Antigenicity of sulfanilamide and its metabolites using fluorescent-labelled compounds

In order to clarify the onset mechanisms of drug-induced allergies, three fluorescent-labelled compounds were synthesized by subjecting sulfanilamide (SA), a base compound for sulfonamides, and its active metabolites, i.e. sulfanilamide hydroxylamine and sulfanilamide nitroso, to dansylation using dansylchloride. In other words, 5-dimethylamino-N-(4-aminobenzyl)-naphthalenesulfonamide (DNS-4ABA), 5-dimethylamino-N-(4-hydroxylaminobenzyl)-1-naphthalenesulfonamide (DNS-4HABA) and 5-dimethylamino-N-(4-nitrosobenzyl)-1-naphthalenesulfonamide (DNS-4NSBA) were synthesized as model haptens. When analysed by HPLC, a conjugate of DNS-4HABA and glutathione (GSH) with nucleophilic amino acids had two peaks (P-1 and P-2). FAB-MS and 1H-NMR revealed that the DNS-4HABA-GSH conjugate consisted of sulphinamide and semimercaptal. The reactivity of GSH to DNS-4ABA, DNS-4HABA and DNS-4NSBA was quantified by HPLC using an oxidization system (horseradish peroxidase/H2O2). The results show that production of DNS-4NSBA-GSH-conjugate was four to eight times higher than that of DNS-4HABA-GSH conjugate, but that DNS-4ABA did not bind with GSH. Skin reactions were assessed using guinea pigs, and strong delayed erythema was seen with DNS-4NSBA, which bound most strongly with GSH, whereas weak delayed erythema was seen with DNS-4ABA, which did not bind with GSH. This suggests a correlation between GSH conjugate production and skin reactions. DNS-4HABA enzymatically bound with proteins in rat and guinea pig liver cytosol and microsomal fractions. The proteins that bound to DNS-4HABA were purified by HPLC and then subjected to N-terminal amino acid analysis. Ubiquitin (10 kDa) and fatty acid binding protein (30 kDa) were detected in the rat liver cytosol fraction; retinol-dehydrogenase (35 kDa) in the rat microsomal fraction; and glutathione-S-transferase B (mmu) (25 kDa) in the guinea pig liver cytosol fraction. When DNS-4HABA or DNS-4NSBA binds to proteins that play important roles in the body, unexpected adverse reactions may occur. Furthermore, by utilizing our technique using model compounds, it may be possible to identify the carrier proteins of various compounds, including pharmaceutical agents.     

Antigenicity of sulfanilamide and its metabolites using fluorescent-labelled compounds

In order to clarify the onset mechanisms of drug-induced allergies, three fluorescent-labelled compounds were synthesized by subjecting sulfanilamide (SA), a base compound for sulfonamides, and its active metabolites, i.e. sulfanilamide hydroxylamine and sulfanilamide nitroso, to dansylation using dansylchloride. In other words, 5-dimethylamino-N-(4-aminobenzyl)-naphthalenesulfonamide (DNS-4ABA), 5-dimethylamino-N-(4-hydroxylaminobenzyl)-1-naphthalenesulfonamide (DNS-4HABA) and 5-dimethylamino-N-(4-nitrosobenzyl)-1-naphthalenesulfonamide (DNS-4NSBA) were synthesized as model haptens. When analysed by HPLC, a conjugate of DNS-4HABA and glutathione (GSH) with nucleophilic amino acids had two peaks (P-1 and P-2). FAB-MS and ¹H-NMR revealed that the DNS-4HABA-GSH conjugate consisted of sulphinamide and semimercaptal. The reactivity of GSH to DNS-4ABA, DNS-4HABA and DNS-4NSBA was quantified by HPLC using an oxidization system (horseradish peroxidase/H₂O₂). The results show that production of DNS-4NSBA-GSH-conjugate was four to eight times higher than that of DNS-4HABA-GSH conjugate, but that DNS-4ABA did not bind with GSH. Skin reactions were assessed using guinea pigs, and strong delayed erythema was seen with DNS-4NSBA, which bound most strongly with GSH, whereas weak delayed erythema was seen with DNS-4ABA, which did not bind with GSH. This suggests a correlation between GSH conjugate production and skin reactions. DNS-4HABA enzymatically bound with proteins in rat and guinea pig liver cytosol and microsomal fractions. The proteins that bound to DNS-4HABA were purified by HPLC and then subjected to N-terminal amino acid analysis. Ubiquitin (10?kDa) and fatty acid binding protein (30?kDa) were detected in the rat liver cytosol fraction; retinol-dehydrogenase (35?kDa) in the rat microsomal fraction; and glutathione-S-transferase B (mμ) (25?kDa) in the guinea pig liver cytosol fraction. When DNS-4HABA or DNS-4NSBA binds to proteins that play important roles in the body, unexpected adverse reactions may occur. Furthermore, by utilizing our technique using model compounds, it may be possible to identify the carrier proteins of various compounds, including pharmaceutical agents.     

