【作者】 王俏；

【导师】 曾苏；

【作者基本信息】 浙江大学 ， 药物分析学， 2007， 博士

【摘要】 天麻素（gastrodin,Gas）为名贵药材天麻（Gastrodia elata B1.）的有效单体,具有镇痛、镇静、抗惊厥、抗癫痫,增加脑血流量,保护神经细胞等功能。临床上,广泛用于神经衰弱、眩晕、头痛的治疗及癫痫的辅助治疗。最近发现,Gas对血管性痴呆有效。Gas现有肌注、静滴和口服制剂,其中注射液最为常用。脑是Gas的作用靶器官,由于其为水溶性药物,透过血脑屏障比较困难。早期有人认为,Gas本身并无中枢作用,而是其代谢产物天麻苷元（gastrodigenin,p-hydroxybenzyl alcohol,HBA）在起作用;后来发现,Gas和HBA都对神经有保护作用,有利于记忆的巩固和恢复,具有抗氧化和清除自由基等活性。Gas注射后入脑的量比较少,临床上为达到治疗效果,给药量比较大。另外,因Gas代谢迅速,大多在2小时内排泄,为维持疗效,临床使用时需频繁给药。虽然动物毒性试验表明Gas的毒性较低,但是频繁注射给药会增加病人的痛苦,口服给药时也有恶心等胃肠道副反应。Gas是作用于脑部而发挥药效的药物,所以对其进入脑部的量及在脑部的分布、代谢的研究尤为重要。至今,有关这方面的报道很少。为此,本工作较系统地研究了Gas的体内外代谢、脑药动学和提高其脑靶向性的给药方法,并对其代谢产物HBA的脑药动学及两者的细胞转运特性也进行了研究。1.天麻素在大鼠脑、肝、肾及脑不同区域组织匀浆中的代谢（1）天麻素在大鼠脑、肝、肾组织匀浆中的代谢采用Sprague-Dawley（SD）雄性大鼠的脑、肝、肾组织匀浆,研究了Gas在这些组织匀浆中的代谢特征。建立了高效液相色谱测定方法,分析柱:C18column（Diamonsil,4.6 min×250mm,5μm,Dikma）;流动相:乙腈—水（2.5:97.5,v/v）;流速:1.0 mL/min;检测波长:221 nm;柱温:33℃。方法对检测HBA的专属性较好,HBA在0.30—18.96μg/mL脑组织匀浆、0.30—9.48μg/mL肝组织匀浆、0.59—75.86;μg/mL肾组织匀浆的浓度范围内有良好的线性关系（n=3,r²＞0.999）。HBA在三种组织匀浆中的日内精密度均小于9.6%,日间精密度均杏?2.1%,准确度为97.5%—106.9%,脑、肝、肾组织匀浆中HBA的定量下限分别为0.290±0.045μg/mL,0.307±0.030μg/mL,0.627±00.047μg/mL（n=5）。稳定性良好。Gas在SD雄性大鼠组织匀浆中的体外代谢研究表明,Gas的代谢产物为HBA,其在大鼠脑、肝和肾匀浆中的生成速率常数分别为0.0305、0.0121和0.1409μg/mL/min。Gas在大鼠脑、肝和肾匀浆中的清除率分别为29.7±2.1、10.4±0.8和299.5±22.7 L/min/g(×10^-6)。Gas在脑、肝匀浆中代谢较慢,在肾匀浆中能被迅速代谢。（2）天麻素在大鼠脑不同区域组织匀浆中的代谢研究了Gas在大鼠脑组织不同区域（小脑、丘脑、脑桥与延脑、脑皮层、海马区、纹状体）匀浆中的代谢特征。结果表明:Gas被代谢生成HBA的反应速度常数以小脑、脑桥与延脑、丘脑三个区比较高,皮层、纹状体、海马三个区比较低,前者为后者的约1.5倍。Gas在大鼠小脑、丘脑、皮层中的清除率分别为45.9±7.4、39.6±5.6和24.4±3.0 L/min/g(×10^-6)。体外实验结果表明Gas在非酶体系中稳定,在肝、肾、脑组织匀浆中Gas可被代谢为HBA,在组织中的代谢,以肾最快,脑次之,肝最小;在脑中,Gas在小脑、丘脑、脑桥与延脑区域代谢速度快于皮层、纹状体、海马区。2.天麻素静注给药的脑药动学建立的高效液相色谱测定方法,测定血、脑脊液时采用的流动相:乙腈—水（2.5:97.5,v/v）,测定脑微透析液采用的流动相:乙腈—水（5:95,v/v）。方法对检测Gas和HBA的专属性良好。Gas在0.28-571.70μg/mL血浆,0.16-40.02μg/mL脑脊液,0.07-17.86μg/mL透析液中线性关系良好（n=5,r²＞0.999）。Gas的定量下限为血浆0.269±0.028μg/mL;脑脊液0.147±0.011μg/mL;透析液0.072±0.008μg/mL（n=6）。Gas的日内日间RSD＜12.4%,血浆、脑脊液、透析液中的平均准确度分别为101.4%、96.6%、103.8%。HBA在0.15-2.36μg/mL血浆,0.07-1.18μg/mL脑脊液,0.04-0.59μg/mL透析液中线性关系良好（n=3,r²＞0.999）。HBA的定量下限为血浆0.139±0.015μg/mL,脑脊液0.072±0.006μg/mL,透析液0.038±0.005μg/mL（n=6）。HBA的日内日间RSD＜13.7%,血浆、脑脊液、透析液中的平均准确度分别为99.7%、98.8%、101.6%。稳定性良好。SD雄性大鼠静注给予200 mg/kg Gas,尾静脉取血,延髓池取脑脊液,四个脑区进行微透析[皮层（AP:2.1;ML:2.0,DV:-0.9 mm）,海马区（AP:-6.0;ML:-4.5,DV:-3.0 mm）,或丘脑（AP:-3.0;ML:1.0,DV:-4.5 mm）和小脑（AP:-11.0;ML:-1.3,DV:-2.0 mm）]。微透析探针的平均回收率为0.209±0.018（流速2.5μL/min,37℃）。大鼠Gas静注给药后,血中药物浓度下降较快,进入脑的速度也很快,但是入脑量(AUC_brain/AUC_plasma)不高,脑脊液、皮层、海马、丘脑和小脑的AUC分别为血浆AUC的4.8±2.4,3.3±1.2,3.0±0.7,3.3±1.3和6.1±1.9%,小脑AUC明显高于其它三个脑区（P＜0.05）。在脑和血浆中有HBA代谢生成,但量很低,浓度下降很快。3.天麻素十二指肠给药的脑药动学SD雄性大鼠在十二指肠给予200 mg/kg Gas,收集血、脑脊液和脑微透析液。微透析探针的平均回收率为0.233±0.021（流速2.0μL/min,37℃）。