药物药代动力学和毒理学评估的肠道/肝脏微生理系统
Summary
我们向对乙酰氨基酚（APAP）暴露了含有肠道和肝脏器官的微生理系统（MPS）。本文介绍了MPS中器官生产和APAP药代动力学和毒理学属性评估的方法。它还描述了验证结果所需的组织功能分析。
Abstract
最近引进的培养人体器官的微生理系统（MPS）在药物开发过程的临床前测试阶段有望比动物表现更好，因为它们是遗传人类，并重述组织之间的相互作用。在这项研究中，将人类肠道屏障（由Caco-2和HT-29细胞的共培养物模拟）和肝脏当量（由分化的肝细胞和人类肝硬质细胞的球类模拟）整合到双器官芯片（2-OC）微流体装置中，以评估一些乙酰氨基酚（APAP）药理动力学（PK）和毒理学特性。MPS有三个组件：肠道只有2-OC，肝脏只有2-OC，和肠/肝脏2-OC与相同的介质细读两个器官。对于 PK 评估，我们在通过肠道屏障（模拟口腔路线）或介质（模拟静脉路线）后，在介质中以预设的时间点对 APAP 进行加注，分别值 12 μM 和 2 μM。通过反相高压液相色谱（HPLC）对介质样品进行分析。对器官分析为基因表达，TEER值，蛋白质表达和活性，然后收集，固定，并提交一组形态学评估。MTT 技术在评估器官生存能力方面表现良好，但高含量分析 （HCA） 能够检测出非常早期的毒性事件，以响应 APAP 治疗。我们验证，介质流没有显著影响APAP吸收，但它显著改善肝脏等效功能。APAP人类肠道吸收和肝代谢可以在MPS中模拟。MPS 数据与硅建模之间的关联在提高体外方法的可预测性方面有很大的潜力，并且比在药代动力学和毒理学研究中的动物模型具有更高的准确性。
Introduction
由于基因组和蛋白质组的差异，动物模型对几种人类结果的预测价值有限。此外，它们既费时又昂贵，而且在道德上也值得怀疑。MPS 是一项相对较新的技术，旨在提高预测能力，减少临床前测试的成本和时间。它们是在媒体流下培养器官（器官的人工模仿功能单元）的微流体装置，促进器官-器官通信。由人类细胞制造的有机体增加转化相关性2，3，4。MPS 预期性能优于动物试验，因为它们是基因人类，并重述组织之间的相互作用。当完全正常运行时，MPS 将提供更有意义的结果，以更高的速度和更低的成本和风险 4。许多团体正在开发MPS，用于多种目的，特别是疾病模型，以测试药物的疗效。
暴露水平是评估药物疗效和毒性的最关键参数之一5，6，7，8，9，10，11，12。MPS 允许有机体整合，模拟全身暴露，预期性能优于传统的 2D 人体组织培养。该技术可显著提高复合肠道吸收和肝代谢的预测。
考虑到这两个器官在药物生物利用度和全身暴露13、14、15等的中心作用，整合肠道和肝脏等量模型的MPS是一个很好的起点。APAP是一种有吸引力的药物，用于研究没有肾脏等价物的MPS，因为它的代谢主要由肝脏16，17完成。
2-OC是一种双室微流体装置，适用于由微通道16连接的两种不同人体等效组织/器官的培养。为了模拟体外人类口服/静脉注射药物，并评估肠道和肝脏等价物之间的交代对APAP药代动力学的影响， 除了器官的功能和生存能力外，还进行了三种不同的MPS组件：（1） 一个”肠2-OC MPS”，由一个肠道等效物组成，其培养物中含有一个包含Caco-2+HT-29细胞共培养的培养物，并集成到2-OC装置中;（2） 由肝球类组成、由 HepaRG + HHSteC （人类肝状斯特拉特细胞） 组成的”肝脏 2-OC MPS”，集成在 2-OC 器件中;和 （3） 由一个设备隔间中的肠道等效物组成的”肠/肝脏 2-OC MPS”，通过介质流经微流体通道与肝脏等效物通信。
由于机械刺激（压缩、拉伸和剪切）对细胞的生存能力和功能18、19、20的影响，所有测定都是在静态（无流动）和动态（带流量）条件下进行的。本文介绍了APAP口服/静脉注射给药模拟的方案，以及含有人类肠道和肝脏等效模型的2-OC MPS中各自的吸收/代谢和毒理学分析。
Protocol
1. 生产组织等价物，用于2-OC的栽培 小肠屏障当量生产 使用肠道等效介质维持 Caco-2 和 HT-29 细胞：DMEM 辅以 10% FBS、1% 青霉素和链霉素，以及 1% 非必需氨基酸，本手稿中称为”DMEM S”。 取出培养基，用1x DPBS洗涤两次，并加入8 mL的0.25%的尝试素/EDTA，以分离细胞培养瓶中生长的Caco-2细胞（175厘米2）。在37°C下孵育5分钟，通过添加至少双量的三辛抑制剂来停止反应。对HT-29?…
Representative Results
要在 2-OC MPS 中执行 PK APAP 测试，第一步是制造人肠和肝脏等价物（器官）。在开始PKAPAP检测之前，它们被集成到2-OC微流体装置（图1A）24小时。第二天，介质被更改，模型将暴露在 APAP。图 1显示了放置在 2-OC 设备（图1B）和APAP PK 实验时间过程（图1C）内的肠道和肝脏等价物。我们在 2D 培养物和 3D 器官中进?…
Discussion
准确、可靠地评估研究性新药的药理特性，对于降低以下开发步骤的风险至关重要。MPS 是一项相对较新的技术，旨在提高预测能力，减少临床前测试的成本和时间。我们的团队正在推进对铅优化所需的药代动力学和毒理学特性的评估。我们与2-OC微流体装置合作，该装置有两个腔室，允许两个器官的整合。APAP之所以被选中，是因为它拥有大量高质量的人类数据，大部分是肝脏代谢的，并且也表现?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
我们感谢克里斯蒂·古根-吉卢佐博士、INSERM 522单元的菲利普·格里邦博士和271 INSERM单元的克里斯蒂安·特雷波博士使用生物材料（Hepa RG细胞），并随后提供给我们进行学术研究。
Materials
| 1x DPBS | Thermo Fisher Scientific | 14190235 | No calcium, no magnesium |
| 2-OC | TissUse GmbH | Two-organ chip | |
| 384-well Spheroid Microplate | Corning | 3830 | Black/Clear, Round Bottom, Ultra-Low Attachment |
| 4% Paraformaldehyde | Use to fix cell | ||
| Acetaminophen | Sigma Aldrich | A7085 | Use to MPS assays |
| Acetonitrile | Tedia | Used to perform HPLC | |
| Alexa Fluor 647 phalloidin | Thermo Fisher Scientific | confocal experiment | |
| Ammonium acetate | Sigma Aldrich | Used to perform HPLC | |
| Caco-2 cells | Sigma Aldrich | 86010202 | |
