戒毒治疗与护理
关键词：颅痛定 药物耐受 吗啡依赖
Abstract To study effects of rotundine on morphine tolerance, physical dependence and psychic dependency, the pain is measured by mouse hot-plate test. The physical and psychic dependences are evaluated by naloxone-precipitated withdrawal in rats and conditioned place preference in mice, respectively. Female mice were given 10 mg/kg(i. p. ) of morphine every day for 9 days. The antinociceptive effect of morphine was decreased each day, after the 6th day morphine had no antinociceptive effect, indicating significant tolerance to morphine. Rotundine (20 mg/kg, s. c. ) significantly inhibited the tolerance development. Naloxone (4 mg/kg, i. p. )precipitated a series of abstinence symtoms in morphine-addicted rats. Rotundine remarkably suppressed some behavioral signs of withdrawal of morphine such as jumping, teeth chattering, diarrhea and irritability, but not wet-dog shakes and ptosis. Rotundine also significantly attenuated the conditioned place preference of morphine. It is obtained rotundine significantly inhibited morphine tolerance, psychic dependence and partial abstinence symtoms.
Key words rotundine drug tolerance morphine dependence
0 Introduction
Major problem in the use of opiate analgesic drugs is the addiction, which is manifestated in tolerance, physical dependence and psychic dependence. Although the treatment of opiate dependence has been investigated for many years and some treatments have been applied, such as methadone, clonidine, naltrexone and buprenorphine methods, there are still some defects, especially for the treatment of psychic dependence. Numerous studies have demonstrated that dopaminergic system was involved in opiate dependence［1-5］. Naloxone administration to morphine-dependence animals reduces extracellular dopamine concentrations in the ventral tegmental area［6］ and nucleus accumbens［7］. Furthermore, clonidine pretreatment that prevent opiate withdrawal symptoms, also eliminates the decrease in accumbal dopamine levels. Dopamine agonists significantly attenuated somatic withdrawal symptoms, especially dopamine-2 selective agonist .［8］ More recently,Maldonado et al.［9］reported that dopamine-2 receptor plays a crucial role in the motivational component of morphine addiction. Rotundine is 1-optical isomer of tetrahydropalmatine, which is an active alkaloid isolated from the stem tubers of the chinese medicinal herb Corydalis ambigua or Yanhuso. It was found to have analgesic, sedative and tranquilizing effects ［10,11］. The biochemical studies proved that rotundine was a dopamine-2 receptor blocker［12,13］.Recently, rotundine is being tried in the treatment of morphine tolerance and dependence in China. However the experimental evidence is lacking. The aim of the present paper was to investigate effects of rotundine on morphine tolerance and dependence.
1 Materials and Methods
1.1 Animals
Kunming strain mice weighting 18-22 g were used in tolerance (female) and psychic dependence (male) experiments. Male Sprague-Dawley rats weighting 190-210 g were used in physical dependence experiment. The animals were housed in a room with controlled temperature (23+1) ℃ and a 12 h light-dark cycle. Animals were housed 5 to a cage in tolerance experiments,and were housed individually in dependence experiments. Food and water were made available continuously.
1.2 Chemicals
Rotundine［α=-289°］ was isolated from Corydalis B(dl-tetrahydropalmatine) which was obtained through extracting and hydrogenizing palmatine existed in Fibraurea tinctoria, Lour, by department of phytochemistry, Shanghai Institute of Materia Medica, Chinese Academy of Sciences. Morphine sulfate (5H2O) and naloxone hydrochloride were purchased from MacFarlan Smith Ltd.(Edinburgh,Great Britain) and DUPONT,respectively.
1.3 Morphine analgesic tolerance
The analgesic effect was measured by hot-plate test as described before［14］. The plate temperature was (55±1)℃.The latency of hind-paw lick was as index of pain sensitivity.A 60s cut-off was imposed to avoid tissue damage. Rotundine was given subcutaneously (s.c.) 10 min before morphine injection (i.p.).Hot-plate latencies were measured before and 20 min after morphine injection. The basal hind-paw lick latencies were subtracted from the effects induced by the drug for each mouse.Percentage maximum possible effect(% MPE) was calculated using the following formula:
1.4 Morphine physical dependence
The physical dependence was evaluated essentially as the method in Ref.8.Morphine dependence was induced in rats by repeated triplicate daily i.p. injection of morphine, (at 8:30,14:30 and 20:30) for 6 days. The morphine doses were started at 10 mg/kg, and increased by 10 mg/kg every day until maintained at 50 mg/kg. On day 7 only one injection of morphine at 50 mg/kg was in morning. Control rats were treated with saline under the same conditions. Rotundine was given subcutaneously (s.c.) at a dose of 20 mg/kg, 20 min before naloxone injection. Withdrawal was precipitated only once in each animal by receiving naloxone (4 mg/kg,i.p.) 3 h after the last morphine administration. Rats were placed individually into round boxes(30 cm diameter, 40 cm height) with a white floor after naloxone injection. The behavioral signs of withdrawal were evaluated immediately after injection for 30 min.The number of jumping and wet-dog shakes was counted. Writhing, teeth chattering, diarrhea, ptosis and vocalizing on touch were evaluated over periods of 5 min, one point being given for the presence of each sign during each period. The number of periods showing the sign was then counted (maximum score of 6). Body weight was determined before and 30 min after naloxone injection.
