Biochemical and pharmacological characterization of ICI 198,615: a peptide leukotriene receptor antagonist

European Journal of Pharmacology, 159 (1989) 73-81

73

Elsevier EJP 50573

Biochemical and pharmacological characterization of ICI 198,615: a peptide leukotriene receptor antagonist R . D . K r e l l *, E.J. K u s n e r , D. A h a r o n y a n d R . E . G i l e s ICl Pharmaceuticals Group, ICI Americas Inc'., Department of Pharmacology, Pulmonary Pharmacology Section, Wilmington, DE 19897, U.S.A.

Received 26 May 1988, revised MS received 30 August 1988, accepted 27 September 1988

ICI 198,615 is one representative of a new chemical class of peptide leukotriene receptor antagonists that are the most potent and selective described to date. ICI 198,615 antagonized LTC4-, LTD 4- and LTE4-induced increases in cutaneous vascular permeability in the guinea pig, with i.v. EDs0 values of 0.083, 0.11 and 0.067/~mol/kg, respectively. Against LTD 4, ICI 198,615 was 615 and 415 times more potent than LY 171883 and FPL 55712, respectively. L-Serine borate, an inhibitor of the metabolism of LTC 4 to LTD 4, did not influence the antagonism by ICI 198,615 of LTC4-induced increases in cutaneous vascular permeability. The compound both inhibited and reversed aerosol ovalbumin antigen-induced increases in pulmonary resistance in passively sensitized guinea pigs, but demonstrated little ability to inhibit or reverse ovalbumin antigen-induced decreases in dynamic lung compliance. At concentrations ranging from 10 -s to 10 -5 M, ICI 198,615 had no significant effect on either the spontaneous or ovalbumin antigen-induced release of histamine, peptide leukotrienes, thromboxane B2 or 6-keto-prostaglandin F~ from chopped guinea pig lung. At 10 ~tM, the compound did not inhibit 5-, 12- or 15-1ipoxygenase. Finally, ICI 198,615 antagonized LTDa-induced increases in TxB 2 release from chopped guinea-pig lung. Leukotriene receptor antagonists; ICI 198,615; LTC4; LTD4; LTE 4

1. Introduction Slow-reacting substance of anaphylaxis was discovered 50 years ago (Feldberg and Kellaway, 1938). It was not until 1979 that the structure of these substances were finally elucidated ( M u r p h y et al., 1979) and the materials r e n a m e d leukotriene Ca, D 4 and E 4. In the years since their structural elucidation, a vast quantity of circumstantial evidence has been amassed suggesting an etiologic role for these materials in various pathophysiologic states including allergic asthma. Consequently, a concerted effort has been put forth to discover agents that are capable of inhibiting either

* To whom all correspondence should be addressed.

leukotriene biosynthesis or the action of leukotrienes at the end organ, i.e., receptor antagonists. The first leukotriene antagonist, F P L 55712, was discovered by Augstein et al. (1973) but was neither particularly potent nor selective and as well lacked a viable biologic half life in vivo. This c o m p o u n d was however an extremely important tool in the structural elucidation of the leukotrienes. In 1985 the first of the ' s e c o n d generation' peptide leukotriene antagonists, LY 171883, was discovered (Fleisch et al., 1985). In 1987 two new classes of leukotriene antagonists were reported: (1) S K F 104,353, a structural analog of the leukotrienes ( H a y et al., 1987), and (2) I C I 198,615, a completely novel chemical structure (Snyder et al., 1987; Krell et al., 1987; A h a r o n y et al., 1987). The latter c o m p o u n d is, thus far, the most potent

0014-2999/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)

74 and selective leukotriene antagonist described to date. This communication provides additional biochemical and pharmacological characterization of ICI 198,615 ([1-[[2-methoxy-4-[[(phenylsulfonyl)amino]carbonyl]phenyl]methyl]-lH-indazol-6-yl]carbonic acid cyclopentyl ester) which indicate that this compound is a valuable new pharmacologic tool for evaluating the role of peptide leukotrienes in normal and pathophysiologic processes.

2. Materials and methods

2.1. General Adult male albino Hartley guinea pigs weighing 200-600 g were used. Animals were allowed free access to food and water except for pulmonary mechanics experiments when food was withheld for 16-18 h prior to experimentation. Animals were anesthetized with 1.5 g / k g (i.p.) of urethane with supplementation as required.

