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In vitro Studies: Inhibition of Nevirapine Metabolism by Nortriptyline in Hepatic Microsomes
体外研究:肝脏微粒体中去甲替林对奈韦拉平新陈代谢的抑制   

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参见作者原研究论文

本实验方案简略版
Antimicrobial Agents and Chemotherapy
Dec 2014

Abstract

One of the most prevalent and interfering psychosocial comorbidities of HIV infection is clinical depression (22 to 45%). For this reason, a study of a possible interaction between the nonnucleoside reverse transcriptase inhibitor nevirapine (NVP) and the tricyclic antidepressant nortriptyline (NT) was carried out. In vitro studies with rat and human hepatic microsomes showed a marked inhibition of NVP metabolism by NT being more intense in rat than in human. The extrapolation of these results to humans suggests increased NVP side effects when both drugs are coadministered, but additional in vivo human studies are required to evaluate the clinical implication of this interaction.

This protocol describes a technique for detecting and measuring the inhibition of the nevirapine metabolism by nortriptyline in hepatic microsomes.

Keywords: Nevirapine (抗艾滋病毒药物的治疗), Nortriptyline (去甲替林), Metabolism inhibition (代谢抑制), Drug interaction (药物相互作用), Hepatic microsomes (肝微粒体)

Materials and Reagents

  1. 10 ml polypropylene tube 16 x 95 mm (Deltalab, catalog number: 400900 )
  2. 5 ml ultracentrifuge tube 13 x 51 mm (Beckman Coulter, catalog number: 326819 )
  3. 1.5 ml screw cap vial 32 x 11.6 mm
  4. 7.5 ml screw cap tube 13 x 100 mm
  5. Wistar rat livers
  6. Saline solution (Laboratorios ERN, catalog number: 999790 )
  7. Protein Standard (2 mg BSA) (BD Biosciences, Falcon®, catalog number: P5619 )
    Note: Currently, it is “Sigma-Aldrich, catalog number: P5619”.
  8. Na2CO3 (EMD Millipore Corporation, catalog number: 106392 )
  9. Copper(II) sulfate pentahydrate (CuSO4.5H2O) (Sigma-Aldrich, catalog number: 209198 )
  10. Potassium sodium tartrate tetrahydrate (Sigma-Aldrich, catalog number: 217255 )
  11. Folin-Ciocalteu’s phenol reagent (EMD Millipore Corporation, catalog number: 109001 )
  12. Potassium phosphate monobasic (KH2PO4) (BD Biosciences, Falcon®, catalog number: 60229 )
    Note: Currently, it is “Sigma-Aldrich, catalog number: 60229”.
  13. Sodium phosphate dibasic (Na2HPO4) (Sigma-Aldrich, Falcon®, catalog number: 56814 )
  14. KCl (EMD Millipore Corporation, catalog number: 104936 )
  15. Ethylendiaminetetraacetic acid disodium salt-2-hydrate (EDTA) (Riedel-de Haën, catalog number: 34549 )
    Note: Currently, it is “Sigma-Aldrich, FLUKA, catalog number: 34549 ”.
  16. Sucrose (BD Biosciences, Falcon®, catalog number: 84100 )
    Note: Currently, it is “Sigma-Aldrich, catalog number: 84100”.
  17. K2HPO4.3H2O (AppliChem GmbH, catalog number: 122333 )
  18. Nevirapine (Viramune) (Boehringer Ingelheim)
  19. Methanol, HiPerSolv CHROMANORM for HPLC-Gradient Grade (VWR International, Prolabo, catalog number: 20864.320 )
  20. Dimethyl sulfoxide (DMSO) (minimum 99.5% GC, plant cell culture tested) (Sigma -Aldrich, catalog number: D4540 )
  21. Nortriptyline hydrochloride, minimum 98% TLC (Sigma-Aldrich, catalog number: N7261 )
  22. D-Glucose 6-phosphate disodium salt hydrate (Sigma-Aldrich, catalog number: G7250 )
  23. β-Nicotinamide adenine dinucleotide phosphate disodium salt (β-NADP) (Sigma-Aldrich, catalog number: 93205 )
  24. Glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides (Sigma-Aldrich, catalog number: G5760 )
  25. MgCl2.6H2O, BioReagent, suitable for cell culture, suitable for insect cell culture (Sigma-Aldrich, catalog number: M2393 )
  26. Acetonitrile (VWR, Prolabo, catalog number: 83639.320 )
  27. Human liver microsomes pooled from 50 different individual donors (Thermo Fisher Scientific, InvitrogenTM, catalog number: HMMCPL )
  28. Sodium hydroxide (extra pure, pellets) (Scharlab,S.L., catalog number: SO0420005P )
  29. Ethyl acetate (extra pure) (Scharlab,S.L., catalog number: AC01431000 )
  30. NaH2PO4.2H2O (Guinama, catalog number: 90790 )
  31. Triethylamine (Sigma-Aldrich, catalog number: T0886 )
  32. 50 mM sodium-potassium phosphate buffer (see Recipes)
  33. 0.1 M potassium phosphate buffer (see Recipes)
  34. Lowry reagent (see Recipes)
  35. 50 mM sodium phosphate monobasic buffer (see Recipes)

