The inhibitory effect of anti-allergic agent suplatast tosilate (IPD-1151T) on methacholine- and allergen-induced bronchoconstriction in sensitized mice. asakazu@med.showa-u.dc.jp.

The influence of an anti-allergic agent, suplatast tosilate (IPD-1151T; (+/-)-[2-[4-(3-ethoxy-2-hydroxypropoxy)phenyl-carbamoyl]-ethyl] dimethylsulfonium p-toluenesulfonate) on allergic bronchoconstriction induced by allergen and methacholine (MCh) were examined in mice. BALB/c mice were sensitized by intraperitoneal injection of dinitrophenylated-keyhole limpet hemocyanin (DNP-KLH) mixed with A1(OH)3 (DNP-KLH). IPD-1151T was administered orally once a day for either 5 or 14 days in doses of 10, 30 or 100 mg/kg. Bronchoconstriction was measured 24h after the final drug administration. IPD-1151T inhibited both antigen- and MCh-mediated bronchoconstriction in actively sensitized mice. The inhibition induced was closely related to the dose and frequency of oral administration of the agent. We also examined the effect of IPD-1151T on IgE production in response to DNP-KLH immunization. IPD-1151T inhibited dose-dependently both total and specific IgE concentrations in serum prepared from mice 15 days after immunization. These results strongly indicate that IPD-1151T inhibits IgE production in vivo and results in attenuating effect on bronchoconstriction.


Introduction
Bronchial hyp er-responsiveness (BHR) is an important feature of bronchial asthma and the degre e of BHR is belie ve d to reflec t the se ve rity of the dise ase. 1 ,2 The ultimate task of asthma treatme nt is the relief of symptoms to permit a normal style of life. The main me asures to achieve this foc us on the de crease of airflow obstruction and the shift of BHR tow ards a normal range . 3,4 To ac complish these objec tives, se veral type s of anti-allergic drugs w ith bronchodilating activity have be en de ve loped and are used in the tre atment and manageme nt of bronchial asthma. Although the mechanisms of BHR are not fully understood, there is an established conce pt that IgE is a prime mediator in de ve lopment of BHR, e spec ially alle rgen-induce d BHR. 4 -7 That is, IgE antibodies bind to spec ific membrane rece ptors on mast c ells and basophils. Combination of spe cific c ell-bound antibody w ith antige n trigge rs a series of events leading to re lease of vasoactive amines and othe r pharmac ologic ally active substanc es responsible for clinic al manifestation of BHR. 8 Re cently IgE antibody rec eptors have also bee n demonstrate d on a subpopulation of e osinop hils and plate le ts, among othe rs. 9 Binding of IgE antibody to rec eptors on these c ells results in the enhancement of their function and the re lease of various chemical me diators responsible for allergic inflammation. 1 0 -12 From the se standp oints, drugs spe cifically inhibiting IgE antibody formation w ould be highly fe asible for the therapy of allergic patients. How e ver, such drugs are not available now adays.
During immunopharmacologic al studie s on dimethylsulfonium compounds, it is found that suplatast tosilate (IPD-1151T) suppresses Th2 type cytokine production, IgE synthe sis, and che mical mediator release from mast ce lls in the mouse and guine a pig mode ls of asthma. 13 -1 7 IPD-1151T has also be en reporte d to suppress the produc tion of Th2 type cytokines and IgE by human periphe ral blood leukocytes w hen the c ells w e re obtained from asthmatic patients and c ultured w ith spec ific antige ns in the presenc e of the agent. 1 8,19 Furthermore, it is observed that oral administration of IPD-1151T c ould attenuate eosinophilia and hyperimmunoglobulinemia E in patients w ith Kimura's disease. 2 0 From the se reports, there is a p ossibility that IPD-1151T may be a new antialle rgic drug that could prove use ful for tre atment and preve ntion of asthma. How e ve r, there are no rep orts of the influence of IPD-1151T on bronchoconstriction.
In this study, the refore , w e investigate d the effect of IPD-1151T on alle rgic airw ay hyper-re sponsivene ss induc ed by ex posure of ke yhole lympe t he moc yanin (KLH)-se nsitized mic e to re petitive antige n and to me thacholine (MCh ).

