THE INFLUENCE OF ANESTHESIA ON THE EVALUATION OF ANTITUSSIVE EFFECT

THE

INFLUENCE

EVALUATION YOSHITOSHI

OF OF

KASE,

ANESTHESIA ANTITUSSIVE

TOMOKAZU

YUIZONO

ON

THE

EFFECT AND GO KITO

Departmentof Chemico-Pharmacology, Facultyof Pharmaceutical Sciences,KumamotoUniversity,Kumamoto Received for publication September 9, 1968

Studies on the antitussive agents have been advanced with the development of bio assay for them. At the present time, a large number of non-narcotic synthetic antitussives are employed in the management of cough instead of narcotic antitussives. The evaluation method for antitussive activity was first reported by Ernst in 1938 (1), that is, coughing was induced by pinching the trachea of a cat which had previously been made to sero-fibrinous pleurisy by means of intrapleural injection of Lugol solution. Two years later, Eichler and Smiatek (2) reported that coughing could be induced in the guinea pig by an inhalation of aerosolized sulfuric acid. However, these two methods have not been accepted widely for the evaluation of antitussive agents because of uncertainty of cough production. In 1952, Kase devised two methods (3, 4) : 1) electrical stimulation of the superior laryngeal nerve and 2) mechanical stimulation on the tracheal mucosa in the unanesthetized dog. At the same time, Domenjoz (5) reported a method, in which coughs were able to be induced by electric stimulation of the superior laryngeal nerve in the anes thetized cat. Kase as well as Domenjoz insisted that their own methods were suitable for the antitussive evaluation and the results obtained from the animal experiments were in good accordance with clinical effects. Since then, about thirty methods have been pre sented one after another for the evaluation of antitussive activity. However, they are classified in three groups after all: 1) mechanical stumulation method (3, 4, 6-13), 2) chemical one (2, 6, 8, 10-12, 14-20), and 3) electrical one (3, 5, 21-26). Anesthesia is used in most of the methods described above. In general, anesthesia has been used as one of routine procedures in animal experiments, but cough reflex is af fected by it to a great extent. If anesthesia is an inevitable procedure in the evaluation of antitussives, the influence of anesthesia on antitussive activity should be studied in detail. As far as we know, there has been no report on such study until the present . In the present paper, the influence of anesthesia on antitussive effect was studied in detail and a few remarks necessary for making use of anesthesia in the evaluation of an titussive agents were described. MATERIALSAND METHODS A.

Materials Animals: The mongrel dogs weighing 5-15 kg, cats weighing 2-3 kg, and guinea

加 瀬  佳 年 ・由 井薗   倫 一 ・鬼 頭  剛

pigs weighing 400-600 g, of both sexes were used. Test drugs: a) Antitussives. Seven drugs used for the present experiments are di vided in the following four groups. I-Group. Potent centrally-acting antitussive agents with analgesic and other CNS activities-morphine hydrochloride and thiambutene hydrochloride [3-dime thylamino-1, 1-di(thien-2-yl) but l -ene hydrochloride, OhtonR]. II-Group. Regarded as a standard antitussive agent possessing moderate antitussive and CNS actions-codeine phosphate. III-Group. Antitussive activity is equal to or slightly weaker than that of codeine, and the drugs have no analgesic activity and their actions on the CNS are rather excitatory than depressive-dextromethorphan hydrobromide, noscapine hydrochloride, and 1 methyl-3-di(thien-2-yl)methylenepiperidine citrate (Asverin%. IV-Group. Antitussive action is said to be due to selective local anesthesia of the alveolar stretch receptors of the lungs-benzonatate (TessalonR). b) Anesthetic: Pentobarbital sodium (Nembutal®-) was used as a representative anesthetic. B.

Methods Coughs were artificially induced by the following methods. 1. Mechanical stimulation of the tracheal mucosa The "coughing dog and cat" methods (3, 9, 27) were used. The dog was used without anesthesia, while the cat was lightly anesthetized with pentobarbital sodium (20 mg/kg, i.p.). Coughs were induced by mechanical stimulation with a bristle stimulator on the mucosa of the tracheal bifurcation through a chronic tracheal fistula which had previous ly been built. Stimulator: twenty bristles of a pig were radially tied around one end of a wire about 25-30 cm in length (for the dog), while fifteen whiskers of a rabbit were tied similarly (for the cat). The stimulator was moved up and down 10 times for 20 seconds with an intensity as constant as possible and stimulation was repeated every 5 minutes or at suitable intervals. The criterion of evaluation was as follows: When the amplitude and/or the frequency of the cough curves on a smoked paper were decreased by more than 20% as compared with the pretreatment control and when such decreases lasted for more than 20 minutes after injection of the drug, the effect was considered to be significant. For the guinea pig, the method of Takagi et al. (11) was used. A guinea pig was lightly anesthetized with pentobarbital sodium (15 mg/kg, i.p.) and fixed on its back. The trachea was exposed and a small hole was cut in it. Through this hole a stimulating bristle (a hog bristle 5 cm long) was inserted into the trachea to a depth of 3 cm with a gradient of about 30° to the longitudinal axis of the trachea for 1 second and then taken out. Violent coughing occurred upon stimulation. When no coughing occurs even once in 5 trials at 15, 30, 60, 90 and 120 minutes after injection, the drug to be tested was regarded as effectvie. 2. Chemical stimulationof the tracheal mucosa Sulfur oxide gas produced from sodium thiosulfate and concentrated sulfuric acid was

