ANALYSIS AND DIFFERENTIATION OF THE MECHANISM IN DEVELOPMENT OF DRUG TOLERANCE

RYUICHI KATO Department of Pharmacology and Department of Toxicology, National Institute of Hygienic Sciences, Setagaya-ku, Tokyo

Received for publication February 16, 1967

Repeated administration of many drugs, especially central acting drugs, cause a de velopment of tolerance to same or related drugs. The development of drug tolerance is an important problem for the evalution of drug actions and for the determination of a schedule of drug administration. However, the mechanism of development of drug tolerance is still in obscure. On the other hand, it is recently well demonstrated by several investigators that single or repeated administration of various drugs induces an increase in the activities of drug metabolizing enzymes of liver microsomes (1-4). Thus, the actions of the same drugs or other drugs given successively are markedly decreased through the increased rates of drug metabolisms (2, 4, 5). It has been well known that most of the central acting drugs are the inducers of drug-metabolizing enzymes of liver microsomes (2, 4, 5). The purpose of present communication, therefore, is to analyse and differentiate the mechanism of the development of drug tolerance. The results of the present investigation showed that there are two kind of drug toler ances and the first one would be called as apparent toleranceand the second one would be called as real tolerance. The development of the first one is mainely due to the induction of drug-metabolizing enzymes of liver microsomes and the second one is mainely due to the decreased tissue sensitivity to the drug. A simple and effective method to differentiate the two kinds of drug tolerance is the joint injection of inhibitors of enzyme protein synthe sis, such as ethionine, with the designed drugs.

MATERIALSAND METHODS Female rats of Sprague-Dawley strain were used. Meprobamate was suspended in 0.5% carboxymethylcellose solution, while pentobarbital sodium and morphine sulphate were dissolved in distilled water. All drugs were given intraperitoneally and pretreat ments were carried out once for day as described in the legends of table and experiments. Determinationof drug effects: Meprobamate paralysis was determined by using an inclinated plate (about 30°), the rats could not stay for 10 seconds on the plate were considered as still in the paralysis, and pentobarbital narcosis was determined by the loss

加 藤 隆 一 of righting reflex. Morphine analgesia was determined by the measurment of pain reaction on a hot plate at every 10 minutes interval. Determinationof in vivometabolism of drug: In vivometabolism of meprobamate, pento barbital and morphine were determined by measurment of the brain and serum concen tration of the drugs after the intraperitoneal administration of drugs as described in the legends of tables and figures. Enzyme assays: The rats were killed by decapitation and their livers removed immediately and homogenized with 4 volumes of 1.15% KCl in a Potter-Elvenhjem type Teflon homogenizer. The homogenate was centrifuged at 9,000 x g for 15 minutes to sediment nuclei and mitochondria. 1) Oxidation of drugs : A typical incubation mixture for oxidation of drugs con sisted of 3 ml of 9,000 x g supernatant, 20 icmoles of glucose-6-phosphate, 0.4 iimoles of NADP, 50 /tmoles of nicotinamide, 50 pmoles of MgCl,, 1.4 ml of 0.2 M phosphate buffer, pH 7.4 and various substrates (meprobamate, 1.5 /tmoles; pentobarbital 1.5 umoles; morphine, 5 /cmoles) and the final volume is 5 ml. The mixture was incubated at 37-C for 30 minutes under air. Meprobamate and pentobarbital oxidations were determined by the disappearance of the drugs after the incubation and morphine N-demethylation was determined by the formation of formaldehyde as described in chemical assay. 2) Glucuronidation of morphine: The 9,000 x g supernatant was centrifuged at 105,000 x g for 60 minutes and the microsomes were suspended in 1.15% KC1 solution. A typical incubation mixture for the glucuronidation of morphine consisted of 0.5 ml of microsomes (equivalent to 0.5 g liver), 0.6 pmoles of UDP-glucronic acid, 25 ,umoles of MgC12j 250 pmoles of phosphate buffer, pH 7.4 and 0.4 moles of morphine sulfate and final volumes is 4.0 ml. The mixture was incubated at 37°C for 20 minutes under air. The reaction was stop ped by 0.5 ml of 20°n TCA and 2 ml of supernatant was used for the determination of morphine. Chemicalassay: Meprobamate and pentobarbital was determined according to the method of Hoffman and Ludwig (6) and Brodie et al. (7), respectively. Formaldehyde was determined according to the method of Nash (8). Amount of morphine in the incubation mixture, and brain and serum was determined according to the method of Fujimoto, Way and Hine (9) and Kupferwerg, Burkhalter and Way (10), respectively.