| Cacodylate buffer | |||
| Cell culture flasks | Sarstedt | ||
| Confocal Fluorescence microscope | Leica | DMI6000 | |
| Cryostat | Leica | CM1950 | |
| DMEM high glucose | Thermo Fisher Scientific | 12800017 | Add supplements: 10% fetal bovine serum, 100 units per mL penicillin, 100 µg/mL streptomycin, and 1% non-essential amino acids |
| DMSO | Sigma Aldrich | D4540 | Add 2% to HepaRG media |
| Ethanol | Synth | ||
| Fetal Bovine Serum | Thermo Fisher Scientific | 12657029 | |
| Freezing medium OCT | Tissue-Tek | Tissue-Tek® O.C.T.™ Compound is a formulation of watersoluble glycols and resins, providing a convenient specimen matrix for cryostat sectioning at temperatures of -10°C and below. | |
| Hematoxylin & Eosin | |||
| HepaRG cells | Biopredic International | HPR101 | Undifferentiated cells |
| HHSTeC | ScienCell Research Laboratories | 5300 | Cells and all culture supplements |
| Hoechst 33342 | HCA experiments | ||
| HT-29 cells | Sigma Aldrich | 85061109 | |
| Human Insulin | Invitrogen – Thermo Fisher Scientific | 12585014 | |
| Hydrocortisone | Sigma Aldrich | H0888 | |
| Isopropanol | Merck | 278475 | |
| Karnovsky’s fixative | |||
| L-glutamine | Thermo Fisher Scientific | A2916801 | |
| Luna C18 guard column SS | Phenomenex | Used to perform HPLC | |
| Microscope | Leica | DMi4000 | |
| Microtome | Leica | RM2245 | |
| Millicell 0.4 µm pore size inserts | Merck | PIHP01250 | |
| Millicell ERS-2 meter | Merck | MERS00002 | Used to TEER measurement |
| MitoTracker Deep Red | HCA experiments | ||
| MTT | Thermo Fisher Scientific | M6494 | |
| MX3000P system | Agilent Technologies | ||
| Neubauer chamber | Counting cells | ||
| Operetta High Content Imaging System | Perkin Elmer | Used to perform HCA | |
| P450-Glo CYP3A4 Assay with Luciferin-IPA | Promega | Cat.# V9001 | |
| Penicillin/Streptomycin | Thermo Fisher Scientific | 15070063 | Cell culture |
| Permount | Thermo Fisher Scientific | Histology | |
| Primers | RT-qPCR | ||
| PVDF membrane | BioRad | ||
| PVDF Syringe filter | 0.22 μm pore size | ||
| Reversed-phase Luna C18 column | Phenomenex | Used to perform HPLC | |
| Shaker (IKA VXR Basic Vibrax) | IKA Works GmbH & Co | 2819000 | Used for spheroids to improve MTT assay |
| Stellate Cell Media (STeC CM) | ScienCell | 5301 | Add STeC CM supplements |
| SuperScriptIITM Reverse Transcriptase | Thermo Fisher Scientific | ||
| SYBR Green PCR Master Mix | Thermo Fisher Scientific | ||
| TRizol TM reagent | Thermo Fisher Scientific | Trizol is a monophasic solution of phenol and guanidine isothiocyanate. | |
| Trypsin/EDTA solution | Thermo Fisher Scientific | R001100 | |
| Ultra-low-attachment plates | Corning | CLS3471-24EA | 6 wells |
| Vectashield plus DAPI mounting media | |||
| White Opaque 96-well Microplate | PerkinHelmer | ||
| Wide-bore tips | |||
| Williams E | Pan Biotech | P04-29510 | Add supplements: 10% fetal bovine serum, 2 mM L-glutamine, 100 units per ml penicillin, 100 µg/mL streptomycin and 5 µg/mL human insulin |