1.5 Morphine psychic dependence
The psychic dependence was performed by the conditioned place preference. The method was similar［9］. The conditioning time was counted for 15 min, and the size of compartments was 15 cm×15 cm×15 cm. The experiments were conducted during the light phase of the dark/light cycle. The place preference condition schedule consisted of three phases. (1) Preconditioning phase,during which mice were placed in the middle of the box and their location time was recorded for the 15 min. After the session, animals were randomized to be paired to drug or saline ad
Fig.1 Effect of rotundine on development of tolerance to morphine in hot-plate test in mice (+SD,n=8)
Mice were injected with morphine (Mor. 10 mg/kg,i.p.),rotundine (Rot.20 mg/kg,s.c.) or morphine (10 mg/kg,i.p.) plus rotundine(20 mg/kg,s.c.)every day for 9 days.%MPE=［(test latency-control latency)/(60-control latency)］×100.*P＜0.05；**P＜0.01 versus morphine group
ministration and to be assigned to a compartment.(2)Conditioning phase,during which mice were treated for 5 consecutive days with alternate injections of drug or saline. Doors matching the walls of the compartment allowed the confinement of the mice for 50 min immediately after drug or saline injections. Mice received the drug in morning and saline in afternoon. Control animals received saline each time. The two treatment sessions were at least 4 h apart.(3) Postconditioning phase, which was conducted exactly as the preconditioning phase. Animals were accessed to each compartment for 15 min in morning on day 7.The amount of time spent in the compartment associated with morphine during a 15 min test period was recorded.
1.6 Statistical analysis
Fig.2 Effect of rotundine on behavioral signs of abstinence and weight loss during naloxone precipitated morphine withdrawal syndrome in rats (+SD,n=5)
Opiate dependence was induced by repeated injection i.p.of morphine triplicate daily (at 8:30,14:30 and 20:30) for 6 days. The morphine doses were started at 10 mg/kg, and increased by 10 mg/kg every day until maintained at 50 mg/kg. Rotundine was given subcutaneously once at a dose of 20 mg/kg,20 min before naloxone injection (4 mg/kg,i.p.). The number of jumping and wet-dog shakes was counted. Teeth chattering, diarrhea, vocalizing, writhing and ptosis were evaluated over periods of 5 min, one point being given for the presence of each sign during each period. The number of periods showing the sign was then counted (maximum score of 6).*P＜0.05;**P＜0.01;***P＜0.001 versus morphine group
The results are expressed as means percentages MPE±S.D. The differences bwtween the individual mean values in different group were analyzed by Student's unpaired t-test (two-tailed,Microsoft Excel). A value of P＜0.05 was considered to be significant.
2 Result
2.1 Effect of rotundine on tolerance to morphine
The effect of rotundine on the antinociception induced by morphine is shown in Fig.1. The animals were administrated 10 mg/kg of morphine every day for 9 days. The antinociceptive effect of morphine was decreased each day, indicating significant tolerance to morphine. On the 6th day, morphine had not antinociceptive effect (P＞0.05 versus the control).Rotundine by itself in dose of 20 mg/kg did not alter the hindpaw-lick latency and thus did not produce antinociceptive effect. However, pretreatment of rotundine (20 mg/kg,s.c.) significantly inhibited the tolerance development of morphine.Between chronic morphine-treated group and chronic(morphine+rotundine)-treated group there were significant differences on 3th and 4th days(P＜0.05)，and very significant differences on 5th, 6th and 7th days(P＜0.01).
