Preclinical Pharmacology of Bilastine,??a New Selective Histamine??H1 ??Receptor Antagonist

Drugs R D 2005; 6 (6): 371-384ORIGINAL RESEARCH ARTICLE 1174-5886/05/0006-0371/$34.95/0

2005 Adis Data Information BV. All rights reserved.

Preclinical Pharmacology ofBilastine, a New SelectiveHistamine H1 Receptor AntagonistReceptor Selectivity and in Vitro Antihistaminic Activity

Reyes Corcostegui, Luis Labeaga, Ana Innerarity, Agustin Berisa and Aurelio Orjales

Department of Research, FAES FARMA, SA, Leioa, Spain

Objective: This study aimed to establish the receptor selectivity and antihistamin-Abstractic activity of bilastine, a new selective antihistamine receptor antagonist.Design and methods: In vitro experiments were conducted using a receptorbinding screening panel and guinea-pig and rat tissues. Antihistaminic activitywas determined using H1 receptor binding studies and in vitro H1 antagonismstudies conducted in guinea-pig tissues and human cell lines. Receptor selectivitywas established using a receptor binding screening panel and a receptor antago-nism screening conducted in guinea-pig, rat and rabbit tissues. Inhibition ofinflammatory mediators was determined through the Schultz-Dale reaction insensitised guinea-pig ileum.Results: Bilastine binds to histamine H1-receptors as indicated by its displace-ment of [3H]-pyrilamine from H1-receptors expressed in guinea-pig cerebellumand human embryonic kidney (HEK) cell lines. The studies conducted on guinea-pig smooth muscle demonstrated the capability of bilastine to antagoniseH1-receptors. Bilastine is selective for histamine H1-receptors as shown inreceptor-binding screening conducted to determine the binding capacity of bilas-tine to 30 different receptors. The specificity of its H1-receptor antagonisticactivity was also demonstrated in a series of in vitro experiments conducted onguinea-pig and rat tissues. The results of these studies confirmed the lack ofsignificant antagonism against serotonin, bradykinin, leukotriene D4, calcium,muscarinic M3-receptors, α1-adrenoceptors, β2-adrenoceptors, and H2- and H3-receptors. The results of the in vitro Schultz-Dale reaction demonstrated thatbilastine also has anti-inflammatory activity.Conclusions: These preclinical studies provide evidence that bilastine has H1-antihistamine activity, with high specificity for H1-receptors, and poor or noaffinity for other receptors. Bilastine has also been shown to have anti-inflam-matory properties.

372 Corcostegui et al.

N

N

CH2CH2OCH2CH3

N (CH2)2 C COOHCH3

CH3

Fig. 1. Chemical structure of bilastine.

F-96221-BM1, bilastine (2-[4-(2-(4-(1-(2-ethox- Many first-generation antihistamines had the dis-advantage of having sedative effects. Although theyethyl) bencimidazole-2-yl) piperidine-1-yl) ethyl)newer, second-generation antihistamines are gener-phenyl]-2-methylpropanoic acid), is a new antihista-ally devoid of this adverse effect, some are associat-mine drug currently in clinical development byed with adverse cardiovascular effects.[2]FAES FARMA, SA (Spain), for symptomatic treat-

This paper describes the preclinical pharmacolo-ment of seasonal or perennial allergic rhinitis andgy of bilastine with respect to its receptor selectivitychronic idiopathic urticaria. Phase III studies ofand in vitro antiallergic effects, includingbilastine are currently being conducted. Its chemicalH1-antagonism and anti-inflammatory activities.structure is shown in figure 1.

Histamine is a major clinical mediator in the Materials and Methodspathophysiology of the allergic response.[1] Hista-mine is stored in mast cells and basophilic granulo-

Antihistaminic Activitycytes, and is released by non-immunological andimmunological stimuli. After its release, the effects

H1-Receptor Binding Studiesof histamine are mediated through actions on a

H1-Receptor Binding in Guinea-Pig Cerebellum[3]multiplicity of receptors, including the histamine

Adult male guinea pigs weighing 400–600g wereH1-, H2-, H3- and H4- receptors. The biologicalused. The animals were killed by decapitation andeffects of histamine related to the H1-receptors in-the whole brain including the cerebellum was quick-clude, among others, smooth muscle contraction,ly removed and subsequently dissected, weighedbronchospasm, increased vascular permeability, andand frozen at –70ºC. Cerebellum that was used forstimulation of sensory nerves and cough receptors.the binding experiments was homogenised with an

Histamine H1-receptor antagonists are the main Ultra-Turrax (setting 5 for 20 sec) in 30 volumes ofpharmacological agents used in the treatment of ice-cold 50mM phosphate buffer (pH 7.5) and cen-allergic conditions, preventing histamine from ex- trifuged at 30 000g for 10 minutes (4ºC). The result-erting some of these inflammatory effects. In addi- ing pellet was resuspended in 30 volumes of ice-tion to blocking the interaction of histamine with the cold 50mM phosphate buffer (pH 7.5), and thenH1-receptors, some antihistamines can inhibit the centrifuged three more times at 30 000g for 10 min-release of histamine or other inflammatory utes (4ºC). The final pellet was resuspended in 10mediators, which contributes to their antiallergic volumes of ice-cold 50mM phosphate buffer (pHeffects. Antihistamines are considered first-line 7.5) and was stored at –70ºC until use. At the time oftreatment for immunoglobulin E-related allergic the experiment, the membranes were diluted in theconditions, including mild, intermittent seasonal or same ice-cold buffer (final dilution 1 : 80, weight/perennial allergic rhinitis as well as mast cell volume). Competition assays were performed indegranulation-mediated diseases, such as chronic triplicate, in a final volume of 1mL. Each assay tubeidiopathic urticaria. contained the following: 0.1mL of the different con-

