Inhibition of antigen-induced airway hyperresponsiveness by a thromboxane A2 receptor antagonist (AA-2414) in Ascaris suum-allergic dogs

Prostaglandins

46:301-318, 1993

I N H I B I T I O N OF A N T I G E N - I N D U C E D A I R W A Y H Y P E R R E S P O N S I V E N E S S BY A T H R O M B O X A N E A 2 R E C E P T O R A N T A G O N I S T (AA-2414) IN A S C A R I S S U U M - A L L E R G I C DOGS T. M a t s u m o t o

a n d Y. A s h i d a

P h a r m a c e u t i c a l R e s e a r c h Laboratories R e s e a r c h Division, Takeda Chemical Osaka 532, Japan

II, P h a r m a c e u t i c a l Industries, Ltd.

ABSTRACT We studied changes in airway responsiveness to acetylcholine (ACn) a f t e r a n t i g e n i n h a l a t i o n in A s c a r z s suum ( A. suum ) - a l l e r g i c dogs. Airway responsiveness was determined by obtaining a dose-response curve of lung resistance p l o t t e d against increasing concentrations of ACh aerosol before and a f t e r i n h a l a t i o n of A. suum a n t i g e n . To d e t e r m i n e the r o l e of thromboxane A~ (TXA~) in the airway response, we tested the effect of a TXA2 receptor antagonist, AA-2414, in A. suum - a l l e r g i c dogs. The procedure was repeated in each dog at an interval of 2 weeks to evaluate the effect of AA-2414 in a crossover manner. The dogs showing an airway response to antigen showed an increase in airway responsiveness to ACh 2, 4 and 6 h a f t e r a n t i g e n i n h a l a t i o n . The increase in airway responsiveness was s i g n i f i c a n t l y i n h i b i t e d by administration of AA-2414 (5 mg/kg, i . v . ) before antigen i n h a l a t i o n . These r e s u l t s suggest t h a t TXA2 may be involved in antigen-induced airway hyperresponsiveness (AHR) in dogs. INTRODUCTION Airway hyperresponsiveness (AHR) is a c h a r a c t e r i s t i c of bronchial asthma and is c l o s e l y associated with the s e v e r i t y and frequency of asthmatic episodes (20). AHR can result from a v a r i e t y of factors, including exposure to c e r t a i n chemicals (14,24). The development of AHR is often associated with inflammatory c e l l i n f l u x and mediator release (8), e p i t h e l i a l damage ( 1 8 ) , v a s c u l a r leak ( I i ) , and a l t e r a t i o n s in mucus c o m p o s i t i o n and secretion (5). The release of eicosanoids from lung tissue in response to immunological s t i m u l i has been described previously (30). Among the eicosanoids, TXA2, which is produced by p l a t e l e t s , mononuclear leukocytes, lung mast c ells and o t h e r c e l l s , has been c o n s i d e r e d one of the i m p o r t a n t me d ia t o r s in inflammatory r e s p i r a t o r y diseases such as asthma because of i t s potent c o n t r a c t i l e effects on pulmonary airways and blood vessels (27). Furthermore, U-46619, a TXAz mimetic drug, increases the airway response to ACh in the i s o l a t e d bronchial preparations of dogs (33). I t has also been suggested t h a t TXAz plays a c r i t i c a l r o l e in the AHR induced by ozone (2) and inflammatory mediators such as leukotriene B4 (28) and p l a t e l e t - a c t i v a t i n g f a c t o r (9). However, the r o l e of t h i s mediator in the AHR induced by immunological stimuli in v i v o has not yet been demonstrated s a t i s f a c t o r i l y .

Copyright © 1993 Butterworth-Heinemann

302

Prostaglandins

We have reported that some quinone derivatives show TXA2 antagonistic a c t i v i t y and that one of these compounds, AA-2414 inhibited antigen-induced immediate bronchoconstriction in actively sensitized guinea pigs (4). In this study, to investigate the possible participation of TXA2 in asthmatic responses, especially in antigen-induced immediate bronchoconstriction and AHR, we studied changes in airway responsiveness to ACh a f t e r antigen inhalation and the effect of the TXA~ receptor antagonist AA-2414 on the airway response to antigen challenge in A. su~m -allergic dogs.

