基于微透析技术的苦参碱凝胶经皮给药的药动学研究

目的 建立同步测定经皮给药制剂在皮肤、血液中药动学的方法,研究苦参碱凝胶在体内的药动学行为。方法 应用经体外和体内回收率校正建立的基于微透析探针的皮肤血液双位点同步微透析系统,通过在皮肤和颈静脉植入探针,在大鼠腹部脱毛部位给予苦参碱凝胶,连续收集探针中12 h的透析液,并采用LC-MS微量检测技术测定探针透析液中的药物浓度。结果 本研究成功构建了双位点同步微透析药动学评价系统,大鼠皮肤给予苦参碱凝胶后,皮肤中的药物浓度、AUC值、半衰期均显著高于血液中药物浓度。结论 本研究建立的微透析结合LC-MS的取样及检测技术为经皮给药制剂的药动学研究提供了新的技术平台。     

青藤碱微透析体外回收率研究

目的考察浓度、流速对回收率和损失率的影响,为体内青藤碱微透析实验提供参考依据。方法利用增量法和减量法测定回收率,采用超高效液相测定青藤碱含量。结果流速相同时,探针的回收率和损失率与周围药物浓度无关;探针的回收率随着流速的增加呈下降趋势,增量法和减量法测定的回收率与损失率在各流速下相近。结论微透析法可用于青藤碱体内药动学研究,反向透析法可用于青藤碱微透析体内回收率的测定。     

京尼平苷体内外微透析回收率的研究

目的 探究京尼平苷在自制线性微透析探针上的体内外回收率与灌流速度、药物浓度、透析膜长之间的关系。方法 采用HPLC测定微透析样品浓度,考察不同灌流速度、不同药物浓度和不同透析膜长对体外正向和体外反向回收率、体内反向回收率的影响。结果 自制探针性质稳定;探针回收率与京尼平苷的药物浓度无关,与灌流速度成反比,随透析膜长增加而增加;体外的正向与反向探针回收率有显著性差异（P<0.01）,而体内反向回收率低于体外反向回收率,与体外正向回收率无显著性差异;新探针体内回收率大于使用过1次的探针体内回收率（P<0.05）。结论 在京尼平苷的皮肤药动学研究中,宜采用体内反向回收率或体外正向回收率作为药物的探针回收率来校正。     

芒柄花素、芍药苷血液和脑在体微透析方法的建立

目的:建立芒柄花素、芍药苷血液和脑局部在体微透析方法。方法:采用增量法考察流速对芒柄花素和芍药苷回收率的影响,结合实际操作,确定流速和取样间隔;利用浓度差法(增量法和减量法)研究流速、浓度、温度对微透析探针体外回收率和损失率的影响,探讨反透析法用于探针体内回收率测定的可行性;将血液探针和脑探针分别植入大鼠颈静脉和脑纹状体,考察探针在10h内体内回收率的稳定性。结果:芒柄花素,芍药苷血液和脑探针体外回收率随着流速的增高而降低,综合考虑取样时间和取样体积,确定流速为1.5μL·min^-1,取样时间间隔为20min;在相同流速和温度下,探针增量法回收率RR与减量法损失率RL近似相等,且探针回收率与药物浓度无关,说明可用反透析法进行体内回收率的测定;在大鼠体内微透析回收率考察中,血液和脑探针10h回收率保持相对稳定,但不同探针相对回收率之间存在一定的差异性。结论:所建立的芒柄花素、芍药苷血液和脑微透析方法可用于大鼠体内和脑局部药动学研究,反透析法可作为同时研究芒柄花素,芍药苷探针体内回收率的测定方法。     