RESULTS 1. Effectof repeatedadministration of meprobamateon the duration of paralysis and brain and serum concentrationof meprobamateat the end of the paralysis The duration of paralysis by the administration of meprobamate was decreased by the second injection of meprobamate and the shortenness in the duration of paralysis be came more marked after the repeated injection of meprobamate (Table 1). For example, TABLE 1. Effect of repeated administration of meprobamate on the duration of paralysis and brain and serum concentrations of meprobamate at the end of the paralysis.

the duration of paralysis was decreased by 34% and 740/, respectively, in the rats pre treated successively for 2 days or 4 weeks. On the other hand, the concentrations of meprobamate in the brain and serum at the end of the paralysis was not significantly altered after the administration of 200 mg/kg

TABLE 2. Effect of repeated administration of pentobarbital on the duration of narcosis and brain and serum concentration of pentobarbital at the end of narcosis. of meprobamate successively for 1, 2 or 3 days or 1 week , but the concentrations of meprobamate were significantly higher in the rats pretreated successively for 2 and 4 weeks. Moreover, the ratios of the concentrations of meprobamate in the brain and serum were not altered by the repeated administration of meprobamate. The evidence in the lack of the alternation in the concentrations of meprobamate in the brain and serum at the end of the paralysis in the tolerant animals indicates that the development of the tolerance in the rats treated successively for 1 week or less is not related to a decreased sensitivity of the central nervous system to the drug, but it is mainly due to the increased breakdown of meprobamate by the liver microsomes. However, it is probable that the sensitivity of the central nervous system is decreased after the daily treatment for 2 or 4 weeks. 2. Effect of repeatedadministration of pentobarbitalon the duration of narcosisand brain and serum concentrationof pentobarbital at the end of the narcosis The duration of narcosis by pentobarbital was progressively decreased after repeated injection of pentobarbital (Table 2). However, the concentrations of pentobarbital in the brain and serum at the end of narcosis were almost same by the treatment for 1 week or less and slightly increased after the daily treatment for 2 weeks or 4 weeks. These re

FiG. 1. Effect of repeated injection of meprobamate on the duration of paralysis, meprobamate concentration in brain and activity of meprobamate oxidase. One hundred sixty-eight rats, weighing about 100g, were divided in 7 groups and treated with meprobamate (200 mg/kg/day) successively for 1, 2 or 3 days or 1, 2 or 4 weeks. Twenty-four hours after the last injections 16 rats of each group were treated with 200 mg/kg of meprobamate and the duration of paralysis was determined in 8 rats and the other 8 rats were killed 3 hours later and the meprobamate concentration in brain were determined. The other 8 rats of each group were killed 24 hours after the last injections and the metabolism of meprobamate by the liver microsomes were determined. suits were almost similar to the results obtained from the studies on the development of meprobamate tolerance. 3. The relationshipsbetween the duration of meprobamateparalysis, in vivo amd in vitro metabolism of meprobamate Fig. 1 shows that the duration of meprobamate paralysis was progressively decreased by the repeated injection of meprobamate and the concentration of meprobamate in the brain at 3 hours after the last administration of meprobamate was decreased as parallel as the duration of paralysis in the rats treated for 1 week or less. However, further de crease in the concentration of meprobamate in the brain was not demonstrated by the treatment for 2 or 4 weeks. The activity of oxidation of meprobamate by liver microsomal enzyme system was progressively increased by the repeated injections of meprobamate, but the further in crease was not demonstrated by the treatment for 2 or 4 weeks. The alternation of the in vitro metabolism of meprobamate were in accodance with that of the meprobamate concentration of in the brain. These results confirmed the view that the development of meprobamate tolerance was closely related to the increased ca pacity of liver microsomal enzyme system to oxidize meprobamate to hydroxymeprobamate

