混合探针底物法测定五味子成分对大鼠肝脏细胞色素P450同工酶的影响

研究大鼠多次口服五味子成分和体外对肝脏细胞色素P450酶(CYP450)6种同工酶的影响。采用超速离心法制备正常及多次口服五味子醇/水提物的大鼠肝微粒体并与探针药进行体外温孵,应用液相色谱?串联质谱分析方法测定CYP450的6种同工酶特异性探针底物非那西丁(CYP1A2)、右美沙芬(CYP2D2)、双氯芬酸钠(CYP2C6)、美芬妥英(CYP2C11)、氯唑沙宗(CYP2E1)、咪达唑仑(CYP3A1/2)在大鼠肝微粒体的代谢产物生成(对乙酰氨基酚、右啡烷、4-羟基双氯芬酸钠、4-羟基美芬妥英、6-羟基氯唑沙宗、1-羟基咪达唑仑)以反映各CYP450同工酶活性。五味子醇提物(28～120μg.mL-1)体外对大鼠肝微粒体CYP450的6个同工酶均有不同程度的抑制作用。大鼠多次口服五味子醇提物(1.5 g.kg-1,qd×7d)对肝脏CYP3A1/2和CYP2E1有显著诱导作用,对CYP2D2和CYP2C11有明显抑制作用,而对CYP2C6和CYP1A2无明显影响。五味子水提物(100～500μg.mL-1)体外对大鼠肝脏CYP450同工酶亦有抑制作用;体内多次给药(1.5 g.kg-1,qd×7d)对肝脏CY...     

甲基原薯蓣皂苷对人肝微粒体中7种CYP450酶活性的影响

目的研究甲基原薯蓣皂苷对CYP450酶的7种亚型酶活性的影响。方法将MPD和CYP450酶7种亚型的特异性探针底物咖啡因(CYP1A2)、右美沙芬(CYP2D6)、甲苯磺丁脲(CYP2C9)、s-美芬妥因(CYP2C19)、氯唑沙宗(CYP2E1)、香豆素(CYP2A6)及咪达唑仑(CYP3A4)与人肝微粒体进行孵化反应,采用HPLC和LC-MS/MS法测定对应的7种代谢产物(1,7-二甲基黄嘌呤、去甲右美沙芬、4-羟基甲苯磺丁脲、4-羟基美芬妥因、6-羟基氯唑沙宗、7-羟基香豆素和1-羟基咪达唑仑)的浓度,通过与对照组比较,确定MPD对以上7种酶活性的影响。结果MPD在1~10μmol.L-1时对7种酶均无明显抑制作用,在100μmol.L-1时对CYP2D6有抑制趋势,但对其他6种酶无抑制作用,均无统计学意义(P>0.05)。结论MPD在与以上6种酶(CYP1A2、CYP2E1、CYP2C19、CYP3A4、CYP2C9和CYP2A6)代谢的药物联合用药时,发生药物相互作用的可能性较小。     

临床治疗剂量氯氮平人体内代谢机制研究

目的本课题主要探讨临床治疗剂量氯氮平的代谢机制，为临床合理用药提供指导。方法15例精神分裂症男性住院病人，单用氯氮平治疗，达到稳态浓度后，采用自身前后对照设计试验，研究氟西汀抑制前后氯氮平及其代谢产物药代动力学参数的变化及其与酶活性的相关性。CYP1A2、CYP3A4和CYP2136的活性分别用咖啡因、咪达唑仑和右美沙芬探测。体内氯氮平及其代谢产物、咪哒唑仑及其代谢产物、尿中右美沙芬及其代谢产物用HPLC-MS测定。血中咖啡因及其代谢产物用HPlC-UV测定。数据用SPSS软件进行统计分析。结果氯氮平合用氟西汀后，氯氮平的Cmax、AUC0-24显著增加，t1/2增加趋势，但没有显著性差异。代谢产物去甲氯氮平的AUC0-24显著降低，Cmax和t1／2无显著性差异。N-氧化氯氮平的Cmax和AUC0-24显著性降低，t1／2没有显著性差异。合用氟西汀前后CYP1A2活性无差异，CYP3A4和CYP2D6活性显著降低。合用氟西汀前后CYP1A2活性分别与合用氟西汀前后氯氮平的AUC0-24以及去甲氯氮平的AUC0-24相关，与N-氧化氯氮平的AUC0-24无显著相关。CYP3A4和CYP2D6活性与氯氮平、去甲氯氮平、N-氧化氯氮平的AUC0-24均无显著相关，但是合用氟西汀后CYP3A4活性变化与N-氧化氯氮平的AUC0-24变化显著相关，CYP2D6活性变化与去甲氯氮平的AUC0-24变化显著相关。结论临床剂量氯氮平的代谢途径为去甲基化和N-氧化，其中去甲基代谢为主要代谢途径主要由CYP1A2催化。CYP3A4和CYP2D6不是催化CLZ代谢的主要酶，但是CYP3A4参与了CLZN-氧化代谢，CYP2D6参与去甲基代谢。     

