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.     

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.     

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.     

The physical dependence liability of butorphanol: a comparative study with morphine.

In these studies, the physical dependence liability of butorphanol, a mixed 'agonist/antagonist' opioid analgesic, was compared to that of morphine. Male, Sprague-Dawley rats received i.c.v. infusions of saline (1 microliter/h), or an equimolar dose of butorphanol or morphine (52.3 nmol/h) for 3 days. The physical dependence liabilities of these two compounds were then compared by assessing both behavioral withdrawal signs and weight loss following naloxone-precipitated withdrawal. Body weight loss was also evaluated following abrupt (cessation of infusion) withdrawal from butorphanol or morphine. In animals receiving i.c.v. infusions of butorphanol or morphine, naloxone administration (5 mg/kg s.c.) induced an equivalent degree of body weight loss compared to saline-treated animals. In addition, the ED50 of naloxone to produce wet shakes, escape behavior, teeth chattering, urination and defecation was equivalent in rats receiving butorphanol or morphine. Infusions of butorphanol or morphine also produced an equivalent degree of weight loss in animals undergoing abrupt withdrawal. These results demonstrate then that a substantial degree of physical dependence had developed in rats which received a large dose of butorphanol.     

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.     

Antinociception and physical dependence produced by [D-Arg2] dermorphin tetrapeptide analogues and morphine in rats.

1. The antinociceptive effects of [D-Arg2] dermorphin tetrapeptide analogues, H-Tyr-D-Arg-Phe-Gly-NH2 and H-Tyr-D-Arg-Phe-beta-Ala-OH when administered subcutaneously (s.c.) in rats were measured by the tail-flick test. In addition, the appearance of typical withdrawal signs upon cessation of administration or on subsequent treatment with naloxone were measured after chronic administration of either peptide or morphine. 2. The dose of peptides and of morphine in the physical dependence test was determined from the AD50 to inhibit the tail-flick test in rats. Doses from 4 to 64 times the AD50 doses were employed in the s.c. administration schedules. 3. The intensity of the antinociception induced by either peptide was greater than that produced by morphine. Moreover, the antinociception induced by the peptides was of much longer duration than that produced by morphine. 4. Abrupt withdrawal after chronic administration of either peptide produced only slight loss of body weight. In contrast, morphine withdrawal produced sharp loss of body weight. 5. Naloxone precipitated withdrawal signs after chronic administration of either peptide were less intense than those after chronic morphine. 6. These results suggest that the antinociception produced by these peptides is more intense and of longer duration than that produced by morphine. It is also interesting to note that the physical dependence produced by these peptides is less marked than that produced by morphine.     

Rapid induction and quantitation of morphine dependence in the rat by pellet implantation

Four schedules of subcutaneous morphine pellet implantation were developed to render rats rapidly physically dependent on morphine. The schedules included implantation of four morphine pellets over a 3-day period (schedule 1), six morphine pellets over a 3-day period (schedule 2), six pellets over a 7-day period (schedule 3), and ten pellets over a 10-day period (schedule 4). Each morphine pellet contained 75 mg of morphine base. The degree of morphine dependence was quantitated by determining the median effective dose (ED₅₀) of naloxone required to induce the stereotyped jumping response. Hypothermia and weight loss, during abrupt and naloxone-induced withdrawal, were also measured. Rats on schedule 4 exhibited a high degree of dependence on morphine as evidenced by an extremely low naloxone ED₅₀ for the precipitated withdrawal jumping response, whereas schedules 1 and 2 produced a low degree of dependence as shown by high naloxone ED₅₀'s. Further evidence for a high degree of physical dependence on morphine is schedule 4 rats was indicated by their greater loss in body weight and greater hypothermic response after abrupt and after naloxone precipitated withdrawal compared with these responses in the rats in the other three schedules. A correlation was found to exist between naloxone ED₅₀ for the jumping response, body weight loss, and hypothermia observed during naloxone-induced withdrawal in morphine-dependent rats. These studies suggest that the implantation of four morphine pellets in the rat produces a mild degree of dependence and that caution should be exercised when making generalized conclusions about the biochemical correlations involved when four or less number of pellets, each containing 75 mg of morphine base, are used to induce morphine dependence in the rat.     

