© 1990 S. Karger AG, Basel 0030-3747/90/0226-0359S2.75/0

Ophthalmie Res 1990;22:359-364

Pharmacodynamics of Amlexanox (AA-673) in Normal and Anaphylactic Rat Conjunctiva and Its Effect on Histamine Concentration George Rankov3 b, Kazuyuki Sasaki a , Masamichi Fukuda a a Department of Ophthalmology, Kanazawa Medical University, Uchinada Ishikawa, Japan; bSofia District Hospital, Clinic of Ophthalmology, Bulgaria

Key Words. Anti-allergic agent • Conjunctiva • Anaphylaxis • Histamine ■Mast cell

Introduction Type I IgE-mediated hypersensitivity reactions occur in several kinds of ocular conjunctival diseases. The major mecha­ nisms and factors involved in anaphylaxis are well known. Potent chemical mediators derived from mast cells are responsible for both immediate anaphylaxis and late-phase reactions [1,2], among which are histamine, serotonin, platelet-activating factor and arachidonic acid metabolites. Inhibition of me­ diator release is the main therapeutic ap­

proach in handling ocular anaphylactic con­ dition. Amlexanox (AA-673) 0.25% ophthal­ mic solution is a newly developed ocular anti-allergic drug which has been reported to inhibit histamine release [3-5], and also slow-reacting substance of anaphylaxis (SRS-A), mainly by affecting the lipooxygenase pathway [5], This study was designed to investigate the dynamics of AA-673 in ana­ phylactic conjunctiva and to evaluate the direct effect on the major mediator of ana­ phylaxis, histamine, utilizing an in vivo model of ocular anaphylaxis.

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Abstract. The pharmacodynamics of Amlexanox (AA-673), an azoxanthone derivative recently developed as an ocular anti-allergic drug, were studied in normal and anaphylactic rat conjunctiva. Ocular anaphylaxis was induced by topical application of dithiothreitol and antigen challenge (egg albumin). The drug concentration was measured at 5, 30, 60 and 120 min after instillation of AA-673 0.25% ophthalmic solution. Histamine concentrations in the pretreated and untreated anaphylactic conjunctiva were compared. In both groups the drug concentration decreased exponentially with time, showing a marked delay in the ana­ phylactic group. There was a significant difference in histamine values between the pre­ treated and untreated groups.

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360

Fig. 1. Chemical structure of Amlexanox (AA-673): 2-amino-7isopropyl - 5 - oxo - 5H - ( 1)benzopyrano(2,3-6)-pyridinc-3-carboxylic acid.

The study was divided into two experiments: (I) dynamics of AA-673 0.25% ophthalmic solution (Senju Pharmaceutical Co. Ltd.) in normal and ana­ phylactic rat conjunctiva, and (II) evaluation of the drug efficacy on conjunctival histamine during ocular anaphylaxis. Experiment 1 A topically induced model of ocular anaphylaxis was employed [6-8]. Fifty male albino (180-200 g) Sprague-Dawley rats were immunized by intraperito­ neal injection of 100 pg egg albumin (EA) plus 20 mg alum in 1 ml PBS (pH 7.4). After 2 weeks the right eyes were pretreated with 10 pi M dithiothreitol in PBS, followed 15 min later by 10 pi EA (100 mg/ml) to induce anaphylaxis. Drug Administration and Time Measurement. 10 pi of AA-673 0.25% ophthalmic solution (fig. 1) was instilled in the cul-de-sac (right eyes). Concentra­ tion of AA-673 in the conjunctiva was determined at 5, 30, 60 and 120 min after instillation. In the ana­ phylactic group eyes underwent antigen challenge 5 min prior to drug administration. Sample Processing. Rats were sacrificed by decap­ itation. The bulbar conjunctiva was excised and weighed. The tissue was then cut into small pieces, placed in 5 ml methanol, shaken for 30 min and left overnight [9], After centrifugation (3,000 rpm. 10 min), 4 ml of the supernatant was removed and evaporated to dryness ex vacuo. After 0.5 ml of the mobile phase was added and mixed, 20 pi was in­ jected for HPLC analysis. 4-Methylumbelliferone 0.1 ml 100 ng/ml (Wako Co.) was used as an internal standard. Standard samples with different drug con­ centrations were processed in the same way. Standard curves were obtained for the drug/IS ratio (r = 0.9989).