2.2. Cutaneous vascular permeability induced by peptide leukotrienes The methods used were similar to those described by Rinkema et al. (1984). Briefly, the backs of guinea pigs were shaved 20-24 h prior to experimentation. Following anesthesia, a 20 ga catheter was inserted into the jugular vein for administration of test compounds. Experimental compounds followed by Evans blue dye (30 mg/kg) were administered i.v. 10 and 2 min, respectively, prior to the intradermal (i.d.) injection of LT. Various doses of LTC 4, LTD 4 or LTE 4 (in 0.1 ml of phosphate-buffered saline; 10 mM, pH = 7.4 in 0.9% NaC1) were injected i.d. into the shaved dorsal skin of the guinea pig. To counteract the vasoconstrictor activity of LT, prostaglandin E 2 (PGE 2, 10 -5 M) was included in the solution as suggested by Peck et al. (1981). In preliminary experiments, this concentration of PGE2was determined to be optimal. A control i.d. injection consisting of 0.1 ml of PBS with 10 -5 M PGE 2 was administered to each animal. Thirty minutes after the injection of LT, the animals were killed by intracardiac injection of 0.5 ml of a

500 m g / m l solution of urethane. The injected area was reflected and the mean orthogonal diameter of each reaction area measured with a caliper. Net reaction was calculated by subtracting the control area (mm 2) from the area of the LT site.

2.3. Chopped guinea pig lung Antigen challenge of chopped lung tissue prepared from actively sensitized guinea pigs was carried out as described previously (Krell and Kusner, 1984). Histamine was assayed by an automated fluorometric procedure using a Technicon AA II as described by Siraganian (1975). Thromboxane B 2 ( T x B 2 ) and 6-keto-prostaglandin F~, (6-keto PGF~,), degradation products of thrombox a n e A 2 and prostacyclin, respectively, were analyzed by radioimmunoassays using commercially available kits (New England Nuclear, MA). Peptide leukotrienes were quantitated by radioimmunoassay as described previously (Aharony et al., 1983). Radioacivity was counted in a Packard Model 2000 CA liquid scintillation spectrometer (Packard Instruments, Downers Grove, IL). Various concentrations of ICI 198,615 or control vehicle were incubated with the tissue for 10 min at 3 7 ° C prior to challenge with the optimal ovalbumin antigen concentration (10 ~g/ml). IC1 198,615 did not interfere with the histamine fluorometric assay or any of the radioimmunoassays.

2.4. Antigen-induced bronchoconstriction in guinea pigs Pulmonary mechanics were measured in anesthetized guinea pigs as described previously (Krell et al., 1987). Antibody for passive sensitization of naive animals was prepared by injecting guinea pigs with 2 mg of ovalbumin (Sigma Grade V, Sigma Chemical Co., St. Louis, MO) in 50% complete Freund's adjuvant; 50% saline i.p., on days 1 and 5. Blood was harvested on day 21 and serum prepared and stored at - 7 0 ° C. Recipient animals received an i.p. injection of 0.2 ml of donor anti-ovalbumin antibody 48 h prior to experimentation. Following anesthesia, a 20 ga cannula was placed into the jugular vein for administration of

75 compounds. Guinea pigs received i.v. injections of pyrilamine (2.0 mg/kg), indomethacin (10 m g / k g ) and dl-propranolol (0.5 mg/kg) 40, 50 and 45 rain, respectively, prior to five breaths of a 40 m g / m l solution of ovalbumin. Phosphate buffered saline was administered in five breath aerosols 10 and 15 rain prior to antigen to stabilize baseline pulmonary mechanics parameters. Aerosols were delivered with a Bird Mark VII respirator with the following settings: inspiratory sensitivity = 25 cm H20; 3 s intervals between breaths. 2.5. Statistics Differences between groups were determined by Student's t-test for paired or unpaired data as appropriate. 2.6. Materials Pyrilamine maleate, prostaglandin E2, soybean 15-1ipoxygenase, dl-propranolol hydrochloride, and indomethacin were obtained from Sigma Chemical Co., St. Louis, MO. ICI 198,615, FPL 55712 and LY 171883 were synthesized at ICI Pharmaceuticals Group (Medicinal Chemistry Department) and structures verified by standard techniques.

