Miho Tanaka^*1, Takafumi Saeki¹, Hiroko Yano¹, Manabu Nozaki ¹, Kazumi Fukagawa^*2,4 Hiroshi , Fujishima^*3.4

1. Overseas Drug Development Group, Research and Development Division, Healthcare Business Unit, Kobayashi Pharmaceutical Co., Ltd., Osaka, Japan

2. Ryogoku Eye Clinic, Tokyo, Japan

3. Department of Ophthalmology, School of Dental Medicine, Tsurumi University, Kanagawa, Japan

4. Keio Allergy Center, Keio University Hospital, Tokyo, Japan

^*Corresponding author: Miho Tanaka,Overseas Drug Development Group, Research and Development Division, Healthcare Business Unit, Kobayashi Pharmaceutical Co., Ltd., Osaka, Japan

Abstract

Background: Cedar pollen-associated allergic inflammation is caused by antigenic proteins released from the pollen wall and the endosporium. The rupture of the pollen wall augments the release of antigenic proteins.Eye washing,particularly with eyewash agents that suppress pollen wall rupture, is one of the most useful preventive measures against pollinosis.In this study, we investigated the effects of the acid level and composition of over-the-counter eyewash solutions on the pollen rupture rate and antigenic protein elution.Methods: Japanese cedar pollen was suspended in various test solutions and the rupture rate was calculated using lightmicroscopy. Cry j 1 and Cry j 2 levels were measured in the supernatant fluid. Test solutions included artificial tears, eyewash, leporine lacrimal fluid,as well as solutions with different pH levels and ingredients that were prepared to determine the factors contributing to pollen rupture.Results: The pollen rupture rate was lower when suspended in eyewash than in artificial tears or leporine lacrimal fluid. The addition of potassium chloride and sodium chloridewas demonstrated to increase the pollen rupture rate.Conclusions: Salts such as sodium chloride and potassium chloride, as well as pH, were involved in pollen wall rupture. The eyewash used in this study had a low pH and did not contain any salts, thus presenting a decreased pollen rupture rate and release of antigenic proteins. Therefore, our results suggest that the use of eyewash suppresses pollen rupture and eliminates cedar pollen.

Keywords: cedar pollen, eyewash, ingredient, lacrimal fluid, pollen wall rupture

Introduction

Antigenic proteins released from the pollen wall and endosporium infiltrate the conjunctival epithelium and bridge the 2 IgE antibody molecules on mastocytes underneath the conjunctival epithelium.This causesthe degranulation of mastocytesand release of chemical mediators,including histamine, which is followed by cedar pollen-induced allergic inflammation. Cry j 1 (molecular weight: 45–50 kDa), which is localized in the pollen wall, and Cry j 2 (molecular weight: 37 kDa), which is localized in starch granules in the pollen, have been identified as the major cedar pollen antigens [1,2]. Their elution is affected by the fluid wherethe pollen granules exist. Antigen elution results from the rupture of the thinnest part of the pollen wall after water absorption. A previous study reported that the rupture rate may increase depending onthepresence of proteins, mucins, and lysozymes in nasal discharge. Furthermore, the physicochemical properties such as alkaline conditions, temperatures close to that of the body, and the presence of in vivo secretioncreate an environment that promotes pollen rupture and elution of antigenic proteins [3].

Similar resultswere reported regarding the relationship between lacrimal fluid and pollen rupture. Astudy on the effects of four eyedrop products for the treatment of allergic conjunctivitis and associated eye dryness on pollen wall ruptureshowed that these eye-drops suppressed antigen elution inlacrimal fluid [4].

Among the body parts exposed to pollens, the eyes are covered with lacrimal fluid and frequently have direct contact with the external environment.Thus, the eyes are more prone to develop symptoms. Although washing out cedar pollen from the eyes to suppress itchinessis one of the useful preventive measures against hay fever [5], agents that not only eliminatethe pollen but also suppress pollen wall rupture are preferable.

We aimed to investigate the effects ofthe acidity and composition of over-the-counter (OTC) eyewash products on pollen wall ruptureand the elution of the major antigenic proteins Cry j 1 and Cry j 2. Furthermore, since as the lacrimal fluid promotes immediatepollen wall rupture and antigenic protein elution, we investigated the importance of washing out pollen residue from the eyes in an in vitro study by measuring Cry j 1 and Cry j 2 levels in the pollen residue after the rupture.

Methods

The following test solutions were used (Table 1): OTC artificial tears (solution 1) atpH7.1, and Eyebon®AL (solution 2) OTC eyewash atpH6.0 (Kobayashi Pharmaceutical Co., Ltd., Osaka, Japan).In addition, an eyewash solution adjusted to pH7.1 with NaOH (solution 3) was prepared to investigate the effect of pH.