METHODS Drugs and chemicals The drugs and chemicals used were: pentobarbital sodium (Abbott, North Chicago,IL), acetylcholine chloride (Tokyo Kasei, Tokyo), pancuronium bromide (Sigma Chemical, St. Louis, MO), Evans blue (Merck, Dermstadt, FRG), Ascar~s s~um extract [100,000 PNU (protein nitrogen unit)/ml; Greer Laboratories, Lenoir, NC] and 0.9% sodium chloride (Otsuka Pharmaceutical Co, Tokyo). AA-2414 [( ± )-7-(3,5,6-trimethyl-1,4-benzoquinon-2- y l ) - 7 phenylheptanoic acid] was synthesized by TakedaChemical Industries, Ltd. General Adult mongrel dogs of either sex weighing between 8 and 16 kg and without cough, rhinorrhea, intestine helminthiasis or microfilaria were chosen for the study. We did not exclude dogs that had been observed to have eggs or dead bodies of Toxocara cants in t h e i r feces. Approximately 60% of the animals were skin test p o s i t i v e (>8 mm in diameter at 20 min) to an intracutaneous injection of 0.1 ml of A. s~um extract (100 PNU/ml). Dogs which were skin test negative did not respond to airway challenge with antigen aerosol and were thus not included in this study of AA-2414. The animals were anesthetized with intravenously administered pentobarbital sodium (25 mg/kg) and paralyzed with pancuronium bromide (0.1 mg/kg i . v . ) ; anesthesia was maintained with a d d i t i o n a l p e n t o b a r b i t a l sodium. An endotracheal tube (10 mm internal diameter) was inserted and connected to a constant-volume respirator (model 613, Harvard Bioscience, South Natick, MA) set at a tidal volume of 15 ml/kg and at a frequency of 20 breaths/min. Airway flow was measured by means of a Fleisch pneumotachographconnected to the endotracheal tube. Tidal volume was obtained by electrical integration of the flow signal. Transpulmonary pressure was measured by a transducer (Validyne model DP-45-16) connected d i f f e r e n t i a l l y to a lateral port at the proximal end of the endotracheal tube and to an intrapleural cannula placed in the f i f t h intercostal space. The signals were displayed on a chart r e c o r d e r and lung r e s i s t a n c e (R L) and dynamic c o m p l i a n c e (C ~y, ) were measured by the method of Amdur and Mead ( 3 ) . A s i g n a l p r o c e s s o r (7T/18A, Nihondenki S a n - e i , Tokyo) was used f o r the c o n t i n u o u s o n - l i n e c o m p u t a t i o n of R L and C ~ n .

Prostaglandins

303

Airway r e s p o n s i v e n e s s to ACh Airway responsiveness was determined by measuring R L in response to doubling concentrations of ACh (0.125 to 128 mg/ml). ACh aerosol was generated using a j e t - n e b u l i z e r (DeVilbiss No.646) with a flow rate of 7 l i t e r / m i n and was delivered via the endotracheal tube and constant-volume r e s p i r a t o r set at a constant t i d a l volume (15 ml/kg) and a constant frequency (20 breaths/min). The ACh aerosols were sequentially inhaled for i min at 2-min intervals. Before ACh was delivered into the airway, baseline R L values were determined using saline aerosol. After each ACh aerosol was inhaled, the peak R L value was measured. Responsiveness was expressed as the concentration of ACh required to increase R L to 200% of the value measured a f t e r saline aerosol inhalation (PC~oo-ACh), calculated by linear interpolation from the concentration-response curve.

Antigen challenge A. s u u ~ antigen aerosol (100,000 PNU/ml) was generated using a j e t - t y p e nebulizer (DeVilbiss No.646) and was inhaled f o r 3 min employing the procedure described f o r the ACh airway responsiveness test. The airway reaction to A. sawm antigen was considered p o s i t i v e i f the R L value increased by 100% from the baseline value.

Skin test Quantitative intracutaneous skin testing was carried out using A. suw~ e x t r a c t . Dogs were anesthetized with pentobarbital sodium, and 1% Evans b l u e was a d m i n i s t e r e d i n t r a v e n o u s l y in a volume of 0.1 m l / k g . Logarithmically increasing d i l u t i o n s (0.01 to 10,000 PNU/ml) of A. suwm extract were then intracutaneously injected in a volume of 0.1 ml. Twenty minutes l a t e r , the skin test reactions were scored positive i f there was a blue wheal of at least 8 mm in diameter at the site of injection. The lowest d i l u t i o n of A. s~m extract producing a positive response was noted.

