Introduction

The nonsedating histamine H1 receptor antagonist (HRA)fexofenadine is the active metabolite of terfenadine andappears to have a better safety profile compared with terfe-nadine [1]. Recently, fexofenadine has been shown to inhibitthe eosinophil-induced release of IL-8, GM-CSF and solubleICAM-1 from isolated human nasal epithelial cells [2]. OtherHRA have been shown to be partial inhibitors of differentFceRI+, histamine-containing cell types [3]. The aim of thepresent study was to investigate the direct effects of fexofe-nadine on the activation of isolated basophil leukocytes toevaluate putative the additional “antiallergic” activity of thecompound.

Materials and methods

All experiments were performed in HEPES buffer or RPMI 1640 [4–6].Anti-IgE antibodies and human recombinant IL-3 (all from Sigma, Deisenhofen, Germany) were employed as basophil secretagogues aspreviously described [4–6]. Fexofenadine hydrochloride was kindlydonated by Hoechst Marion Roussel, Bad Soden, Germany, and wasdissolved in warm (37 °C) ethanol (10 mmol/l) and further diluted (logsteps) in warm (37 °C) buffer. Controls with equivalent serial dilutionsof ethanol did not affect any assay.

Human basophils were isolated from buffy coats of healthy donors[5, 6]. In experiments, where IL-4 production was assessed, basophilswere further purified [4] to a final purity of 70–90%. In a further set ofexperiments, blood was drawn from donors suffering from atopic der-matitis. All donors had a total IgE level of more than 1000 kU/l.

Cells (5 ¥ 105 cells /ml) were resuspended in warm buffer (37 °C)and equilibrated for 10 min at 37 °C. Cells were then incubated for 15min at 37 °C with a serial dilution of fexofenadine before being stimu-lated with anti-IgE (final concentration 0.01% of stock solution) for 30 min. In priming experiments, incubation with fexofenadine was fol-lowed by pretreatment with IL-3 (10 ng/ml) for 10 min and subsequentactivation with anti-IgE antibodies (0.005% of stock solution) for a fur-ther 30 min. All other methods were performed as described before [5, 6].

Histamine release was measured spectrofluorometrically in bothsupernatants and lysed pellets (Technicon Autoanalyzer II, Bran &Luebbe, Hamburg, Germany). IL-4 release was measured using anELISA (Immunotech, Marseille, France).

Protein kinase C (PKC) was isolated from rat brain extracts and par-tially purified as described [5]. In vitro inhibitory activity of fexofena-dine on PKC was determined using a modified cell-free PKC-specificassay system as previously published [5].

Statistical analysis was performed using a Student’s t test for pairedand unpaired data, differences being considered significant when theprobability (p) was p < 0.05 and highly significant when p was < 0.02.

Results and discussion

When isolated human basophils from healthy donors wereincubated with increasing concentrations of fexofenadine, aninhibition of IgE-mediated histamine release was observed ata concentration achieved with normal therapeutic dosing [1; Dr. Höhler, Bad Soden, Germany, personal communica-tion 1999] (Fig. 1). Statistical significance was reached at a

Inflamm. res. 49, Supplement 1 (2000) S13–S141023-3830/00/010S13-02 $ 1.50+0.20/0

© Birkhäuser Verlag, Basel, 2000

Inflammation Research

In vitro studies with fexofenadine, a new nonsedating histamine H1

receptor antagonist, on isolated human basophilsU. Amon1, S. Amon1, B.F. Gibbs2

1 PsoriSol Centre for Dermatology and Allergy, Mühlstrasse 31, 91217 Hersbruck, Germany, Fax +49 9151 72 94 19, e-mail: Amon@psorisol.de2 Medical University of Lübeck, Department of Dermatology, Ratzeburger Allee 160, 23538 Lübeck, Germany

Correspondence to: U. Amon

Fig. 1. Effects of fexofenadine on IgE-mediated histamine release fromhuman basophils. Results are presented as mean values ± SEM (n = 6).Mean control release (anti-IgE-induced histamine release withoutfexofenadine) was 56.3 ± 4.7%. Mean spontaneous histamine releasewas 0.9 ± 0.5 %. ** Represents p < 0.02 showing significant inhibitionof control release.

concentration of 10 mmol/l. Fexofenadine also inhibited theIgE-mediated histamine release from basophils isolated frompatients with acute exacerbation of atopic dermatitis in aconcentration-dependent fashion (Table 1). Maximum inhibi-tion was 84.6 ± 5.5% at 10 mmol/l compared with control.However, the drug did not significantly influence the release of IL-4 after stimulation via the FceRI (Table 1). No signifi-cant modulation of IL-3 primed basophils [7] was observed(Table 1). The gene family of PKC isozymes has been demon-strated to be responsible for many diverse and central cellu-lar functions. These include biochemical events following theaggregation of high-affinity IgE receptors on mast cells andbasophils [5]. To investigate whether fexofenadine exerts itsadditional “antiallergic” effects in vitro via modulation ofPKC, the drug was incubated with the isolated enzyme.However, a broad range of concentrations did not signifi-cantly alter the in vitro activity of PKC (data not shown).