Animals
Spe cific pathoge n-free, normal and athymic BALB/c mic e w ere purchased from Charle s River Japan Inc., Atsugi, Japan. The y w ere all male and 5 w e eks of age at the start of the ex perime nts. Conve ntional Wistar male rats w e re obtained from Nippon Bio-supply Ce nter (Tokyo, Japan ). All animal ex pe rimental proc edure s use d in this study w ere approve d by the Show a University Animal Ethic s Committee, and c arrie d out in accordanc e w ith the guideline s of the Physiologic al Socie ty of Japan.

Agent
IPD-1151T ( Fig. 1 ) w as kindly supplied from Taiho Pharmac eutic al Co. Ltd., Tokyo, Japan, as a prese rvative -fre e pure pow de r. The agent w as dissolve d in distilled w ate r at 5 mg /ml just before use.

Antigens
KLH (Sigma Chemic al Co. Ltd, St Louis, MO, USA) w as coupled w ith dinitrobe nzen-sulfonic acid sodium salt (DNP) (Tokyo Kasei Corp., Tokyo, Japan ) acc ording to the methods of Le e and Se hon. 2 1 Bovine serum albumin (BSA) (Sigma Chemic al Co. Ltd.) w as also coupled w ith DNP in a similar manne r. The y w ere abbreviated DNP-KLH and DNP-BSA, respective ly.

Preparation of cell suspension
To pre pare splee n cell suspe nsion, normal BALB/c mic e w ere killed by intrape ritoneal inje ction w ith pentobarbital sodium (Abbott Lab., North Chicago, IL, USA) at a dose of 60 mg /kg. The splee n w as remove d, poole d from five mice and stored at 4°C until proce sse d. The organs w e re pressed through a 60-gauge ste el me sh and then filte red through a 200-gauge ste e l mesh to remove de bris and ce ll clumps. The cells w e re w ashed five times w ith RPMI-1640 medium (Flow Lab., Irvine, Sc otland ) suppleme nted w ith 10% fe tal c alf se rum (FCS) (Flow Lab., North Ride , Australia ), 5 ´10 -5 M 2 mercaptoethanol, 10 mM N-2-hydrox yethylpipe razine-N'-2-ethanesulfonic ac id, 100 U/ml penic illin and 100 m g /ml stre ptomyc in (RPMI-FCS) and resuspe nded in the fresh medium at a c once ntration of 5 ´10 6 c ells /ml. Splee n c ell suspensions w ere also prep ared from five athymic nude mic e in a similar manne r.

Cell culture
Splee n ce ll suspension (100 m l) w as introduce d into each w ell of 96-w ell flat-bottome d microculture plates (Nunc Inte rmed, De nmark ), w hich c ontaine d 10 m g /ml of e ither lippoporisaccharide (LPS) or concanavalin A (Con A). The plats w ere maintained for 48 h in a humidified atmosphe re w ith 5% CO 2 at 37°C. Ce ll activation w as assessed by adding 1 m Ci/w e ll of 3 H-thymidine (Ame rsham International p lc , Buc ks, UK ) for the final 6 h of culture. The results w ere ex pre sse d as me an c pm ± SD of triplicate cultures. To prepare c ulture supe rnatant, 1 ml of spleen ce ll suspe nsion w as culture d in 24-w ell culture plates (Nunc Interme d ) c ontaining 1 ml of 10 m g /ml of Con A. Afte r 24 h, c ulture supe rnatants w ere obtained after pelleting the c ells by c entrifugation at 2000 g for 10 min at 4°C. The supe rnatant w as stored at -40°C until used.

Immunization
Mic e w ere immunized by intraperitoneal inje ction w ith 5 m g /ml of DNP-KLH mix ed w ith 2 mg of Al(OH) 3 (Wako Pure Chemic al Ind., Os aka, Japan ) in a total volume of 0.5 ml of saline .