祛疣洗剂急性毒性与过敏性试验

目的：对祛疣洗剂进行皮肤急性毒性试验和皮肤过敏性试验,以了解其安全性。方法：祛疣洗剂单次给药或多次给药用于白色豚鼠背部正常或破损去毛区皮肤,观察其产生急性毒性情况;采用致敏与激发接触祛疣洗剂,观察动物过敏情况。结果与结论：祛疣洗剂未产生急性毒性,反复致敏后使用,也无皮肤与全身过敏反应。     

Antigenicity studies on catena-(S)-[mu-[N alpha-(3-aminopropionyl)histidinato(2-)-N1,N2,O:N tau]-zinc]

The antigenicity of Z-103 (catena-(S)-[mu[N alpha-(3-aminopropionyl) histidinato(2-)-N1,N2,O:N tau]-zinc], CAS 107667-60-7) was evaluated using the following assay procedures: 1. active systemic anaphylaxis (ASA) in guinea pigs. 2. passive cutaneous anaphylaxis (PCA) in guinea pigs with serum from guinea pigs sensitized with Z-103, 3. delayed type skin reaction (Maximization Test) in guinea pigs, 4. passive cutaneous anaphylaxis (PCA) in rats with serum from mice sensitized with Z-103 and 5. passive hemagglutination (PHA) with serum from mice sensitized with Z-103. In each test except for Maximization Test, the sera obtained 1 or 6 h (hereinafter designated as 1-h-sera or 6-h-sera) after a single oral administration of 500 mg/kg of Z-103 to the unused rats, guinea pigs or rabbits, were used as the challenge antigen. 1. ASA in guinea pigs: No anaphylaxis reaction was observed in any of the sensitized guinea pigs by elicitation with challenge antigen. 2. PCA in guinea pigs: PCA titer of sera from all the sensitized animals was less than 1 in elicitation with the challenge antigen. 3. Delayed type skin reaction test: No skin reaction was observed in sensitized guinea pigs after intradermal injection or dermal application of Z-103. 4. PCA in rats: PCA titer of sera from BALB/c and C3H/He mice sensitized with Z-103 was less than 5 in elicitation with the challenge antigen. 5. PHA reaction: When erythrocytes coated with challenge antigen were added to sensitized sera, the hemagglutination titer was less than 1.(ABSTRACT TRUNCATED AT 250 WORDS)     

ACE-inhibitor-induced enhancement of spontaneous and IgE-mediated histamine release from mast cells and basophilic leukocytes and the modulatory effect of capsaicin sensitive nerves

Frequently reported adverse inflammatory skin and airway reactions have been reported in subjects being medicated with angiotensin converting enzyme (ACE)-inhibitors. Intradermally evoked wheal and flare reactions to ovalbumin, capsaicin and bradykinin, in ovalbumin sensitized guinea pigs, was previously demonstrated to be enhanced by pretreatment with the ACE-inhibitor MK 422 (the active parent diacid of enalapril). In vitro results from this study demonstrate that the ACE-inhibitor MK 422 degranulated guinea pig lung and skin mast cells as well as human basophils, and enhanced allergen-evoked histamine release. Local capsaicin pretreatment in vivo of guinea pig skin decreased spontaneous and allergen-triggered release of histamine in vitro from skin mast cells. No clear enhancing effect of MK 422 was seen on the allergen-triggered histamine release in vitro from capsaicin pretreated skin, and the spontaneous release was unaffected by the ACE-inhibitor. The allergen-triggered wheal and flare reaction in ovalbumin sensitized guinea pigs was potentiated by MK 422 and the late phase reaction of the inflammatory response was especially augmented. Capsaicin pretreatment of the guinea pigs abolished this late phase reaction as well as the inflammatory enhancing effect of MK 422. Our in vitro results from capsaicin pretreated skin indicate that the reduced inflammatory response in vivo in capsaicin pretreated skin is due not only to capsaicin induced depletion of neuropeptides from sensory nerves, but also to secondary degranulation of mast cells by one or more of these peptides.     

