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复方酮替芬滴眼剂对实验过敏性结膜炎的疗效观察
http://www.100md.com 《眼科研究》 2000年第3期
     作者:谢雷克 张俊杰 陈祖基 徐岩

    单位:河南省科研究所,郑州450003

    关键词:酮替芬;微量激素;过敏性结膜炎

    眼科研究000301

    摘要 目的 观察复方酮替芬滴眼剂(含0.1%酮替芬及0.0005%地塞米松)对实验大鼠急性过敏性结膜炎的治疗作用。 方法 雄性Wistar大鼠27只随机分为3组(复方酮替芬、0.1%酮替芬及地塞米松组),每组9只,各预先腹腔注射卵清蛋白致敏液(100μg卵清蛋白及20mg硫酸铝钾溶于1ml磷酸盐缓冲液,pH7.4),两周后以10%卵清蛋白液(溶于磷酸盐缓冲液)10μl滴眼攻击,引起结膜速发型过敏反应。攻击前15min以1mol.l-1DL-二巯苏糖醇20μl作眼部预处理,临攻击时静脉注射Evans蓝(EB)溶液(约2mg/100g);攻击前60,45,30,15min及攻击后15,30min各组1眼给药,对侧眼为溶媒。攻击后1h处死所有动物,测定各眼EB渗出量,进行比较,计算药物抑制率。 结果 各组药物治疗眼EB外渗量均低于溶媒治疗眼,复方酮替芬组和0.1%酮替芬组显示统计学有显著意义。复方酮替芬抑制率为53.98%±15.57%,显著高于0.1%酮替芬(27.91%±28.36%,P=0.028)及0.0005%地塞米松(11.90%±34.16%,P=0.004)。 结论 结果提示复方酮替芬滴眼剂具有良好的抗过敏作用。
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    分类号 R777.3

    Treatment of experimental allergic conjunctivitis with compound ketotifen eye drops

    Xie Leike,Zhang Junjie,Chen Zuji,Xu Yan

    (Henan Institute of Ophthalmology,Zhengzhou 450003,China)

    Abstract Objective:To examine the effect of compound ketotifen eye drops(CKF,containing 0.1% ketotifen and 0.000 5% dexamethasone) on experimental acute allergic conjunctivitis in rats.Methods:27 male Wistar rats were immunized by intraperitoneal injection of egg albumin and were randomized into three groups: CKF,0.1% ketotifen (KF) and 0.000 5% dexamethasone (DM) Rats in each group received the respective eye drops in one eye and vehicle in the other eye 60,45,30,15 min before and 15,30 min after challenge.Immediately before challenge,the rats were injected intravenously with Evans Blue(EB).The challenge was performed by topical instillation of egg albumin,and 60 min later,the animals were killed and the dye was extracted from the eyes.The intensity of EB extravasation was determined by spectrophotometry at 620nm and inhibitory rates were calculated.Results:CKF,KF and DM suppressed EB extravasation significantly.The inhibitory rate was 53.98%±15.57% for CKF,27.91%±28.36% for KF and 11.90%±34.16% for DM.CKF was more effective than both KF and DM on inhibiting dye vascular leakage in the eyes (P=0.028,0.004,respectively).Conclusion:These data clearly indicate that the compound of ketotifen eye drops has potential as a topical ocular formulation for anti-allergic disease.
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    Key words ketotifen micro-corticosteroid allergic conjunctivitis

    Ketotifen is a potential anti-allergic agent.It has been showed that ketotifen was greatly effective and well tolerated for clinical treatments of seasonal and perennial rhinitis,bronchial asthma and atopic dermatitis[1].In the present study,we evaluated an effectiveness of compound ketotifen eye drops prepared with ketotifen (0.1%) and micro-dexamethasone(0.0005%) on experimental ocular immediate hypersensitivity as compared to each drug administered alone.
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    We used a model of topically induced ocular anaphylaxis in rats,which were immunized and challenged with egg albumin(EA) in the study.This ocular model was quite similar to the human conjunctival allergic disease.The severity of the allergic reaction and efficacy of anti-allergic drugs were assessed objectively by quantifying evans blue(EB) extravasation in ocular tissue.

    MATERIALS & METHODS

    Animals Twenty seven male Wistar rats,weighing 250~300g,were purchased from Henan Experimental Animal Center.
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    Drugs and reagents Ketotifen(Shanghai),Dexamethasone(Shanghai),DL-Dithiothreitol (Shanghai),Egg Albumin (Sigma),and Evans Blue(TCI,Japan) were used.Solutions for making eye drops,including compound ketotifen (CKF),0.1% ketotifen (KF),and 0.0005% dexamethasone(DM),were prepared using the same vehicle.

