眼内药物的药代动力学研究采用兔眼
1Department of Ophthalmology,Seoul National University Bundang Hospital, 2Department of Ophthalmology, College of Medicine,Seoul National University, 3Department of Ophthalmology,Hanyang University Hospital, 4Department of Ophthalmology,Seoul Metropolitan Government-Seoul National University Boramae Medical Center, 5Department of Clinical Pharmacology,Seoul National University Hospital, 6Department of Clinical Pharmacology,Seoul National University Bundang Hospital
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
给药的途径眼压使高浓度的治疗药物的交付，同时尽量减少他们的全身吸收。几种药物给药进入前房或玻璃体和眼内注射已有效地治疗各种眼内疾病。兔眼已广泛用于眼科研究相比，其他哺乳动物的动物是易于处理和经济，兔眼的大小是相似的人眼。使用地下30针，药物可注入兔眼的前房和玻璃体内的空间。然后眼球冷冻直至分析，并且可分为房水，玻璃体和视网膜/脉络膜。玻璃体和视网膜/脉络膜样品可以匀浆并分析前溶解。然后，可以执行免疫测定以测量在每个隔室的眼内药物浓度。合适的药代动力学模型可以是用于计算多个参数，诸如半衰期和药物的最大浓度。兔眼可用于眼内药物的药代动力学研究的良好模式。
Introduction
眼内药物递送的出现之前，药物治疗的眼内疾病的主要关注的是与该药物能渗透到眼睛的效率。血眼屏障防止许多物质，包括药物，扩散进入眼睛。因此，可能不容易获得的高于治疗水平的药物的浓度。眼内药物施用方法，包括前房内和玻璃体内注射，能够直接绕过血眼屏障1-3，使药物治疗浓度可在眼4,5来实现。
因此，玻璃体内给药已成为治疗几种眼疾病5,6-一种流行的方法。例如，玻璃体内注射被广泛用于与年龄有关的黄斑变性，糖尿病性视网膜病变，视网膜静脉阻塞，和眼内感染7-10进行。特别是，由于引进抗VEGF药物，玻璃体内注射的次数已显着地增加了对视网膜疾病的治疗。因此，了解这些药物的眼内药物动力学，用于评估药物治疗的有效性和安全性是重要的。
虽然药物的眼内施用被认为是眼病药物治疗的重大突破，监控眼球内的药物浓度是技术要求高。因为人眼仅含有少量的水状液（约200微升）和玻璃体（约4.5毫升， 表1）的，它在技术上是难以得到足够量的眼内流体的测量药物浓度。此外，被用于获得眼部流体的方法，如玻璃体攻丝或前房穿刺放，可能会损坏眼组织，并导致严重的并发症，例如白内障，眼内炎，或视网膜脱离11,12。因此，动物模型中的常用的眼内药物13的药代动力学研究中使用。在这些动物模型中，兔或猴是最常用的动物。
兔子，这是在家庭兔科的顺序兔形目的小型哺乳动物，在世界一些地区被发现。因为兔子是没有攻击性，他们很容易处理，在实验中使用，并观察。成本较低，动物，类似于眼睛大小，以人类随时可用性和大型数据库的信息进行比较青睐执行使用兔眼药代动力学研究。在本文中，对于眼内药物在兔眼药代动力学研究的协议描述。
Protocol
我们的协议如下首尔国立大学盆唐医院的机构动物护理和使用委员会（IACUC），其批准的所有在该协议提出的动物的程序及动物护理方法的指导方针。该IACUC是完全符合指南的实验室动物的护理和使用（2011）第八版。所有的程序都用坚持协会的研究在视觉与眼科声明对动物眼科和视觉研究使用动物的指导进行。单独的笼子被用于房屋的兔子。另外的手术或制剂在进行该实验前（ 即 ，灭菌）…
Representative Results
用来进行的兔眼用无菌技术感兴趣的药物的玻璃体内注射的过程示于图1。将处理过的眼睛在预定的时间去核并储存在-80℃。对于分析，三个室，房水，玻璃体和视网膜/脉络膜，从冷冻兔眼分离，如在图2中表现出来。所述隔室的样品用于ELISA制备。与第二抗体温育后，光密度在96孔板，其中包含了玻璃体内注射后收集在多个时间点是从三个隔为标?…
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 |
References
- Urtti, A. Challenges and obstacles of ocular pharmacokinetics and drug delivery. Adv Drug Deliv Rev. 58, 1131-1135 (2006).