Anti-allergic effects of 1-(2-ethoxyethyl)-2-(4-methyl-1-homopiperazinyl)benzimidazole fumarate (KB-2413)

The effects of KB-2413 on four types of allergic reactions classified by Coombs and Gell were investigated. KB-2413 inhibited homologous passive cutaneous anaphylaxis and passive anaphylactic bronchoconstriction in guinea pigs mediated by IgE-like antibody, and ED50 values were 0.0017 mg/kg, p.o., and 0.022 mg/kg, p.o., respectively. KB-2413 also inhibited IgG-mediated anaphylactic bronchoconstriction in guinea pigs actively sensitized with egg albumin. Both complement-dependent immune hemolysis and complement-independent hypotonic hemolysis were inhibited by KB-2413 in a concentration-dependent manner. KB-2413 had no effect on the Forssman systemic reaction. The passive Arthus reaction in guinea pigs sensitized with anti-egg albumin rabbit serum was unaffected by KB-2413. However, the early stage of the active Arthus reaction in rabbits sensitized with egg albumin was inhibited. KB-2413 had an inhibitory effect on the efferent phase of delayed-type hypersensitivity induced by picryl chloride (PC-DTH) in mice. On the other hand, the afferent phase of PC-DTH in mice was unaffected. These results suggest that KB-2413 strongly suppresses type I allergic reactions, and it slightly suppresses type II, III and IV allergic reactions.     

Skin sensitization potential of trisodium ethylenediaminetetraacetate

A sodium salt of ethylenediaminetetraacetate (Na3EDTA) and ethylenediamine (EDA) were subjected to a repeated insult patch test on Hartley albino guinea pigs (10 per compound). All guinea pigs (10 of 10) receiving EDA were sensitized. None of the guinea pigs (0 of 10) was sensitized to Na3 EDTA. Likewise, none of the guinea pigs sensitized to EDA reacted positively when challenged with Na3 EDTA. Based on these results, it is concluded that EDTA is not likely to be a sensitizer to humans, and would not likely cross-sensitize with EDA. In addition, the presence of very small amounts of the sodium salts of EDTA in cosmetic and pharmaceutical preparations does not represent an appreciable risk of human skin sensitization.     

咪康唑莫米松皮肤成膜凝胶皮肤用药安全性试验

目的对咪康唑莫米松皮肤成膜凝胶进行皮肤刺激性试验和过敏性试验,为,临床用药安全提供依据。方法采用家兔进行皮肤刺激试验,分为完整皮肤组和破损皮肤组,连续给药7d后观察;采用豚鼠皮肤进行主动过敏性试验（ACA）,观察咪康唑莫米松皮肤成膜凝胶是否引起豚鼠皮肤或全身过敏反应。结果家兔皮肤刺激性试验,完整皮肤组和破损皮肤组均未出现红斑、水肿等现象;豚鼠皮肤主动过敏性试验,未引起皮肤过敏反应或全身过敏反应。结论咪康唑莫米松皮肤成膜凝胶未引起家兔皮肤刺激性反应和豚鼠皮肤过敏性反应,其临床用药剂量安全可靠。     

Skin Sensitization Potential of Trisodium Ethylenediaminetetraacetate

ABSTRACT

A sodium salt of ethylenediaminetetraacetate (Na₃EDTA) and ethylenediamine (EDA) were subjected to a repeated insult patch test on Hartley albino guinea pigs (10 per compound). All guinea pigs (10 of 10) receiving EDA were sensitized. None of the guinea pigs (0 of 10) was sensitized to Na₃ EDTA. Likewise, none of the guinea pigs sensitized to EDA reacted positively when challenged with Na EDTA. Based on these results, it is concluded that EDTA. is not lhcely to be a sensitizer to humans, and would not likely cross-sensitize with EDA. In addition, the presence of very small amounts of the sodium salts of EDTA in cosmetic and pharmaceutical preparations does not represent an appreciable risk of human skin sensitization.     