2005 Adis Data Information BV. All rights reserved. Drugs R D 2005; 6 (6)

Bilastine: Receptor Selectivity and Antihistaminic Preclinical Studies 373

centrations of the displacer drugs (0.1mL of vehicle from IC50 values by the Cheng and Prusoff equa-if no competing drug was added) and 0.1mL of [3H]- tion.[6]

pyrilamine (NEN, 23 Ci/mmol) in buffer (final con-In Vitro H1-Antagonism Studiescentration 1nM). Nonspecific binding was deter-

mined using 10µM of cold astemizole. H1-Antagonism in Guinea-Pig IleumThe binding experiment was initiated by addition Fragments of distal ileum (2–3cm length) were

of 0.8mL of membrane suspension (600µg of prote- obtained from adult male guinea pigs (after cervicalin/mL). After incubation for 30 minutes at 25ºC, the dislocation and exsanguination) and vertically sus-reaction was stopped for vacuum filtration through pended from isotonic lever transducers (HSE TypeWhatman GF/B glass filters, using a Brandel Cell B 368) under 1.2g resting tension in a 20mLHarvester, followed by two washes with 5mL of ice- Hoechst-type organ bath containing Tyrode solutioncold 50mM phosphate buffer (pH 7.5). Filters were at 37ºC and oxygenated with carbogen (95% O2, 5%placed in scintillating polyethylene vials (with 5mL CO2). The physiological solution was changed sev-scintillation cocktail), equilibrated, and filter-re- eral times during a 45- to 60-minute equilibrationtained radioactivity measured in a liquid scintilla- period before addition of the drugs. Contractionstion counter (Kontron Beta V). IC50 (50% inhibitory were recorded using a Watanabe 3101 polygraph.concentration) and Ki (inhibition constant) values For each ileum fragment, consecutive histaminewere calculated using the computer program EBDA (10nM–328µM) cumulative concentration-responseas described by McPherson.[4] For each drug, 3–6 curves were obtained until uniformity in the re-independent experiments were performed. sponse was attained. After washing, the preparation

was allowed to equilibrate for 30 minutes and thenH1-Receptor Binding in Human H1-Histamine different concentrations of the tested drugs (bilas-Receptor Expressed in Human Cells[5]

tine, cetirizine and fexofenadine; one per fragment)Human embryonic kidney (HEK) 293 cells were

were added to the bath 30 minutes before histaminestably transfected with the human recombinant

cumulative concentration-response curves wereH1-receptor clone. Competition assays were per-

again recorded. Bilastine was assayed at 30, 100,formed in duplicate using [3H]-pyrilamine as radi-

300 and 1000nM final concentrations. Drug para-oligand at 3nM final concentration. Nonspecific

meters (pA2 [log [CR – 1] – log [B], where [B] is thebinding was determined using 1µM cold pyrilamine.

molar concentration of the antagonist and CR is theBinding conditions were as follows: 60 minutes

concentration ratio of the EC50 values in the pres-incubation period and 22°C temperature. Filter-re-

ence and absence of antagonist) and pD′2 (log [ER –tained radioactivity was measured in a liquid scintil-

1] – log [B], where [B] is the molar concentration oflation counter.

the antagonist and ER is the effect ratio of the EmaxThe specific ligand binding to the receptors was value in the absence and presence of antagonist)

defined as the difference between the total binding were calculated by standard methods.[7,8] For eachand the nonspecific binding determined in the pres- drug, 3–4 independent experiments were performed.ence of an excess of unlabelled ligand. The results

H1-Antagonism in Guinea-Pig Tracheaare expressed as the percentage of control specificbinding obtained in the presence of bilastine. The Adult male guinea pigs were killed by cervicalIC50 values were determined by nonlinear regres- dislocation and one segment of the trachea wassion analysis of the competition curves using Hill dissected. Next, tracheal strips were prepared ac-equation curve fitting. Ki values were computed cording to Constantine.[9] The strips were mounted



in a 20mL Hoechst-type organ bath containing microsurgery instruments. The spiral was placed inKrebs-Henseleit solution pH 7.4 and maintained at a 20mL Hoechst-type organ bath containing Krebs-37ºC, and oxygenated with carbogen. Subsequently, Henseleit solution pH 7.4 oxygenated with carbogenthey were attached to a force displacement isometric and maintained at 37ºC. The upper ends of thetransducer (FS30) coupled to a Watanabe 3101 re- preparations were attached with fine surgical suturecorder. For each tracheal strip, consecutive hista- thread to isometric transducers (F30) such that themine (0.1µM–1mM) cumulative concentration-re- circular vascular musculature was parallel to thesponse curves were constructed until a homogenous thread attached to the transducer, which was thenresponse was achieved. After washing, the prepara- connected to a linear recorder (WR3310). After thetion was allowed to equilibrate for 30 minutes and preparations were mounted, a tension of 0.5g wasthen different concentrations of the test compounds applied and adjusted every 15 minutes after each(bilastine, cetirizine and fexofenadine; one per strip) wash.were added to the bath 30 minutes before histamine Following 60-minute stabilisation, a 10µM con-cumulative concentration-response curves were centration of serotonin (5-HT) was added to verifyagain recorded. Bilastine was assayed at 30, 100 and the contractile potency of the preparation. After300nM final concentrations. Drug parameters (pA2 recovering the original tension (minimum 30 min-and pD’2) were calculated by standard methods.[7,8] utes), two cumulative curves separated by 60 min-For each drug, 3–4 independent experiments were utes were constructed with serotonin, which wasperformed. added in a geometric progression by a factor of three

