【作者】 雍政；

【导师】 宫泽辉；

【作者基本信息】 中国人民解放军军事医学科学院 ， 药理学， 2008， 博士

【摘要】 目的和意义:随着吸毒人口逐年倍增,毒品滥用已经成为全球性的社会和医学问题。由于95%以上的脱毒者在短期内复吸造成戒了再吸,吸了再戒,周而复始的恶性循环,使得戒毒治疗的长期效果不能令人满意。因此,预防复吸已是全世界物质依赖防治医学中公认的最重要的课题。开发和研制既有脱毒防复吸作用,又能治疗稽延症状,减少心理渴求的新药物已成为当今医学研究的重点、难点和热点。然而,目前国内外均缺乏理想的防复吸药物。常用的戒毒药纳曲酮虽能阻断阿片类毒品与受体结合,但不能缓解稽延症状和心理渴求,使服药者的顺应性差;又因有效时间不够长,停药后24h即可复吸,致使6个月的操守率只有服药病例的20%-30%。美沙酮维持治疗在替代海洛因毒瘾的同时,会使病人产生对美沙酮的新依赖,这种混合毒品形成的依赖比单一毒品更难戒掉。近年来,阿片受体部分激动剂丁丙喏啡(Buprenorphine,Bup)在戒毒临床应用的疗效较好,且有效时间长,不但能缓解稽延症状,而且能阻断毒品的欣快感。但由于Bup口服吸收较差,本身也具有一定的依赖潜能,使得Bup难以成为一个理想的戒毒良药。噻吩诺啡是我所自行设计合成的具有自主知识产权的Bup的衍生物。本研究室前期工作发现,噻吩诺啡是一种新型非选择性阿片受体部分激动剂。噻吩诺啡镇痛效能比丁丙诺啡强、口服吸收好、具有抗吗啡诱导的精神依赖的作用及安全性高等特点,而且在有效剂量仅为纳曲酮的1/10-1/20的情况下,其拮抗吗啡的生物效应竟长达约14 d(t1/2为108 h,约为纳曲酮的26倍)。这一长效特性使其可降低服药的脱失率,最大限度地提高脱毒后的操守率。噻吩诺啡最突出的特点是:在前期大量依赖试验中噻吩诺啡均未表现出具有精神依赖和躯体依赖特性,说明该药依赖性极低。上述特点决定了噻吩诺啡有望成为一个较理想的防止脱毒后复吸的药物,具有诱人的应用前景。一旦上市将可能取代纳曲酮和丁丙诺啡,占领绝大部分的戒毒与防复吸的临床治疗市场,使我国的戒毒成功率取得突破性进展,并有可能打入国际市场,为世界戒毒工作作出贡献。有关噻吩诺啡低依赖性的机制目前尚未阐明。本实验室前期研究结果表明,噻吩诺啡对μ,δ,κ三种阿片受体的选择性没有差别,结合速率均较快,解离速度缓慢;在35S-GTPγ-S实验中,噻吩诺啡对μ,κ受体的激动活性较强,对δ受体的激动活性较弱;用噻吩诺啡慢性处理CHO-μ细胞,可使膜阿片受体数量剂量依赖性下调;这些结果尚不足以解释噻吩诺啡的低成瘾性。本课题根据目前已揭示的与依赖相关的神经生物学基础,如与躯体依赖相关的中枢蓝斑核去甲肾上腺素功能;与精神依赖相关的奖赏环路中的单胺递质;与神经可塑性变化等为切入点,寻找噻吩诺啡与阿片类药物对中枢神经系统影响的不同点,以便进一步了解噻吩诺啡低成瘾性的机制。这不仅能拓展药物依赖的神经精神原理,而且能够指导噻吩诺啡的临床合理应用,探索以噻吩诺啡为主药的戒毒复方的研究,具有重大的学术价值和重要的实际意义。研究内容和方法:1、观察噻吩诺啡的药理学特性与对中枢阿片受体亚型选择性的关系。本部分根据κ受体激动可以拮抗μ受体激动所引起的躯体依赖的理论,探讨噻吩诺啡低依赖性是否与受体亚型的选择有关。拟利用不同受体拮抗剂为工具药,在小鼠疼痛和躯体依赖模型上观察噻吩诺啡对中枢μ受体和κ受体功能的影响。2、考察噻吩诺啡对依赖相关脑区单胺类递质含量及相关酶类活性的影响。本部分拟利用清醒动物脑微透析技术结合高效液相-电化学检测(HPLC-ECD)等方法,以吗啡为阳性对照药物,观察噻吩诺啡急、慢性给药(纳络酮催促戒断)对大鼠蓝斑核、伏隔核、纹状体等药物依赖密切相关脑区单氨类神经递质释放的影响,同时测定递质代谢限速酶单胺氧化酶(Monoamine oxidase,MAO)活性的变化,比较分析噻吩诺啡与吗啡急、慢性给药后不同脑区神经系统的单胺类递质释放和MAO活性的变化,进一步从递质水平探讨噻吩诺啡低依赖性的神经机制。3、评价噻吩诺啡对突触可塑性的影响。该部分采用透射电镜技术,定性观察与突触可塑性变化密切相关的突触界面结构参数;用Western blot的方法检测位于突触囊泡膜上的突触素(Synaptophysin,SYP)一种钙结合蛋白。观察并测量噻吩诺啡慢性处理大鼠伏隔核,海马CA1区神经元超微结构、突触界面结构参数的改变;检测突触素在成瘾相关脑区含量的变化,探讨该药对大鼠突触结构可塑性的影响,并通过比较分析噻吩诺啡与吗啡给药后上述指标的异同变化,进一步分析噻吩诺啡的低依赖性的突触机制。主要结果与结论:1.在小鼠乙酸扭体模型上,脑室注射κ受体特异性拮抗剂Nor-BNI可以抑制噻吩诺啡的镇痛效果,使其镇痛强度从100%下降至70.94%;在小鼠热辐射甩尾模型上,Nor-BNI使噻吩诺啡的可能最大镇痛百分率从69.79%下降至41.19%。用μ受体特异性拮抗剂纳洛肼也能部分抑制噻吩诺啡的镇痛效果,使其镇痛强度分别下降38.24%和44.11%,略强于κ受体拮抗剂Nor-BNI。在小鼠躯体依赖模型形成实验中,Nor-BNI+噻吩诺啡组、噻吩诺啡组和噻吩诺啡+吗啡组给与纳洛酮催促以后,均没有产生跳跃等躯体依赖的症状。结果提示,噻吩诺啡镇痛作用与其激动中枢μ和κ受体均有关;但噻吩诺啡的低成瘾性与激动中枢κ受体的关系尚不明确,可能是κ受体在中枢的分布与μ受体不同,不能干预蓝斑核μ受体诱导的躯体依赖效应。2.噻吩诺啡急、慢性(纳洛酮促催戒断)处理不影响大鼠蓝斑核内去甲肾上腺素(Norepinephrine,NE)的含量,也不影响伏隔核和纹状体内多巴胺(Dopamine,DA)的含量,各脑区神经递质的含量与盐水对照组动物相同(P>0.05),而吗啡慢性(纳洛酮促催戒断)处理后NE和DA的含量增加是其依赖性的神经基础,这可能是噻吩诺啡依赖性低的神经机制之一。与盐水对照组和吗啡组比较,噻吩诺啡慢性处理大鼠给予纳洛酮催促后纹状体内DA的代谢产物3,4-二羟基苯乙酸(3,4-hihydroxyphenylacetic acid,DOPAC)、高香草酸(Homovanillic acid,HVA)明显升高,同时MAO活性显著增强,提示在该脑区噻吩诺啡可能是通过增强MAO的活性,加速了DA的代谢,阻断了依赖形成神经通路的DA递质含量的显著增加,进而不表现出依赖特性。3.噻吩诺啡慢性处理对大鼠伏隔核突触素含量有升高作用,对大鼠海马区突触素含量无影响,而吗啡对海马区突触素含量的有降低作用,提示噻吩诺啡对学习记忆的影响显著小于吗啡。噻吩诺啡组与盐水对照组相比,伏隔核突触活性区长度及突触后致密质厚度显著减小(P<0.01),海马CA1区突触活性区长度减小(P<0.01);与吗啡组相比,伏隔核区突触间隙减小(P<0.05)及突触活性区长度增厚(P<0.05),海马CA1区突触活性区长度(P<0.05)和突触后致密物厚度均增加(P<0.05)。上述结果提示噻吩诺啡和吗啡均可以降低大鼠伏隔核和海马CA1区神经元突触的传递效能,但突触结构参数的变化反映出的认知功能的损害程度上噻吩诺啡明显低于吗啡,这可能是噻吩诺啡的低依赖性而区别于吗啡的突触机制。综上所述,本研究从受体、神经递质、突触可塑性三个方面入手,探讨了噻吩诺低成瘾性的可能机制,为全面深入阐明噻吩诺啡的作用特点及其合理应用并进一步开发提供实验依据。更多还原