reserved in a vinyl plastic bag with two tubes, each of which is provided with a rubber stopper. The gas was freshly prepared and reserved every day. A needle of syringe was inserted through the stopper and a certain amount of SO, was taken into the syringe. A fine polyethylene tube, to which the needle of syringe was connected, was inserted into the trachea deeply through a branch of a tracheal cannula and the gas was quickly blown into the trachea to evoke coughing (in most cases, a single cough). In many instances, volumes of the gas necessary to evoke coughing were 2-5 ml for dogs and 1-2 ml for cats. The criterion of evaluation is similar to that described in the mechanical stimulation. 3. Electric stimulation of the superior laryngeal nerve The superior laryngeal nerve was stimulated electrically according to the method of Kase in the dog (3) and that of Domenjoz in the cat (5). The parameter of stimulus was as follows: Rectangular wave; pulse duration, 1 msec; frequency, 20 cps; voltage, 0.1 5.0 Volt. The duration of stimulation was 10 seconds. The antitussive effect was de termined from the elevation of threshold stimulus requirement and the decreases in am plitude and/or frequency of tracing of coughs induced by a stimulus of moderate intensity. The depth of anesthesia was determined by the method of Sakamoto (28), which is a modified one of Magnus and Girndt method (29), and classified in five degrees such as 0, I, II, III, IV and V. The degree 0 means wakeful state, I the most superficial anesthetic state, and V the deepest. Other experimental methods will be described in each part concerned. RESULTS 1. Relation between the antitussiveactivity and the depth of anesthesia The principles of this study is as follows : A fixed dose of thiambutene, which had previously been determined as an ineffective one when given intravenously to test animals under conscious state, was administered repeatedly to the same test animals under various states of anesthesia and antitussive effect of the drug was determined at suitable intervals during the course of anesthesia. The "coughing dog and cat" methods were used. The antitussive effect of a fixed dose of thiambutene (0.64 mg/kg for dogs, and 2.0 mg/kg for cats, intravenously) was deter mined before anesthesia, and it was confirmed that no effect was seen with respective doses under unanesthetized state. Pentobarbital sodium (20 mg/kg) was given intraperitoneal ly to the animals. The same dose of thiambutene as before was given intravenously at 1, 3, 5 and 7 hour intervals and the antitussive effects which might be considerably affected by the anesthesia were checked at each interval. The results are shown in Fig. 1. 1) The time-course of depth of anesthesia in the dog and cat was as follows : At the first hour after the administration of pentobarbital, the depth of anesthesia was at III IV degrees; at the 3rd hour, I-II degrees; at the 5th hour, I-->O degrees (between I and O degrees, nearly awake) ; at the 7th hour, the animals returned to the pretreatment state (0 degree).

FiG. 1. Relationship between the antitussive effect and the time-course of anesthesia in the dog. The depth of anesthesia was classified in five degrees. All the arrows indicate the intravenous administration of thiambutene HC1 0.64 mg/kg. Coughs were induced by mechanical stimulation of the tracheal mucosa ("coughing dog" method).

2)

Antitussive effect of the same dose of thiambutene, shown by decreases in am

plitude and frequency of the cough curves, decreased in parallel with awakening from anesthesia. 3) The duration of antitussive effect described above also shortened in parallel with the decrease in the depth of anesthesia. The results above definitely show that change in the depth of anesthesia has signifi cant influences not only on the potency of antitussive effect but also on the duration of the effect of the test drug in both animal species used. 2.