Gas十二指肠给药后,血T_max与脑脊液T_max相似,分别为55.6和57.5 min,四个脑区以小脑中C_max最高（5.3±1.2μg/mL）,AUC最大（491.2±220.6 minμg/mL）。脑脊液、皮层、海马、丘脑和小脑的AUC分别为血浆AUC的4.9±1.2,2.4±1.0,2.5±0.8,2.6±0.8和4.7±2.2%。Gas小脑的AUC明显高于其它三个脑区（P＜0.05）,与静注相似。脑和血浆中代谢生成的HBA量也很低。Gas血浆的绝对生物利用度为55.0%。相同剂量给药,口服Gas后代谢产生的HBA要比静注稍多,血中HBA的AUC为静注的1.4倍（但无显著性差异,P＞0.05）,而脑脊液AUC约为静注2.3倍（P＜0.01）。结果显示Gas十二指肠给药后,其药动学特性与静注相似。4.天麻素静注与鼻腔给药的药动学比较建立的高效液相色谱测定方法,对检测在脑脊液和血样中的Gas有较好的专属性,Gas在0.16-9.98μg/mL脑脊液,0.39-99.85μg/mL血浆范围线性关系良好（n=5,r²＞0.999）。日内日间RSD＜6.4%,平均准确度脑脊液和血浆分别为100.4、100.6%。定量下限为脑脊液0.148±0.008μg/mL,血浆0.397±0.020μg/mL（n=6）。稳定性良好。SD雄性大鼠鼻腔或静注给予50 mg/kg Gas。Gas鼻腔给药与相同剂量的静注比较,鼻腔给药后进入血中的量比较低,而相应地进入脑脊液中的量比较高,脑脊液中Gas的AUC与静注的相似。鼻腔给药的血浆AUC与静注AUC的比值为8.8%,而脑脊液AUC与静注AUC比值为105.5%,从而可使脑靶向指数达12.3。提示,通过鼻腔给药可提高Gas的脑靶向性。5.天麻苷元与天麻素静注给药的药动学比较建立的高效液相色谱测定方法,对于检测脑脊液中HBA的流动相:乙腈—水（4:96,v/v）;对于检测血中HBA的流动相:乙腈—水（3:97,v/v）。方法对检测脑脊液和血浆中的HBA有较好的专属性,HBA在0.07-37.73μg/mL脑脊液,0.23-30.34μg/mL血浆范围内线性关系良好（n=3,r²＞0.999）。定量下限为脑脊液0.074±0.007μg/mL,血浆0.258±0.016μg/mL（n=5）。脑脊液、血浆中的日内日间RSD＜9.4%,平均准确度分别为100.5%、105.5%。稳定性良好。SD雄性大鼠静注给予50 mg/kg HBA。Gas静注给药（50 mg/kg）同第4部分内容。研究了HBA直接静注后的药动学,结果表明HBA血和脑脊液中的t_1/2分别为9.2±1.7和12.4±3.1min,明显短于Gas,表明HBA的消除比Gas更快。脑脊液中HBA的C_max和AUC高于血浆。6.天麻苷元静注与鼻腔给药的药动学比较SD雄性大鼠鼻腔或静注给予10 mg/kg HBA。HBA同剂量鼻腔给药与静注给药相比,都表现出脑脊液中AUC和C_max高于血浆的特点。两种途径给药,HBA都可迅速进入脑脊液,鼻腔给药的脑脊液或血浆AUC与静注的比较无显著性差异。提示,HBA鼻腔给药后能快速完全地进入体循环,再透过血脑屏障,鼻腔给药可望替代静注给药。7.天麻素和天麻苷元在Caco-2、MDCK-MDR1和MDCK细胞的转运研究采用Caco-2细胞、MDCK-MDR1和MDCK细胞进行Gas和HBA的转运研究。Gas和HBA在Caco-2细胞转运研究结果表明Gas口服吸收利用度可能不高,这与其自身脂溶性较差有关;而HBA的吸收较好。Gas和HBA在MDCK-MDR1和MDCK细胞转运研究结果表明Gas较难透过,Gas的R值小于2,不是P-gp的底物;HBA易透过,R值也小于2,对Rho123的外排也无显著影响,不是P-gp的底物,也不是P-gp的抑制剂。两个细胞转运试验结果表明,Gas的跨膜转运较差,不是P-gp底物,可能存在非P-pg介导的外排。HBA的跨膜转运好,也不是P-gp底物或抑制剂,低浓度时有较弱的非P-pg介导的外排机制存在,这种外排机制可为高浓度的HBA逆转。更多还原

【Abstract】 Gastrodin（Gas） is the major and bioactive component in Tianma（Gastrodia elata Bl.） and has sedative,anticonvulsive and neuroprotective effects.It has been approved as a drug for the treatment of neurasthenia,dizzy,headache and adjunctive therapy to epilepsy in China.The results of recent clinical trials showed that it was efficient in treatment of patients with vascular dementia.Currently,the marketed preparations of Gas are intramuscularly,intravenously or orally administered,with injection as the most common dosage form.The target organ of Gas is brain,but it is difficult to pass blood-brain barrier （BBB） because of its water-solubility.There has a controversy about the active form of Gas.Some experts believe only the metabolite of Gas（p-hydroxybenzyl alcohol, HBA） can permeate through the blood-brain barrier（BBB） and have pharmacologic effect.Others think both of Gas and HBA have pharmacologic effect.But the dose of Gas is high in clinic in order