The effect of metiamide in in vitro and in vivo canine models of type I hypersensitivity reactions

European Journal o f Pharmacology, 44 (1977) 35--44

35

© Elsevier/North-Holland Biomedical Press

T H E E F F E C T O F M E T I A M I D E IN IN V I T R O A N D IN V I V O C A N I N E M O D E L S O F T Y P E I HYPERSENSITIVITY REACTIONS * ROBERT D. KRELL AND LAWRENCE W. CHAKRIN Smith Kline and French Laboratories, Department o f Biological Research, Section on Respiratory Diseases, 1500 Spring Gatden Street, Philadelphia, Pennsylvania 19101, U.S.A.

Received 2 November 1976, revised MS received 19 January 1977, accepted 15 March 1977

R.D. KRELL and L.W. CHAKRIN, The effect o f metiamide in in vitro an in vivo canine models o f type I hypersensitivity reactions, European J. Pharmacol. 44 (1977) 35--44. The histamine H2-receptor antagonist metiamide was evaluated in in vitro and in vivo canine models of immediate-type hypersensitivity reactions. At concentrations of 2 and 4 × 10 -4 M, the antagonist significantly increased the amount of histamine present in the medium surrounding passsively sensitized canine lung fragments which had been challenged with ascaris antigen. In contrast, the compound was without effect on the release of a slow reacting substance of anaphylaxis. On the basis of these observations metiamide was investigated in an in vivo canine model of allergic asthma. At doses of 10, 30 and 50 pmoles/kg, the antagonist did not enhance the ascaris antigen-induced pulmonary pathophysiology. Similarly, at 10 and 50 pmoles/kg metiamide did not alter histamine-induced increases in pulmonary resistance or decreases in dynamic lung compliance. Insofar as the canine model of allergic asthma may be predictive of the human disease, it could be anticipated that the use of histamine H2-receptor antagonists may not be deleterious to allergic asthmatics. H2-antagonist

Immediate hypersensitivity

Histamine

1. I n t r o d u c t i o n Histamine is capable o f inhibiting antigeni n d u c e d h i s t a m i n e release f r o m isolated human leukocytes presumably by a mechanism involving elevation o f intracellular levels o f cyclic AMP ( B o u r n e et al., 1 9 7 1 ) . Following t h e d i s c o v e r y o f a series o f c o m p o u n d s capable o f b l o c k i n g the a c t i o n o f h i s t a m i n e at H : - r e c e p t o r sites (Black et al., 1 9 7 2 ; 1 9 7 3 ; Burland et al., 1 9 7 5 ) , it was d e m o n s t r a t e d t h a t this e f f e c t o f h i s t a m i n e is m e d i a t e d b y h i s t a m i n e H2-type r e c e p t o r s ( L i c h t e n s t e i n and Gillespie, 1 9 7 3 ; 1 9 7 5 ) . On the basis o f these o b s e r v a t i o n s it was suggested t h a t h i s t a m i n e

* Portions of this work were presented at the Fall Meeting of the American Society for Pharmacology and Experimental Therapeutics, Pharmacologist 18, 204 (1976).

Pulmonary studies

released d u r i n g allergic episodes m a y serve to m o d u l a t e the i n t e n s i t y o f the p a t h o p h y s i o l o gic response. These findings were e x t e n d e d t o lung tissue as the H2-antagonist m e t i a m i d e appeared to enhance immunologically i n d u c e d histamine release f r o m primate, b u t n o t rat, lung in vitro (Chakrin et al., 1 9 7 4 ) . The e f f e c t in p r i m a t e lung was m e d i a t o r selective as the c o m p o u n d had little e f f e c t on the release o f a slow reacting substance o f anaphylaxis. If h i s t a m i n e i n t e r a c t i o n With H2-receptors does serve t o m o d u l a t e allergic reactions, H2-receptor a n t a g o n i s t s c o u l d negate this f u n c t i o n and t h e r e b y intensify a s t h m a t i c episodes. T o evaluate this h y p o t h e s i s , the e f f e c t o f the H2-receptor a n t a g o n i s t m e t i a m i d e was e x a m i n e d in in vitro and in vivo canine m o d els o f i m m e d i a t e - t y p e h y p e r s e n s i t i v i t y reactions.

