併用甲基安非他命及嗎啡引發行為變異及其分子作用機制之研究

Abstract

依據藥物濫用流行病學的統計資料顯示，在藥物成癮者尿液陽性的檢體中，以安非他命類及嗎啡類藥物最常被使用。我們也發現有些多重藥物濫用者，經常會合併使用甲基安非他命及嗎啡此二種藥物，有時合併使用者甚至比單獨使用者多。當甲基安非他命及嗎啡同時被合併使用時，此二種藥物會產生交互作用，進而引起各種神經行為的改變，其藥理及毒理的分子作用機制，與分別單獨使用一種藥物截然不同。甲基安非他命是一種中樞神經興奮劑，之所以會引起行為改變，加強活動量和造成情緒激昂，進而引起興奮提神作用的機制理論觀點，目前認為是由於甲基安非他命作用於中樞神經多巴胺系統，一方面在神經末端增加多巴胺的釋放，進而刺激活化多巴胺受體。同時甲基安非他命也會抑制多巴胺轉運子及多巴胺的代謝，進而增加細胞外多巴胺的濃度，促進多巴胺系統的神經傳導。但最近的研究指出，甲基安非他命的藥理作用，與增加興奮性胺基酸麩胺酸的釋放及刺激麩胺酸受體密切相關。總之，甲基安非他命的藥理作用機制並非如此單純，目前主張多元因素相互作用結果的假說，有待更進一步研究闡明。有關嗎啡的藥理作用機制也是眾說紛云，中樞神經的類鴉片受體有許多種，包括mu (μ), delta (δ), kappa (κ), epsilon (ε) 和 sigma (σ)。嗎啡主要作用在mu鴉片受體，並且此受體的活化與嗎啡引起的行為改變，包括欣快感和成癮性及止痛作用密切相關。另外，mu鴉片受體的細胞訊息傳遞也具多樣性相當複雜，例如降低細胞內鈣離子，減少細胞環腺苷單磷酸或打開細胞膜鉀離子通道等。在相互關連的神經網路中，藥物成癮的機制涉及許多腦部區域，其中以中腦邊緣路徑及黑質紋狀路徑最為重要。並且渴求藥物行為的表現與促進多巴胺及血清素神經系統的傳導密切相關，然而這些神經系統也接受其他神經傳遞物質的調控，包括類鴉片、麩胺酸及γ-胺基丁酸(GABA)。嗎啡會藉由活化mu鴉片受體，間接作用於多巴胺及血清素神經系統，進而影響神經末端傳遞物質的釋放及代謝，如此更加強嗎啡藥理作用的複雜性。本研究論文使用動物實驗鼷鼠模式，分別觀察單獨或合併使用甲基安非他命及嗎啡對鼷鼠行為改變的影響，進而探討其行為改變的中樞神經分子作用機制。實驗第一部分，以腹腔注射方式，重複藥物處理雄性鼷鼠，在不同組別單獨或合併注射甲基安非他命及嗎啡，測量與藥物成癮性密切相關的情境喜好行為、水平距離活動量及刻板動作行為變化。並且利用腦部微透析方法，取得腦細胞外微透析液，然後以高效液相層析儀分析多巴胺和血清素，以及二者的主要代謝物濃度，進而探求合併使用此二種成癮藥物，對多巴胺及血清素傳導系統的神經化學分子作用機制。實驗結果顯示：嗎啡會加強甲基安非他命引起的情境喜好行為，合併注射此二種藥物引起的情境喜好時間，比較於單獨注射任何一種藥物不僅更高，並且情境喜好時程維持更久。利用相同的藥物注射流程，證實併用甲基安非他命及嗎啡引起情境喜好時間增加的行為變異，與增加中腦邊緣路徑伏腹核細胞外的多巴胺及血清素濃度，以及抑制多巴胺的代謝轉變相關，但對於血清素的代謝轉變沒有影響。另一方面重複合併注射甲基安非他命及嗎啡，會增加鼷鼠刻板行為的表現，而此種刻板行為敏感性與增加紋狀體細胞外多巴胺及血清素的濃度，以及抑制多巴胺及血清素的代謝轉變有關。實驗第二部分，研究mu鴉片受體對於多巴胺及血清素代謝作用的調控角色。以腹腔注射方式，處理正常基因雄性鼷鼠或mu鴉片受體基因剔除鼷鼠。比較急性單次注射、重複注射及停藥一段時間之後，再次注射甲基安非他命，對於腦部紋狀體多巴胺及血清素代謝作用的影響。實驗結果顯示：單次注射、重複注射及停藥後再次注射甲基安非他命，對腦部紋狀體多巴胺及血清素代謝物濃度的影響不同。經過重複注射或停藥後再次注射藥物，比單次注射更明顯抑制多巴胺及血清素的代謝，而此種抑制作用具有劑量反應關係。除此之外，當重複注射較高劑量的甲基安非他命時，相對於較低劑量組，具有濃度較低的多巴胺代謝物基礎值。在mu鴉片受體基因剔除鼷鼠，給予單次注射、重複注射及停藥後再次注射甲基安非他命，多巴胺代謝物的濃度與正常鼷鼠明顯不同。注射甲基安非他命後，mu鴉片受體基因剔除鼷鼠具有較高的多巴胺代謝物濃度；因此mu鴉片受體剔除之後，會減低甲基安非他命對多巴胺代謝的抑制作用。當給予較高劑量甲基安非他命注射時，在正常與mu鴉片受體基因剔除鼷鼠之間，血清素代謝物的濃度始具有明顯差異性；mu鴉片受體剔除之後，也會減低甲基安非他命對血清素代謝的抑制作用。此實驗結果也顯示，注射甲基安非他命之後，mu鴉片受體基因剔除鼷鼠，腦部紋狀體多巴胺及血清素的代謝物濃度，回復至未注射藥物前基礎值的速率，較快速於正常鼷鼠。綜合以上實驗結果，證實在中樞神經系統甲基安非他命及嗎啡產生交互作用，嗎啡不僅會協同加強甲基安非他命引起的情境喜好行為，同時與甲基安非他命共同作用，進而引起刻板行為敏感性。而這些行為改變的作用機制，發生在中腦邊緣路徑和黑質紋狀體傳導路徑的影響，則是藉由增加伏腹核及紋狀體多巴胺及血清素的釋放，以及更加強抑制多巴胺及血清素的代謝轉變。除此之外，此二種藥物交互作用，也具有影響細胞外多巴胺及血清素代謝物濃度的作用；急性、重複及再次注射甲基安非他命對多巴胺及血清素代謝作用不同，停藥後再次注射對甲基安非他命引起降低多巴胺及血清素代謝物的作用最為明顯。mu鴉片受體可以調控甲基安非他命對腦部紋狀體多巴胺及血清素的代謝，當mu鴉片受體基因剔除之後，會減低甲基安非他命抑制多巴胺及血清素的代謝作用，同時也會加速多巴胺及血清素代謝物的回復速率。