本文選題：YQA-14 + 藥物代謝動(dòng)力學(xué)　； 參考：《中國(guó)人民解放軍軍事醫(yī)學(xué)科學(xué)院》2014年博士論文

【摘要】：藥物成癮是一種長(zhǎng)期使用成癮藥物而誘發(fā)的慢性精神類疾病，盡管有嚴(yán)重的不良后果，但患者仍持續(xù)地強(qiáng)迫性覓藥和用藥。多巴胺是參與獎(jiǎng)賞行為的重要調(diào)節(jié)分子，通過(guò)調(diào)節(jié)分布于不同腦區(qū)的多巴胺受體亞型，參與藥物成癮等生命活動(dòng)。D3R是目前DA受體亞型的研究熱點(diǎn)， D3R主要分布在中腦邊緣系統(tǒng)，在帕金森、精神分裂癥以及藥物依賴等疾病的生理過(guò)程中扮演著重要角色。近年來(lái)隨著選擇性D3R配體的研發(fā)，發(fā)現(xiàn)多巴胺D3R選擇性拮抗劑能顯著降低成癮藥物的獎(jiǎng)賞效應(yīng)并且可以抑制復(fù)吸的發(fā)生。 BP897、SB-277011A、NGB2904及PG-01037均是目前國(guó)際制藥公司近年來(lái)發(fā)現(xiàn)的具有較為領(lǐng)先的藥理學(xué)參數(shù)及較好選擇性的D3R拮抗劑。SB-277011A曾為美國(guó)GSK公司開(kāi)發(fā)的新藥，雖然在藥理實(shí)驗(yàn)中發(fā)現(xiàn)其能夠顯著的減弱可卡因、尼古丁、甲基苯丙胺和四氫大麻酚增加的腦獎(jiǎng)賞效應(yīng)，但是在PK研究中發(fā)現(xiàn)SB-277011A在靈長(zhǎng)類動(dòng)物體內(nèi)的代謝時(shí)間不足20分鐘，是肝臟醛氧化酶的底物，目前已停止臨床開(kāi)發(fā)。NGB2904是美國(guó)Neurogen公司開(kāi)發(fā)全新化合物，是目前選擇性最好的D3R拮抗劑。但NGB2904水溶性較差，影響藥物在體內(nèi)的吸收、分布、代謝及排泄。PG-01037Neurogen公司在NGB2904的基礎(chǔ)上修飾得到的另一化合物，其在NGB2904的基礎(chǔ)上改善了水溶性，但卻被發(fā)現(xiàn)為外排轉(zhuǎn)運(yùn)體P-gp的特異性底物。 軍事醫(yī)學(xué)科學(xué)院毒物藥物研究所以優(yōu)勢(shì)結(jié)構(gòu)為指導(dǎo)原則，自行設(shè)計(jì)并合成苯并VA唑啉-2-酮-甲酰胺類化合物YQA-14。藥理學(xué)研究發(fā)現(xiàn)YQA-14與D3R間具有兩個(gè)親和力位點(diǎn)，Ki分別為0.68×10-4nM和2.11nM，但與D2R只有一個(gè)競(jìng)爭(zhēng)位點(diǎn)，Ki為335.3nM，選擇性為分別為4×10-6和150倍。YQA-14是目前鑒定出的具有國(guó)際上最強(qiáng)親和力和選擇性的D3R拮抗劑。鑒于YQA-14良好的藥理學(xué)表現(xiàn)，軍事醫(yī)學(xué)科學(xué)院毒物藥物研究所擬將該化合物作為候選化合物進(jìn)行開(kāi)發(fā)，國(guó)際制藥公司也對(duì)該化合物表示了濃厚的興趣，有很強(qiáng)的合作意向。 鑒于現(xiàn)有高選擇性D3R拮抗劑的ADME性質(zhì)普遍不理想，并因此嚴(yán)重影響了此類藥物的研發(fā)進(jìn)程，因此對(duì)YQA-14進(jìn)行早期的ADME性質(zhì)評(píng)價(jià)及人體DMPK性質(zhì)預(yù)測(cè)具有十分重要的意義。 本研究主要進(jìn)行了以下工作： 1.生物樣品中YQA-14的LC-MS-MS檢測(cè)方法的建立建立了生物樣品中YQA-14的LC-MS-MS檢測(cè)方法。所建立的方法可以適用于肝微粒體、肝胞質(zhì)液、大鼠血漿、比格犬血漿、人血漿、大鼠腦組織、小鼠腦組織等多個(gè)生物基質(zhì)中YQA-14的含量測(cè)定，滿足相應(yīng)試驗(yàn)的需求。在上述基質(zhì)中，YQA-14具有適宜的LLOQ及線性范圍，并在線性范圍內(nèi)線性良好�？