in concentrations ranging from 10nM to 0.3µM untilReceptor Selectivity Studies the maximal effect was attained. Finally, a specific

concentration of the test drugs (bilastine, cetirizine,fexofenadine and ketanserin) was added and follow-Receptor Binding Screeninging 15-minute incubation new cumulative curves forAn in vitro profiling screen was performed toserotonin were recorded. On completion of the as-examine the specificity of the binding capacity ofsay, the most significant parameters were deter-bilastine to a range of 30 different receptors using amined depending on the type of antagonism ob-customised Pharmacology Screening technique de-served.[7,8] For each drug, 3–4 independent experi-veloped by MDS Pharma Services. Specific radio-ments were performed.ligands were used for each receptor in competition

with bilastine 10µM. The percentage of displace-Bradykinin Antagonism in Guinea-Pig Ileum

ment was subsequently determined.The method used was similar to that described in

detail above. Briefly, segments of guinea-pig ileumSerotonin Antagonism in the Rat Caudal Artery

were placed in a 20mL tray containing a solution ofWistar rats weighing 250–300g and sacrificed byTyrode pH 7.4 oxygenated with carbogen and main-asphyxia in an atmosphere of CO2 and exsangui-tained at 37ºC. The segments were allowed tonated by carotid artery section were used in thestabilise for 30–45 minutes under an effectiveexperiment. The upper end of the caudal artery waspreload of 1.2g. The contractions induced by addi-then located and cannulated with a fine wire (0.3mmtion of bradykinin in a cumulative mannerdiameter) lubricated with petrolatum. Following(1.25nM–2.5µM) were recorded isotonically.careful removal of the connective tissue adhering to

the artery, a spiral of about 2cm in length and 2mm When homogenous bradykinin curves werein width (one per artery) was obtained using achieved, the test compounds (bilastine, cetirizine



Calcium Antagonism in Guinea-Pig Ileumand fexofenadine) were added at a concentrationThe method used was similar to that described inof 0.1mM. Concentration-response curves for

detail above. Briefly, segments of guinea-pig ileumbradykinin were recorded following a 10-minutewere placed in a 20mL tray containing a calcium-incubation period. After completing the assay, char-free Tyrode solution (EDTA 88.8 mg/L) pH 7.4acteristic parameters were calculated depending onoxygenated with carbogen and maintained at 37ºC.the type of antagonism observed.[7,8] For each drug,The tissues were allowed to stabilise for 30 minutes3–4 independent experiments were performed.under an effective preload of 1.2g. This solution was

Leukotriene D4 Antagonism in Guinea-Pig Trachea then replaced by a potassium-rich depolarisingThe method used was similar to that described in Tyrode solution (KCl 120nM, NaCl 20mM), main-

detail above. Briefly, guinea-pig trachea spirals taining the ileum in solution for another 15 minuteswere placed in 20mL trays containing Krebs-Hen- with solution changes every 5 minutes. The contrac-seleit solution pH 7.4 oxygenated with carbogen and tions induced by cumulative added calcium chloridemaintained at 37ºC. To this solution, 1µM (CaCl2 ) [10µM–20mM] were then recorded isoton-diclofenac sodium was added to inhibit pros- ically. When the maximal effect was attained, thetaglandin synthesis via cyclo-oxygenase (COX-1). preparations were washed with calcium-free TyrodeAfter attaching the upper end to an F30 isometric for 15 minutes and then a further 15 minutes withtransducer, the spiral was submitted to an initial depolarising Tyrode (all washes were performedtension of 2g and allowed to stabilise for 60–120 every 5 minutes). A new cumulative curve wasminutes with solution changes and tension adjust- constructed with CaCl2.ments every 15 minutes. After this period, two suc- When uniform control curves were obtained, bi-cessive contractions separated by 60 minutes were lastine was added at final concentrations of 30µMinduced with 20µM histamine to apply the appro- (n = 2) or 100µM (n = 3). Following 15 minutes’priate amplification factor. After a new 60-minute incubation, the concentration-response curve forstabilisation period and recovery of baseline values, CaCl2 was recorded again. The experiment was alsoa single concentration-response curve was recorded conducted with cetirizine and fexofenadine. Afterfor leukotriene (LT)-D4 added in a cumulative man- completing the assay, characteristic parameters werener in concentrations ranging from 0.1–100nM in calculated depending on the type of antagonismthe presence of 3mM of L-cysteine in order to observed.[7,8]

prevent conversion of LTD4 to LTE4. The contrac-tile responses obtained with each concentration of Muscarinic M3-Antagonism in Guinea-Pig Ileum

LTD4 were expressed as a percentage of the maxi- The method used was similar to that described inmum response obtained with 20µM histamine. detail above. Briefly, segments of guinea-pig ileum

