经鼓膜药物治疗耳毒性的临床研究
Summary
本文提出了一种通过鼓膜入耳蜗进行药物局部化管理的技术。通过这条途径运送药物不会影响化疗药物如顺铂的抗癌功效。
Abstract
保护剂治疗药物性耳毒性的系统管理受到限制, 因为这些保护剂可能会干扰初级药物的化疗效果。这种情况尤其适用于药物顺铂, 其抗癌作用因抗氧化剂而减弱, 为听力丧失提供了足够的保护。其他当前或潜在的 otoprotective 代理可能会造成类似的问题, 如果系统管理。将各种生物制品或保护剂直接应用于耳蜗, 可以使这些药物的高水平在局部具有有限的系统性副作用。在本报告中, 我们展示了一种跨鼓膜的方法, 提供各种药物或生物试剂的耳蜗, 这应该加强对耳蜗的基础科学研究, 并提供一个简单的方法指导使用 otoprotective 剂在诊所。本报告详细介绍了一种跨鼓膜药物传递方法, 并举例说明了该技术如何成功地应用于实验动物治疗顺铂耳毒性。
Introduction
外周听觉系统非常敏感的药物, 如顺铂和氨基糖苷类抗生素。顺铂是一种广泛使用的化疗剂, 用于治疗各种实体肿瘤, 如卵巢, 睾丸, 头颈癌。使用这种药物的耳毒性是剂量限制和相当常见的, 影响75-100% 的患者治疗1。其他药物, 如卡铂和奥沙利铂, 已成为替代顺铂2,3,4,5, 但它们的用处仅限于少数癌症。
早期研究表明活性氧 (ROS) 对顺铂和氨基糖苷类产生的耳毒性有重要作用。随后的研究表明, NOX3 异构体氧化酶是耳蜗中 ROS 的主要来源, 并由顺铂6,7激活。ROS 的产生危及细胞的抗氧化缓冲能力, 导致细胞膜脂质过氧化增加8。此外, 顺铂增加了羟基自由基产生剧毒醛 4-hydroxynonenal (4-他), 一个引发细胞死亡9,10。根据这些发现, 对治疗顺铂耳毒性的几种抗氧化剂进行了研究。这些包括 n-乙酰半胱氨酸 (NAC), 硫代硫酸钠 (STS), 汀和 d-蛋氨酸。然而, 抗氧化剂治疗的一个主要关注是, 这些抗氧化剂可以降低顺铂化疗的功效时, 系统地管理11通过与硫醇组的相互作用的抗氧化剂分子。
鉴于这些问题的抗氧化剂治疗, 本研究的目的是检查传递抗氧化剂和其他药物的耳蜗, 以减少听力损失的跨鼓膜路线。下面描述的药物和短干涉 (si) RNA 的跨鼓膜路线似乎特别有希望。
Protocol
雄性大鼠是按照国家卫生研究院的动物使用指南和由南伊利诺伊大学医学院实验室动物护理和使用委员会批准的协议处理的。在药物管理局麻醉前和72小时后, 对大鼠进行听觉脑干反应 (ABR) 检查, 以验证其对鼓膜药物传递的影响。 1. 听觉脑干反应 (ABR) 注意:通过听觉测试设备和软件采集 ABR 测量结果。ABR 代表诱发电位或高频波产生的第八颅神经 …
Representative Results
在顺铂治疗后三天大鼠的 ABR 反应显示阈值显著升高。通过鼓膜 (r) n-phenylisopropyladenosine (r-PIA), 腺苷 A1受体激动剂15, 顺铂前, 这些阈值的升高明显减少。R-PIA 在腺苷 A1受体上的作用的特异性表明, 它是由 8-环戊基-13-dipropylxanthine (DPCPX) 对抗,一个1 受体特定拮抗剂16。这种药物增强顺铂诱发的 ABR ?…
Discussion
通过鼓膜管理途径, 可以将药物和其他药物的局部运送到耳蜗, 如果系统地进行管理, 可能会产生明显的系统性副作用。这种药物管理方法允许药物迅速进入行动地点, 其剂量要比通过系统途径所能达到的要高得多。在这里和发表的结果表明, 通过鼓膜管理的 [R] n-苯基异丙腺苷 (r-PIA) 保护耳蜗从顺铂诱导的外毛细胞损失 (图 1)19和诱导炎症的表现,…
Divulgations
The authors have nothing to disclose.