梢詫�(duì)生物樣品進(jìn)行準(zhǔn)確，快速，敏感的定量分析。此外YQA-14在上述各生物基質(zhì)中具有良好的長(zhǎng)期放置穩(wěn)定性及反復(fù)凍融穩(wěn)定性，可以保證所分析樣品結(jié)果的準(zhǔn)確。 2.與ADME性質(zhì)相關(guān)的理化性質(zhì)研究 YQA-14具有較差的水溶性（400μg mL-1）和較好的脂溶性（LogD7.4=2.15）。Caco-2細(xì)胞模型的透過(guò)性研究結(jié)果顯示YQA-14的Papp(AL→BP)值為19.90×10-6cm s-1，屬于吸收較好的化合物。YQA-14在全血與血漿中的分配系數(shù)研究發(fā)現(xiàn)，YQA-14與血紅蛋白的結(jié)合率適中，并且在大鼠、比格犬和人三個(gè)種屬中不存在種屬差異。 3. YQA-14的體外代謝穩(wěn)定性研究 YQA-14在靈長(zhǎng)類及非靈長(zhǎng)類動(dòng)物的肝臟胞質(zhì)液中均保持穩(wěn)定，說(shuō)明其不是醛氧化酶的底物。YQA-14的微粒體穩(wěn)定性具有一定的種屬差異。在大鼠肝微粒體中代謝較快，比格犬最慢，人居中。通過(guò)IVIVE外推發(fā)現(xiàn)，YQA-14在大鼠和人體內(nèi)的代謝可能具有更大的相似性，肝臟的首過(guò)抽提率分別為45.14%和40.23%，在比格犬體內(nèi)的代謝較慢，，肝臟首過(guò)抽提率約為13.73%。YQA-14在重組酶中的孵育發(fā)現(xiàn)YQA-14的代謝是多酶介導(dǎo)的，其中CYP3A4和CYP2D6為最主要的代謝酶。 4.YQA-14在不同種屬動(dòng)物體內(nèi)的PK性質(zhì)研究 大鼠靜脈給予25mg kg-1的YQA-14后，清除率CL為29.7mL min-1kg-1，半衰期t1/2為1.9h。表觀分布容積Vd為4.8L kg-1，說(shuō)明YQA-14在大鼠體內(nèi)主要全身分布在血管外(Vd0.7L kg-1)。口服給予25mg kg-1的YQA-14后，tmax約為0.5h，吸收較快，但生物利用度僅為15.6%。比格犬靜脈給予5mg kg-1的YQA-14后，清除率CL為8.3mL min-1kg-1，半衰期t1/2為2.9h。表觀分布容積Vd為2.1L kg-1�？诜o予5mg kg-1的YQA-14后，tmax約為0.5h，生物利用度為45.9%。 5. YQA-14與不同生物基質(zhì)的蛋白結(jié)合率研究 YQA-14的肝微粒體中結(jié)合率為64%，屬于中度結(jié)合，因此不容易受到其它高結(jié)合率藥物的影響，而改變其肝臟代謝速率。在大鼠、比格犬及人血漿的血漿蛋白結(jié)合率均約為99%，且不存在濃度依賴性。 6.基于生理藥動(dòng)學(xué)模型對(duì)YQA-14在人體內(nèi)的PK性質(zhì)預(yù)測(cè) 以GastroplusTM軟件為平臺(tái)，建立了大鼠和比格犬的PBPK模型。在該模型上模擬動(dòng)物體內(nèi)靜脈及口服給藥，對(duì)比實(shí)測(cè)數(shù)據(jù)證實(shí)所建立的模型可以較為真實(shí)的模擬動(dòng)物體內(nèi)的真實(shí)情況。在驗(yàn)證模型建立成功的基礎(chǔ)上進(jìn)一步應(yīng)用于YQA-14在人體內(nèi)的PK情況及口服吸收情況進(jìn)行了擬合和預(yù)測(cè)。YQA-14在人體內(nèi)的代謝速率適中，t1/2約為2.5h，呈全身性分布。人體口服YQA-14溶液后吸收較快，tmax約為0.4h，但吸收程度及生物利用度與給藥劑量相關(guān)：當(dāng)給予287mg Human-1時(shí)，約吸收54.7%，生物利用度約為16.9%；當(dāng)給藥劑量將為57.4mg Human-1時(shí)，約能吸收89.7%，生物利用度提高到35.1%。