Comparison of tolerance development and dependence capacities of morphine, β-endorphin,and [D-MET2, PRO5]-enkephalinamide

The tolerance-development capacities of -endorphin, [D-Met², Pro⁵]-enkephalinamide, and morphine were compared in rats, and the dependence capacity of morphine was compared with that of the enkephalin analogue in mice. Tolerance to the analgesic effect, as measured by the tail-flick test, developed somewhat more rapidly in the [D-Met², Pro⁵]-enkephalinamide-treated group than in the others. A similar relationship was found for the dependence capacity. Considering that the enkephalin analogue displayed the strongest analgesic activity, the wellknown correlation between antinociceptive and tolerance development/dependence capacities of opiates seems to be valid for opioid peptides as well.     

New in vivo evidence for narcotic agonistic property of leucine-enkephalin

Administration of leucine-enkephalin or morphine to mice rendered dependent on morphine by pellet implantation inhibited the naloxone-precipitated abstinence syndrome. The withdrawal jumping response was inhibited by morphine or leucine-enkephalin; however, both failed to inhibit withdrawal defecation and rearing behavior. On a molar basis, leucine-enkephalin was half as potent as morphine in inhibiting the abstinence syndrome. New in vivo pharmacological evidence for narcotic agonist-like activity of leucine-enkephalin is provided.     

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.     

Synthesis of a potent enkephalin analog,Tyr-D-Thr-Gly-Phe-Δ3Pro-NH2, and some of its biological activities

The enkephalin analog, H-Tyr-D-Thr-Gly-Phe-Δ³Pro-NH₂ x HCl (VI)([D-Thr², Δ³Pro⁵]-enkephalinamide), has been synthesized by conventional methods in solution and purified to homogeneity by reversed phase HPLC. The analog is a potent analgesic agent. For evaluation of some of its biological activity a related compound [D-Thr², Thz⁵]-enkephalinamide (XI) was also synthesized in solution. Anti-diarrhea activity was evaluated in mice by the intravenous route for anti-DL-5-hydroxytryptophan (5-HTP) induced diarrhea activity. Analgesic activity was assayed by the method of Nilsen in mice using the intravenous route, and by a modified tail flick test in rats and the acetic acid writhing test in mice following subcutaneous administration. Within the constraints of the assays the two analogs are approximately equipotent. Both are less active than [D-Ala², MePhe⁴, Met(0)ol]-enkephalinol (XII). Earlier receptor binding studies of compound XI indicated enhanced affinity for the μ receptor and little for the δ receptor. By comparison this may also be the case for compound VI.     

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.     

An electromyographic method for the assessment of naloxone-induced abstinence in morphine-dependent rats

Summary The electromyographic (EMG) activities of suprahyoideal muscle were recorded to measure naloxone-precipitated abstinence signs in morphine-dependent rats anesthetized with urethane (1 g/kg). Rats were rendered dependent on morphine by implanting 2 morphine pellets (75 mg each) and abstinence signs were induced by intravenous injections of various doses of naloxone at different times after pellet implantation. Three precipitated abstinence signs, a) myoclonic twitch activity (MTA), b) mastication, and c) body shakes were observed on EMG recordings after the injection of naloxone. Of these symptoms, only the MTA induced by naloxone (10 g/kg) occurred 4 h after pellet implantation and its sensitivity to naloxone increased with prolonged pellet implantation. Both mastication and precipitated shakes could be induced at 24 h. However, higher doses of naloxone were required to produce the shakes than is required to induce mastication. There appears to be a positive correlation between the intensity of naloxone-induced MTA and the degree of physical dependence on morphine. Since the MTA and mastication can be induced by low doses of naloxone in morphine-dependent rats, we suggest that these two parameters may be used to detect morphine abstinence signs in this species.Deceased February 13, 1979     

The effect of aspartic acid on the intensity of physical dependence in morphine dependent mice

The mice (Balb/C strain) given 2% aspartic acid in 5% saccharose solution or only saccharose solution (p. os) starting 12 hr before the sc morphine pellet implantation until the removal of pellet were rendered physically morphine dependent. During the development of morphine dependence and after the removal of pellets (on 3-d day) spontaneous motor activity and analgesic threshold were measured as reliable abrupt withdrawal signs. Aspartic acid prevented to some extent the appearance of symptoms of physical morphine dependence.     