然而這些神經化學的作用是否與mu鴉片受體調控甲基安非他命引起的行為變異相關，值得進一步的研究探討。本論文的研究發現及實驗結果，提供了對於多重藥物濫用者，為何喜好合併使用甲基安非他命及嗎啡，進而引起行為改變的基礎原理，以及合併使用藥物成癮性相關的神經生化分子作用機制。尤其在中樞神經系統，mu鴉片受體對於多巴胺及血清素神經系統的影響，闡明其作用於神經傳遞物質的濃度及其代謝。未來的研究展望及實驗工作，將進一步探討與藥物成癮性及酬償行為密切相關的中腦邊緣神經傳導路徑，麩胺酸受體的作用。同時對於合併甲基安非他命及嗎啡引起的神經毒性，及其關聯於神經細胞凋亡及壞死、鈉鉀腺苷三磷酸酶活性、氧自由基產生量、脂質過氧化、細胞內鈣離子濃度變化、一氧化氮合成量及一氧化氮合成酶表現等，探究更詳細的分子作用機制。進而嘗試以麩胺酸受體拮抗劑，來測試神經保護作用或解毒藥物，對於合併使用甲基安非他命及嗎啡是否不同，期許能將此動物模式所獲得的研究資訊推廣應用於病人。

According to the survey of abused drugs, methamphetamine (MA) and morphine (M) are the most common drugs of abuse in Taiwan and worldwide. In addition, the epidemiologic data of abused drug screen with urine samples have indicated that the incidence of concurrent abuse of MA and M is more often than either drug alone. The interaction between MA and M may result in neurobehavioral alterations when they are abused together, such as euphoria, agitation, violent behavior, hyperactivity, and psychosis among the polydrug abusers. The pharmacological effects of MA are mainly dependent on the combined ability of the drug to release dopamine (DA), to block DA reuptake by DA transporters, and to inhibit DA metabolism by monoamine oxidase (MAO) in the nerve terminals. Moreover, recent lines of evidence have suggested that enhancement of excitatory amino acid, glutamate release and potentiation of glutamatergic neurotransmission are considered to be associated with MA abuse. Opiates have several characterized forms of the opioid receptor (OR) including, mu (μ), delta (δ), kappa (κ), epsilon (ε) and sigma (σ). In particular, the μ-OR is thought to mediate the analgesic and euphoric effects of M, as well as the addictive properties. Drug addiction is mediated by multiple brain regions and neurotransmitter systems. The DA and serotonin (5-HT, 5-hydroxytryptamine) systems in the CNS are thought to play critical roles in the development of addictive behavior and are strongly modulated by other neurotransmitter systems, including the opioidergic, glutamatergic, and γ-aminobutyric acid (GABA-ergic) systems. Furthermore, topographic evidence has demonstrated the overlap of dopaminergic and opioidergic neurons in the ventral tegmental area, substantial nigra and striatum, suggesting an interaction between these two neuronal systems. However, little is known of the μ-OR influence on DA and 5-HT neurotransmission