參數(shù)敏感性分析發(fā)現(xiàn)YQA-14在體內(nèi)的吸收是一非線性過(guò)程，隨給藥劑量增加吸收程度和生物利用程度均會(huì)降低；而溶解度和透膜性的提高均可為藥物在體內(nèi)的吸收與利用產(chǎn)生正面影響，尤其是溶解度的影響更為顯著，研究結(jié)果提示，在后續(xù)開(kāi)發(fā)的制劑學(xué)研究中，應(yīng)該著重于藥物在胃腸道中溶解度的提高。 7. YQA-14的靶組織分布研究 穩(wěn)態(tài)條件下（血藥濃度≈250ng mL-1），YQA-14在大鼠各個(gè)腦區(qū)中的藥物濃度均小于25ng g-1，不到血藥濃度的10%。在小鼠單次腹腔注射后，YQA-14在小鼠腦組織中的藥物濃度明顯低于在全血中的濃度，AUCbrain/AUCblood僅為3.97%，YQA-14難以進(jìn)入小鼠腦組織中。 8.甲基苯丙胺成癮模型中YQA-14的靶組織分布研究 腹腔注射藥物后，正常動(dòng)物組中YQA-14的AUCbrain/AUCblood為5.62%，甲基苯丙胺成癮動(dòng)物中AUCbrain/AUCblood為5.83%，YQA-14在兩組動(dòng)物中腦組織分布并未發(fā)現(xiàn)明顯變化。 9.基于藥物轉(zhuǎn)運(yùn)體的特異性分布機(jī)制研究 通過(guò)體外（Caco-2細(xì)胞）和體內(nèi)（小鼠）兩種模型進(jìn)行YQA-14在腦組織中特異性分布的機(jī)制研究。溫度對(duì)YQA-14在Caco-2細(xì)胞模型上的雙向轉(zhuǎn)運(yùn)的影響研究發(fā)現(xiàn)，YQA-14可能是外排轉(zhuǎn)運(yùn)體的底物，且在轉(zhuǎn)運(yùn)過(guò)程中需要能量支持，認(rèn)為YQA-14在Caco-2細(xì)胞上的外排是由ATP結(jié)合盒式蛋白介導(dǎo)的。通過(guò)使用不同的特異性抑制劑，發(fā)現(xiàn)YQA-14是外排轉(zhuǎn)運(yùn)體P-gp的底物，不是BCRP的底物。YQA-14在腦內(nèi)分布較差主要是由于P-gp對(duì)其外排造成的。
[Abstract]:Drug addiction is a chronic mental disease induced by long-term use of addictive drugs. Although there are serious adverse consequences, the patient continues to forage drugs and drugs. Dopamine is an important regulator involved in rewarding behavior. It participates in drug addiction and other life activities by regulating the subtype of dopamine receptor distributed in different brain regions. .D3R is the current research focus of the DA receptor subtype. D3R is mainly distributed in the mesencephalic marginal system and plays an important role in the physiological processes of Parkinson, schizophrenia, and drug dependence. In recent years, with the development of selective D3R ligands, it is found that dopamine D3R selective antagonists can significantly reduce the reward effect of addictive drugs. And it can inhibit the occurrence of relapse.