to achieve pharmacologic activity because the amount of Gas permeation through BBB to brain is too low.The elimination of Gas is fast and the accumulation is very low in body.In order to keep effect,it is needed to administer several times each day in clinic.Although the toxicity of Gas is low,the frequent injection can increase the patient’s pain and oral administration has some gastrointestinal side effect.Since little was known about the neuropharmacokinetics and brain metabolism of Gas,it would be meaningful to investigate the brain pharmacokinetics of Gas and HBA and their brain target in Sprague-Dawley（SD） male rat.In this paper,we also studied the cell transport of Gas and HBA. 1.Metabolism in vitro（1） The metabolism of Gas in rat brain,liver and kidney homognateThe metabolism of Gas in brain,liver and kidney was studied in rat tissue homogenate.A HPLC method was established to determine the metabolite,HBA concentration after incubation.HBA was analyzed on Diamonsil C18 column（4.6 mm×250mm,5μm）,with a mobile phase consisting of methanol-water（2.5:97.5,v/v）, and detected with UV detector at 221nm.The flow rate was set at 1.0 mL/min.The temperature of the column oven was maintained at 33℃.The assay was linear over the concentration range of 0.30—18.96μg/mL in brain homogenate,0.30—9.48μg/ mL in liver homogenate and 0.59—75.86μg/mL in kindey homogenate（n=3, r²＞0.999）.The intra-and inter-day precision over these ranges were not more than 9.6%and 12.1%,respectively.The Accuracy was in the range of 97.5%—106.9%. The lower limit of quantification（LLOQ） was 0.290±0.045μg/mL,0.307±0.030μg/mL and 0.627±00.047μg/mL in brain,liver and kidney homogenate（n=5）, respectively.The results showed the velocity constants of formation metabolite,HBA,were 0.0305,0.0121 and 0.1409μg/mL/min in brain,liver and kidney,respectively.The clearance rates of Gas were 29.7±2.1,10.4±0.8 and 299.5±22.7 L/min/g(×10^-6) in the brain,liver and kidney,respectively.The metabolism rate of Gas was slow in brain and liver,while it was fast in kidney.（2） The metabolism of Gas in rat different brain regionsThe metabolism of Gas was studied in six different brain regions（the cerebellum,thalamus,pons and medulla oblongata,frontal cortex,hippocampus and striatum）.In brain homogenate,the formation velocity of HBA was fast in the cerebellum, thalamus,pons and medulla oblongata,and slow in the frontal cortex,hippocampus and striatum.The velocity constants of the formers were about 1.5 times higher than those of the latters.Clearance rates of Gas were 45.9±7.4,39.6±5.6 and 24.4±3.0 L/min/g(×10^-6) in cerebellum,thalamus and cortex,respectively.The results suggested