微透析技术应用于Gefitinib药动学采样的可行性研究

目的：本研究旨在考察微透析采样技术用于Gefitinib体内药动学研究的可行性,为后续探讨Gefitinib肿瘤局部药动学及药效学研究奠定方法学基础。方法： 采用浓差法（增量法、减量法）测定Gefitinib体外回收率（RG）及释放率（RL）,探讨流速、pH、浓度、温度对回收率的影响,及体内外回收率与释放率的稳定性,建立Gefitinib微透析采样方法。结果： 反透析法测得Gefitinib的RG与RL呈良好一致性,与灌流液pH及探针外液温度存在线性关系,对浓度变化保持恒定,且体内外日内RG与RL稳定性良好。结论：微透析采样技术可以实现Gefitinib体内药物浓度动态监测,研究所得微透析采样参数可为该法进行Gefitinib肿瘤局部药动学研究提供参考。     

钩藤碱微透析探针回收率测定及影响因素研究

目的:考察体内外微透析探针回收率的稳定性和重现性,为进一步的体内微透析试验提供依据。方法:采用增量法和减量法考察浓度、流速对探针回收率的影响。结果:微透析探针回收率受流速影响较大,基本不受浓度影响,且体内回收率稳定性良好。结论:微透析技术可用于钩藤碱的体内药代动力学研究,反透析法可作为体内研究钩藤碱探针回收率的测定方法。     

在体皮肤微透析的建立及对乌头碱经皮给药药代动力学研究

目的:建立大鼠在体皮肤微透析技术,并用于乌头碱经皮给药药代动力学研究。方法:采用HPLC-MS/MS联用技术建立大鼠血浆和微透析样品中乌头碱的分析方法。体外回收率的测定采用浓差法(增量法、减量法),体内回收率的测定采用减量法,考察流速、浓度对回收率的影响。5只SD大鼠在麻醉状态下,作皮肤微透析预处理后,将乌头碱凝胶涂于探针所在皮肤表面,进行皮肤微透析和颈动脉采血,应用HPLC-MS/MS测定透析液样品及血浆中乌头碱浓度,并绘制血药浓度-时间曲线,并计算药动学参数。结果:乌头碱在检测要求范围内线性关系良好,色谱的专属性、回收率、精密度等测定结果均符合生物样品测定要求。增量法及减量法测定的回收率一致;回收率随灌流速度的增大而减小,探针的回收率与溶媒中乌头碱的浓度无关。体内皮下探针对乌头碱的回收率为(34.48±3.05)%,乌头碱在皮下探针中6 h回收率的变化基本上保持稳定。乌头碱经皮给药后,在皮肤中最大药物浓度为(2723.8±848.8)mg.L-1,AUC(0～t)为(18212.4±4749.1)mg.h.L-1,体内血药浓度比较稳定。结论:本研究提示体内研究的反透析法可作为微透析研究中乌头碱回收率的测定...     

血液微渗析技术研究酮洛芬在大鼠体内的药代动力学

目的考察酮洛芬微渗析体内外回收率及影响因素，研究酮洛芬静脉给药后非结合型药物在大鼠体内的药代动力学。方法大鼠颈静脉插入探针后，依次用不同浓度的灌注液对探针进行灌注，测定酮洛芬体内回收率及非结合型酮洛芬在大鼠体内的药代动力学。以高效液相色谱法测定微渗析液中药物浓度。体外回收率的测定采用浓差法。结果增量法及减量法测定的回收率一致。以浓差法测定的体外回收率为28.75%；反渗析法测定体内回收率为(40.3±2.7)%。酮洛芬静脉给药后非结合型药物的T_1/2，AUC和CL分别为(181±16) min，(112±27) μg·min·mL^-1和(0.22±0.05) min^-1。结论血液微渗析技术可用于研究非结合型酮洛芬在大鼠体内的药代动力学。     

反向透析法用于体内经皮吸收微透析探针回收率的测定

目的考察反向透析法用于体内经皮吸收过程中微透析探针回收率校正的可行性.方法在体外试验中用零净流量法测定探针的回收率和传递率,在体内试验中用反向透析法测定探针的回收率;以小猪为试验动物,观察模型药雷公藤内酯醇软膏的体内经皮吸收过程.结果体外试验中,微透析探针的回收率为(64.29±2.34)%,回收率和传递率一致;体内经皮微透析实验中探针的回收率在实验前为(51.47±3.51)%,实验结束后为(51.96±4.97)%.雷公藤内酯醇软膏经皮吸收后,药物在皮内浓度很快达到稳态,去除药物后皮内药物浓度则迅速下降.结论反向透析法测定的探针回收率在实验过程中稳定,可以用于测定体内经皮吸收微透析探针回收率.     