Opiate-like action of methionine-enkephalin in inhibiting morphine abstinence syndrome

Intracerebroventricular administration of methionine-enkephalin or morphine sulfate immediately prior to naloxone administration inhibited the precipitated withdrawal jumping response in mice rendered dependent on morphine by the pellet implantation method. Both methionine-enkephalin and morphine sulfate failed to inhibit withdrawal defecation and rearing behavior. Morphine sulfate was found to be four times as potent as methionine-enkephalin, on molar basis, in inhibiting the abstinence syndrome. These data provide new in vivo pharmacologic evidence for the opiate-like action of methionine-enkephalin.     

Time course of the effects of naturally occurring cannabinoids on morphine abstinence syndrome

The effects of a single intraperitoneal injection (10 mg/kg) of delta9-tetrahydrocannabinol, delta8-tetrahydrocannabinol and 11-hydroxy-delta8-tetrahydrocannabinol on abstinence syndrome were investigated in mice rendered dependent on morphine by pellet implantation. In morphine dependent mice from which the pellets had been removed, delta9-tetrahydrocannabinol inhibited the naloxone-precipitated jumping response as evidenced by an increase in the ED50 of naloxone. This inhibition was evident for 24 hr, the most pronounced effect being produced between two to four hr after delta9-tetrahydrocannabinol administration. Withdrawal defecation was also inhibited for two hours. Similarly, in mice from which pellets were not removed, delta9-tetrahydrocannabinol suppressed the jumping response; however, the intensity of effect was less than when the pellets were removed. delta8-tetrahydrocannabinol and 11-hydroxy-delta8-tetrahydrocannabinol were not effective in suppressing morphine abstinence syndrome two hr following their administration. The suppression of jumping response was specific, since, the vertical jumping behavior induced by coadministration of amphetamine and l-dopa was not affected by cannabinoids. These results demonstrate that single injection of delta9-tetrahydrocannabinol is effective in controlling morphine abstinence syndrome for 24 hr, and that the drugs related to cannabinoids may show promise in narcotic detoxification.     

内啡肽-morphiceptin 的构象和量子力学研究

本文用分子动力学方法对内啡肽morphiceptin(Tyr¹-Pro²-Phe³-Pro⁴-NH₂)进行了构象研究。所得结果与¹H,¹³CNMR和其它实验方法的结果一致,证明我们的构象搜索方法有效。量子力学计算表明此多肽和吗啡在空间和电性上非常相近。因此,它们应作用于同一受体。     

Differential modification of morphine and methadone dependence by interferon alpha

Subcutaneous implantation of a pellet of methadone was presented as a novel method for the establishment of physical dependence upon this agent and it was compared to (1) the state of physical dependence induced by multiple injections of methadone, administered over several days, and (2) the dependence established by injections of morphine and the implantation of a morphine pellet. Comparable signs of drug dependence were observed in rats treated with both morphine and methadone following the administration of the opiate antagonist naloxone. The administration of interferon-alpha significantly attenuated the severity of the withdrawal syndrome in dependent rats after chronic exposure to morphine and to a lesser extent after morphine and methadone in combination. In contrast, alpha interferon did not affect 6 of the 7 abstinence signs in animals dependent upon methadone alone. The observations suggest that the states of physical dependence upon morphine and methadone may be separate phenomena that involve different physiological mechanisms. Thus, interferon may be a useful adjunct in the treatment of subjects dependent upon morphine but not in those dependent on methadone.     

Cerebral cortical 5-HT1, and 5-HT2, receptors of morphine tolerant-dependent rats

The effect of chronic administration of morphine to rats on 5-HT₁ and 5-HT₂ receptors in the cerebral cortex was determined. Male Sprague-Dawley rats were implanted subcutaneously with 6 pellets of morphine (each containing 75 mg of morphine free base) during a 7 day period. Animals which served as controls were implanted with placebo pellets. The procedure for implantation of pellets produced a high degree of tolerance to and physical dependence on morphine in the rat. The tolerance to the analgesic and hyperthermic effects of morphine was demonstrated by decreased responses in the rats implanted with morphine pellet in comparison to the placebo-treated controls. The physical dependence was shown by the greater weight loss after removal of the pellet in the rats implanted with morphine pellets when compared to rats implanted with placebo pellets. The pellets were removed (withdrawn) and, after 6–8 h, the rats were sacrificed and the cerebral cortex was isolated. In another experiment the pellets were left in place (tolerant-dependent rats). The 5-HT₁ and 5-HT₂ receptors were characterized by using [^3H]5-HT and [^3H]spiroperidol as the ligands and unlabelled 5-HT and ketanserin, respectively, to determine non-specific binding. The [^3H]5-HT bound to 5-HT₁ receptors on membranes from the cerebral cortex of rats implanted with placebo pellets, at a single high affinity site, with a B_max of 102 ± 10 fmol/mg protein and a K_d of 6.02 ± 0.98 nM. Implantation of morphine pellets, followed by removal of the pellets resulted in a 50% increase in the B_maxvalue of [^3H]5-HT but the K_d values did not change. In rats from which the pellets were not removed, the B_max and K_d values of [^3H]5-HT in placebo- and morphine-treated groups did not differ. [^3H]Spiroperidol bound to 5-HT₂ receptors on cortical membranes of rats implanted with placebo pellet at a single high affinity site with B_max and K_d values of 131 ± 5 fmol/mg protein and 0.22 ± 0.01 nM, respectively. The implantation of pellets of morphine followed by removal of the pellets did not alter the characteristics of 5-HT₂, receptors, however in rats with the pellets in place, the B_max for 5-HT₂ receptors in placebo- and morphine-treated groups did not differ but the K_d values were much smaller in morphine-treated rats compared to rats implanted with placebo pellets. It is concluded that the development of tolerance to, and physical dependence on, morphine by implantation of pellets results in up-regulation of 5-HT₂ receptors whereas in morphine-abstinent rats there is a selective up-regulation of 5-HT₁ receptors on the membranes in the cerebral cortex.     