Drug administration
Mic e w ere orally administe red w ith either 10, 30 or 100 mg /kg of IPD-1151T in a volume not ex c ee ding 0.5 ml. Administration w as performed once a day on the follow ing sche dules: one group of mice w ere tre ate d for 5 days from Day 9 of sensitization and the othe rs for 14 days from Day 0. Control mice re ceive d distilled w ater only in a volume not ex c ee ding 0.5 ml.

Assay for IgE antibody
Mic e w ere ble d by cardiac puncture . Se rum w as isolated by ce ntrifugation and stored at -40°C until used. Total c onc entration of IgE antibody in serum w as assaye d by c ommercially prepared mouse IgE enzyme-linked immunosorbent assay (ELISA) te st kit (Yamasa Shoyu Co. Ltd, Chiba, Japan ) acc ording to the manufacture r's re comme nde d proc edure . Briefly, w ells of a 96-w ell microtite r plate c oated w ith antimouse IgE monoclonal antibody re ce ive d 100 m l of te st samples and of standards, separately. After inc ubation at 25°C for 30 min, the w ells w e re w ashed 5 times w ith w ashing buffer. Antimouse IgE enzyme conjugate (100 m l/w ell) w as then introduce d into each w ell and further inc ubate d for 30 min at 25°C. After w ashing, 200 m l of substrate, te trame thylbe nzidine, w as dispensed, and the re ac tion proce eded at 25°C for 15 min. Absorbance at 450 nm w as dete rmine d w ith an ELISA reade r (MRP A4i) (Tosoh Co. Ltd, Tokyo, Japan ) afte r adding 50 m l of 2N HCl. The ELISA w as p erformed in dup lic ate and mean absorbanc e w as obtaine d. Se rum antibody conce ntrations w ere calculated from standard c urve and the results w ere ex pre sse d as mean ng /ml ± SE of five individual mice. The conc entration of DNP-spec ific IgE antibody in se rum w as assaye d by passive cutaneous anaphylax is (PCA) in Wistar rats. An intradermal inje ction of 0.1 ml of se rum dilutions w as given on the shave d back of rats. Sensitized rats w ere challenge d by 1 mg of DNP-BSA in 1 ml of 0.5% Evans blue dye. Re ciprocals of the highest dilution w e re re corde d as the tite r of the spe cific IgE antibody. The results w ere ex pressed as the mean PCA tite r ± SE of five individual samples.

Assay for IL-4
IL-4 conte nts in c ulture supe rnatants w as assayed by the comme rc ially available mouse IL-4 ELISA kit (GENZYME TECHNE Corp., Minneapolis, MN, USA) according to the manufacturer's recomme ndation. The ELISA w as done in duplicate and the re sults w ere ex pre sse d as me an pg /ml ± SE of five individual mic e.

Measurement of airway reactivity
Tw enty-four hours afte r the final drug administration, mic e w e re anesthe tize d w ith intrape ritoneal inje ction of 1.8 mg /kg body w e ight of pentobarbital sodium (Abbott Lab.). Whe n an appropriate plane of anesthesia w as induc ed, the trachea w as cannulate d and conne cted to a c onstant-volume respirator for small animals (SN-480 -3; Shinano Se isakusho, Tokyo, Japan ) that provided ventilation at a tidal volume of 0.5 ml/100 g body w eight and a rate of 60 stroke s / min. 2 1 Under the se c onditions, w e found that arterial ox ygen tension of the mice w as kept betw ee n 70 and 80 torr as me asure d w ith a transc utaneous ox ygen te nsion meter (OXV-7101 ) (Nihon Koden Co. Ltd, Tokyo, Japan ). The flow at the outle t of the intratracheal cannula w as me asure d using a differential transducer (TP-602 ) (Nihon Kode n Co. Ltd ). One end of the diffe re ntial pressure w as connec te d to the outle t of the tracheal cannula, the other end being ex pose d to atmosphe ric pressure . The re spiratory resis tance w as dete rmined by the method of Konze tt and Rossler 2 2 from transpulmonary pressure, airflow and re spiratory volume measures. Airw ay response s to KLH w e re measured by intravenous administration of 500 m g /ml of KLH in a volume of 0.1 ml. 2 1 In the case of ex amining the influenc e of a bronchoconstric tor agonist on airw ay re sponse s, mice w ere injected intravenously w ith inc re asing quantitie s of MCh (10 -6 M to 10 -2 M) in a volume of 0.1 ml. The results w ere ex p re sse d as mean volume (ml) of air overflow ± SE of five mice .