豆腐果苷对人肝微粒体CYP450酶体外抑制作用研究

目的:通过评价豆腐果苷在体外对人肝微粒体CYP450酶的7种亚型酶活性的影响,预测服用豆腐果苷可能出现的食物-药物及药物-药物代谢的影响。方法:将豆腐果苷与CYP450酶7种亚型的特异性探针底物咖啡因(CYP1A2)、右美沙芬(CYP2D6)、甲苯磺丁脲(CYP2C9)、S-美芬妥因(CYP2C19)、氯唑沙宗(CYP2E1)、香豆素(CYP2A6)及咪达唑仑(CYP3A4)与人肝微粒体进行孵育反应,采用HPLC和LC-MS/MS法测定对应的7种代谢产物(1,7-二甲基黄嘌呤、去甲右美沙芬、4-羟基甲苯磺丁脲、4-羟基美芬妥因、6-羟基氯唑沙宗、7-羟基香豆素和1-羟基咪达唑仑)的浓度,与对照组比较,确定豆腐果苷对以上7种亚酶活性的影响。结果:豆腐果苷在1～100μmol.L-1时对7种酶的抑制作用均无明显统计学意义(P>0.05)。结论:豆腐果苷可能不会引起有临床意义的CYP450酶抑制现象的发生。     

补骨脂素和异补骨脂素对体外细胞色素P450酶活性的抑制和诱导作用

目的探讨补骨脂素(PSO)和异补骨脂素(IPSO)对细胞色素P450(CYP)活性的抑制和诱导作用。方法将人肝微粒体或鼠肝微粒体与PSO或IPSO以及CYP特异性探针底物共孵育30 min,分别以非那西丁O-脱乙基、甲苯磺丁脲4-羟基化、美芬妥因-4-羟基化、右美沙芬O-脱甲基化和咪达唑仑1'-羟基化为同工酶CYP1A2,CYP2C9,CYP2C19,CYP2D6(大鼠2D2)和CYP3A4(大鼠3A1/2)代谢活性的标志,用HPLC-MS/MS法检测相应代谢产物的生成量并计算相应的IC50值,评价PSO和IPSO对5种CYP同工酶的潜在抑制作用。将PSO和IPSO或阳性诱导剂与"三明治"培养大鼠肝原代细胞共孵育72 h后,再加入CYP探针底物孵育1 h,检测相应代谢产物的生成量,与阳性诱导剂组比较,评价二者对CYP1A和CYP3A的诱导作用。结果 PSO和IPSO对人肝微粒体和鼠肝微粒体的CYP1A2均有较强的抑制作用,在人肝微粒体中的IC50值分别为0.17和0.13μmol·L-1;在鼠肝微粒体中的IC50值分别为0.47和0.36μmol·L-1。二者对人肝微粒体的CYP2D6也有中等强度的抑制作用,IC50值分别为3.59和9.51μmol·L-1。PSO和IPSO 100μmol·L-1可分别将大鼠肝细胞的CYP3A活性提高1.18和0.96倍,有一定的诱导作用。结论 PSO和IPSO能显著抑制CYP1A2酶活性,对CYP3A有一定的诱导作用。     

