Cetirizine in horses: Pharmacokinetics and pharmacodynamics following repeated oral administration

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The Veterinary Journal The Veterinary Journal 177 (2008) 242–249 www.elsevier.com/locate/tvjl

Cetirizine in horses: Pharmacokinetics and pharmacodynamics following repeated oral administration Lena Olse´n

a,*

, Ulf Bondesson

b,c

, Hans Brostro¨m d, Hans Tja¨lve a, Carina Ingvast-Larsson

a

a

d

Division of Pathology, Pharmacology and Toxicology, Department of Biomedical Sciences and Veterinary Public Health, Swedish University of Agricultural Sciences, SE-750 07 Uppsala, Sweden b Division of Analytical Pharmaceutical Chemistry, Biomedical Center, Uppsala University, SE-751 23 Uppsala, Sweden c Department of Chemistry, National Veterinary Institute, SE-751 89 Uppsala, Sweden Division of Medicine and Surgery, Department of Clinical Sciences, Swedish University of Agricultural Sciences, SE-750 07 Uppsala, Sweden Accepted 28 March 2007

Abstract The pharmacokinetics of the histamine H1-antagonist cetirizine and its effect on histamine-induced cutaneous wheal formation were studied in six healthy horses following repeated oral administration. After three consecutive administrations of cetirizine (0.2 mg/kg body weight, bw) every 12 h, the trough plasma concentration of cetirizine was 16 ± 4 ng/mL (mean ± SD) and the wheal formation was inhibited by 45 ± 23%. After four additional administrations of cetirizine (0.4 mg/kg bw) every 12 h, the trough plasma concentration was 48 ± 15 ng/mL and the wheal formation was inhibited by 68 ± 11%. The terminal half-life was about 5.8 h. A pharmacokinetic/ pharmacodynamic link model showed that the maximal inhibition of wheal formation was about 95% and the EC50 about 18 ng/mL. It is concluded that cetirizine in doses of 0.2–0.4 mg/kg bw administered at 12 h intervals exhibits favourable pharmacokinetic and pharmacodynamic properties without causing visible side effects, and the drug may therefore be a useful antihistamine in equine medicine. Ó 2007 Elsevier Ltd. All rights reserved. Keywords: Horse; Antihistamine; Cetirizine; PK/PD link model

Introduction Histamine is an important chemical mediator, which is involved in the aetiology of various hypersensitivity manifestations via interactions with histamine H1-receptors. Specific IgE antibodies attached to mast cells cross-bind antigens and induce release of histamine and other immunological mediators. This results in type 1 allergic reactions, which are characterised by dilatation of vascular smooth muscle, increased permeability of capillary endothelium and contraction of bronchial smooth muscle. In equine therapy, histamine H1-antagonists (antihistamines) such as tripelennamine, promethazine and chlorphentamine have been used in conditions in which histamine is a critical mediator, such as allergic reactions *

Corresponding author. Tel.: +46 18 67 31 76; fax: +46 18 50 41 44. E-mail address: [email protected] (L. Olse´n).

1090-0233/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.tvjl.2007.03.026

to venoms and other antigens and drug-related immunological reactions, including anaphylactic shock (Morrow et al., 1986; Adams, 1995; Rosenkrantz, 1995; Foster et al., 1998). Antihistamines may also be useful to treat the syndrome called ‘‘sweet itch’’, ‘‘summer eczema’’ or ‘‘allergic urticaria’’. This is a worldwide chronic seasonally recurrent pruritic allergic skin disease in horses hypersensitive to antigens in the saliva of biting flies, in particular midges of the genus Culicoides, and sometimes also black flies from the genus Simulium (Baker and Quinn, 1978; Braverman et al., 1983; Quinn et al., 1983; Brostro¨m et al., 1987; Greiner et al., 1988, 1990; Hallorsdottir et al., 1989; Fadok and Greiner, 1990; Anderson et al., 1991, 1993; Marti et al., 1999; Mullens et al., 2005; Baselgia et al., 2006). It is known that IgE plays a role in the pathogenesis of this syndrome (Matthews et al., 1983; Van der Haegen et al., 2001; Wilson et al., 2001, 2006; Ru¨fenacht et al.,