Equipment

  1. Surgical tools
  2. Scissors
  3. Spectrophotometer (Hitachi High-Technologies Corporation, model: U-2900 )
  4. 1 cm polystyrene spectrophotometer cuvette (Sigma-Aldrich, catalog number: C5219 )
  5. Thermostatic water bath
  6. Refrigerated centrifuge (Sigma Laborzentrifugen, model: 2K15 )
  7. Refrigerated ultracentrifuge (130,000 rpm) (Beckman Coulter, model: OptimaTM MAX ultracentrifuge )
  8. Tissue Grinder, Potter-Elvehjem type, 30 ml Glass Vessel and Plain Plunger (VWR, Prolabo, catalog number: 432-0204 and 89026-400 , respectively)

Procedure

  1. Inhibition of the nevirapine metabolism by nortriptyline in rat hepatic microsomes
    1. Isolate rat liver microsomes following these steps:
      1. Sacrifice a total of 5 Wistar rats (280-310 g) by isoflurane inhalation.
      2. Open the abdominal cavity of each rat, excise the liver and place it into a beaker containing saline solution.
      3. Wash the liver with saline solution and place it on a paper towel.
      4. Weigh the liver and cut it into small pieces at 4 °C.
      5. Place the liver into the glass vessel and add 5x its mass in volume of 50 mM sodium-potassium phosphate buffer (pH 7.4) containing 1.15% (wt/vol) KCl, 2 mM EDTA, and 0.25 M sucrose.
      6. Homogenize the liver with the tissue grinder until a complete homogeneous solution is obtained.
      7. Transfer the homogenate to 10 ml polypropylene tubes and centrifuge (4 °C) at 9,000 x g for 10 min.
      8. Transfer the supernatant to ultracentrifuge tubes and centrifuge (4 °C) at 150,000 x g for 1 h.
      9. Resuspend the microsomal pellet in 0.1 M potassium phosphate buffer (pH 7.4).
    2. Determine microsomal protein concentration following the method of Lowry et al. (1951):
      1. Prepare a series of dilutions of 2 mg bovine serum albumin in water, to give concentrations of 0.1 to 1 mg/ml.
      2. Add to 100 µl of each dilution of standard or sample containing microsomes and 2 ml of Lowry reagent to each 10 ml polypropylene tube.
      3. Incubate the tubes in the dark at room temperature for 15 min.
      4. Add 200 µl of an aqueous solution of Folin-Ciocalteu’s phenol reagent (1/2, vol/vol) to each tube.
      5. Incubate the tubes in the dark at room temperature for 30 min.
      6. Measure absorbance at 750 nm in 1 cm spectrophotometer cuvettes.
    3. Prepare a mixture in a 1.5 ml screw cap vial that contains the following compounds (per 100 µl):
      1. 10 µl of a solution of NVP in a mixture of water, methanol, and DMSO (final concentrations: 5 µg/ml NVP, 0.12% methanol, and 0.08% DMSO)
      2. 10 µl of NT solutions in water (final NT concentrations of 100, 200, 300, 500, 1,000, 2,000, 3,000, and 10,000 ng/ml) or 10 µl of H2O in the case of controls.
      3. 5 µl of a 20-mg/ml glucose-6-phosphate aqueous solution
      4. 5 µl of a 20-mg/ml β-NADP aqueous solution
      5. 10 µl of a glucose-6-phosphate dehydrogenase aqueous solution (10 IU/ml)
      6. 5 µl of a MgCl2 aqueous solution (13.4 mg/ml)
      7. Quantity sufficient to 100 µl of 0.1 M potassium phosphate buffer (pH 7.4)
      8. Rat hepatic microsomes previously isolated (equivalent to 0.1 mg of protein). With this addition the metabolic reaction is started
    4. Incubate the test vials for 30 min at 37 °C in a water bath.
    5. Terminate the reaction in an ice bath by adding 100 µl of acetonitrile to the test vials.
    6. Centrifuge the vials at 2,000 x g for 5 min at 4 °C.
    7. Determine the amount of NVP in the supernatant liquid by high performance liquid chromatography (HPLC), using a C18 column and a UV detector set at 240 nm, as described previously (Usach et al., 2014).

