Evaluation of SSR161421, a novel orally active adenosine A3 receptor antagonist on pharmacology models

European Journal of Pharmacology 699 (2013) 172–179

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European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar

Pulmonary, gastrointestinal and urogenital pharmacology

Evaluation of SSR161421, a novel orally active adenosine A3 receptor antagonist on pharmacology models Endre G. Mikus n, Judit Szeredi, Kinga Boer, Ge´za Tı´ma´ri, Michel Finet, Pe´ter Aranyi, Anne-Marie Galzin ´ utca 1–5, Hungary Sanofi Co. Ltd, H-1045 Budapest To

a r t i c l e i n f o

a b s t r a c t

Article history: Accepted 27 November 2012 Available online 5 December 2012

The effects of a novel adenosine A3 receptor antagonist, SSR161421, were examined on both antigen per se and adenosine receptor agonist-increased airway responses in antigen-sensitized guinea pigs. Adenosine (10  5 M) and AB-MECA [N6-(4-aminobenzyl)-adenosine-50 -N-methyl-uronamide dihydrochloride] (10  7 M) increased the antigen response up to 617 3.0% and 887 5.2% of maximal contraction, respectively. The agonists of adenosine A1 and A2 adenosine receptors NECA [1-(6-amino-9H-purin-9-yl)-1-deoxy-N-ethyl-b-D-ribofuranuronamide-50 -N-ethylcarboxamidoadenosine], R-PIA [N6-R-phenylisopropyladenosine], and CGS21680 (10  7 M) were ineffective. In vivo intravenous adenosine (600 mg/kg) and AB-MECA (30 mg/kg) increased the threshold antigen doseinduced bronchoconstriction by 214 7 13.0% and 220 7 15.2%, respectively. SSR161421 in vitro (IC50 ¼ 5.9  10  7 M) inhibited the AB-MECA-enhanced antigen-induced airway smooth muscle contractions and also in vivo the bronchoconstriction following either intravenous (ED50 ¼ 0.008 mg/kg) or oral (ED50 ¼ 0.03 mg/kg) administration in sensitized guinea pigs. Antigen itself could evoke tracheal contraction in vitro and bronchoconstriction in vivo in antigen-sensitized guinea pigs. SSR161421 (3  10  6 M) decreased the AUC of the antigen-induced contraction-time curve to 20.8 75.4% from the 100% control level. SSR161421 effectively reversed the antigen-induced bronchoconstriction, plasma leak and cell recruitment with EC50 values of 0.33 mg/kg p.o., 0.02 mg/kg i.p. and 3 mg/kg i.p., respectively. & 2012 Published by Elsevier B.V.

Keywords: Mast cell Ovalbumin-sensitization Adenosine A3 receptor SSR161421 Bronchoconstriction

1. Introduction Adenosine can be considered as an important pathological factor in asthma as a recently published comprehensive review discusses it (Brown et al., 2008). This statement is based on two main observations. First, adenosine levels are elevated in the lung tissue of asthmatic patients (Driver et al., 1993). Second, asthmatic but not healthy subjects respond to adenosine or adenosine monophosphate (AMP) inhalation with acute bronchoconstriction (Van den Berge et al., 2002; Polosa, 2002). This indicates that asthmatic patients are more sensitive to adenosine than healthy people. Moreover, endobronchial adenosine challenge in asthmatic patients caused significant elevation in mast-cell-derived mediators like histamine, PGD2 and tryptase (Polosa et al., 1995) and adenosine induced in vitro histamine release from human bronchoalveolar lavage mast cells (Forsythe and Ennis, 1999) suggesting that adenosine can also induce mast cell degranulation. Contact of the mast cell with activated T cell membrane causing the expression and release of cytokines, chemokines,

n

Corresponding author. Tel.: þ36 30 2793338. E-mail address: [email protected] (E.G. Mikus).

0014-2999/$ - see front matter & 2012 Published by Elsevier B.V. http://dx.doi.org/10.1016/j.ejphar.2012.11.049

growth factors and adenosine. The released adenosine binds to adenosine A3 receptors (AR3), initiates a complex signaling crosstalk, whereby the adenosine A3 receptor, by coupling to the G-protein Gi3, eventually remains the only adenosine receptor that contributes to ERK1/2 signaling (Baram et al., 2010). So mast cells are under both endocrine and paracrine control of adenosine (Meade et al., 2001). Four adenosine receptors subtypes have been identified: adenosine A1 and adenosine A2a receptors are stimulated by low concentrations of adenosine, and adenosine A2b and adenosine A3 by higher concentrations of adenosine. It is considered that all these adenosine receptors may have a significant role in allergy and asthma. Adenosine A1/A2b receptor antagonists as well as adenosine A2a receptor agonists are currently under development for the treatment of asthma (Fozard and McCarthy, 2002). The role of adenosine A3 receptors is less clear and still controversial in both allergic diseases and inflammatory animal models. This controversy is due to the lack of selective and in vivo active adenosine A3 receptor agonists or antagonists to test in allergic animal models. By the synthesis and biochemical characterisation of a new and highly selective adenosine A3 receptor antagonist SSR161421, we have a potent experimental tool to reveal the role of adenosine A3 receptors in inflammation (Mikus et al., in press).