3. Results

3.1. Peptide leukotriene-induced increases in cutaneous vascular permeability in guinea pigs LTC 4, L T D 4 and LTE4 produced dose-related increases in cutaneous vascular permeability in guinea pigs when the vasodilator PGE 2 (10 -5 M) was coadministered (table 1). All three peptide LT also produced dose-related increases in vascular permeability in the absence of PGE2; however, responses were smaller and more variable. The doses of LTC 4, LTD 4 and LTE 4 necessary to produce a 90 mm 2 net reaction area, an approximate ED~0, were, respectively; 0.012, 0.022 and 0.046, nmol/site. Thus, LTC 4 and LTD 4 were equipotent and, perhaps, slightly more potent than LTE 4 .

Table 2 illustrates the effect of the cyclooxygenase inhibitor indomethacin, the histamine Hi-receptor antagonist, pyrilamine and the serotonin antagonist, cyproheptadine, on vascular responses to LTD 4. None of these agents significantly (P > 0.05) altered responses to LTD 4 indicating that neither cyclooxygenase products nor histamine or serotonin were contributing to LTD4-induced increases in permeability. ICI 198,615 produced dose-related antagonism of the increases in vascular permeability produced by LTC 4 (table 3), LTD 4 (fig. 1) and LTE 4 (table 3). The EDs0 values for ICI 198,615 versus these three agonists were, respectively; 0.083, 0.11 and 0.067 /.tmol/kg. ICI 198,615 was 615 and 415 times more potent than LY 171883 and FPL 55712, respectively. ICI 198,615 and LY 171883 inhibition curves appeared parallel (fig. 1) whereas inhibition by FPL 55712 appeared steep, perhaps owing to the well-documented brief biological half-life in many species (Mead et al., 1981). Experiments in isolated guinea pig tracheal smooth muscle have demonstrated the existence of a distinct LTC 4 receptor which is not susceptible to known peptide-leukotriene antagonists (Snyder and Krell, 1984). To demonstrate the occurrence of this receptor, the metabolism of LTC 4 to LTD 4, and subsequently LTE4, must be prevented with L-serine borate, an inhibitor of y-glutamyl transpeptidase, an enzyme which converts LTC4 to LTD 4 (Aharony et al., 1985). To determine whether a distinct LTC 4 receptor might exist in the cutaneous vasculature, the ability of ICI 198,615 to inhibit LTC4-induced increases in cutaneous vascular permeability in the presence and absence of L-serine borate was investigated. As illustrated in table 4, the inhibition by ICI 198,615 of LTC4-induced increases in cutaneous vascular permeability was independent of L-serine borate. 3.2. Inhibition of ovalbumin antigen-induced bronchoconstriction in guinea pigs by ICI 198,615 Guinea pigs passively sensitized with antiovalbumin antibody obtained from donor animals respond to aerosol administration of ovalbumin antigen with increases in pulmonary resistance (Rp) and decreases in dynamic lung compliance

76 TABLE 1 D o s e - r e s p o n s e r e l a t i o n s h i p for p e p t i d e l e u k o t r i e n e - i n d u c e d i n c r e a s e s in c u t a n e o u s v a s c u l a r p e r m e a b i l i t y in g u i n e a pigs. Dose

LTC 4

(nmol/site) "

( m m 2) h

nd

(mm 2)

LTD 4 n

( m m 2)

LTE 4

0.005 0.01 0.02 0.05 0.20 0.30

68.9 4- 7.0 c 86.0_+11.0 103.0_+ 4.9 1 2 0 . 0 + 7.6 1 3 1 . 0 ± 8.5 1 6 0 . 0 ± 6.7

6 7 10 5 6 5

40.0 ± 6.4 75.9±6.1 99.1 ± 5 . 5 117.0_+6.6 134.0_+6.8 158.0_+6.6

6 7 10 5 6 5

33.7 ± 64.1 ± 78.0± 89.8_+ 119.0± 163.0+

6.7 6.6 3.0 8.0 10.5 7.1

6 7 10 5 6 5

D o s e of L T a d m i n i s t e r e d in 0.1 ml c o n t a i n i n g 10 5 M P G E 2. b N e t r e a c t i o n a r e a . " V a l u e s a r e m e a n s i S . E . M , animals.

a n = n u m b e r of

TABLE 2 Effect of v a r i o u s a g e n t s o n L T D 4 - i n d u c e d i n c r e a s e s in c u t a n e o u s v a s c u l a r p e r m e a b i l i t y in g u i n e a pigs ~. Compound

Dose b (mg/kg)

Route

Control ( m m 2)

nc

Experimental (mm 2)

n

P "

Indomethacin Cyproheptadine Pyrilamine

10 1.0 2.0

i.v. i.v. i.v.