用在体皮肤微透析法研究黄芩苷凝胶经大鼠皮肤吸收的局部药动学

目的建立大鼠在体皮肤微透析技术,研究黄芩苷凝胶经皮吸收局部药动学。方法采用HPLC-MS/MS联用技术测定大鼠皮肤微透析液中黄芩苷的浓度。SD大鼠在麻醉状态下做皮肤微透析预处理,然后将黄芩苷凝胶涂于探针所在皮肤表面,收集皮肤微透析液样品进行黄芩苷浓度测定,绘制黄芩苷浓度-时间曲线,计算经皮吸收局部药动学参数。结果用于定量分析的离子对为m/z 447.3→271.2,黄芩苷在检测浓度范围内线性关系良好,色谱的专属性、精密度等测定结果均符合生物样品测定要求。体内皮下探针对黄芩苷的回收率为（24.40±0.91）%,240 min内各取样点回收率保持稳定;黄芩苷经皮给药后8 h内微透析液中均可检测到黄芩苷的存在,且药物在皮肤组织内浓度持续升高,AUC0-t为（50.04±34.17）（mg·min）/L。结论在体皮肤微透析法可用于黄芩苷经皮吸收局部药动学研究。     

The Design and Validation of a Novel Intravenous Microdialysis Probe: Application to Fluconazole Pharmacokinetics in the Freely-Moving Rat Model

Purpose. The purpose of this study was to design and validate a concentric, flexible intravenous microdialysis probe to determine drug concentrations in blood from the inferior vena cava of a freely-moving animal model. Methods. An intravenous microdialysis probe was constructed using fused-silica tubing and an acrylonitrile/sodium methallyl sulfonate copolymer hollow fiber. The probe was tested in vitro for the recovery of fluconazole and UK-54,373, a fluconazole analog used for probe calibration by retrodialysis. Subsequent in vivo validation was done in rats (n = 7) that had a microdialysis probe inserted into the inferior vena cava via the femoral vein, and the femoral artery was cannulated for simultaneous blood sampling. Comparisons of fluconazole pharmacokinetic parameters resulting from the two sampling methods were performed at 2 and 10 days after probe implantation. Results. There were no statistical differences between the microdialysis sampling and conventional blood sampling methods for the T_1/2, Cl, Vdss, and dose-normalized AUC by paired t-test (p > 0.05) for repeated dosing at day 2 and day 10 after probe placement. The probe recovery, as determined by retrodialysis, significantly decreased over the ten day period. This finding indicates the necessity for frequent recovery determinations during a long-term blood microdialysis experiment. Conclusions. These results show that microdialysis sampling in the inferior vena cava using this unique and robust probe design provides an accurate method of determining blood pharmacokinetics in the freely-moving rat for extended experimental periods. The probe design allows for a simple surgical placement into the inferior vena cava which results in a more stable animal preparation for long-term sampling and repeated-measures experimental designs.     