E.G. Mikus et al. / European Journal of Pharmacology 699 (2013) 172–179

Therefore, the purpose of this study was to determine the role of adenosine A3 receptors in antigen-induced bronchoconstriction in vitro and in vivo in sensitized guinea pigs by using SSR161421 (4-methoxy-N-(benylamino-3-cyano-quinolin-2-yl)-benzamide) a potent and selective receptor antagonist of human adenosine A3 receptor.

173

The ovalbumin contractions were calculated in all the evaluated time points in percentage of the maximal contraction evoked by 3  10  6 M histamine. From this data, area under the curve (AUC ) values were calculated using the following formula in the case of each preparation: AUC ¼

n X

W iXi

i¼1

2. Materials and methods 2.1. Sensitization protocol Male albino guinea pigs HSD POC:DH Harlan-Winkelmann, Germany, weighing 180–220 g were used. The animals were sensitized by 2 subcutaneous injections of ovalbumin (10 mg) in the presence of 1 mg aluminium hydroxide in 1 ml NaCl 0.9% solution, at 15-day intervals. The animals were tested 1 week after the second injection. 2.2. In vitro isolated trachea Presensitized animals were sacrificed by cervical dislocation. The trachea was rapidly removed and placed in Krebs-Henseleit (KH) solution of the following composition (mM): NaCl-118, KCl-4.7, CaCl2  H2O-2.5, KH2PO4-1.2, NaHCO3-25.0, MgSO4  7H2O1.2, glucose 11.1 and 5  10  6 M indomethacin. The trachea was carefully stripped of connective tissue, and cut into approximately 3 mm wide rings. These rings were opened longitudinally and mounted into a 20 ml organ bath containing KH solution, maintained at 37.470.1 1C, and continuously bubbled with 95% O2 and 5% CO2 to obtain pH of 7.470.1. The preparations were preloaded with 0.5 g. The tissues then were allowed to equilibrate for 1.5–2 h, during which time they were washed approximately every 20 min with fresh KH solution, until they developed a stable tracheal tone. A contractile response to histamine (3  10  6 M ) was generated twice, separated by a wash out period until recovery of baseline. After the second histamine challenge and washout, adenosine (10–7, 10  6 or 10  5 M), AB-MECA (N6-(4aminobenzyl)-adenosine-50 -N-methyl-uronamide dihydrochloride) (10–8, 10  7 or 10  6 M) or their vehicle (saline) were added to the organ bath 30 s. before tracheal contractions were induced with ovalbumin (0.5 mg/l). Only one concentration of agonist was used per tissue. In the experiments where the effect of adenosine receptor antagonists was tested, the antagonist was added to the organ bath 20 min before the administration of submaximal concentrations of adenosine or AB-MECA followed by ovalbumin as indicated above. The second histamine-induced contraction was considered as 100% contraction for each tracheal preparation and ovalbumin induced contractions were expressed as a percentage (mean 7S.E.M.). For statistical evaluation, one-way analysis of variance followed by Dunnett’s test was used. 2.3. Effect of SSR161421 on ovalbumin-induced tracheal contraction After equilibration and two histamine challenges (3  10  6 M), vehicle or SSR161421 was added to the organ baths. This histamine dose was selected as reference response because it was calculated that this is the EC50 value of histamine and it evoked significant constrictor response (11007388 mg, n¼17) in other experiments. Finally, 20 min later, all tissues were exposed to ovalbumin at a dose of 700 ng/ml to evoke antigen-induced tracheal contraction. This dose of ovalbumin evoked approximately the same tracheal contraction as 3  10  6 M histamine did.