FiG. 2. Effect of repeated injection of pentobarbital on the duration of narcosis, pentobarbital concentration in brain and activity of pentobarbital oxidase. One hundred sixty-eight rats, weighing about 100 g, were divided in 7 groups and treated with pentobarbital (26 mg/kg/day) successively for 1, 2 or 3 days or 1, 2 or 4 weeks. Twenty-four hours after the last injections two-third of rats of the each group were treated with 26 mg/kg of pento barbital and the sleeping time was determined (8 rats) and 1 hour later the other 8 rats were killed and the pentobarbital concentration in brain were determined. The other one-third of rats were killed 24 hours after the last injections and the metabolism of pentobarbital by the liver microsomes were determined. (11, 12). Thus, the development of the decreased activity of meprobamate paralysis may be called as a development of apparent tolerance. 4. The relationshipsbetween the duration of pentobarbital narcosis, in vivo and in vitro metabolism of pentobarbital The results given in Fig. 2 shows the almost same tendency to the results given in Fig. 1. Thus, the development of pentobarbital tolerance was closely related to the increas ed activity of liver microsomal enzyme system for the oxidation of the drugs. 5. Effect of administration of morphineon the analgestic action, its concentrationin the brain and serum at the end of analgesia and on in vitro metabolismof morphine It is well known that the analgesic action of morphine is markedly decreased by re peated administration. The development of morphine tolerance was clearly observed already after single injection and it became more marked by repeated injection. Table 3 shows that the morphine concentration in the brain and serum at the end of analgesia was markedly higher in the rats treated with morphine for 2 days than in the control rats.

TABLE 3. Effect of morphine pretreatment on the duration of morphine analgesia and the concentrations of morphine in the brain and serum at the end of analgesia.

TABLE 4. Effect of morphine pretreatment on the activities of morphine glucuronidation and N-demethylation. These results indicates that the sensitivity of the central nervous system to morphine likely decreased in the morphine-treated rats. Moreover, the activities of transglucu ronylferase and N-demethylase of morphine in liver microsomes were not significantly altered in the rats treated with morphine (Table 4). These results were accord with the observation of Takemori and Glowacki (13) who observed no alternation of trans glucuronylferase in morphine tolerated rats, and confirmed that the development of mor phine tolrea;ice did not due to the increased metabolism of morphine, but it was due to the development of real tolerance of the central nervous system to morphine. 6. Methodfor the differentiationin developmentof two kinds of dugs tolerance In previous papers we reported that the joint injection of ethionine with the inducing drugs, such as meprobamate and pentobarbital, prevents the increase in the metabolism of these drugs and the development of decrease in the effects of these drugs (4, 11, 12, 14). It seems, therefore, usefull to utilize the inhibitory action of ethionine for the differentiation in the development of the two kinds of drug tolerance. A typical example was shown in Fig. 3. The administration of ethionine prevented the decreases of pentobarbital narcosis and meprobamate paralysis by the pretreatment with the same drugs, but it did not prevent the development of tolerance to morphine by the pretreatment with same drugs.

FiG.3. Effect of ethionine on the development of tolerance to pentobarbital, meprobamate or morphine. Female rats, weighing about 100g, were divided in three groups for the experiments with pentobarbital, meprobamate or morphine. Pentobarbital (26 mg/kg, i.p.) and meprobamate (250 mg/kg, i.p.) were given 48 hours prior to the determination of pentobarbital narcosis (25 mg/kg, i.p.) and meprobamate paralysis (200 mg/kg, i.p.). Morphine (20 mg/'kg, i.p.) were given 24 hours and 48 hours prior to the determination of morphine analgesia. Ethionine (250 mg/kg) was given 48.5 hours prior to the determination of pentobarbital narcosis and meprobamate paralysis, and in the determination of morphine analgesia ethionine (250 mg/kg) was given 48.5 hours and 24.5 hours before. The results were given as the average from 8 rats and ex pressed as the relative activity to control. DISCUSSION