Experimental protocols Protocol 1 :Relationships between airway and cutaneous s e n s i t i v i t y to antigen and airway responsiveness to ACh In order to find a convenient way to select, at a high frequency, dogs t h a t w i l l show an airway response to A. swum antigen from among many mongrel dogs, we have studied the relationships among airway and cutaneous s e n s i t i v i t y to A. suwm antigen and airway responsiveness to ACh in A. swum - a l l e r g i c mongrel dogs. Twenty five dogs were anesthetized and a r t i f i c i a l l y ventilated. They were then tested for skin reactions to A. s~m antigen and airway responsiveness to ACh. About one hour after the tests, the dogs were challenged with A. swum antigen. Antigen aerosols were sequentially inhaled for 3 min at 7-min intervals. After each of the increasing concentrations of A. swum protein (1,000, 10,000, 100,000 PNU/ml) was delivered into the airway, the peak value of R L was measured. The airway reaction to A. swum antigen was scored p o s i t i v e i f the R L value increased by 100% from the baseline value. The lowest d i l u t i o n of the antigen producing a p o s i t i v e response was noted.

Prostagla ndins

304

Protocol 2 :Change in airway responsiveness after antigen inhalation To determine whether there were any changes in airway responsiveness a f t e r a n t i g e n c h a l l e n g e , the ACh responsiveness t e s t was performed 2, 4 and 6 h a f t e r a n t i g e n c h a l l e n g e , in seven responders and s i x nonresponders. They were t h e n t e s t e d f o r s k i n r e a c t i o n s t o A. s~um a n t i g e n and a i r w a y responsiveness to ACh. One hour a f t e r the t e s t s , the dogs were challenged w i t h the antigen (i00,000 PNU/ml), and the peak value of R ~ was measured. Responders were defined as dogs t h a t showed a p o s i t i v e airway r e a c t i o n to A. s~m antigen.

3O0

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responder (n:7) nonresponder (n:6)

~200

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,

u

¢: 10(]1

,

10

c

20

s

!

60 70

20

40

60

TIrne afferantlgeninhalatlon(min)

Fig i . Change in RL and C dy, a f t e r Antigen Challenge in Dogs Responsive and Nonresponsive to Antigen I n h a l a t i o n . The numbers on the o r d i n a t e represent the percent change from baseline value measured before antigen challenge. The data represent the mean± S.E. f o r 6 or 7 animals.

Prostaglandins

305

Protocol 3 :Relationship between immediate bronchoconstrietion and AHR To determine whether there were any correlations between airway response to A. suum antigen and AHR, the airway responsiveness to ACh was determined in sixteen dogs p r i o r to antigen challenge and again 4 h a f t e r antigen challenge. Skin testing and antigen challenge were done as described in protocol 2. Protocol 4 :Effect of h h - 2 4 1 4 on immediate bronehoconstriction and AHR Seven dogs were used in t h i s study of AA-2414. The study was performed in a v e h i c l e - c o n t r o l l e d manner w i t h a c r o s s o v e r design. There was a 2-week i n t e r v a l between t h e s t u d y days. AA-2414 (5 mg/kg) was i n t r a v e n o u s l y a d m i n i s t e r e d 2 min b e f o r e a n t i g e n c h a l l e n g e . Skin t e s t i n g and a n t i g e n c h a l l e n g e were done as d e s c r i b e d in p r o t o c o l 2. Airway responsiveness to ACh was determined p r i o r to a n t i g e n c h a l l e n g e and again 4 h a f t e r a n t i g e n challenge.

Statistics Data a r e p r e s e n t e d as t h e mean± S.E. S p e a r m a n ' s r a n k - c o r r e l a t i o n coefficient was used t o a n a l y z e t h e r e l a t i o n s h i p between v a r i a b l e s . S t a t i s t i c a l s i g n i f i c a n c e from b a s e l i n e responsiveness was determined using S t u d e n t ' s p a i r e d t - t e s t a f t e r l o g a r i t h m i c t r a n s f o r m a t i o n of the data. We considered a P value of less than 0.05 to i n d i c a t e a s i g n i f i c a n t d i f f e r e n c e .