161 ± 11.4 ,l 184 ± 15.8 1 5 9 ± 9.0

7 5 5

152 _+ 5.0 194 _+ 17.5 1 5 4 + 10.4

7 5 5

> 0.05 > 0.05 > 0.05

a L T D 4 w a s at 0.3 n m o l e / s i t e a n d c o n t a i n e d 10 5 M P G E 2. h C o m p o u n d s w e r e a d m i n i s t e r e d 10 m i n p r i o r to agonist. ~ n = numbew of a n i m a l s , a Values a r e m e a n s ± S.E.M. e Level o f s i g n i f i c a n c e cf. c o n t r o l . TABLE 3 I n h i b i t i o n of L T C 4- a n d L T E 4 - i n d u c e d i n c r e a s e s in c u t a n e o u s v a s c u l a r p e r m e a b i l i t y b y ICI 198,615 ~' ICI 198,615 b ( bt m o l / k g )

Agonist LTC4

LTE4

Area c ( m m 2) 0 0.018 0.055 0.18 0.55 EDs0 ( ~ m o l / k g ) g

182.0 159.0 101.0 62.4 10.6 0.083

± 4.5 f ± 2.0 _+ 9.0 ±24.1 ± 8.3

% inhibition

N d

P e

12.9 44.4 65.8 94.2

7 6 6 6 6

< < < <

Area ( m m 2)

0.001 0.001 0.001 0.001

172.0 139.0 81.5 50.4 12.0 0.067

±12.4 _+ll.6 ± 17.2 ±11.6 ± 2.7

% inhibition

n

P

19.3 50.4 70.7 93.0

5 4 4 4 4

> < < <

0.05 0.001 0.001 0.001

a D o s e o f a g o n i s t w a s 0.3 n m o l / s i t e in 0.1 ml P B S c o n t a i n i n g 10 5 M P G E 2. b A d m i n i s t e r e d i.v. 10 m i n p r i o r to a g o n i s t injection. c N e t r e a c t i o n a r e a . d N u m b e r o f a n i m a l s , e Level o f s i g n i f i c a n c e , r V a l u e s are m e a n s ± S.E.M. g D o s e o f ICI 198,615 p r o v i d i n g 50% inhibition. TABLE 4 Effect of ICI 198,615 o n L T C 4 - i n d u c e d i n c r e a s e s in c u t a n e o u s v a s c u l a r p e r m e a b i l i t y in the a b s e n c e a n d p r e s e n c e of L-serine b o r a t e an inhibitor of y-glutamyl transpeptidase a Treatment

Dose

Net area (mm 2)

nc

% inhibition d

Vehicle c o n t r o l I C I 198,615 L-serine b o r a t e b ICI 198,615 + L-serine borate

0.18/t mol/kg 45 m M 0.18/~mol/kg 45 m M

153.0_+ 9.9 f 52.9 ± 19.1 1 4 8 . 0 ± 16.3 38.6 _+ 12.8

6 6 6 6

65.9 73.9

p e

< 0.001 < 0.001

a L T C 4 (0.3 n m o l / s i t e ) w a s a d m i n i s t e r e d w i t h 10 5 M P G E 2. C o m p o u n d s (or vehicles) w e r e a d m i n i s t e r e d i.v. 10 m i n p r i o r to L T C 4 b L-Serine b o r a t e (45 m M ) w a s a d m i x e d w i t h L T C 4 a n d P G E 2. c n = n u m b e r o f a n i m a l s , a % i n h i b i t i o n cf. respective c o n t r o l ¢ Level of significance, f V a l u e s are m e a n s _ S.E.M.

77 Net Area

(ram2 180"

• IC1198,615

160-

• Lg171,883

• FPL 55712

Control

+T

140" 120"

"x,~I271

100" 80" 60" 40" 20" 0 0.01

[651~. ~

[

9

5

]

'

o:1

~

~

lb

{

8

6

]

160

~mole/kg, (i.v.) Fig. 1. I.v. dose-response curve for ICI 198,615, LY 171883 and FPL 55712 against LTD4-induced increases in cutaneous vascular permeability in the guinea pig. Antagonists and Evan's blue dye were administered 10 and 2 rain, respectively, before LTD 4 (0.3 nmol/site, in combination with 10 -5 M PGE2). Cutaneous injection volume was 0.1 ml for L T D 4 / P G E 2. Each point is the mean+S.E.M, of 6-14 animals each receiving 4 identical injections. EDs0 values for ICI 198,615. LY 171883 and FPL 55712 were, respectively; 0.11, 66.4 and 44.8 ktmol/kg. Open symbols represent vehicle control. Bracketed values are % inhibition. * P < 0.01, • * P < 0.001.