References
- Zhang, D., Luo, G., Ding, X., Lu, C. Preclinical experimental models of drug metabolism and disposition in drug discovery and development. Acta Pharmaceutica Sinica B. 2 (6), 549-561 (2012).
- Hynds, R. E., Giangreco, A. The relevance of human stem cell-derived organoid models for epithelial translational medicine. Stem Cells. 31 (3), 417-422 (2013).
- Sivaraman, A., et al. A Microscale In vitro Physiological Model of the Liver: Predictive Screens for Drug Metabolism and Enzyme Induction. Current Drug Metabolism. 6 (6), 569-591 (2005).
- Dehne, E. M., Hasenberg, T., Marx, U. The ascendance of microphysiological systems to solve the drug testing dilemma. Future Science OA. 3 (2), 185 (2017).
- Oleaga, C., et al. Multi-Organ toxicity demonstration in a functional human in vitro system composed of four organs. Scientific Reports. 6, 20030 (2016).
- Maschmeyer, I., et al. A four-organ-chip for interconnected long-term co-culture of human intestine, liver, skin and kidney equivalents. Lab Chip. 15 (12), 2688-2699 (2015).
- Esch, M. B., Mahler, G. J., Stokol, T., Shuler, M. L. Body-on-a-chip simulation with gastrointestinal tract and liver tissues suggests that ingested nanoparticles have the potential to cause liver injury. Lab Chip. 14 (16), 3081-3092 (2014).
- Materne, E. M., et al. The Multi-organ Chip – A Microfluidic Platform for Long-term Multi-tissue Coculture. Journal of Visualized Experiments. , e52526 (2015).
- Esch, M. B., Ueno, H., Applegate, D. R., Shuler, M. L. Modular, pumpless body-on-a-chip platform for the co-culture of GI tract epithelium and 3D primary liver tissue. Lab Chip. 16 (14), 2719-2729 (2016).
- Sung, J. H., Kam, C., Shuler, M. L. A microfluidic device for a pharmacokinetic–pharmacodynamic (PK-PD) model on a chip. Lab on a Chip. 10 (4), 446-455 (2010).
- Viravaidya, K., Sin, A., Shuler, M. L. Development of a Microscale Cell Culture Analog to Probe Naphthalene Toxicity. Biotechnology Progress. 20 (1), 316-323 (2004).
- Sin, A., Chin, K. C., Jamil, M. F., Kostov, Y., Rao, G., Shuler, M. L. The Design and Fabrication of Three-Chamber Microscale Cell Culture Analog Devices with Integrated Dissolved Oxygen Sensors. Biotechnology Progress. 20 (1), 338-345 (2004).
- Tsamandouras, N., et al. Integrated Gut and Liver Microphysiological Systems for Quantitative In vitro Pharmacokinetic Studies. The AAPS Journal. 19 (5), 1499-1512 (2017).
- Graham, G. G., Davies, M. J., Day, R. O., Mohamudally, A., Scott, K. F. The modern pharmacology of paracetamol: Therapeutic actions, mechanism of action, metabolism, toxicity and recent pharmacological findings. Inflammopharmacology. 21 (3), 201-232 (2013).
- Hodgman, M. J., Garrard, A. R. A Review of Acetaminophen Poisoning. Critical Care Clinics. 28 (4), 499-516 (2012).
- Maschmeyer, I., et al. Chip-based human liver-intestine and liver-skin co-cultures – A first step toward systemic repeated dose substance testing in vitro. European Journal of Pharmaceutics and Biopharmaceutics. 95, 77-87 (2015).