HPLC Conditions. Mobile phase: 50 m.W NaH2P 0 4 (pH 8.0): acetonitrile = 3:1; detection wave­ length: Ex.350 nm, Em. 402 nm; flow rate: 1 ml/min; column: Schim-pack Cs, column temperature, 40 °C [ 10].

Experiment II Ocular anaphylaxis was induced in 32 males al­ bino Sprague-Dawley (180-200 g) rats which were divided into two groups - pretreated with AA-673 0.25% and untreated. Drug Administration and Time Measurement. In the untreated group histamine concentration was measured at 5, 30, 60 and 120 min after antigen chal­ lenge. Pretreated rats received 10 pi AA-673 0.25% ophthalmic solution instilled in the cul-de-sac (right eyes), 5 min prior to antigen stimulation. Sample Processing. Histamine was extracted with n-butanol as previously described [11,12], Bulbar con­ junctival tissue was excised, washed with PBS (pH 7.4) and homogenized in 2 ml ice-cold 0.4 M perchloric acid. After centrifugation (3,000 rpm, 10 min) to 1.5 ml of the supernatant, 1.0 ml 5 A' NaOH and 1.0 ml n-butanol were added. After shaking (15 min) and centrifugation, to 0.8 ml of the organic phase. 0.5 ml of NaCl saturated NaOH as added to remove amino acid residues. After shaking and centrifuging again, 0.6 ml of the organic phase as transferred to a tube containing 0.15 ml 0.1 (V HC1 and 1.5 ml ben­ zene. The mixture was shaken for 5 min. After centrif­ ugation, 0.1 ml of the acidic aqueous phase was added to 40 pi 1 N NaOH. After mixing, 1% OPT (ophthalaldehyde) was added to develop fluorescence. After 4 min, 20 pi 4 N HC1 was added to stop the reaction and stabilize the histamine-OPT derivative. 100 pi was injected for HPLC analysis. Standards were pro­ cessed in a similar manner. The present HPLC assay allows the determination of histamine in conjunctival tissue against an external standard. Linear calibration

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Materials and Methods

Amlexanox Dynamics and Effect on Histamine in Anaphylactic Conjunctiva

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graphs were obtained for peak area and the corre­ sponding initial concentration (r = 0.9970). The mini­ mal amount detected was 60 pg. HPLC Conditions. Mobile phase: methanol: 1 N NaCl = 45:55 (pH 3.0); detection wavelength: Ex. 360 nm, Em. 450 nm; flow rate: 0.7 ml/min; column: Schimpack C8; column temperature: 40 °C. The data were analyzed on a Chromatopac C-3RA (Shimadzu Co.). Figure 2 shows the peak and retention time of histamine under the described conditions. Statistics Statistical analyses employed Student’s paired t test.

Results Experiment I

When comparing the pharmacodynamics of AA-673 0.25% ophthalmic solution the peak concentrations occurred 5 min after in­ stillation of the drug in both groups (table 1). The peak values were 9.55 ± 0.83 pg/100mg tissue wet weight (anaphylactic group) and 8.58 ± 6.39 pg/100 mg tissue wet weight (normal group). Drug levels were followed until 2 h after instillation. Both groups exhibited an exponential decay of drug concentration (fig. 3). The rate con­ stants were 1.008 and 1.5 h~1 (anaphylactic and normal, respectively).

Fig. 2. Chromatogram of histamine. Fig. 3. Dynamics of AA-673 0.25% ophthalmic solution in normal and anaphylactic conjunctiva. ▲ = normal c = 10.2" 001681; • = anaphylactic c = 6.46"00251. Significant difference between values oc­ curred at 30, 60 and 120 min (p < 0.05). Points indi­ cate mean values ± SD. n = 6.