Statistics
Statistical signific anc e w as de te rmined by Mann -Whitne y U-te st.

Influence of IPD-1151T on airway hyper-responsiveness
The first se t of ex p eriments w as de signed to ex amine the inf luence of IPD-1151T on airw ay hype r-responsivene ss. To do this, w e ex amined firstly the minimum conc entration of MCh that can c ause significant bronchoconstric tion in non-sensitize d mic e. The data are show n in Fig. 2. Although low dose s of MCh (10 -6 M and 10 -5 M) sc arce ly affe cte d airw ay response s, significant (P< 0.05 ) inc rease in airw ay ove rflow w as obse rved w he n mice w ere injec te d intravenously w ith 10 -4 M MCh. The nex t ex perime nts w ere designe d to ex amine the influe nce of oral administration of IPD-1151T on MCh-induc ed bronchoc onstriction in DNP-KLH-se nsitized mic e. As show n in Fig. 3, intrave nous administration of 10 -4 M MCh into se nsitized mic e caused a signific ant increase in airw ay hyper-re sponsivene ss as compare d w ith non-se nsitized control (P< 0.001). Oral administration of IPD-1151T inhibited MCh-induc ed inc re ase in airw ay responses. The minimum effective dose in 5-day tre atment w as 100 mg /kg and that in 14-day treatme nt w as 10 mg /kg. The influe nce of IPD-1151T on antige n-induce d allergic airw ay responses is show n in Fig. 4. KLH in sensitized mic e caused a strong allergic bronchoconstric tion as compare d w ith non-sensitize d control (P< 0.001 ). Oral administration of IPD-1151T inhibited alle rgic bronchoc onstric tion in a dosedepende nt fashion: as dose and frequenc y of administration w e re inc reased, volume of air ove rflow gradually dec reased (Fig. 4 ).
Influence of IPD-1151T on in vivo IgE production BALB/c mice w ere se nsitized intraperitoneally w ith DNP-KLH. Immune serum w as obtaine d from five individual mice 7, 10, 14, 17, 21 and 25 days afte r se nsitization. Non-immune serum w as also obtaine d from age-matche d, non-sensitize d mic e. As show n in Fig. 5A, total IgE conce ntration in immune serum gradually inc re ased, pe aked on Day 14 and decline d thereafter from Days 17 to 25. Changes in the levels of DNP-spe cific IgE antibody show ed a similar p attern to that obse rved in total IgE conce ntration: the spe cific antibody w as first de te c te d on Day 7, pe aked on Day 14 and de clined control leve ls from Day 17, but sustaine d on Day 25 (Fig. 5B). The influe nce of oral administration of IPD-1151T on total and spe cific IgE conc entration in se rum prepared from mice se nsitized w ith DNP-KLH is show n in Fig. 6. Oral administration of IPD-1151T, starting on Day 9 of se nsitization, significantly inhibited both total and spec ific IgE conc entration in serum obtained 15 days after sensitization (P< 0.05; Fig. 6 ). This inhibitory action of IPD-1151T on w as further strengthe ned w hen administration of age nt w as started on Day 0 of se nsitization (Fig. 6 ). Influence of IPD-1151T on in vitro lymphocyte proliferative response and interleukin (IL)-4 production Influenc e of IPD-1151T on prolife rative activities of sple en ce lls in response to mitoge nic stimulation is show n in Table 1. IPD-1151T inhibited c ell prolifera-tion in dose-depende nt manner w he n the ce lls w ere prepared from nude mice and stimulate d w ith LPS in v itro . How ever, IPD-1151T could not suppre ss T ce ll mitogen (Con A)-induc ed prolife rative response of sple en c ells prepared from normal mic e e ve n w hen the c ells w ere c ulture d in the presenc e of 100 m g /ml of the age nt. IL-4 production by spleen ce lls in response to Con A stimulation in v itro w as significantly inhibited by IPD-1151T. This inhibitory effect on IL-4 production w as dose-de pendent and first note d at a c oncentration of as little as 5 m g /ml ( Table 2 ).

