Attenuating Effect of a Thromboxane Synthetase Inhibitor (OKY-046) on Bronchial Responsiveness to Methacholine Is Specific to Bronchial Asthma

Attenuating Effect of a Thromboxane Synthetase Inhibitor (OKy-046) on Bronchial Responsiveness to Methacholine Is Specific to Bronchial Asthma* Jtlasaki fujinlura, ~\f.D.; Sayuri Sakllnwfo, i\I.D.; and

Tanwtsu Matsuda, M.D.

To determine whether the involvement of thromboxane ~ in bronchial hyperresponsiveness (BUR) is speci6c to asthma, we examined the effects of a selective inhibitor of thromboxane synthetase (OKY-046) and a cyclooxygenase inhibitor (indomethacin) on bronchial responsiveness to methacholine in normal subjects and patients with chronic bronchitis, diffuse bronchiectasis, and intrinsic bronchial asthma. The provocative concentration of methacholine producing a 20 percent fall in forced expiratory volume in 1 s (PCIO-FEV.) was measured before and after oral administration of OKY-046 (2,600 mg over four days) and indomethacin (450 mg over three days) in ten normal, ten bronchitic, nine bronchiectatic, and eight asthmatic subjects, respectively. Baseline values of FEV I and forced vital capacity (FVC) were not altered by OKY-046 or indomethacin. The geometric mean value of PCtD-FEV. increased signi6cantly (p<0.OO5) from 1.78 to 4.27 mglml after OKY-

of the major clinical features of bronchial O neasthma is the increased bronchial responsiveness

to various specific and nonspecific stimuli.· Thromboxane A2 (TXA2 ), a nletabolite of arachidonic acid, is a potent bronchoconstrictor. 2 A selective thromboxane synthetase inhibitor, OKY-046 ([E]-3-[ 4-(1-imidazolylmethyl)phenyl]-2-propenoic acid hydrochloride monohydrate),3 was shown to suppress ainvay anaphylaxis induced by antigen inhalation" and bronchoconstriction caused by leukotriene C 4 , a component of slowreacting substance of anaphylaxis,5.(l in a guinea pig in vivo. 7 Aizawa and coworkersk reported that OKY-046 inhibited the increase of airway responsiveness induced by ozone in dogs. From these aninlal experiments, TXA2 is tholl~ht to be involved in bronchial asthma, especially in bronchial hyperresponsiveness (BHR). Clinically, Walters 9 reported that BHR to histamine

046 in asthmatic subjects, but not in normal, bronchitic, or

bronchiectatic subjects. On the other hand, PC7.0-FEV. increased significantly (p
=

PC.-FEV I provocative concentration of methacholine producing a 20 percent fall in forced expiratory volume in I s;

FVC = forced vital capacity; GSEM = geometric standard error

of the mean; BUR thromboxane As

=bronchial

hyperresponsiveness; T~

=

in patients with asthma was suppressed by the cyclooxygenase inhibitor, indonlethacin. We sho\ved that oral administration of the thromboxane synthetase inhibitor, OKY-046, reduced BHR to acetylcholine but did not alter the baseline pulmonary functions in asthmatic subjects. to Therefore, it has been our hypothesis that subthreshold dose of TXA2 , which has no effect on bronchomotor tone per se, may be involved in BHR in asthma. Well recently demonstrated that bronchial responsiveness to aerosol histamine could be potentiated by subthreshold dose of a TXA2 nlimetic, STA2 ,12 in the guinea pig. Ho\vever, it has never been kno\\'n \vhether the involvenlent of TXA2 in BHR is specific to asthnla or not. This study \vas designed to evaluate it. We examined the effects of OKY-046 and indomethacin on bronchial responsiveness to methacholine in norlnal subjects and patients \vith asthlna, chronic bronchitis, and diffuse bronchiectasis.

*FrOlll the Third DeparhDent of Internal 1\.1t'didnl", Kanazawa University School of Medicine. Kanazawa, Japan. Manuscript received November 6; revision accepted January 2.