BP897, SB-277011A, NGB2904 and PG-01037 are new drugs developed by international pharmaceutical companies in recent years with more leading pharmacological parameters and better selectivity D3R antagonists,.SB-277011A, which have been developed by.SB-277011A for GSK companies in the United States. Although it has been found in pharmacological experiments, it can significantly reduce cocaine, nicotine, methamphetamine and four hydrogen. Cannabinol increases the reward effect of brain, but in PK study, the metabolic time of SB-277011A in primates is less than 20 minutes, and it is the substrate of liver aldehyde oxidase. At present, the clinical development of.NGB2904 is a new compound developed by American Neurogen company, which is the best selective D3R antagonist at present. But the water solubility of NGB2904 is better than that of NGB2904. Poor, the other compound that affects the absorption, distribution, metabolism and excretion of drugs in the body based on NGB2904, which is based on NGB2904, improves water solubility, but is found to be a specific substrate for the exo transporter P-gp.
The research of toxicant drugs in Military Medical Science Academy of the PLA is the guiding principle. The study of YQA-14. pharmacology of benzo VA azoline -2- ketamine compound has been designed and synthesized by ourselves. It is found that there are two affinity sites between YQA-14 and D3R, Ki is 0.68 x 10-4nM and 2.11nM respectively, but there is only one competitive site with D2R, Ki is 335.3nM, selectivity is 4 * 10-6 and 150 times.YQA-14, respectively, are currently identified as the strongest D3R antagonists with the strongest international affinity and selectivity. In view of the good pharmacological performance of YQA-14, the compound of the Military Medical Science Academy of the PLA is intended to develop the compound as a candidate compound, and the international pharmaceutical company has also expressed a strong interest in the compound. It is interesting and has a strong intention to cooperate.
Since the ADME properties of existing high selective D3R antagonists are generally not ideal, and thus seriously affect the development process of such drugs, it is of great significance to evaluate the early ADME properties of YQA-14 and predict the DMPK properties of the human body.
The main work of this study is as follows:
1. LC-MS-MS detection method for YQA-14 in biological samples has been established to establish a LC-MS-MS detection method for YQA-14 in biological samples. The method can be applied to the determination of YQA-14 content in many biological substrates such as liver microsomes, hepatocytes, rat plasma, human plasma, human plasma, rat brain tissue, mouse brain tissue and so on. In the above matrix, YQA-14 has a suitable LLOQ and linear range and is well linear in linear range. It can be used for accurate, rapid and sensitive quantitative analysis of biological samples. In addition, YQA-14 has good long-term stability and repeated freezing and thawing stability in these biological substrates, which can guarantee the analysis of the samples. The result is accurate.
Study on the physical and chemical properties related to the properties of 2. ADME
YQA-14 with poor water solubility (400 mu g mL-1) and better lipid soluble (LogD7.4=2.15).Caco-2 cell model showed that the Papp (AL to BP) value of YQA-14 was 19.90 x 10-6cm s-1. There were no species differences in the three species of rats, Beagles and humans.
Study on the metabolism stability of 3. YQA-14 in vitro
YQA-14 remained stable in the cytoplasm of the liver of primates and non primates, indicating that the microsomal stability of.YQA-14, which is not a substrate for aldehyde oxidase, has certain species differences. The metabolism in rat liver microsomes is faster, the slowest in the Beagle dog and the human medium. The metabolism of YQA-14 in rats and human bodies may be found by IVIVE extrapolation. With greater similarity, the first extraction rate of the liver was 45.14% and 40.23%, respectively, and the metabolism in the Beagle dog was slower. The first extraction rate of the liver was about 13.73%.YQA-14 in the recombinant enzyme. The metabolism of YQA-14 was mediated by multiple enzymes, and CYP3A4 and CYP2D6 were the most important metabolic enzymes.