- Geroski, D. H., Edelhauser, H. F. Drug delivery for posterior segment eye disease. Invest Ophthalmol Vis Sci. 41, 961-964 (2000).
- Ghate, D., Edelhauser, H. F. Ocular drug delivery. Expert Opin Drug Deliv. 3, 275-287 (2006).
- Del Amo, M. E., Urtti, A. Current and future ophthalmic drug delivery systems. A shift to the posterior segment. Drug Discov Today. 13, 135-143 (2008).
- Avery, R. L., et al. Intravitreal injection technique and monitoring: updated guidelines of an expert panel. Retina. 34, S1-S18 (2014).
- Kim, Y. C., Chiang, B., Wu, X., Prausnitz, M. R. Ocular delivery of macromolecules. J Control Release. 190, 172-181 (2014).
- Group, C. R., et al. Ranibizumab and bevacizumab for neovascular age-related macular degeneration. N Engl J Med. 364, 1897-1908 (2011).
- Campochiaro, P. A., et al. Sustained benefits from ranibizumab for macular edema following central retinal vein occlusion: twelve-month outcomes of a phase III study. Ophthalmology. 118, 2041-2049 (2011).
- Brown, D. M., et al. Ranibizumab for macular edema following central retinal vein occlusion: six-month primary end point results of a phase III study. Ophthalmology. 117, 1124-1133 (2010).
- Diabetic Retinopathy Clinical Research Network. Aflibercept, bevacizumab, or ranibizumab for diabetic macular edema. N Engl J Med. 372, 1193-1203 (2015).
- McCannel, C. A. Meta-analysis of endophthalmitis after intravitreal injection of anti-vascular endothelial growth factor agents: causative organisms and possible prevention strategies. Retina. 31, 654-661 (2011).
- Meyer, C. H., et al. Incidence of rhegmatogenous retinal detachments after intravitreal antivascular endothelial factor injections. Acta Ophthalmol. 89, 70-75 (2011).
- Del Amo, E. M., Urtti, A. Rabbit as an animal model for intravitreal pharmacokinetics: Clinical predictability and quality of the published data. Exp Eye Res. 137, 111-124 (2015).
- Hughes, P. M., Krishnamoorthy, R., Mitra, A. K. Vitreous disposition of two acycloguanosine antivirals in the albino and pigmented rabbit models: a novel ocular microdialysis technique. J Ocul Pharmacol Ther. 12, 209-224 (1996).
- Ahn, J., et al. Pharmacokinetics of Intravitreally Injected Bevacizumab in Vitrectomized Eyes. J Ocul Pharmacol Ther. , (2013).
- Park, S. J., et al. Intraocular pharmacokinetics of intravitreal vascular endothelial growth factor-Trap in a rabbit model. Eye (Lond). 29, 561-568 (2015).
- Jager, R. D., Aiello, L. P., Patel, S. C., Cunningham, E. T. Risks of intravitreous injection: a comprehensive review. Retina. 24, 676-698 (2004).
- Durairaj, C., Shah, J. C., Senapati, S., Kompella, U. B. Prediction of vitreal half-life based on drug physicochemical properties: quantitative structure-pharmacokinetic relationships (QSPKR). Pharm Res. 26, 1236-1260 (2009).
- Ahn, S. J., et al. Intraocular pharmacokinetics of ranibizumab in vitrectomized versus nonvitrectomized eyes. Invest Ophthalmol Vis Sci. 55, 567-573 (2014).
- Mochizuki, K., et al. Intraocular kinetics of ceftazidime (Modacin). Ophthalmic Res. 24, 150-154 (1992).
- Bakri, S. J., et al. Pharmacokinetics of intravitreal ranibizumab (Lucentis). Ophthalmology. 114, 2179-2182 (2007).
- Kondo, T., Miura, M., Imamichi, M. Measurement method of the anterior chamber volume by image analysis. Br J Ophthalmol. 70, 668-672 (1986).
- Toris, C. B., Yablonski, M. E., Wang, Y. L., Camras, C. B. Aqueous humor dynamics in the aging human eye. Am J Ophthalmol. 127, 407-412 (1999).
- Remtulla, S., Hallett, P. E. A schematic eye for the mouse, and comparisons with the rat. Vision Res. 25, 21-31 (1985).
- Barza, M., Zak, O., Sande, M. A. Animal models in evaluation of chemotherapy of ocular infections. Experimental Models in Antimicrobial Chemotherapy. , 187-211 (1986).
- Hughes, A. A schematic eye for the rat. Vision Res. 19, 569-588 (1979).