The use of hairless (IAF/HA-HO) guinea pigs for the determination of delayed-type hypersensitivity to tuberculin

Guinea pigs are a classic animal model for studying delayed-type hypersensitivity (DTH) reactions. However, skin irritation due to hair removal can interfere with the evaluation of the modulation of these responses by various mediators. A DTH model using hairless (IAF/HA-HO) guinea pigs, sensitized with complete Freund's adjuvant and repeatedly skin tested with tuberculin, purified protein derivative, (PPD) has therefore been developed. At 10 weeks after sensitization, intradermal PPD elicited minimal erythema at 6 h, which increased over the next 18 h to a maximum at 24 h, and declined by 48 h. The response could be quantified by bioassay using graded doses of PPD. Reactions at 24 h were characterized by predominantly mononuclear cell deep and superficial dermal infiltrates. Dermal DTH in hairless guinea pigs is thus, grossly and histologically similar to that seen in Hartley guinea pigs.     

Antianaphylactic benzophenones and related compounds

The synthesis and biological properties of 85 benzophenones and related compounds are described. The majority of the compounds inhibit the release of leukotrienes (LT) C4 and D4 in vitro from sensitized guinea pig chopped lung. In addition, some of the compounds inhibited the release of LTs from passively sensitized human chopped lung and protected guinea pigs from the effects of anaphylaxis in a modified Herxheimer test.     

Antianaphylactic agents. 1. 2-(Acylamino)oxazoles.

The synthesis and biological properties of 35 2-(acylamino)oxazoles are described. The majority of the compounds inhibit the release of slow-reacting substance of anaphylaxis (SRS-A) in vitro from sensitized guinea pig chopped lung. In addition, several of the compounds inhibited the release of SRS-A from passively sensitized human chopped lung and protected guinea pigs from the effects of anaphylaxis in a modified Herxheimer test.     

Studies on experimental iodine allergy: 3. Low molecular weight elicitogenic antigens of iodine allergy

We hypothesize that iodine allergy is an immune response to iodinated self proteins produced in vivo from various iodine-containing chemicals. Since an antigenic determinant of experimental iodine allergy is diiodotyrosine (DIT), we designed low molecular weight DIT derivatives having provocative antigenicity without sensitizing immunogenicity. Tetraiododityrosine and hexaiodotrityrosine provoked dose-dependent skin reactions in guinea pigs previously immunized with iodine. No guinea pigs immunized with hexaiodotrityrosine showed anaphylactic reaction by i.v. challenge with hexaiodotrityrosine and none of their antisera showed positive passive cutaneous anaphylaxis (PCA) reaction in guinea pigs, indicating the non-immunogenic nature of the compound. Erythrosine, one of the color additives having a structure common with DIT, was assessed for its immunological property. Enzyme-linked immunosorbent assay (ELISA) inhibition studies on erythrosine revealed that the inhibitory activity of erythrosine was stronger than that of DIT. Furthermore, erythrosine provoked a PCA reaction in animals sensitized with anti-iodine antisera. In conclusion, hexaiodotrityrosine is thought to be useful for skin testing of iodine allergy without any fear of sensitization to the allergen. Erythrosine was shown to provoke an experimental iodine allergy and, also, the relationships between the new concept of iodine allergy and features of clinical findings of adverse effects by iodocontrast media are discussed.     

Anti-allergic effects of (E)-3-[p-(1H-imidazol-1-lylmethyl) phenyl]-2-propenoic acid (OKY-046), a specific thromboxane (TX) A2 synthetase inhibitor: effects on type I allergic reactions]

We studied the effects of OKY-046 on type I allergic reactions. OKY-046 (100 mg/kg) given orally suppressed antigen-induced bronchoconstriction and TXB2 generation in broncho alveolar lavage fluid in rats passively sensitized with anti-DNP-As monoclonal IgE. At the dose of 30 mg/kg given intraduodenally, it also inhibited antigen-induced bronchoconstriction in guinea pigs passively sensitized with anti DNP-As serum and actively sensitized with ovalbumin. However, aspirin (30 mg/kg) didn't suppressed them significantly. Azelastine (10 mg/kg) inhibited bronchoconstriction in passively sensitized rats and actively sensitized guinea pigs. In 48 hour homologous PCA reactions of rats and mice, oral administration of OKY-046 (300 mg/kg) and tranilast (100 mg/kg) suppressed the extravasated dye in the skin. OKY-046 decreased histamine release from passively sensitized rat peritoneal exudate cells. There was no effect of OKY-046 on SRS-A and leukotriene release from actively sensitized guinea pig lungs and passively sensitized rats. In conclusion, we think that OKY-046 should be an useful asthmatic drug or anti-allergic drug by oral administration.     