  2. Inhibition of nevirapine metabolite formation by nortriptyline in human hepatic microsomes
    1. Prepare a mixture in a 7.5 ml screw cap tube that contains the following compounds (per 100 µl):
      1. 20 µl of a solution of NVP in a mixture of water, methanol, and DMSO (final concentrations of 5 µg/ml NVP, 0.12% methanol, and 0.08% DMSO)
      2. 20 µl of NT solutions in water (final NT concentrations of 100, 1,000 and 10,000 ng/ml) or 10 µl of H2O in the case of controls.
      3. 10 µl of a 20-mg/ml glucose-6-phosphate aqueous solution
      4. 10 µl of a 20-mg/ml β-NADP aqueous solution
      5. 20 µl of a glucose-6-phosphate dehydrogenase aqueous solution (10 IU/ml)
      6. 10 µl of a MgCl2 aqueous solution (13.4 mg/ml)
      7. Quantity sufficient to 0.2 ml of 0.1 M potassium phosphate buffer (pH 7.4)
      8. Human hepatic microsomes (equivalent to 0.2 mg of protein). With this addition the metabolic reaction is started.
    2. Incubate test tubes for 30 min at 37 °C in a water bath.
    3. Terminate the reaction in an ice bath by adding 25 µl of a 2 N sodium hydroxide solution and 5 ml of ethyl acetate to extract NVP and its metabolites.
    4. Shake the mixture and wait a few minutes until the two phases are completely separated.
    5. Transfer the supernatant to another 7.5 ml screw cap tube and evaporate it in a 40 °C water bath under a nitrogen stream.
    6. Repeat the extraction: Add 3 ml of ethyl acetate, shake the tube and evaporate the supernatant as before.
    7. Dissolve the dried residue into 100 µl of the mobile phase composed of acetonitrile and sodium phosphate monobasic buffer (50 mM [pH 4.6]) containing 0.1% (vol/vol) triethylamine (12/88, vol/vol).
    8. Centrifuge vials at 2,000 x g for 5 min at 4 °C.
    9. Determine the amount of NVP and its metabolites in the supernatant liquid by HPLC, using a C18 column and a UV detector set at 240 nm, as described previously (Usach, et al., 2014).

Representative data


Figure 1. Representative HPLC-UV chromatogram of NVP metabolism in rat liver microsomes (NVP 5 µg/ml). A. Standard of NVP (no metabolism); B. NVP after 30 min at 37 °C; C. NVP and NT (10 µg/ml) after 30 min at 37 °C.


Figure 2. Representative HPLC-UV chromatogram of NVP metabolism in human liver microsomes (NVP 5 µg/ml). A. Standard of NVP (no metabolism); B. NVP after 30 min at 37 °C; C. NVP and NT (10 µg/ml) after 30 min at 37 °C.

Notes

Solutions and materials used for the isolation of rat microsomes have to be kept on ice (4 °C).