RESULTS Relationships between airxay and cutaneous s e n s i t i v i t y to antigen and airway responsiveness to ACh A. suum a n t i g e n i n h a l a t i o n caused a t r a n s i e n t i n c r e a s e in R L and a decrease in C ~y. which reached a maximal l e v e l i t o 6 min a f t e r a n t i g e n i n h a l a t i o n (Fig 1). About an hour a f t e r i n h a l a t i o n , b a s e l i n e R L and C dyo values were again observed. There was a s i g n i f i c a n t p o s i t i v e c o r r e l a t i o n between a i r w a y and cutaneous s e n s i t i v i t y t o A. suum a n t i g e n ( r = 0 . 6 5 , P
Change in airway responsiveness after antigen inhalation We measured the airway responsiveness to ACh before and 2, 4 and 6 h a f t e r A. suum p r o t e i n i n h a l a t i o n in two groups (7 responders and 6 nonresponders) ( T a b l e 1). B a s e l i n e values of R L b e f o r e and 2, 4 and 6 h a f t e r a n t i g e n inhalation in r e s p o n d e r s were 3 . 5 0 ± 0 . 2 4 c m H z O / I / s e c , 3 . 9 5 ± 0 . 4 7 c m H 2 0 / I / s e c , 4 . 3 0 ± 0 . 5 7 c m H ~ O / I / s e c and 4 . 2 9 ± 0 . 5 8 c m H 2 0 / I / s e c , r e s p e c t i v e l y , and b a s e l i n e values o f R L b e f o r e and 2, 4 and 6 h a f t e r antigen i n h a l a t i o n in nonresponders were 3.08 ± 0 . 2 6 cmHzO/I/sec, 3 . 7 9 ± 0 . 4 1 c m H z O / I / s e c , 3 . 6 3 ± 0 . 4 6 c m H 2 0 / I / s e c and 3 . 6 9 ± 0 . 6 0 cmHzO/I/sec,

Prostaglandins

306

(A) r=0.65

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lo

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Airway Sensitivity to A. suum antigen (PNU/ml)

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' 1000

, 10000

, 100000

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Airway sensitivity to A s c a r i a s u u m antigen (PNU/ml)

Fig 2. Relationships between Airway and Cutaneous S e n s i t i v i t y to the Antigen (A) and Airway S e n s i t i v i t y to Antigen and Airway Responsiveness to ACh (B) in A. s~m - A l l e r g i c Dogs. PCzoo-ACh is the concentration of ACh provoking an increase in the R L of 100 %. Values of airway and cutaneous s e n s i t i v i t y to the antigen i n d i c a t e the lowest concentration of A. s~m antigen producing p o s i t i v e airway and cutaneous response, r e s p e c t i v e l y . NR indicates no response to the antigen aerosol (i00,000 PNU/ml). P
Prostaglandins

307

respectively.

Table I. Change in Airway Responsiveness to ACh a f t e r Antigen Challenge in Dogs Responsive and Nonresponsive to A. s~m Antigen. Dog No.

Pre

PCzoo-ACh (mg/ml) I 2 h 4 h

6 h

Cutaneous Response2 (PNU/ml)

No. 1 5.37 4.07 No. 2 14.4 3.37 No. 3 32.5 18.8 No. 4 5.52 2.14 No. 5 25.2 13.3 No. 6 2.52 1.65 No. 7 1.16 0.389 Mean±SE 12.4 ± 4 . 6 6.25±2.64m

Responders 1.89 0.727 4.27 10.1 13.7 12.0 3.64 4.30 11.3 30.4 1.85 0.777 0.247 0.484 5.27 ± 1 . 9 5 m 8.40 ± 4 . 0 7

100 100 100 100 100 100 1

No. 8 45.0 73.5 No. 9 163 191 No. IO 94.4 64.0 No. l l 52.0 35.8 No.12 32.0 18.5 No.13 6.66 4.74 Mean±SE 65.5 ±22.8 64.6 ±27.4

Nonresponders 97.5 123 208 98.7 69.5 248 52.1 70.7 33.5 58.7 6.78 6.99 77.9± 28.9 1 0 1 ±33.5

100 100 1000 1000 10 1000

1) See Fig 2. 2) The lowest concentration of A.s~m antigen inducing positive cutaneous response, m P<0.05 vs. PCzoo-ACh value before antigen challenge (paired t -test).

In responders, mean R L and C d,o were changed an average of 226% and 50% f i v e minutes after antigen challenge, respectively. In nonresponders, mean R L and C dyo were only changed an average of 22% and 15% f i v e minutes a f t e r antigen challenge, respectively (Fig 1). In responders, the log (PC2oo -ACh) values before and 2, 4 and 6 h after A. s~m antigen inhalation were O. 86 ± 0.20, 0 . 5 2 ± 0 . 2 1 (P
Prostaglandins

308 little o r no change in a i r w a y r e s p o n s i v e n e s s responsiveness to A. suum antigen.