【Abstract】 Objective and significance:With the number of drug users increasing yearly, drug abuse has become a global social and medical problem. However, detoxification treatment has so far failed to achieve satisfactory long-term results, so much so that the relapse rate in drug use tops 95 % among patients undergoing detoxification treatment. Post-detoxification relapse prevention has become a focus to addictive medicine worldwide. Medical researchers are faced with the important task to develop a new medicine for the prevention of opioid addiction, reduction of psychological curving and post-detoxification relapse prevention.However, the available anti-opioid relapse drugs are insufficient at home and abroad. As a commonly used anti-opioid relapse agent, naltrexone calls for large dosage, but its effective period is so short that its medication’s failing rate is running high. Only 20-30% of all the detoxification patients treated with naltrexone can manage to remain drug free for six months. In recent years, a partial opioid agonist known as Buprenorphine (Bup) has been proved effective for its good and long detoxification effects in alleviating the protracted withdrawal syndrome, and obstructing drug craving. However, for its poor oral absorption and some dependence potentials, Bup can hardly be accepted as a desirable detoxificaion agent.Thienorphine (Thien) is a Bup derivant with independent intellectual property rights synthesized by our institute. It has been proved that Thien is a new type of non-selective opioid receptor partial agonists which has stronger analgesic effect than buprenorphine, good oral absorption, a long term of treatment and clinical safety. Moreover, at an effective dosage only 1/10-1/20 that of naltrexone, Thien can antagonize the effects of morphine for as long as 14 days. Its t1/2 was 108 h (26 times longer than that of naltrexone). This prolonged anti-morphine effect, which helps cut medication’s failing rate and maximize the post-detoxification abstinence rate. Another prominent pharmacological characteristic of Thien is its low psychological and physical dependence. Those features determine that Thien may become a choice medicine in dealing with opioid relapse. Once introduced to clinic use, Thien will be widely used in the medical treatment of drug abuse and will be likely to replace naltrexone and Bup. China’s drug rehabilitation rate will be increased by our breakthroughs. Thien will be also likely to enter the world market and contribute to worldwide anti-drug efforts.However, the mechanisms