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2005; Hellberg et al., 2006; Wagner et al., 2006; Ferroglio et al., 2006). IgE and mast cell activation are also involved in the pathogenesis of equine heaves, chronic bronchitis/ bronchiolitis, chronic obstructive pulmonary disease (COPD) and recurrent airway obstruction (RAO). These syndromes show similar clinical signs, but can be mediated by varying immunological mechanisms (McGorum et al., 1993; Halliwell et al., 1993; Schmallenbach et al., 1998; Franchini et al., 1998; Olszewski et al., 1999; Eder et al., 2000; Bowles et al., 2002; Van der Haegen et al., 2005; Horhov et al., 2005). Antihistamines may also be useful in treating these conditions. Cetirizine, a metabolite of hydroxyzine, is a non-sedative second-generation histamine H1-antagonist, widely used in human medicine to treat seasonal/perennial allergic rhinitis and chronic idiopathic urticaria (Zuberbier and Henz, 1999; Simons, 2001). In man, cetirizine is well tolerated and has been shown not to alter memory, attention, alertness or performance (Pagliara et al., 1998; Benedetti et al., 2001; Curran et al., 2004; Theunissen et al., 2004, 2006). Cetirizine also displays high and selective affinity for cloned human H1-histamine receptors and it has very little or no anti-cholinergic activity (Gillard et al., 2002; Orzechowski et al., 2005). We have recently studied the pharmacokinetics of cetirizine in horses given a single oral dose of the drug (Olse´n et al., 2007). The result showed that cetirizine had a favourable pharmacokinetic profile. Previously examined antihistamines, such as clemastine and fexofenadine, were shown to have very low oral bioavailability (To¨rneke et al., 2003; Olse´n et al., 2006). The aim of the present study was to examine the pharmacokinetics and the antihistaminic effect of cetirizine in horses following repeated oral administrations. Materials and methods Horses Six healthy Standardbred trotters (mares), 5–21 years old, weighing 490–570 kg were used. They were fed hay, straw, sugar-beet and oats during the experiment. Water was available ad libitum. The study protocol was approved by the Animal Ethics Committee, Uppsala, Sweden (C6503).

Study design For oral administration, racemic cetirizine dihydrochloride (Cetirizin Biochemie, tablets 10 mg) were mixed with sugar-beet and given to the horses. Cetirizine was administered at times 0, 12, 24, 36, 48, 60 and 72 h (i.e. at 12 h intervals). For the first three administrations the dose was 0.2 mg/kg body weight (bw), and for and the last four administrations the dose was 0.4 mg/kg bw. Blood samples (10 mL; collected in heparinised test tubes) were taken by direct jugular venepuncture at time 0 (pre-dose), and immediately prior to the administrations at time-points 12, 24, 36, 48, 60 and 72 h (i.e. just before the administration of a new dose). Following the last oral administration (at 72 h) blood was also taken via an intravenous catheter at the post-administration intervals 0.5, 1, 1.5, 2, 3, 5, 7, 11, 24 and 30 h. The blood was centrifuged at 1500 g for 15 min and the plasma was separated and stored at 20 °C until analyses.

