反相离子对高效液相色谱法测定血浆吗啡及其代谢物M3G的含量

目的:建立反相离子对高效液相色谱法测定血浆吗啡及其代谢物3-葡萄糖醛酸吗啡(M3G)的浓度。方法:血浆样品以乙酸乙酯提取,以纳洛酮为内标。应用HPLC-UV方法测定吗啡浓度,分析柱DiamonsilTMC18(250mm×4.6mm),流动相为0.01mol·L-1磷酸二氢钾缓冲液(含0.5mmol·L-1的十二烷基磺酸钠)-乙腈(71:29),检测波长210nm,流速0.65ml·min1。血浆样品经β-葡萄糖醛酸苷酶水解测定其代谢物含量。结果:吗啡标准曲线线性范围宽(10-4000μg·L-1),线性关系良好;最低检测限10μg·L-1;高、中、低浓度的回收率在85·3%以上。血浆M3G在250-8250μg·L-1浓度范围内与水解增加吗啡浓度呈线性关系。结论:本法简单,快速,血浆中杂质不干扰样品的测定,满足生物样品分析要求。     

戒毒康胶囊对吗啡依赖性大鼠戒断症状的治疗作用评价

目的：评价戒毒康胶囊对吗啡依赖性大鼠戒断症状的治疗作用。方法：采用吗啡依赖性大鼠催促戒断模型和自然戒断模型。结果：（1）在吗啡依赖性大鼠催促戒断治疗试验中,戒毒康胶囊0．25、0．5和1．0g／kg剂量均可显著降低吗啡依赖性大鼠的催促戒断症状分值（P〈0．01）,0．5和1．0g／kg剂量抑制体重下降（P〈0．05或P〈0．01）。（2）在吗啡依赖性大鼠自然戒断治疗试验中,戒毒康胶囊0．25、0．5和1．0g／kg剂量,均可明显抑制吗啡依赖性大鼠自然戒断后的体重下降（P〈0．05或P〈0．01）。结论：戒毒康胶囊能显著降低吗啡依赖性大鼠催促戒断和自然戒断后所产生的戒断反应,具有明显的脱毒治疗作用。     

γ-氨基丁酸对大鼠吗啡戒断症状的影响及其作用机理

目的研究γ-氨基丁酸(GABA)对大鼠吗啡戒断症状的影响及其可能的机理。方法建立大鼠吗啡依赖模型,在戒断症状高峰出现前30min侧脑室(icv)注入GABA或生理盐水(0.9%),观察其戒断症状的改变。用分光光度法测定戒断时大鼠血清及脑组织中一氧化氮(NO)含量、一氧化氮合酶(NOS)活性。结果GABA可缓解吗啡戒断时理毛、湿抖、扭体、咬牙、舔阴等运动反应,减少活动次数,抑制植物神经系统症状。Icv注入10μl(500μg·10μl-1)的GABA可抑制吗啡依赖大鼠血清及脑组织NO含量和血清中NOS活性的增高(P<0.05)。结论GABA可缓解大鼠吗啡戒断症状,其作用机理可能与阿片受体、中枢神经系统中NO的表达机制有关。     

异博定对幼年大鼠吗啡戒断症状的治疗作用

目的··:观察异博定对幼年大鼠吗啡成瘾后戒断症状的治疗作用。方法··:将50只SD幼鼠分为异博定治疗组(n=20)、实验对照组 (n=20)和阴性对照组 (n=10) ,建立吗啡成瘾幼鼠动物模型和吗啡戒断动物模型 ,用异博定对纳洛酮诱发的吗啡戒断幼鼠的戒断症状进行治疗 ,并分别对异博定治疗组和试验对照组吗啡成瘾及戒断症状进行观察和比较。结果··:给成瘾动物单次ip异博定 (10mg·kg-1)能明显缓解由纳洛酮诱发的吗啡戒断症状 ,并对吗啡戒断引起的体重下降有减轻作用。而连续3d治疗能明显加速纳洛酮诱发的戒断症状的缓解 ,且与试验对照组比较有显著性差异 (P<0.01)。结论··:异博定对纳洛酮诱发的幼年大鼠吗啡戒断症状有一定治疗作用。     