(Cdy.): indices of large and small airway constriction, respectively. This response is mediated in large part via mast cell histamine release. Previous studies have demonstrated that pretreatment with pyrilamine, a histamine Hi-receptor antagonist, dl-propranolol, a fl-adrenoceptor antagonist and indomethacin, a cyclooxygenase inhibitor, gives rise to a smaller bronchoconstriction in response to aerosol ovalbumin, but one that is mediated, at least in part, by peptide leukotrienes (Ritchie et al., 1981). The ability of ICI 198,615 to inhibit this response was investigated. As illustrated in fig. 2, pretreatment of animals with 1 /xmol/kg (i.v.) of ICI 198,615 provided a significant (P < 0.05) reduction in aerosol ovalbumin-induced increases i n R p . On the other hand, antigen-induced decreases in Cdy n w e r e not significantly ( P > 0.05) affected. The ability of ICI 198,615 to reverse an antigen-induced bronchospasm was also investigated. I.v. administration of 1 /xmol/kg of ICI 198,615 immediately following a m a x i m a l R p increase induced by aerosol ovalbumin antigen produced a significant (P < 0.05), near total reversal of R p increases (fig. 3). In contrast, the compound demonstrated little to no

ability to reverse small airway spasm (fig. 3).

(Cdyn) broncho-

3.3. Inhibition of ovalbumin antigen-induced mediator release from actively sensitized guinea pigs Chopped lung prepared from guinea pigs immunized to ovalbumin antigen release a variety of substances on in vitro exposure to the antigen including, but not limited to, histamine, peptide leukotrienes, thromboxane A 2 (TxA 2) and prostacyclin (PGI2). The latter two products are measured as respective degradation products TxB2 and 6-keto PGF1,. The ability of ICI 198,615 to alter either the spontaneous or antigen-induced release was assessed. At concentrations of ICI 198,615 ranging from 10 -8 to 10 -5 M, the compound did not alter either the spontaneous (P > 0.05) or antigen-induced (P > 0.05) release of histamine, LTD4, TxB 2 or 6-keto PGFI~ (data not shown).

3.4. Inhibition by 1(21 198,615 of lipoxygenases ICI 198,615 (10 #M) did not inhibit 5-, 12- or 15-1ipoxygenases partially purified from guinea

78 • 2 % PEG vehicle control (n = 9) : ICi 198,615 1.0 Iamole/kg, i.v. (n = 9) * p < 0.05

I 500 -

* p -: 0.05 400 -

400-

300-

~" 300-

200 -

200 c =~ O o~

100-

J

_

/

_, x

(8)

xT,.~... , ~" T

"I-___i_ --

T --

(7)

T (8)

.

100J¢ o o~

-10-20-

• 2% PEG vehicle control (n ~ 8) IC1198,615, 1.0 9mole/kg, i.v. (n = 6)

500-

-10-20-

T

-30 -40-

-30 -40 -

T.__r

g -5o-

-50-

8

-60 -70-

k

T T r_.---~----v~(~ I

-60 -70 -80 -90-

-80-90-

-4 -;

-2

Peak 2 4 6 8 1'0 1'2 Time (Min) From Peak Rp increase

1'4

Fig. 2. Inhibition of aerosol ovalbumin antigen-induced bronchospasm by ICI 198,615. All animals were pretreated i.v. with pyrilamine (2.0 m g / k g ) , dl-propranolol (0.5 m g / k g ) and indomethacin (10 m g / k g ) at 40, 50 and 45 min prior of antigen provocation. ICI 198,615 (1 ~tmol/kg) or vehicle was administered i.v. 35 rain prior to ovalbumin aerosol. Five breaths of PBS were administered 15 and 10 rain prior to five breaths of aerosol ovalbumin (40 m g / m l ) to stabilize pulmonary resistance (Rp) and dynamic lung compliance (Cdy~) baseline values. The time of maximal Rp increase was considered 0 and Rp and Cdyn changes at 2 rain intervals before and after peak change were calculated. Symbols with vertical lines represent the means+_S.E.M, of n animals. N u m b e r s in parentheses associated with data points refer to the number of animals (n) for that point only. * P < 0.05.