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Recording of the pharmacodynamic effect The antihistaminic effect of cetirizine was recorded using an intradermal test. Intra-dermal skin testing is an accepted method for detecting allergen hypersensitivity in atopic humans. Determination of the inhibition of cutaneous reactions following intra-dermal histamine-injections is used to estimate the effect of antihistamines. The protocol applied in the present study was adopted from investigations in humans and has been optimised to horses and used in several previous studies as well as in allergen hypersensitivity detections at horse clinics in Sweden (Simons et al., 1993; To¨rneke et al., 2003; Olse´n et al., 2006; K. Bergwall, personal communication). The horses were shaved with electric clippers on the lateral neck prior to intra-dermal injections with 7 lg/site of histamine hydrochloride (0.1 mg/mL, Apoteket AB) using 27-gauge needles. The diameter of the skin reaction was measured after 20 min using Vernier callipers. Histamine-injections before administration of cetirizine served as positive controls. Histamine-injections were then performed immediately before each of the consecutive 12 h administrations of cetirizine and in addition at 1, 3, 5, 7, 11 and 24 h after the last cetirizine-administration. Three injections were performed on each occasion and the median value was used for further calculations. Sterile saline (0.07 mL served as a negative control. The negative control mean value was calculated from at least 11 administrations for each horse.

Analysis of cetirizine Cetirizine dihydrogen chloride (Sigma-Aldrich) and internal standard [2H4]-cetirizine (C/D/N isotopes) were used as reference standards. Stock solutions of cetirizine and [2H4]-cetirizine were prepared in methanol– water (50:50) and stored at 20 °C until used. The water was of Millipore quality and had a resistance of 10.0 MX cm. All other solvents and reagents were at least of analytical grade. The protocol was adopted from Olse´n et al. (2007). Briefly, [2H4]-cetirizine (9.6 ng base in 100 lL methanol) was added to each plasma sample (1.0 mL), which after the addition of 0.2 M sodium acetate buffer pH 4.0 (1.0 mL) was applied to Bond Elut C18 solid phase extraction columns (3 mL, 200 mg sorbent mass). The columns were sequentially preconditioned with methanol (2 mL), deionised water (2 mL) and 0.2 M sodium acetate buffer pH 4.0 (1.5 mL) and washed with deionised water (2 mL), methanol–water (50:50, v/v, 2 mL) and methanol (1 mL). After a drying period of 2 min, cetirizine and [2H4]-cetirizine were eluted from the columns with 50 mM triethylamine in methanol (2.5 mL) and the eluates were evaporated to dryness under nitrogen. The extraction procedure was performed by Gilson ASPEC XL4 instrumentation (Pretech). The residue in each vial was reconstituted in 100 lL of methanol and 0.1 M acetic acid in water (4:6, v/v). The reconstituted samples were quantified by liquid chromatography electrospray tandem mass spectrometry (LC–ESI-MS/MS) using a Finnigan TSQ Quantum Ultra mass spectrometer (Thermo Electron Corporation), with a Surveyor MS pump. The separation was performed with a Zorbax Eclipse XDB C18 (Agilent) chromatographic column (length 50 mm, inner diameter 2.1 mm and particle diameter 5 lm). The samples were eluted using a gradient created by mixing phases from two reservoirs comprising respectively of 0.1% acetic acid in water and methanol. The injection volume was 5.0 lL and the volumetric flow-rate 0.2 mL/min. The ionisation technique was electrospray (ESI) in positive mode. The ESI source voltage was set at 3.5 kV and sheath gas flow-rate and auxiliary gas were 50 and 2 arbitrary units, respectively. When running the collisioninduced dissociation (CID) procedure, argon was used as collision gas at a pressure of 1.5 mTorr. For the Selected Reaction Monitoring (SRM) mode, the following transitions were recorded: cetirizine [M+H+] m/z 389 ! 201 and [2H4]-cetirizine [M+H+] m/z 393 ! 201 using collision energy 25 V. Different concentration-intervals in the calibration curve were used for different samples: one for 0.11–11 ng/mL and one for 11– 179 ng/mL. For evaluation of the accuracy, the quantification precision as percentage relative standard deviation (RSD) and the linearity of the method quality control (QC) samples in three different concentrations (1.0,

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8.1 and 108 ng/mL) were prepared by spiking blank plasma. The accuracy was 97–101%; RSD varied between 1.9% and 5.2% (n = 10 for each QC level); the correlation was 0.9994–0.9997. The standards and the QC samples were treated in the same way as the plasma samples obtained from the horses given cetirizine.