where wi are the trapezoidal weights (% values); xi are the values measured at the time point i (i¼1,2,y,n). wi ¼ ðt i t i1 Þ=2 where t0, t1, t2,y, tn are time points. The ovalbumin responses in the different groups are expressed as mean7SD (AUC). Percentage of inhibitions were calculated from these group-mean values. For statistical evaluation, one-way analysis of variance followed by Dunnett’s test was used. 2.4. Effect of adenosine receptor agonists on antigen-induced bronchoconstriction A modified Konzett–Rossler method was used (Konzett and Rossler, 1940) for the measurement of bronchoconstriction. Presensitized guinea pigs were anaesthetized with urethane (1 g/kg i.p.). Thereafter the jugular vein and the trachea were cannulated. The tracheal cannula was connected to a respiratory pump (Ugo Basile, Italy) for artificial respiration (60 strokes/min, insufflated volume 1 ml/100 g). The initial pressure was adjusted to 10 cm H2O. Spontaneous breathing was arrested with suxamethonium chloride (5 mg/kg s.c.). The bronchial basal increased overflow was measured continuously by a bronchospasm transducer (Ugo Basile, Italy) and registered on a chart recorder. When the basic overflow was stabilized, 100% overflow was determined by clamping the tracheal cannula for two consecutive respiratory cycles. The clamp was released and when the basal overflow was stabilized again the following experiments were carried out. 2.5. Adenosine and AB-MECA-enhanced antigen-induced bronchoconstriction Animals were injected i.v. by the vehicles, saline or 1-methy-l2-pyrrolydinone/PEG400/water (1/4/5; v/v/v) mixture (control) or the adenosine receptor agonist and bronchoconstriction was induced by 0.3 mg/kg i.v. ovalbumin, dissolved in saline and given in a volume of 1 ml/kg. This dose of ovalbumin was choosen to evoke reproducible threshold antigen response. 2.6. Inhibition of adenosine or AB-MECA-enhanced antigen-induced bronchoconstriction In these experiments two different control groups were used. In the first control group (Group 1) the animals were treated i.p./p.o. with the vehicle of the test compound, 1-methy-l-2pyrrolydinone/PEG400/water (1/4/5; v/v/v) mixture and saline (vehicle of adenosine and AB-MECA) in a volume of 1 ml/kg. In the second control group (Group 2) the vehicle of the test compound was administered first and thereafter 0.6 mg/kg adenosine (submaximal dose) or 30 mg/kg AB-MECA (submaximal dose) was injected intravenously. In the test compound groups (Group 3) the animals were treated by the drug followed by 0.6 mg/kg adenosine or 30 mg/kg AB-MECA. Finally the antigen-induced bronchoconstriction was induced by ovalbumin (0.3 mg/kg i.v.) in all groups. This dose of ovalbumin was selected to induce, in the presence of an adenosine receptor agonist, a half maximal bronchoconstriction. The net increase of the bronchoconstriction

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(Group 2–Group 1) was considered as a 100% effect. The effect of the adenosine A3 receptor antagonists (Group 3) was expressed as percentage of inhibition of bronchoconstriction (mean7S.E.M.). For statistical evaluation, one-way analysis of variance followed by Dunnett’s test was used. Dose-response and statistical analyses were made using computer programme SAS V8.2 for Sun Solaris.

2.7. Ovalbumin-evoked bronchoconstriction in guinea-pigs The SSR161421 (0.03–10 mg/kg) or vehicle pretreated animals were anaesthetized and prepared as described above. The antigen-induced bronchoconstriction was evoked by i.v. injection of 1.5 mg/kg ovalbumin in both groups and the airway obstruction was continuously monitored.

2.10. Materials AB-MECA HCl salt (N6-(4-aminobenzyl)-adenosine-50 -N-methyluronamide dihydrochloride) and SSR161421 (4-methoxy-N-(benylamino-3-cyano-quinolin-2-yl)-benzamide was synthetized in Chinoin Co. Ltd. as described (WO-02096879). Adenosine, NECA [1-(6-Amino-9H-purin-9-yl)-1-deoxy-N-ethylb-D-ribofuranuronamide-50 -N-ethylcarboxamidoadenosine], CGS21680 [N6-cyclopentyl-9-methyladenine (N-0840), 2-p-(2-carboxyethyl)-phenethylamino-50 -N-ethylcarboxamido-adenosine], R-PIA [N6-R-phenylisopropyladenosine], MRS1220 [N-(9-chloro-2-furan2-yl-[1,2,4]triazolo[1,5-c]quinazolin-5-yl)-2-phenyl-acetamide], PEG400 [polyethylene glycol 400] were purchased from Sigma (St. Louis, MO).