-2 Peak 2

4

6

8

1'0

12

1'4 1'6

Time (Min) From Peak Rp increase

16

Fig. 3. Reversal by ICI 198,615 of aerosol ovalbumin-induced bronchospasm in passively sensitized guinea pigs. ICI 198,615 (1 ~ m o l / k g i.v.) or vehicle was administered immediately after peak Rp increase and the rate of return of Rp and Cdy,1 to baseline monitored. See fig. 2 for additional details.

concentration-dependent increase in the release of TxB 2 (fig. 4). As expected the cyclooxygenase inhibitor indomethacin (10 ~M) produced an ap9000 '

I

I

I

I

8000~ .Control . ~1~ T • IC1198,615 (100 nM) I ~ I "/I 7000- •lndomelhacin(10~M)/~ 1 I~ T ~ 60005000-

pig polymorphonuclear leukocytes, human platelets and soybeans, respectively (data not shown).

4000~ 3000~20~-

3.5. Inhibition by ICI 198,615 of LTD4-induced release of TxB 2 from chopped guinea pig lung LTD 4 has been shown to release TxA 2 from chopped guinea pig lung by a peptide leukotriene receptor mediated process (Folco et al., 1981; Mong et al., 1987). It was of interest to determine whether ICI 198,615 could inhibit this process. Incubation of varying concentrations of L T D 4 with chopped guinea pig lung tissue provoked a

0 0.1

1.0

tO.O

100.0

10~.0

1001)11

LTD, [nMl

Fig. 4. I n h i b i t i o n o f L T D 4 - i n d u c e d release T x A 2 f r o m chopped guinea pig lung tissue by ICI 198,615. Tissue was incubated with either 1CI 198,615 (100 nM), indomethacin (10 /~M) or vehicle for l0 min prior to the addition of various concentrations of L T D 4. Results shown are from a typical experiment conducted in triplicate. Symbols with vertical lines are the m e a n s + S.E.M. of triplicate observations.

79 proximate 84% inhibition. ICI 198,615 (10 7 M) produced a substantial rightward (about 5000 fold), apparently, non-parallel shift, in the LTD4 curve. The high degree of variability in the LTD 4induced release of TxB 2 along with the bell-shaped dose-response curve obviated attempts to determine a meaningful K B value for the compound.

4. Discussion

ICI 198,615 is a novel, potent (pA 2 = 10.1 vs. LTE 4 in guinea pig trachea) and highly selective antagonist of the effects of LTD 4 and LTE4 on the respiratory system of the guinea pig both in vitro and in vivo (Snyder et al., 1987; Krell et al., 1987; Aharony et al., 1987). This investigation served to expand the pharmacologic profile of IC! 198,615. Previous studies have documented the ability of LT to increase vascular permeability in skin (Rinkema et al., 1984) and airways (Woodward et al., 1983). In the present investigation, all three peptide leukotrienes, i.e., LTC4, LTD 4 and LTE 4, produced dose-related increases in cutaneous vascular permeability in the guinea pig and were approximately equipotent. The response to LTD 4 was not dependent upon the release of histamine, serotonin or products of the cyclooxygenase pathway of arachidonic acid metabolism. These observations are in agreement with those of Rinema et al. (1984). On the other hand, LTD4-induced increases in cutaneous vascular permeability were inhibited in a dose-related manner by ICI 198,615 >> FPL 55712 > LY171883. ICI 198,615 also inhibited LTC 4- and LTE4-induced increases in cutaneous vascular permeability with essentially equal potency to that displayed against L T D 4. Previous studies in guinea pig treacheal smooth muscle have indicated that LTC4 interacts with a receptor distinct from the L T D a / L T E 4 receptor (Snyder and Krell, 1984). With currently available antagonists, this receptor can only be distinguished when the metabolic conversion of LTC4 to L T D 4 and subsequently LTE4 is inhibited by L-serine borate complex. However, when L-serine borate was co-administered with LTC 4 into the skin, the antagonism of LTC 4 displayed by ICI