The study demonstrates that fexofenadine, as the mainmetabolite of terfenadine, also partially inhibits direct activa-tion of isolated human basophils. The mechanism by whichHRA inhibit mediator release is still unknown. Both mem-brane stabilising effects and regulation of calcium mobilisa-tion and influx have been suggested [3]. We and others havehypothesised that a membrane stabilising activity is respon-sible for the “antiallergic” effects observed in in vitro experi-ments with HRA [3, 8]. PKC as a central element of signaltransduction is not inhibited by fexofenadine. Additionalexperiments focusing on other activation mechanisms of allergic effector cells are necessary to further evaluate theantiallergic profile of fexofenadine. However, inhibition ofICAM-1 expression [2], in combination with strong antihis-taminergic effects, as well as suppression of IgE-mediatedactivation of basophils, can explain the documented clinicalefficacy of the drug in urticaria and allergic rhinitis [1].

References

[1] Markham A, Wagstaff AJ. Fexofenadine. Drugs 1998; 55: 269–74.[2] Abdelaziz MM, Devalia JL, Khair OA, Bayram H, Prior AJ, Davies

RJ. Effect of fexofenadine on eosinophil-induced changes in epithe-lial permeability and cytokine release from nasal epithelial cells ofpatients with seasonal allergic rhinitis. J Allergy Clin Immunol1998; 101: 410–20.

[3] Okayama Y, Church MK. Drugs modifying the response of mastcells and basophils. In: Foreman JC, editors. Immunopharmacologyof mast cells and basophils. London: Academic Press, 1993:139–52.

[4] Gibbs BF, Vollrath IB, Albrecht C, Amon U, Wolff HH. Inhibitionof interleukin-4 and interleukin-13 release from immunologicallyactivated human basophils due to the actions of anti-allergic drugs.Naunyn-Schmiedeberg’s Arch Pharmacol 1998; 357: 573–8.

[5] Amon U, Dieckmann D, Nitschke M, Gibbs BF, Wolff HH. Hetero-geneity of signal transduction mechanisms in human basophils andhuman skin mast cells. I. Pharmacological investigations withactivators and inhibitors of protein kinase C. Skin Pharmacol 1996;9: 211–20.

[6] Noll T, Dieckmann D, Gibbs BF, Nitschke M, Albrecht C, VollrathI, et al. Heterogeneity of signal transduction mechanisms in humanbasophils and human skin mast cells. Biol Signals 1997; 6: 1–10.

[7] Nitschke M, Sohn K, Dieckmann D, Gibbs BF, Wolff HH, Amon U.Effects of basophil-priming and stimulating cytokines on histaminerelease from isolated human skin mast cells. Arch Dermatol Res1996; 288: 463–8.

[8] Ramachers U, Hiller C, Nitschke M, Dieckmann D, Zhang MQ,Timmerman H, et al. In vitro Untersuchungen mit Terfenadin undverschiedenen Derivaten zur Mediatorfreisetzung aus mensch-lichen Hautmastzellen, eosinophilen und basophilen Granulozyten.Allergo J 1996; 5: 447–52.

S14 Inflamm. res., Supplement 1 (2000)

Table 1. Effects of fexofenadine on immunologically induced mediator release from human basophils. Results are presented as mean values ± SEMfor the number of experiments noted.

Fexofenadine IgE-mediated histamine release (%) IgE-mediated histamine release (%) IgE-mediated IL-4 release (pg/106 cells)(mmol/l) from basophils of atopic patients from IL-3 primed basophils from basophils

0.001 36.02 ± 9.28 68.43 ± 3.06 355.55 ± 178.580.01 34.97 ± 7.11 76.42 ± 7.05 387.78 ± 187.740.1 35.51 ± 7.02 60.11 ± 3.17 337.21 ± 193.791 28.06 ± 6.66 62.48 ± 1.93 336.85 ± 169.4110 8.01 ± 2.73 34.98 ± 10.92 271.15 ± 136.57Positive control 1 42.92 ± 8.94 71.13 ± 7.02 367.51 ± 193.22Spontaneous release 20.12 ± 8.72 1.13 ± 1.13 0.84 ± 0.84n 6 3 6

1 The following positive controls were used: anti-IgE antibodies (0.01% of stock solution) for histamine release from basophils of atopic patients orIL-4 release from basophils of healthy donors, anti-IgE (0.005%) plus IL-3 (10 ng/ml) for histamine release from primed basophils.