0

10

20

30

40

50

60

70

80

90

100

OLO 0.3 OLO 3.0 BEP 0.3 BEP 3.0 ALCAF 0.3 ALCAF 3.0%

IN

HIB

ITIO

NDRUG CONC (mM)

FMLP

IL-5

GM-CSF

* *

*

*

#

#

0

10

20

30

40

50

60

NO DRUG OLO 0.3 OLO 3.0 BEP 0.3 BEP 3.0 ALCAF 0.3 ALCAF 3.0

ED

N R

EL

EA

SE

(%

TO

TA

L)

DRUG CONC (mM)

UNS

FMLP

IL-5

GM-CSF

*

* * *

*

*

*

#

#

Eosinophils pre-incubated with olopatadine released

less EDN in response to FMLP, IL-5 and GM-CSF.

Average % inhibition by olopatadine ranged from 42 to

78% over both concentrations tested (0.3 and 3.0 mM).

Bepotastine had a variable effect, inhibiting EDN

release in 2 subjects at 3.0 mM. However, bepotastine

also increased spontaneous release in those subjects.

Alcaftadine had a variable effect, inhibiting EDN release

in 2 subjects at the 0.3 mM concentration.

Alcaftadine increased EDN release at the 3.0 mM

concentration even from unstimulated eosinophils

Rationale: Eosinophils and eosinophil-derived mediators in tears and

conjunctival biopsy specimens are associated with both acute and chronic

ocular allergic inflammation. Our previously published research demonstrated

that olopatadine inhibited mast cell-mediated and FMLP-stimulated eosinophil

degranulation in vitro. The purpose of this study was to compare the direct

effects of three ocular allergy drugs (olopatadine, alcaftadine, and bepotastine)

on eosinophil degranulation in response to allergic cytokines and FMLP in vitro.

Methods: Peripheral blood eosinophils were pre-incubated with either media,

olopatadine, alcaftadine, or bepotastine (0.3 and 3 mM) and challenged with

either media, IL-5, GM-CSF, or FMLP for 4 hrs. Supernates were harvested and

eosinophil derived neurotoxin (EDN) was measured by commercial ELISA as an

indicator of degranulation.

Results: Olopatadine pre-treatment (both concentrations) resulted in complete

inhibition of EDN release from eosinophils challenged with IL-5, GM-CSF and

FMLP. Pre-treatment with alcaftadine and bepotastine (both concentrations)

failed to inhibit EDN release in response to any of the stimuli, and, in the case

of alcaftadine (3 mM), enhanced EDN release from both unstimulated and

stimulated eosinophils.

Conclusions: The ability of olopatadine to inhibit eosinophil degranulation in

response to allergic cytokines may contribute to its proven efficacy in the

treatment of ocular allergic inflammation.

.

Ellen B. Cook‡#, James L. Stahl‡#, Elizabeth A. Schwantes*, Neal P. Barney‡#, and Sameer K. Mathur*

‡Department of Ophthalmology & Visual Sciences, #McPherson Eye Research Institute; *Division of Allergy, Pulmonary, and Critical Care Medicine, Department of Medicine

University of Wisconsin School of Medicine and Public Health, Madison, WI 53792

Abstract

Acknowledgments

Clinical Relevance

This project was supported by Alcon Labs, Fort Worth, TX, the National Heart, Lung, and

Blood Institute of the National Institutes of Health through Grant Number HL088594. and an

unrestricted grant from Research to Prevent Blindness.

Summary

Purpose Results

Background

IL-5, GM-CSF and FMLP-Stimulated Eosinophil Degranulation is Inhibited by Olopatadine,

but not Other Drugs in the Same Class

The purpose of this study was to compare the

direct effects of three ocular allergy drugs

(olopatadine, alcaftadine, and bepotastine) on

eosinophil degranulation in response to allergic

cytokines and FMLP in vitro.

Media

Eosinophils were pre-incubated for 15 mins at

37°C with either media alone, olopatadine,

bepotastine or alcaftadine at 0.3 and 3.0 mM

concentrations.

Drug Eosinophils were then put in 96 well plates (1x106 /ml)

and stimulated for 4 hrs at 37°C with either

FMLP (10-7 M), IL-5 (1.0ng/ml) or GM-CSF (1.0ng/ml).

Unstimulated (Media) Stimulated

Supernates were collected and analyzed

for Eosinophil Derived Neurotoxin (EDN)

by ELISA.

3

4

2

Peripheral blood was obtained from normal or allergic and/or

asthmatic donors ranging in age from 18 to 55 years with equal

gender distribution. Informed consent was obtained before

participation and the study was approved by the University of

Wisconsin Health Sciences Institutional Review Board (DHHS

Multiple Project Assurance ID # M128501).

Effect Of Ocular Anti-allergic Compounds On

Eosinophil Degranulation: EDN Release

Effect Of Ocular Anti-allergic Compounds on

Eosinophil Degranulation: Percent Inhibition

Methods

Eosinophils in Acute and Chronic Allergic Conjunctivitis:

Eosinophils and eosinophil-derived mediators in tears and

conjunctival biopsy specimens are associated with both acute and

chronic ocular allergic inflammation (1).