- Maurice, D. M., Mishima, S. . Ocular pharmacokinetics. 69, (1984).
- Greenbaum, S., Lee, P. Y., Howard-Williams, J., Podos, S. M. The optically determined corneal and anterior chamber volumes of the cynomolgus monkey. Curr Eye Res. 4, 187-190 (1985).
- Ruby, A. J., Williams, G. A., Blumenkranz, M. S. Vitreous humor. Foundations of Clinical Ophthalmology. , (2006).
- Jaffe, G. J., Ashton, P., Andrew, P. . Intraocular Drug Delivery. , (2006).
- Iyer, M. N., et al. Clearance of intravitreal moxifloxacin. Invest Ophthalmol Vis Sci. 47, 317-319 (2006).
- Fauser, S., et al. Pharmacokinetics and safety of intravitreally delivered etanercept. Graefes Arch Clin Exp Ophthalmol. 242, 582-586 (2004).
- Scholes, G. N., O’Brien, W. J., Abrams, G. W., Kubicek, M. F. Clearance of triamcinolone from vitreous. Arch Ophthalmol. 103, 1567-1569 (1985).
- Stastna, M., Behrens, A., McDonnell, P. J., Van Eyk, J. E. Analysis of protein composition of rabbit aqueous humor following two different cataract surgery incision procedures using 2-DE and LC-MS/MS. Proteome Sci. 9, 8 (2011).
- Sinapis, C. I., et al. Pharmacokinetics of intravitreal bevacizumab (Avastin(R)) in rabbits. Clin Ophthalmol. 5, 697-704 (2011).
- Gaudreault, J., Fei, D., Rusit, J., Suboc, P., Shiu, V. Preclinical pharmacokinetics of Ranibizumab (rhuFabV2) after a single intravitreal administration. Invest Ophthalmol Vis Sci. 46, 726-733 (2005).
- Maurice, D. Review: practical issues in intravitreal drug delivery. J Ocul Pharmacol Ther. 17, 393-401 (2001).
- Laude, A., et al. Intravitreal therapy for neovascular age-related macular degeneration and inter-individual variations in vitreous pharmacokinetics. Prog Retin Eye Res. 29, 466-475 (2010).
- Christoforidis, J. B., Carlton, M. M., Knopp, M. V., Hinkle, G. H. PET/CT imaging of I-124-radiolabeled bevacizumab and ranibizumab after intravitreal injection in a rabbit model. Invest Ophthalmol Vis Sci. 52, 5899-5903 (2011).
- Sangwan, V. S., Pearson, P. A., Paul, H., Comstock, T. L. Use of the Fluocinolone Acetonide Intravitreal Implant for the Treatment of Noninfectious Posterior Uveitis: 3-Year Results of a Randomized Clinical Trial in a Predominantly Asian Population. Ophthalmol Ther. 4, 1-19 (2015).
- Bajwa, A., Aziz, K., Foster, C. S. Safety and efficacy of fluocinolone acetonide intravitreal implant (0.59 mg) in birdshot retinochoroidopathy. Retina. 34, 2259-2268 (2014).
- Sanford, M. Fluocinolone acetonide intravitreal implant (Iluvien(R)): in diabetic macular oedema. Drugs. 73, 187-193 (2013).
- Haller, J. A., et al. Dexamethasone intravitreal implant in patients with macular edema related to branch or central retinal vein occlusion twelve-month study results. Ophthalmology. 118, 2453-2460 (2011).
- Boyer, D. S., et al. Three-year, randomized, sham-controlled trial of dexamethasone intravitreal implant in patients with diabetic macular edema. Ophthalmology. 121, 1904-1914 (2014).
- Patel, S. R., et al. Targeted administration into the suprachoroidal space using a microneedle for drug delivery to the posterior segment of the eye. Invest Ophthalmol Vis Sci. 53, 4433-4441 (2012).
- Makadia, H. K., Siegel, S. J. Poly Lactic-co-Glycolic Acid (PLGA) as Biodegradable Controlled Drug Delivery Carrier. Polymers (Basel). 3, 1377-1397 (2011).
Cite This Article
Ahn, S. J., Hong, H. K., Na, Y. M., Park, S. J., Ahn, J., Oh, J., Chung, J. Y., Park, K. H., Woo, S. J. Use of Rabbit Eyes in Pharmacokinetic Studies of Intraocular Drugs. J. Vis. Exp. (113), e53878, doi:10.3791/53878 (2016).