LC-MS/MS法研究Cocktail探针药物咖啡因、右美沙芬、奥美拉唑、咪达唑仑及其代谢产物在大鼠血浆中的药代动力学

目的建立灵敏、快速、稳定的测定大鼠血浆中CYP450探针药物/代谢产物,咖啡因/副黄嘌呤、奥美拉唑/5-羟基奥美拉唑、右美沙芬/右啡烷、咪达唑仑/1'-羟基咪达唑仑的LC-MS/MS方法,并用于其在大鼠体内的药代动力学研究。方法 100μL血浆样品加入内标地西泮及乙酸铵溶液,乙酸乙酯进行液液萃取,离心取上清挥干复溶,LC-MS/MS检测。选用Agilent Eclipse Plus-C_(18)色谱柱(50 mm×2.1mm,3.5μm),乙腈和0.01%甲酸(1 mmol·L~(-1)甲酸铵)作为流动相,梯度洗脱,流速为0.3 m L·min~(-1),进样量10μL。采用电喷雾电离源(ESI),扫描方式为多反应正离子监测模式(MRM+),咖啡因m/z:195.0/138.1;副黄嘌呤m/z:181.1/124.1;奥美拉唑m/z:346.1/198.1;5-羟基奥美拉唑m/z:362.1/214.1;右美沙芬m/z:272.2/147.1;右啡烷m/z:258.1/157.1;咪达唑仑m/z:326.1/291.1;1'-羟基咪达唑仑m/z:342.1/324.1;内标地西泮m/z:285.1/154.0。结果咖啡因、副黄嘌呤、奥美拉唑、5-羟基奥美拉唑、右美沙芬、右啡烷、咪达唑仑和1'-羟基咪达唑仑的线性范围分别为1.95~2 000、0.98~250、0.48~2 000、0.98~250、0.98~2 000、0.48~125、1.95~2 000和1.95~250μg·L~(-1)。RSD<15%且无基质效应。结论该方法快速、灵敏、重现性好,可用于血浆中上述探针药物/代谢产物浓度的测定,便于深入进行代谢酶CYP1A2、CYP2C19、CYP2D6和CYP3A4的代谢相关研究。     

六神丸对CYP活性的诱导作用研究

目的:研究六神丸对CYP活性的诱导作用。方法:体外传代培养人肝癌Hep G2细胞。以3、1、0.5μg/ml(以丸质量计,下同)六神丸培养细胞48 h后,采用实时荧光聚合酶链反应(RT-PCR)法测定细胞CYP1A2、CYP2C9、CYP2C19、CYP2D6、CYP2E1、CYP3A4 m RNA的表达;采用Western blot法测定细胞CYP3A4、CYP1A2、CYP2C19、CYP2C9蛋白的表达,采用高效液相质谱法测定咪达唑仑代谢产物1-羟基咪达唑仑的含量,以咪达唑仑生成速率代表CYP3A4活性。上述试验均设阴性对照(0.3%二甲基亚砜)组、阳性对照(30μmol/L利福平、100μmol/L地塞米松)组。将18只Wistar大鼠随机均分为空白对照[等容羧甲基纤维素钠(CMC-Na)]组、六神丸(1.613 mg/kg)组、地塞米松(50 mg/kg)组,连续6 d ig给药后测定大鼠肝微粒1-羟基咪达唑仑生成速率以判定CYP3A1、CYP3A2活性;采用RT-PCR法测定CYP3A m RNA的表达。结果:3、1、0.5μg/ml六神丸培养细胞48 h后,与阴性对照比较,Hep G2细胞中CYP2C9、CYP2C19、CYP2E1、CYP3A4 m RNA表达增强,CYP3A4蛋白表达增强,CYP3A4活性增强,差异均有统计学意义(P<0.05)。与空白对照组比较,大鼠肝微粒体CYP3A1、CYP3A2活性增强,CYP3A1、CYP3A2 m RNA表达增强,差异均有统计学意义(P<0.05)。结论:六神丸对CYP具有一定的诱导作用,其机制可能与增强CYP m RNA的表达作用有关。     