of Thien’s low dependence remain largely unknown. Previous available results obtained from our institute indicated that Thien had no selective affinity toμ、κandδopioid receptors, and can easily bind to the three receptors. Nevertheless, its dissociation toμ,κopioid receptors was quite slow. We also found that Thien had stronger capability of activatingμandκopioid receptors than that ofδopioid receptors. Thien can down-regulate the number of MOR in the cell membrane in a dose-dependent manner after incubating with CHO-μcells. But these results are still insufficient to explaining the mechanism of Thien’s low dependence. So we try to explain the mechanisms of Thien’s low dependence based on current mechanism of opioid dependence including the function of Norepinephrine (NE) in the locus coeruleus (LC) related with physical dependence, the release of monoamine neurotransmitter in the reward system related with psychological dependence and the change of synaptic plasticity. To clarify this question is valuable not only for developing nerves theory of drug dependence but also guiding clinical practice and synthesizing more and better anti-opioid relapse agents using Thien as the leading compound.Content and Methodology:1. Study relationship between Thien’s pharmacological properties and its activation of central opioid receptor in vivo. We utilize mu and kappa receptor antagonist to oppose Thienorphine antinociceptive effects and establish mice physical dependence model and pretreat with kappa receptor antagonist to observe dependence symptoms. The aim of this part is to investigate receptor mechanism of Thien’s low dependence on the basis of the theory on activation ofκreceptor against dependence induced by activation ofμreceptor.2. Investigate the effect of Thien on the content of monoamine neurotransmitter and activity of enzymes in the brain region correlatd with dependence. We analysis the release course of monoamine neurotransmitter in the Locus coeruleus (LC), Nucleus accumbens (NAc) and Striatum of dialyzed rats by HPLC-ECD method and measure activity of Monoamine oxidase (MAO) after Thien acute and chronic administration. Our objective of this part is to approach mechanisms of Thien’s low dependence from neuromechanism level compared with Mor.3. Explore the possible role of Thien-induced synaptic plasticity. We observe the ultra-microstructure of neurons and the synaptic interface structure in NAc and hippocampus CA1 (Hip CA1) using transmission electron microscope and measure the activity of Synaptophysin (SYP) in the brain region correlatd with dependence of rats treated with Thien administration by Western blot way. Our purpose of this part is to get the mechanisms of Thien’s low dependence from synaptic plasticity aspect.Results and conclusions:1.Nor-BNI (a kappa receptor