Physical dependence on morphine, phenobarbital and diazepam in rats by drug-admixed food ingestion.

To produce physical dependence on morphine, phenobarbital and diazepam in rats, these drugs were mixed with powder form of rat food in concentrations of 0.5 mg/g, 1 mg/g and 2 mg/g of food. One group of rats (the lower dose group) was continuously exposed for 1 week to two morphine-admixed foods with morphine to food ratios of 0.5 mg/g and 1 mg/g in a cage. The other group (the higher dose group) could choose between two morphine-admixed foods with morphine to food ratios of 1 mg/g and 2 mg/g. After 1 week, morphine-admixed foods were replaced with morphine free food for 2 days. Both groups of rats showed greatly reduced body weight and food intake after the first 24-48 hr withdrawal. The body weight decrease was greater for rats in the higher dose group. Control groups of morphine dependent rats were kept on the morphine added food diets and showed the same body weight increase as well as normal control rats during the course of these experiments. Physical dependence on phenobarbital and diazepam was produced using the same dosage schedules as with morphine. Both the lower and higher dose groups showed significant decrease in body weight due to withdrawal after 1 week of drug-food exposure. Levallorphan (0.5, 1, 3 and 5 mg/kg, s.c.) administered to morphine dependent rats had dose-dependent effects on the intensity of abstinence symptoms (e.g., diarrhea, piloerection and wet shakes phenomena), maximal decrease in body weight and duration of decreased body weight. Cross-physical dependence between phenobarbital and diazepam was demonstrated by this method.     

Comparison of development of drug dependence in naive and drug dependence-experienced rats]

Phenobarbital, chordiazepoxide, diazepam and/or morphine were repeatedly administered to both male and female rats (N equals 10) for 4 similar to 6 weeks. The drug dose was gradully increased from 5 to 10, 20, 40, 80 and 160 mg/kg once daily (p.o) at seven day intervals. In the case of morphine, the last dose was 40 mg/kg. The drugs were constantly withdrawn for 24 hr at 8 day intervvals. None of the rats were given drugs for 16 days after administration of the last scheduled dose in order to recover their initial weight (Exp. I). Onset of dependence formation, decrease in body weight and food intake, days required to reach the maximum decrease in body weight and duration of withdrawal signs were observed throughout this experiment. The rats (drug dependence-experimented rats) who survived the first stage of this experiment were continuously subjected to re-administration by the same dosage schedule as in Exp. I (seven days of drug administration, 48hr of withdrawal). The re-administered rats showed a more rapid onset of dependence formation and a longer duration of decreas in boy weight during 16 days withdrawal than did the naive rats. It is concluded, that in addition to the decrease in body weight by withdrawal plus duration of the withdrawal signs, the onset of drug dependence formation is also a specific factor.     

Aspects of abstinence after morphine ingestion

Sprague-Dawley male rats were intoxicated with morphine, using an ingestion method where exposed and control rats received equivalent amounts of calories and nutrients. The degree of physical dependence on morphine was demonstrated by studying and quantifying abstinence symptoms after withdrawal or after administration of opiate antagonists. The aims of the study were (1) to further enlighten the specificity and validity of the intoxication method concerning physical dependence, and (2) to determine whether some of the abstinence signs might be of value to facilitate quantitation of the degree of physical dependence on morphine, with diet and fluid intake being maintained under control. Withdrawn rats showed a decreased fluid diet intake and a body weight loss, the latter partly due to anorexia. Other mild abstinence signs were irritation, tremor and some motor excitation. The body weight loss during the first day of morphine withdrawal was proportional to the accumulated drug dose (between 25 and 300 mg morphine PO/kg b.wt.). However, prolonged morphine treatment on one dose (340 mg/kg b.wt.) did not reinforce the body weight changes caused by morphine withdrawal. The succeeding weight gain some days after morphine withdrawal was not entirely dependent on the amount of fluid diet intake. Methadone was shown to partially block the decrease in diet intake and the weight loss seen during morphine withdrawal. The naloxone-precipitated withdrawal symptoms were motor excitation, cholinergic signs, body weight loss, diarrhoea and decreased diet intake. The weight loss 2 hr after naloxone administration to long-term intoxicated rats was proportional to the naloxone dose.(ABSTRACT TRUNCATED AT 250 WORDS)     