Stimulation of brain-stem protein synthesis by morphine

Rats were intoxicated with morphine as intraperitoneal (i.p.) single doses, or for 4 days (final dose 130 mg/kg b.w.) or for 13 days (final dose 340 mg/kg b.w.) using an ingestion method where intoxicated and control rats received the same amount of calories and fluid. The intoxicated groups showed different degrees of physical dependence, demonstrated by variously expressed abstinence symptoms after withdrawal of the drug or after administration of the opiate receptor antagonist naloxone. Soluble protein synthesis was measured in vivo in brain stem by double labelling with 3H and 14C valine and followed over time in the various rat groups after i.p. morphine injection in different doses. Protein synthesis in astroglial-enriched primary cultures from brain stem and secretion of labelled protein to the serum free incubation medium was also evaluated after morphine treatment. There were dose- and time-dependent effects of morphine on brain stem protein synthesis with an initial decrease and a later increase, 1-3 hr after a single dose of morphine administration. Following a morphine single dose of 25 mg/kg b.w. the stimulation was more rapid in onset and more pronounced in rats with a higher degree of physical dependence. Specific protein fractions including one with a subunit M.W. of approx. 80,000 were identified by electrophoretic separation of labelled proteins. Some similar protein fractions increased in synthesis and were released to the serum-free incubation medium when separating astroglial primary culture proteins after morphine treatment. It might be that the biphasic changes in protein synthesis after morphine administration underlie adaptive phenomena such as tolerance/physical dependence development and that some of the identified proteins including proteins synthesized in astroglial cells and secreted to the incubation media participate in these processes.     

A new approach for assessment of narcotic physical dependence using urinary sex-dependent low molecular weight proteins in male rats

Attempts have been made to examine the relationship between urinary excretion of sex-dependent low molecular weight proteins found only in male rats (LMWP) and morphine physical dependence. Chronic administration of morphine produced a dose-related decrease in urinary LMWP excretion, which was correlated to the intensity of withdrawal signs including body weight loss and abnormal behaviors recognized after naloxone challenge. Furthermore, a statistically high correlation was obtained between the decrease in urinary LMWP excretion and the loss of body weight precipitated by naloxone challenge. LMWP was identified immunologically in the livers, kidneys, and sera using an antibody against purified LMWP. The serum level of LMWP was increased rapidly following bilateral nephrectomy. After chronic treatment with morphine, the LMWP content in the livers, kidneys, and sera were decreased. These findings indicate that the decrease in urinary LMWP excretion induced by chronic administration of morphine can be a useful parameter to assess the development of physical dependence on narcotics on the peripheral level without requiring drug withdrawal and naloxone challenge. This decrease in urinary LMWP may be caused by the inhibition of LMWP synthesis in the liver.     

Comparison of peripheral and central administration of naloxone in precipitating abstinence in morphine-dependent rats

Although it has been known that a morphine abstinence syndrome can be induced by naloxone administered centrally or peripherally, data on a detailed qualitative and quantitative comparison are not available. In the present study morphine pellets were implanted into rats and naloxone was administered intracerebroventricularly (i.c.v.) or subcutaneously (s.c.) 72 h later. A full array of abstinence signs with similar latency, duration, and intensity was seen in morphine-dependent rats following naloxone by either route. There were no major differences in the spectrum of withdrawal signs or in the proportion of rats showing the individual signs. In terms of body weight and temperature, the highest doses tested by each route produced similar quantitative effects. Our results demonstrate that naloxone given i.c.v. can precipitate the full morphine abstinence syndrome in rats at about 1/3 the dose needed for comparable effects when the antagonist is administered s.c.     

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.     

Morphine analgesia, tolerance and physical dependence in the adrenalectomized rat

1. Adrenalectomy reduced the median antinociceptive dose (AD50) of morphine in male Sprague-Dawley rats. The antinociceptive effect was assessed by the tail-flick method of D'Amour & Smith (1941).2. Tolerance to the antinociceptive effect of morphine developed in adrenalectomized and sham-operated rats after chronic exposure to morphine. Development of tolerance did not significantly alter the increased sensitivity of adrenalectomized rats to the antinociceptive effect of morphine.3. Adrenal weights were not increased in rats rendered physically dependent on morphine by subcutaneous implantation of a morphine pellet. Withdrawal, induced by intraperitoneal injection of naloxone hydrochloride, 4 mg/kg, or by removal of the implanted pellet, resulted in a rapid increase in adrenal weight.4. In morphine-dependent animals, the incidence of abstinence signs and body weight loss during precipitated withdrawal did not appear to be significantly influenced by adrenalectomy or by corticosterone-pretreatment.     