Statistical analysis For pharmacodynamic data an ANOVA was used followed by Dunnet’s post hoc test, comparing wheal areas before and after treatment. The significance level was set at 0.95 (P < 0.05). The software was Minitab, release 14 (Minitab Ltd.).

Pharmacokinetic calculations For each horse, all plasma concentrations of cetirizine were plotted versus time and the data were analysed using the software program Win Nonlin 5.0.1 (Pharsight Corporation). Different models and weighting factors were assessed by visual inspection of the curve fits and the residuals’ scatter plots, and combined with the goodness of fit measures incorporated in the software, including the Akaike Information Criterion and Schwartz criteria, to decide the most suitable model. The data were analysed and best fitted to a two-compartment model with first-order input, first-order output using the equation: CðtÞ ¼ Aðeat Þ þ Bðebt Þ þ CðeK01t Þ where C(t) is the plasma concentration at time t, K01 is the absorption rate and a, b the declining rates. The appropriate weighing factor was found to be 1/Y, were Y is the observed concentration. The primary pharmacokinetic parameters were calculated from the yaxis intercepts (A  C) and the exponents of the respective phase (a, b and K01). Secondary parameters calculated for the 0.2 mg/kg dose (1st administration) were the area under the plasma concentration–time curve (AUC) from time zero to infinity, the time (Tmax0.2) to obtain the maximal plasma concentration (Cmax0.2) and the Cmax0.2. The a- and b-half-lives were calculated by dividing ln 2 by a and b, respectively. The apparent total body clearance (Cl/F) was determined by dividing the dose by the AUC. The apparent volume of distribution (V/F) was determined by dividing Cl by the declining rate b. After the last administration of cetirizine (0.4 mg/kg bw, at 72 h) the Tmax0.4 and Cmax0.4 were read from the plotted concentration–time curve of each individual animal. Also the trough plasma concentrations were read from the plotted concentration– time curves. Median and range or means and standard deviations (SD) were calculated for all investigated parameters.

Pharmacodynamic calculations The diameters of each wheal were determined by taking the mean of two perpendicular diameters. The wheal areas were then calculated and expressed as percentage inhibition of wheal formation. For each horse the effect of cetirizine on wheal areas were calculated by comparing the effect of histamine before and after administration of the drug. 0% inhibition was defined as the wheal area after histamine injection only (before administration of cetirizine) and 100% inhibition as the wheal area after injection of sterile saline.

Pharmacokinetic/pharmacodynamic link model A simultaneous pharmacokinetic/pharmacodynamic (PK/PD) link model was applied using pharmacodynamic data from a concentration/ response equation and pharmacokinetic data from the two-compartmental model. Using the PK/PD link model, the maximal effect (Emax), which is the plasma concentration producing 50 % of the maximal effect (EC50), and the rate constant for the elimination of drug from the effect compartment (Ke0) were calculated (Toutain et al., 1994; Toutain and Lees, 2004). At equilibrium, the rates of drug transfer between the central and effect compartment are equal. The concentration/response equation used was E ¼ ðEmax  C ce Þ=ðC ce þ EC c50 Þ where Ce is the apparent effect compartment concentration. Gamma (c) is the slope (Hill coefficient) expressing the sigmoidicity of the concentrationeffect relationship.

Results No visible adverse reactions, such as sedation or gastrointestinal disturbances, were seen in any of the treated horses. It was possible to determine the concentration of cetirizine in plasma for 30 h after the last administration. The main pharmacokinetic parameters obtained are presented in Table 1. The repeated oral administrations of cetirizine caused a significant inhibition of the histamine-induced wheal areas (ANOVA, P < 0.0001; Fig. 1a). The maximal observed inhibition of wheal formation was 84 ± 7% and occurred 5 h after the last administration. At 11 h after the last administration, the inhibition still remained >55% (Dunnet’s post hoc test, P < 0.05). In Fig. 1b the plasma concentrations of cetirizine are plotted. The trough (minimum) plasma concentration of cetirizine at 36 h, (i.e. after three doses of 0.2 mg/kg bw) was 16 ± 4 ng/mL (mean ± SD). At this time the wheal formation was inhibited by 45 ± 23% (mean ± SD). The trough plasma concentration of cetirizine at 72 h (i.e. after three doses of 0.4 mg/kg bw) was 48 ± 15 ng/mL. At this