Table 1. Concentrations of AA-673 0.25% ophthalmic solution in normal and anaphylactic conjunctiva

Anaphylactic Normal

Time 5 min

30 min

60 min

120 min

9.55 + 0.83 8.58 + 6.39

5.64 ± 0.91 2.47 ± 1.20

4.03 ±0.91 0.93±0.50

1.32 ± 0.6 0.43 ±0.12

Values expressed as pg/100 mg tissue wet weight. Each value represents the mean ± SD. Significant differ­ ence at 30, 60 and 120 min (p < 0.05)

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Drug concentrations

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Experiment II

In the untreated group histamine levels were generally higher (table 2) and there was a distinct peak in histamine concentration (11.57 ± 3.54 ng/mg tissue wet weight) oc­ curring 30 min after induction of ocular ana­ phylaxis (fig. 4). The pretreated group, in addition to having lower conjunctival hista­ mine levels, did not show any sharp rise. At

Fig. 4. Effect of AA-673 0.25% ophthalmic solu­ tion on histamine during ocular anaphylaxis. ■ = Pre­ treated group; • = untreated group. Significant differ­ ence between values occurred at all time points (p < 0.05). Points indicate mean values ± SD. n = 4.

30 min the histamine concentration in the pretreated group was 4.81 ± 2.29 ng/mg tis­ sue wet weight.

Discussion Therapy for type I IgE-mediated reac­ tions includes avoidance of the inducing an­ tigen, inhibition of mediator release, inhibi­ tion of mediator action, generalized suppre­ sion of inflammation and desensitization immunotherapy. Amlexanox (AA-673) was developed as an agent to inhibit mediator release from the mast cell. It influences hista­ mine release and SRS-A generation by inhib­ iting the lipooxygenase pathway [3, 4]. This distinguishes AA-673 from disodium cromoglycate (DSCG) - another inhibitor of me­ diator release. However, it is not clear whether AA-673 action is mainly due to sup­ pression of SRS-A generation or histamine release. It may be possible that a combina­ tion of these two effects makes the drug 50 times more potent than DSCG in inhibiting IgE-mediated PCA in rats [3]. It is signifi­ cant to note that in this study there was a marked delay in drug elimination in the ana­ phylactic rat conjunctiva. This finding could

Table 2. Concentrations of histamine in pretreated and untreated groups

Untreated Pretreated

Time 5 min

30 min

60 min

120 min

5.62± 1.36 3.59±0.76

11.57 ±3.54 4.81 ±2.29

9.33 ± 1.66 5.13 ± 1.55

7.83±0.46 3.90±0.83

Values expressed as ng/mg tissue wet weight. Each value represents the mean ± SD. Significant difference at all time points (p < 0.05).

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Drug concentration

Amlexanox Dynamics and Effect on Histamine in Anaphylactic Conjunctiva

It should be noted that despite the low conjunctival histamine levels in the pre­ treated group, clinical symptoms such as redness, edema and lacrimation were appar­ ent, implying other crucial factors in the pathogenesis of ocular type I hypersenitivity reactions that require further investigation.

References 1 Lewis, R.: Mast cell dependent immediate hyper­ sensitivity response. Prog. clin. biol. Res. 213: 457-470(1986). 2 Gronneberg, R.: Inhibition of the late phase reac­ tion to anti-IgE by previous mast cell activation with compound 48/80. Allergy 39: 119-123 (1984). 3 Saijo, T.; Kuriki, H.; Ashida, Y.; et al.: Mecha­ nism of action of Amoxanox (AA-673) an orally active antiallergic agent. Int. Archs Allergy appl. Immun. 75: 43-50 (1985). 4 Saijo, T.; Makino, H.; Tamura, S.; et al.: The antiallergic agent Amoxanox suppresses SRS-A generation by inhibiting lipooxygenase. Int. Archs Allergy appl. Immun. 79: 231-237 (1986). 5 Sakuma, Y.; Mita, H.: Inhibitory effect of AA-673 eye drops on ocular provocation for Japanese Ce­ dar pollinosis. Allergy 38: 93-97 (1989). 6 Trocme, S.; Allansmith, M.; Bloch, K.; et al.: Top­ ically induced ocular anaphylaxis in rats immu­ nized with egg albumin. Ophthalmic Res. 18: 6874 (1986). 7 Allansmith, M.; Baird. R.; Greiner, J.; et al.: Late phase reactions in ocular anaphylaxis in the rat. J. Allergy clin. Immunol. 73: 49-55 (1984). 8 Allansmith, M.; Bloch, K..; Baird, R.; et al.: Ocular anaphylaxis: induction by local injection of anti­ gen. Immunology 44: 623-627 (1981). 9 Ogawa, T.; Ohira, M.; Ikejiri, Y.; et al.: Intraocu­ lar penetration of AA-673 ophthalmic solution, an anti-allergic agent. Folia Ophthalmol, jap. 39: 633-637 (1988). 10 Walker, A.; Bloch, K.; Isselbachcr, K.: Immuno­ logic control of soluble protein absorption from the small intestine: A gut surface phenomenon. Am. J. clin. Nutr. 27: 1434-1440 (1974).