    Instruments Spectrophotometer UV/V 7530-G(Shanghai,China)and electronic analytical balance AE 260(Mettler,Switzerland)were used.

, http://www.100md.com     Immunization and challenge All rats were injected intraperitoneally with 100μg EA plus 20mg of alum in 1ml of phosphate buffered saline(PBS),pH7.4 for immunization.14d later,rats were challenged topically with 10μl EA 10% in PBS.To obtain a remarkable ocular reaction,20μl of 1mol/L DL-Dithiothreitol,a mucolytic agent,in PBS(freshly prepared before use)was applied to each eye 15min before topical challenge as described by Calonge et al[2] and Trocme et al[3].EB(2mg/100g) in 1ml of PBS was intravenously injected immediately prior to challenge.Animals were anesthetized with intraperitoneal 5% chloral hydrate(about 0.2ml/100g).
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    Drug administration Animals were divided into three groups(CKF,KF and DM)of nine each.One eye of each animal received one drop of relevant drug and the fellow eye vehicle 60,45,30,15min before and 15,30min after challenge.

    Evaluation procedure All rats were killed by exsanguination 1h after challenge.Globes and adnexa were exenterated,weighed,and then immersed in an extracting solution of 5ml sodium sulfate(0.5%) plus acetone(3∶7,v/v),shaken strongly,and kept at room temperature.24h later,solutions were centrifuged at 1000rpm for 10min.The color intensity of supernatant was detected spectrophotometrially at 620nm.
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    Standard curve was performed to transform absorbance unit in microgram of EB per milliliter of solution (μg/ml) and EB extravasation in ocular tissues was calculated.Drug efficacy were expressed as percent inhibition of the reaction on the vehicle treated eye.IR(%)=(CV-CD)/CV;IR:drug inhibitory rate;CD is EB content in drug treated eye and CV in the untreated control eye.Data were analyzed by paired and unpaired,two-tailed student t-test.A P value of 0.05 or less was considered significant.
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    RESULTS

    In three groups,drug-applied eyes exhibited lower evans blue extravasation [(6.50±2.34)μg/ml.g for CKF,(6.76±2.32)μg/ml.g KF and (11.22±3.24)μg/ml.g DM]than those of the relevant vehicle-applied eyes[(14.94±4.49)μg/ml.g,(10.46±5.15)μg/ml.g and (13.74±3.87)μg/ml.g,respectively].The statistical differences between drug-and vehicle-treated eyes were significant in CKF (P=0.0007) and KF (P=0.043) groups.(See Tab.1)
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    Tab.1 Evans blue(EB)extravasation in ocular tissues Drugs

    EB extravasation in eyes (μg/ml.g)±s

    P value

    1

    2

    3

    4

    5

    6
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    7

    8

    9

    CKF

    4.38

    4.68

    7.20

    5.15

    6.27

    7.08

    11.90

    7.31

    4.58
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    6.50±2.34

    Veh

    23.97

    7.32

    14.26

    11.95

    16.83

    15.98

    16.89

    14.29

    13.02

    14.94±4.49

    0.0007
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    KF

    4.46

    4.75

    4.56

    4.95

    10.44

    7.96

    6.33

    7.54

    9.88

    6.76±2.32

    Veh

    5.50
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    3.65

    9.09

    19.06

    15.27

    6.81

    7.74

    12.35

    14.72

    10.46±5.15

    0.043

    DM

    8.21

    6.16
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    13.45

    9.62

    12.53

    10.48

    17.43

    12.24

    10.82

    11.22±3.24

    Veh

    6.28

    17.57

    15.73

    11.20
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    19.38

    14.90

    11.63

    14.34

    12.67

    13.74±3.87

    0.16

    CKF:Compound ketotifen eye drops;KF:0.1% Ketotifen eye drops;DM:0.0005% Dexamethasone eye drops;Veh:Vehicle

    EB leakage significantly decreased by 53.98%±15.57% in CKF treated-eyes,when compared with either KF(27.91%±28.36%,P=0.028)or DM(11.90%±34.16%,P=0.004) treated eyes.The differences were statistically significant.(See Tab.2)
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    Tab.2 Inhibition rates of the drugs on evans blue leakage in eyes(%)

    Inhibition of EB extravasation in eyes±s

    1

    2

    3

    4

    5

    6

    7

    8
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    9

    CKF

    81.37

    36.06

    49.51

    56.90

    62.74

    55.69

    29.54

    48.84

    64.82

    53.98±15.57

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    18.91

    -30.14

    49.83

    74.03

    31.63

    16.89

    18.22

    38.95

    32.88

    27.91±28.36

    DM

    -30.73
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    64.94

    14.49

    14.15

    35.34

    29.62

    -49.87

    14.64

    14.52

    11.90±34.16

    CKF had a significantly high inhibition rate,when compared with either KF (P=0.028)or DM(P=0.004).
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    DISCUSSION