Nonresponders I

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Fig 3. Change in Airway Responsiveness to ACh a f t e r Antigen Challenge in Dogs Responsive ( r i g h t ) and Nonresponsive ( l e f t ) to Antigen I n h a l a t i o n . The numbers on the o r d i n a t e represent the l o g a r i t h m i c a l l y transformed values of PC2oo-ACh values measured before and a f t e r antigen challenge. PC2oo-ACh values are defined in the legend f o r Fig 2. The h o r i z o n t a l bars represent the mean values of each group (n=6 or 7). $ P<0.05, $ #P < 0 . 0 1 , (Student's paired t - t e s t ) . N.S. stands f o r not s i g n i f i c a n t (Student's paired t - t e s t ) . R e l a t i o n s h i p between i m e d i a t e b r o n c h o c o n s t r i c t i o n and ARR In 16 responders, we measured the airway responsiveness to ACh before and 4 h a f t e r a e r o s o l i n h a l a t i o n o f A. suum a n t i g e n (100,000 PNU/ml). We assessed the r a t i o (PC2oo-ACh , r . /PC2oo-ACh 4 , ) values as the degree of AHR a f t e r a n t i g e n i n h a l a t i o n . There was a s i g n i f i c a n t c o r r e l a t i o n between maximal increase in R L and AHR f o l l o w i n g A. suum antigen i n h a l a t i o n (r=0.68, P
Prostaglandins

309

the responders were 3 . 2 6 ± 0.34 cmH20/I/sec and r e s p e c t i v e l y and the d i f f e r e n c e was s i g n i f i c a n t However, t h e r e was not a s i g n i f i c a n t c o r r e l a t i o n and the increase in b a s e l i n e R L value (data not

~

10

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,:

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200

400 600 800 % Increase In R c

1000

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Fig 4. R e l a t i o n s h i p between Airway Hyperresponsiveness to ACh and Maximal Increase in R ~ in Antigen Inhalation-Responsive Dogs. Animals inhaled the antigen at a concentration of i00,000 PNU/ml f o r 3 min. The r a t i o means PC2oo-ACh o . , d i v i d e d by PC2oo-ACh4 , value. E f f e c t of AA-2414 on immediate b r o n c h o c o n s t r i c t i o n and AHR B a s e l i n e values of RL and C dy~ in t h e v e h i c l e group were 2 . 6 6 ± 0.26 cmH20/I/sec and 0.0402 ±0.0046 I/cmHzO, r e s p e c t i v e l y , and those in the AA2414 g r o u p were 2 . 9 7 ± 0.28 c m H 2 0 / I / s e c and 0.0451 ± 0 . 0 0 9 3 I/cmH20, respectively. In the v e h i c l e group, the mean values of the maximal % change in R L and C dy~ were 5 4 5 ± i i 0 % and 6 2 . 7 ± 3.7 %, r e s p e c t i v e l y . In the AA2414 group, the mean values of the maximal % change in R L and C d , , were 263±83 % and 46.6 ± 6 . 7 %, r e s p e c t i v e l y . The maximal % changes in R L and C dyo in the AA-2414 group were less than those in the v e h i c l e group, but the d i f f e r e n c e s were not s i g n i f i c a n t (Fig 5, Table 2). In t h e v e h i c l e group, PC2oo-ACh v a l u e s b e f o r e and 4 h a f t e r a n t i g e n i n h a l a t i o n were s i g n i f i c a n t l y d i f f e r e n t (P
310

Prostaglandins

not significantly different (Fig 6). The ratio (PC2oo-ACh b. . . . . /PC2oo-ACh 4,) in the vehicle group and in the AA-2414 group was s i g n i f i c a n t l y different (P
500 IITT

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i N,I]]T , AA-24,4,m., ., .v. --

300

~o~ o

20

c

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20 40 60 Time a~erantlgenlnhaletlon(mln)

Fig 5. E f f e c t of AA-2414 on Antigen-Induced B r o n c h o c o n s t r i c t i o n in A. s~umA l l e r g i c Dogs. Animals i n h a l e d the a n t i g e n t w i c e a t an i n t e r v a l of 2 weeks and a t a c o n c e n t r a t i o n of 100,000 PNU/mI. AA-2414 was administered i n t r a venously a t a dose of 5 mg/kg 2 min b e f o r e a n t i g e n i n h a l a t i o n in a v e h i c l e - c o n t r o l l e d , crossover manner. The numbers on the o r d i n a t e are expressed as the percent changes in R L and Cdyn from b a s e l i n e a f t e r antigen challenge. The data r e p r e s e n t the mean± S.E. f o r 7 animals.

Prostaglandins

311

1.5

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P<0.01

I

I f

N.S.___:.