36 2. Materials and methods Adult male mongrel dogs weighing 10 to 20 kg were used.

2.1. Screening for dogs cutaneously hypersensitive to ascaris antigen Dogs were anesthetized with 35 mg/kg pentobarbital (i.v.) and injected with 5 ml of 0.5% Evan's Blue Dye (i.v.) dissolved in 0.9% NaC1. Duplicate intracutaneous 0.1 ml injections of serial dilutions of a 1% v/v solution of standard ascaris antigen (SAA) were made into a lightly pigmented, shaved area of the body. Antigen was diluted in phosphate-buffered saline (PBS, 10 mM phosphate buffer, pH 7.4 in 0.9% NaC1) the latter served as the injection control. Injected sites were evaluated 30 min later. Animals with wheals and dye infiltration at sites injected with dilutions of 1 : 1000 or greater were considered ascaris sensitive. SAA was prepared as described previously (Krell and Chakrin, 1976).

2.2. Preparation and challenge of lung fragments Dogs cutaneously sensitive to ascaris antigen were anesthetized with 35 mg/kg pentobarbital (i.v.) and killed by an intracardiac injection of Beuthanasia (300 mg/ml pentobarbital, Burns-Biotectic Labs, Oakland, Calif.). The lungs were removed and thoroughly rinsed in modified Tyrode solution of the following composition (mM): NaC1 137; KC1 3.4; CaC12 1.3; MgC12 0.1; NaH2PO4 11.9; and glucose 5. The tissue was manually fragmented with a scalpel blade into cubes of approximately 3 × 3 × 3 mm avoiding large sections of airway smooth muscle wherever possible. The fragments were thoroughly rinsed with Tyrode solution until the effluent was visible free of blood and then passively sensitized by incubating for 90 min at 37°C with a 1 : 5 dilution (in Tyrode) of high titer antiascaris serum prepared from ascaris sensitive donor dogs. Passive sensitization of frag-

R.D. KRELL, L.W. CHAKRIN ments is necessary to insure adequate mediator release upon antigen challenge. After passive sensitization the sections were thoroughly rinsed and randomly divided into groups of approximately 5--6 collectively weighing 225--250 mg and placed in 2 ml of Tyrode solution. For some experiments, the same weight of tissue was placed in 4 or 6 ml of Tyrode solution. Samples were challenged by incubating with a 1% v/v concentration of SAA for 30 min at 37 °C. At the end of the challenge period the Tyrode solution was decanted and acidified (pH - 6) for assay of histamine or alkalinized (pH - 9) for assay of a slow reacting substance of anaphylaxis-like material (SRS-A). Samples were stored frozen (--20°C) until the day of assay. Metiamide (SK&F 92058) was incubated with the fragments at 22°C for 5 min prior to the addition of antigen and was present throughout the challenge period. All treatments were carried out in triplicate. Histamine and slow reacting substance of anaphylaxis were assayed by methods described previously (Krell and Chakrin, 1976). Metiamide, at concentrations equal to those in experimental samples diluted for SRS-A bioassay, had no effect on the ability of canine SRS-A to contract the guinea-pig ileum.

2.3. Measurement of pulmonary mechanics, ascaris antigen-challenge and analysis of drug effects The techniques used have been described previously (Krell et al., 1975; 1976). Briefly, dogs were anesthetized with pentobarbital (40 mg/kg, i.p.) and the trachea intubated. Airflow was measured by a heated Fleisch pneumotachograph (Instrumentation Associates, Inc., New York) connected to a Statham PM5E differential pressure transducer (Statham Instruments Inc., Calif.). Transpulmonary pressure was measured with a Statham PM5E differential pressure transducer which monitored the pressure difference between a side-arm adapter of the endotracheal tube and an intrapleural cannula placed in the fifth or