To investigate the effects of the ingredients contained in artificial tears and eyewash, potassium chloride and sodium chloride were added to eyewash solution(solutions 4 and 5, respectively).Potassium chloride/sodium chloride (solution 7), dipotassium glycyrrhizinate (solution 8), sodium chondroitin sulfate (solution 9), chlorpheniramine maleate (solution 10), epsilon aminocaproic acid (solution 11), polysorbate 80 (solution 12), and polysorbate 80/l-menthol/d-borneol (solution 13) were added to a basic boric acid solution (solution 6, Table 1). The pH of solutions that were numbered 4 to 13 was adjusted to 7.1.

Rabbit lacrimal fluid (pH 7.8) was obtained from the Drug Safety Testing Center Co., Ltd. (Saitama, Japan). They were collected from forced open eyes using a lid retractor under general anesthesia was used as a control.

Measurement of pollenrupture rate

Japanese cedar pollen (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan)was added to each test solutionat a concentration of10mg/mL and was then incubated at 37°C. After 5, 30, and 60min, 10 μL of each test solution was placed on a glass slide and covered with a cover slip. The number of ruptured pollen grains as well as the total number of pollen grains (≥200) were counted using a light microscope (ECLIPSE E600; Nikon, Tokyo, Japan). The pollen rupture rate was calculated using the following equation:pollen rupture rate (%) = number of ruptured pollen/total number of pollen grains×100.The meansof thethree repeated procedures were used.

Microscopic observations of ruptured pollen

Microscopic images(100× magnification)were captured after 5 and 60 minusing a light microscope (ECLIPSE E600; Nikon).

Quantification of antigenic proteins

Japanese cedar pollen (FUJIFILM Wako Pure Chemical Corporation) was added to each test solution at a concentration of10 mg/mL to measure Cry j 1, and 200 mg/mL to measure Cry j 2,before incubating at 37°C. The solutions were immediately cooled using ice water after 5 min. The solutions were then centrifuged at 11,000 × g at 4°C for 3 minbefore removing the supernatant, which was also centrifuged. The supernatant was analyzed using Cry j 1 and Cry j 2 enzyme-linked immunosorbent assay (ELISA) kits (Institute of Immunology Co., LTD, Tokyo, Japan) by a sandwich assay.

Extraction of antigenic proteins contained in pollen residue after rupture

Japanese cedar pollen (FUJIFILM Wako Pure Chemical Corporation) was added to an artificial tear solution at a concentration of10 mg/mL to measure Cry j 1, and at 200 mg/mL to measure Cry j 2,before incubating at 37°C for 3 h. The pollen rupture rate was measured to confirm that the percentageof ruptured pollen was ≥95%. The solutions were immediately cooled using ice water and centrifuged at 11,000 × g and 4°C for 3 min before removing the supernatant, which was stored at 4°C as the post-rupture elution solution. Toremove the remaining pollen residue, the solution was washed three times via centrifugation with artificial tears, disrupted with an ultrasonic disruptor Handy Sonic UR-20P(Tomy Seiko Co.,Ltd., Tokyo, Japan) for 15 min, and centrifuged at 11,000 × g and 4°C for 3 minbefore removing the supernatant, which was stored at 4°C as the disrupted pollen residue solution. The post-rupture elution solution and the disrupted pollen residue solution were analyzed using Cry j 1 and Cry j 2 ELISA kits (Institute of Immunology Co., LTD) by a sandwich assay.

Statistical analysis

Rate tests were conductedto assess pollen rupture rates, and unpaired Student's t-tests were performed to compareantigenic protein levels. The level of significance for statistical analyses was 5% (two-sided).

Results

Micrographs of pollen rupture in artificial tear (pH7.1) and eyewash (pH6.0) solutionsare shown in Figure1.The changes observed in cedar pollen rupture ratesin eyewash(pH6.0), artificial tears(pH7.1), and leporine lacrimal fluid(pH7.8)over time areshown in Figure 2.The rupture rates at 5, 30, and 60 min were 44.6%, 81.6%, and89.9%, respectively,in artificial tears; 68.5%, 80.8%, and 90.3%, respectively, inleporine lacrimal fluid;and only 1.6%, 4.0%, and 7.1%, respectively, in eyewash.

Figure 1.Micrographs showing pollen in each solution.(A) Few pollen ruptures are observed after 5 min in eyewash solution (pH 6.0). (B) Limited pollen rupture is observed after 60 min in eyewash solution (pH 6.0). (C) Limited pollen rupture is observed after 5 min in artificial tear solution (pH 7.1). (D) Most of the pollens rupture after 60 min in artificial tear solution (pH 7.1).

Figure 2.Change in cedar pollen rupture rates. The rupture rate is significantly lower in eyewash at pH 6.0 (●) between 5 and 60 min (especially, 60 min) compared with that in artificial tear solution at pH 7.1 (▲) and leporine lacrimal fluid at pH 7.8 (○); n = 3, *p<0.05.