The curve of LTD4 was performed in the pres- were placed in 20mL trays containing a solution ofence of the study compounds or their vehicles and Tyrode pH 7.4 oxygenated with carbogen and main-following a 30-minute incubation period. Bilastine, tained at 37ºC. The tissues were allowed to stabilisecetirizine and fexofenadine were tested at a concen- for 30–45 minutes under an effective preload oftration of 100µM each. For each drug, 3–4 indepen- 1.2g. The contractions induced by acetylcholine ad-dent experiments were performed. After completing ded in a cumulative manner (2.5nM– 0.1µM) werethe assay, the most representative parameters were recorded isotonically. When uniform acetylcholinecalculated depending on the type of antagonism curves were obtained, the test compounds (bilastine,observed.[7,8] cetirizine and fexofenadine) were added at different



concentrations (one per ileum segment and 15-min- were placed in 20mL trays containing Krebs-Hen-ute incubation) and concentration-response curves seleit solution pH 7.4 oxygenated with carbogen andfor acetylcholine were recorded. After completing maintained at 37ºC. Diclofenac sodium (1µM) wasthe assay, characteristic parameters were determined then added to inhibit prostaglandin synthesis viadepending on the type of antagonism observed.[7,8] cyclo-oxygenase (COX-1). The spiral was submit-For each drug, 3–4 independent experiments were ted to an initial tension of 1g and allowed to stabiliseperformed. for 120 minutes with solution changes every 15

minutes. After this period and every 60 minutes, theα1-Adrenoceptor Antagonism in Rabbit

contractions induced by 20µM acetylcholine wereThoracic Aortarecorded isometrically until a homogenous responseMale New Zealand rabbits, weighing 2–2.5kgwas achieved. When the maximal effect was at-and anaesthetised and exsanguinated prior to remov-tained (around the fourth or fifth contraction), relax-al of the thoracic aorta, were used in this experiment.ation curves with isoproterenol were recorded in theAfter removal of any adhering tissue, aorta rings ofabsence (control curve) or presence (15-minute in-3–4mm in length were obtained and placed in acubation) of bilastine, cetirizine and fexofenadine.20mL Hoechst-type organ bath containing Krebs-Bilastine was added at a concentration of 100µM.Henseleit solution pH 7.4 oxygenated with carbogenAfter completing the assay, the most representativeand maintained at 37ºC. Ascorbic acid (0.113mM)parameters were calculated depending on the type ofand 1µM of the nonspecific β-adrenoceptor antago-activity observed.[7,8] For each drug, 3–4 indepen-nist propranolol were added to the Krebs-Henseleitdent experiments were performed.solution. The rings were allowed to stabilise for

2 hours under a tension of 2g with solution changesHistamine H2-Antagonism in Guinea-Pigevery 15 minutes. After this period, 30µM of theRight Atriacatecholamine reuptake inhibitor cocaine was addedSpontaneously beating right atria obtained fromto the tray and the contractions induced by cumula-

tive addition of noradrenaline were recorded isomet- Dunkin Hartley guinea pigs were placed in a 20mLrically, starting at 1nM (geometric progression by a Hoechst-type organ bath containing Krebs-Hense-factor of 2) until the concentration-response curve leit solution pH 7.4 oxygenated with carbogen andwas obtained. A second concentration-response maintained at 37ºC. Tissues were allowed tocurve was recorded 1 hour later. stabilise for 45 minutes under an initial tension

The test drugs (bilastine and cetirizine) were of 1g. The positive chronotropic effect of anadded 15 minutes before performing the third con- H2-specific agonist, dimaprit, added cumulatively tocentration-response curve. Bilastine was tested at a the tray, was then isometrically recorded by routingconcentration of 100µM. After completing the as- the signal to a heart-rate coupler. When uniformsay, the most significant parameters were calculated dimaprit curves were obtained, the test compoundsdepending on the type of antagonism observed.[7,8] were added at different concentrations (30-minuteFor each drug, 3–4 independent experiments were incubation) and concentration-response curves forperformed. dimaprit were constructed.

Bilastine was tested at a concentration of 100β2-Adrenoceptor Antagonism inGuinea-Pig Trachea µmol/L (n = 4). After completing the assay, charac-

teristic parameters were calculated depending on theThe method used was similar to that described ineffect observed. Cetirizine was also tested at a simi-detail above. Briefly, guinea-pig trachea spirals



lar concentration. Fexofenadine was tested at 100 Inhibition of Inflammatory Mediators

and 200µM.

Antiallergic Effects in Immediate-TypeHistamine H3-Antagonism inHypersensitivity: Schultz-Dale Reaction in

Electrically-Stimulated Guinea-Pig Jejunum Sensitised Guinea-Pig IleumFragments of guinea-pig jejunum (2–3cm in Male guinea pigs were immunised by intraper-

length) were placed in a 20mL Hoechst-type organ itoneal injection of 1 mg/mL of ovoalbumin solutionbath containing Krebs-Henseleit solution pH 7.4 and aluminium hydroxide saline solution at days 1with carbogen and maintained at 37ºC. Mepyramine and 7. After 7–12 days, animals were exsanguinated

and the ileum was removed. Fragments of ileum(1µM), an H1-antagonist, was added to avoid inter-(20mm, 3–4 per guinea pig) were maintained atference from histamine H1-receptor activation. Tis-37°C in Tyrode solution at pH 7.4, constantly oxy-sues were submitted to an initial tension of 1g andgenated with carbogen. Next, the fragments were

allowed to stabilise for 60 minutes, with solutionexposed to 1µM histamine until contractions of ho-

changes and tension adjustment every 15 minutes. mogeneous intensity were recorded.The contractions induced by electrical stimulation

Bilastine at different concentrations (30, 100, 300(0.1Hz, 0.5mA, maximum voltage [15 ± 3v]) by and 1000nM) was then added and only one contrac-F2H field electrodes were isometrically recorded tion with ovoalbumin (0.5 µg/mL, final concentra-(F30). Once a uniform contractile response (twitch) tion) was registered, comparing its intensity with the

histamine-induced contraction detected in the samewas attained (approximately 30 min), the selectivefragment. A similar test was conducted using ce-H3-agonist (R)-α-methyl-histamine (1nM–10µM)tirizine and fexofenadine. For each drug, 2–3 inde-or bilastine (100µM) were added and their agonistpendent experiments were performed. The IC50 val-

effect recorded. If the twitch was inhibited by bilas-ues were determined graphically by linear regres-

tine, the curve was repeated in the presence of a sion analysis.[10]

selective H3-antagonist (thioperamide 10nM,

15-minute incubation). These concentration-re-Drugssponse curves were repeated following a rest period