3. Results 2.8. Ovalbumin-induced plasma leakage in trachea of sensitized guinea-pigs One week after the last ovalbumin injection, the animals were treated intraperitoneally with SSR161421 (0.003–1 mg/kg) or its vehicle. Saline-treated pre-sensitized animals were used as a second control group. The jugular veins of anaesthetized animals (urethane 1.6 g/kg i.p.) were cannulated. 30 min after the treatment with SSR161421 or its vehicle, 20 mg/kg Evans blue (0.1 ml/100 g b.w. in saline) were injected in the jugular veins, followed by 100 mg/kg ovalbumin (0.1 ml/100 g b.w. in saline). Ten minutes later, the animals were exsanguinated by cutting the carotid artery. The vasculature was then infused with 50 ml saline and the trachea was dissected and weighed. The tissue samples were placed in 1 ml formamide (for 24 h, 50 1C) to extract the dye. Its amount was evaluated by spectrophotometry at 620 nm wavelength.

2.9. Ovalbumin-induced eosinophil migration to bronchoalveolar lavage fluid in sensitized guinea-pigs One week after the second ovalbumin injection, the animals were treated i.p. with SSR161421 (0.03–10 mg/kg) or vehicle (control group). 30 min later, the animals set in a chamber, inhaled ovalbumin (10 mg/ml in saline, Devilbis nebulizer, airflow 140 l/h, 10 min). The treatment with SSR161421 or vehicle was repeated 30 min later. In order to prevent the collapse due to ovalbumin-induced anaphylaxis, the animals were treated with the histamine H1 blocker mepyramine 30 min before the first drug or vehicle treatment. Saline-challenged and pre-sensitized animals were used as vehicle-control group. Broncho-alveolar lavage was performed 6 h after antigen challenge. The tracheas of anaesthetized animals (Nembutal, 50 mg/kg i.p.) were cannulated and connected to a syringe. The lungs were washed with 3  2 ml Hank’s balanced salt solution (HBSS). The recovered fluid collections were usually 75–90% of the instilled volume. The lavage fluid was centrifuged (600 rpm, 4 1C, 10 min). The resulted cell pellet was suspended in 100 ml HBSS and the cell number was counted using a Burker-chamber with Turk-stain. Smears were prepared, fixed with Diff-Quick Fix and stained with Diff-Quick I–II. Differential counts on 3  100 cells were done by using standard morphologic criteria to classify cells as eosinophilic, neutrophilic and mononuclear cells. The numbers of the different cell types (cells/bronchoalveolar lavage) were calculated from the percentage distribution of the different cell types. The protocols used have been approved by the Ethical Committee for Laboratory Animals of Sanofi-Synthe´labo Recherche and was carried out in accordance with European Directive 86/609/EEC.

3.1. Effect of adenosine and adenosine receptor agonists on antigeninduced tracheal contraction in vitro Exposure to adenosine enhanced the antigen-induced tracheal contractions in a concentration-dependent manner (Fig. 1A). This effect was statistically significant at the 10  5 M concentration. A selective adenosine A3 receptor agonist (AB-MECA) also increased the antigen-induced tracheal contractions in a concentration-dependent manner (Fig. 1A). In contrast, neither the non selective adenosine A1/A2 receptor agonist NECA, nor the selective adenosine A1 receptor agonist R-PIA or the adenosine A2a receptor agonist CGS21680 significantly enhanced the antigen-induced tracheal contractions at the 10  7 M concentration (Fig. 1B). These agonists per se did not induce tracheal contraction. The time of exposure to adenosine rceptor agonists before the ovalbumin challenge was found to be an important parameter to control. If antigen was applied 30 s or 15 min after 10  5 M adenosine or 10  7 M AB-MECA, a significant contractile response (higher than in the control group) was obtained (data not shown). However, if antigen was applied 60 min after the adenosine receptor agonists, no significant effect of agonist was observed on the antigen induced contraction. Based on these results, a 30 s AB-MECA or adenosine preincubation period was chosen for adenosine A3 receptor antagonist measurements. 3.2. Enhancement of antigen-induced bronchoconstriction by adenosine and AB-MECA in vivo Adenosine or AB-MECA enhanced the antigen-induced bronchoconstriction in a dose-dependent manner when antigen was injected i.v. immediately after the adenosine receptor agonist (Fig. 2) in line with the in vitro results. Here again the time elapsed between adenosine administration and antigen challenge had a strong influence on the bronchoconstriction enhancing effect of adenosine receptor ligands. Challenging the animals immediately or 10 min after adenosine by antigen resulted in a more severe bronchoconstriction than what was evoked by the antigen alone. Increasing the adenosine pretreatment time further up to 30 min or 60 min evoked a reduced bronchoconstrictor response to antigen challenge than observed in the control group. Similar results were obtained with adenosine and with AB-MECA (data not shown). 3.3. Effect of SSR161421 on adenosine or AB-MECA-enhanced ovalbumin-induced bronchoconstriction in vitro Due to the lack of effect of adenosine A1 and adenosine A2 receptor agonists on the ovalbumin induced bronchoconstriction in sensitized

E.G. Mikus et al. / European Journal of Pharmacology 699 (2013) 172–179

*** ***

% increase of bronchoconstriction

% tracheal contraction

70 AB-MECA

100

80 §

60

40

control for AB-MECA control for adenosine

adenosine AB-MECA

20

0.5 µg/L 10-8 ovalbumin no AB-MECA

10

-7

10

-6

10

-5

10

-4

***

60 adenosine

*** ***

50

***

*

40 30

** ***

**

20

***

10

ova. contr. without AB-MECA

0

ova. contr. without adenosine

0 1

AB-MECA or adenosine (M)

3

10

30

100

300

1000

-1

dose (µg kg i.v.)