川芎嗪对小鼠吗啡戒断症状的抑制作用

目的:研究川芎嗪(Lig)对小鼠吗啡戒断症状的影响。方法:以剂量递增法形成吗啡依赖模型,用纳洛酮催促戒断。结果:sc给药,Lig 2 0mg/kg抑制“湿狗”样抖动、打洞、前爪震颤症状;Lig 4 0mg/kg抑制跳跃、“湿狗”样抖动、上睑下垂、前爪震颤、体重下降等症状;Lig 80mg/kg抑制“湿狗”样抖动、打洞、上睑下垂、前爪震颤、体重下降等症状。结论:Lig能抑制大部分吗啡戒断症状。     

氯胺酮对大鼠吗啡戒断症状的影响及其作用机理

目的　研究氯胺酮对大鼠吗啡戒断症状的影响及其可能的机理。方法　建立大鼠吗啡依赖模型 ,在用纳洛酮催瘾前 2min给予不同剂量的氯胺酮 ,观察其戒断症状的改变 ;用分光光度法测定戒断时大鼠一氧化氮 (NO)含量、一氧化氮合酶 (NOS)活性 ,用放射免疫法测定环鸟苷酸 (cGMP)含量。结果　 3个剂量的氯胺酮 (5、10和 2 0mg·kg- 1)均可缓解吗啡戒断时探究、扭体、湿狗样抖动、跳跃等运动反应 ,减少活动次数 ,抑制植物神经系统症状。10、2 0mg·kg- 1氯胺酮可显著减轻吗啡戒断所致的体重下降 ,小剂量氯胺酮 (5mg·kg- 1)可抑制吗啡依赖大鼠前额叶皮质、小脑的NOS活性和NO、cGMP含量的增高。结论　氯胺酮可缓解大鼠吗啡戒断症状 ,其作用机理可能与减弱大鼠吗啡戒断时NMDA NO cGMP通路效应有关。     

清风胶囊对吗啡依赖大鼠戒断症状治疗作用的研究

目的：本实验通过制作吗啡依赖的大鼠催促戒断、自然戒断的动物模型,观察清风胶囊对吗啡依赖大鼠催促戒断、自然戒断症状的治疗作用。方法：通过复制吗啡依赖大鼠催促戒断和自然戒断模型,评价中药制剂清风胶囊对动物吗啡戒断症状的抑制作用,并与阳性药可乐定进行比较。结果：在大鼠催促戒断模型上,清风胶囊三个剂量组（0．5,1．0,2．0g／kg）ig给药均能部分控制戒断症状,并能有效抑制大鼠的体重下降。在吗啡依赖大鼠自然戒断模型上,清风胶囊中、高两个剂量组（1．0,2．0g／kg）控制体重下降作用均优于可乐定。清风胶囊中、高剂量组能明显控制吗啡依赖大鼠的自然戒断症状,与模型组比较有显著性差异（P〈0．05）。结论：清风胶囊对吗啡依赖大鼠戒断症状有肯定的脱毒治疗效果。     

达尔康对吗啡依赖大鼠戒断症状的影响

目的 :观察中药达尔康对吗啡依赖大鼠戒断症状的抑制作用。方法 :采用剂量递增法皮下注射吗啡 14d和3 0d ,分别建立大鼠吗啡依赖模型 ,观察达尔康大、中、小 3个剂量对吗啡依赖大鼠纳洛酮催促和自然戒断的戒断症状及体重的影响。结果 :达尔康能显著减轻吗啡依赖大鼠ip纳洛酮引起的催促戒断症状 (P <0 .0 5 )和体重下降(P <0 .0 1) ,能抑制自然戒断大鼠体重下降 (P <0 .0 1和P <0 .0 5 )。结论 :达尔康对吗啡依赖大鼠戒断反应有明显的抑制作用     