that Gas is stable in non-enzyme system and can be metabolized to HBA in brain,liver and kidney homogenate in vitro.And Gas is metabolized most rapidly in the kidney homogenate.The metabolism rates of Gas in the cerebellum,thalamus,pons and medulla oblongata are faster than other three brain regions.2.Brain pharmacokinetics and metabolism of Gas after intravenous administrationA HPLC method was established to determine Gas and its metabolite HBA after Gas intravenous（i.v.） administration（200 mg/kg）.The samples were analyzed on a Diamonsil C18 column（5μm,250 mm×4.6 mm） with a mobile phase consisting of acetonitrile-water（5%acetonitrile for brain microdialysate,2.5%acetonitrile for plasma and cerebrospinal fluid（CSF））,and detected with a UV detector at 221 nm.No peaks interfered with the analytes in the chromatograms of plasma and brain samples.The calibration curves of Gas were linear over the concentration range of 0.28-571.70μg/mL in plasma,0.16-40.02μg/mL in CSF and 0.07-17.86μg/mL in microdialysate（n=5,r²＞0.999）.The LLOQ of Gas was 0.269±0.028μg/mL in plasma,0.147±0.011μg/mL in CSF and 0.072±0.008μg/mL in microdialysate （n=6）.The intra-and inter-day precision of Gas over these ranges were not more than 12.4%.The Accuracy was 101.4%,96.6%,103.8%in plasma,CSF and microdialysate,respectively.The calibration curves for HBA were also linear over the range of 0.15-2.36μg/mL in plasma,0.07-1.18μg/mL in CSF and 0.04-0.59μg/mL in brain microdialysate（n=3,r²＞0.999）.The LLOQ of HBA was 0.139±0.015μg/mL,0.072±0.006μg/mL in CSF and 0.038±0.005μg/mL in brain microdialysate（n=6）.The intra-and inter-day precision of HBA over these ranges were not more than 13.7%. The Accuracy was 99.7%,98.8%,101.6%in plasma,CSF and microdialysate, respectively.SD male rats were administered with Gas at a dose of 200 mg/kg via the femoral vein.The blood sample was taken from the tail vein and CSF sample was collected using the cistern puncture.The microdialysis was applied in the frontal cortex （coordinates:AP 2.1,ML 2.0,DV 1.0） and hippocampus（AP-6.0,ML-4.6,DV 3.0）, or thalamus（AP-3.0,ML 1.0,DV 4.5） and cerebellum（AP-11.0,ML-1.3,DV 2.0） according to the Paxinos and Watson atlas.Each probe was subjected to in vitro recovery studies before in vivo experiments for validation.The mean value of Gas recovery for all of the microdialysis probes was 0.209±0.018 at a microdialysate flow rate of 2.5μl/min at 37℃.The results from distribution of Gas in rat showed that the levels of Gas declined rapidly after drug administration and the entry of Gas into the brain was rapid. However,the ratios of AUC_brain/AUC_plasma were not high.The individual ratios of