METIAMIDE IN HYPERSENSITIVITY REACTIONS sixth intercostal space. Pulmonary resistance (Rp) and dynamic lung compliance (Cdyn) were calculated on a breath-by-breath basis by an on-line analog computer (Buxco Electronics, Inc., Sharon, Conn.) and recorded on a Beckman Type RM 8-channel dynograph (Beckman Instruments, Inc. Schiller Park,

ill.). Dogs cutaneously sensitive to ascaris antigen were challenged at two week (biweekly) intervals with appropriate dilutions of SAA in PBS. Animals demonstrating reproducible pulm o n a r y responses to aerosol antigen on three successive biweekly challenges were considered suitable for drug evaluation. Because of marked interanimal variability, biweekly antigen challenges in the presence of the drug were alternated with vehicle alone so that each dog served as its own control. As reported previously (Krell et al., 1975), the time interval between antigen provocation and peak increase in Rp varies considerably from challenge to challenge even within the same animal. For example, peak Rp increases occurred 5.5 (2--9) min (mean and range of 100 responses in 10 dogs) after antigen administration. Further, Rp and Cdyn do not consistently reach m a x i m u m responses at the same time. In order to avoid this variability, the responses are analyzed as a function of peak Rp increase without regard to time of antigen administration or peak decrease in Cdyn.

2. 4. Histamine dose--response curves Partial i.v. histamine dose--response curves weye obtained on Rp and Cdyn in control dogs. Each dose of histamine was administered after the effect of the previous dose had waned and Rp and Cdyn had returned to preinjection control levels. After larger doses of histamine, the return to baseline was not always complete, however, baseline values could usually be restored by two or three lung hyperinflations. After the first dose--response curve, the animal was treated with either 10 or 50 gmoles/kg of metiamide i.v. or an equal

37 volume of vehicle and 5--15 min later a second dose--response curve was obtained.

2. 5. Drugs and solutions For the in vivo studies, metiamide was dissolved in an appropriate volume of polyethylene glycol 200--0.9% NaC1 (1 : 1) and administered i.v. 10 min prior to antigen administration.

3. Results

3.1. Fragmented canine lung Passively sensitized lung fragments prepared from dogs cutaneously sensitive to ascaris antigen released a net 4.96% (1.13 + 0.35 pg/g wet wt, mean + S.E.M., n = 12) of the total tissue histamine content of 22.8 + 2.5 pg/g wet wt, (mean ± S.E.M., n = 12) following challenge with 1% v/v SAA. Spontaneous histamine release, i.e. release which occurred in the absence of antigen, was 0.14 + 0.04 pg/g wet wt (mean ± S.E.M. n = 12). Gross SRS-A released following antigen provocation was 125 + 30.2 units/g wet wt, (mean ± S.E.M., n = 9) compared to a " s p o n t a n e o u s " release of 52.8 -+ 8.13 units/g wet wt, (mean ± S.E.M. n = 9). At the two highest concentrations studied, 2 and 4 X 10 -4 M, metiamide did not alter the " s p o n t a n e o u s " release of histamine or SRS-A. Consequently, the effects of lower concentrations were not investigated. Metiamide was w i t h o u t effect on the antigen-induced release of histamine at concentrations of 2 X 10 -s M and 2 X 1 0 -6 M (fig. 1). However, at 2 and 4 X 10 -4 M, metiamide appeared to enhance 09 < 0.05) the release of histamine in a concentration-related manner. Although there was a t e n d e n c y toward enhancement of the antigen-induced release of SRS-A at the highest concentration (4 X 10 -4 M), metiamide lacked significant (p > 0.05) effect on the release of this material (fig. 1).

38

R.D. K R E L L , L.W. C H A K R I N

Metiamide

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100

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Fig. 1. E f f e c t o f m e t i a m i d e o n a n t i g e n - i n d u c e d release o f h i s t a m i n e ( e --e) and SRS-A (o . . . . . -c~) from canine lung in vitro. Passively sensitized lung f r a g m e n t s p r e i n c u b a t e d (22°C for 5 rain) with varying c o n c e n t r a tions o f m e t i a m i d e were challenged w i t h 1% v/v SAA ( 3 7 ° C for 30 min). N u m b e r s in p a r e n t h e s e s refer to the n u m b e r o f animals. Each e x p e r i m e n t was c o n d u c t e d in triplicate. Values are m e a n s + S.E.M. * p < 0.05.