(no stimulation) of 30 minutes. It was then ascer-Bilastine was synthesised by FAES FARMA,tained whether bilastine 30µM (15-minute incuba-

SA, and used dissolved in 0.1M aqueous tartariction) was able to significantly displace the twitchacid. Cetirizine was obtained from Impex Quimicareduction curve registered for (R)-α-methyl-hista-(Barcelona, Spain), and was dissolved in distilled

mine. Agonistic values were calculated as pD2 (–logwater. Fexofenadine was extracted from marketed

EC50, where EC50 is the concentration of the agonist Telfast (Laboratorios Marion Merrell SA, Madrid,that produces 50% of its maximum response and Spain)1 and was dissolved in methanol. Agonists,antagonistic values as pA2 or pD′2).[7,8] For each radioligands and reference drugs were obtaineddrug, 3–4 independent experiments were performed. from commercial sources.

1 The use of trade names is for product identification purposes only and does not imply endorsement.



Results

Antihistaminic Activity

H1-Receptor Binding Studies

H1-Receptor Binding in Guinea-Pig Cerebellum

Bilastine inhibited in a dose-dependent manner

the specific binding of [3H]-pyrilamine in the exper-

iments conducted in guinea-pig cerebellum, with

mean Ki and pKi values of 44.15 ± 6.08nM and 7.37

± 0.06nM, respectively. The Ki values obtained

from the radioligand-receptor binding assays are

depicted in figure 2.

H1-Receptor Binding in Human H1-Histamine

Receptor Expressed in Human Cells

Bilastine exerted a dose-dependent and specific

inhibition of [3H]-pyrilamine binding to human re-

100

80

60

40

20

0

Spe

cific

bin

ding

(%

)

10-9 10-8 10-7 10-6 10-5

Ki = 44.15 ± 6.08nMpKi = 7.37 ± 0.06

mmol/L

H1 antagonist (number of experiments)

Bilastine (n = 4)

Cetirizine (n = 4)

Ketotifen (n = 3)

Pyrilamine (n = 3)

Diphenhydramine (n = 3)

Fexofenadine (n = 4)

Astemizole (n = 2)

Ki ± SEM (nM)

44.15 ± 6.08

143.12 ± 16.35

0.46 ± 0.041

0.90 ± 0.065

25.28 ± 1.51

246 ± 40.7

30.9 ± 1.6

pKi

7.37

6.85

9.34

9.05

7.60

6.62

7.51

Fig. 2. Histamine H1-receptor binding in guinea pig cerebellum:determination of Ki and pKi values for bilastine, cetirizine, ketotifen,pyrilamine, diphenhydramine, fexofenadine and astemizole.

combinant histamine H1-receptors. The IC50 valuesIn Vitro Histamine H1-Antagonistic Activity Studiesfor bilastine and pyrilamine (reference compound)

were 180nM and 2.8nM, respectively. Ki values of Human H1-Antagonism in Guinea-Pig Ileum64 ± 1.15nM and 1 ± 0.07nM were calculated for Bilastine exhibited mixed H1-antagonist beha-bilastine and pyrilamine, respectively (figure 3). viour in this test: competitive up to 33nM and non-

100

120

80

60

40

20

0

Spe

cific

bin

ding

(%

)

10-9 10-8 10-7 10-6 10-5 10-4

IC50 = 180nMKi = 64nM

Bilastine (mol/L)

Fig. 3. Histamine H1-receptor binding in human H1-receptors expressed in human cells: determination of Ki value for bilastine. IC50 = 50%inhibitory concentration.



Histamine H1-Antagonism in Guinea-Pig Tracheacompetitive from 100nM (pA2 = 8.0, pD′2 = 6.2).Bilastine exhibited non-competitive H1-antagon-Cetirizine showed identical but less potent beha-

ism in this test (pD′2 = 7.1). Cetirizine showedviour (pA2 = 7.3, pD′2 = 5.2) with dominance of theidentical but less potent behaviour (pD′2 = 6.3).competitive over the non-competitive componentFexofenadine showed a dominant non-competitive(figure 4). Bilastine was approximately 5.5 timescomponent starting from 30nM (pD′2 = 6.9, data notmore potent than cetirizine as a competitive antago-shown). The results for bilastine and cetirizine arenist and approximately 10 times more potent thanexpressed graphically in figure 5.cetirizine as a non-competitive antagonist. Fexo-

fenadine showed a dominant non-competitive com-Receptor Selectivity Studiesponent starting from 100nM (pD′2 = 6.2, data not

shown).Receptor Binding Screening

An in vitro profiling screen was performed toexamine the specificity of the binding capacity ofbilastine at a range of common receptor sites. The

Con

trac

tion

(%)

100

80

60

40

20

0

10-8 10-7 10-6 10-5 10-4 10-3

Control30 nmol/L100 nmol/L300 nmol/L1000 nmol/L

100

80

60

40

20

0

10-8 10-7 10-6 10-5 10-4 10-3

Control100 nmol/L300 nmol/L1000 nmol/L

b

a

Histamine (mol/L)

pA2 = 7.3pD'2 = 5.2

pA2 = 8.0pD'2 = 6.2

Spe

cific

bin

ding

(%

)

Fig. 4. Histamine H1-receptor antagonism in guinea-pig ileum: de-termination of pA2 and pD′2 values for (a) bilastine and (b) ce-tirizine.