Study on PK properties of 4.YQA-14 in different species of animals
After the rat vein was given YQA-14 of 25mg kg-1, the clearance rate was 29.7mL min-1kg-1, the half life t1/2 was 1.9h. apparent distribution volume Vd was 4.8L kg-1, indicating that YQA-14 was mainly distributed outside the blood vessels in rats. After intravenous administration of YQA-14 of 5mg kg-1, the clearance rate of CL was 8.3mL min-1kg-1, and the half life t1/2 was 2.9h. apparent distribution volume Vd was 2.1L kg-1..
Study on protein binding rate of 5. YQA-14 with different biological substrates
The binding rate of YQA-14 in liver microsomes is 64%, which belongs to moderate binding, so it is not easily affected by other high binding drugs and changes its liver metabolic rate. In rats, the binding rate of plasma protein in Beagle and human plasma is about 99%, and there is no concentration dependence.
6. prediction of PK properties of YQA-14 in human body based on physiological pharmacokinetic model
On the platform of GastroplusTM software, the PBPK model of rat and beagle was established. The model was simulated on the model of animal body veins and oral administration. Compared with the measured data, it was proved that the model could be more true in simulating the real situation in the animal body. On the basis of the successful validation of the model, the model was further applied to the human body in the human body. The PK situation and oral absorption were fitted and predicted that the metabolic rate of.YQA-14 in the human body was moderate, t1/2 was about 2.5h, and was generalized. The absorption of YQA-14 solution was faster and Tmax was 0.4h, but the degree of absorption and bioavailability were related to the dosage: when 287mg Human-1 was given, it absorbed about 54.7% and bioavailability. It is about 16.9%; when the dosage is 57.4mg Human-1, it can absorb about 89.7%, and the bioavailability is increased to 35.1%. parameter sensitivity analysis. It is found that the absorption of YQA-14 in the body is a nonlinear process, with the increase of the dosage and the degree of bioavailability, and the increase of dissolution and membrane permeability can be used as a drug in body. The absorption and utilization of internal absorption and utilization have a positive effect, especially the effect of solubility. The results suggest that the improvement of the solubility of drugs in the gastrointestinal tract should be emphasized in the subsequent development of preparation studies.
Study on target tissue distribution of 7. YQA-14
The drug concentration of YQA-14 in all brain regions of rats was less than 25ng g-1 under steady condition (250ng mL-1). The concentration of YQA-14 in the brain tissue of mice was significantly lower than that in the whole blood after single intraperitoneal injection of 10%. in mice. The concentration of AUCbrain/AUCblood was only 3.97%, and YQA-14 was difficult to enter the mouse brain group. Weave.
Target tissue distribution of YQA-14 in methamphetamine addiction model 8.
After intraperitoneal injection, the AUCbrain/AUCblood of YQA-14 in the normal animal group was 5.62%, and the AUCbrain/AUCblood in methamphetamine addicts was 5.83%. The distribution of brain tissue in the two groups of animals did not change significantly.
9. based on the specific distribution mechanism of drug transporters
Study on the specific distribution mechanism of YQA-14 in brain tissue by two models in vitro (Caco-2 cells) and in vivo (mice). The effect of temperature on the bidirectional transport of YQA-14 on the Caco-2 cell model shows that YQA-14 may be the substrate of the outer transporter and requires energy support during the transport process. It is believed that YQA-14 is on Caco-2 cells. The outer row is mediated by ATP combined with the box protein. By using different specific inhibitors, YQA-14 is found to be the substrate of the outer row transporter P-gp, and the poor distribution of.YQA-14 in the brain, not the substrate of BCRP, is mainly due to the effect of P-gp on its outer row.
【學(xué)位授予單位】：中國(guó)人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：R965

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