Table 1 Pharmacokinetic variables and parameters calculated from drug plasma concentrations using a two-compartmental model after repeated oral administration of cetirizine at three initial doses of 0.2 mg/kg bw at timepoints 0, 12 and 24 h, followed by four additional doses of 0.4 mg/kg bw at time-points 36, 48, 60 and 72 h (n = 6) Parameter

AUCinf (ng h/mL) Cmax0.2 (ng/mL) Tmax0.2 (h) Cmax0.4 (ng/mL)a Tmax0.4 (h)a t1/2K01 (h) t1/2a (h) t1/2b (h) Cl/F (L/h/kg) V/F (L/kg)

Cetirizine Median

Range

379 58 0.8 132 1.0 0.23 0.07 5.8 0.49 2.9

347–453 40–76 0.7–1.0 118–168 – 0.12–0.65 0.06–0.24 4.1–5.9 0.44–0.58 1.2–3.7

AUC: area under the concentration time curve from time 0 to infinity for a 0.2 mg/kg bw dose (1st administration); Cmax0.2: peak plasma concentration for 0.2 mg/kg bw; Tmax0.2: time to peak plasma concentration for 0.2 mg/kg bw dose; Cmax0.4: peak plasma concentration for the 0.4 mg/kg bw dose; Tmax0.4: time to peak plasma concentration for the 0.4 mg/kg bw dose; t1/2K01 absorption half-life; t1/2a: distribution half-life; t1/2b: terminal elimination half-live; Cl/F: apparent total body clearance; V/F: apparent volume of distribution. a Values read from the plotted concentration–time curve after the last administration.

Effect (%)

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Discussion

100 90 80 70 60 50 40 30 20 10 0

___________ a _____________

0

12

24

36

48

60

72

84

96

108

72

84

96

108

Time (h)

Conc (ng/mL)

245

160 140 120 100 80 60 40 20 0 0

12

24

36

48

60

Time (h)

Fig. 1. Six horses were given cetirizine orally at an initial dose of 0.2 mg/ kg bw and this dose was repeated after 12 and 24 h. The dose was then increased to 0.4 mg/kg bw at 36, 48, 60 and 72 h. (a) Histamine-induced inhibition of wheal area formation in the horses given cetirizine (mean ± SD). The intra-dermal test with histamine (0.7 lg/injection site) was conducted immediately before each administration of cetirizine and at 1, 3, 5, 7, 11, and 24 h after the last administration of cetirizine. 100 % effect designates total inhibition of the wheal formation. aSignificant differences compared with controls (P < 0.05). (b) Plasma concentrations in the horses given cetirizine (mean ± SD). Frequent blood samples were taken only after the last administration (at 72 h). The indicated cetirizine levels determined at 12–72 h represent the trough plasma concentrations immediately before each successive cetirizine dose.

time the wheal formation was inhibited by 68 ± 11%. It was possible to link the pharmacodynamic effect versus time profile to two-compartment pharmacokinetic model (Table 2, Fig. 2).