the AUC in the CSF,frontal cortex,hippocampus,thalamus and cerebellum to the AUC in the plasma were 4.8±2.4%,3.3±1.2%,3.0±0.7%,3.3±1.3%and 6.1±1.9%, respectively.The AUC in the cerebellum was significantly higher than that in other brain regions（P＜0.05）.The concentrations of HBA,the main metabolite of Gas,were very low in both of the CSF and plasma.3.Brain pharmacokinetics and metabolism of Gas after duodenum admininstraionSD male rats were administered with Gas via duodenum at a dose of 200 mg/kg and the samples of blood,CSF and brain microdialysate were collected at the indicated time points.The mean value of Gas recovery for the microdialysis probes was 0.233±0.021 at a microdialysate flow rate of 2.0μl/min at 37℃. The results showed T_max in plasma and CSF was similar.The C_max and AUC were the biggest in cerebellum among four brain regions.The individual ratios of the AUC in the CSF,frontal cortex,hippocampus,thalamus and cerebellum to the AUC in the plasma were 4.9±1.2%,2.4±1.0%,2.5±0.8%,2.6±0.8%and 4.7±2.2%, respectively.The absolute bioavailability of Gas in plasma after duodenum admininstration was 55.0%.The metabolism of Gas in both of brain and plasma were low.The AUC of HBA in brain and plasma after oral was higher than that after i.v.. The AUC of HBA in CSF and plasma were 2.3 and 1.4 times compared with those after i.v..4.Brain pharmacokinetics of Gas after intranasal and intravenous admininstraionThe pharmacokinetic behavior of Gas in rat plasma and CSF after intranasal （i.n.） and intravenous（i.v.） administration（50 mg/kg） was investigated.A HPLC method for the determination of Gas in rat CSF and plasma was developed and validated.The assay was linear over the concentration range of 0.156—9.985μg/mL in CSF and 0.390-99.85μg/mL in plasma（n=5,r²＞0.999）,and intra-and inter-day precision over these range were not more than 6.4%.The LLOQ in CSF and plasma were 0.148±0.008μg/mL and 0.397±0.020μg/mL（n=6）, respectively.The mean accuracy in plasma and CSF were 100.4%and 100.6%, respectively.Intranasal administration of Gas provided a comparable AUC in CSF compared with the intravenous administration.But Gas level in plasma was very low.The ratios of AUC values of intranasal to intravenous administration were 8.8%and 105.5%in plasma and CSF,respectively.The drug targeting index（DTI） was 12.3. Intranasal administration of Gas is a promising alternative to traditional administration.Olfactory mucosa did present another pathway for transport Gas to the brain. 