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 , Administration of this drug to rats by the oral, intraperitoneal and intrave nous routes significantly inhibited a passive anaphylaxis response , Additionally, topical administration of lo doxamide, via aerosol, to atopic asthmatics provided sig nificant protection from bronchoconstriction in an aller gen challenge trial . 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) . 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 Downloaded by: Kings's College London 126.96.36.199 - 10/27/2017 8:28:36 AM 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  and have been reported to be effective in suppressing allergic conjuncti vitis in animal systems . 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 . 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 Downloaded by: Kings's College London 188.8.131.52 - 10/27/2017 8:28:36 AM 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 , 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 Downloaded by: Kings's College London 184.108.40.206 - 10/27/2017 8:28:36 AM 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 , 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 ; however, the relevance of this activity to acute classical type-I immediate hypersensitivity reactions remains to be determined. Secchi et al.  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 . 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 . Fur thermore, lodoxamidc did not antagonize histamine-in duced cutaneous wheal and flare reactions in human clinical trials . 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. 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