A study on drug dependence using fast acting drugs]

Physical dependence on narcotics is induced in laboratory animals by intermittent parenteral administration (2 approximately 3 times daily). However, inducing of dependence on pethidine has been unsuccessful using the parenteral method. Recently, it has been reported that physical dependence on pethidine can be induced by continuous infusion methods (5.6). In the present experiment, pethidine was administered to rats (n=5 approximately 6) by ingestion of pethidine-admixed food preparations (0.5 approximately 4.0 mg/g of feed). The results indicated that (a) when rats are allowed free access to two food preparations (0.5 mg/g vs. 1 mg/g of food) for 7 weeks, spontaneous intake ratios of food (1 mg/g of food) gradually increased from 15% to 30% after 3 weeks. (b) Abrupt withdrawal for 48 hr after a 10 day administration period (2 mg/g of food on day 1 approximately 3 and 4 mg/g of food on day 4 approximately 10) resulted in a loss of body weight in the next 24 hr, and the prewithdrawal level of body weight was recovered in 48 hr. (c) The time course of body weight and food intake during the first 24 hr withdrawal period demonstrated the characteristic pattern of abstinence syndrome of pethidine, viz. early onset (12 approximately 13 hr) and rapid recovery (within 48 hr), as compared to morphine withdrawal. (d) Suppression of pethidine abstinence of both a single injection of morphine (10 mg/kg, s.c.) and substitution for morphine-admixed food was also realized. (e) When levallorphan (5 mg/kg, s.c.) was administered to both pethidine and morphine dependence rats, the maximal decrease in body weight was less than that in morphine dependent rats. These data indicate that pethidine possesses about one fifth the dependence liability of morphine and the maximal abstinence syndrome appears within 24 hr after withdrawal. Conclusively, application of a drug-admixed food preparation in drug dependence tests in rats has proven to be a useful method, particularly in the case of pethidine-like drugs which rapidly disappear from the blood.     

Eating pattern of morphine dependent rats

To analyze the drug ingestion patterns of rats in the course of dependence development while on the drug-admixed food (DAF) method, an automatic food intake measuring apparatus was developed. Rats were put on morphine-admixed food, and the food ingestion patterns were recorded with the apparatus in the course of dependence development, during drug withdrawal and at the time of challenge with levallorphan. The naive rats ate the regular diet intermittently at night, and the eating time of morphine-treated rats was longer than that of naive rats. The treated rats also exhibited a frequent eating behavior after 4--5 days on the morphine treatment. During morphine withdrawal, the animal gradually ate the regular diet at about 1-hour intervals, even after evolvement of abstinence signs. When the morphine-dependent rats were given levallorphan, they neither ate nor approached to the food for the first 2--3 hours, but after this time, showed abrupt increases in these activities. Thus, the drug intake pattern of rats in the course of morphine dependence development suggests a correlation between the stage of development of physical dependence and the stage when the animals frequently eat the drug-admixed food.     

Characteristics of precipitated withdrawal from spinal morphine: changes in [Met5]enkephalin levels.

This investigation was carried out to study the development of physical dependence on spinally administered morphine, and it was determined if this phenomenon is associated with altered levels of [Met5]enkephalin. Morphine was infused continuously into the intrathecal space of rats for three or six days. In morphine-dependent animals, an intrathecal naloxone challenge produced increased reaction to nociceptive stimuli, hypertension, hyperthermia, decreased urinary output, and loss of body weight. Chronic spinal infusion of morphine alone failed to alter levels of [Met5]enkephalin in sacral and lumbar spinal cord. However, 24 h after the naloxone challenge, there was a significant increase in spinal enkephalin levels in morphine-dependent animals. It is concluded that spinal morphine treatment leads to the development of physical dependence. Certain characteristics of this phenomenon, as reflected in the naloxone-precipitated withdrawal signs, differ from those associated with dependence on systemic morphine.     

中药戒得安对吗啡依赖性大鼠的脱毒疗效及药物身体依赖性的实验研究

本文研究自拟中药戒得安对吗啡依赖大鼠戒断综合征的治疗效果，并对其是否存在药物身体依赖性作出评价。结果表明，戒得安能明显缓解吗啡依赖大鼠的戒断综合征，戒断症状综合积分及体重减轻情况与无治疗戒断组相比有显著性差异（Ｐ＜０．０１）。实验亦证实戒得安本身无身体依赖性。     

A comparison of the cardiovascular and the behavioral changes following naloxone as measures of the degree of physical dependence on morphine in rats