针刺对吗啡依赖大鼠戒断症状及M—Enk表达的影响

目的探讨针刺对吗啡依赖大鼠戒断症状和蓝斑（Locus ceruleusL C）甲硫氨酸脑啡肽（methionine—enkephalin，M—Enk）表达的影响。方法采用剂量递增方法，建立吗啡依赖模型。针刺“神门”穴干预，测定针刺对吗啡戒断大鼠蓝斑M—Enk的表达。结果针刺可减轻依赖大鼠的戒断症状，并使吗啡戒断大鼠蓝斑组织M—Enk表达增高（P〈0．01）。结论针刺能调动戒断大鼠脑内阿片肽的表达。     

Pharmacokinetics and pharmacodynamics of morphine-3-glucuronide in rats and its influence on the antinociceptive effect of morphine

In this study the pharmacokinetics and pharmacodynamics of morphine-3-glucuronide (M3G) were investigated in rats after i.v. administration as a bolus dose (86.7 μmol kg^?1) and as a constant rate infusion (2.9 μmol h^?1) over 5 days. After the bolus dose, the clearance (Cl) was 12.1 ± 0.6 ml min ^?1* kg, the volume of distribution at steady state (V_ss) 1.68 ± 0.89 1 kg^?1, the half-life of the first phase 13.2 ± 1.8 min and the halflife of the second phase 11.6 ± 7.7 h. After the constant rate infusion, Cl was 10.5 ± 1.7 ml min^?1*kg. The antagonistic effect of M3G on the antinociceptive effect of a bolus dose of morphine (35 μmol kg^?1) was tested during steady state concentrations of M3G on day 4 and to M3G naïve rats. No antinociceptive, hyperalgesic or withdrawal effects were observed as a result of M3G administration, but a significantly lower antinociceptive effect of morphine was found in the M3G infusion group compared to the control group. Systemically administered M3G antagonized the antinociceptive effect of morphine, but this cannot be the only explanation to the tolerance development observed after morphine administration.     

High levels of morphine-6-glucuronide in street heroin addicts

Rationale In the body, heroin is rapidly transformed to 6-acetylmorphine (6-AM) and then to morphine, that in turn is mainly metabolized to morphine-3-glucuronide (M3G) and, at lesser extent, to morphine-6-glucuronide (M6G). Unlike M3G, M6G is a potent opioid agonist. Intravenous heroin abusers (IHU) are exposed to a wide array of drugs and contaminants that might affect glucuronidation. Objectives We assessed plasma and urine concentrations of M3G and M6G in four groups of subjects: the first two included long-term IHU either exposed to street heroin (n=8) or receiving a single IV injection of morphine (n=4), while the other two groups included non-IHU patients receiving acute IV (n=8) or chronic oral (n=6) administrations of morphine. Methods After solid phase extraction plasma and urine concentrations of morphine metabolites were determined by HPLC analyses. Results M3G accounted for the greater part of morphine glucuronides detected in body fluids of non-IHU patients treated with morphine. This pattern of metabolism remained stable across 15 days of oral administration of incremental doses of morphine. In contrast, the two groups of IHU (street heroin taking or morphine-treated subjects) showed a reduction of blood and urine M3G concentrations in favor of M6G. Consequently, M6G/M3G ratio was significantly higher in the two IHU groups in comparison with the non-IHU groups. Conclusions Chronic exposure to street heroin causes a relative increase in concentrations of the active metabolite, M6G. Since the pattern of M6G action seems closer to heroin than to morphine, the increased synthesis of M6G observed in IHU may prolong the narrow window of heroin effects.     