Changesin 50% antitussiveeffectivedose and in the duration of antitussiveeffectof various test drugs due to anesthesia As the antitussive activities were remarkably affected by the time-course of anesthesia as mentioned above, to the next, it was studied what kinds of antitussives are more pro foundly affected by anesthesia. In order to examine this, antitussive effects of the test drugs under unanesthetized state were compared with those under a constant level of ane sthesia. Dogs, cats and guinea pigs were used. The determination of antitussive effect under anesthesia was carried out within 1-2 hours after the administration of pentobarbital sodi um (20 mg/kg, i.p. for dogs and cats; 15 mg/kg, i.p. for guinea pigs). More than 4 groups of animals were used and each group consisted of more than 5 animals. From the effects of the drugs shown by intravenous administration (dogs and cats) or intraperitoneal one (guinea pigs), 50% antitussive dose (abbreviated as AtD5o) and its fiducial limits (p=0.05) were calculated by the method of Litchfield and Wilcoxon.

The test drugs were given intravenously to dogs and cats, intraperitoneally to guinea pigs. Twenty mg/kg, intraperitoneally for dogs and cats ; 15 mg/kg, intraperitoneally for guinea pigs. AtD50 (I) /AtD50 (II ). *Excitation occurred . **Excitation and convulsions occurred. sig.-significant, ins ig.-insignificant.

A. 50% Antitussive effectivedose 1) Mechanical stimulation i) In three animal species used, the antitussive effects were increased by anesthesia. In the dog and cat, significant increases were observed with the test drugs except bent zonatate (in the dog and cat) and noscapine (in the cat). Particularly in case of potent centrally-acting antitussive drugs (I-Group), potentiation was more marked, that is, mor phine became 1.80 (cat) to 4.68 (dog) times and thiambutene 1.66 (cat) to 3.39 (dog) times more potent. Although guinea pigs were so lightly pentobarbitalized (15 mg/kg, i.p.) that they were able to be awaked perfectly from anesthesia 3 hours later, they were also influenced more or less by anesthesia. However, the influence seemed to be surely less than those in the dog and cat. ii) Regardless of kind of antitussive agents, the influence of anesthesia was more

marked

in the dog than in the cat and guinea pig, and this tendency

most remarkable iii)

was, in particular,

as seen in the dogs used for the drugs of I-Group.

Antitussive effect of dextromethorphan

was apt to be modified to a great extent

TABLE 1-B. Comparison of 50% antitussive dose determined by chemical stimulation method.

TABLE 1-C. Comparison superior laryngeal

of 50% nerve.

antitussive

dose

determined

by

electrical

stimulation

of the

by anesthesia. It was difficult to demonstrate a clear-cut antitussive effect of the drug in unanesthetized dogs even when such a large dose as 28.8 mg/kg (severe convulsions oc curred and occasioned death) was given intravenously, in contrast to this, a definite anti tussive effect was able to be demonstrated with such a smaller intravenous dose as 10.2 mg/kg under anesthesia. iv) Effect of codeine in the dog coincided approximately with that in the cat whether or not they were anesthetized. v) As shown in Table 1, toxic symptoms such as excitation, convulsions and so forth, often occurred even with AtD50 of some drugs, were able to be observed only in unanes thetized animals, in other words, anesthesia prevents the observation of such symptoms. 2) Chemicalstimulation Depressing effects of the test drugs on chemically-induced coughs coincided approxi mately with those obtained by mechanical stimulation method. Furthermore, influence of anesthesia is also of the same quality and of the same quantity as those seen in mechani cal stimulation. 3) Electrical stimulation The effects obtained by electric stimulation method coincided with those by mechani cal or chemical stimulation one. B. The duration of antitussiveeffect As the second experiment, the test drugs were given intravenously to the dog and cat, TABLE 2. Comparisonof the duration of antitussiveeffectin the dog and cat.

Coughs were induced by mechanical stimulation of tracheal mucosa. Pentobarbital sodium 20 mg/kg. i.p. §§ Duration (11) /Duration (I) . * Excitation occurred . ** Excitation and convulsions occurred.

