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Original Paper
Int Arch Allergy Immunol 1993;101:102-106
Allcrgy/Inflammation Research,
Alcon Laboratories Ine.,
Fort Worth, Tex., USA
Key Words
Lodoxamide
Immediate hypersensitivity
Allergic conjunctivitis
Conjunctiva
Mast cells
Allergic mediator release
Effect of Lodoxamide on in vitro
and in vivo Conjunctival
Immediate Hypersensitivity
Responses in Rats
Abstract
The antiallergic compound, lodoxamide, was evaluated for its abilities to atten­
uate a local allergic reaction in rat conjunctiva in vivo and to inhibit rat con­
junctival mast cell mediator release in vitro. Topically applied lodoxamide
(0.01, 0.10 and 1.0%, w/v) dose-dependently reduced the allergic response (23,
43 and 72%, respectively) in vivo. Lodoxamide was more effective than cromo­
lyn sodium, N-acetyl aspartyl glutamic acid (Naaxia®) and levocabastine, and
25 (7-200) times more potent than nedocromil sodium in direct comparisons.
Addition of lodoxamide (10 pg/ml) to sensitized conjunctival tissue in vitro im­
mediately prior to antigen challenge significantly reduced the amount of hista­
mine released by the tissue. These data suggest that lodoxamide’s in vivo anti­
allergic activity in the conjunctiva is associated with its ability to prevent al­
lergic mediator release from mast cells contained in this same tissue.
Lodoxamide, 2,2'-[(2-chloro-5-cyano-l,3-phenylene)diimino]bis(2-oxo-acetic acid) tromethamine salt, is an ac­
tive antiallergic agent capable of inhibiting histamine re­
lease from rodent mast cells in vitro [1], Administration of
this drug to rats by the oral, intraperitoneal and intrave­
nous routes significantly inhibited a passive anaphylaxis
response [2], Additionally, topical administration of lo­
doxamide, via aerosol, to atopic asthmatics provided sig­
nificant protection from bronchoconstriction in an aller­
gen challenge trial [3]. Because of the noted topical activ­
ity, studies were undertaken to evaluate the efficacy of an
ophthalmic preparation of lodoxamide for use in allergic
conjunctivitis. A preliminary report indicated that the
compound was effective as an inhibitor of passive conjunc­
tival anaphylaxis in rats when administered topically onto
the eye in concentrations ranging between 0.25 and 2.0%
(w/v) [4]. The present report describes the preclinical ef­
fects of lodoxamide in models of immediate ocular hyper­
sensitivity and compares these effects with those of other
antiallergic drugs.
The in vivo effectiveness of topically applied com­
pounds was assessed in a passive anaphylaxis assay per­
formed in rat conjunctiva. Briefly, male Sprague-Dawley
rats (6/group) were passively sensitized by subconjunctival
injection of rat serum (10 pi) containing heat-labile IgE
specific for ovalbumin (OA). Twenty-four hours after sen­
sitization, 20 pi of test ophthalmic preparations were ap­
plied onto the sensitized eye and animals were immedi­
ately challenged intravenously via the lateral tail vein with
1.0 ml of a solution containing OA (1.0 mg) and Evans blue
Correspondence to: I)r. John M. Yanni
Alcon Laboratories Inc.
6201 South Freeway
Fort Worth, TX 76134-2099 (USA)
© 1993S.Karger AG, Basel
1018-2438/93/1 (H1-0102
S 2.75/0
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J.M. Yanni
L.K. Weimer
R.
L. Glaser
L.S. Lang
S. M. Robertson
J.M. Spellman
challenge produced concentration-dependent inhibition
of the allergic response. Significant inhibition was ob­
served with the 0.1 and 1.0% concentrations. Similarly,
ncdocromil inhibited the response in a concentrationdependent manner. At concentrations of 0.3 and 3.0%
this compound significantly attenuated the response. A
relative potency determination between lodoxamide and
ncdocromil performed using the data presented in table 1
indicates that lodoxamide is 25 (7-200) times more potent
than ncdocromil in inhibiting the allergic response
(table 1).
Topical ocular administration of cromolyn sodium
(0.4%) immediately prior to antigen challenge signifi­
cantly reduced (35%) the response. However, none of the
other concentrations tested effectively prevented the hy­
persensitivity response. Lcvocabastine and Naaxia also
failed to inhibit the allergic response at the concentrations
tested. The (1-agonist albuterol (0.1%) reduced the re­
sponse by 51% in these experiments (table 1).