Heroin-using drivers: importance of morphine and morphine-6-glucuronide on late clinical impairment

Objective To evaluate the relationship between major heroin metabolites (morphine, morphine-6-glucoronide), pattern of drug use, and late impairment of psychomotor functions.Methods From the database of the Norwegian Institute of Public Health, Oslo, blood morphine concentration in samples from heroin users (n=70) containing only morphine were correlated with results of the clinical test for impairment (CTI). For comparison, test results were explored in individuals without any positive analytical finding in blood samples (n=79) selected from the same database.Results In the “no drug” cases, 86% were judged as not impaired and 14% as impaired. In the morphine only cases, 20% were judged as not impaired, and 80% as impaired. Both daily users and non-daily users had the same proportion of impaired cases. Median blood morphine concentration (M) was 0.09 μmol/l in the “not impaired” group and 0.15 μmol/l in the “impaired” group (P=0.067). For morphine-6-glucuronide (M6G), the median blood concentration was 0.09 μmol/l in the “not impaired” group and 0.14 μmol/l in the “impaired” group (P=0.030). A significant correlation between concentration quartiles and number of cases determined as “impaired” was found for M6G (P=0.018) and for the sum M+M6G (P=0.013).Conclusion In our population of heroin-drugged drivers, blood concentrations of M6G and the sum M+M6G appeared to have concentration-dependent effects on the CNS that may lead to impairment as judged from a CTI. Variations in pattern of use did not seem to have any bearing on the judgement of impairment.     

Uptake of morphine-3-glucuronide by choroid plexuses in vitro

The uptake of morphine-3-glucuronide (morphine glucuronide) by choroid plexuses of rabbits was investigated in vitro. The uptake of morphine glucuronide could not be saturated by increasing the concentration of morphine glucuronide in the medium and the tissue to medium concentration ratio of morphine glucuronide was less than unity. These results indicate that the choroid plexus does not transport morphine glucuronide by an active process as it does morphine.     

Non-opioid induction of morphine-6-glucuronide synthesis is elicited by prolonged exposure of rat hepatocytes to heroin

Background

Liver metabolism of morphine leads to the formation of morphine-3-glucuronide (M3G) and morphine-6-glucuronide (M6G), the latter possessing strong opioid activity that however differs from that of the parent compound. In previous studies conducted in rats we have shown that repeated in vivo exposure to phenanthrene class of mu opioid receptor (MOR) agonists or antagonists (heroin, morphine, and naltrexone), but not to non-phenanthrene class of MOR agonist methadone, affects morphine glucuronidation by liver microsomes.

Methods

In the present study, we measured the in vitro formation of M3G and M6G by rat hepatocytes incubated for 120 min with morphine (0.1–1.0 mM) after 72 h pre-incubation with one of the following MOR agonists: heroin (3.3 or 6.6 μM), morphine (7.8 μM), or methadone (12 μM). The MOR antagonist naltrexone (10 or 25 μM) was also tested, alone or in combination with heroin. The amount of M3G and M6G synthesized was then measured by HPLC method.

Results

Heroin inhibited M3G synthesis and induced the formation of M6G, which under basal conditions is not synthesized in rats. Heroin effects were not blocked by naltrexone. Morphine, but not methadone, produced effects similar to those of heroin but more modest in intensity. Pre-incubation with naltrexone alone slightly increased M3G synthesis, but had no effect on M6G formation.

Conclusions

These results are in agreement with those of previous ex vivo studies and indicate that exposure to heroin or, to a lesser extent, morphine, can affect morphine glucuronidation via direct non-opioid actions on the hepatocytes.     

Pharmacokinetics of morphine-6-glucuronide following oral administration in healthy volunteers