5.Brain pharmacokinetics of HBA after intravenous administraionA HPLC method for the determination of Gas in rat cerebrospinal fluid and plasma was developed and validated.A mixture of acetonitrile-water was employed as a mobile phase,with a flow rate of 1.0 mL/min（3%acetonitrile for the plasma and 4%acetonitrile for the CSF）.The assay was linear over the concentration range of 0.07-37.73μg/mL in CSF and 0.23-30.34μg/mL in plasma（n=3,r²＞0.999）.The intra-and inter-day precision over these ranges were not more than 9.4%.The mean accuracy were 100.5%and 105.5%in CSF and plasma,respectively.The LLOQ in CSF and plasma were 0.074±0.007μg/mL and 0.258±0.016μg/mL（n=5）, respectively.SD male rats were administered with HBA or Gas at 50mg/kg via i.v..The result of pharmacokinetics of HBA after i.v.（50 mg/kg） showed that t_1/2 in CSF and plasma was 12.4±3.1 and 9.2±1.7 min,respectively,which was only 35%of those of Gas with same dosage.The concentration of HBA was declined faster than that of Gas.The C_max and AUC of HBA in CSF were higher than those in plasma,while the C_max and AUC of Gas in CSF were much lower than those in plasma.6.Brain pharmacokinetics of HBA after i.n.and i.v.admininstraionSD male rats were administered with HBA at a dose of 10 mg/kg via i.v.or i.n.. The results showed that HBA rapidly enters CSF after administration.There was no significant difference in AUC_CSF or AUC_plasma between i.n.and i.v.administration.In conclusion,HBA can be absorbed into the systemic circulation rapidly and completely after i.n.administration.Intranasal HBA is a promising alternative to intravenous administration. 7.The cell transport of Gas or HBA on Caco-2,MDCK and MDCK-MDR1 cellsThe cell transport of Gas or HBA was studied on Caco-2,MDCK and MDCK-MDR1 cells which were seeded on polycarabonate microporous membrane filters and allowed to grow to confluence as an in vitro model to assess the membrane permeability properties of drugs.The result from the transport experiment of Caco-2 cells showed that oral absorbed availability of Gas may be poor and related to its poor liposolubility,while HBA can be absorbed well.The result from the transport experiment of MDCK and MDCK-MDR1 cells showed Gas permeated cells very difficult.The R ratio of Gas was lower than 2 and it suggested that Gas is not the substrate of P-gp.HBA permeated cells easily.HBA had no significant effect on the efflux ratio of Rho123 and its R ratios was also lower than 2,which suggested HBA is not the substrate or inhibitor of P-gp.The results from two cells transport experiments showed that Gas is not the substrate of P-gp.HBA can be absorbed well and not the substrate or inhibitor of P-gp.The mechanism of non-P-pg mediated effiux may be existed at low HBA concentration and can be reversed by high concentration of HBA.更多还原