Lodoxamide inhibited the release of histamine from
passively sensitized rat conjunctival tissue upon antigen
challenge in vitro (fig. 1). Significant inhibition (46%) was
obtained with 10 ,ug/ml of lodoxamide. The reference com­
pound theophylline (180 ¡jg/ml) also reduced (56%) hista­
mine release from the conjunctiva. Radioligand binding
studies, performed with lodoxamide at concentrations as
high as 10 uM, clearly show an absence of interaction with
adenosine 1 and 2, a- and (1-adrenergic, excitatory and in­
hibitory amino acids, Ca2+, K+, CL channel proteins, sero­
tonin 1 and 2, histamine 1, dopamine 1 and 2, cholinergic,
prostanoid, and opiate receptors (table 2).
The data obtained in vivo demonstrate that the refer­
ence drug albuterol significantly reduced the allergic re­
sponse in the rat conjunctiva. Beta agonists inhibit mast
cell mediator release from human tissue [6] and have been
reported to be effective in suppressing allergic conjuncti­
vitis in animal systems [7]. Of the other antiallergic drugs
evaluated only lodoxamide and ncdocromil consistently
inhibited the immediate hypersensitivity response in this
model. Both lodoxamide and nedocromil have been re­
ported to be effective in allergic conjunctivitis patients [8,
9]. Nedocromil has also been reported to inhibit mast cell
mediator release [10]. However, the present data demon­
strate that lodoxamide is 25 (7-200) times more potent an
inhibitor of immediate hypersensitivity than nedocromil
sodium.
Cromolyn sodium failed to significantly attenuate al­
lergic conjunctivitis in these experiments. Similar nonsig­
nificant inhibition was noted with topical administration
of cromolyn sodium by other investigators [11,12] in mod-
103
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dye (2.5 mg). Thirty minutes after antigen challenge, ani­
mals were killed, skin was reflected, and the size of the re­
sulting wheal and the intensity of the extravasated dye
were determined. The wheal area multiplied by the dye in­
tensity produced the individual response score. Scores for
each group of animals were compared with the scores of
the saline-treated group using Dunnett’s t test [5], Linear
regression was used to determine dose response and rela­
tive potency.
The ability of lodoxamide to inhibit antigen-stimulated
release of histamine from passively sensitized rat conjunc­
tival tissue in vitro was also evaluated. The conjunctival
tissues of male Sprague-Dawley rats were passively sensi­
tized with anti-OA antiserum as described above. Twentyfour hours following sensitization, animals were killed and
the conjunctivae were removed and placed in Tyrode’s
buffer supplemented with 1 mg/ml bovine serum albumin.
Test compounds were added to the tissue either immedi­
ately (lodoxamide) or 15 min (theophylline) prior to OA
challenge (100 ,ug/ml). Tissues were then incubated with
OA for 60 min at 37 °C. Supernatants were collected and
analyzed for histamine content using a quantitative ra­
dioimmunoassay (AMAC Inc., Westbrook, Me., USA).
Data were analyzed using Dunnett’s t test.
Lodoxamide’s potential for interaction with 35 physio­
logically relevant receptors was investigated utilizing
PROFILE™ (Nova Pharmaceutical Corporation. Balti­
more, Md., USA) radioligand binding assays.
Lodoxamide (Alcon Laboratories Inc., Fort Worth,
Tex.), ncdocromil sodium (prepared by Alcon Laborato­
ries Inc.), and cromolyn sodium (Sigma Chemical Co.,
St. Louis, Mo.) were prepared as solutions in sterile dis­
tilled water (Elkins-Sinn Inc., Cherry Hill, N.J., USA) im­
mediately prior to topical administration. Commercially
available preparations of N-acetyl aspartyl glutamic acid
(Naaxia®, Laboratoires Allergan Dulcis, Monaco) and Levocabastine (Janssen Pharmaceutical, Beerse, Belgium)
were obtained and concentrations adjusted by dilution
with water or a sterile 0.9% NaCl solution when appropri­
ate. The reference compounds albuterol (Research Bio­
chemicals Inc., Natick, Ma., USA) and theophylline
(Sigma Chemical Co.) were prepared as solutions in 0.9%
NaCl in water immediately prior to use. For in vitro use,
lodoxamide was prepared as a stock solution in sterile dis­
tilled water and appropriate dilutions made with 0.9%
sterile saline prior to addition to the conjunctival tissues.
All w/v solutions were prepared to reflect the percentage
of free acid or base.
In the in vivo allergic conjunctivitis model, topical ad­
ministration of lodoxamide immediately prior to antigen
Table 1. Relative activity of lodoxamide
and reference compounds in a passive an­
aphylaxis in rat conjunctiva model
Compound
Dose
%, w/v
Wheal area x intensity
(x±SD )
% change
Relative potency
95% confidence limits
254 ±33
Saline
Albuterol
0.1
125 ±51*
-51
Lodoxamide
0.01
0.1
1.0
196 ±44
143 ±67*
70 ±20*
-22
-43
-72
Nedocromil
0.03
0.3
3.0
244 ±37
165 ±47*
152 ±59*
-4
-35
-40
Cromolyn
0.04
0.4
2.0
4.0
223 ±49
167 ±63*
204 ±95
192 ± 74
-12
-34
-20
-24
Levocabastine
0.05
216 ± 53
-15
Naaxia
0.5
5.0
220 ±80
216 ±59
-13
-15
1
0.04
(0.005-0.15)
The compounds were administered immediately prior to antigen challenge.
