drugs tested at the dose ranges shown in Table I on both conditioning and extinction indicated that only those drugs which interfered with normal motor activity (e. g., amobarbital, meprobamate, and 2-ethyl-2-(3-methyl)-butylmalonic acid diethylester) and those drugs with anorexic properties (e. g., dextro-amphetamine sulfate) had significant effects when compared to the controls. While the t tests demonstrate that these several drugs do affect beFvior significantly, these results cannot be construed to mean that those drugs had any significant effect on the contingent learning process becaw of the associated effects of these compounds. On the other hand, it would be erroneous to suggest that these drugs had no effect on psycho-
Vol. SO, No. 10, Ortober 1961
827
logical processes. Rather i t is suggested that a
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refinement of technique is required to differentiate between the various effects. In the course of these experiments, it was further noted that the drugs used in this study had more pronounced pharmacological effects on the animals as compared to the animals used in avoidance-escape training which were allowed to feed ad libitum.
J o ( ~ ~ & ~ n $ $&5~i~&e'o~ M. D., and
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( 2 ) Pfeiffer, C . C . , et a l . , A n n . N . Y . Acad. Sci., 67, 734 (1 957). E . , and Weiss, H . , J . B i d . Chem., 214, 807
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Therap., 100, 27(1950).
( 5 ) Berger, F. M., ibid., 104, 229(1952). (6) Toman, J . E. P., el a l . , i b i d . , 106, 419(1952). (7) Summa, A. F . , and Jannke, P. J . , THISJOURNAL, 46,
363(1957). ( 8 ) Edwards, A. L., -Experimental ~~~i~~ in psycho. logical Research," Rinehart & Co., New York, N . Y., 1954, pp. 150, 151.
In Vitro Tests for Measuring Antibacterial Activity of Toilet Soap and Detergent Bars By L. J. VINSON, E. L. AMBYE, A. G. BENNETT, W. C. SCHNEIDER, and J. J. TRAVERS Two new simple in witro screening tests are described which have proved valuable in predicting the degerming effectiveness of germicidal toilet bars under regular use conditions. The tests, skin disk substantivity and finger imprint tests, measure the residual antibacterial activity of skin treated with soap solutions followed by a water rinse. Thus, a soap germicide is evaluated on the basis of its capacity to become adsorbed to skin and its ability, at low concentrations, to inhibit the growth of bacteria. In witro test data are described on 3,4',~-tribromosalicylanilide and other germicides incorporated into soap and detergent bars. Good correlation between the new in vitro test results and regular hand washing results (serial basin wash test) is achieved.
are being employed I more and more ingermicides toilet bars, contributing imN RECENT YEARS
portant properties not possessed by plain soaps. Although antiseptic agents have been used in soaps for many years it was only with the introduction of soap germicides like the bisphenolics (1, Z ) , tetramethylthiuramdisulfide (3, 4), and trichlorocarbanilide ( 5 ) that a substantial degerming of skin was achieved in regular washing. These germicides all possessed in common two properties to varying degrees: ( a ) antibacterial action a t low concentrations against Grampositive bacteria (micrococci), normal residents of skin which are not destroyed by exposure to plain soap washing; ( b ) an affinity for skin such that during washing with a soap, the germicide is adsorbed on the epidermis and is not washed out during the rinse operation. The presence of trace amounts of an effective germicide on the skin has the effect of suppressing the growth of the resident bacteria that feed on skin detritus. Received March 8, 1961, from Lever Brothers Co., Research and Development Division, Edgewater, N . J Accepted for publication March 17, 1SG1.
Thus, the typical "body odor" which arises from bacterial growth can be reduced. Standard bacteriological procedures like the phenol coefficient test or toxic dilution tests, employing seeded agar plates or broth tubes, are not good methods for predicting the effectiveness of germicides in toilet bars. A preparation may be effective at very low concentrations as demonstrated in such tests but may not be substantive to skin, or i t may prove incompatible with the vehicle. The practical usefulness of germicidal soaps is determined in the serial basin wash test which provides antibacterial data in terms of per cent reduction in hand bacterial counts after regular use of the test bar. This method, however, is tedious and time consuming and care must be taken in interpreting results with small panels (6). Another practical test for assessing germicidal bars is to determine the inhibitory effect on perspiration odor development of washed axillae (3). In the screening of a new germicide simple tests are necessary to determine not only the minimum effective concentration in various