+ovalbumin (0.5 µg/L) 100

*** 80

Fig. 2. Effect of adenosine and AB-MECA on ovalbumin-induced (0.3 mg/kg i.v.) bronchoconstriction in anaesthetized ovalbumin presensitized guinea pigs. The time between adenosine/AB-MECA and ovalbumin was 10 min. Results are shown as mean 7 S.E.M. (n¼ 3–4, nP o0.05, nnPo 0.01, nnnP o 0.001, one-way analysis of variance followed by Dunnett’s test).

60

40 120

20

A AB

-M

EC

A PI

A EC N

C

G

S2

co

16

nt

ro

80

l

0

10-7 M ovalbumin

% value of tracheal contraction

% tracheal contraction

175

Fig. 1. Effect of adenosine and AB-MECA (A) and other adenosine receptor agonists (10  7 M) (B) on ovalbumin-induced tracheal contraction in preparations derived from ovalbumin presensitized guinea pigs. Ovalbumin (0.5 mg/l) was administered 30 s after the agonist. Percentage of maximum contraction induced by 3  10  6 M histamine. Results are shown as mean 7 S.E.M., one way analysis of variance followed by Dunnett’s test, nnnPo 0.001, yP o 0.05 compared to matching control for adenosine and AB-MECA, respectively. Sample number was n¼ 6–15 in panel A, while n¼5–6 in panel B.

100 80 *

60 ***

40 20

***

0 3·10-7 M

10-7 M

10-6 M

3·10-6 M

concentrations (M) 100

guinea pigs, in our conditions, it appears likely that the antigeninduced tracheal contractions probably occurred through activation of A3 receptors. This hypothesis was tested using the selective adenosine A3 receptor antagonist SSR161421. SSR161421 inhibited AB-MECA-enhanced ovalbumin-induced tracheal contractions in a concentration-dependent manner (IC50 ¼ 5.9  10  7 M) (Fig. 3A). SSR161421 also inhibited the adenosine-enhanced ovalbumin-induced tracheal contractions (Table 1). In addition, MRS1220, a selective adenosine A3 receptor antagonist (Jacobson et al., 1998), also inhibited the enhanced antigen response in a concentration dependent way (Fig. 3B). The inhibitory effect of both SSR161421 and MRS1220 (belonging to different chemical series) on adenosine receptor agonist-enhanced response further supported the involvement of adenosine A3 receptors in both adenosine and AB-MECA enhanced antigeninduced tracheal contractions.

% tracheal contraction

80

60

*

40

20

***

0 MRS1220

- -

3·10 M

- -

10 M

- -

3·10 M

AB-MECA 10 M

- + +

- + +

- + +

ovalbumin

+ + +

++ +

+ + +

Fig. 3. Inhibitory effect of SSR161421 (A) and MRS1220 (B) on 10  7 M AB-MECA enhanced ovalbumin-induced tracheal contractions in isolated guinea pig preparations. Data are expressed as mean 7 S.E.M., nP o 0.05, nnnPo 0.001 one-way analysis of variance followed by Dunnett’s test. Sample number was n¼9–12 in panel A, while n ¼8–12 in panel B.

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Table 1 Inhibitory effect of SSR161421 on 10  5 M adenosine enhanced ovalbumininduced tracheal contractions in isolated guinea pig preparations. Treatment

% Tracheal constriction (mean 7SD)

Ovalbumin Ovalbumin þ ADE Ovalbumin þ ADE þSSR (3  10  8 M) Ovalbumin þ ADE þSSR (10  7 M)

26.2 77.5 66.1 77.0 64.7 76.7 24.2 73.9n

Data are expressed as mean 7S.E.M., n¼ 8–10. n P o0.001 one-way analysis of variance followed by Dunnett’s test compared to ovalbumin þADE group. 100% contraction was evoked by 3  10  6 M histamine. ADE-adenosine (10  5 M), ovalbumin–ovalbumin (0.5 mg/l), SSR161421.