Aim After oral administration, morphine-6-glucuronide (M6G) displays an atypical absorption profile with two peak plasma concentrations. A proposed explanation is that M6G is hydrolysed to morphine in the colon, which is then absorbed and subsequently undergoes metabolism in the liver to morphine-3-glucuronide (M3G) and M6G. The aims of this study were to confirm and elucidate the biphasic absorption profile as well as clarify the conversion of M6G to morphine after a single oral administration of M6G in healthy volunteers. Methods The study was conducted accordingly to a nonblinded, randomised, balanced three-way crossover design in eight healthy male subjects. The subjects received 200 mg oral M6G, 50 mg oral M6G and 30 mg oral morphine. Blood samples were collected until 72 h after M6G administration and until 9 h after morphine administration. Paracetamol and sulfasalazine were coadministered with M6G as markers for the gut contents reaching the duodenum and colon, respectively. Results The plasma concentration peaks of M6G were seen at 4.0 (2.0–6.0) and 18 (12.0–24.0) h after 200 mg M6G and at 3.5 (2.0–6.0) and 21.3 (10.0–23.3) h after 50 mg M6G, which was in agreement with previously published results. The K_{M6G_abs}/K_{M6G_M6G} ratio was found to be 10. Conclusion The pharmacokinetic profile of M6G after oral administration was confirmed and with the presence of M3G and morphine in plasma after oral administration of M6G, proof seems to be found of the constant and prolonged absorption of M6G. The K_{M6G_abs}/K_{M6G_M6G} ratio of 10 indicates that the second absorption peak of M6G consists of approximately 10 times more absorbed M6G than reglucuronidated M6G. However, further studies are required to determine the precise kinetics of the second absorption peak.     

The effect of different routes of administration on the metabolism of morphine: The disposition of morphine and its metabolites after topical application

The disposition of morphine (MOR) and its metabolites in the rabbit was measured after topical administration of its hydrochloride salt (MOR · HCl), and their time course was compared with those after intravenous and oral administration. The area under the plasma concentration—time curve (AUC) ratio of metabolites/MOR after the topical application of MOR · HCl was similar to that after intravenous injection, but differed from that after oral administration. Pharmacokinetic parameters of the disposition of MOR and its metabolites were obtained by a general curve fitting of the time course of plasma concentrations of these compounds after intravenous injection of MOR · HCl and its metabolites, respectively. On the other hand, the time courses of plasma concentrations of the metabolites after intravenous, oral, and topical administration of MOR · HCl were simulated using a simple compartment model without consideration of enterohepatic circulation and the pharmacokinetic parameters obtained as above. The resulting curves of the metabolites agreed well with the observed values except for those after oral administration. These results suggest that no first-pass metabolism of MOR · HCl occurs after percutaneous administration, and that topical administration of this salt is more advantageous than oral administration in terms of bioavailability.     

Effect of repeated administrations of heroin, naltrexone, methadone, and alcohol on morphine glucuronidation in the rat

Rationale Heroin is rapidly metabolized to morphine that in turn is transformed in morphine-3-glucuronide (M3G), an inactive metabolite, and morphine-6-glucuronide (M6G), a potent mu-opioid receptor (MOR) agonist. We have found that heroin addicts exhibit higher M6G/M3G ratios relative to morphine-treated control subjects. We have also shown that heroin-treated rats exhibit measurable levels of M6G (which is usually undetectable in this species) and reduced levels of M3G. Objective We investigated the role of MOR in these effects of heroin, by examining the effects of methadone, a MOR agonist, and of naltrexone, a MOR antagonist, on morphine glucuronidation. We also investigated the effects of alcohol, which is known to alter drug metabolism and is frequently coabused by heroin addicts. Methods Morphine glucuronidation was studied in liver microsomes obtained from rats exposed daily for 10 days to saline, heroin (10 mg/kg, i.p.), naltrexone (20–40 mg/kg, i.p.), heroin + naltrexone (10 mg/kg+20–40 mg/kg, i.p.), methadone (5–20 mg/kg, i.p.), or 10% ethanol. Results Heroin induced the synthesis of M6G and decreased the synthesis of M3G. Naltrexone exhibited intrinsic modulatory activity on morphine glucuronidation, increasing the synthesis of M3G via a low-affinity/high-capacity reaction characterized by positive cooperativity. The rate of M3G synthesis in the heroin + naltrexone groups was not different from that of the naltrexone groups. Methadone and ethanol induced a modest increase in M3G synthesis and had no effect on M6G synthesis. Conclusion The effects of heroin on morphine glucuronidation are not shared by methadone or alcohol (two drugs that figure prominently in the natural history of heroin addiction) and do not appear to depend on the activation of MOR.     