3.2. The effect o f altering the volume o f the incubation medium on ascaris antigen-induced release of histamine In an e f f o r t to determine w h e t h e r a relationship exists b e t w e e n extracellular concentrations of histamine and the total a m o u n t of histamine released f r om tissues on antigen challenge, experiments were c o n d u c t e d using larger volumes of T y r o d e solution for a fixed tissue weight. Table 1 illustrates that increasing the incubation medium by two or threefold, and t h e r e b y reducing the final extracellular c o n c e n t r a t i o n of histamine, did n o t alter the a m o u n t of histamine released from tissues challenged with ascaris antigen.

3.3. Histamine dose--response curves To d eter min e w h e t h e r an H2-receptor antagonist could a t t e n u a t e the constrictive effect o f histamine on large (Rp) and small (Cdyn) airways, the effect of metiamide on histamine (i.v.) dose--response curves was evaluated. To insure a reproducible baseline

between consecutive curves, only partial dose--response curves were obtained in the presence and absence of 10 and 50 pmoles/kg of metiamide (fig. 2). The m odest shifts in the histamine dose--response curves obtained in the presence of either dose of metiamide were

TABLE 1 The e f f e c t of altering the v o l u m e o f the i n c u b a t i o n m e d i u m on ascaris a n t i g e n - i n d u c e d release of histamine 1. Medium volume (ml)

n 2

Net h i s t a m i n e release (pg/g)

% Change histamine release

2.0 ( c o n t r o l ) 4.0 2.0 ( c o n t r o l ) 6.0

6 6 3 3

1.48 1.58 2.51 2.41

_ +6.7 ---4.0

± 0.68 3 + 0.60 _+ 1.12 ± 1.23

1 Passively sensitized canine lung f r a g m e n t s collectively weighing 2 2 5 - - 2 5 0 mg were placed in 2, 4 or 6 ml o f T y r o d e and challenged with 1% v/v SAA. 2 n = n u m b e r o f animals; each e x p e r i m e n t was u n d e r taken in triplicate. 3 Means ± S.E.M.

METIAMIDE

IN H Y P E R S E N S I T I V I T Y

REACTIONS

1800

39

3.4. In vivo canine model o f allergic asthma

1600

The effect of metiamide in a canine model of allergic asthma was studied at doses ranging from 10 to 50 pmoles/kg, equivalent to approximately 3--16 times the EDs0 for inhibition of histamine-induced gastric acid secretion in this species (Black et al., 1973). Most of the animals studied demonstrated comparatively moderate increases in Rp as increases

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Fig. 2. E f f e c t o f m e t i a m i d e o n p a r t i a l h i s t a m i n e dose--response c u r v e s . S e q u e n t i a l h i s t a m i n e (i.v.) dose--response curves were obtained on Rp and C d y n . F o l l o w i n g t h e first d o s e - - r e s p o n s e c u r v e a seco n d c u r v e w a s o b t a i n e d in t h e s a m e a n i m a l 5 - - 1 5 m i n a f t e r a d m i n i s t r a t i o n o f 10 o r 5 0 p m o l e s / k g o f m e t i amide or an equivalent volume of vehicle (control curves not illustrated), n = number of animals. Numb e r s in p a r e n t h e s e s a s s o c i a t e d w i t h d a t a p o i n t s r e f e r to the number of observations for that point only. V a l u e s a r e m e a n s ± S . E . M . R p = p u l m o n a r y resistance. Cdyn = dynamic lung compliance, o o, C o n t r o l ( n = 6); e - ~,, m e t i a m i d e ( 1 0 p m o l e s / k g , n = 6); A A, c o n t r o l ( n = 6); • •, metia m i d e ( 5 0 p m o l e s / k g , n = 6).

similar to those obtained in the presence of the polyethylene glycol : saline vehicle (not shown). The 50 pmoles/kg dose of metiamide appeared to enhance the 30 pg/kg dose of histamine; however, complete dose--response curves must be obtained to determine the validity of this observation.