Subjects

cine, KaooZllwa University School of .\Ieclicirw, 1:3-1 ltlkara-11uu·hi. Kaooultva 920, Japan

Twenty-seven patients (16 IDen and 11 WOlDen) and ten Ilonnal healthy subje(.·ts (one Juan and nine w(ul1en) participated in the study (Tahle I). Individuals had no other disease. Ten patients had

1990. Re1J1int requests: Dr. Fujilnura. 3nl Deptlrt"wnt of lntl'r"a/ .\I(Jdi-

656

MATERIALS AND METII()DS

OKY-046 Effect on Bronchial Responsiveness (Fujimura, Sakamoto, Matsuda)

Table I-Characteristics of Subjects· Feature

Bronchial Asthrua

Diffuse Bronchiectasis

Chronic Bronchitis

No. AJte, yr Male:fernale FVC, %pred FEV I , %pred FEV/FVC, %

R 55± 14 4:4 109± 15 95± 15 73±9

58± 15 5:4 84±21 69±23 67±11

9

10 6O± 11 7:3 96± 16 82±17 72±13

Normal Control

10 21 ± 1 1:9

105± 12 101 ± II 88±7

*Oata are presented as mean ± SO. Abbreviations: FVC = forced vital capacity; FEV. = forced expiratory volume in I s. chronic bronchitis alone. 13 Nine patients had sinobronchial syndrorne H \\rith diffuse bronchiectasis,llIi sputum more than 20 mVday (mean, 65 mVday; range, 20 to 200 mVday), and chronic sinusitis. Diffuse bronchiectasis was diaJtnosed based on chest roentgenogranl, chest {.'omputed tomo~raphic scan, bronchofibroscopy, and br()ncho~raphy.Chronic sinusitis was dia~nosed by ()tolaryn~()lowsts based on symptoms (postnasal drip, nasal dischar~e, and nasal ohstnlction), physical examinations, and r()ent~en()gram. Their sputa \\'ere all punllent and HaenuJ1Jhilus influenz.ae, Streptococcus prwunwniae, Pseudomonas aeruginosa, and Escherichia coli ~rew from the sputa in 4, I, 3, and 1 patient, respectively. Eight patients had mild stable intrinsic asthma based on the follo\\'ing three criteria: (I) a history ofrecurrent episodes ofwheezing, (2) reversihle airway obstruction previously documented by a physician, and (3) no familial history of allergic diseases, no positive skin test to ten (.'onuuon allergens, and no increased levels ofspecific IgE antibodies. Ten nonnal subjects were chosen from 24 nonatopic healthy students of the Sch
Method.4; The methacholine test was performed \\,hen their symptoms were nlild and stahle while they were receiving oral bronchodilators (theophylline and ~2-adrenerJtic agonists) and mucolytic agents but not steroids. Treatment with all medication was stopped at 9 PM the previous day to allow a washout time of 12 hours before the measurement of hronchial responsiveness at 10 AM on the test day. All the medication was maintained without any modification durin~ the study period. Bronchial responsiveness was evaluated by methacholine challenge. lfi Methacholine chloride was dissolved in physiolowc saline solution to make solutions of 0.04, 0.08, 0.16, 0.31,0.63, 1.25,2.5, 5, 10, 20, 40, 80, and 160 mWml. Saline solution and methacholine were inhaled from a nebulizer (DeVilbiss 646, DeVilbiss Co, Soruerset, Pa) operated by (.'()mpressed air at 5 Umin. The nebulizer output was 0.28 mV2 min. Saline solution was inhaled first for m'o minutes and forced expiratory volume in 1 s (FEV.) was measured