after the combination of MA and M. To explore the behavioral alterations induced by polydrug abuse of combined MA and M, we designed an animal model to investigate behavioral effects of repeated intraperitoneal injections of MA and M alone or in combination on addictive liability and behavioral sensitization in mice. In addition, to further identify the neurochemical mechanisms of combined abuse of MA and M, we used the in vivo microdialysis method to elucidate the role of μ-ORs in modulation of MA-induced DA and 5-HT release in free moving mice and their metabolism within μ-OR knockout mice in the striatum. The results showed that systemic M increased MA-induced conditioned place preference (CPP), as revealed by higher CPP values which were also maintained for a longer duration compared with those induced by an identical dose of MA or M alone. Subsequent to challenge with combined MA and M, mice exhibited an increase of stereotyped behavior, which appeared to be associated with elevation of extracellular concentration of DA and inhibition of DA turnover in the striatum. On the other hand, subsequent to either acute, repeated or challenged intraperitoneal administration of MA, wild-type mice revealed decreases in extracellular concentrations of 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA) and 5-hydroxyindoleacetic acid (5-HIAA) in a dose-dependent manner. Moreover, wild-type mice had reductions in basal concentrations of DOPAC and HVA following repeated MA treatment with a higher dose. The effects of acute, repeated or challenge MA administration upon extracellular levels of DOPAC, HVA and 5-HIAA within μ-OR knockout mice significantly differed from the wild-type controls. The duration of recovery to the basal levels of extracellular DA and 5-HT metabolites induced by MA were much longer in wild-type mice than for μ-OR knockout mice. These findings provide insight into the behavioral and neurochemical basis responsible for the combined abuse liability of MA and M. Moreover, the possible mechanisms of MA-induced behavioral alterations as modulated by μ-OR are mediated by changes in DA and 5-HT release and metabolism in the mouse striatum. These results are useful for the establishment of animal model to test the drug design and new therapy modality in the management of polydrug abuse.