Chronopharmacological study of physical dependence on morphine in rats

Relationship between withdrawal time or naloxone injection time and withdrawal signs were examined in morphine-treated rats. Sixty-five rats were treated chronically with morphine-admixed food (1 mg/g food) for 7 days and were divided into 13 groups. The rats of 4 groups were abruptly withdrawn from morphine, and the rats of another 4 groups were given naloxone (3 mg/kg, s.c.) at 20:00 on the 8 th day and 2:00, 8:00 and 14:00 on the 9 th day after the morphine administration, respectively. Withdrawal signs were observed at intervals of 2 hr. After each naloxone injection, abnormal behaviors were observed for 60 min, and body weight was measured for 3 hr at intervals of 15 or 30 min. In the withdrawal test, weight loss at 24 hr after withdrawal in each group was approximately 10%, and there was no difference between each group. However, the body weight of non-treated rats and morphine-treated rats increased during the night period (20:00-8:00) and decreased during the daytime (8:00-20:00). Therefore, body weight reached the minimum at 20:00, and then this time is appropriated for withdrawal. In the naloxone test, withdrawal signs in the night period were more potent than that in the daytime. The withdrawal signs induced by naloxone at 8:00 showed the maximum magnitude. Plasma morphine levels in rats treated with morphine-admixed food were high in the night period and low in the daytime. These results suggest that the magnitude of naloxone-precipitated withdrawal signs depends on the amount of morphine in the plasma.     

Morphine dependence and preference. (I). Morphine preference in naive and morphine-experienced rats]

The spontnaeous morphine intake ratio (M-SIR) under free access to morphine-admixed food and quinine-admixed food conditions was measured for 3 weeks in naive and morphine-experienced rats. In the case of morphine (0.5 mg/g of food) vs. quinine (0.5 mg/g of food), naive rats gradually increased M-SIR from 17% to 77%. Using a higher level of morphine- and quinine-admixed food (1 mg/g vs. 1 mg/g of food), M-SIR was more rapidly increased than that in the lower group. Thus while on the 10 approximately 60 mg/kg/day dose range, the M-SIR was gradually increased dose dependently in naive rats due mainly to the positive reinforcing properties of morphine. Morphine-experienced rats showed a significant increase in M-SIR for the first 4 days specifically as compared with naive rats. Morphine dependent rats thus obtained morphine in sufficient amounts to maintain dependent states only after the first 2 approximately 3 days. This choice behavior revealed the psychological aspects of morphine dependence in rats and the preference for morphine was also observed after withdrawal for more than 2 weeks as secondary abstinence syndrome.     

Assessment of morphine-type physical dependence liability in mice using the drug-admixed food method

Physical dependence on morphine-type drugs (morphine, codeine and pethidine) in mice were examined by the drug-admixed food method. Mice were treated with drug-admixed food of increasing concentration (1, 2 and 3 mg/g food) every third day for 9 days. Morphine- and codeine-treated mice showed withdrawal signs when they were given naloxone (5 mg/kg, s.c.), while pethidine-treated mice did not show the withdrawal signs. However, mice treated with pethidine-admixed food (1-6 mg/g food) for 28 days showed naloxone precipitated withdrawal signs. Thus, the data obtained with mice indicate that pethidine produces a weak physical dependence. On the other hand, codeine (40 mg/kg, s.c.) and pethidine (100 mg/kg, s.c.) administration suppressed the abrupt withdrawal signs of morphine-dependent mice that were treated with morphine-admixed food, while the withdrawal signs were completely suppressed in mice administered only 5 mg/kg morphine. These results suggest that the physical dependence liability of morphine type drugs can be predicted by the drug-admixed food method.     