on u dry wedge spirometer (Transfer test, ~K. Mor~lm Ltd, EnJtland). Sinee the chan~e in FEV I from the haseline after inhalation of saline solution was 10 percent or less in all subjects, inhalation of methacholine was started. Methacholine \\'a.~ inhaled f()r two minutes hy tidal hreathjn~ with a nose clip, and this was fi)lIowed imnlediately hy spin)metry. Increasin~ (.'Oncentrations were inhaled until a fall of 20 per<''enl or more in FEV. occurred. The meusured values were plotted on selnilo~arithmic~raph paper and tht" metha(.'holine provocative (,()Il{.'entration producing a 20 percent fall in FEV. (PC 2ll,-FEV.) was calculated. OKY-046 (Kissei Phannaceutical Co, Ltd, Matsumoto, and Ono Pharmaceuticill Co, Ltd, Osaka, Japan) was given orally in a dose of 200 mg finn times a day for three days plus 200 nlg in the mornin~ on the fourth day (test day). Bronchial responsiveness to methacholine was then measured at 10 AM. Indomethacin \\'as given orally at a dose of 50 mg filur times a day for two days plus 50 m~ in the mornin~ on the third day (test day). A methacholine challen~e test was carried out at 10 AM. Infonned (..'()nsent \\ras obtained from all subjects after the purpose of the test had heen explained. No information on the test dru~s and their pharmacolowc actions was ~iven to the subjects or the technic.l1 staff who performed the methacholine inhalation test and calculated PC~,-FEV•. This study was approved by the ethics (.'()mmittee of our university. Methacholine PC 2ll,-FEV. values were expressed as ~eometric means with the ~e()metric standard error of the mean (GSEM) expressed as a factor. Values for baseline FVC and FEV I were reported as arithmetic means and stundard errors of the mean (SE~t).

Geometric mean PC~I- FEV I values were compared by the pe'lired t test. A p value of 0.05 or less was taken as significant.

RESULTS

There was no significant difference in the FEV. or FVC baseline values before and after each drug in the normal, bronchitic, bronchiectatic, or asthmatic group. Mean baseline FVC and FEV. values are shown before and after the administration of OKY-046 and indomethacin in Table 2. The PC 20-FEV. values before and after oral dosing with OKY-046 in each group are shown in Figure 1. There was a significant increase in PC 2o -FEV. (p
Table 2-Effects ofOKY-046 and Indomethacin on Baseline FVC and FEV. Values· FVC, L

Asthma (n = 8) Diffuse bronchiectasis (n = 9) Chronic bronchitis (0 = 10) Normal eontrol (0 = 10)

Control

OKY-046

Indomethacin

Control

OKY-046

Indomethacin

3.44± 1.01 2.61 ±0.90 3.17±0.88 3.63±0.77

3.45± 1.03 2.43±0.81 3.15±0.88 3.67±0.69

3.39± 1.04 2.50± 1.04 3.09±O.81 3.68±0.73

2.47±0.72 1.73±0.60 2.26±0.77 3.17±0.71

2.46±O.70 1.66±0.52 2.28±0.77 3.21 ±0.65

2.45±O.77 1.65±0.59 2.26±O.75 3.16±0.62

*Data are presented as mean ± SD. CHEST I 98 I 3 I SEPTEMBER. 1990

657

NS

P( 0 .005

E

NS

NS

~

100

~

.........

~

I

I~I

10

~I 0

~

1~i :=::::::,

~

til

~

o .1

o .01

OKY

Cont

BA

OKY

Cont

OKY

Cont

BE

Cant

OKY

NC

CB

FIC;URE 1. Effects of oral administration of ()KY-046 (OKY) on methacholine PC21t-FEV I (the provocative concentration of methacholine reducin~ FEV. hy 20 percent) and ten normal, ten hronchitic, nine hronchiectatic, and ei~ht asthnlatic suhjects. NC = normal <..'ontrol; CB = chronic hronchitis; BE = diffuse hronchiectasis; BA = hronchial asthma. Group values are presented as ~eometric means with the geometric standard error of the mean.

P( 0 .005

NS

E

100

.........

--~

>~

10

NS

NS

~

I~I

I

0

til

~

~

o .1

~

Cont

10M

BA

Cont

10M

BE

Cont

10M

Cont

10M

CB

FIGURE 2. Effects of oral adrninistration of indolnethacin (10M) on methacholine PC 2lt-FEV I (see

Fi~

1

le~end) in ten 1l0rnla1, ten hronchitic, nine bronchiectatic, and ei~t asthmatic subjects. NC = normal

control; CB = chronic hronchitis; BE = diffuse hronchiectasis; BA = bronchial asthma. Group values are presented as geonlctric means "rith the ~eometric standard error of the mean.

