新型多巴胺D3受体拮抗剂YQA-14的早期ADME性质评价

发布时间：2018-05-21 12:39

本文选题：YQA-14 + 药物代谢动力学　；参考：《中国人民解放军军事医学科学院》2014年博士论文

【摘要】：药物成瘾是一种长期使用成瘾药物而诱发的慢性精神类疾病，尽管有严重的不良后果，但患者仍持续地强迫性觅药和用药。多巴胺是参与奖赏行为的重要调节分子，通过调节分布于不同脑区的多巴胺受体亚型，参与药物成瘾等生命活动。D3R是目前DA受体亚型的研究热点， D3R主要分布在中脑边缘系统，在帕金森、精神分裂症以及药物依赖等疾病的生理过程中扮演着重要角色。近年来随着选择性D3R配体的研发，发现多巴胺D3R选择性拮抗剂能显著降低成瘾药物的奖赏效应并且可以抑制复吸的发生。 BP897、SB-277011A、NGB2904及PG-01037均是目前国际制药公司近年来发现的具有较为领先的药理学参数及较好选择性的D3R拮抗剂。SB-277011A曾为美国GSK公司开发的新药，虽然在药理实验中发现其能够显著的减弱可卡因、尼古丁、甲基苯丙胺和四氢大麻酚增加的脑奖赏效应，但是在PK研究中发现SB-277011A在灵长类动物体内的代谢时间不足20分钟，是肝脏醛氧化酶的底物，目前已停止临床开发。NGB2904是美国Neurogen公司开发全新化合物，是目前选择性最好的D3R拮抗剂。但NGB2904水溶性较差，影响药物在体内的吸收、分布、代谢及排泄。PG-01037Neurogen公司在NGB2904的基础上修饰得到的另一化合物，其在NGB2904的基础上改善了水溶性，但却被发现为外排转运体P-gp的特异性底物。军事医学科学院毒物药物研究所以优势结构为指导原则，自行设计并合成苯并VA唑啉-2-酮-甲酰胺类化合物YQA-14。药理学研究发现YQA-14与D3R间具有两个亲和力位点，Ki分别为0.68×10-4nM和2.11nM，但与D2R只有一个竞争位点，Ki为335.3nM，选择性为分别为4×10-6和150倍。YQA-14是目前鉴定出的具有国际上最强亲和力和选择性的D3R拮抗剂。鉴于YQA-14良好的药理学表现，军事医学科学院毒物药物研究所拟将该化合物作为候选化合物进行开发，国际制药公司也对该化合物表示了浓厚的兴趣，有很强的合作意向。鉴于现有高选择性D3R拮抗剂的ADME性质普遍不理想，并因此严重影响了此类药物的研发进程，因此对YQA-14进行早期的ADME性质评价及人体DMPK性质预测具有十分重要的意义。本研究主要进行了以下工作： 1.生物样品中YQA-14的LC-MS-MS检测方法的建立建立了生物样品中YQA-14的LC-MS-MS检测方法。所建立的方法可以适用于肝微粒体、肝胞质液、大鼠血浆、比格犬血浆、人血浆、大鼠脑组织、小鼠脑组织等多个生物基质中YQA-14的含量测定，满足相应试验的需求。在上述基质中，YQA-14具有适宜的LLOQ及线性范围，并在线性范围内线性良好。可以对生物样品进行准确，快速，敏感的定量分析。此外YQA-14在上述各生物基质中具有良好的长期放置稳定性及反复冻融稳定性，可以保证所分析样品结果的准确。 2.与ADME性质相关的理化性质研究 YQA-14具有较差的水溶性（400μg mL-1）和较好的脂溶性（LogD7.4=2.15）。Caco-2细胞模型的透过性研究结果显示YQA-14的Papp(AL→BP)值为19.90×10-6cm s-1，属于吸收较好的化合物。YQA-14在全血与血浆中的分配系数研究发现，YQA-14与血红蛋白的结合率适中，并且在大鼠、比格犬和人三个种属中不存在种属差异。 3. YQA-14的体外代谢稳定性研究 YQA-14在灵长类及非灵长类动物的肝脏胞质液中均保持稳定，说明其不是醛氧化酶的底物。YQA-14的微粒体稳定性具有一定的种属差异。在大鼠肝微粒体中代谢较快，比格犬最慢，人居中。通过IVIVE外推发现，YQA-14在大鼠和人体内的代谢可能具有更大的相似性，肝脏的首过抽提率分别为45.14%和40.23%，在比格犬体内的代谢较慢，，肝脏首过抽提率约为13.73%。