198,615 did not decline as it does under similar experimental conditions in the guinea pig trachea. While other explanations or interpretations are equally plausible, these findings suggest that LTC4, L T D 4 and LTE 4 may interact with a common receptor in the skin. Moreover, these results indicate that this unique chemical class of leukotriene antagonists, represented by ICI 198,615, are capable of blocking the effect of peptide leukotrienes in non-pulmonary tissues, and that the greater potency for ICI 198,615, as compared with FPL 55712 and LY 171883, apparent in respiratory tissue carried over to a non-respiratory leukotriene responsive system. ICI 198,615 inhibited ovalbumin antigen-induced bronchospasm in a guinea pig model of 'asthma' in which, through pharmacologic manipulation, endogenous peptide leukotrienes are prominent mediators of the bronchospasm (Ritchie et al., 1981). The ability of ICI 198,615 to inhibit the effect of aerosol ovalbumin antigen of large airways (Rp) was notably more prominent than the effect of the compound on small airways (Cdyn). Likewise, i.v. administration of ICI 198,615 at the peak of the antigen-induced bronchospasm produced a substantial reversal of large airway constriction, but again, produced little to no effect on the small airway constriction. One possibility is that LTC 4 released by antigen was acting on a distinct LTC 4 airway receptor (Snyder and Krell, 1984), which is not susceptible to blockade by ICI 198,615. However, IC1 198,615 does inhibit pulmonary responses to exogenously administered LTC 4 in the guinea pig indicating that LTCa is rapidly metabolized to L T D 4 a n d / o r LTE 4 in guinea pig lung in vivo (Krell et al., 1987). These findings suggest that mediators other than L T D a / L T E 4 may be involved in the constriction of small airways. ICI 198,615 at concentrations ranging from 10 -s to 10 -5 M did not alter either the spontaneous or antigen-induced release of histamine, peptide LTs, TxB2 or 6-keto PGF1a indicating that the compound does not interfere with antigen-induced mast cell degranulation, biosynthesis of peptide leukotrienes or phospholipase A2, cyclooxygenase, thromboxane synthetase or prostacyclin synthetase. Similarly, the compound, at

80

high concentrations, had no effect on 5-, 12- or 15-1ipoxygenase. Consequently, ICI 198,615 should be a valuable pharmacologic tool for assessing the role of peptide leukotrienes in models of allergy as well as other models in which peptide LT are thought to play a role. Indeed ICI 198,615 has been evaluated in a Rhesus monkey model of allergic asthma where it apparently lacks efficacy (Patterson et al., unpublished observations). Lastly, peptide LT have been shown to stimulate formation of TxB 2 in isolated guinea pig lung (Folco et al., 1981; Mong et al., 1987). The peptide LT antagonist, SKF 104,353 inhibited this response with a K B value identical to the K~ value determined for inhibition of [3H]LTD4 binding in guinea pig lung parenchymal membranes. In our hands, LTD4-induced TxB 2 release from chopped guinea pig lung was highly variable and consistently resulted in bell-shaped dose-response curves. While ICI 198,615 clearly antagonized the effect of LTD4, variability prevented determination of a KB value. The lack of effect of ICI 198,615 on TxA 2 release by antigen indicates that LTD 4 released by antigen does not serve as a positive stimulus under these experimental conditions. In summary, ICI 198,615 is the most potent and selective peptide leukotriene receptor antagonist described thus far. The pharmacologic profile of this compound indicates that it lacks significant pharmacology in a variety of other systems. Consequently, at the present time, it appears to offer utility as a selective pharmacologic tool for investigating the role of peptide leukotrienes in a variety of pathophysiologic states.

Acknowledgements The authors wish to express their appreciation to Ms. R. Walton and D. Zakielarz for their assistance in the preparation of this manuscript and especially to Ms. D. Dea and R. Marks for their highly skilled technical assistance.

References Aharony, D., P. Dobson, P.R. Bernstein, E.J. Kusner, R.D. Krell and J.B. Smith, 1983, Determination of SRS-A re-