The concentration of morphine in serum of rats made dependent using a drug-admixed food method

The present study reports on the induction of physical dependence in rats using morphine-admixed food and addresses the question of the resulting concentration of morphine in serum. The stability of morphine in food is good, since no decrease in concentration could be observed. The concentration of morphine in serum during the experiment was measured using a radioimmunoassay technique. A correlation was found between the food intake during a 7-hour period and the concentration of morphine in the serum at the end of that period, both for a 1 g/kg and a 2 g/kg batch of morphine-admixed food. The concentration of morphine in serum was also found to be dose-related during a period of 6-23 days when the rats were fed for a prolonged period. After long-term administration of 1 g/kg morphine in food a steady-state level of about 0.5 mg/l serum was obtained. Similarly with 2 g/kg morphine in food a steady-state level of 0.8-1.1 mg/l serum was reached. After withdrawal of morphine the serum concentration of morphine dropped to 0.1 mg/l within 24 hours and to below the detection limit within 48 hours. During the withdrawal period sharp drops were noted in body weight (20%) and food intake (50%) after one day.     

Induction of physical dependence in rats by short interval medication

Rats were intermittently medicated at one hour intervals through an implanted intravenous cannula. Physical dependence on morphine and codeine was developed rapidly and it was detectable with the maintenance dose as low as 9.6 mg/kg/day. Physical dependence on pentazocine was also developed with the maintenance dose of 96 mg/kg/day, but was not with 9.6 mg/kg/day. In the pentazocine-treated rats, body weight loss was observed after the abrupt withdrawal, and abstinence signs were precipitated by naloxone 1 mg/kg. Cross physical dependence between morphine and pentazocine was demonstrated. Pentazocine suppressed the abstinence signs of rats weakly dependent on morphine, and morphine suppressed those of pentazocine-dependent rats. ID-1229, a new benzomorphan analgesic, did not produce dependence in this test and did not suppress the abstinence signs of morphine- and pentazocine-dependent rats.     

Physical dependence and tolerance development after chronic exposure to low levels of morphine in the rat

A sustained-release delivery system containing ¹⁴C-morphine was implanted subcutaneously in rats. Measurement of urinary excretion of ¹⁴C suggested a steady state release of approximately 640 μg ¹⁴C morphine/day during a 10-day test period. Tolerance developed rapidly to the analgetic effects produced by an injected ED₉₅ dose of morphine sulfate in implanted rats tested on the hot-plate. Physical dependence, determined by naloxone-precipitated abstinence behavior, was evident in rats at 24 hr. Morphine dosage was estimated to be as low as 2.5 mg/kg/day. Peak abstinence behavior was observed on Day 4. However, naloxone-precipitated withdrawal signs were markedly diminished by Day 6 and essentially absent by Day 8. These results are discussed with reference to the suggestion that metabolic changes, occuring during chronic exposure to morphine, may explain the lack of abstinence behavior during a time when maximal concentrations of urinary morphine were observed and a high degree of tolerance was manifest.     

Effect of p-chloroamphetamine on morphine withdrawal syndrome

Adult male rats were injected four times a day with increasing doses of morphine sulfate until a dose of 405 mg/kg/day was tolerated. After 5 days of maintenance at that dose, withdrawal signs were observed at 24, 48, and 72 hr of withdrawal. Given 4 or 18 hr after last morphine injection, a single administration (3–12 mg/kg) of p-chloroamphetamine (PCA) blocked withdrawal “wet shakes” and ptosis during the following withdrawal period. Dose-dependent reduction in withdrawal hypothermia was observed only at 24 hr of withdrawal. Writhing was blocked only at higher doses. Morphine-withdrawal aggression measured at 72 hr of withdrawal and loss in body weight were not blocked by any delayed action of PCA. However, withdrawal aggression was blocked by PCA when given 1 hr before measurement at a dose of 3 mg/kg. Mice that were made morphine dependent by repeated injections of morphine given in large doses, exhibited withdrawal jumping either after cessation of morphine injections, or on injection of naloxone. PCA (3–12 mg/kg) given 2 hr before naloxone blocked this jumping in a dose-dependent manner. It also blocked withdrawal jumping in morphine-deprived mice when injected 2 hr, but not 14 or 17 hr before measurement. Jumping induced by α-naphthyloxyacetic acid (α-NOAA) was not blocked. The effect of PCA on different signs of morphine withdrawal is time dependent and may be related to its similarly time-dependent effect on brain serotonin and dopamine.     