Table 2 Time course of the inhibitory effect of 0.03 mg/kg p.o. SSR161421 on 30 mg/kg i.v. AB-MECA enhanced ovalbumin-induced bronchoconstriction in ovalbumin presensitized guinea pigs. Time after oral SSR161421 treatment (h)

Percentage inhibition of bronchoconstriction

0.5 1.0 2.0 3.0 4.0 8.0

14.97 8.9 42.67 11.2n 47.97 4.5nnn 30.07 3.3nn 33.87 8.1nn 10.97 6.3

Data are expressed as mean 7 S.E.M., n ¼4–9. n

Po 0.05. Po 0.001. nnn P o 0.001 one-way analysis of variance. nn

% values of bronchoconstriction

120 100

Table 3 Inhibitory effect of i.v. MRS1220 on 30 mg/kg i.v. AB-MECA enhanced ovalbumininduced bronchoconstriction in ovalbumin presensitized guinea pigs.

80 60

Dose of MRS1220 (mg/kg i.v.)

Percentage inhibition of bronchoconstriction

40

0.03 0.1 0.3 1.0

11.3 71.2 23.9 74.2n 40.7 71.2n 68.2 73.9n

20 0 0.001

Data are expressed as mean 7 S.E.M., n ¼3–7.

0.003

0.01

0.03

0.1

0.3

n

Po 0.001 one-way analysis of variance followed by Dunnett’s test.

SSR161421 (mg/kg)

administration as well. Potency and efficacy of SSR161421 (administered i.v.) were equivalent whatever the agonist (Fig. 4B ). MRS1220 was also effective after i.v. (ED50 ¼0.3 mg/kg) administration but both potency and efficacy were weaker than those of SSR161421 (Table 3).

% value of bronchoconstriction

120 100 80

3.5. Effect of SSR161421 on ovalbumin-induced tracheal contraction

60

SSR161421 decreased ovalbumin-induced tracheal contractions in a concentration-dependent manner. 3  10  6 M concentration of SR161421 decreased the AUC of the contraction-time curve to 20.8 75.4% from the 100% control level (Fig.5A).

40 20 0 0.0003

0.001

0.003

0.01

0.03

3.6. Ovalbumin-evoked bronchoconstriction in guinea-pigs

SSR161421 (mg/kg i.v.) Fig. 4. Inhibitory effect of SSR161421 on 30 mg/kg i.v. AB-MECA enhanced ovalbumin-induced bronchoconstriction following intravenous (10 min pretreatment) or oral (120 min pretreatment) administration (A) and the inhibitory effect of i.v. SSR161421 on 30 mg/kg i.v. AB-MECA and 600 mg/kg i.v. adenosine (B) enhanced ovalbumin-induced bronchoconstriction in ovalbumin presensitized guinea pigs. Data are expressed as mean 7 S.E.M., nnPo 0.01, nnnPo 0.001 one-way analysis of variance followed by Dunnett’s test. Sample number was n¼4–6 in panel A, while n¼4–9 in panel B.

The effect of SSR161421 on early response after allergen challenge was studied in anaesthetized, ovalbumin presensitized guinea pigs. 1.5 mg/kg ovalbumin injection evoked approximately 50% increase in airway overflow. Time-course experiments showed that the most pronounced inhibitory activity of SSR161421 on airway overflow was achieved after a 2 h oral pretreatment (Fig. 5C). SSR161421 effectively reversed the ovalbumin induced bronchoconstriction administered orally with an ED50 value of 0.33 mg/kg (Fig. 5B).

3.4. Effect of SSR161421 on AB-MECA-enhanced ovalbumin-induced bronchoconstriction in vivo SSR161421 inhibited the AB-MECA (30 mg/kg i.v.) enhanced ovalbumin-induced bronchoconstriction after both i.v. (ED50 ¼ 0.008 mg/kg) and p.o. (ED50 ¼0.03 mg/kg) administration (Fig. 4A). Following oral treatment, the inhibition was still significant 4 h after administration (Table 2). In addition, SSR161421 inhibited adenosineenhanced bronchoconstriction following i.v. (ED50 ¼0.006 mg/kg)

3.7. Ovalbumin-induced plasma leakage in trachea of sensitized guinea-pigs SSR161421 dose-dependently (0.01–1 mg/kg) inhibited the plasma leakage in trachea, after intraperitoneal administration, this inhibition reaching a maximum about 65% at 1 mg/kg i.p. (Fig. 6A). The calculated ED50 value is 0.02 mg/kg i.p.