Recipes

  1. 50 mM sodium-potassium phosphate buffer (pH 7.4) [containing 1.15% (wt/vol) KCl, 2 mM EDTA, and 0.25 M sucrose]
    Mix 102 mg KH2PO4 with 603 mg Na2HPO4
    Add 1.15 g KCl, 74 mg EDTA and 8.5 g sucrose
    Add dH2O to 100 ml
  2. 0.1 M potassium phosphate buffer (pH 7.4)
    Mix 1.83 g K2HPO4.3H2O with 0.27 g KH2PO4
    Add dH2O to 100 ml
  3. Lowry reagent
    Mix 49 ml of reagent A with 0.5 ml of reagent B and 0.5 ml of reagent C:
    Reagent A: 2% Na2CO3 in 0.1 N NaOH
    Reagent B: 1% CuSO4.5H2O in water
    Reagent C: 2% potassium sodium tartrate tetrahydrate in water
  4. 50 mM sodium phosphate monobasic buffer (pH 4.6) [containing 0.1% (vol/vol) triethylamine]
    Add 7.8 g NaH2PO4.2H2O and 1 ml trimethylamine
    Add dH2O to 1 L
    Adjust pH to 4.6

Acknowledgments

This protocol was recently described and applied by Usach et al. (2014). Isolation of rat microsomes and composition of mediums used in the inhibition assay were adapted from Kawashima et al. (1999) and Choi et al. (2008), respectively. Extraction of NVP and its metabolites from human microsomes was adapted from Erickson et al. (1999). I. Usach has been granted a predoctoral fellowship from the Atracció de Talent (VLC-CAMPUS) program of University of Valencia.

References

  1. Choi, Y. H., Chung, S. J. and Lee, M. G. (2008). Pharmacokinetic interaction between DA-8159, a new erectogenic, and metformin in rats: competitive inhibition of metabolism via hepatic CYP3A1/2. Br J Pharmacol 153(7): 1568-1578.
  2. Erickson, D. A., Mather, G., Trager, W. F., Levy, R. H. and Keirns, J. J. (1999). Characterization of the in vitro biotransformation of the HIV-1 reverse transcriptase inhibitor nevirapine by human hepatic cytochromes P-450. Drug Metab Dispos 27(12): 1488-1495.
  3. Kawashima, K., Hosoi, K., Naruke, T., Shiba, T., Kitamura, M. and Watabe, T. (1999). Aldehyde oxidase-dependent marked species difference in hepatic metabolism of the sedative-hypnotic, zaleplon, between monkeys and rats. Drug Metab Dispos 27(3): 422-428.
  4. Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J. (1951). Protein measurement with the Folin phenol reagent. J Biol Chem 193(1): 265-275.
  5. Usach, I., Melis, V., Gandia, P. and Peris, J. E. (2014). Pharmacokinetic interaction between nevirapine and nortriptyline in rats: inhibition of nevirapine metabolism by nortriptyline. Antimicrob Agents Chemother 58(12): 7041-7048.

简介

HIV感染的最普遍和干扰的心理社会共病是临床抑郁症(22%至45%)。 为此,进行了非核苷逆转录酶抑制剂奈韦拉平(NVP)和三环抗抑郁药去甲替林(NT)之间可能的相互作用的研究。 使用大鼠和人肝微粒体的体外研究显示在大鼠中NT比在人中更强烈的NVP代谢的显着抑制。 将这些结果外推到人表明当两种药物共同给药时,NVP副作用增加,但是需要另外的体内人体研究来评价这种相互作用的临床意义。
该方案描述了 检测和测量肝微粒体中去甲替林对奈韦拉平代谢的抑制的技术。