1.0

÷

÷-

0.5

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-0.5 before

Vehicle

4 h

before

4 h

AA-2414

Fig 6. Effect of AA-2414 on Airway Hyperresponsiveness to ACh in A. s~m Allergic Dogs. Animal experiments were carried out as described in the legend for Fig 5. The numbers on the ordinate represent the logarithmically transformed values of PC2oo-ACh values measuredbefore and after antigen challenge. PC2oo-ACh values are defined in the legend for Fig 2. The data represent the mean ± S.E. for 7 animals. P
DISCUSSION AHR is a characteristic of bronchial asthma and is closely associated with the s e v e r i t y and frequency of asthmatic episodes (20). I t has been suggested that TXA2 plays a c r i t i c a l role in the AHR observed in animals such as primates (26), dogs (10) and sheep ( i ) . In this study, to determine the role of TXA2 in AHR, we tested the effect of a TXA2 receptor antagonist, AA-2414, on antigen-induced AHR in seven A. s~m - a l l e r g i c dogs. The

312

Prostaglandins

procedure was repeated in each dog at an interval of 2 weeks to evaluate the effect of AA-2414 in a crossover manner, and we observed that AA-2414 remarkably inhibited the development of airway hyperresponslveness in some dogs, but the inhibitory effect was not great in other dogs. In this way, the degree of inhibition of antigen-induced AHR by AA-2414 varied among the dogs; however, the degree of AHR in each AA-2414-treated dog was less than that in each vehicle-treated dog without exception (Table 2). Furthermore AA-2414 did not affect airway contractile response to ACh in dogs in v~ao or zR v~t~o (data not shown). Therefore, the observations in the present study suggest that TXA2 plays a pivotal role in the development of AHR in dogs, and although there was a difference in the degree of the contribution of TXAe to the AHR among the dogs, AA-2414 could i n h i b i t more or less the antigen-induced AHR in almost a l l the dogs. Furthermore, Jones et aL. indicated that a TXA2 mimic (U-46619) has the a b i l i t y to induce AHR to methacholine in human subjects (19). Also, a TXA~ antagonist, AA-2414, has been reported to have a reducing effect on AHR in mild asthmatic subjects (15). These observations in dogs and humans suggest that TXA2 is a mediator for AHR in human asthma and that TXA2 antagonists such as AA-2414 may be useful in the treatment of asthma.

Table 2. Effect of AA-2414 on Maximal Increase in R L and AHR upon Antigen Challenge in A. s~um -Allergic Dogs. Vehicle-treated Dog Max. Increase PC2ooAChI No. in R L (%) pre 4 h K-9 N-8 N-11 K-25 K-2 K-3 K-4

207 401 864 950 560 604 228

Mean 545 ±SE ±110

5.43 10.8 10.7 4.64 2.97 2.26 2.48

3.54 6.50 3.98 1.89 0.525 0.693 1.85

AA-2414-treated Max. Increase PC2ooAChI Ratio 2 in R L (%) pre 4 h 1.53 1.66 2.69 2.46 5.66 3.26 1.34

5.61 2.71 ~ ~ 2.66 ±1.40 ±0.80 ±0.57

Ratio 2

366 393 18.4 164 143 653 104

5.42 5.28 8,18 4.12 5.99 3.87 2.10

5.51 5.61 8.33 1.82 1.86 1.55 1.84

0.984 0.941 0.982 2.26 3.22 2.50 1.14

263 ±83

4.99 ±0.72

3.79 ±1.02

1.72. ±0.35

1) PC2oo-ACh values (mg/ml) are defined in the legend for Fig 2. 2) Ratio means PCsoo-ACh ~ . divided by PC2oo-ACh~,. P <0.05 vs. the ratio in the vehicle-treated group (paired ~ -test). t t P <0.01 vs. the PC2oo-ACh value before antigen challenge (paired t - t e s t ) .