LC-MS/MS法测定比格犬血浆中芬太尼浓度及药动学研究

目的 建立比格犬血浆中芬太尼的液相色谱-串联质谱（LC-MS/MS）测定方法,并用于药动学研究。方法 采用固相萃取（SPE）法从血浆中提取芬太尼和内标芬太尼-d5,建立比格犬血浆中芬太尼的LC-MS/MS测定方法,进行特异性、准确度、精密度、基质效应、灵敏度、稀释可靠性、稳定性方法学验证;8只比格犬,分别单次iv给予芬太尼的生理盐水溶液400 mg/只,用LC-MS/MS测定给药后血浆中芬太尼浓度,并用WinNonLin软件计算药动学参数。结果 芬太尼的线性范围为2～1 000 pg/mL,精密度、准确度、基质效应、灵敏度、稀释可靠性、稳定性均符合生物样品分析要求。比格犬体内芬太尼药动学参数：t_1/2为（4.53±0.748）h,AUC_0-t为（19 659±3 889）h·ng/mL,CL为（2 259±284）mL/（h·kg）,符合二室开放模型。结论 建立的LC-MS/MS分析方法准确灵敏,适用于芬太尼的药动学研究。     

Assessment of in vitro and in vivo recovery of gallamine using microdialysis

The application of microdialysis technique for the investigation of pharmacokinetics and pharmacodynamics of drugs requires careful assessment of probe performance to ensure validity of the data obtained using this technique. The aim of this study was to establish and validate the microdialysis technique for investigation of the pharmacokinetics and pharmacodynamics of the neuromuscular blocker, gallamine. In vitro recovery of gallamine from the microdialysis probe when different perfusion flow rates were employed was evaluated leading to selection of a flow rate of 2 microl/min with 15-min sampling intervals for the subsequent studies. In vitro recovery of gallamine from the microdialysis probe was independent of concentration, stable over an 8-h period and reproducible. Comparable in vitro recoveries were obtained by different established approaches including recovery estimation by gain, loss and the zero-net flux (ZNF) method. Recovery by loss was used to study the in vivo recovery of gallamine from rat muscle tissue. The in vivo recovery was stable over a 5.5-h sampling period. In vitro performance of the probe subsequent to the in vivo study remained stable supporting reusage of the probe. These data highlight the importance of a systematic examination of microdialysis probe validation.     

Brain Penetration and In Vivo Recovery of NMDA Receptor Antagonists Amantadine and Memantine: A Quantitative Microdialysis Study

Purpose. To determine free brain concentrations of the clinically used uncompetitive NMDA antagonists memantine and amantadine using microdialysis corrected for in vivo recovery in relations to serum, CSF and brain tissue levels and their in vitro potency at NMDA receptors. Methods. Microdialysis corrected for in vivo recovery was used to determine brain ECF concentrations after steady-state administration of either memantine or amantadine. Additionally CSF, serum, and brain tissue were analyzed. Results. Following 7 days of infusion of memantine or amantadine (20 and 100 mg/kg/day respectively) whole brain concentrations were 44-and 16-fold higher than free concentrations in serum respectively. The free brain ECF concentration of memantine (0.83 ± 0.05 M) was comparable to free serum and CSF concentrations. In case of amantadine, it was lower. A higher in vivo than in vitro recovery was found for memantine. Conclusions. At clinically relevant doses memantine reaches a brain ECF concentration in range of its affinity for the NMDA receptor and close to its free serum concentration. This is not the case for amantadine and different mechanisms of action may be operational.     

Fluconazole Distribution to the Brain: A Crossover Study in Freely-moving Rats Using In Vivo Microdialysis

Purpose. The purpose of this study was to determine if the microdialysis sampling technique is feasible to study the central nervous system distributional kinetics of a novel triazole antifungal agent, fluconazole, in an awake, freely-moving rat model, and to determine fluconazole distribution to the extracellular fluid (ECF) of the brain. Methods. The relative recovery of the microdialysis probes (CMA-12) was determined in vitro and in vivo by retrodialysis using UK-54,373, a fluorinated analog of fluconazole. Sprague-Dawley rats received 10 mg/kg and 20 mg/kg fluconazole IV bolus doses in a crossover design, and brain extracellular fluid fluconazole concentrations were monitored using microdialysis and on-line HPLC analysis. The plasma fluconazole concentration vs. time data were determined using sequential blood sampling and HPLC analysis. Results. There was no statistical difference between relative probe recoveries for both fluconazole and UK-54,373, either in vitro or in vivo, and probe recoveries did not change during the course of the in vivo crossover experiment. Fluconazole rapidly distributes into in the brain ECF and the average brain distribution coefficient (brain/plasma AUC ratio) was 0.60 ± 0.18 and was independent of dose. Plasma pharmacokinetic parameters were linear in the dose range studied. Conclusions. Fluconazole rapidly reaches a distributional equilibrium between brain extracellular fluid and plasma, and the distribution to the brain is substantial and not dependent on dose over a two-fold range. Furthermore, the results indicate that microdialysis utilizing UK-54,373 as the in vivo retrodialysis probe calibrator is a feasible method to study the transport of fluconazole into the central nervous system.     