Tolerance, dependence and lethality in morphine-dependent mice after repeated oral administration of methadone

Mice were rendered tolerant to and dependent on morphine via a morphine pellet implantation. Three days later methadone hydrochloride was administered at a dose of 100mg/kg per os 3 hours after pellet removal and then daily for a total of 5--6 days. This dose of methadone was shown to exhibit a high efficacy for the blockade of morphine abrupt withdrawal jumping and only minimal toxicity. Under these conditions, the level of analgetic tolerance with respect to morphine and methadone and the level of dependence as measured by the naloxone ED50 were initially elevated by the morphine treatment. However, upon substitution with oral methadone these levels declined with time at a rate which did not differ from that of a group of mice receiving only water after morphine pellet removal. Despite these findings, the methadone treatment was associated with an increasing tolerance to methadone lethality during the administration of this narcotic which was nearly double that of a similarly treated water control group by the sixth day. This observation could not be explained by an elevation in the level of cellular tolerance rendered by the methadone treatment since the morphine LD50 was not elevated following identical treatment with morphine and then methadone. The significance of these results is discussed with respect to the role of methadone administration and its metabolism in the modification of tolerance and dependence.     

HEXAHYDRO DERIVATIVE OF (D-MET2, PRO-NH25]-ENKEPHALIN GIVES RISE TO PHYSICAL DEPENDENCE

Filippi et al. (1979) have claimed that substitution of cyclohexylalanine for phenylalanine in leucine-enkephalin and its L-Ser³ derivative resulted in synthetic analogs which were non-addicting. We have examined the hexahydro derivative of [D-Mer², Pro-NH⁵₂]-enkephalin, a potent analgesic agent, to determine if dependence liability can be separated from short-term opiate actions after hydrogenation of the phenylalanine residue. In contrast to Filippi et al.'s observations, we found that the hexahydro derivative of [D-Met², Pro-NH⁵₂]-enkephalin gives rise to physical dependence and that its short-term potencies generally paralleled its long-term ability to produce physical dependence.     

Studies on the physical dependence potential of analgesics

Influence of morphine on the fine structure of mitochondria in the cell of zona fasciculata of adrenal cortex in mice treated chronically with morphine pellets was studied using the electron microscope. The trnasformation of mitochondrial structure was observed 12 hours after morphine pellet implantation and the degree of transformation reached a maximum at 48 hours. These changes, however, disappeared within 4 days. On the 4th day after implantation, removal of the pellet or levallorphan challenge resulted in alteration of the mitochondria with evidence of withdrawal syndrome. Reinjection of morphine to the mice immediately after removal of the pellet, however, prevented the appearance of such mitochondrial transformation. Chlorpromazine or sodium pentobarbital did not affect on the transformation of mitochondria.     

