眼薬の薬物動態試験でウサギの眼の使用
Summary
Rabbits are widely used to study the pharmacokinetics of intraocular drugs. We describe a method for conducting pharmacokinetic studies of intraocular drugs using rabbit eyes.
Abstract
彼らの全身吸収を最小限に抑えながら、薬物投与の眼内経路は、治療薬の高濃度の配信を可能にします。いくつかの薬剤は、前房または硝子体内に投与され、眼内注射は、様々な眼疾患を硬化させるのに有効でした。動物は、他の哺乳類に比べて取り扱いが容易かつ経済的であるように、ウサギの目は広く、眼科研究のために使用されており、ウサギの眼の大きさは、人間の目と同様です。 30 G針を使用して、薬物は、ウサギの眼の房内および硝子体内空間に注入することができます。眼球は、分析まで凍結され、房水、硝子体、及び網膜/脈絡膜に分けることができます。硝子体および網膜/脈絡膜サンプルを均質化し、分析の前に可溶化することができます。そして、イムノアッセイは、各区画内に、眼内の薬物の濃度を測定するために行うことができます。適切な薬物動態学的モデルは、することができますこのような半減期および薬物の最大濃度などのいくつかのパラメータを計算するために使用されます。ウサギの眼は、眼内の薬物の薬物動態学的研究のための良いモデルとなり得ます。
Introduction
眼内薬物送達の出現する前に、眼内疾患に対する薬物療法の主な関心事は、薬物が眼に浸透可能性があると効率でした。血液眼関門は、眼への拡散が、薬物を含む多くの物質を、防ぐことができます。したがって、治療レベルより上である薬物の濃度を容易に得ることがないかもしれません。薬物の治療濃度は、眼4,5で達成することができるように房内および硝子体内注射を含む眼内薬物投与の方法は、直接、血液眼関門1-3をバイパスすることができます。
したがって、硝子体内薬物送達は、いくつかの眼疾患5,6のための治療の一般的な方法となっています。例えば、硝子体内注射が広く、加齢性黄斑変性症、糖尿病性網膜症、網膜静脈閉塞、眼内感染7-10に対して行われます。具体的には、以来、抗VEGF薬の導入は、硝子体内注射の頻度は著しく網膜疾患の治療のために増加しています。したがって、薬物療法の有効性および安全性を評価するためのこのような薬物の眼内薬物動態を理解することが重要です。
薬物の眼内投与は、眼疾患のための医学的治療における画期的な成果であると考えられているが、眼球内薬物濃度を監視することは技術的に厳しいです。人間の目は唯一の房水の少量（およそ200μL）および硝子体（約4.5 mlであり、 表1）を含有するので、薬物濃度を測定するために、眼の流体の十分な量を得ることは技術的に困難です。さらに、このような硝子体タッピングまたは前房の穿刺などの眼の流体を得るために使用される方法は、眼組織に損傷を与え、例えば白内障、眼内炎、又は重篤な合併症をもたらし得ます網膜剥離11,12。従って、動物モデルは、一般的に使用される眼内薬物13の薬物動態学的研究において使用されています。これらの動物モデルの中で、ウサギやサルは、最も頻繁に使用される動物です。
家族のウサギ科で注文ウサギ目の小さな哺乳動物であるウサギは、世界のいくつかの部分で発見されています。ウサギは攻撃的ではないので、取り扱いが容易で、実験に使用し、観察します。低コスト、人間に対する動物、同様の目の大きさが容易に入手できる、と比較好意がウサギの眼を使用して、薬物動態学的研究を行うための情報の大規模なデータベース。本論文では、ウサギの眼における眼薬の薬物動態試験のためのプロトコルが記載されています。
Protocol
Representative Results
Discussion
With the increasing use of intraocular drugs, such as anti-vascular endothelial growth factor (VEGF) agents, for the treatment of diverse ocular diseases, knowledge of the tissue distribution and clearance of the drug after the intraocular injection is important. Understanding the pharmacokinetics of intraocular drugs is important for understanding the efficacy and safety of drugs, determining the optimal dosage of the drugs, and minimizing systemic or intraocular complications. However, detailed pharmacokinetic studies …
Disclosures
The authors have nothing to disclose.
Acknowledgements
We would like to thank Ms. Ji Hyun Park and Ji Yeon Park for their technical assistance in the animal experiments. This work was supported by a grant from the Seoul National University Bundang Hospital Research Fund (grant number: Grant No. 14-2014-022) and from a grant (CCP-13-02-KIST) from the Convergence Commercialization Project of the National Research Council of Science and Technology, Seoul, Korea.
Materials
| Zoletil | Virbac Laboratories, Carros Cedex, France | ||
| Xylazine hydrochloride | Fort Dodge Laboratories, Fort Dodge, IA | ||
| Proparacaine hydrochloride (Alcaine) | Alcon laboratories, Fort Worth, TX | ||
| Phenylephrine hydrochloride and tropicamide | Santen Pharmaceutical, Co., Osaka, Japan | ||
| Recombinant Human VEGF 165 | R&D systems | 293-VE-050 | |
| Carbobate-Bicarbonate buffer | SIGMA | C3041-50CAP | |
| NUNC MICROWELL 96F W/LID NUNCLON D SI | Thermo SCIENTIFIC | 167008 | 96 well plate |
| Bovine Serum Albumin (BSA) 25grams(Net) | BOVOGEN | BSA025 | |
| Phosphate Buffered Saline (PBS) pH7.4 (1X), 500mL | gibco | 10010-023 | |
| Sheep anti-Human IgG Secondary Antibody, HRP conjugate | Thermo SCIENTIFIC | PA1-28652 | |
| Goat Anti-Human IgG Fc(HRP) | abcam | ab97225 | |
| Goat anti-Human IgG, Fab'2 Secondary Antibody, HRP conjugate | Thermo SCIENTIFIC | PA1-85183 | |
| CelLytic MT Cell Lysis Reagent | SIGMA | C3228-50ML | lysis buffer |
| 100 Scalpel Blades | nopa instruments | BLADE #15 | |
| 100 Scalpel Blades | nopa instruments | BLADE #10 | |
| FEATHER SURGICAL BLADE STAINLESS STEEL | FEATHER | 11 | |
| 1-StepTM TMB-Blotting substrate solution, 250mL | Thermo SCIENTIFIC | 34018 | |
| Stable Peroxide Substrate Buffer (10X), 100mL | Thermo SCIENTIFIC | 34062 | |
| Softmax Pro | Molecular Devices | v.5.4.1 | software for generating standard curve |
| SAAM II | Saam Institute, Seattle, WA | software for pharmacokinetic modeling | |
| Phoenix WinNonlin | Pharsight, Cary, NC | v. 6.3 | software for pharmacokinetic modeling |
| Avastin (bevacizumab) | Genentech |
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