    Allergic conjunctivitis is a response resulting from a reaction of allergen with IgE antibodies on mast cells in the conjunctival stroma.This reaction triggers a cascade of events resulting in the degranulation of mast cells,and releasing chemical mediators(e.g.,histamine,prostaglandins and leukotrienes) into the conjunctival stroma and tears.These mediators cause vasodilation and increasing in vascular permeability,which induce ocular itching,burning,conjunctival injection,chemosis and limbal hyperemia.[4,5]
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    Corticosteroids,histamine H1-receptor antagonists,mast cell stabilizers and non-steroidal anti-inflammatory drugs,which exert antiallergenic effects by different pharmacological mechanism,have been used to treat anaphylactic ocular disorders[4,5].

    Ketotifen is the second generation histamine H1-receptor antagonist and also possesses stabilizing mast cell activity,which prevents the mast cell degranulation and chemical mediators release in the conjunctiva,and reduces the ocular symptoms and signs[1,6].Hagihara[7] has clinically evaluated oral ketotifen for the treatment of 22 cases with efficacy rate of 80%.No serious drug side effect was found in his study.Mikuni[8] determined quantitatively the therapeutic effect of 0.08% ketotifen eye drops in 10 volunteers suffering from seasonal cedar pollinosis provocated during a quiescent stage.The efficacy rate of the preparation was found to be 80%.
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    It is well-known that corticosteroids have a pronounced antianaphylactic action.The application of topical corticosteroids is now an indispensable modality in the treatment of severe ocular allergic conditions.Unfortunately,topical ocular corticosteroids may cause serious adverse effects,such as glaucoma,cataract formation,diminished epithelial wound healing,and the risk of ocular infection,which make long-term corticosteroid use invadisable.However,there was evidence that an administration of micro-dexamethasone could not only offer its desired pharmacological activity,but also minimize the adverse effects[9].
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    In order to avoid the corticosteroid side effects and retain the antiallergenic effect,we conducted micro-dexamethasone and prepared topical ophthalmic formulation with 0.1% ketotifen and 0.0005% dexamethasone in this study.The micro-dexamethasone-applied eyes revealed lower EB extravasation than that of the control eyes,although no statistically significant difference was revealed between the two treated groups.The combination of 0.1% ketotifen and 0.0005% dexamethasone were a synergic effect on ocular anaphylaxis,inhibiting remarkably ocular evans blue extravasation in the model of acute allergic conjunctivitis with an inhibitory ratio of 53.98%,which was considerably higher than that of each drug used alone(27.91% and 11.90% for 0.1% ketotifen and 0.0005% dexamethasone,respectively).The differences were statistically significant.The results of this study suggested the potential of compound ketotifen eye drops in the treatment of allergic conjunctivitis.
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    REFERENCES

    1,Grant SM,Goa KL,Fitton A,et al.Ketotifen.A review of its pharmacodynamic and pharmacokinetic properties,and therapeutic use in asthma and allergic disorders.Drugs,1990,40(3)∶412

    2,Calonge MC,Pastor JC,Herreras JM,et al.Pharmacologic modulation of vascular permeability in ocular allergy in the rat.Invest Ophthalmol Vis Sci,1990,31(1)∶176

    3,Trocme SD,Trocme MC,Bloch KJ,et al.Topically induced ocular anaphylaxis in rats immunized with egg albumin.Ophthalmic Res,1986,18(1)∶68
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    4,Jaanus SD,Hegeman SL,Swanson MW.Antiallergy drugs and decongestants.In:Bartlett JD,Jaanus SD,eds.Clinical Ocular Pharmacology.3rd ed.Boston:Butterworth-Heinemann,1995.337-53

    5,Abelson MB,Sloan JE,Mooshian M.Basis of ocular allergy and mechanisms for successful management.Ophthalmoic Practice,1993,11(6)∶278

    6,Heyman SN,Karmeli F,Brezis M,et al.The effect of ketotifen on nitric oxide synthase activity.Br J Pharmacol,1997,120(8)∶1545
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    7,Hagihara M,Sakashita M,Inoue A.Clinical evaluation of ketotifen (zaditen) of the treatment of allergic conjunctival disorders.Folia Ophthalmol Jpn,1988,39∶392

    8,Mikuni L,Sato K,Togawa K,et al.A quantitative tear fluids determination of therapeutic efficacy for allergic conjunctivitis.Tokai J Exp Clin Med,1984,9(1)∶35

    9,Chen ZJ.Practical Ocular Pharmacology.Beijing:Scientific & Technological Press of China,1993.212-34

    (收稿:1999-09-15 修回:2000-01-08), 百拇医药