Desired clinical properties of an antihistamine are a fast onset of action, sufficient oral bioavailability, long duration of action, high affinity for the H1-receptor, adequate therapeutic effect, no or few side effects and low inclination to participate in drug interactions. These properties are partly related to the pharmacokinetic parameters. The short absorption half-life (0.23 h) and the short time to reach peak plasma concentration (Tmax 0.8 h) show that cetirizine is rapidly absorbed in the horses. A rapid absorption is often correlated with a rapid onset of action. With regard to the observed half-life and the observed duration of the antihistaminic effect we consider that two daily administrations of cetirizine would be suitable in horse. Although the absolute bioavailability was not assessed, our results indicate that, in contrast to the previously examined antihistamines clemastine and fexofenadine (To¨rneke et al., 2003; Olse´n et al., 2006), the oral bioavailability of cetirizine is sufficient in horses. At a pH between 3.5 and 7.5 cetirizine exists nearly exclusively as a zwitterion, which in addition appears to form partial intramolecular charge neutralisation in folded conformers of lower polarity (Pagliara et al., 1998). This implies that cetirizine probably to a considerable extent is absorbed by passive diffusion. Studies in man have shown that after oral administration of cetirizine the urinary excretion of the unchanged drug during 24 h was about 60% in adults and about 40% in children (Desager et al., 1993). Lefebvre et al. (1988) reported about 65% urinary excretion of unchanged cetirizine during 32 h in young adults, whereas elderly subjects excreted 60% during this period. Studies with the Renantiomer levocetirizine have shown that there is an almost complete absorption after oral administration and

Table 2 Simultaneous pharmacokinetic/pharmacodynamic link model in horses given cetirizine orally Parameter

Cetirizine Median

Range

Emax (%) EC50 (ng/mL) c Ke0 (h1) t1/2Ke0 (h)

95 18 1.5 2.7 0.3

66–100 8–36 0.8–3.6 0.5–9.9 0.1–1.4

The parameters are calculated from drug plasma concentrations and effects at each time point for each horse (n = 6). Emax: maximum effect; EC50: plasma concentration of cetirizine at steady state conditions required to produce one-half of the drug induced maximal effect; c: slope of line, shape parameter expressing the sigmoidicity of the concentration-effect relationship; Ke0 rate constant of drug loss from the effect compartment; t1/2Ke0 half-life of the drug loss from the effect compartment.

Fig. 2. Results obtained in the simultaneous PK/PD link model. Mean plasma concentrations (s = observed concentrations; - - - = predicted concentrations) and effects on histamine-induced cutaneous wheal formation (e = observed effects; — = predicted effects) in six horses given cetirizine orally. The symbols represent the observed effects or the observed concentrations; the lines connecting the symbols are the predicted values calculated from the PK/PD model. The y-axis represents both the concentration (ng/mL) and the effect (%). A 100% effect designates total inhibition of the wheal formation. The dose of cetirizine at time 0, 12 and 24 h was 0.2 mg/kg bw and at time 36, 48, 60 and 72 h 0.4 mg/kg bw.

