Fluoroquinolones對於人類角膜表皮細胞之細胞毒性和作用機轉

The cytotoxicity and mechanism of fluoroquinolones on human corneal epithelial cells

蔡紫筠(Tzu-Yun Tsai)

指導教授 ： 胡芳蓉

國立臺灣大學/醫學院/臨床醫學研究所/碩士(2010年)

https://doi.org/10.6342/NTU.2010.01543

摘要

細菌性角膜炎是常見的眼部感染疾病，若無適當的處理會造成視力損傷或永久失明。然而根據臺大醫院在2004年所發表的十年統計報告中顯示，配戴隱形眼鏡是造成細菌性角膜炎最常見的原因，佔所有病例的44%；其中，Pseudomonas species是最常見的致病菌種，所幸若能及早依據潰瘍檢體的抹片及細菌培養結果，投予合適的抗生素治療，有七成以上的患眼能單純僅以點藥方式痊癒1。
在1960年代Nalidixic acid上市後，衍生自non-fluorinated nalidixic acid的Fluoroquinolones (FQs) 類抗生素開始合成並廣泛被使用；FQs是屬於廣效的抗生素並適合用於單一藥物的治療，作用機轉為抑制細菌去氧核糖核酸旋轉酶（DNA gyrase）及排列異構酶IV（topoisomerase IV），進而影響細菌的去氧核糖核酸形成，最終造成細菌死亡，現今已逐漸為細菌性角膜炎的治療首選藥物。然而隨著抗藥性菌種的增加，以及逐漸被報告對於角膜表皮細胞有不等程度的細胞毒性，FQs在臨床使用上也有其限制。根據國內外文獻發表，FQs眼藥水會造成角膜表皮細胞的細胞毒性，目前所知主要來自於藥水中的防腐劑，benzalkonium chloride（BAC）2,3。因此近年推出的第四代FQs眼藥水製劑宣稱相較於前三代的FQs（levofloxacin、ciprofloxacin、ofloxacin、norfloxacin）有更強的殺菌力，更良好的藥物穿透能力，還可減少抗藥性菌種的產生，特別是第四代的moxifloxacin更強調其中不含防腐劑benzalkonium chloride，可達到最低的角膜上皮毒性。
但根據本實驗室先前針對不同的FQs對於細胞毒性的初步比較，我們採用MTS assay比較市售FQs眼科製劑（commercial FQs ophthalmic solutions）、不同濃度的BAC、以及不含防腐劑的FQs針劑型（standard powder form），初步結果發現細胞毒性在前兩者為Cip＞Gat＝0.005% BAC＞Mox＝Nor＞Ofl＝Lev；而在不含防腐劑的針劑型藥劑中，moxifloxacin則表現最強的細胞毒性4。因此讓我們感興趣的是究竟角膜上皮細胞的細胞毒性是來自於防腐劑，又或與不同的FQs本身藥性也有相關。
另一個令我們感興趣的，FQs在臨床上被發表最多的併發症是造成肌腱病變，特別是針對阿基里斯腱斷裂（Achilles tendon rupture），相關的研究自2000年開始有許多文獻發表5-9。目前認為使用全身性FQs藥物造成肌腱炎或肌腱斷裂主要來自於藥物本身造成細胞的氧化壓力（oxidative stress）增加，影響細胞內粒線體的活性，進一步導致肌腱細胞的凋亡（apoptosis），並且實驗中也發現不同的FQs藥物本身會造成不等程度的傷害6。另一些更新的研究提出FQs藥物本身有促進癌細胞走向細胞凋亡10或是有增進抗癌藥物的作用11。因此我們也希望能進一步了解FQs藥物對於角膜上皮細胞毒性的作用機轉，這在過去文獻中迄今尚未有人發表的。
我們的結果發現， benzalkonium chloride是細胞毒性的來源之一，其作用機轉會導致反應性氧化物的增加，並進一步造成細胞凋亡。但相較於前幾代的fluoroquinolones，最新第四代的moxifloxacin，儘管市面上有不含防腐劑的劑型，但其藥物本身卻會造成更明顯的細胞毒性，並在長時間的浸潤下，其細胞毒性甚至超越benzalkonium chloride，並產生大量反應性氧化物，增加細胞的氧化壓力，在早期明顯導致細胞走向細胞凋亡的途徑。但我們實驗的限制在於這是in vitro的研究，並使用永久的人類角膜上皮細胞株來取代活體的研究，我們期望未來能針對其作用機轉更進一步深入研究，也期望下一步能以活體的研究方式來研究各種fluoroquinolones的毒性差異。我們的實驗結果提供臨床藥物一個相當重的選擇，尤其是運用在角膜表皮受損的個案，不論是受傷或是手術後病人，藥物的細胞毒性可能造成角膜上皮缺損，甚至是角膜穿孔都需要被慎重考慮。