Con

trac

tion

(%)

100

80

60

40

20

0

10-7 10-6 10-5 10-4 10-3

Control0.03 mmol/L0.1 mmol/L0.3 mmol/L

a

pD'2 = 7.1

100

80

60

40

20

0

10-7 10-6 10-5 10-4 10-3

Control0.1 mmol/L0.3 mmol/L1 mmol/L

b

pD'2 = 6.3

Histamine (mol/L)

Fig. 5. Histamine H1-receptor antagonism in guinea-pig trachea:determination of pD´2 values for (a) bilastine and (b) cetirizine.



results are shown in table I. Bilastine selectively and α1- and β2-adrenoceptors (table II). In general,binds to the histamine H1-receptor, since no binding cetirizine and fexofenadine presented a similar re-to other receptors was observed. ceptor antagonism profile to bilastine, and showed

only a weak competitive antagonist behaviour (pA2Receptor Antagonism Screening

= 4.9 and 5.2, respectively, for the 5-HT2A receptorsAt a concentration of 100µM, bilastine did not

from rat caudal artery). Moreover, the calcium ant-significantly modify the concentration-response

agonism showed by cetirizine and fexofenadine incurves induced by serotonin, bradykinin, LTD4, cal-

depolarised guinea-pig ileum contracted by calciumcium chloride, acetylcholine, noradrenaline andchloride was slightly higher than that of bilastineisoproterenol in different isolated organs from rats,(table II). In all isolated organ preparations the spe-guinea pigs and rabbits. Therefore, bilastine did notcific antagonists used as reference drugs showed theshow antagonistic activity on 5-HT2A receptors,highest pA2 values characteristic of their potency asbradykinin B1-receptors, LTD4 receptors, N-type

voltage-dependent calcium receptors, M3-receptors, competitive antagonists.

Table I. Receptor binding capabilities of bilastine using a customised in vitro profiling screening test

Receptor Ligand Tissue Displacement (%) 10µM

Adenosine A1 [3H]DPCPX Rat brain 7

Adenosine A2 [3H]CGS-21680 Rat striatum 5

α1-Adrenoceptor [3H]Prazosin Rat brain 35

α2-Adrenoceptor [3H]Rauwolscine Rat cerebral cortex 15

β1-Adrenoceptor [125H]Cyanopindolol Recombinant, human 7

β2-Adrenoceptor [3H]CGP-12177 Recombinant, human –1

Calcium channel (L) [3H]Nitrendipine Rat cerebral cortex –13

Dopamine D1 [3H]SCH23390 Recombinant, human –2

Dopamine D2L [3H]Spiperone Recombinant, human –4

Estrogenic [3H]Estradiol Cow uterus 7

GABA-A agonist [3H]Muscinol Rat brain 21

GABA-A chloride [3H]TBOB Rat cerebral cortex –7

Glucocorticoid [3H]Dexamethasone Jurkat cells, human –7

Phencyclidine [3H]TCP Rat cerebral cortex 2

Glutamate, non-selective [3H]L-Glutamate Rat brain –9

Glycine, strychnine [3H]Strychnine Rat medulla –14

Insulin [3H]Insulin Rat liver –3

Muscarinic M2 [3H]NMS Recombinant, human 4

Muscarinic M3 [3H]NMS Recombinant, human 8

Opiate δ [3H]DPDPE Guinea-pig brain 17

Opiate κ [3H]U-69593 Guinea-pig brain –11

Opiate µ [3H]DAMGO Guinea-pig brain 4

Phorbol ester [3H]PDBu Mouse brain 18

Potassium channel [3H]Gliburide Hamster, HIT-T15 11

Progesterone [3H]R-5020 Cow uterus 3

5-HT1 [3H]5-HT Rat cerebral cortex 19

5-HT2 [3H]Ketanserine Rat brain 11

Sigma-1, non-selective [3H]DTG Guinea-pig brain 20

Sodium channel, site 2 [3H]Batracotoxine Rat brain 25

Testosterone [3H]Mibblerone Rat prostate 2



Table II. Receptor antagonism screening: determination of pA2 values for bilastine, cetirizine and fexofenadine in different isolated organassays

Agonist Receptor Tissuea Antihistamine drug Specific antagonist

bilastine cetirizine fexofenadine

Serotonin 5-HT2A Rat caudal artery <4.0 4.9 5.2 Ketanserin pA2 = 9.6

Bradykinin B1 Guinea-pig ileum <4.0 <4.0 <4.0

Leukotriene D4 LTD4 Guinea-pig trachea <4.0 <4.0 <4.0 LY-171883 pA2 = 6.8

Calcium chloride N-type Guinea-pig ileum <4.0 4.1 4.8 Verapamil pA2 = 8.4

Acetylcholine M3 Guinea-pig ileum <4.0 <4.0 <4.0 Atropine pA2 = 8.9

Noradrenaline α1 Rabbit thoracic aorta <4.0 <4.0 Prazosin pA2 = 8.8

Isoproterenol β2 Guinea-pig trachea <4.0 <4.0 <4.0 Propranolol pA2 = 8.7

a See Material and Methods section.

Histamine H2-Antagonism in Guinea-Pig nificantly displaced the concentration-responseRight Atria curve of α-methyl-histamine, and therefore theyAt a concentration of 100µM, bilastine lacked

significant H2-antagonist activity, as the concentra-tion-response curve for dimaprit was not significant-ly altered (figure 6). Similar results were obtainedwith cetirizine and fexofenadine. A slight reductionin heartbeat frequency was induced by the additionof the three compounds at a concentration of 100µM(37%, 13% and 15%, respectively) [data notshown].