In chronic diseases (atopic and vernal keratoconjunctivitis), which

can result in corneal scarring and loss of vision:

Eosinophil granule proteins are found in serum, tears and

corneal plaques and are correlated with disease severity.

Conjunctival eosinophils are activated as evidenced by

increased expression of cytokines (e.g., IL-5, GM-CSF) and

surface receptors (e.g., ICAM-1, HLA-DR).

A study has shown that the activation state of eosinophils

(surface receptor & cytokine expression), rather than number

correlates with disease severity (2).

Our previously published research demonstrated that olopatadine

inhibited mast cell-mediated and FMLP-stimulated eosinophil

degranulation in vitro (3).

In chronic allergic conjunctivitis, changes in eosinophil activation

rather than number correlates with disease severity (2).

Therefore, the ability of olopatadine to inhibit eosinophil

degranulation may be important in treatment of chronic allergic

conjunctivitis, where it has been shown to be efficacious in vernal

keratoconjunctivitis (4).

In animal models of acute allergic conjunctivitis, both bepotastine

and alcaftadine have been shown to inhibit eosinophil recruitment to

conjunctival epithelium, so the inability to inhibit eosinophil

degranulation may not be as relevant in acute disease (5,6).

1. Leonardi A, De Dominicis C, Motterle L. Immunopathogenesis of ocular allergy: a schematic approach to different clinical entities.

Curr Opin Allergy Clin Immunol 2007;7(5):429-345.

2. Hingorani M, Calder V, Jolly G, et al. Eosinophil surface antigen expression and cytokine production vary in different ocular allergic

diseases. J Allergy Clin Immunol 1998;102:821–830.

3. Cook EB, Stahl JL, Sedgwick JB, Barney NP, Graziano FM. The promotion of eosinophil degranulation and adhesion to conjunctival

epithelial cells by IgE-activated conjunctival mast cells. Ann Allergy Asthma Immunol 2004;92:65-72.

4. Corum I, Yeniad B, Bilgin LK, Ilhan R. Efficiency of olopatadine hydrochloride 0.1% in the treatment of vernal keratoconjunctivitis

and goblet cell density. J Ocul Pharmacol Ther 2005;21(5):400-5.

5. Ono SJ, Lane K. Comparison of effects of alcaftadine and olopatadine on conjunctival epithelium and eosinophil recruitment in a

murine model of allergic conjunctivitis. Drug Des Devel Ther 2011;5:77-84.

6. Kida T, Fujii A, Sakai O, Iemura M, Atsumi I, Wada T, Sakaki H. Bepotastine besilate, a highly selective histamine H(1) receptor

antagonist, suppresses vascular hyperpermeability and eosinophil recruitment in in vitro and in vivo experimental allergic

conjunctivitis models. Exp Eye Res 2010;91(1):85-91

References

olopatadine

1 Eosinophils were isolated from heparinized peripheral blood using

magnetic bead negative selection, as described previously (3).

* p < 0.05 compared to NO DRUG

# p < 0.10 compared to NO DRUG

Eosinophils pre-incubated with olopatadine released less EDN in response to FMLP, IL-5 and GM-CSF. Bepotastine (3.0 mM) and

alcaftadine (0.3 mM) had variable effects, inhibiting EDN release in 2 subjects. Alcaftadine increased EDN release at the 3.0 mM

concentration even from unstimulated eosinophils. There was no effect of drug diluent controls (data not shown). (OLO = olopatadine; ALCA = alcaftadine; BEP = bepotastine; EDN = eosinophil derived neurotoxin; N = 4-5 separate donors)

Percent inhibition of EDN release by olopatadine was significant for FMLP (0.3 and 3.0 mM), IL-5 and GM-CSF (3.0 mM) stimulated

cells. In contrast, bepotastine and alcaftadine did not significantly inhibit EDN release. There was no effect of drug diluent

controls (data not shown). (OLO = olopatadine; ALCA = alcaftadine; BEP = bepotastine; EDN = eosinophil derived neurotoxin; N = 4-5 separate donors)

0.0

5.0

10.0

15.0

20.0

25.0

30.0

35.0

40.0

45.0

50.0

0.001 0.01 0.1 1 10

EDN

REL

EASE

(% T

OTA

L)

CONC (mM)

FMLP STIMULATED EOSINOPHILS

OLO

BEP

ALCAF

* *

0.0

10.0

20.0

30.0

40.0

50.0

60.0

0.001 0.01 0.1 1 10

EDN

REL

EASE

(% T

OTA

L)

CONC (mM)

IL-5 STIMULATED EOSINOPHILS

OLO

BEP

ALCAF

0.0

10.0

20.0

30.0

40.0

50.0

60.0

0.001 0.01 0.1 1 10

EDN

REL

EASE

(% T

OTA

L)

CONC (mM)

GM-CSF STIMULATED EOSINOPHILS

OLO

BEP

ALCAF

#

* p < 0.05 compared to NO DRUG

# p < 0.10 compared to NO DRUG

* p < 0.05 compared to NO DRUG

# p < 0.10 compared to NO DRUG

*