Prostaglan

dins

313

AHR e x i s t i n g before antigen challenge however is f u r t h e r enhanced by antigen provocation and has been thought to be an important f a c t o r that leads to severe and chronic asthma ( 7 ,1 2 , 2 0, 3 4 ) . In a d d i t i o n to dogs, antigen-induced AHR has also been observed in A. suum - a l l e r g i c sheep (22), s e n s i t i z e d g u i n e a p i g s (16) and r a b b i t s ( 2 5 ) . A l t h o u g h a i r w a y responsiveness was increased only 2 to 3 - f o l d a f t e r antigen challenge in this study (Fig 3), the antigen-induced AHR was reproducible and s i g n i f i c a n t , and the degree of the AHR was s i m i l a r to that in human asthma (7,12,34). 8rusasco et al. also (7) demonstrated a p o s i t i v e c o r r e l a t i o n between degree of increased airway responsiveness and maximal f a l l in the parameter of pulmonary f u n c t i o n (FEVI.o v a l u e ) in human a s t h m a t i c s , which is compatible with our r e s u l t s (Fig 4). The degree of enhancement of airway responsiveness caused by antigen challenge in this study was less than that observed in other animal studies (10,25). Chung et al. observed that airway responsiveness was increased l l - f o l d 6 h a f t e r antigen aerosol challenge in ragweed-sensitized dogs (10). This d i f f e r e n c e could be explained by our results (Fig 4), because the maximal increase in R L in Chung's study was more than t h a t in our study (about a 1000% increase from the b a s e l i n e value). These observations suggest t h a t antigen-induced AHR is a common r e a c t i o n in humans and animals, that there is some degree of s i m i l a r i t y among the factors that cause AHR in dogs and humans and that this AHR dog model is v a l i d f o r i n v e s ti g a ti n g the underlying mechanism of AHR. However, pathological investigations have revealed the presence of a large number of eosinophils in the airway a f t e r antigen challenge in humans (7) and guinea p i g s ( 1 6 ) , w h i l e an i n c r e a s e i n t h e number o f n e u t r o p h i l s in bronchoalveolar lavage f l u i d s has been dominantly observed in dogs (10). The d i f f e r e n c e in c e l l population might suggest differences in the mechanisms of asthmatic response among species, but further pathological investigation of the airway and lung tissue w i l l be required. In this study, we did not examine the recruitment of inflammatory c e l l s into the airways, but t h i s po i n t is very important to c l a r i f y the mechanism of AHR and is now under investigation. In a d d i t i o n to the c o n t r i b u t i o n of airway inflammation to AHR (5,6), i t has been p o s t u l a t e d t h a t a i r w a y n a r r o w i n g may p a r t i c i p a t e in t h e pathogenesis of AHR in human asthma (17). In the present study, baseline R L values were increased 2 h, 4 h and 6 h a f t e r antigen challenge. However, there was not a s i g n i f i c a n t c o r r e l a t i o n between the degree of AHR and the increase in baseline R L value, and t h e r e f o r e t h i s observation suggests t h a t the i n c r e a s e in b a s e l i n e R L v a l u e does not c o n t r i b u t e to the enhancement of airway responsiveness to ACh a f te r antigen inhalation in dogs. In order to f i n d a convenient way to select, at a high frequency, dogs that w i l l show an airway response to A. suum antigen from among many mongrel dogs, we have s t u d i e d the r e l a t i o n s h i p s among a i r w a y and cutaneous s e n s i t i v i t y to A. suum antigen and airway responsiveness to ACh in A. suum a l l e r g i c mongrel dogs. The results in this study demonstrated that i f doqs

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showed either a cutaneous response to a low concentration of the antigen (less than 100 PNU/ml) or an airway response to a low concentration of ACh (less than 40 mg/ml), they would show airway response to antigen at a high frequency. In fact, as shown in Fig 2, thirteen of the seventeen dogs which satisfied both of these conditions described above showed a positive airway response to antigen challenge (100,000 PNU/ml). Although the data is not shown, the dogs that maintain both skin sensitivity to antigen and airway responsiveness to ACh during the experimental period show reproducible airway response to antigen. These results demonstrated that these two responses are good markers for selecting dogs that w i l l show airway response to antigen and for monitoring the condition of airway responsiveness in the dogs. Therefore, in this study of AA-2414, we used only dogs that maintain both skin sensitivity to antigen and airway responsiveness to ACh during the experimental period. In general, asthmatic patients show remarkable AHR to nonspecific stimuli such as ACh and histamine as comparedto non-asthmatics. In this study, dogs responsive to antigen provocation showed enhanced airway responsiveness after antigen challenge, and before antigen challenge, PC2oo-ACh values in dogs responsive to antigen were also s i g n i f i c a n t l y lower than those in nonresponsive dogs (Table 1). Sears et a~. recently demonstrated that even in children who have been asymptomatic throughout t h e i r lives and have no history of atopic disease, AHR appears to be closely linked to an a l l e r g i c diathesis, as reflected by the total serum IgE level (31). Although A. s~m - a l l e r g i c mongrel dogs did not show symptoms of asthma without inhaling antigen, the dogs that displayed cutaneous and airway responsiveness to a low dose of a n t i g e n showed a tendency to have enhanced a i r w a y responsiveness to ACh, and furthermore we confirmed that the cutaneous response to A. s~m antigen depends on the serum IgE level by the P-K reaction test using a l l e r g i c dog serum (data not shown). These results demonstrate the s i m i l a r i t y of the f a c t o r s t h a t c o n t r o l airway responsiveness in dogs and humans. Although the underlying mechanism that causes enhanced airway responsivevness to ACh without antigen inhalation is not yet known, sensitization alone might induce AHR as has been described in mice (23). I t has been proved that TXAz plays a role in the immediate asthmatic reaction in guinea pigs (4), and there are some reports related to the role of TXA~ in asthmatic responses in dogs. Kleeberger et ~L . reported that the TXA2 synthase inhibitors OKY-046 and UK-37248 reduced antigen-induced immediate bronchoconstriction in A. s~m - a l l e r g i c mongrel dogs (21). Richards et a~. indicated that the TXA2 antagonist AH23848 s i g n i f i c a n t l y inhibited the immediate asthmatic response in beagle dogs sensitized with A. s~m but that the TXA2 synthase i n h i b i t o r U-63557A did not (29). Also, Chung and coworkers reported that OKY-046 did not suppress immediate bronchoconstriction in ragweed-sensitized mongrel dogs (10). In the present study, the TXA2 antagonist AA-2414 inhibited the antigen-induced immediate