Comparative effects of thyrotropin releasing hormone,MK-771 and DN-1417 on morphine abstinence syndrome

The effect of thyrotropin releasing hormone (TRH) were compared with two of its analogs, l-N-(2-oxopiperidin-6-yl-carbonyl)-l-histidyl-l-thiazolidine-4-carboxamide (MK-771) and -butyrolactone-4-carboxyl-histidylprolineamide (DN-1417) on the abrupt and naloxone-precipitated abstinence symptoms in morphine-dependent male Swiss-Wester mice. Mice were made physically dependent on morphine by subcutaneous implantation for 3 days of a pellet containing 75 mg morphine free base. Control mice were implanted with placebo pellets. Intracerebral administration of TRH (10 ng-10 g per mouse) immediately after removal of placebo pellets had no effect on the basal temperature of mice. Mice implanted with morphine pellets exhibited a characteristic hypothermic response following the removal of the pellets. TRH at all doses employed prevented the hypothermia observed during abrupt withdrawal of morphine (pellet removal). DN-1417 and MK-771 (10 ng-10 g per mouse) on the other hand produced a short lived hyperthermic response in mice from which placebo pellets had been removed. However, both TRH analogs produced long-lasting antagonism of withdrawal hypothermia in mice from which morphine pellets had been removed. TRH and its analogs had no effect on the body weight loss observed during abrupt withdrawal of morphine. Intracerebral administration of 10 g TRH and its analogs inhibited the naloxone-induced jumping response as evidenced by increases in naloxone ED₅₀ values to elicit this response. It is concluded that TRH and its analogs may be useful in combating some of the withdrawal symptoms in opiate-dependent subjects.     

Effects of MK 801 on morphine physical dependence: attenuation and intensification.

It has previously been reported that the noncompetitive NMDA receptor antagonists ketamine and dextromethorphan suppressed the naloxone-induced morphine abstinence syndrome. In addition, the previous blockade by ketamine and dextromethorphan of NMDA receptors has been shown to intensify the naloxone-elicited morphine abstinence syndrome. On the basis of this information, another noncompetitive NMDA receptor antagonist, (+)-5-methyl-10,11-dihydro-5H-dibenzo-a,d-cyclohepten-5,10-imine maleate (MK 801), was administered to rats in which two morphine-containing (75 x 2 morphine base) pellets had been implanted. The naloxone-precipitated abstinence syndrome in rats injected with 0.3 mg/kg MK 801 36 h after pellet implantation was found significantly more intense than controls whereas the abstinence syndrome in rats that received 0.1 mg/kg MK 801 before naloxone injection was less intense. The intensification by MK 801 given 36 h following pellet implantation was attributed to the further increase in upregulation and supersensitivity of NMDA receptors caused by morphine. The attenuation was explained by the blockade by MK 801 of NMDA receptors as occurred in the case of ketamine and dextromethorphan.     

Influence of Plasma-protein Binding on Analgesic Effect of Methadone in Rats with Spontaneous Withdrawal

The effect of spontaneous withdrawal on α₁-acid glycoprotein (AAG) levels and methadone protein binding has been studied in the rat. Animals were made physically dependent on morphine by providing morphine HCl in drinking water for three weeks. The natural opiate withdrawal was induced in rats by substituting the morphine solution with drinking water. The severity of the abstinence syndrome was assessed at various time intervals. After 12 h of withdrawal, the animals showing abstinence signs and low morphine levels were injected with intravenous methadone (0.35 mg kg^?1) and the analgesic effect was measured by the tail-flick method and compared with animals receiving water. The oral administration of morphine produced an increase in AAG levels from 0.64 ± 005 g L^?1 in control animals to 1.47 ± 0.92 g L^?1 in experimental animals at the point of withdrawal and 1.21 ± 009 g L^?1 24 h after withdrawal. The percentage of methadone unbound was significantly lower in morphine-treated than in control animals. A significant correlation between AAG levels and percentage of methadone bound was observed. A parallel analgesic effect after intravenous methadone, as measured by AUC in the tail-flick test, was less in abstinence animals than in control (287.6 ± 24.8 compared with 401.0 ± 37.06 s min). We suggest that in the withdrawal syndrome an adjustment of methadone dose may be necessary because of changes in protein binding.     

Tachykinin antagonists inhibit the morphine withdrawal response in guinea-pigs

Summary This study was an investigation of the effects of the tachykinin antagonists, spantide and (d-Pro⁴, d-Trp^{7, 9, 10})substance P 4-11, injected intracerebroventricularly (ICV), on the locomotor and behavioural responses of guinea-pigs to substance P (SP) injected ICV and to naloxone-induced morphine withdrawal. SP, 50 nmol, produced increased locomotor activity and behaviour that mimicked the response induced by injection of naloxone hydrochloride, 15 mg/kg, in guinea-pigs treated 2 h previously with morphine sulphate, 15 mg/kg. Spantide or (d-Pro⁴, d-Trp^{7, 9, 10})SP4-11, 10 nmol, reduced the locomotor and behavioural responses to SP and to morphine withdrawal. The results support the suggestion that SP or a related tachykinin might be a mediator of the opioid withdrawal response in the central nervous system as has been proposed for the enteric nervous system.     

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.