大鼠肝细胞色素P4503A活性测定及其孵育条件的优化

目的:测定大鼠肝微粒体中细胞色素P4503A(CYP3A)的活性,并对其体外孵育条件进行优化。方法:采用1’-羟基咪哒唑仑的生成量表示肝中CYP3A的活性,高效液相色谱法测定肝微粒体中1’-羟基咪哒唑仑的浓度,用单因素试验法对其体外孵育条件进行优化,用线性Lineweaver-Burk双倒数作图法研究肝CYP3A酶促动力学。结果:1’-羟基咪哒唑仑在18.20~1 820.00μg.L-1范围内线性关系良好;体外优化的孵育条件为5μmol.L-1咪哒唑仑、0.102mg肝微粒体、孵育15min;肝CYP3A酶促动力学参数Km为1.67μmol.L-1,Vmax为95.15pmol.min-1.mg-1。结论:HPLC法操作简便、灵敏、快速,适用于肝微粒体中1’-羟基咪哒唑仑的浓度测定,肝CYP3A孵育条件及其酶促动力学研究为研究经CYP3A代谢的药物相互作用及其他物质对CYP3A酶的影响提供理论依据。     

评价HPPH对细胞色素P450酶体外代谢活性的影响

目的:评价HPPH在体外对大鼠及人肝微粒CYP450酶的6种亚型酶活性的影响,预测使用HPPH可能出现的药物相互作用。方法:将注射用HPPH与CYP450酶的6种亚型的特异性探针底物非那西汀(CYP1A2)、甲苯磺丁脲(CYP2C9)、S-美芬妥因(CYP2C19)、右美沙芬(CYP2D6)、氯唑沙宗(CYP2E1)、咪达唑仑(CYP3A4)和睾酮(CYP3A4)与大鼠及人肝微粒进行孵育反应,采用HPLC-MS/MS法测定对应的7种代谢产物(对乙酰氨基酚、羟基甲苯磺丁脲、4-羟基美芬妥因、O-去甲基右美沙芬、6-羟基氯唑沙宗、1′-羟基咪达唑仑、6β-羟基睾酮)的浓度。结果:在本实验条件下,HPPH浓度为1.00~50.00μmol·L-1时,未发现其对大鼠的CYP1A2、CYP2C9、CYP2D6、CYP2E1、CYP3A4产生抑制作用。在本实验条件下,HPPH浓度为0.50~10.00μmol·L-1时,未发现其对人CYP1A2产生抑制作用;但对人CYP2C9、CYP2C19、CYP2D6、CYP2E1均存在抑制作用;对人CYP3A4,对底物咪达唑仑存在抑制作用,对底物睾酮未发现抑制作用。结论:HPPH对CYP450酶的抑制作用存在种属差异,在人体内HPPH与CYP450酶作用有待体内实验进一步证明。     

Cocktail探针药物法评价威麦宁胶囊对大鼠体内CYP450活性的影响

目的采用Cocktail探针药物法评价威麦宁胶囊对大鼠体内6种CYP450亚型酶活性的影响。方法分别选用甲苯磺丁脲、氯唑沙宗、茶碱、咪达唑仑、奥美拉唑和右美沙芬作为CYP2C6、CYP2E1、CYP1A2、CYP3A2、CYP2D1和CYP2D2的探针底物。大鼠每日灌胃威麦宁胶囊1.6 g·kg-1,采用LC-MS/MS测定给药前后大鼠体内6种混合探针的血药浓度,计算药动学参数。结果威麦宁胶囊连续给药2周后,与给药前相比,奥美拉唑ρmax、AUC0-t、tmax及AUC0-∞显著升高(P<0.05);咪达唑仑ρmax、AUC0-t和AUC0-∞显著升高(P<0.01或P<0.05);右美沙芬ρmax、AUC0-t升高(P<0.05);甲苯磺丁脲t1/2、AUC0-∞和tmax显著升高(P<0.01或P<0.05),而CL/F显著降低(P<0.01),ρmax、AUC0-t无显著改变(P>0.05);氯唑沙宗ρmax升高(P<0.05),CL/F降低(P<0.05),AUC0-t升高但无显著差异(P>0.05);茶碱ρmax、AUC0-t升高但无显著差异(P>0.05)。表明奥美拉唑、咪达唑仑和右美沙芬代谢明显减慢(均P<0.05),茶碱、甲苯磺丁脲、氯唑沙宗的代谢无显著差异;威麦宁胶囊对大鼠体内CYP2D1、CYP3A2、CYP2D2酶有抑制作用,对CYP1A2、CYP2C6、CYP2E1酶的活性无显著影响。结论当威麦宁胶囊与CYP2C19、CYP3A4、CYP2D6酶的底物药物合用时,需要调整给药剂量,避免因药物相互作用使体内血药浓度过高产生毒副作用。     