2.2 Effect of rotundine on physical dependence on morphine(Fig.2)
Naloxone administration (4 mg/kg,i.p.) precipitated the behavioral signs of withdrawal in morphine-treated group and did not trigger behavioral changes in saline-injected control group. However, when rotundine was given subcutaneously(s.c)at a dose of 20 mg/kg, 20 min before naloxone injection in morphine-treated animals, animals did not appear jumping response and irritability. Moreover, the other signs of withdrawal, such as teeth chattering, writhing, diarrhea and body weight loss, also were significantly attenuated (P＜0.05 versus morphine alone group). But the behavioral changes of wet-dog shakes and ptosis were similar between with and without rotundine in morphine-treated animals.
2.3 Effect of rotundine on morphine psychic dependence
As shown in Fig.3, rotundine alone (20 mg/kg.s.c.) did not affect the spent time in the drug-associated compartment (P＞0.05 versus the preconditioning phase). Morphine administration (10 mg/kg,i.p.) induced a clear place preference, indicated by a significant increase in the time spent in the drug-associated compartment during the postconditioning phase (P＜0.05 versus the preconditioning phase). However, the coadministration of morphine (10 mg/kg,i.p.) and rotundine(20 mg/kg,s.c.) did not induced place preference (P＞0.05 versus the preconditioning phase). There is a very significant difference between morphine group and (morphine+rotundine) group when compared with postconditioning time spent in the drug-associated compartment(P＜0.01), showing inhibition of rotundine for morphine-induced a place preference.
3 Discussion
The present study demonstrated that rotundine in dose of 20 mg/kg inhibited the development of tolerance to the analgesic effect of morphine in the mice. However,the mechanisms by which rotundine inhibites the morphine tolerance development is not as yet understood. Morphine analgesia undoubtedly involves multiple neurotransmitter systems at spinal and supraspinal levels. There in now considerable evidence that chronic administration of morphine, which results in the development of tolerance to many of its pharmacologic actions, is associated with alterations in the activity of brain dopaminergic neuron［15］. Previous report has shown that rotundine is a dopamine receptor antagonist［12］. Therefore, the alteration of morphine tolerance by rotundine may be involved in dopamine receptors.
Morphine physical dependence was induced by intraperitoneal injections of morphine triplicate daily for 6 days. This method induces a severe degree of dependence in rats as shown by the remarkable withdrawal syndrome after naloxone administration. We have evaluated eight characterized behavioral signs of morphine abstinence in rats:jumping, wet-dog shakes, teeth chattering, writhing, diarrhea, ptosis, vocalizing and body weight loss. A direct relationship between the appearance of jumping behavior or teeth chattering and the severity of the abstience syndrome is found in some studies［16,17］. They have been designated as dominant signs of the withdrawal syndrome in rodents. Rotundine significantly inhibited jumping, teeth chattering and irritability. However, withdrawal-induced wet-dog shakes and ptosis were less effectively antagonizied by rotundine than the other symptoms. Catecholaminergic activation has been directly related to the expression of withdrawal, and particularly to the number of jumps［18,19］.The biochemical effects on the rat monoaminergic neuronal system suggested that rotundine is a short-acting monoamine depletor［20］.Thus, the decrease in catecholaminergic activity induced by rotundine administration could be involved in inhibition of morphine withdrawal syndrome. The disappearance of jumping behavior and vocalizing on touch in rats induced by rotundine during morphine withdrawal may be also due to the tranquilizing action properties of rotundine.
In the physical dependence experiment of this study, rotundine (20 mg/kg,s.c.) was given once 20 min before naloxone injection.The chronic treatment(t.i.d.) of rotundine in the dose used in the absence of morphine did not display any overt toxicity. However, the chronic coadministration of rotundine with morphine,the inhibition of morphine physical dependence in the rats by rotundine was associated with serious toxicity such as higher weight loss and occasional death.
Fig.3 Effect of rotundine on morphine-induced place preference in mice(+SD,n=5)
Mice received morphine (Mor.10 mg/kg,i.p.),rotundine (Rot.20 mg/kg,s.c.)or morphine (10 mg/kg,i.p.) plus rotundine (20 mg/kg,s.c.).The time spent in the drug-associated compartment during the preconditioning (white bars) and the postconditioning phases (black bars) was determined. *P＜0.05 versus the preconditioning phase;**P＜0.01 versus postconditioning phase in morphine group
The effect of rotundine on morphine psychic dependence had also been investigated in this study, using place preference test. The results showed that rotundine by itself did not induced place preference, and could antagonize the morphine-induced conditioned behavior. Recently, Maldonado et al［9］. had report that dopamine-2 receptor plays a crucial role in the motivational component of morphine addiction using the knockout technology of dopamine-2 receptor gene in mice.The binding study revealed that the affinity of rotundine toward dopamine-2 receptor was 211 times higher than that toward dopamine-1 receptor［12］. Therefore,the mechanism in which rotundine inhibited morphine induced psychic dependence may be involved in blockade of dopamine-2 receptor.