Physical dependence potential of an enkephalin analog, EK-399, in rats

The physical dependence potential of Tyr-D-Met(O)-Gly-EtPhe-NHNHCOCH3.AcOH (EK-399), a novel enkephalin analog with a potent analgesic effect, was assessed in rats. The animals were given EK-399 (0.008, 0.032, 0.125, or 0.5 mg/kg), morphine (0.125, 0.5, or 2 mg/kg), pethidine (2 or 8 mg/kg), or pentazocine (2 or 8 mg/kg) every hour through an implanted intravenous cannula. After 3 days of treatment, precipitated withdrawal tests were conducted: naloxone (5 mg/kg) was administered subcutaneously. Rats treated with morphine showed withdrawal signs such as hyperirritability, salivation, diarrhea, and weight loss. Rats treated with pethidine, pentazocine, or EK-399 showed similar signs, but they were less evident than those in morphine-treated rats. In abrupt withdrawal tests after 7 days of treatment, rats treated with morphine, pethidine, or pentazocine showed weight loss, whereas rats treated with EK-399 showed little or no weight loss. In substitution tests, EK-399 suppressed the withdrawal signs in morphine-dependent rats, and vice versa. These results show that EK-399 has a morphine-like physical dependence potential that is weaker than that of morphine, pethidine, or pentazocine in rats.     

Mode of antagonistic action of levallorphan in morphine-dependent rats and assessment of physical dependence liability

The antagonistic mode of levallorphan in rats dependent on morphine or codeine was studied from the viewpoints of the doses of morphine and the lengths of administration and also from the standpoint of timing of the challenge. In morphine-dependent rats on morphine-admixed food (60--100 mg/kg/day) for 1, 3 and 6 weeks, the rate of maximum weight loss on application of levallorphan (2 mg/kg, s.c.) did not correlate with the length of morphine treatment. The rate of weight loss on single application of levallorphan 0, 6, 12 or 24 hours after withdrawal or morphine was lower with the passage of time after the withdrawal. Rats which were given levallorphan 3 times in succession, i.e., at 0, 5 and 10 hours after morphine withdrawal showed such a pattern of weight loss that the first application of levallorphan resulted in 7% loss, while with the second and third applications there was little weight loss. Despite the continued withdrawal, the animals began to gain body weight as early as 14 hour, and body weight was totally recovered before the withdrawal in 24 hour. In conclusion, it is advisable to challenge with levallorphan at 0 hour of withdrawal to obtain qualitative and reproducible results. In addition, the application of levallorphan to morphine-dependent rats at adequate intervals provides for the early recovery of abstinence signs.     

Physical dependence liability test of ifenprodil in rats (author's transl)

A single administration of ifenprodil at the doses of 100, 200 and 400 mg/kg (p.o.), and 50 and 100 mg/kg (i.m.) produced a moderate CNS depression in rats, such as, sedation, ptosis, systemic muscle relaxation and decrease in motor activity. These symptoms appeared dose-dependently and persisted for about 4 hours following administration. In a direct physical dependence test, 5 groups of rats were fed the ifenprodil-admixed food together with drinking water ad libitum for 24 hours daily for 53 approximately 103 days (mean ifenprodil intake, 43--240 mg/kg/day), on the gradedly increased dosage schedule with a dosage level of 0.5 vs. 1 mg/g food to 4 mg/g food. In the natural withdrawal following administration, no significant withdrawal signs were observed in any group. In a substitution test either for phenobarbital or morphine, no suppression of withdrawal signs during the period of cross-administration of ifenprodil and no maintenance of dependence were observed. In a physical dependence-producing test, the rats fed ifenprodil never manifested withdrawal signs such as diarrhea, "wet shakes", sudden loss of body weight as in the levallorphan precipitation test. Ifenprodil apparently has no physical dependence liability.     