and differencein the durationof antitussiveeffectsshownby'the samedoseof the drugs betweenunanesthetizedand 'anesthetizedstates was also studied. 1) Mechanical stimulation Theresults obtained from We dog and cat are as follows i) Prolongationin the duration of effectof the I-Group drugs by anesthesiawas more markedin dogs [.5 (thiambutene)to 6.3 (morphine)times] than in cats [2.4 (thiambutene)to 5.8 (morphine) times]. The prolongationwas definitelysignificant with a probabilityof 0.001or 0.05 (thiambutenein the cat). ii) $enzonatate,"a peripherally-actingantitussive,was not affected by anesthesia at all in the dog and cat. iii) In the dog and cat, not onlycodeine(possessing weakcentraldepressingactions) but also noscapine(possessingrather central stimulatingactions) exhibitedsignificant prolongation(about 2 times)in antitussiveeffect. iv) Asverin,belongingto III-Group,showedsignificantprolongationwhichis equal to the duration of I-Group drugs. v) The duration of effectof dextromethorphen,which was able to manifestanti tussiveeffectonlywith the aid of anesthesiain the dog as describedabove,was prolonged by anesthesia to about 4 times in the cat. 2) Chemicaland electrical stimulations Approximately the same results as those shown by mechanical stimulation method were obtained, that is, the effects of antitussives possessing central depressing action such as morphine and thiambutene were evidently prolonged by anesthesia, on the other hand, that of benzonatate was not modified at all. DISCUSSION Although anesthesia is widely used as a routine procedure in pharmacological ex periments involving the bioassay for antitussives, the depth of anesthesia is particulary important not only for the evaluation of various actions on the CNS but also for that of antitussive effect. It seems to be best to perform the evaluation of antitussive effect under conscious state, but it is not always able to be carried out in every animal species. When we are obliged to use anesthesia for antitussive evaluation, it is very necessary to have a thorough knowledge of influence of anesthesia on antitussive effect. May and Widdicombe (8)_demonstrated that pentobarbital sodium per se has antitus sive activity if a large amount of the drug was given, and Chakravarty et al. (26) did so in case of thiopental, too. These suggest that deep anesthesia itself interferes with the production of artificially-induced cough, so one must be careful to avoid deep anesthesia. However, no reports have been presented since then on how antitussive effect is varied with the depth of anesthesia. The-first experiment of the present -study, in which thiambutene was used as a short acting antitussive and pentobarbital sodium as a routine anesthetic, seems to be able part ly to solve the problem. Not only the antitussive effect of thiambutene but also the dura

tion of such an effect varied to a large extent with the depth of anesthesia, in other words, with the time-course of anesthesia. There is the possibility of misjudgement of evalua tion that a drug may be regarded as an ineffective antitussive when the drug was tested under unanesthetized or slightly anesthetized states, in contrast to this, it may be regarded as a potent one under deep anesthesia. Therefore, it is a problem when the evaluation should be done during a given anesthesia. In the second experiment, the influence of anesthesia on antitussive evaluation was studied from such points of view as antitussive effective dose and the duration of effect, in order to know what kinds of antitussives, experimental procedures, and animal species are mostly affected by anesthesia. The conclusions obtained from the experiments are as follows. As it had previously been expected, the centrally-acting antitussives (especially those have potent actions on the CNS such as morphine and thiambutene) were affected strong ly by anesthesia, on the contrary, the peripherally-acting ones were little affected, and other antitussives which show moderate activities on the CNS such as codeine, noscapine, and dextromethorphan were also influenced to some extent. Furthermore, the tendency described above had nothing to do with the experimental procedures, that is, it was out of question what kinds of stimuli were used for the production of cough. Potentiation of antitussive effect by anesthesia was most marked in the dog, followed by the cat and guinea pig. This indicated that the dog should be used under unanesthe tized state and the cat and guinea pig are allowed to be used under light anesthesia such as intraperitoneal administration of pentobarbital sodium, 20 mg/kg for the cat and 15 mg/kg for the guinea pig. In this case, however, it should be kept in mind that effect should be evaluated at a suitable time after the injection of an anesthetic agent, that is, in a suitable depth of anesthesia. According to the present authors' experience, evaluation within 2 hours after the administration of the respective dose (described above) of an anesthetic seems to be best. When conscious dogs are used for evaluation, it is of great advantage to know toxic symptoms which often appear with antitussive effective dose. SUMMARY The influence of anesthesia on the evaluation of antitussive activity was studied in the dog, cat and guinea pig. Four groups of antitussives, consisted of seven drugs possessing different sites of action and pharmacological properties such as morphine, thiambutene; codeine; dextromethor phan, noscapine, AsverinJ benzonatate, were used as test drugs, and pentobarbital was used as a representative anesthetic. 1. Both antitussive effect and duration of the effect were closely related to the depth of anesthesia. Therefore, when the evaluation is carried out under anesthetic state, it should be done during the period of time, in which the depth of anesthesia seems to be constant. 2. The effect and its duration of centrally-acting antitussives such as morphine and

thiambutene

were

trary,

of peripherally-acting

those 3.

Regardless

sive agents, than

4.

Toxic

animals,

were

guinea

remarkably

of the kinds

the influence

in the

5.

most

cat

and

pig are

of stimuli

allowed

and

prolonged

as benzonatate

used

by anesthesia, were

little

for the production

on antitussive

activity

of cough

was more

on the con

affected.

marked

and

antitus

in the dog

pig.

which

often

to be overlooked

It is recommended

such

of anesthesia

guinea

symptoms, apt

potentiated one

that

the

to be used

appear

with

antitussive

effective

very

often

during

anesthesia.

dog

must

be used

without

under

lightly

anesthetized

dose

anesthesia;

in conscious

the cat and

state.

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