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about 86% of the parent drug is excreted in the urine during 48 h (Benedetti et al., 2001). Our results showed low inter-individual variability in plasma concentrations in the horses. This is a characteristic for drugs which have limited biotransformation. With reference to this observation one may assume that cetirizine in horse, like in man, is metabolised to a relatively low extent. This is advantageous for an antihistamine since it minimises metabolic drug–drug interactions. The apparent volume of distribution (V/F) in the present study ranged from 1.2 to 3.7 L/kg which is considerably larger than in man where the volume (V/F) is 0.49–0.7 L/ kg (Simons et al., 1987, 1993; Desager et al., 1993). In the present study the horses showed apparent Cl/F of 7.3–9.6 mL/min/kg. This is higher than in man where there are reports of Cl/F from 0.5 to 1.5 mL/min/kg (Lefebvre et al., 1988; Desager et al., 1993; Simons et al., 1993; Urien et al., 1999). These data indicate that in horse there is a more incomplete F and/or a large Vd in combination with a high Cl compared to man. In the present study cetirizine was given at 12 h intervals to the horses mixed with sugar-beet and there was no restriction of the food-supply during the experiment. In our previous study (Olse´n et al., 2007), cetirizine was given as a single dose via a nasogastric tube and the horses were not fed 12 h before and 3.5 h after the administration (although they were bedded on straw). The results of the present study showed that after a dose of 0.2 mg/kg bw the calculated median plasma concentration (Cmax) was 58 ng/mL, which is lower than in the previous study (132 ng/mL) when cetirizine was given as a single oral dose of 0.2 mg/kg bw. On the other hand similar values were observed in the two studies for the calculated AUC. In addition the calculated median terminal half-life in the present study (5.8 h) was longer than in our previous study (3.4 h). These data indicate that food present in the stomach and in the proximal intestine will delay the absorption of cetirizine and influence the Cmax and the terminal halflife (flip-flop phenomena), but the impact of the total extent of the absorption is of minor importance. This phenomenon also enlarges the value of V/F. Cetirizine in doses of 0.2 and 0.4 mg/kg were adequate to significantly reduce the histamine-induced cutaneous wheal formation. The PK/PD link model showed that cetirizine has a high efficacy, the maximal inhibition of wheal formation being about 95% and the inhibitory effect remaining over 55% for at least 11 h after the last dose. Also the potency was high, the EC50-value for the antihistaminic effect being about 18 ng/mL. The other antihistamines examined in horses at our department, clemastine and fexofenadine, show EC50 values of 3.3 ng/mL and 16 ng/mL, respectively (To¨rneke et al., 2003; Olse´n et al., 2006). In humans, Gillard et al. (2005) reported that inhibition of histamine-induced wheal formation by cetirizine was 100% at 4 h and 60% at 24 h, at plasma concentrations of 28 and 4 nM (11 and 1.5 ng/mL), respectively. The EC50 value for the wheal inhibition was not calculated in that

study. Devalia et al. (2001) reported a maximal inhibition of wheal formation in humans of 79%, occurring 6 h after the administration of cetirizine. In that study the measured plasma concentrations were approximately 78 ng/mL at 4 h and 53 ng/mL at 8 h after dosage. De Vos et al. (1987) reported that the doses of cetirizine reducing histamine-induced skin-reactions by 50% (ED50) were 4.2 lmol/kg bw (1.6 mg/kg) in rats, 0.14 lmol/kg (0.05 mg/kg) in mice and 0.40 lmol/kg (0.16 mg/kg) in guinea pigs. Studies by Urien et al. (1999) in humans given an oral dose of 10 mg (approximately 0.14 mg/kg) of cetirizine showed an EC50 for wheal inhibition of about 40 ng/mL and an Emax of about 60% 3–5 h after administration. In dogs cetirizine inhibited the histamine-induced skin-reactions by 49–80% (at 2–24 h) at a dose of 0.22 lmol/kg (0.85 mg/kg). In the dogs this dose also inhibited the immediate hypersensitivity reaction induced by Ascaris extracts, which contain histamine and also other inflammatory/ allergenic agents (De Vos et al., 1987). In the present study the slope factor (Hill coefficient) was 1.9 (median and range 0.8–3.6). Steep slope (c > 1) indicates good selectivity i.e. the drug could achieve full efficacy without significant side effects (Toutain and Lees, 2004). Cheng et al. (2006) showed that cetirizine inhibits the release of granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-8 (IL-8) secretion in human airway epithelial cells. It was therefore suggested that the drug may exert clinically relevant effects in inflammatory airways disorders in man. Cetirizine has also a down-regulating effect on tumour necrosis factor alpha (TNF-a)induced hyperactivation of the nuclear factor kappa-B (NF-jB) in human endothelial cells and in a human endothelial pulmonary cell line, indicating that the drug may interfere in a central key mechanism in the cell trafficking and bronchial inflammation (Rihoux et al., 1999). It is known that there is an increased amount of IL-8 and interferon gamma (INF-c) in the airway secretion in the chronic phase of COPD and RAO in horses (Franchini et al., 1998, 2000; Gigue`re et al., 2002; Ainsworth et al., 2003, 2006; Laan et al., 2006). Increased levels of both TNF-a and NF-jB activities have been shown in horses susceptible to heaves (Bureau et al., 2000a,b; Gigue`re et al., 2002). These reports may imply that cetirizine could be used in horses for the treatment of disorders such as heaves, COPD and RAO. In our study no side effects were seen in any of the horses. Cetirizine has a wide safety margin in humans and has no or very minor anti-cholinergic effects (Orzechowski et al., 2005). This is an important property for a drug which potentially may be used in horses. The main side effects of the first-generation antihistamines are sedation, drowsiness and impaired performance. Cetirizine, like other second-generation antihistamines, enters the brain only to a low extent and has low incidence of these side effects (Snyder and Snowman, 1987; Pagliara et al., 1998; Tashiro et al., 2004). P-glycoprotein (P-gp) mediated