A comprehensive evaluation of metabolic activity and intrinsic clearance in suspensions and monolayer cultures of cryopreserved primary human hepatocytes

Primary human hepatocytes are widely used for metabolic stability evaluations. However, there are limited data directly comparing phase I and phase II drug‐metabolizing enzymes in fresh and cryopreserved hepatocytes prepared from the same human donor liver. We evaluated the metabolic competency of human hepatocytes prepared from seven donor tissues before and after cryopreservation. Temporal‐dependent enzyme activity in suspension and matched adherent cultures of primary human hepatocytes was also assessed. Cryopreservation of hepatocytes resulted in statistically significant increases in activities of CYP1A2, CYP2B6, CYP2C9, CYP2D6, and CYP3A but not CYP2C8, CYP2C19, FMO, UGT, and SULT, relative to fresh hepatocytes. In suspension cultures of hepatocytes, enzyme stabilities were as follows: UGT相似文献   

Comparison of inducibility of CYP1A and CYP3A mRNAs by prototypical inducers in primary cultures of human, cynomolgus monkey, and rat hepatocytes

This study was conducted to investigate the effects of treatment with the prototypical inducers rifampicin (Rif), dexamethasone (Dex), and omeprazole (Ome) on the mRNA levels of drug-metabolizing enzymes in primary cultures of cryopreserved human, cynomolgus monkey, and rat hepatocytes. Analysis was performed by quantitative real-time RT-PCR using primers and TaqMan probes. Treatment with Ome substantially increased the mRNA levels of both CYP1A1 and CYP1A2 in human hepatocytes, but increased only the mRNA level of CYP1A1 in monkey hepatocytes, whereas it had no marked effect on the mRNA levels of CYP1A1 or CYP1A2 in rat hepatocytes. Treatment with Rif or Dex did not markedly affect the mRNA level of CYP1A in any of the hepatocyte cultures under the conditions used. All three inducers increased the mRNA level of CYP3A8 in monkey hepatocytes (in the order Rif>Dex>or=Ome), and a similar profile was observed for the mRNA level of CYP3A4 in human hepatocytes, but the potency of induction was markedly attenuated. In contrast, only Dex substantially increased the mRNA level of CYP3A1 in rat hepatocytes, with Rif and Ome showing no effects. These results indicate that the molecular mechanisms responsible for the regulation of CYP1A2 genes differ between humans and cynomolgus monkeys, although the regulatory mechanisms for CYP1A1 and CYP3A genes are similar.     

Cytochrome p450 expression of cultured rat small hepatocytes after long-term cryopreservation.

Small hepatocytes (SHs) are hepatic progenitor cells that can be cryopreserved for a long time. After thawing, the cells can proliferate and, when treated with Matrigel, they can differentiate into mature hepatocytes (MHs). In this study, we investigated whether cryopreserved SHs could express cytochromes P450 (P450s), whether P450 expression was induced by appropriate inducers, and whether P450 activities were measurable. 3-Methylcholanthrene (3-MC), phenobarbital (PB), pregnenolone-16alpha-carbonitrile (PCN), and ethanol were used as inducers for CYP1A, 2B, 3A, and 2E, respectively. Immunoblot analysis indicated that cryopreserved SHs constitutively expressed CYP1A1/2, CYP2E1, and CYP3A2 as much as 26 days after plating. Significant expression of CYP1A1/2 and 3A2 in the cells treated with Matrigel was induced by 3-MC and PCN, respectively. Although Matrigel did not up-regulate the enzymatic activity of CYP1A, CYP3A and CYP2E activities increased. Induction of CYP1A and CYP3A activities by each inducer was observed in cryopreserved cells treated with Matrigel. Although the expression of CYP2B1 could be detected in subcultured SHs treated with PB, it was not detected in cryopreserved SHs. The activity of NADPH-cytochrome P450 reductase was measured in both subcultured and cryopreserved SHs, although the activities in both were approximately 30% of that of MHs. Profiles of (14)C-testosterone metabolites were examined in cultured MHs and in cryopreserved SHs by high-performance liquid chromatography. Similar peaks for testosterone metabolites in MHs and SHs were observed in the same elution time. These results indicate that, although induction of CYP3A and 2B in cryopreserved SHs is inferior to that in subcultured ones, SHs can maintain the expression and activities of P450s after long-term cryopreservation.     