关键字:抗艾滋病毒药物的治疗, 去甲替林, 代谢抑制, 药物相互作用, 肝微粒体

材料和试剂

  1. 10ml聚丙烯管16×95mm(Deltalab,目录号:400900)
  2. 5ml超速离心管13×51mm(Beckman Coulter,目录号:326819)
  3. 1.5 ml螺帽瓶32 x 11.6 mm
  4. 7.5ml螺旋盖管13×100mm
  5. Wistar大鼠肝脏
  6. 盐水溶液(Laboratorios ERN,目录号:999790)
  7. 蛋白标准品(2mg BSA)(BD Biosciences,Falcon ,目录号:P5619)
    注意:目前,它是"Sigma-Aldrich,目录号:P5619"。
  8. Na 2 CO 3(EMD Millipore Corporation,目录号:106392)
  9. 硫酸铜(II)五水合物(CuSO 4·5H 2 O·5H 2 O)(Sigma-Aldrich,目录号:209198)
  10. 酒石酸钾钠四水合物(Sigma-Aldrich,目录号:217255)
  11. Folin-Ciocalteu的酚试剂(EMD Millipore Corporation,目录号:109001)
  12. 磷酸二氢钾(KH 2 PO 4)(BD Biosciences,Falcon ,目录号:60229)
    注意:目前,它是"Sigma-Aldrich,目录号:60229"。
  13. 磷酸氢二钠(Na 2 HPO 4)(Sigma-Aldrich,Falcon ,目录号:56814)
  14. KCl(EMD Millipore Corporation,目录号:104936)
  15. 乙二胺四乙酸二钠盐-2水合物(EDTA)(Riedel-deHa?n,目录号:34549)
    注意:目前,它是"Sigma-Aldrich,FLUKA,目录号:34549"。
  16. 蔗糖(BD Biosciences,Falcon ,目录号:84100) 注意:目前,它是"Sigma-Aldrich,目录号:84100"。
  17. (AppliChem GmbH,目录号:122333)
  18. 奈韦拉平(Viramune)(Boehringer Ingelheim)
  19. 甲醇,用于HPLC-梯度级的HiPerSolv CHROMANORM(VWR International,Prolabo,目录号:20864.320)
  20. 二甲基亚砜(DMSO)(最小99.5%GC,测试的植物细胞培养物)(Sigma-Aldrich,目录号:D4540)
  21. 盐酸去甲替林,最少98%TLC(Sigma-Aldrich,目录号:N7261)
  22. D-葡萄糖-6-磷酸二钠盐水合物(Sigma-Aldrich,目录号:G7250)
  23. β-烟酰胺腺嘌呤二核苷酸磷酸二钠盐(β-NADP)(Sigma-Aldrich,目录号:93205)
  24. 来自肠膜明串珠菌的葡萄糖-6-磷酸脱氢酶(Sigma-Aldrich,目录号:G5760)
  25. 适合于细胞培养的适于昆虫细胞培养的生物试剂(Sigma-Aldrich,目录号:M2393)的MgCl 2·6H 2 O,BioReagent,
  26. 乙腈(VWR,Prolabo,目录号:83639.320)
  27. 汇集来自50个不同个体供体(Thermo Fisher Scientific,Invitrogen TM,目录号:HMMCPL)的人肝微粒体
  28. 氢氧化钠(超纯的颗粒)(Scharlab,S.L。,目录号:SO0420005P)
  29. 乙酸乙酯(超纯)(Scharlab,S.L。,目录号:AC01431000)
  30. 2(Guinama,目录号:90790)。
  31. 三乙胺(Sigma-Aldrich,目录号:T0886)
  32. 50 mM磷酸钠 - 钾缓冲液(见配方)
  33. 0.1 M磷酸钾缓冲液(见配方)
  34. Lowry试剂(参见配方)
  35. 50 mM磷酸二钠缓冲液(见配方)

设备

  1. 外科工具
  2. 剪刀
  3. 分光光度计(Hitachi High-Technologies Corporation,型号:U-2900)
  4. 1cm聚苯乙烯分光光度计比色杯(Sigma-Aldrich,目录号:C5219)
  5. 恒温水浴
  6. 冷冻离心机(Sigma Laborzentrifugen,型号:2K15)
  7. 冷冻超速离心机(130,000rpm)(Beckman Coulter,型号:Optima TM MAX超速离心机)
  8. Tissue Grinder,Potter-Elvehjem型,30ml玻璃容器和平柱塞(分别为VWR,Prolabo,目录号:432-0204和89026-400)