Histamine H3-Antagonism inElectrically-Stimulated Guinea-Pig Jejunum

Neither bilastine, cetirizine nor fexofenadine at aconcentration of 30µM (15-minute incubation) sig-

100

80

60

40

20

0

-20

-10

Chr

onot

ropi

sm in

crea

se

10-730 min 10-4 10-310-510-6

Control0.1 mmol/L

Dimaprit (mol/L)

Fig. 6. Lack of significant histamine H2-receptor antagonistic activi-ty in guinea-pig right atria.

Twitc

h re

duct

ion

(%)

60

a

50

30

40

20

10

0

10-9 10-8 10-7 10-6 10-5

Control10 nmol/L

pA2 = 8.7

60

70

b

50

30

40

20

10

0

10-9 10-8 10-7 10-6

Control30 nmol/L

(R)a-methyl-histamine (mol/L)

Fig. 7. Lack of significant histamine H3-receptor antagonism inelectrically stimulated guinea-pig jejunum with (a) thioperamidecompared with (b) bilastine.



ed an intermediate affinity for H1-receptors. Ascompared with other H1-antagonist compounds, theH1-receptor affinity observed for bilastine was in-ferior compared with that calculated for ketotifenand pyrilamine, similar to that of astemizole anddifenhydramine, and superior to that of cetirizineand fexofenadine. In addition, the results of bindingexperiments conducted on human recombinantH1-receptors expressed in HEK 293 cells demon-strated that bilastine shows similar affinity values.

Although the binding studies did not determinethe agonist or antagonist properties of a compound,the studies performed using isolated organs demon-strated the capability of bilastine to antagoniseH1-receptors. The experiments conducted onsmooth muscle (guinea-pig ileum and trachea)

100

80

60

40

20

0

OA

inhi

bitio

n

10-8 10-7 10-6 10-5

BilastineCetirizineFexofenadine

Antihistamine drug (mmol/L)

Compund

Bilastine

Cetirizine

Fexofenadine

IC50 (nM)

95.5

759

282

Fig. 8. Schultz-Dale reaction in sensitised guinea-pig ileum: IC50

(50% inhibitory concentration) values for bilastine, cetirizine andfexofenadine. OA = ovoalbumin.

showed that bilastine inhibits, with a moderate tolacked H3-antagonist activity in this experimental potent strength, the contractions induced by hista-model (pA2 values <4.5) [figure 7]. The pA2 value mine, an effect mediated by the H1-receptors locatedfor the H3-/H4-antagonist thioperamide was 8.7 (fig- in those tissues.ure 7). The calculated pA2 and pD′2 values required to

inhibit the ileum and trachea contractions showedInhibition of Inflammatory Mediators

that bilastine has a mixed H1-antagonistic behaviour(competitive and non-competitive). In guinea-pig

Antiallergic Effects in Immediate-Typeileum, bilastine showed a remarkable residual ef-Hypersensitivity: Schultz-Dale Reaction infect, since after consecutive washes of the prepara-Sensitised Guinea-Pig Ileum

tion, histamine did not recover its initial contractileIn the absence of treatment, the response toability (in the absence of bilastine). Cetirizine andovoalbumin was 97.96 ± 2.46% (n = 26) of that

recorded for histamine 1µM. Bilastine, in the con- fexofenadine, H1-antagonists that were used in thecentration range analysed, exhibited a potent and experiment as reference compounds, showed a mix-concentration-dependent anti-anaphylactic activity, ed antagonism, but were less potent in this respectwith an IC50 value of 95.5nM. The results for bilas- than bilastine.tine, fexofenadine and cetirizine are shown in figure Mast cells contain a variety of vasoactive and8. bronchoconstrictor mediators that can induce, after

their release, a rapid alteration in pulmonary func-Discussion

tion and a prolonged inflammatory response. Therelease of these mediators, which include amongBinding studies conducted on H1-receptors in theothers histamine and leukotrienes, synthesised andguinea-pig cerebellum membranes showed that bi-stored in the tissue mast cells, is called mast celllastine significantly displaces the [3H]-pyrilaminedegranulation. This is a complex process mediatedradioligand. The calculated mean pKi value indicat-



by calcium ions, changes in phospholipid metabo- did not significantly displace other radioligandslism and modifications in the cAMP/cGMP ratio. used in the experiment.

Experimental analysis of mast cell degranulation In guinea-pig ileum, bilastine, at concentrationsis difficult because the response to degranulation up to 10mM, was unable to diminish the contrac-stimuli is highly variable based on animal species tions induced by acetylcholine, and it therefore lacksand cell type selected for the experiment. Other M3-antagonist activity. The experiment also re-factors such as the environmental pH and the solu- vealed that cetirizine and fexofenadine also lackedbility of the drug also alter this response. Under anticholinergic activity in this preparation.these conditions, bilastine significantly reduced the Experiments conducted using rabbit thoracic aor-response of sensitised guinea-pig ileum against the ta rings showed that bilastine and cetirizine, atcontractions induced by the antigen (ovoalbumin) in 100µM, do not significantly shift the concentration-the in vitro Schultz-Dale reaction. From the results response curve for noradrenaline, and therefore itof this experiment it can be concluded that bilastine can be concluded that both compounds lack affinityis three to eight times more potent than fexofenadine for vascular α1-adrenoceptors. Likewise, neither bi-and cetirizine, respectively. The anti-inflammatory lastine nor cetirizine nor fexofenadine, at the sameproperties of bilastine will be described in detail in concentration, modified the relaxation curve in-another publication in the near future. duced by the β2-adrenoceptor agonist isoproterenol