- Bessems, J. G. M., Vermeulen, N. P. E. Paracetamol (acetaminophen)-induced toxicity: Molecular and biochemical mechanisms, analogues and protective approaches. Critical Reviews in Toxicology. 31 (1), 55-138 (2001).
- Hirt, M. N., et al. Increased afterload induces pathological cardiac hypertrophy: A new in vitro model. Basic Research in Cardiology. 107 (6), 307 (2012).
- Fink, C., et al. Chronic stretch of engineered heart tissue induces hypertrophy and functional improvement. The FASEB journal official publication of the Federation of American Societies for Experimental Biology. 14 (5), 669-679 (2000).
- Zhang, B., Peticone, C., Murthy, S. K., Radisic, M. A standalone perfusion platform for drug testing and target validation in micro-vessel networks. Biomicrofluidics. 7 (4), 044125 (2013).
- Araújo, F., Sarmento, B. Towards the characterization of an in vitro triple co-culture intestine cell model for permeability studies. International Journal of Pharmaceutics. 458 (1), 128-134 (2013).
- Cadena-Herrera, D., et al. Validation of three viable-cell counting methods: Manual, semi-automated, and automated. Biotechnology Reports. 7, 9-16 (2015).
- Gripon, P., et al. Infection of a human hepatoma cell line by hepatitis B virus. Proceedings of the National Academy of Sciences of the United States of America. 99 (24), 15655-15660 (2002).
- Guillouzo, A., et al. The human hepatoma HepaRG cells: A highly differentiated model for studies of liver metabolism and toxicity of xenobiotics. Chemico-Biological Interactions. 168 (1), 66-73 (2007).
- Wagner, I., et al. A dynamic multi-organ-chip for long-term cultivation and substance testing proven by 3D human liver and skin tissue co-culture. Lab on a Chip. 13 (18), 3538-3547 (2013).
- Forrest, J. A. H., Clements, J. A., Prescott, L. F. Clinical Pharmacokinetics of Paracetamol. Clinical Pharmacokinetics. 7 (2), 93-107 (1982).
- Dollery, C. . Therapeutic Drugs. , (1999).
- Shah, V. P., et al. Bioanalytical method validation-a revisit with a decade of progress. Pharmaceutical research. 17 (12), 1551-1557 (2000).
- Marin, T. M., et al. Shp2 negatively regulates growth in cardiomyocytes by controlling focal adhesion kinase/src and mTOR pathways. Circulation Research. 103 (8), 813-824 (2008).
- Linkert, M., et al. Metadata matters: Access to image data in the real world. Journal of Cell Biology. 189 (5), 777-782 (2010).
- OECD. OECD TG 491 – Short Time Exposure In vitro Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage. OECD. 14, (2018).
- Fernandes, M. B., Gonçalves, J. E., Tavares, L. C., Storpirtis, S. Caco-2 cells permeability evaluation of nifuroxazide derivatives with potential activity against methicillin-resistant Staphylococcus aureus (MRSA). Drug Development and Industrial Pharmacy. 41 (7), 1066-1072 (2015).
- OECD. OECD TG 492 – Reconstructed human Cornea-like Epithelium (RhCE) test method for identifying chemicals not requiring classification and labelling for eye irritation or serious eye damage. OECD. 35, (2018).
- OECD, O. E. C. D. OECD TG 431 – In vitro skin corrosion: reconstructed human epidermis (RHE) test method. OECD. 8, (2016).
- OECD. OECD TG 439 – In vitro Skin Irritation: Reconstructed Human Epidermis Test Method. OECD. 21, (2015).
- Marin, T. M., et al. Acetaminophen absorption and metabolism in an intestine/liver microphysiological system. Chemico-Biological Interactions. 299, 59-76 (2019).
- Maass, C., Stokes, C. L., Griffith, L. G., Cirit, M. Multi-functional scaling methodology for translational pharmacokinetic and pharmacodynamic applications using integrated microphysiological systems (MPS). Integrative Biology (United Kingdom). 9 (4), 290-302 (2017).
- Sung, J. H., Wang, Y., Shuler, M. L. Strategies for using mathematical modeling approaches to design and interpret multi-organ microphysiological systems (MPS). APL Bioengineering. 3 (2), 021501 (2019).
Citer Cet Article
Marin, T. M., Indolfo, N. d. C., Rocco, S. A., de Carvalho, M., Dias, M. M., Vasconcelos Bento, G. I., Bortot, L. O., Schuck, D. C., Lorencini, M., Pagani, E. An Intestine/Liver Microphysiological System for Drug Pharmacokinetic and Toxicological Assessment. J. Vis. Exp. (166), e60184, doi:10.3791/60184 (2020).