E.G. Mikus et al. / European Journal of Pharmacology 699 (2013) 172–179

% inhibition of Evans blue dye extravasation

rel. AUCs for ovalbumin-induced contractions

140 120 100 80 60 40 20 0

100 ***

80 *** ***

60 40

***

***

20 0 0.001

0.01

0.1

1

SSR161421 (mg/kg i.p.)

100

total cell number eosinophil cell number

100 80

90

% inhibition of the cell migration

% inhibition of bronchoconstriction

177

60

40

20

0 0.03

0.1

0.3

1

3

*** ***

******

80 *

70 60 50 **

40 30 20 10

10

0

SSR161421 (mg/kg, p.o.)

1

3

10

30

SSR161421 (mg/kg i.p., b.i.d) % inhibition of bronhoconstriction

100 Fig. 6. Inhibitory effect of SSR161421 on ovalbumin induced plasma leakage (A) and on eosinophilic cell migration (B) in sensitized guinea pig trachea following i.p. administration. The extent of plasma leakage following antigen challenge in sensitized guinea pigs was measured by detecting the amount of extracted Evans blue from trachea. SSR161421 reached a maximal inhibition about 65% at 1 mg/kg i.p. Data are expressed as mean 7 S.E.M., P o0.001. Sample number was n¼5–5 in panel A, while n ¼4–5 in panel B.

80

60

40 Table 4 Ovalbumin-induced leukocyte migration to the broncho-alveolar space of ovalbumin presensitized guinea pigs.

20

0 0.5 1.0

2.0

3.0 4.0 pretreatment (hours)

8.0

Fig. 5. Inhibitory effect of SSR161421 on ovalbumin-induced tracheal preparation derived from ovalbumin presensitized guinea pigs (A) and both the dose-response (B) and time duration (C) on ovalbumin-induced bronchoconstriction in ovalbumin presensitized guinea pigs following oral administration. The Data are expressed as mean 7 S.E.M., nnnP o0.001 one-way analysis of variance followed by Dunnett’s test. Sample number was n¼8–9 in panel A, n ¼5–6 in panel B and n¼ 5–5 in panel C.

3.8. Ovalbumin-induced eosinophil migration to bronchoalveolar lavage fluid in sensitized guinea-pigs To assess the influence of SSR161421 on an allergy based inflammation model, the effect of the compound was tested on ovalbumin inhalation evoked leukocyte migration, in vivo. The amount of total cells and eosinophils in bronchoalveolar lavage

Saline inhalation controls Ovalbumin inhalation controls

Total cell number (  105)

Eosinophil cell number (  105)

8.8 7 1.2 35.6 7 7.8n

1.9 7 0.7 18.3 7 7.3n

Data are expressed as mean 7 S.E.M., n¼5–5. n

P o0.001.

fluid after SSR161421 pretreatment was determined. The allergen provocation produced a 4.3-fold increase in total cell number (eosinophils, neutrophils and mononuclear cells) and a 6.2-fold increase in eosinophil number (Table 4). SSR161421 dosedependently inhibited the leukocyte recruitment to the bronchoalveolar space of antigen challenged animals. The ED50 values of inhibition of SSR161421 concerning total cell number and eosinophil cell number are respectively equal to 3 mg/kg and 2.4 mg/kg (Fig. 6B).