Interactions between morphine and the morphine-glucuronides measured by conditioned place preference and locomotor activity

After intake of heroin or morphine, active metabolites are formed in the body. The two most important morphine metabolites are morphine-6-glucuronide (M6G) and morphine-3-glucuronide (M3G). M6G and M3G are present for longer time periods and in higher concentrations than the parent drug, but their potential contribution to reward and to development of dependence and addiction is not clear.We tested the effects of morphine and M6G separately (doses of 10, 20, 30 and 50 µmol/kg), administered together, and also in combination with with 200 µml/kg M3G in male C57BL/6J-Bom mice. M3G in doses of 50, 100, 200, 300 and 400 µmol/kg were also tested alone. We evaluated the rewarding effects in a conditioning place preference (CPP) model and the psychomotor stimulating effects by recording locomotor activity.Mice were subjected to three consecutive conditioning days with drugs or saline before testing. Changes in locomotor activity from conditioning day one to day three were also compared to the expression of CPP on the test day.This study revealed that coadministration of morphine and M6G induced CPP of similar magnitude to the sum of equimolar doses of these compounds alone, and different ratios of the two drugs did not affect the results. M3G did not cause CPP and reduced the CPP induced by both morphine and M6G when coadministered with these drugs. Morphine induced locomotor activity was reduced by coadministration of M3G, but this was not seen when M3G was co-injected with M6G. The changes in locomotor activity during the conditioning periods did not correlated with the expression of CPP.This study revealed that the morphine-glucuronides in different and complex ways can influence the pharmacological effects of psychomotor activation and reward observed after intake of morphine.     

Morphine, morphine-6-glucuronide and morphine-3-glucuronide pharmacokinetics in newborn infants receiving diamorphine infusions

1The pharmacokinetics of morphine, morphine-6-glucuronide (M6G) and morphine-3-glucuronide (M3G) were studied in 19 ventilated newborn infants(24–41 weeks gestation) who were given a loading dose of 50 μg kg⁻¹ or 200 μg kg⁻¹ of diamorphine followed by an intravenous infusion of 15 μg kg⁻¹ h⁻¹ of diamorphine. Plasma concentrations of morphine, M3G and M6G were measured during the accrual to steady-state and at steady state of the diamorphine infusion. 2Following both the 50 μg kg⁻¹ or 200 μg kg⁻¹ loading doses the mean steady-state plasma concentration (±s.d.) of morphine, M3G and M6G were 86±52 ng ml⁻¹, 703±400 ng ml⁻¹ and 48±28 ng ml⁻¹ respectively and morphine clearance was found to be 4.6±3.2 ml min⁻¹ kg⁻¹. 3M3G formation clearance was estimated to be 2.5±1.8 ml min⁻¹ kg⁻¹, and the formation clearance of M6G was estimated to be 0.46±0.32 ml min⁻¹ kg⁻¹. 4M3G metabolite clearance was 0.46±0.60 ml min⁻¹ kg⁻¹, the elimination half-life was 11.1±11.3 h and the volume of distribution was 0.55±1.13 l kg⁻¹. M6G metabolite clearance was 0.71±0.36 ml min⁻¹ kg⁻¹, the elimination half-life was 18.2±13.6 h and the volume of distribution was 1.03±0.88 l kg⁻¹. 5No significant effect of the loading dose (50 μg kg⁻¹ or 200 μg kg⁻¹) on the plasma morphine or metabolite concentrations or their derived pharmacokinetic parameters was found. 6We were unable to identify correlations between gestational age of the infants and any of the determined pharmacokinetic parameters. 7M3G:morphine and M6G:morphine steady-state plasma concentration ratios were 11.0±10.8 and 0.8±0.8, respectively. 8The metabolism of morphine in neonates, in terms of the respective contributions of each glucuronide pathway, was similar to that in adults.