Discussion
The prese nt study show s that IPD-1151T inhibits MCh-induc ed increase in airw ay re sponses in pre-se nsitized mic e. IPD-1151T has an ill-de fine d mode of action, and various eleme nts c ould acc ount for this prote c tive effe ct. Func tional antagonism tow ards MCh-induc ed bronchoconstriction does, how ever, not ac count for the findings in the present study, sinc e the MCh re sponsivene ss in normal c ontrol animals is not alte red (Fig. 3 ). It is reporte d that mouse has airw ay contractile tissue that responds w hen ex posed to muscarinic agonists such as MCh by the vascular route. 23 The re is also e videnc e that airw ay resp onse to musc arinic agonists is pre sumably   me diated by specific muscarinic rece ptor ac tivation. 2 4 Therefore, the data in Fig. 3 may be interprete d as show ing that IPD-1151T inhibits muscarinic rec eptor ac tivation that is induced by se nsitization w ith DNP-KLH and results in therapeutic effe cts on KLH-induce d inc re ase in airw ay response to MCh. The importance of IgE-re late d reactions in te rms of the ir ability to influence airw ay function has be en appare nt from clinical and ex perime ntal studies. Sensitization of mice by inhalation of alle rgen c ause d not only IgE hyper-produc tion but also increase d airw ay re ac tivity. 4 ,5 Histological ex amination of the nose and lungs including low er and upp er airw ays prepared from se nsitized mic e re ve ale d an absenc e of ne utrophils, e osinop hils and mast ce lls at the time of the incre ased airw ay hyper-responsiveness. 4 It is also reported that passively transfe rred rabbit serum containing allergen-sp ecific IgE is capable of sensitizing naive rabbits so that the y develop airw ay obstruction, 2 5 as w ell as inc reased airw ay hyperresponsive ness after ex posure to spec ific allergen. 2 6,27 The se reports strongly suggest that alte rations in airw ay func tion such as hyp erresponsive ness to allergens are close ly linked to the presenc e of allerge n-spe cific IgE. The pre sent study revealed the inhibitory effe ct of IPD-1151T on levels of both total and spe cific IgE w hen the mic e w ere administe red orally w ith the age nt (Fig. 6 ). It is also show ed that inhibitory e ffec t of IPD-1151T on IgE produc tion is due to its suppressive action on B-ce ll prolife ration (Table 1 ) and IL-4 production from T-cells (Table 2 ). Taken toge the r, a sec ond possible ex planation for the protec tive e ffec t of IPD-1151T on antigen-induce d airw ay hype r-responsiveness (Fig. 3 ) w ould be an e ffec t on IgE production.
Upon antige nic stimulation, c ross-linking of surfac e IgE bound to rec eptors ex pressed on mast ce lls and basophils caused se cretion of substanc es store d in granule s such as histamine and se rotonin, etc. 2 8,2 9 and new ly produc ed physiologic ally ac tive substanc es such as le ukotrien and platelet ac tivating factor. 3 0, 3 1 These chemic al mediators are re ported to provoke alteration of smooth muscle in airw ays and result in bronchoconstric tion. 32 , 33 Matsuura e t a l. 1 5 re ve ale d the inhibitory action of IPD-1151T on degranulation of mast ce lls in response to antigenic stimulation in v itro . From these observations, there is a possibility that IPD-1151T may func tion as a me mbrane stabilizer and atte nuate bronchoconstric tion induce d by MCh and spec ific antige n.
Since mice administe re d IPD-1151T for 14 days (100 mg /kg /day ) did not show w e ight loss, ruffled fur and a hunched posture , compared w ith the c ontrol, IPD-1151T could be ex pe cted to develop into a new anti-asthmatic drug.