Lodoxamide regression line: (y = 63x + 74); t value slope = -4.65, p <0.001; r = 0.758.
Nedocromil regression line: (y = 46x + 163); t value slope = -3.16, p <0.001; r = 0.620.
* p <0.05.
104
Yanni/Weimer/Glaser/Lang/Robertson/
Spellman
Lodoxamide’s Effect on Allergic
Conjunctivitis
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Fig. 1. Effects of 10 pg/ml of lodoxamide
and 180 ug/ml of theophylline on antigen-in­
duced histamine release from conjunctival
tissue.
els of allergic conjunctivitis. Levocabastine, an antihistaminic compound, failed to reduce the local allergic reac­
tion when applied topically immediately prior to antigen
challenge. Although histamine does not play a significant
role in vascular permeability responses in rats [131, levoca­
bastine has been reported effective against allergen-in­
duced vascular permeability effects in rats following oral
administration 1 h before allergen challenge [14], This sug­
gests that a longer time interval between dose and allergen
challenge may be required for topical efficacy with this
compound. The proported antiallergic agent Naaxia was
devoid of inhibitory activity in the model of IgE-mediated
immediate hypersensitivity at concentrations as high as
5.0% (w/v). It has been reported that Naaxia prevents an
immune complex-mediated inflammatory reaction [15];
however, the relevance of this activity to acute classical
type-I immediate hypersensitivity reactions remains to be
determined. Secchi et al. [16] reported that Naaxia inhibits
histamine release from guinea-pig choroidal tissue chal­
lenged with compound 48/80, a nonspecific, IgE-independent mast cell secretagoguc capable of causing hista­
mine release in the absence of calcium and glucose [17].
The relevance of this finding to IgE-mediated mast cell ac­
tivation in conjunctival tissue is undetermined.
The data obtained in the present studies indicate that
topically applied lodoxamidc effectively attenuates the al­
lergic reaction occurring in the conjunctiva. Measure­
ments of histamine release from this target tissue in vitro
suggest that inhibition of mast cell mediator release by lodoxamide is responsible for the in vivo antiallergic activity
noted above. The lack of significant interaction with rele­
vant receptors (histamine, serotonin, prostanoid, beta) in
vitro further supports the finding that lodoxamide’s anti­
allergic efficacy in the conjunctivitis model is associated
with mast cell mediator release inhibition and not endorgan antagonism of released or synthesized pro-inflam­
matory mediators. The lack of affinity for the receptors
noted in the radioligand binding studies confirms the pre­
viously reported lack of end-organ antagonism for hista­
mine, serotonin, bradykinin and acetylcholine [2]. Fur­
thermore, lodoxamidc did not antagonize histamine-in­
duced cutaneous wheal and flare reactions in human
clinical trials [18]. Data presented in this communication
clearly demonstrate the effectiveness of topically applied
lodoxamide in models of allergic conjunctivitis and sug­
gest that this drug is superior in these rodent models to the
other ocular antiallergic drugs evaluated.
Table 2. Evaluation of lodoxamidc (10 p/VQ in radioligand bind­
ing assays
% inhi­
bition1
Receptor/
selectivity
Adenosine
Adenosine 1
Adenosine 2
Adrenergic
Alpha 1
Alpha 2
Beta
Excitatory amino acids
Glycine
Quisqualate
Kainate
NMDA
PCP
Inhibitory amino acids
Glycine
GABA a
GABA,j
Benzodiazepine
Channel proteins
Calcium (types T, L)
Calcium (type N)
Potassium
Chloride
1
7.5
15.2
-7.4
-0.8
-0.7
-12.7
-6.0
23.5
-6.0
5.4
-0.9
5.2
-7.1
-1.9
Receptor/
selectivity
% inhi­
bition1
Biogenic amines
-3.2
Dopamine 1
Dopamine 2
4.3
11.9
Serotonin 1
Serotonin 2
11.3
Histamine 1
15.3
Cholincrgics
2.2
Muscarinic 1
Muscarinic 2
36.5
Nicotinic
-19.7
Opiate
-1.4
MU
-1.4
Delta
Kappa
2.1
-0.4
Sigma
Prostanoids
-4.8
Leukotriene B4
-1.8
Leukotriene D4
Thromboxane A,
5.6
12.2
7.0
16.1
3.8
Lodoxamide was evaluated at concentrations of 10"5,10~7 and lO-1'
M . Data are shown only for 10'5M . Percent inhibition represents an
average of 2 samples. Negative inhibition values reflect a slight en­
hancement of radioligand binding.
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