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be attributed to the microcirculatory changes that occur during anaphylaxis (platelet aggregation, stasis, etc.). There was a significant difference in histamine concen­ tration between the pretreated and untreated anaphylactic groups. The peak value in the untreated group was observed at 30 min af­ ter inducing anaphylaxis. This peak, as well as the entire time course of histamine con­ centration, corresponds with morphological studies related to conjunctival mast cell de­ granulation [7, 8], However, the effect of AA-673 in this study cannot be explained solely by inhibition of histamine release. Ho­ mogenization in ice-cold perchloric acid de­ stroys membranes, including those of mast cell granules. Since it is well established that histamine formation increases during ana­ phylaxis [13-15], these findings leave open the possibility that AA-673 may inhibit his­ tamine formation capacity, i.e. the rate at which histamine is formed in tissues by his­ tidine decarboxylase [13]. The present study of AA-673 mechanisms differs from pre­ vious experiments in several aspects. Instead of using mast cell culture techniques, an in vivo model of ocular anaphylaxis was em­ ployed, involving topical application of anti­ gen, thereby simulating anaphylactic reac­ tions [6]. Moreover, this study examined conjunctival histamine levels, while the ma­ jority of previous reports utilize rat perito­ neal mast cells. It should be stressed that mast cells vary considerably in histological appearance, biochemical composition and physiological properties. Not only are there significant differences in the mast cell population among different species, but also between tissues within the same species [ 16]. HPLC proved to be a wellsuited technique for tissue histamine mea­ surement.

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11 Tsuruta, Y.; Kohashi K.: Determination of hista­ mine in plasma by high speed liquid chromatogra­ phy. J. Chromatogr. 146: 490-493 (1978). 12 Skofitsch, G.; Saria, A.: Histamine in tissue: De­ termination by HPLC after condensation with ophthaldialdehyde. J. Chromatogr. 226: 53-59 (1981). 13 Schayer, R.: Enzymatic formation of histamine from histidine. Handb. Exp. Pharmacol. 18: 688— 725 (1966). 14 Schayer, R.; Ganley, OH.: Adaptive increase in mammalian histidine decarboxylase activity in re­ sponse to non-specific stress. Am. J. Physiol. 197: 721-724 (1959). 15 Kahlson, G.; Rosengreen, E.: Accelerated hista­ mine formation in hypersensitivity reactions. Lancet i: 782-784 (1966).

Rankov/Sasaki/Fukuda

16 Galli, S.: Mast cell heterogenity: Can variation in mast cell phenotype be explained without postu­ lating the existence of distinct mast cell lineages; in Befus, Bienenstock, Denburg, Mast cell differ­ entiation and heterogenity, pp. 167-181 (Raven Press, New York 1986).

Received: January 9, 1990 Accepted: April 26, 1990 K. Sasaki Department of Ophthalmology Kanazawa Medical University Uchinada, Ishikawa 920-02 (Japan)

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364

Pharmacodynamics of Amlexanox (AA-673) in normal and anaphylactic rat conjunctiva and its effect on histamine concentration.

The pharmacodynamics of Amlexanox (AA-673), an azoxanthone derivative recently developed as an ocular anti-allergic drug, were studied in normal and a...
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