It was extensively studied through different investigators that repeated administration of many drugs, especially central acting drugs, causes a decreases in the effect of same drugs or related drugs. For example, the repeated administration of morphine markedly decreases in the analgesic effect of morphine and the repeated administration of pentobarbi tal or barbital markedly decreases in the duration of narcosis by pentobarbital or barbital. These phenomena were called as development of drug tolerance. Several years before, Remmer demonstrated that single administration of phenobarbital or other bar biturates markedly increased the activities of liver microsomal enzyme for the metabolism of barbiturates, thus the duration of narcosis by hexobarbital markedly decreased 24-48 hours after the single injection of phenobarbital (15). Successively, Kato showed that the administration of meprobamate and other drugs increased the metabolism of meprobamate by liver microsomes and the duration of paralysis by meprobamate was significantly decreased (12, 16, 17). However, Kato also showed that the concentration of meprobamate in the serum and brain at the end of the paralysis in the rats pretreated with meprobamate was almost same as in the control (11). Similary, Remmer showed that the repeated administration of pentobarbital to rabbits decreased the duration of narcosis, but the concentration of pentobarbital in the serum and brain at the end of the narcosis in the rabbits pretreated with pentobarbital was almost same as in the control (18). In the present investigation it was demonstrated that the single or repeated administration of meprobamate or pentobarbital increased the metabolism of meprobamate or pentobarbital by liver microsomes. However, the concentration of meprobamate or pentobarbital in the serum and brain at the end of the paralysis or narcosis was not significantly altered in the rats treated with meprobamate or pentobarbital. The repeated administration of meprobamate or pen tobarbital for more than two weeks did not cause further increase in the metabolism of meprobamate or pentobarbital. However, the duration of meprobamate paralysis or pentobarbital narcosis was continuousely decreased in the rats treated more than one week. Moreover, the concentration of meprobamate or pentobarbital in the serum and brain at the end of paralysis or narcosis was significantly increased in the rats treated with mepro bamate or pentobarbital for two weeks. These results indicated that the decrease in the effects of the drugs after single or sever al injection of meprobamate or pentobarbital is mainely due to the increased activity of drug metabolism by liver microsomes, and it is not due to decreased sensitivity of the central nervous systems of the treated rats. Moreover, the repeated injection of mepro bamate or pentobarbital for 2 or 4 weeks resulted in a decrease in the sensitivity of the central nervous systems, and it is probable that the decreased sensitivity of the central nervous systems play an important role for the development of the tolerance. On the other hand, it was also demonstrated that administration of morphine markedly decrease the duration of morphine analgesia, but the metabolism of morphine by liver microsomes was not altered. Therefore, it was clearly demonstrated in the present investigation that there are two kind of mechanism for the development of drug tolerance. The first one is related to decrease in the sensitivity of tissue to the drugs, the second one is related to increase in the activity of drug metabolism by liver microsomes. The first type of tolerance is called as real toleranceand represented by the morphine tolerance, and the second type is called as apparent toleranceand represented by the pentobarbital tolerance. The administration of ethionine prevented the increase in the activities of drug-me tabolizing enzymes of liver microsomes. The duration of pentobarbital narcosis or mepro bamate paralysis, therefore, was not decreased by the joint injection of ethionine with pentobarbital or meprobamate. In contrast, the duration of morphine analgesia was decreased by the joint injection of ethionine with morphine. These results, therefore, indicated that the use of ethionine may be an effective tool for the differentiation of the drug tolerances, whether they are due to an increased metabo lism of drugs or not. The real mechanism for the development of morphine tolerance is not yet elucidated, but the lack of effect of ethionine to prevent the development of mor phine tolerance indicated that de novoprotein synthesis likely not involved in the develop ment of morphine tolerance. Furthermore, it was also observed that the administration of some drugs, such as barbital, which is not practically metabolized in rats, resulted in a tolerance after the repeated injection for 3 weeks without any significant increase of the drug metabolism (19).

SUMMARY Single or repeated administration of pentobarbital, meprobamate or morphine caused marked decrease in the duration of pentobarbital narcosis, meprobamate paralysis or mor phine analgesia, respectively. The decrease of morphine analgesia is likely due to a decrease in the sensitivity of the central nervous system (real tolerance),while the decrease of pentobarbital narcosis or mepro bamate paralysis may be mainely due to an increase in the metabolism of the drugs (apparenttolerance) and a real tolerance likely developed only after 2 weeks of the repeated injection. The use of ethionine as an inhibitor of de novoenzyme synthesis together with tolerance induced drugs is an effective method for the differentiation of the tolerance, whether they are real or apparent one.

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