658

OKY-Q46 Effect on Bronchial Responsiveness (Fujimura, Sakamoto, Matsuda)

tatic group, respectively. The PC2()-FEV I values before and after the administration of indol1lethacin in each group are shown in Fi~ure 2. There was a significant increase in PC2()FEV. (p
Throlnhoxane synthetase inhibitors have been found to suppress bronchoconstriction induced by antigen and some bronchoconstrictive agents in the guinea pig in vitro l ";, and in vivo. 4 ,7,lH Aizawa and coworkersH sho\\'ed that a selective thromboxane synthetase inhibitor, OKY-046,:l inhibited the increase of aif\\'ay responsiveness to acetylcholine induced by ozone and a TXA2 nlimetic, U-46619, in subthreshold doses caused an increase in ainvay responsiveness to acetylcholine in dogs. Well also demonstrated that subthreshold dose of inhaled STA2 , a TXA2 mimetic, potentiated bronch'al responsiveness to aerosol histamine in guinea pigs. In addition, TXA2 has been shown to be released from human lung parenchyma at the time of anaphylaxis. 19 These findings suggest that TXA 2 plays a part in bronchial asthlna, especially in the development ofBHR. Our previous studylO showed that oral administration of OKY-046 si~nificantly reduced BHR to acetylcholine in patients with intrinsic asthma. Yukawa and coworkers20 also reported the inhibitory effects of OKY-046 on BHR to acetylcholine and leukotriene D 4 in extrinsic asthmatic subjects. In this study, we confirmed the role of TXA2 in BHR to methacholine in asthma. Althou~h the above-mentioned findings indicate that TXA2 is involved in BHR in asthma, it has never been kno\\'n \vhether the involvement ofTXA2 in BHR is specific to asthnlu or not. In the present study, we exalnilled the effects of OKY-046 and indonlethacin on bronchial responsiveness to methacholine in normal, bronchitic, bronchiectatic, and asthmatic subjects. Patients \"ith chronic bronchitis and diffuse bronchiectasis were studied as disease controls who have chronic airn'ay inflammation as well as asthmatic patients. In our results, bronchial responsiveness to methacholine and baseline pulmonary function were not altered by OKY-046 in normal, bronchitic, or bronchiectatic subjects. In asthmatic subjects, OKY-

046 significantly reduced bronchial responsiveness to methacholine but did not change haseline puhnonary function. On the other hand, bronchial responsiveness was significantly reduced by indolllethacin only in bronchiectatic subjects having expectoration of Inure than 20 mVday. From these results, it is su~ested that the significant involvement of TXA 2 in BHR Inay he specific to asthlna. The follo\ving hvo Inechanisllls might be considered to be responsible for the specific role of TXA2 in asthma: (1) difference in aitway inflammatory cell populations releasing infhllllinatory mediators, namely lymphocytes, neutrophils, eosinophils, and mast cells in asthnla vs neutrophils and lymphocytes in bronchitis and bronchiectasis; and (2) acceleration of the thromboxane synthetase in asthlna re~ardless of difference in ainvay inflammatory cells. However, we do not know what mechanislll operates for TXA2 production in asthma. Further studies are needed in \vhich measurernents of cyclooxygenase products in airway tissue and bronchoalveolar lava~e fluid and TXA2 -releasing capacity of recovered cells from airway tissue would be carried out in patients with asthma in comparison to those in norlllal subjects and those with other chronic inOalnlnatory hronchial diseases. It is surprising that three days' adlllinistratioll of indomethacin (200 mg/day) si~nificantly reduced bronchial responsiveness in bronchiectatic subjects. Tamaoki and coworkers 21 showed that two weeks' inhalation of indomethacin reduced sputuln production and concentration of prostaglandin E:z and F2t1 but not thromboxane B2 and 6-keto-prosta~landinFlo in sputum in patients with excessive sputum. However, short-term oral administration of indomethacin did not affect amount of expectoration, clinical synlptorns, or baseline pulmonary functions in this study. Consequentl); improvement of the bronchial conditions, especially reduction of inflammatory edelna of bronchial mucosa, after the administration is not thou~ht to be responsible for the indomethacin-induced bronchial hyporesponsiveness. We speculate that hronchial responsiveness of bronchiectasis mi~ht be potentiated by inflammatory release of bronchoconstrictor prostaglandins, for example pr()sta~landin O 2 and F:z.n , except for TXA2 • In conclusion, these results indicate that the involvement of TXA2 in BHR may he specific to asthlna. However, it is known that OKY-046 has some potentiating effect on prostacyclin (PGI 2 ) production as well as inhibitory effect on thromboxane synthesis.:l Parsargiklian and Bianco22 reported that inhaled PGI 2 gave an excellent degree of protection against ultrasonic H 2 0 mist and physical exercise-induced bronchoconstriction but had no effect on allergen-induced bronchoconstriction. They22 and Hardy and co\\'orkers2.1 showed that inhaled PGI 2 had no consistent bronchoCHEST I 98 I 3 I SEPTEMBER, 1990