Effect of propranolol on antinociceptive and withdrawal characteristics of morphine

Propranolol at a dose (10 mg/kg) which did not alter tail-flick latency by itself, did not alter the ED50 of morphine when given 10 min prior to the narcotic. Propranolol at doses of 10 and 25 mg/kg given 10 min prior to naloxone challenge did not significantly alter the frequency of naloxone induced jumping 72 hr after morphine pellet implantation. The ED50 of naloxone in morphine pelleted mice was not altered by treatment with propranolol at 0, 24, and 48 hr after pellet implantation. Naloxone caused hyperactivity in mice when administered 72 hr after morphine pellet implantation. An injection of 25 mg/kg propranolol 10 min prior to naloxone did not block this hyperactivity. In addition, administration of 10 mg/kg of propranolol every 8 hr to rats during withdrawal from morphine failed to alleviate the withdrawal syndrome as evidenced by changes in either body weight or water intake. These data suggest that the beta-adrenergic blocking agent, propranolol, does not alter the antinociceptive activity or lessen the withdrawal syndrome of morphine in rodents.     

A method for computer control of morphine ingestion by rats

In this paper, we describe a method for controlling the administration of liquid diet and morphine to sixteen rats using a computer. Morphine ingestion treatment was performed with 6 feeding occasions per 24 hr, all experimental animals receiving similar drug doses. The amount of drug was individual and based on body weight at each feeding occasion. Control and experimental animals were kept under isocaloric conditions. Corrections of drug doses in order to compensate for changes in body weight were made every 24 hr. Sensors registered the exact time of complete drug and diet consumption and prevented overdistribution. Rats were administered 103 mg/kg b.wt. morphine during 24 hr. In another experiment rats were administered 191 mg/kg b.wt. morphine during 48 hr, and no weight loss or decrease in fluid diet intake was registered during the time of drug administration in either of the experiments. After exclusion of morphine from the fluid diet, the body weight loss was 6.1% and 8.3%, respectively, and the liquid diet intake decreased by 12.4 ml and 13.4 ml, respectively, compared with control animal intake. This demonstrates the induction of physical drug dependence. A major advantage of using computer-aided administration of morphine-admixed, fluid diet is the stepwise, small dose increments provided several times a day, resulting in higher drug dose per unit time when compared with ingestion procedures using one feeding occasion per day. The method enables rats to rapidly ingest large morphine doses under standardized conditions.     

Quantitative relationships among measures of morphine tolerance and physical dependence in the rat

The influence of treatment dose on a number of characteristics of opiate tolerance was studied in male Sprague-Dawley rats treated with daily intraperitoneal (IP) injections of morphine sulfate. Zero, 7.5, 15, 25 or 45 mg/kg/day was given for 34 consecutive days and the degree of morphine effect on four different tests was periodically assessed. Dose-related effects on tailflick latency (tail immersion test, 1.5 hr post injection), swimming test (2 hr post injection), and body weight gain revealed the development of tolerance; there was a non-dose-related hyperthermia (1.5 hr post injection) to which rapid sensitization occurred. All changes reached asymptote and the rate and extent of change varied with the test. Plots of log response vs test day for tailflick and swimming indicated an early steep component and a later less steep component of decline. Subsequent testing indicated that the log-dose/response (LDR) curves for tailflick latency and time to maximum hyperthermia shifted to the right by an amount dependent on the treatment dose; there was no change in the curve for hyperthermia duration. In high dose groups no further shift occurred, but the tailflick LDR curves became flattened. The tailflick LDR curve changes were replicated in rats treated for 24 days with 0, 8, 24, 48, 96, or 240 mg/kg/day. Subsequently, a constellation of withdrawal signs precipitated by naloxone HCl (1 mg/kg, IP) was measured. On the basis of the relation between treatment dose and the magnitude of the various measures, there was a parallel between analgesia tolerance and some, but not all, signs of physical dependence.     