關鍵字

氟化奎林酮 ； 角膜上皮細胞 ； 細胞毒性 ； 細胞凋亡 ； 活性氧

並列摘要

Bacterial keratitis is a common ocular infection and a leading cause of ocular morbidity and blindness worldwide. However, according to our previous report regarding 10-year experience of microbial keratitis at the National Taiwan University Hospital, contact lens-related pseudomonal keratitis was the most common form of microbial keratitis in Taiwan. Effective topical therapy, using fortified antimicrobials or monotherapy commercial ophthalmic preparation selected based on the results of diagnostic corneal smears and cultures, is essential for management of patients with microbial keratitis. Above seventy percent of offending eyes could be successfully treated topically.
Fluoroquinolones (FQs) were derived from the non-fluorinated drug nalidixic acid, which was developed during the early 1960s. FQs are potent antimicrobial agents with a broad antibacterial spectrum and are suitable for monotherapy. They act rapidly by inhibiting bacterial DNA gyrase and topoisomerase IV, which are selective for bacterial cells. FQs are nowadays widely used in clinical practice to treat ocular infection, with intravitreal topical and systemic routes of administration. In fact, several investigators have reported bacterial keratitis resistant to FQs and delays in the healing rate and corneal perforation after administration of FQs preparation. Moreover, many eye drops contain preservatives known to cause severe side effects to the ocular surface. Benzalkonium chloride (BAC) is the preservative currently used the most, and many in vivo and in vitro studies have showed its toxicity on corneal epithelial cells.
Base on our preliminary report regarding to the toxicity toward the corneal epithelial cells between different FQs, the cytotoxicity observed with FQ eye drops seems to be caused mainly by the preservative. However, we found that the new generation of FQs, moxifloxacin, no less cytotoxicity towards corneal epithelial cells than the old generation preparations. Therefore, the purpose of this study is to compare and identify the specific types of cytotoxic damage that might occur with exposure to various FQs and BAC.
Besides, fluoroquinolones antimicrobial agents are widely used in clinical practice as broad-spectrum antibiotics with good bioavailability. However, they have been reported to induce tendinopathy, and the main target is Achilles tendon rupture. All the studies began during 2000 and showed that tendinitis and tendon rupture during treatment with fluoroquinolone antibiotics is thought to be mediated via oxidative stress and mitochondrial damage. Some innovative studies showed fluoroquinolones could modulate cycle cell progression and apoptosis in cancer cells or enhance the function of chemotherapeutic agents in cytotoxicity in tumor-derived cells. Therefore, we want to identify the mechanism of cytotoxicity of fluoroquinolones on human corneal epithelial cell which had not been investigated before in literatures.
Our study showed that the main source of cytotoxicity from commercial fluoroquinolone ophthalmic solutions came from preservatives, benzalkonium chloride. The mechanism is through increased the level of reactive oxygen species, and then induces cell apoptosis. However, the latest four generation of fluoroquinolones, moxifloxacin, also showed obvious cytotoxicity. Significant cell toxicity was found in the moxifloxacin group after 3 hours incubation than benzalkonium chloride, which induced oxidative stress and cell apoptosis in early stage. The limitation of our study was the in vivo setting and the use of immortalized human corneal epithelial cell line instead of primary cultures of corneal epithelial cells. The preliminary results of the our study provided us a new way and more information for further new methods in studying the mechanism of cytotoxicity of fluoroquinolones.

並列關鍵字

Fluoroquinolone ； corneal epithelial cell ； cytotoxicity ； apoptosis ； reactive oxygen species

參考文獻

1. Fong CF, Tseng CH, Hu FR, et al. Clinical characteristics of microbial keratitis in a university hospital in Taiwan. Am J Ophthalmol 2004;137:329-36.

2. Denoyer A, Ossant F, Arbeille B, et al. Very-high-frequency ultrasound corneal imaging as a new tool for early diagnosis of ocular surface toxicity in rabbits treated with a preserved glaucoma drug. Ophthalmic Res 2008;40:298-308.

3. Dutot M, Pouzaud F, Larosche I, et al. Fluoroquinolone eye drop-induced cytotoxicity: role of preservative in P2X7 cell death receptor activation and apoptosis. Invest Ophthalmol Vis Sci 2006;47:2812-9.

4. Tsai TH, Chen WL, Hu FR. Comparison of fluoroquinolones: cytotoxicity on human corneal epithelial cells. Eye 2010;24:909-17.

5. Shakibaei M, Stahlmann R. Ultrastructure of Achilles tendon from rats after treatment with fleroxacin. Arch Toxicol 2001;75:97-102.