Effect of cryopreservation on cytochrome P-450 enzyme induction in cultured rat hepatocytes.

In the present study, we evaluated the inducibility of cytochrome P-450 (CYP) CYP1A, CYP2B, CYP3A, and CYP4A by beta-naphthoflavone, phenobarbital, dexamethasone, and clofibric acid, respectively, in primary hepatocyte cultures prepared from both fresh and cryopreserved rat hepatocytes. Rat hepatocytes were successfully thawed and cultured after cryopreservation in liquid nitrogen for up to 1 month. Percentage of total recovery, viable cell recovery, and final viability of the cells were 68%, 72%, and 85%, respectively. Regardless of whether they were cryopreserved or not, cultured hepatocytes exhibited near-normal morphology. Treatment of cryopreserved hepatocytes with beta-naphthoflavone caused an 8-fold increase in 7-ethoxyresorufin O-dealkylase (CYP1A1/2) activity, with an EC50 of 1.5 microM; treatment with phenobarbital caused a 26-fold increase in 7-pentoxyresorufin O-dealkylase (CYP2B1/2) activity, with an EC50 of 10 microM; treatment with dexamethasone caused a 10-fold increase in testosterone 6beta-hydroxylase (CYP3A1/2) activity, with an EC50 of 1.3 microM, whereas treatment with clofibric acid caused a 3-fold increase in lauric acid 12-hydroxylase (CYP4A1-3) activity, with an EC50 of 170 microM. The induction of CYP1A, CYP2B, CYP3A, and CYP4A enzymes by these inducers was confirmed by Western immunoblotting. The patterns of P-450 induction in cryopreserved rat hepatocytes, in terms of concentration response, reproducibility, magnitude, and specificity of response, were similar to those observed in freshly isolated hepatocytes. Additionally, the magnitude and specificity of induction was similar to that observed in vivo in rats. In conclusion, under the conditions examined, cryopreserved rat hepatocytes appear to be a suitable in vitro system for evaluating xenobiotics as inducers of P-450 enzymes.     

Comparison of the inducibility of CYP mRNA exposed to typical inducers in fresh and cryopreserved cynomolgus monkey hepatocytes

Herein, we evaluated CYPs and their nuclear receptor mRNA induction by exposure to typical inducers, omeprazole, rifampicin, and phenobarbital in cynomolgus monkey hepatocytes. Six freshly-isolated hepatocytes and 6 cryopreserved hepatocytes from cynomolgus monkey liver were prepared for a 14-day monolayer culture, 28-day co-culture with feeder cells, and 28-day 3D spheroid culture with feeder cells. Omeprazole and rifampicin respectively induced CYP1A1 and CYP3A8 mRNAs, while phenobarbital induced CYP2C43, CYP2C75, and CYP3A8, and slightly induced CYP2B6. The nuclear receptors AHR, PXR, and CAR mRNA levels, which were activated by omeprazole, rifampicin, and phenobarbital, respectively, tended to decrease via exposure to inducers despite the increase in CYP mRNA levels. These trends were similar for all three culture methods. No evident difference was observed in CYP mRNA induction between fresh and cryopreserved hepatocytes. Based on mRNA levels, the co-culture and 3D spheroid culture methods are more reasonable than monolayer culture for CYP evaluation, because the use of feeder cells can reduce the number of hepatocytes, improve the cell adhesion, and maintain the mRNA expression levels. In addition, co-culture method is more cost-effective, as common culture plates can be used.     