Physical dependence liability test of 4-(o-benzylphenoxy)-N-methylbutylamine hydrochloride (MCI-2016) in rats

An acute administration of MCI-2016 at the doses of 30, 100 and 300 mg/kg (p.o.), and 10, 20 and 30 mg/kg (i.p.) produced a slight CNS depression in rats, such as, sedation, ptosis, decrease in motor activity and systemic muscle relaxation. In a direct physical dependence test, rats were fed the MCI-2016-admixed food together with drinking water ad libitum for 24 hours daily for 51-71 days (mean MCI-2016 intake 29.9-210.7 mg/kg/day), on the gradedly increased dosage schedule with a dosage level of 0.25 and 0.5 mg/g food to 4 mg/g food. In a natural withdrawal following administration of MCI-2016, no significant withdrawal signs were observed in any group. In a naloxone-precipitation test the rats that were treated with MCI-2016-admixed food did not show any withdrawal signs. In a substitution test in either morphine or barbital dependent rats, no suppression of withdrawal signs or maintenance of dependence were observed by cross-administration of MCI-2016. In conclusion, MCI-2016 was considered to have no physical dependence potential.     

Matricaria chamomilla extract inhibits both development of morphine dependence and expression of abstinence syndrome in rats

The effect of Matricaria chamomilla (M. chamomilla) on the development of morphine dependence and expression of abstinence was investigated in rats. The frequencies of withdrawal behavioral signs (paw tremor, rearing, teeth chattering, body shakes, ptosis, diarrhea, and urination) and weight loss induced by naloxone challenge were demonstrated in morphine-dependent rats receiving M. chamomilla extract or saline. The withdrawal behavioral manifestations and weight loss were inhibited significantly by chronic co-administration of M. chamomilla extract with morphine. Administration of a single dose of M. chamomilla before the naloxone challenge in morphine-dependent animals abolished the withdrawal behavioral manifestations. The dramatic increase of plasma cAMP induced by naloxone-precipitated abstinence was prevented by chronic co-administration of M. chamomilla extract with morphine. These results suggest that M. chamomilla extract inhibits the development of morphine dependence and expression of abstinence syndrome.     

Assessment of precipitated abstinence in morphine- dependent rats

An experimental model is described for quantifying the precipitated abstinence syndrome in morphine-dependent rats. Male rats were made dependent on morphine by subcutaneous implantation of a morphine pellet and the abstinence syndrome precipitated by intraperitoneal injection of naloxone hydrochloride. A ranking system, based on the median effective dose of naloxone for abstinence signs, quantitatively related the incidence of certain precipitated signs to the dose of naloxone. The time course for the development of dependence was shown to be maximal 70–74 h after pellet implantation. Food or water deprivation for 48 h dissociated the body weight loss during abstinence from the behavioral signs of precipitated withdrawal. Ganglionic blockade did not significantly modify abstinence behavior. An evaluative procedure which ranks abstinence signs is proposed for quantifying physical dependence on morphine.     

A water lick conflict paradigm using drug experienced rats

Narcotics and other drugs were injected into the rat once an hour for about 1 week. Morphine and codeine showed physical dependency with a maintenance dose as low as 9.6 mg/kg/day. Development of dependence was also recorded in rats treated with cyclazocine (9.6 mg/kg/day) and pentazocine (96 mg/kg/day), and suspected in the levallorphan-treated (9.6 mg/kg/day) rats. Dependence on pethidine, wich is known to be difficult to detect by the usual method in rats, also developed in this experiment (96 mg/kg/day), but it was estimated to be of lesser degree than that of codeine. Dependence on allazocine (9.6 mg/kg/day) and aminopyrine (96 mg/kg/day) did not develop. Barbital dependence (96 mg/kg/day) was induced, but it was distinguished from morphine-like drugs by the naloxone precipitation test and by substitution experiments. Cross-dependence between morphine and dependence-inducing drugs was investigated. Withdrawal weight loss in the morphine-dependent rats was suppressed or attenuated by pentazocine, pethidine, and codcine. Withdrawal weight loss in the rats dependent on cyclazocine, pentazocine, pethidine, or codeine was suppressed by morphine. Intermittent infusion of pentazocine at longer intervals induced dependence not as severe as that induced by 1-h infusion.