Microdialysis Sampling for the Investigation of Dermal Drug Transport

Microdialysis perfusion in vivo has the potential to be a powerful sampling technique in dermal and transdermal drug delivery studies. Characterization of a commercially available microdialysis probe in vitro considering relevant physiological parameters is a vital first step in the evaluation of microdialysis as a dermal sampling technique. In previous microdialysis studies, analyte concentration and neutrality have been implicated in altering microdialysis recovery. The recovery of a model compound 5-fluorouracil (5-FU) was investigated at several pH values and donor concentrations. The relative recovery of 5-FU by the microdialysis probe was affected by pH but not by donor concentration. To confirm further that the changing concentration and pH profile presented by the flux of 5-FU was not significantly altering microdialysis recovery, an experiment comparing direct and microdialysis sampling of a Franz diffusion cell receptor compartment was performed. Although the 5-FU concentration (0-686 ng/ml) and pH (7.40-7.24) changed substantially, the recovery of 5-FU was not adversely affected. To demonstrate the feasibility of dermal microdialysis, the flux of a commercial preparation of 5-fluorouracil was monitored utilizing a microdialysis probe implanted in excised rat skin in vitro. The results from the dermally implanted probe demonstrate the potential of the technique while establishing the limitations of the current microdialysis system.     

精氨酸双糖苷检测方法的建立及药动学研究

目的 建立快速、灵敏、高效的精氨酸双糖苷的测定方法并研究精氨酸双糖苷在大鼠体内的药动学。方法 以乌苯美司作为内标,采用LC-MS/MS检测方法测定血浆中精氨酸双糖苷浓度,利用DAS2.0软件计算药动学相关参数,并分析方法的可靠性。结果 空白血浆对检测物质不产生干扰,在20~20 000 ng/mL符合线性关系,方法学中RSD以及RE值均小于15%。结论 采用LC-MS/MS方法检测精氨酸双糖苷方法合理可靠,精氨酸双糖苷的绝对生物利用度为（4.50±3.75）%。     

Dermal Microdialysis Sampling in Vivo

Microdialysis sampling of the dermis in vivo was accomplished using a linear microdialysis probe. In contrast to previous studies using a commercial cannula-style microdialysis probe, the linear probe had no effect on the flux of drug through the skin in vitro. The extent of tissue damage in vivo due to probe implantation was evaluated by histological examination and microdialysis delivery studies. Tissue damage due to implantation of the linear probe was minimal with no bleeding or edema observed. Infiltration of lymphocytes into the tissue was observed beginning 6 hours after probe implantation with scar tissue beginning to form after approximately 32 hours. The infiltration of lymphocytes had no effect on the behavior of implanted microdialysis probes. Delivery of 5-fluorouracil was between 20 and 25% for six different probes implanted in six different animals demonstrating good probe-to-probe and implantation-to-implantation reproducibility. Constant delivery was maintained for at least 24 hours in all cases indicating that experiments of at least 24 hour duration are feasible. The dermal concentration of topically applied 5-FU cream, Efudex^®, was continuously monitored by an implanted microdialysis probe demonstrating the feasibility of this technique as for monitoring skin drug levels in vivo. The dermal concentration of 5-FU following topical application was approximately 40-fold higher for in vitro excised skin than for in vivo intact skin.