In conclusion, the present studies demonstrated that the pretreatment of rotundine significantly inhibited the tolerance development of morphine and significantly attenuated morphine-induced a conditioned place preference in mice. In addition, rotundine could suppress some withdrawal symptoms such as writhing, teeth chattering and diarrhea, but not other signs of physical dependence in rats. Therefore, rotundine in appropriate dose will be useful for morphine addicts-treatment. Further study of the relationship between the action of rotundine and dopamine receptor subtype in central nervous system is required.
References：
1 Spyraki C, Fibiger H C, Phillips A G. Attenuation of heroin reward in rats by disruption of the mesolimibic dopamine system. Psychopharmacology,1983;79:278-283
2 Imperato A, Di Chiara G. Trans-striatal dialysis coupled to reverse phase high performance liquid chromatography with electrochemical detection:a new method for the study of the “in vivo” release of endogenous dopamine and metabolites. J Neurosci,1984;4:966-977
3 Acquas E, Carboni E, Di Chiara G.Profound depression of mesolimbic dopamine release after morphine withdrawal in dependent rats. Eur J Pharmacol,1991;193:133-134
4 Maldonado R, Stinus L, Gold L, Koob G F.Role of different brain structures in the expression of the physical morphine withdrawal syndrome. J Pharmacol Exp Ther,1992;261:669-677
5 Bozarth M A.Physical dependence produced by central morphine infusions: an anatomical mapping study. Neurosci Biobehav Rev,1994;18:373-383
6 Rossetti Z L, Hmaidan Y, Gessa G L. Marcked inhibition of mesolimbic dopamine release: a common feature of ethanol, morphine, cocaine and amphetamine abstinence in rats. Eur J Pharmacol,1992;221:227-234
7 Pothos E, Rada P, Mark G P, Hoebel B G. Dopamine microdialysis in the nucleus accumbens during acute and chronic morphine,naloxone-precipitated withdrawal and clonidine treatment.Brain Res,1991;566:348-353
8 Harris G C, Aston-Jones G. Involvement of D2 dopamine receptors in the nucleus accumbens in the opiate withdrawal syndroms. Nature, 1994;371:155-157
9 Maldonado R, Saiardi A, Valverde A O, Samad T A, Roques B P, Borrelli E. Absence of opiate rewarding effects in mice lacking dopamine D2 receptors.Nature, 1997;388:586-589
10 Hsu B, Kin K C. Some pharmacological properties of corydalis B (tetrahydropalmatine) and its related compounds. Scientia Sinica, 1964;8:605-609
11 Kin K C, Zhen X F, Hsu B. Studies on the pharmacological actions of corydalis:XII.The effects of optical isomers of tetrahydropalmatine on central nervous system.Acta Physiologica Sinica, 1964;27:47-58
12 Xu S X, Yu L P, Han Y R, Chen Y, Jin G Z. Effects of tetrahydroprotoberberines on dopamine receptor subtypes in brain. Acta Pharmacologica Sinica, 1989;10:104-110
13 Sun B C, Huang K X, Jin G Z. Comparison of effects of tetrahydropalmatine enantiomers on firing activity of dopamine neurons in substantia nigra pars compacta.Acta Pharmacologica Sinica, 1992;13:292-297
14 Chao Y, Tsoh S Y. A modified “hot-plate” method for the evaluation of analgesics.Acta Pharmaceutica Sinica, 1956;4:97-105
15 Bhargave H N. Cyclo(leucycglycine) inhibits the development of morphine induced analgesic tolerance and dopamine receptor supersensitivity in rats. Life Sci, 1980;27:117-123
16 Marshall I, Weinstock M. Quantitative method for assession one symptom of the withdrawal syndrome in mice after chronic morphine administration. Nature, 1971;234:223-224
17 Blasig J,Herz K,Reinhold K,Zieglgansberger S.Development of physical dependence on morphine in respect to time and dosage and quantification of the precipitated withdrawal syndrome in rats. Psychopharmacologia, 1973;33:19-38
18 Simonato M.The neurochemistry of morphine addiction in the neocortex. Trends Pharmacol Sci,1996;17:410-415
19 Ushijima J, Mizuki Y, Yamada M, Glaving G. Neuronal mechanisms involved in drug-induced jumping behavior in mice.Eur J Pharmacol,1985;112:225-229
20 Liu G-Q, Algeri S, Garattini S. DL-Tetrahydropalmatine as monoamine depletor. Arch Inter Pharmacodyn Ther,1982;258:39-50