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efflux at the blood–brain barrier has been shown to limit the brain exposure of ceterizine (Chen et al., 2003; Polli et al., 2003). In our previous study (Olse´n et al., 2007) it was shown that a 12 h pre-treatment interval the antiparasitic macrocyclic lactone ivermectin increases the AUC of orally administered cetirizine by 60%. This effect may be related to the decreased urinary secretion of cetirizine due to ivermectin-induced inhibition of P-gp in the proximal tubular cells in the kidney. In that study no clinical side effects were seen in any of the horses pre-treated with ivermectin. Conclusions Our data show a favourable pharmacokinetic profile of cetirizine in horses. Oral administrations of the drug in doses of 0.2–0.4 mg/kg given at 12 h intervals are adequate to significantly reduce histamine-induced cutaneous wheal formation. Cetirizine may therefore be a useful antihistamine in equine medicine. Acknowledgements This study was supported by the Swedish Foundation for Equine Research and by the Hippocampus program of the Swedish University of Agricultural Sciences. References Adams, R.H., 1995. Histamine, serotonin, and their antagonists. Veterinary Pharmacology and Therapeutics, seventh ed. Iowa State University Press, pp. 401–411. Ainsworth, D.M., Gru¨nig, G., Matychak, M.B., Yong, J., Wagner, B., Erb, H.N., Antczak, D.F., 2003. Recurrent airway obstruction (RAO) in horses is characterized by IFN-gamma and IL-8 production in bronchoalveolar lavage cells. Veterinary Immunology and Immunopathology 96, 83–91. Ainsworth, D.M., Wagner, B., Franchini, M., Gru¨nig, G., Erb, H.N., Tan, J.Y., 2006. Time-dependent alterations in gene expression of interleukin-8 in the bronchial epithelium of horses with recurrent airway obstruction. American Journal of Veterinary Research 67, 669– 677. Anderson, G.S., Belton, P., Kleider, N., 1991. Culicoides obsoletus (Diptera: Ceratopogonidae) as a causal agent of Culicoides hypersensitivity (sweet itch) in British Columbia. Journal of Medical Entomology 28, 685–693. Anderson, G.S., Belton, P., Kleider, N., 1993. Hypersensitivity of horses in British Columbia to extracts of native and exotic species of Culicoides (Diptera: Ceratopogonidae). Journal of Medical Entomology 30, 657–663. Baker, K.P., Quinn, P.J., 1978. A report on clinical aspects and histopathology of sweet itch. Equine Veterinary Journal 10, 243– 248. Baselgia, S., Doherr, M.G., Mellor, P., Torsteinsdottir, S., Jermann, T., Zurbriggen, A., Jungi, T., Marti, E., 2006. Evaluation of an in vitro sulphidoleukotriene release test for diagnosis of insect bite hypersensitivity in horses. Equine Veterinary Journal 38, 40–46. Benedetti, M.S., Plisnier, M., Kaise, J., Maier, L., Baltes, E., Arendt, C., McCracken, N., 2001. Absorption, distribution, metabolism and excretion of [14C]levocetirizine, the R enantiomer of cetirizine, in healthy volunteers. European Journal of Clinical Pharmacology 57, 571–582.

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