程序

  1. 在大鼠肝微粒体中去甲替林的奈韦拉平代谢的抑制
    1. 按照以下步骤分离大鼠肝微粒体:
      1. 通过异氟醚吸入总共牺牲5只Wistar大鼠(280-310g)
      2. 打开每只大鼠的腹腔,切除肝脏,并将其放入含有盐水溶液的烧杯中
      3. 用盐溶液洗肝脏,并将其放在纸巾上。
      4. 称重肝脏,并在4°C切成小块
      5. 将肝脏放入玻璃容器中,加入其体积的5倍 的50mM磷酸钠 - 钾缓冲液(pH 7.4),其含有1.15% (wt/vol)KCl,2mM EDTA和0.25M蔗糖
      6. 用组织研磨机匀化肝脏,直到获得完全均匀的溶液
      7. 将匀浆转移到10ml聚丙烯管中,并以9000×g离心10分钟(4℃)。
      8. 将上清液转移到超速离心管中,并在150,000xg下离心(4℃)1小时。
      9. 在0.1M磷酸钾缓冲液(pH 7.4)中重悬微粒体沉淀。
    2. 按照Lowry等人的方法(1951)确定微粒体蛋白浓度:
      1. 制备一系列稀释的2mg牛血清白蛋白的水溶液,使浓度为0.1至1mg/ml。
      2. 加入10 ml聚丙烯管100μl的每个稀释的标准品 ?或含有微粒体的样品和2ml的Lowry试剂。
      3. 在黑暗中室温孵育试管15分钟。
      4. 向管中加入200μlFolin-Ciocalteu的酚试剂(1/2,vol/vol)的水溶液。
      5. 在黑暗中室温孵育试管30分钟。
      6. 在1cm分光光度计比色杯中测量750nm处的吸光度。
    3. 在含有以下化合物(每100μl)的1.5ml螺帽小瓶中制备混合物:
      1. 10μlNVP在水,甲醇和DMSO的混合物中的溶液 ?(终浓度:5μg/ml NVP,0.12%甲醇和0.08%DMSO)
      2. 将10μlNT水溶液(最终NT浓度为100,200, ?300,500,1,000,2,000,3,000和10,000ng/ml)或10μl的H 2 O在 ?控制的情况。
      3. 5μl20mg/ml葡萄糖-6-磷酸水溶液
      4. 5μl的20-mg/mlβ-NADP水溶液
      5. 10μl葡萄糖-6-磷酸脱氢酶水溶液(10IU/ml)
      6. 5μlMgCl 2水溶液(13.4mg/ml)
      7. 数量足以使100μl的0.1M磷酸钾缓冲液(pH 7.4)
      8. 先前分离的大鼠肝微粒体(相当于0.1mg 蛋白)。通过该添加,开始代谢反应
    4. 在37℃水浴中孵育试管30分钟
    5. 通过向试验瓶中加入100μl乙腈,在冰浴中终止反应
    6. 在4℃下,以2000×g离心样品瓶5分钟。
    7. 通过高确定上清液中NVP的量 高效液相色谱(HPLC),使用C18柱和UV 检测器设置在240nm,如先前所述(Usach,,2014)。

  2. 人肝脏微粒体中去甲替林对奈韦拉平代谢物形成的抑制
    1. 在含有以下化合物(每100μl)的7.5ml螺旋盖管中制备混合物:
      一个。 20μlNVP在水,甲醇和DMSO的混合物中的溶液 ?(终浓度为5μg/ml NVP,0.12%甲醇和0.08%DMSO) b。将20μlNT水溶液(最终NT浓度为100, 1,000和10,000ng/ml)或10μlH 2 O。
      C。 10μl20mg/ml葡萄糖-6-磷酸水溶液
      d。 10μl的20-mg/mlβ-NADP水溶液
      e。 20μl葡萄糖-6-磷酸脱氢酶水溶液(10IU/ml)
      G。量足以使0.2ml的0.1M磷酸钾缓冲液(pH 7.4)
      H。人肝微粒体(相当于0.2mg蛋白质)。加入后,开始代谢反应
    2. 在37℃水浴中孵育试管30分钟
    3. 在冰浴中通过加入25μl的2N终止反应 氢氧化钠溶液和5ml乙酸乙酯萃取NVP, 其代谢物。
    4. 摇动混合物,等待几分钟,直到两相完全分离
    5. 将上清液转移到另一个7.5螺旋盖管中,并在40℃水浴中在氮气流下蒸发。
    6. 重复萃取:加入3ml乙酸乙酯,摇动试管,如前所述蒸发上清液
    7. 将干燥的残余物溶解在100μl的流动相中 的乙腈和磷酸二氢钠缓冲液(50mM [pH4.6]) 含有0.1%(vol/vol)三乙胺(12/88,vol/vol)
    8. 在4℃下,以2000×g离心样品瓶5分钟。
    9. 确定上清液中NVP及其代谢物的量 液相,使用设置在240nm的C18柱和UV检测器, (Usach,,2014)。