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4. Discussion Adenosine A3 receptor is widely expressed in human tissues with the most abundant expression in the lung and the liver. Numerous studies have provided data showing that adenosine A3 receptors are primarily expressed on eosinophils in human lung (Kohno et al., 1996; Walker et al., 1997). The expression of functional adenosine A3 receptors is also demonstrated on other human inflammatory cells i.e., neutrophils (Gessi et al., 2002), dendritic cells (Fossetta et al., 2003), and lymphocytes (Gessi et al., 2004). A3 transcripts but also A3 binding sites and their functions were selectively increased after activation in human CD4þ cells (Gessi et al., 2004). A3 expression and function on human mast cells is less clear because of the difficulty to obtain human differentiated mast cells from tissular origin and due to differences in phenotype and function of mast cells between different tissues. Studies in animals suggest that depending on species and models, adenosine A1, A2b and A3 receptor activation could lead to bronchospasm. Isolated tracheal preparations contain tissue mast cells, smooth muscle and nerve elements so it is considered as a good experimental model organ to examine both mast cell dependent and independent effect of adenosine ligands. It has also been reported in guinea pigs that sensitization by ovalbumin reveals a tracheal constrictor response to adenosine (D’Agostino et al., 1999). In this preparation, adenosine induced moderate tracheal contractions at low doses but significant relaxation at higher doses (Martin and Broadley, 2002a, 2002b, Farmer et al., 1988). We have also observed similar biphasic effect of adenosine on isolated guinea pig tracheal preparation (data not shown). The direct tracheal contracting effect of adenosine is relatively weak compared to its antigen-induced mast cell degranulation enhancing effect at similar doses. Similar to what was observed on RBL2H3 cells (Ramkumar et al., 1993) adenosine potentiates in vitro antigen-induced release of histamine (Welton and Simko, 1980; Dexter et al., 1999) and enhances antigen-induced bronchoconstriction and histamine release in rat isolated lungs (Post et al., 1990). Adenosine A3 receptor agonist AB-MECA also increased the plasma histamine levels in mice after i.v. administration (Mikus et al., in press). Adenosine had a biphasic effect on in vitro sensitized lung fragments derived from patients; it potentiated antigen-induced mediator release at low concentrations but at high concentrations inhibited the release (Konnaris et al., 1996). Increased circulating adenosine levels induced by intravenous administration of adenosine potentiated the antigen-induced immediate bronchospasm and bronchoconstrictor mediator release in sensitized guinea pigs (Huszar et al., 1998). This synergism can be explained by the fact that both crosslinking of the mast cell membrane-bound IgE receptors and adenosine A3 receptor activation increase the enzymatic production of intracellular inositol-1,4,5-triphosphate and 1,2-diacylglycerol resulting Ca2 þ mobilization, cell activation and the release of mast cell-derived smooth muscle constrictor mediators release (Wilkinson and Hallam, 1994; Ramkumar et al., 1993; Jacobson et al., 1998). In fact adenosine and the selective adenosine A3 receptor agonist AB-MECA (Jacobson et al., 1998; Forsythe and Ennis, 1999) effectively enhanced the antigen-induced tracheal contractions, the greater efficacy of AB-MECA suggesting an involvement of adenosine A3 receptors in this model. Our experimental data further supported by the lack of effect of the adenosine A1 and A2 receptor agonists NECA, R-PIA and CGS21680 at enhancing the contractions in vitro. In line with these results, the selective adenosine A3 receptor antagonist SSR161421 dose-dependently inhibited both AB-MECA and adenosine enhanced smooth muscle contractions both in vitro and in vivo, with similar potency against both agonists after i.v.

administration. MRS 1220, another potent adenosine A3 receptor antagonist of human receptors (Jacobson et al., 1997) also inhibited AB-MECA-enhanced allergic reaction, although less active than SSR161421. Huszar et al. (1998) mentioned the potential involvement of adenosine A3 receptors during adenosine-induced mast cell degranulation and bronchoconstriction in vivo. We also found that an adenosine A3 receptor antagonist, SSR161421, inhibited the ovalbumin-evoked bronchoconstriction in sensitized guinea pigs. Current therapeutic approaches to the treatment of asthma pay great attention to its inflammatory component. After allergen challenge, permeability of the blood vessel walls of airways increases and this results in protein transmigration and plasma exudation. In addition we investigated the effect of SSR161421 on allergen-induced plasma leakage in sensitized guinea pigs. After intravenous ovalbumin injection, SSR161421 effectively blocked the increase in plasma leakage – the marker of oedema formation – in trachea removed from sensitized guinea pigs. This result confirms the – possibly indirect – role of adenosine A3 receptors expressed on mast cells in the development of asthmatic symptoms. Leukocyte migration to the airways is a specific marker of airway inflammation. Spruntulis and Broadley (2001) demonstrated that MRS-1220, a selective adenosine A3 receptor antagonist, blocked the leukocyte infiltration into the lungs of allergen-sensitized guinea pigs after inhalation of the adenosine derivative 50 -AMP, indicating that this response is mediated via adenosine A3 receptors. Our results with SSR161421 prove that the allergen inhalationinduced leukocyte influx to the bronchoalveolar space can be blocked with an adenosine A3 receptor antagonist. With these results, we showed for the first time that an adenosine A3 receptor antagonist blocks not only the early response but even the eosinophil migration during the late phase allergic reaction after allergen exposure. Blocking the accumulation of eosinophils in the bronchoalveolar lavage after the allergen provocation indicates that the effect of SSR161421 in guinea pig asthma models is beneficial at both levels. In the allergic asthmatic model induced by ovalbumin in guinea pigs, SSR161421 inhibits early bronchoconstriction, tracheal oedema and late phase eosinophilic recruitment. Moreover, SSR161421 appears very potent against ovalbumin-induced bronchoconstriction as oral preventive administration, which could be very important in the context of asthmatic treatment, where orally active drugs without side effects are somewhat lacking. In conclusion, SSR161421 as a potent and selective antagonist against human adenosine A3 receptors could be an interesting tool to delineate the role of adenosine A3 receptors in human pathologies, and if confirmed, could be a new treatment for asthma.

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