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Fig. 3. E f f e c t o f m e t i a m i d e o n a n t i g e n - i n d u c e d pulm o n a r y r e s p o n s e s in d o g 1 1 0 2 9 . M e t i a m i d e ( 1 0 pmoles/kg) or an equivalent volume of vehicle was a d m i n i s t e r e d i.v. 10 r a i n p r i o r t o 4 i n h a l a t i o n s o f a 1 : 10 dilution of SAA. A, baseline values obtained immediately prior to antigen administration, for control: C = 2 . 3 5 ± 0 . 1 6 a n d f o r m e t i a m i d e : M = 2 . 2 5 +_ 0 . 3 2 ; B, C = 8 9 . 8 + 3 . 8 4 a n d M = 9 9 . 3 ± 1 0 . 1 . n = n u m b e r of observations. Values are means ± S.E.M. Where S.E.M. are omitted they were smaller than the symbol for the mean. Rp = pulmonary resistance. Cdyn = dynamic lung compliance. • ¢,, C o n t r o l (n = 4 ) ; o ...... o, m e t i a m i d e ( n = 4).

40

R . D . K R E L L , L.W. C H A K R I N

ranging from 500 to 1000% can be obtained in many animals (Krell et al., 1975). The increases in Rp obtained in most animals would have, therefore, presumably allowed for an enhancement of the response. At the lowest dose investigated (10 pmoles/ kg), the H2-antagonist was without significant effect on the anaphylactic reaction in one dog (fig. 3) and significantly (p < 0.05) inhibited peak increases in Rp in a second (fig. 4). Further, in this animal there was also a significant

800

A. Rp(cm H20/LPS)

(p < 0.05) reduction in Cdyn decreases at one time period (fig. 4). At a dose of 30 pmoles/kg, metiamide was without significant (p > 0.05) effect on Rp increases in three dogs although, in two of the animals, there was a slight, but insignificant, tendency toward enhancement of peak Rp increases. An experiment representative of this series is illustrated in fig. 5. Ascaris antigen-induced decreases in Cdyn were not influenced by metiamide. When metiamide was evaluated at a dose of 50 pmoles/kg in an additional three animals, two of which are illustrated in figs. 6 and 7,

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Fig. 4. E f f e c t o f m e t i a m i d e o n a n t i g e n - i n d u c e d pulm o n a r y r e s p o n s e s in d o g N 8 7 8 . M e t i a m i d e ( 1 0 t l m o l e s / k g ) or a n e q u i v a l e n t v o l u m e o f vehicle w a s a d m i n i s t e r e d i.v. 10 r a i n p r i o r t o 3 i n h a l a t i o n s o f a 1 : 10 d i l u t i o n o f S A A . N u m b e r s in p a r e n t h e s e s associated with data points refer to the n u m b e r of observ a t i o n s f o r t h a t p o i n t o n l y . * p < 0 . 0 5 . See fig. 3 f o r a d d i t i o n a l details. • ~, C o n t r o l (n = 3); ©. . . . . . o, m e t i a m i d e ( n = 4). A, C = 1.91 ~ 0 . 2 0 ; M = 1 . 9 6 * 0 . 1 1 . B, C = 1 0 6 _+ 1 4 . 1 ; M = 8 1 . 8 ~ 5.80.

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Fig. 5. E f f e c t o f m e t i a m i d e o n a n t i g e n - i n d u c e d pulm o n a r y r e s p o n s e s in d o g Y 1 6 7 4 . M e t i a m i d e ( 3 0 p m o l e s / k g ) or an equivalent v o l u m e of vehicle was a d m i n i s t e r e d i.v. 10 rain p r i o r t o 5 i n h a l a t i o n s o f a 1 : 10 d i l u t i o n o f S A A . See figs. 3 a n d 4 f o r a d d i t i o n a l detail. • e , C o n t r o l ( n = 4); o . . . . . . ©, m e t i a m i d e ( n = 3 - - 4 ) . A, C = 2.35 ± 0 . 1 7 ; M = 2.29 +~ 0.39. B, C = 57.7 + 1.65; M = 54.3 + 8.29.