Behavioral and cardiovascular changes evoked by naloxone (0.5 mg/kg) were evaluated in unrestrained rats made physically dependent by a constant infusion of morphine at increasing doses over 7 days. The relationship between the duration of morphine administration and the naloxone-induced cardiovascular changes was investigated to determine the reliability and sensitivity of blood-pressure measurements in assessing the degree of physical dependence produced in these animals. No significant behavioral or cardiovascular changes from preaddiction levels were observed during morphine infusion. Arterial pressure increased significantly in a dose- and time-dependent manner in response to naloxone injected on days 1, 3, 5, or 7 of the morphine infusion schedule. Heart rate changes which accompanied the increase in blood pressure, however, were similar in magnitude over the course of increasing physical dependence. The frequency of counted behavioral signs precipitated by naloxone over the same infusion schedule indicated a significant, progressive increase only for escape attempts, whereas withdrawal body shakes and writhing peaked early in the schedule and actually declined to very low frequencies by day 7. A high dose of morphine (50 mg/kg) was injected following naloxone-precipitated withdrawal in an attempts to reverse the abstinence symptoms. The only symptoms significantly inhibited by morphine were the increase in arterial pressure and the occurrence of diarrhea. These results indicate that the absolute increase in arterial blood pressure produced by naloxone in opiate-dependent animals may serve as a simple, objective, and sensitive measurement, along with traditional behavioral signs, in the assessment of narcotic dependence liability.     

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.     

An implanted reservoir of morphine solution for rapid induction of physical dependence in rats

1. Rats were dosed continuously with morphine hydrochloride by giving a daily dose through tubes connected to small, subcutaneously implanted reservoirs. Morphine was withdrawn by washing out the reservoir with drug vehicle. The daily dose of morphine, or substitute drug received by each rat was determined by difference by estimating the drug remaining in reservoir washings.2. Withdrawal symptoms were more pronounced after 9 days than after 4 days of dosing with morphine.3. Body weight loss, maximal at 24 h, and increased defaecation during the first 7 h were the chief physiological signs of morphine withdrawal. The body weight loss was the result of hypodipsia and anorexia exacerbated by increased defaecation.4. When substituted for morphine in the reservoir, methadone and codeine completely prevented body weight loss and increased defaecation, while pethidine was effective against increased defaecation, but not against 24 h body weight loss. The opiate-antagonist analgesics pentazocine, nalorphine and cyclazocine either had no effect on withdrawal symptoms or increased their severity.5. In morphine dependent rats under continued morphine administration subcutaneous doses of the opiate-antagonists nalorphine, cyclazocine and naloxone all precipitated the withdrawal symptoms of body weight loss and increased defaecation. The weak antagonist pentazocine caused a significantly increased defaecation, but no significant change in body weight, while the opiates pethidine, codeine and methadone had no significant effect on body weight or defaecation.6. The advantages of inducing dependence by this method of dosing are discussed.     

Pharmacodynamics and kinetics of loss of tolerance and physical dependence on morphine induced by pellet implantation in the rat.

The decay characteristics of tolerance and physical dependence on morphine induced by a pellet implantation procedure were determined in male Sprague-Dawley rats. Rats were implanted subcutaneously with 6 morphine pellets during a 7-day period. The pellets were removed, and at various times thereafter tolerance to the analgesic and hyperthermic effects of morphine was measured by determining the response in rats implanted with morphine and placebo pellets. Similarly, the physical dependence was assessed by monitoring withdrawal signs following an injection of naloxone. A high degree of tolerance developed to the analgesic and hyperthermic effects of morphine. Similarly, a high degree of physical dependence also developed as evidenced by a high incidence of jumping response, teeth chattering and production of fecal boli induced by injections of naloxone. In addition, loss of body weight and body temperature also occurred. The analgesic and hyperthermic response to morphine recovered very gradually. There was no significant difference in the analgesic and hyperthermic responses to morphine on day 4 after the pellet removal in rats implanted with morphine and placebo pellets. The decay of tolerance was linear with time for the analgesic effect (r = 0.98) and for the hyperthermic effect (r = 0.93). The change in symptoms of physical dependence on morphine with time depended on the specific symptom monitored. The average number of jumps and teeth chattering decreased with time in a linear fashion with r values of 0.98 and 0.99, respectively. However, the number of fecal boli and wet dog shakes increased linearly with time (r = 0.97). The recovery of loss of body weight was also linear with time. Thus, it is clear that fecal boli and wet dog shakes, which increase in number as the dependence decays, are signs of a low degree of dependence. The results suggest that different central or peripheral mechanisms may be operating in different withdrawal symptoms. These studies may prove to be useful when studying the mechanisms involved in the induction and reversibility of tolerance and dependence processes, and in long-term effects of opiates at a time when the tolerance and physical dependence is no longer evident.