Acknowledgements
本文所述的工作得到了 RO1 CA166907、NIDCD RO1-DC 002396 和 RO3 DC011621 的支持。
Materials
| Ketathesia (100 mg/ml) 10 ml | Henry Schein | 56344 | Controlled substance |
| AnaSed Injection/Xylazine (20 mg/ml) 20 ml | Henry Schein | 33197 | |
| 2.5 mm disposable ear specula | Welch Allyn | 52432 | |
| Surgical Scope | Zeiss | ||
| 29 G X 1/2 insulin syringe | Fisher Scientific | 14-841-32 | Can be purchased through other vendors |
| cis-Diammineplatinum(II) dichloride | Sigma Aldrich | P4394 | TOXIC – wear proper PPE |
| Harvard 50-7103 Homeothermic Blanket Control Unit | Harvard Apparatus | Series 863 | |
| Excel International 21 G X 3/4 butterfly needle | Fisher | 14-840-34 | Can be purchased through other vendors |
| BSP Single Speed Syringe Pump | Brain Tree Sci, Inc | BSP-99 | |
| Pulse Sound Measurement System | Bruel & Kjaer | Pulse 13 software | |
| High-Frequency Module | Bruel & Kjaer | 3560C | |
| 1/8″ Pressure-field Microphone —-Type 4138 | Bruel & Kjaer | bp2030 | |
| High Frequency Transducer | Intelligent Hearing System | M014600 | |
| Opti-Amp Power Transmitter | Intelligent Hearing System | M013010P | |
| SmartEP ABR System | Intelligent Hearing System | M011110 | |
| Disposable Subdermal EEG Electrodes | CareFusion | 019-409700 | |
| 16% Formaldehyde, Methanol-free | Fisher Scientific | 28908 | TOXIC – wear proper PPE |
| 7 mL Borosilicate Glass Scintillation Vial | Fisher Scientific | 03-337-26 | Can be purchased through other vendors |
| EDTA | Fisher Scientific | BP118-500 | Can be purchased through other vendors |
| Sucrose | Fisher Scientific | S5-500 | Can be purchased through other vendors |
| Tissue Plus OCT Compound | Fisher Scientific | 4585 | |
| CryoMolds (15 mm x 15 mm x 5mm) | Fisher Scientific | 22-363-553 | Can be purchased through other vendors |
| Microscope Slides (25mm x 75mm) | MidSci | 1354W | Can be purchased through other vendors |
| Coverslips (22 x 22 x 1) | Fisher Scientific | 12-542-B | Can be purchased through other vendors |
| Poly-L-Lysine Solution (0.01%) | EMD Millipore | A-005-C | Can be purchased through other vendors |
| HM525 NX Cryostat | Thermo Fischer Scientific | 956640 | |
| MX35 Premier Disposable Low-Profile Microtome Blades | Thermo Fischer Scientific | 3052835 | |
| Wheaton™ Glass 20-Slide Staining Dish with Removable Rack | Fisher Scientific | 08-812 | |
| Super Pap Pen Liquid Blocker | Ted Pella, Inc. | 22309 | |
| Normal Donkey Serum | Jackson Immuno Research | 017-000-121 | Can be purchased through other vendors |
| TritonX-100 | Acros | 21568 | Can be purchased through other vendors |
| BSA | Sigma Aldrich | A7906 | Can be purchased through other vendors |
| Phospho-Stat1 (Ser727) antibody | Cell Signaling | 9177 | |
| VR1 Antibody (C-15) | Santa Cruz | sc-12503 | |
| DyLight 488 Donkey anti Rabbit | Jackson Immuno Research | 711-485-152 | Discontinued |
| DyLight 488 Donkey anti Goat | Jackson Immuno Research | 705-485-003 | Discontinued |
| Rhodamine (TRTIC) Donkey anti Rabbit | Jackson Immuno Research | 711-025-152 | Discontinued |
| ProLong® Diamond Antifade Mountant w/ DAPI | Thermo Fisher | P36971 | |
| (−)-N6-(2-Phenylisopropyl)adenosine | Sigma Aldrich | P4532 | |
| 8-Cyclopentyl-1,3-dipropylxanthine | Sigma Aldrich | C101 | |
| siRNA pSTAT1 | Qiagen | Custome Made | Kaur et al. 201120 |
| siRNA NOX3 | Qiagen | Custome Made | Kaur et al. 201120 |
| Scrambled Negative Control siRNA | Qiagen | 1022076 | Kaur et al. 201120 |
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Citer Cet Article
Sheehan, K., Sheth, S., Mukherjea, D., Rybak, L. P., Ramkumar, V. Trans-Tympanic Drug Delivery for the Treatment of Ototoxicity. J. Vis. Exp. (133), e56564, doi:10.3791/56564 (2018).