Cryopreserved rat, dog and monkey hepatocytes: measurement of drug metabolizing enzymes in suspensions and cultures

Metabolism in fresh and cryopreserved (CP) rat, dog and monkey hepatocyte suspensions and cultures was measured using midazolam (CYP3A), tolbutamide (CYP2C), dextromethorphan (CYP2D) and p-nitrophenol (glucuronosyl S-transferases (UGT), sulphotransferases (ST)). CYP3A, CYP2C9, CYP2D6, UGT and ST enzyme functions in fresh and CP rat, dog and monkey hepatocyte suspensions were retained - CP rat hepatocytes lost some CYP2C activity but this was restored by adding NADPH or by placing the cells in culture, suggesting that the enzyme was still functional. Phase 2 activities were equivalent in fresh and CP hepatocyte suspensions. In some cases, incubation conditions increased the rate of metabolism, possibly reflecting de novo cofactor synthesis. However, this effect was substrate and species dependent and was not always the same in fresh and CP cells. CYP3A, CYP2C, CYP2D, UGT and ST activities at 24 hours of culture of rat and monkey hepatocytes were not compromised by cryopreservation. CYP3A, CYP2D but not CYP2C were lower in 24-hour cultures of CP dog hepatocytes than in fresh cells. Despite being lower than fresh cells, UGT activity in dog CP hepatocytes did not decrease from 0 to 24 hours of culture. Species-specific metabolism of p-nitrophenol could be demonstrated in both CP cell suspensions and cultures. In conclusion, these data suggest that the enzyme characteristics of fresh and CP hepatocytes from each species and under specific incubation conditions should be considered when carrying out metabolism studies of new compounds.     

Cryopreserved primary hepatocytes as a constantly available in vitro model for the evaluation of human and animal drug metabolism and enzyme induction

The use of primary hepatocytes is now well established for both studies of drug metabolism and enzyme induction. Cryopreservation of primary hepatocytes decreases the need for fresh liver tissue. This is especially important for research with human hepatocytes because availability of human liver tissue is limited. In this review, we summarize our research on optimization and validation of cryopreservation techniques. The critical elements for successful cryopreservation of hepatocytes are (1) the freezing protocol, (2) the concentration of the cryoprotectant [10% dimethyl-sulfoxide (DMSO)], (3) slow addition and removal of DMSO, (4) carbogen equilibration during isolation of hepatocytes and before cryopreservation, and (5) removal of unvital hepatocytes by Percoll centrifugation after thawing. Hepatocytes of human, monkey, dog, rat, and mouse isolated and cryopreserved by our standard procedure have a viability > or = 80%. Metabolic capacity of cryopreserved hepatocytes determined by testosterone hydroxylation, 7-ethoxyresorufin-O-de-ethylase (EROD), 7-ethoxycoumarin-O-deethylase (ECOD), glutathione S-transferase, UDP-glucuronosyl transferase, sulfotransferase, and epoxide hydrolase activities is > or = 60% of freshly isolated cells. Cryopreserved hepatocytes in suspension were successfully applied in short-term metabolism studies and as a metabolizing system in mutagenicity investigations. For instance, the complex pattern of benzo[a]pyrene metabolites including phase II metabolites formed by freshly isolated and cryopreserved hepatocytes was almost identical. For the study of enzyme induction, a longer time period and therefore cryopreserved hepatocyte cultures are required. We present a technique with cryopreserved hepatocytes that allows the induction of testosterone metabolism with similar induction factors as for fresh cultures. However, enzyme activities of induced hepatocytes and solvent controls were smaller in the cryopreserved cells. In conclusion, cryopreserved hepatocytes held in suspension can be recommended for short-term metabolism or toxicity studies. Systems with cryopreserved hepatocyte cultures that could be applied for studies of enzyme induction are already in a state allowing practical application, but may be further optimized.     

Comparison of inducibility of sulfotransferase and UDP-glucuronosyltransferase mRNAs by prototypical microsomal enzyme inducers in primary cultures of human and cynomolgus monkey hepatocytes