KC-404: A POTENTIAL ANTI-ALLERGIC AGENT WITH ANTAGONISTIC ACTION AGAINST SLOW REACTING SUBSTANCE OF ANAPHYLAXIS

The mode of action of a novel compound, 3-isobutyryl-2-isopropylpyrazolo [1,5-a]pyridine (KC-404), as a potential anti-allergic agent has been investigated. KC-404 was shown to have a direct bronchodilator activity in guinea pig trachea in vitro and in anesthetized guinea pig in vivo. In addition, KC-404 had a fairly selective antagonistic action against slow reacting substance of anaphylaxis (SRS-A) on guinea pig ileum in vitro. In anesthetized guinea pigs, ED50 values for intravenously and intraduodenally injected KC-404 to inhibit SRS-A-induced bronchoconstriction were 0.0014 and 0.0065 mg/kg, respectively. Much higher doses were required to inhibit bronchospasms produced by histamine or particularly by acetylcholine. Orally administered KC-404, 0.001 to 0.1 mg/kg, also showed a selective inhibitory effect on increased vascular permeability by intradermal SRS-A in guinea pigs and rats. KC-404 inhibited the immunological release of mediators, notably SRS-A from sensitized guinea pig chopped lung in vitro at 10⁻⁸ to 10⁻⁴ g/ml. In vivo, the release of SRS-A, but not of histamine, mediated by a nonreaginic antibody in the peritoneal cavity of sensitized rats was inhibited by KC-404 at oral doses above 3 mg/kg. In a similar anaphylactic reaction but mediated by a reaginic antibody, KC-404 also inhibited SRS-A release at intraperitoneal doses of 2.5 to 10 mg/kg. The inhibitory activity on histamine release was less than half of that on SRS-A release. These results indicate that KC-404 is an orally active compound with a unique mode of action to inhibit preferentially both the effects and immunological release of SRS-A.     

IMMUNOPHARMACOLOGICAL STUDIES OF SODIUM COPPER CHLORPHYLLIN (SCC)

Effect of sodium copper chlorophyllin (SCC) on experimental allergic reaction was investigated. IgE antibody mediated reactions, homologous passive cutaneous anaphylaxis (PCA) in rats and the release of anaphylactic mediators (histamine and/or slow reacting substance of anaphylaxis (SRS-A)) from sensitized guinea pig lung tissues or rat peritoneal mast cells classified as a Type I reaction were clearly inhibited by SCC at a similar potency as N-(3’,4’-dimethoxy cinnamoyl) anthranilic acid (N-5’). The increase of vascular permeability in rat skin caused by autacoids or enzymes that participate in the Type I reaction was also inhibited by SCC. Type II or III, complement dependent, reactions including reversed cutaneous anaphylaxis (RCA) in rats and Forssman cutaneous vasculitis (FCV) in guinea pigs were inhibited by SCC. Prednisolone inhibited RCA in rats, but did not inhibit FCV in guinea pigs. Two experimental types of glomerulonephritis, nephrotoxic serum (NTS) nephritis in rats and immune complex nephritis in (NZW×NZB) F₁, mice, in which Type II and III reactions might participate in the onset and the development of the disease, were slightly inhibited by SCC in terms of the biochemical changes of blood and urine parameters and histopathological scores. A moderate remission of the onset and development of these two experimental types of nephritis was recognized by the administration of prednisolone. Delayed hypersensitivity reaction as a Type IV reaction caused by sheep red blood cells (SRBC) in sensitized mouse footpad was not affected by SCC. Prednisolone clearly inhibited the SRBC induced footpad reaction in mice. IgM antibody production in mice and IgE antibody production in rats were not influenced by daily injection of SCC.     