YQA-14在重组酶中的孵育发现YQA-14的代谢是多酶介导的，其中CYP3A4和CYP2D6为最主要的代谢酶。 4.YQA-14在不同种属动物体内的PK性质研究大鼠静脉给予25mg kg-1的YQA-14后，清除率CL为29.7mL min-1kg-1，半衰期t1/2为1.9h。表观分布容积Vd为4.8L kg-1，说明YQA-14在大鼠体内主要全身分布在血管外(Vd0.7L kg-1)。口服给予25mg kg-1的YQA-14后，tmax约为0.5h，吸收较快，但生物利用度仅为15.6%。比格犬静脉给予5mg kg-1的YQA-14后，清除率CL为8.3mL min-1kg-1，半衰期t1/2为2.9h。表观分布容积Vd为2.1L kg-1。口服给予5mg kg-1的YQA-14后，tmax约为0.5h，生物利用度为45.9%。 5. YQA-14与不同生物基质的蛋白结合率研究 YQA-14的肝微粒体中结合率为64%，属于中度结合，因此不容易受到其它高结合率药物的影响，而改变其肝脏代谢速率。在大鼠、比格犬及人血浆的血浆蛋白结合率均约为99%，且不存在浓度依赖性。 6.基于生理药动学模型对YQA-14在人体内的PK性质预测以GastroplusTM软件为平台，建立了大鼠和比格犬的PBPK模型。在该模型上模拟动物体内静脉及口服给药，对比实测数据证实所建立的模型可以较为真实的模拟动物体内的真实情况。在验证模型建立成功的基础上进一步应用于YQA-14在人体内的PK情况及口服吸收情况进行了拟合和预测。YQA-14在人体内的代谢速率适中，t1/2约为2.5h，呈全身性分布。人体口服YQA-14溶液后吸收较快，tmax约为0.4h，但吸收程度及生物利用度与给药剂量相关：当给予287mg Human-1时，约吸收54.7%，生物利用度约为16.9%；当给药剂量将为57.4mg Human-1时，约能吸收89.7%，生物利用度提高到35.1%。参数敏感性分析发现YQA-14在体内的吸收是一非线性过程，随给药剂量增加吸收程度和生物利用程度均会降低；而溶解度和透膜性的提高均可为药物在体内的吸收与利用产生正面影响，尤其是溶解度的影响更为显著，研究结果提示，在后续开发的制剂学研究中，应该着重于药物在胃肠道中溶解度的提高。 7. YQA-14的靶组织分布研究稳态条件下（血药浓度≈250ng mL-1），YQA-14在大鼠各个脑区中的药物浓度均小于25ng g-1，不到血药浓度的10%。在小鼠单次腹腔注射后，YQA-14在小鼠脑组织中的药物浓度明显低于在全血中的浓度，AUCbrain/AUCblood仅为3.97%，YQA-14难以进入小鼠脑组织中。 8.甲基苯丙胺成瘾模型中YQA-14的靶组织分布研究腹腔注射药物后，正常动物组中YQA-14的AUCbrain/AUCblood为5.62%，甲基苯丙胺成瘾动物中AUCbrain/AUCblood为5.83%，YQA-14在两组动物中脑组织分布并未发现明显变化。 9.基于药物转运体的特异性分布机制研究通过体外（Caco-2细胞）和体内（小鼠）两种模型进行YQA-14在脑组织中特异性分布的机制研究。温度对YQA-14在Caco-2细胞模型上的双向转运的影响研究发现，YQA-14可能是外排转运体的底物，且在转运过程中需要能量支持，认为YQA-14在Caco-2细胞上的外排是由ATP结合盒式蛋白介导的。通过使用不同的特异性抑制剂，发现YQA-14是外排转运体P-gp的底物，不是BCRP的底物。YQA-14在脑内分布较差主要是由于P-gp对其外排造成的。
[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.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R965

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