Serotonin, bradykinin and leukotrienes are also in preparations of guinea-pig trachea that had beenimportant mediators released by mast cells during previously contracted using acetylcholine.the allergic reaction. In the experiments conducted Dimaprit, a selective H2-agonist, induced a con-in a rat caudal artery in vitro model, bilastine did not centration-dependent positive chronotropic effect.modify the concentration curves induced by seroto- Its concentration-response curve was not signifi-nin, an effect mediated by 5-HT2A receptors. Ce- cantly shifted after incubation with bilastine, ce-tirizine and fexofenadine showed a very weak com- tirizine or fexofenadine at 100µM. Therefore, at thispetitive antagonistic behaviour. concentration, the three compounds lacked the abili-

The results of in vitro experiments conducted ty to antagonise the effect of dimaprit, which isusing guinea-pig ileum showed that neither bilas- mediated by H2-receptors.tine, cetirizine nor fexofenadine reduced The H3-agonist (R)-α-methyl-histamine inhib-bradykinin-induced contractions in guinea pig ile- ited the electrical twitch in guinea-pig ileum in aum. At high concentrations (100µM), these com- concentration-dependent manner. Thioperamide, atpounds also lacked the capacity to competitively 10nM, competitively shifted towards the right theantagonise the contractions induced by LTD4 in (R)-α-methyl-histamine curve with a pA2 value ofguinea-pig trachea. When contractions were induced 8.7. Addition of bilastine or cetirizine induced aby CaCl2 in depolarised guinea-pig ileum, bilastine decrease in the twitch of 60%, 66% and 49.20%,lacked significant inhibitory activity, while ce- respectively, but it was not modified by previoustirizine and fexofenadine, although very weak, incubation with thioperamide. Therefore, the inhibi-showed competitive antagonist activity. tory effect of the twitch observed with bilastine

The in vitro profiling screen performed to inves- could not have been related to an agonist action attigate the binding capacity of bilastine to 30 differ- the H3-receptors. On the other hand, bilastine, atent receptors other than the histamine H1-receptor 30µM, did not significantly shift the curve of (R)-α-showed that at a concentration of 10µM, bilastine methyl-histamine, ruling out a H3-antagonist effect.



3. Benavides J, Schoemaker H, Dana C, et al. In vitro and in vivoThe binding affinity of bilastine for H4-receptorsinteraction of the novel selective histamine H1 receptor antag-

is still under study. However, the results of recent onist mizolastine with H1 receptors in rodents. ArzneimittelForschung 1995; 45 (5): 551-8studies of the binding affinity of histamine H1-, H2-

4. McPherson GA. A personal computer-based approach to theand H3-receptor ligands at the human H4-receptoranalysis of radioligand binding experiments. Comput Pro-

lead us to believe that bilastine lacks binding affinity grams Biomed 1983; 17: 107-14for this specific receptor.[11]

5. Smit JM, Timmerman H, Hijzelendoorn JC, et al. Regulation ofthe human histamine H1 receptor stably expressed in Chinesehamster ovary cells. Br J Pharmacol 1996; 117 (6): 1071-80Conclusions

6. Cheng YC, Prusoff WH. Relationship between the inhibitionconstant (K1) and the concentration of inhibitor which cause

The results of in vitro preclinical studies provide 50 per cent inhibition (IC50) of an enzymatic reaction. Bio-chem Pharmacol 1973; 22 (23): 3099-108evidence that bilastine has antihistamine activity

7. Arunlakshana O, Schild HO. Some quantitative uses of drugwith high specificity for H1-receptors and poor or noantagonists. Br J Pharmacol 1959; 14 (1): 48-58affinity for other receptors. Bilastine was also

8. Van Rossum JM. Cumulative dose-response curves: II. Tech-shown to have anti-inflammatory properties and to nique for the making of dose-response curves in isolatedbe more potent than cetirizine and fexofenadine in organs and the evaluation of drug parameters. Arch Int

Pharmacodyn Ther 1963; 143: 299-330an in vitro model of allergy.9. Constantine JW. The spirally cut tracheal strip preparation. J

Pharmacol 1965; 17: 384-5Acknowledgements 10. Fukuda T, Saito T, Tajima S, et al. Antiallergic effect of

1-(2-ethoxyethyl)-2-(4-methyl-1-homopiperazinyl) benzimi-This work has been funded in part by the Ministry of dazole difumarate (KB-2413). Arzneimittel Forschung 1984;

34 (7): 805-10Industry, Tourism and Commerce (formerly Ministry of Sci-ence and Technology) of Spain (PROFIT) and by the Depart- 11. Lim HD, van Rijn RM, Ling P, et al. Evaluation of histamine

H1-, H2-, and H3-receptor ligands at the human histamine H4ment of Industry, Commerce and Tourism of the Basquereceptor: identification of 4-methylhistamine as the first potentGovernment (INTEK).and selective histamine H4 receptor agonist. J Pharmacol ExpAll the authors are employees of FAES FARMA, SA.Ther 2005 Jun 9 [Epub ahead of print]

References Correspondence and offprints: Dr Aurelio Orjales, Depart-1. Slater JW, Zechnich AD, Haxby DG. Second-generation anti-

ment of Research, FAES FARMA, SA, Maximo Aguirre, 14,histamines: a comparative review. Drugs 1999; 57 (1): 31-4748940 Leioa, Spain.2. Yap YG, Camm AJ. The current cardiac safety situation with

antihistamines. Clin Exp Allergy 1999; 29 Suppl. 1: 15-24 E-mail: [email protected]


Preclinical Pharmacology of Bilastine,??a New Selective Histamine??H1 ??Receptor Antagonist

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