METIAMIDE

400

IN HYPERSENSITIVITY

REACTIONS

41

A. Rp(cm H20/LPS)

A. Rp (cm H20/LPS)

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Fig. 6. E f f e c t o f m e t i a m i d e o n a n t i g e n - i n d u c e d p u l m o n a r y r e s p o n s e s in d o g N 8 7 8 . M e t i a m i d e ( 5 0 / x m o l e s / k g ) o r a n e q u i v a l e n t v o l u m e o f v e h i c l e w a s a d m i n i s t e r d i.v. 1 0 r a i n p r i o r t o 7 i n h a l a t i o n s o f a 1 : 10 d i l u t i o n o f S A A . * p < 0 . 0 5 . S e e fig. 3 f o r a d d i t i o n a l d e t a i l . $-t,, C o n t r o l ( n = 4); o . . . . . -o, m e t i a m i d e (n = 4). A , C = 2 . 8 8 ± 0 . 1 4 ; M = 2 . 4 2 -+ 0 . 2 7 . B, C = 7 3 . 4 + 9 . 8 8 ; M = 8 5 . 6 ± 7.0.

again there was no significant alteration in Rp increases at any time period. In two of these animals, exemplified by fig. 6, there was an apparent t e n d e n c y toward inhibition of Rp increases. A significant (p < 0.05) inhibition of Cdyn decreases was seen in only one of the animals (fig. 6) with no effect in the other two. It should be noted that dog N878 (fig. 6) is the same animal in which a significant inhibition of the pulmonary response was obtained at a dose of 10/~moles/kg.

-2

I 0

I 2

I 4

10

Minutes before (-) and after peak Rp increase Fig. 7. E f f e c t o f m e t i a m i d e o n a n t i g e n - i n d u c e d p u l m o n a r y r e s p o n s e s in d o g 1 1 2 7 5 . M e t i a m i d e ( 5 0 pmoles/kg) or an equivalent volume of vehicle was a d m i n i s t e r e d 10 r a i n p r i o r t o 7 i n h a l a t i o n s o f 1 : 10 d i l u t i o n o f S A A . See figs. 3 a n d 4 f o r a d d i t i o n a l detail. • t,, C o n t r o l (n = 4 ) ; o . . . . . -o, m e t i a m i d e ( n = 4). A , C = 2 . 4 9 +- 0 . 2 9 ; M = 2 . 1 3 ± 0 . 1 2 . B,C = 88.7 ± 11.6;M= 79.7 ± 5.90.

4. Discussion Passively sensitized canine lung fragments released histamine and a slow reacting substance of anaphylaxis-like material following challenge with ascaris antigen. Previous studies have shown that the immunologic release of these substances can be manipulated by a variety of agents including fl-agonists, phosphodiesterase inhibitors and cholinergic agonists (Krell and Chakrin, 1976). Thus, the nature of the mediators released from canine lung on immunologic challenge, as well as the influence of pharmacologic agents on the release process, resembles the characteristics

42 of both primate (Chakrin et al., 1974; Ishizaka et al., 1970; 1971) and h u m a n lung (Assem and Schild, 1969; Orange et al., 1971; Kaliner et al., 1972). Consequently, canine lung was considered an appropriate experimental system in which to evaluate the effects of an H2-receptor antagonist. At high concentrations, metiamide appeared to enhance the immunologic release of histamine from canine lung with only modest effects on the release of SRS-A. These results are quantatively and qualitatively similar to observations obtained in fragmented primate lung (Chakrin et al., 1974). Whether the increased a m o u n t of histamine present in the medium following challenge represents a true enhancement of release or an inhibition of metabolism and/or tissue accumulation by metiamide subsequent to release cannot be determined from these experiments. H2-antagonists have been shown to be capable of inhibiting the degradation of histamine by histamine N-methyl transferase (Taylor, 1973) as well as the accumulation of histamine into some tissues (Fantozzi et al., 1973; 1975). These processes have not been thoroughly studied in canine lung. As well, the apparent enhancement of histamine release by metiamide is compatible with the concept that histamine may serve a m o d u l a t o r y role in allergic reactions by activating mast cell H2-receptors leading to an elevation of cyclic AMP and inhibition of histamine release (Lichtenstein and Gillespie, 1973; Lichtenstein and Gillespie, 1975). Metiamide, by blocking histamine interaction with the H2-receptor, would negate this m o d u l a t o r y influence and thereby enhance release. Most experimental evidence for this hypothesis has been obtained from studies on partially purified h u m a n basophils. Whether a similar mechanism is operative in lung remains to be established. Experiments designed to test this hypothesis in canine lung by reducing amine concentrations in the medium through variation in the incubation volume surrounding the fragments failed to yield results consistent with the hypothesis.