代表数据


图1.大鼠肝微粒体(NVP5μg/ml)中NVP代谢的代表性HPLC-UV色谱图。 A.NVP标准(无代谢)。 B.在37℃下30分钟后的NVP; C.NVP和NT(10μg/ml)在37℃下30分钟后

图2.人肝微粒体(NVP5μg/ml)中NVP代谢的代表性HPLC-UV色谱图。 A。 NVP标准(无代谢); B.在37℃下30分钟后的NVP; C.NVP和NT(10μg/ml)在37℃下30分钟后。

笔记

用于分离大鼠微粒体的溶液和材料必须保持在冰上(4℃)。

食谱

  1. 50mM磷酸钠 - 钾缓冲液(pH7.4)[含有1.15%(wt/vol)KCl,2mM EDTA和0.25M蔗糖] 将102mg KH 2 PO 4与603mg Na 2 HPO 4混合物混合 加入1.15g KCl,74mg EDTA和8.5g蔗糖 将dH <2> O添加到100 ml
  2. 0.1M磷酸钾缓冲液(pH7.4) 将1.83g K 2 H 2 HPO 4+与3.27g KH 2混合物混合在一起> PO 4
    将dH <2> O添加到100 ml
  3. Lowry试剂
    将49ml试剂A与0.5ml试剂B和0.5ml试剂C:
    混合 试剂A:在0.1N NaOH中的2%Na 2 CO 3溶液
    试剂B:在水中的1%CuSO 4+。在水中的H 2/H 2 O 试剂C:2%的酒石酸钾钠四水合物在水中
  4. 50mM磷酸钠一碱性缓冲液(pH4.6)[含有0.1%(vol/vol)三乙胺] 加入7.8g NaH 2 PO 4 .0H 2 2H 2 O和1ml三甲胺。
    将dH <2> O添加到1 L
    将pH调节至

致谢

该协议最近由Usach等人(2014)描述和应用。用于抑制测定的大鼠微粒体的分离和培养基的组成分别改变自Kawashima等人(1999)和Choi等人(2008)。从人微粒体提取NVP及其代谢物是从Erickson等人(1999)。 I. Usach已经被授予瓦伦西亚大学的人才培养计划(VLC-CAMPUS)的前行奖学金。

参考文献

  1. Choi,Y.H.,Chung,S.J.and Lee,M.G。(2008)。 DA-8159,一种新的勃起功能药物和二甲双胍在大鼠中的药代动力学相互作用:代谢途径的竞争性抑制hepatic CYP3A1/2。 Br J Pharmacol 153(7):1568-1578。
  2. Erickson,D.A.,Mather,G.,Trager,W.F.,Levy,R.H。和Keirns,J.J。(1999)。 人肝细胞色素的HIV-1逆转录酶抑制剂奈韦拉平的体外生物转化的表征P- Drug Metab Dispos 27(12):1488-1495。
  3. Kawashima,K.,Hosoi,K.,Naruke,T.,Shiba,T.,Kitamura,M.and Watabe,T。(1999)。 镇静催眠,扎来普隆,猴子之间的肝脏代谢中的醛氧化酶依赖性标记物种差异大鼠。 Drug Metab Dispos 27(3):422-428。
  4. Lowry,O.H.,Rosebrough,N.J.,Farr,A.L.and Randall,R.J。(1951)。 使用Folin酚试剂进行蛋白质测量。J Biol Chem 193(1):265-275
  5. Usach,I.,Melis,V.,Gandia,P.and Peris,J.E。(2014)。 大鼠中奈韦拉平和去甲替林之间的药代动力学相互作用:去甲替林对奈韦拉平代谢的抑制。 em> Antimicrob Agents Chemother 58(12):7041-7048。
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引用:Usach, I. and Peris, J. (2015). In vitro Studies: Inhibition of Nevirapine Metabolism by Nortriptyline in Hepatic Microsomes. Bio-protocol 5(19): e1607. DOI: 10.21769/BioProtoc.1607.
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