antagonist) can inhibite Thien’s antinociceptive effects which inhibition rate decreased from 100% to 70.94% (writhing model) and from 69.79% to 41.19% (P<0.05) (tail flick model); Naloxonazine(a mu receptor antagonist) can also antagonize Thien analgesic effect which inhibition rate dropped from 79.26% to 41.02% (P <0.05) (writhing model) and from 86.08% to 41.97% (P < 0.05) (tail flick model). In mice model of physical dependence, Mice pretreated with Nor-BNI on Thien group did not show jumping and other physical dependence symptoms. Thien can activate kappa receptors to take its effect in vivo. It is not certain that relationship between Thien’s kappa receptors activation and its low dependence. Maybe distribution ofκopioid receptor in the central nervous system (CNS) is different with that ofμopioid receptor, so Thien’s kappa receptors activation can not interfere with the physical dependence induced byμopioid receptor in the locus coeruleus.2.Compared with saline group, Thien’s acute and chronic administration did not affect the content of NE in the LC and the content of DA in the NAC and the Striatum of freely moving rats using microdialysis, while Mor increased the content of NE and DA. These results may display one of neuromechanism of Thien’s low dependence. Compared with saline and Mor group, Thien’s chronic administration increased the content of DOPAC and HVA (DA metabolites) and reinforce the activity of MAO in the Striatum of rats. The results indicated that Thien may accelerate DA metabolism course owing to strengthening the activity of MAO and block the change of neurotransmitter and did not show dependence.3. Thien’s chronic administration increased the content of SYP in the NAc of rats, but did not affect the content of SYP in the hippocampus CA1 area (Hip CA1) of rats, while Mor decreased the content of SYP in the Hip CA1 of rats. These results showed Thien’s effect on neural plasticity is lower than that of Mor. Length of synaptic active zone (NAc and Hip CA1) and the post-synaptic density (NAc) of Thien-treated rats were significantly lower than that of saline group. Length of synaptic active zone in NAc and Hip CA1 of Thien-treated rats were greater than that of Mor group, but the width of synaptic cleft and the post-synaptic density have an opposite result. These result indicated Thien and Mor can decrease synaptic transmission efficiency, but decrease level of Thien was lower than that of Mor. This might be one of mechanisms of Thien’s low dependence from synaptic plasticity aspect.In a word, we investigate the possible mechanism of Thien’s low dependence from three directions including receptor, neurotransmitter and synaptic plasticity. These results provide experimental foundation for explaining of Thien’s pharmacological characteristic and clinical practice.更多还原