We investigated the change of the mRNA levels of sulfotransferase and UDP-glucuronosyltransferase isoforms by the prototypical microsomal enzyme inducers rifampicin (Rif), dexamethasone (Dex), and omeprazole (Ome) in primary cultures of cryopreserved human and cynomolgus monkey hepatocytes. Real-time RT-PCR analysis was performed using primers and TaqMan probes. Rif, Dex, and Ome increased SULT2A1 mRNA level in both human and cynomolgus monkey hepatocytes in dose-dependent manner, but not SULT1A1 mRNA level. Rif, Dex, and Ome increased the mRNA level of UGT1A1 in both human and cynomolgus monkey hepatocytes, Ome more potently in humans and Rif and Ome more potently in monkeys. They also increased the mRNA levels of UGT1A6 and UGT1A9 in cynomolgus monkey hepatocytes, though the extent of elevation of UGT1A6 and UGT1A9 mRNA levels was smaller than that of UGT1A1 mRNA level. Furthermore, these inducers scarcely affected UGT1A6 and UGT1A9 in human hepatocytes. Rif, Dex, and Ome also showed no remarkable effect on the mRNA levels of UGT2Bs in human or cynomolgus monkey hepatocytes. We also studied in detail the time course of mRNA expression of these enzymes in primary cultures of hepatocytes. In conclusion, the results of the present study show that primary cultures of hepatocytes isolated from the cynomolgus monkey liver are as useful as human hepatocytes for evaluating the induction of drug-metabolizing enzymes in preclinical studies.     

Determination of cytochrome P450 1A2 and cytochrome P450 3A4 induction in cryopreserved human hepatocytes

Freshly prepared human hepatocytes are considered as the 'gold standard' for in vitro testing of drug candidates. However, several disadvantages are associated with the use of this model system. The availability of hepatocytes is often low and consequently the planning of the experiments rendered difficult. In addition, the quality of the available cells is in some cases poor. As an alternative, cryopreserved human hepatocytes were validated as a model to study cytochrome P450 1A2 (CYP1A2) and cytochrome P450 3A4 (CYP3A4) induction. In a single blinded experiment, hepatocytes from three separate lots were incubated with three concentrations of different compounds, and compared to non-treated cells and cells incubated with omeprazole or rifampicin. CYP1A2 and CYP3A4 induction was determined by measuring 7-ethoxyresorufin-O-deethylation activity and 6beta-hydroxytestosterone formation, respectively. CYP1A2 and CYP3A4 mRNA and protein expression were analyzed by TaqMan QRT-PCR and immunodetection. Cells responded well to the prototypical inducers with a mean 38.8- and 6.2-fold induction of CYP1A2 and CYP3A4 activity, respectively. Similar as with fresh human hepatocytes, high batch-to-batch variation of CYP1A2 and CYP3A4 induction was observed. Except for 1 and 10 microM rosiglitazone, the glitazones did not significantly affect CYP1A2. A similar result was observed for CYP3A4 activity although CYP3A4 mRNA and protein expression were dose-dependently upregulated. In conclusion, cryopreserved human hepatocytes may be a good alternative to fresh hepatocytes to study CYP1A and 3A induction.     

Retention of transporter activities in cryopreserved, isolated rat hepatocytes.

The success of cryopreservation of isolated hepatocytes with existing methodologies is assessed with respect to the retentivity of cell integrity/viability (defined by trypan blue) and metabolic activities upon thawing in comparison to those of freshly prepared cells. But the ability of the cryopreserved cells to transport xenobiotics relative to that of freshly prepared cells has not been investigated. In this study, we optimized our previous methodology for cryopreservation and evaluated the metabolism and transport of thawed hepatocytes. Half of the freshly, isolated rat hepatocytes prepared by collagenase perfusion were immediately used for studies of transport of [(14)C]taurocholate, [(3)H]estrone sulfate and [(3)H]estradiol 17beta-D-glucuronide (1 microM) and metabolism of 7-hydroxy-4-(trifluoromethyl)-coumarin (100 microM), (3,4-difluorobenzyloxy)-5,5-dimethyl-4-(4-methylsulfonylphenyl)-(5H)-furan-2-one (250 microM), bufuralol (100 microM), and tolbutamide (100 microM), probes for UDP-glucuronyl transferase (UGT) and CYP3A, CYP2D, and CYP2C, respectively. The remaining half was cryopreserved using an optimized, programmed-freezing protocol, which was developed to minimize the prolonged release of latent heat during freezing. With the exception of the UGT probe, no significant difference (P > 0.05) was found in both metabolism and transport with freshly isolated versus cryopreserved hepatocytes upon thawing. In conclusion, we have demonstrated for the first time that thawed rat hepatocytes cryopreserved by a programmed-freezing protocol retain drug transport activities.