659

dilator effect in either normal subjects or asthmatic patients. We investigated that oral administration of a PGI 2 mimetic, OP-41483,24 failed in reducing BHR to acetylcholine in asthmatic subjects. 2.'; It is likely therefore that the inhibitory effect of OKY-046 on BHR is due to a reduction in TXA 2 production rather than increased PGI 2 production. Further studies using TXA2 receptor antagonists are needed to confirm the conclusion. ACKNO\VLEDGMENT: \Ve wish to thank Kissei Pharmacentkal Co~ Ltd~ Matsumoto~ and Ono Phannaceutical Co, Ltd, Osaka, Japan~ f(~r.kin~ly suppl~'ing OKY-046, and u.lso to express thanks to the parhclpahn~ technical staff, ~t rs. Yumie Karnio, for performin~ the measurements of hronchial responsiveness to methacholine. REFERENCES 1 Boushey HA, Holtzman ~fJ~ Sheller JR, Nadel JA. Bronchial hyperreactivity. Anl Rev Respir Dis 1980; 121:389-413 2 Svensson J, Strandber~ K, TuvenlO T, Harnher~ M. Thromhoxane A2 : effects on airway and vascular smooth muscle. Prosta~landins 1977; 14:425-36 3 Naito J~ Komatsu H~ Ujiie A, Hamano A, Kubota T, Tsuhoshima M. Effects ofthromboxane synthetase inhihitors on a~re~ation of rabbit platelets. Eur J Pharmacol1983; 91:41-8 4 Fujimura M, Koshino K, Nishioka S, Matsuda T. Involvement of thromboxane A2 in SRS-A mediated hronchoconstriction induced by aerosol antigen in the guinea pi~. Kokyu lh Junkan 1984; 3:1066-71 5 Murphy Re, Hammarstronl S, Samuelsson B. A slow reacting suhstance from murine mastocytoma cells. Proc Natl Acad Sci USA 1976; 76:4275-79 6 Samuelsson B, Hammarstrom S. Nomenclature for leukotrienes. Prostaglandins 1980; 19:645-48 7 Fujirnura M, Miyake Y, Uotani K, Kanarnori K~ Matuda T. Secondary release of thromhoxane A2 in aerosolleukotriene C..induced broncho(.,. (lnstriction in guinea pigs. Prostaglandins 1988; 35:427-37 8 Aizawa H, Cht1n~ KF, LeikaufGD, Ueki I, Bethel RA O'Bvrne PM, et al. Significance of thrornboxane generation ~n oz~)ne­ induced airway hyperresponsiveness in dogs. J Appl Physiol 1985; 59:1918-23 9 \Valters Ell. Pr()sta~landinsand the (..'()ntrol of airn'ays responses to histamine in normal and asthmatic subjects. Thorax 1983; 38:188-94 10 Fujilnura M~ Sasaki F, Nakatsunli Y, Takahashi Y, Hifumi S, Taga K, et al. Effects of a thromboxune synthetase inhibitor (OKY046) and a lipoxygenase inhibitor (AA-861) on hronchial responsiveness to acetylcholine in asthmatic subjects. Thorax 1986; 41:955-59 11 Fujimura M, Saito M, Kuntshima K, Miyake Y, Sakamaoto S,

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OKy-046 Effect on Bronchial Responsiveness (Fujimura, Sakamoto, Matsuda)