lease from guinea pig lungs by radioimmunoassay, Biochem. Biophys. Res. Commun. 117, 574. Aharony, D., P.T. Dobson and R.D. Krell, 1985, In vitro metabolism of [3H]peptide leukotrienes in human and ferret lung: a comparison with the guinea pig, Biochem. Biophys. Res. Commun. 131, 892. Aharony, D., R.C. Falcone and R.D. Krell, 1987, Inhibition of 3H-leukotriene D 4 binding to guinea pig lung receptors by the novel leukotriene antagonist ICl 198,615, J. Pharmacol. Exp. Ther. 243, 921. Augstein, J., J.B. Farmer, T.B. Lee, P. Sheard and M.L. Tattersall, 1973, Selective inhibitor of slow reacting substance of anaphylaxis, Nature New Biol. 245,215. Feldberg, W. and C.H. Kellaway, 1938, Liberation of histamine and formation of lysolecthin-like substances by cobra venom, J. Physiol. (London) 94, 187. Fleisch, J.H., L.E. Rinkema, K.D. Haisch, D. Swanson-Bean, T. Goodson, P.P.K. Ho and W.S. Marshall, 1985, LY 171883, l-2-hydroxy-3-Propyl-4 < 4-(lH-tetrazol-5-yl) butoxy > phenyl > ethanone, an orally active leukotriene D 4 antagonist, J. Pharmacol. Exp. Ther. 233, 148. Folco, G., G. Hansson and E. Granstrom, 1981, Leukotriene C4 stimulates TxA 2 formation in isolated sensitized guinea pig lungs, Biochem. Pharmacol. 30, 2491. Hay, D.W.P., R.M. Muccitelli, S.S. Tucker, L.M. Vickery-Clark, K.A. Wilson, J.G. Gleason, R.F. Hall M.A. Wasserman and T.J. Torphy, 1987, Pharmacologic profile of SK&F 104353: a novel, potent and selective peptidoleukotriene receptor antagonist in guinea pig and human airways, J. Pharmacol. Exp. Ther. 243, 474. Krell, R.D., R.E. Giles, Y.K. Yee and D.W. Snyder, 1987, In vivo pharmacology of ICI 198,615: a novel, potent and selective peptide leukotriene antagonist, J. Pharmacol. Exp. Ther. 243. 557. Krell, R.D. and E.J. Kusner, 1984, Differential effect of cyclooxygenase inhibition on antigen- and ionophore-induced release of slow reacting substance from fragmented guinea pig lung, Br. J. Pharmacol. 82, 871. Mead, B., L.H. Patterson and D.A. Smith, 1981, The disposition of FPL 55712 acid (7-[3-(4-acetyl-3-hydroxy-2-propylphenoxy)-2-hydroxypropoxy)-4-uxo-8-propyl-4H-benzopyran-2-carboxylic acid) in rat and dog, J. Pharm. Pharmacol. 198, 682. Mong, S., H.-L. Wu, J. Miller, R.F. Hall, J.G. Gleason and S.T. Crooke, 1987, SKF 104,353, A high affinity antagonist for human and guinea pig lung leukotriene D 4 receptor, blocked phosphatidylinositol metabolism and thromboxane synthesis induced by leukotriene 04, Mol. Pharmacol. 32, 223. Murphy, R.C., S. Hammarstr/Sm and B. Samuelsson, 1979, Leukotriene C; a slow-reacting substance from murine mastocytoma cells, Proc. Natl. Acad. Sci. (U.S.A.) 76, 4275. Peck, M.J., P.J. Piper and TJ. Williams, 1981, The effect of leukotrienes C4 and D 4 on the microvasculature of the guinea pig skin, Prostaglandins 21,315. Rinkema, L.E., K.G. Berris and J.H. Fleisch, 1984, Production and antagonism of cutaneous vascular permeability in the

81 guinea pig in response to histamine, leukotrienes and A23187, J, Pharmacol. Exp. Ther. 230, 550. Ritchie, D.M., J.N. Sierchio, R.J. Capetola and M.E. Rosenthale, 1981, SRS - a mediated bronchospasm by pharmacologic modification of lung anaphylaxis in vivo, Agents Actions 11, 396. Siraganian, R.P., 1975, Refinements in the automated fluorometric histamine analysis system, J. lmmunol. Meth. 7, 283. Snyder, D.W. and R.D. Krell, 1984, Pharmacological evidence for a distinct leukotriene C4 receptor in guinea pig trachea, J. Pharmacol. Exp. Ther. 231, 616.

Snyder, D.W., R.E. Giles, R.A. Keith, Y.K. Yee and R.D. Krell, 1987, In vitro pharmacology of 1CI 198,615: a novel, potent and selective peptide leukotriene antagonist, J. Pharmacol. Exp. Ther. 243, 548. Woodward, D.F., M.A. Wasserman and B.M. Weichman, 1983. Investigation of leukotriene involvement in the vasopermeability response associated with guinea pig tracheal anaphylaxis: comparison with cutaneous anaphylaxis, European J. Pharmacol. 93, 9.