R.D. KRELL, L.W. CHAKRIN The lack of effect may be accounted for if it is assumed that the concentrations of histamine required to suppress release are not related to those present in the medium but rather to those in the immediate milieu of the cells being inhibited. When considering the feasibility of this hypothesis for lung tissue, it is n o t e w o r t h y that the ability of metiamide to influence the release of SRS-A does not parallel its effects on histamine. In contrast, other agents which inhibit histamine release, presumably by alterations in cyclic nucleotide levels, e.g., isoproterenol and papaverine, demonstrate an apparent equal potency for inhibition of SRS-A release (Krell and Chakrin, 1976). Thus, if the hypothesis is functional, it would appear that this effect of histamine is unique. Such selectivity would at least suggest the possibility that SRS-A may originate from other than mast cells, a hypothesis currently receiving some attention (Bach and Brashler, 1974). Regardless of the mechanism, enhancement of histamine release by metiamide during allergic reactions, on the basis of current hypothesis, could exacerbate pathophysiologic responses. When evaluated in an in vivo canine model, at doses ranging from 3 to 16 times the EDs0 for histamine-induced gastric acid secretion in the dog (Black et al., 1972), metiamide did not consistently influence antigen-induced changes in pulmonary mechanics. Significant alterations occasionally seen were in the direction of inhibition of the response, not enhancement. This lack of effect of metiamide in vivo may be due to one or more of the following circumstances: (1) the inability of histamine released on anaphylaxis to achieve the concentrations necessary to elevate mast cell cyclic AMP. As yet unknown mechanisms of tissue accumulation and metabolism as well as diffusion and removal by the circulation may prevent the attainment of sufficient concentrations of histamine at the biophase of the H2-receptor. (2) Assuming adequate amine concentrations, metiamide, at the doses employed, may not have achieved concentrations

METIAMIDE IN HYPERSENSITIVITY REACTIONS sufficient to antagonize the effect of histamine on H2-receptors. Conceivably, greater doses of metiamide might enhance histamine release, however, the t h e r a p e u t i c relevance o f h i g h e r d o s e s w o u l d be d o u b t f u l . A n d f i n a l l y (3) this h i s t a m i n e selective p h e n o m e n a m a y h a v e b e e n f u n c t i o n a l in vivo a n d as s u c h may, therefore, reflect the relative unimport a n c e o f t h i s a m i n e p e r se as a m e d i a t o r o f ascaris a n t i g e n - i n d u c e d p u l m o n a r y a n a p h y l a x i s in t h e d o g . This a l t e r n a t i v e is c o m p a t i b l e w i t h m o r e r e c e n t o b s e r v a t i o n s (Krell, 1 9 7 7 ) . The relevance of the present experimental r e s u l t s t o m a n is d e p e n d e n t on t h e d e g r e e o f s i m i l a r i t y b e t w e e n h u m a n allergic a s t h m a a n d t h e c a n i n e m o d e l o f t h i s disease. While p h y s : iologic and immunologic characteristics a p p e a r to parallel the h u m a n disease (Krell et al., 1 9 7 5 ) t h e r e is y e t a p a u c i t y o f i n f o r m a t i o n as t o t h e p h a r m a c o l o g i c a n d b i o c h e m i c a l chara c t e r i s t i c s o f t h e in vivo c a n i n e m o d e l . While s o m e p h a r m a c o l o g i c d i f f e r e n c e s have b e e n e l u c i d a t e d ( K r e l l e t al., 1 9 7 5 ) f u r t h e r c o m p a r a t i v e s t u d i e s are n e c e s s a r y .

Acknowledgements The authors wish to thank the following individuals for providing skillful technical assistance: P. Christian, E. Giannone, L. Howe, J. McCoy, J. Mengel, R. Osborn and D. Young.

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