Anti-asthmatic effect of ASP3258, a novel phosphodiesterase 4 inhibitor

ASP3258 is a potent and selective PDE4 inhibitor and exerts a wide-range of anti-inflammatory effects with low emetic potential, a major adverse effect of PDE4 inhibitors. Here, we investigated the anti-asthmatic potency of ASP3258 as compared with those of two representative PDE4 inhibitors: roflumilast and cilomilast. Orally administered ASP3258, roflumilast, and cilomilast all inhibited ovalbumin (OVA)-induced eosinophil infiltration into the airway of sensitized Brown Norway rats with ED(50) values of 0.81, 0.46, and 4.4 mg/kg, respectively. Histological examination also revealed a decreasing trend in inflammatory cell infiltration into the lung following ASP3258 administration. In vitro investigation of bronchodilatory activities showed that these compounds (10(-8)-10(-6) M) concentration-dependently inhibited OVA-induced contraction of trachea isolated from sensitized guinea pigs but had no effect on spasmogen-precontracted tracheal tension prepared from non-sensitized guinea pigs up to 10(-6) M. In vivo experiments using sensitized guinea pigs showed that these orally administered compounds inhibited OVA-induced increases in airway resistance with ED(50) values of 2.2, 0.35, and 12 mg/kg, respectively. Further, orally administered ASP3258 (0.1 and 1 mg/kg), roflumilast (0.1 and 1 mg/kg), and cilomilast (10 mg/kg) significantly suppressed airway hyperresponsiveness caused by OVA exposure. ASP3258's potent inhibition of antigen-induced bronchoconstriction and airway hyperresponsiveness, two characteristic symptoms of bronchial asthma, suggests that this compound will be useful in treating asthma.     

Antiallergic effects of major metabolites of astemizole in rats and guinea pigs.

Antiallergic effects of astemizole (CAS 68844-77-9) and its metabolites were studied using rats and guinea pigs. All the metabolites of astemizole tested i.e., desmethylastemizole, 6-hydroxydesmethylastemizole and norastemizole, were more active than astemizole in inhibiting the contraction of the ileum as well as the bronchoconstriction induced by histamine in guinea pigs. Desmethylastemizole was about the same as the parent compound in inhibiting the 3H-mepyramine binding in guinea pig cerebellum. In heterologous passive cutaneous anaphylaxis (PCA) and homologous PCA, the metabolites caused almost equipotent inhibition to that seen in astemizole. On the other hand, in the studies of histamine release from rat peritoneal mast cells induced by compound 48/80 or from lung fragments in actively sensitized guinea pigs, desmethylastemizole, 6-hydroxydesmethylastemizole and norastemizole were much less potent than was astemizole. No H2-antagonistic activity was observed with either astemizole or desmethylastemizole.     

Treatment of dermatitis caused by the sesquiterpene lactone helenin

Many sesquiterpene lactones, terpenoid compounds present in members of the Compositae (Asteraceae) family of plants, are known to cause allergic contact dermatitis. There is no treatment of this skin disease. We report on the use of an amino acid, L-cysteine, to control dermatitis in guinea pigs sensitized to the sesquiterpene lactone, helenin. The cysteine treatment of allergic reactions induced by helenin reduced the recovery time of the animals by about 50%. The effect of cysteine treatment might be explained by: cysteine reaction with free helenin, and substitution of skin proteins from the protein-helenin complex by cysteine through competitive reactions. Cysteine treatment present a new promising way for control of this type of allergy in humans.     

Effect of azelastine hydrochloride on vascular permeability in hypersensitivity reaction skin site in guinea pig

4-(p-Chlorobenzyl)-2-(hexahydro-1-methyl-1H-azepine-4-yl)-1-(2H)-phthalazinone hydrochloride(azelastine) is a new compound promising anti-allergic effect, which was tested on increased vascular permeability in two types of hypersensitivity skin reaction in guinea pigs. The reversed passive Arthur (RPA) reaction was induced by bovine serum albumin and its heterologous or homologous antibody IgG. A monophasic permeability response, shown by amount of extravasated Evan's blue, was considerably suppressed by pretreating the animals orally with azelastine at different concentrations. In contrast, antihistamines such as triprolidine, chlorpheniramine and pyrilamine had no effect on the vascular response. Thus the potent antiexudative action of azelastine was demonstrated for the RPA reactions in guinea pigs as the model. However, the vascular response in tuberculin reaction in guinea pigs was not influenced by azelastine suggesting a different permeability mechanism.