Prostaglandins

315

bronchoconstriction in A. suum - a l l e r g i c dogs, but the i n h i b i t o r y e f f e c t was not s i g n i f i c a n t . These data f o r TXA~ a n t a g o n i s t s suggest t h a t TXAz is involved in immediate bronchoconstriction in dogs. However, the r e s u l t s of the studies using the TXA2 synthase i n h i b i t o r s are not in agreement. The reasons f o r the i n e f f e c t i v e n e s s of TXA2 synthase i n h i b i t o r s f o r asthmatic responses are not yet c l e a r , but i t is thought t h a t the s u b s t r a t e PGH2 accumulates as a r e s u l t of the i n h i b i t i o n of TXA~ synthesis and acts as a TXA2 agonist at TXA2 receptors (35). Also, the differences in s e n s i t i z a t i o n ( n a t u r a l s e n s i t i z a t i o n vs. a c t i v e s e n s i t i z a t i o n using an adjuvant) and in t h e p o p u l a t i o n of a n i m a l s ( b e a g l e dogs vs. mongrel dogs) m i g h t be r e s p o n s i b l e f o r the c o n f l i c t i n g r e s u l t s . Furthermore, the present study showed t h a t the immediate b r o n c h o c o n s t r i c t i o n was reduced by AA-2414 in four of the seven dogs but not in the others (Table 2), which suggests t h a t the degree of the c o n t r i b u t i o n of TXA2 in the airway response varies among dogs. This v a r i a b i l i t y might also lead to the c o n f l i c t i n g results. There are also some r e p o r t s concerning the r o l e of TXA2 in the human asthmatic response. Pharmacological studies with cyclooxygenase i n h i b i t o r s (13) and s t u d i e s on a r a c h i d o n i c a c i d m e t a b o l i t e s in plasma (32) and bronchoalveolar lavage f l u i d s have suggested the c o n t r i b u t i o n of c o n t r a c t i l e prostanoids i n c l u d i n g TXA2 in immediate b r o n c h o c o n s t r i c t i o n in asthmatic p a t i e n t s ; however, t h e r e are some d i s c r e p a n t r e p o r t s (36). T h e r e f o r e , f u r t h e r study is needed to c l a r i f y the exact r o l e of TXAz in the immediate bronchoconstriction in asthmatic patients. In summary, AHR to ACh was observed a f t e r antigen i n h a l a t i o n in A. su~m a l l e r g i c mongrel dogs. A d m i n i s t r a t i o n of the TXA2 receptor antagonist AA2414 before antigen i n h a l a t i o n reduced the AHR in the dogs. The r e s u l t s of t h i s study o f f e r f u r t h e r evidence t h a t TXA2 is involved in AHR in asthma and suggest that AA-2414 may be useful in the treatment of human asthma.

ACKNOWLEDGEMENTS We acknowledge the excellent technical assistance of Yoichi Ishisaka. Thanks also to J e f f r e y A. Hogan for his valuable aid in preparing the manuscript. REFERENCES i

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Received: 5-26-93

Accepted: 9-13-93