Inhibition of histamine, leukotriene CJD4, and thromboxane B2 release from human leukocytes and human chopped lung mast cells by the allergic mediator release inhibitor, CI-949 M. C. Conroy, PhD, J. A. Kennedy, BA, J. C. Chestnut, MS, C. D. Wright, PhD, R. L. Adolphson, PhD, and D. O. Thuesono PhD Ann Arbor, Mich. The novel antiallergy compound, 5-methoxy-3-(1-methylethoxy)-l-phenyl-N-1H-tetrazol-5-yl-lHindole-2-carboxamide, L-arginine salt (CI-949), inhibited mediator release from human basophilic leukocytes and from human chopped lung mast cells challenged with anti-lgE. In leukocytes, CI-949 was a more potent inhibitor of leukotriene C4/D4 and thromboxane B2 release (concentration of drug that inhibits mediator release by 50% [IC5o] 0.5 and 0.1 la~nol/L, respectively) than of histamine (IC5o, 11.4 lamol / L ) when anti-l gE was the challenging stimulus. In human lung, inhibition of release of all three mediators occurred at approximately equal concentrations (ICsosfor histamine, 16.6 I.tmol/L; for leukotriene C4/D4, 7.1 lamol/L; and for thromboxane B2, 6.2 lamol/L). The inhibition of histamine release from basophils by CI-949 was further characterized using a variety of stimuli. Challenge with anti-lgE, histamine-releasing factor derived from lymphocytes, N-formyl-L-methionyl-L-leucylL-phenylalanine, and concanavalin A revealed potent inhibition (IC5o, 10 to 15 lamol / L ). CI-949 was less potent versus calcium ionophore A23187, phorbol myristate acetate (12-o-tetradecanoylphorbol-13-acetate ), and C5a (ICsos, 30, 54, and 60 lamol / L, respectively). These results suggest that diverse pathways of cell activation-excitation coupling exist for different stimuli in basophils. Furthermore, the activity and potency of CI-949 in inhibiting release of histamine, leukotrienes, and thromboxane from both human basophils and mast cells suggest that the compound will be effective clinically for indications in which these mediators are implicated, including asthma and allergic rhinitis. ( J ALLERGYCLIN IMMUNOL 1990;86: 902-8.)

It is well established that inhalation challenge with histamine, peptidoleukotrienes, thromboxane, prostaglandin D2, or platelet-activating factor can cause immediate bronchoconstriction in man.l-6 These mediators, produced by basophils, mast cells, and other inflammatory cells, can interact to promote a synergistic response or sensitize bronchial tissue to other mediators under the appropriate conditions. Arm From the Immunopharmacology Section, Department of Experimental Therapy, Parke-Davis Pharmaceutical Research Division, Warner-Lambert Co., Ann Arbor, Mich. Received for publication June 1, 1989. Revised July 10, 1990. Accepted for publication July 26, 1990. Reprint requests: M. C. Conroy, PhD, Parke-Davis Pharmaceutical Research, Warner-Lambert Co., 2800 Plymouth Road, Ann Arbor, MI 48105. 111/24309

902

et al. v have reported that inhalation of LTE4, but not methacholine, selectively enhances histamine responsiveness in subjects with asthma. Phillips and Holgate 8 reported small increases in responsiveness to prostaglandin Dz and histamine after LTC4 inhalation by subjects with asthma. Furthermore, the contribution of multiple mediators to bronchial smooth muscle contraction in vitro 9 or in vivo 1~ has been inferred from studies with specific mediator antagonists and specific inhibitors of mediator synthesis. Our goal in drug development was therefore to find a compound that inhibited the production/release of multiple mediators from basophils and mast cells and that exhibited antiallergic activity in in vivo models of immediate hypersensitivity. We anticipated that this profile of activity would optimize the discovery of a clinically useful new chemical entity. 11, lz In this article, we report the inhibitory effects of such a c o m -

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Abbreviations used CI-949:

/OCW(CW3)z

5-Methoxy-3-(1methylethoxy)- 1-phenyl-N1H-tetrazol-5-yl- 1H-indole-2carboxamide, L-arginine salt

FMLP: N-Formyl-l-methionyl-lleucyl-l-phenylalanine Con A: Concanavalin A HEPES: N-2-hydroxyethylpiperazineN'-2-ethanesulfonic acid HRF: Histamine-releasing factor derived from lymphocytes A23187: Calcium ionophore A23187 PMA: Phorbol myristate acetate (12o-tetradecanoylphorbol-13 acetate) IC~o: Concentration of drug that inhibits mediator release by 50% LTE,, LTC4, LTD4: Leukotrienes E4, C4 and D4 RIA: Radioimmunoassay TXB~: Thromboxane B2

pound, CI-949 (Fig. 1), on release o f histamine, leukotriene, and thromboxane from both human basophils and mast cells. We studied human cells because there are clear differences between animal and human cells in respect to pharmacologic modulation. 13 CI949, but not cromolyn sodium, ketotifen, or nedocromil, inhibited the release of all three mediators from both mast cells and basophils stimulated with anti-IgE. We have also examined the ability of CI949 to inhibit histamine, L T C J D 4 , and TXB2 release from human basophilic leukocytes challenged with F M L P because o f the potential importance o f nonIgE-dependent triggers in allergic and asthmatic reactions.14-16 CI-949 inhibited the release of all three mediators in response to FMLP. Additional experiments characterized the differential potency of CI-949 to inhibit histamine release after challenge of human leukocytes with receptor-mediated stimuli and more distally acting activators.

MATERIAL AND METHODS The following reagents were purchased: hydroxyethyl starch (Hespan), American Critical Care, Division of American Hospital Supply Corp., Irvine, Calif.; HEPES, Con A, PMA, and FMLP, Sigma Chemical Co., St. Louis, Mo.; Human serum albumin, Miles Laboratories, Elkhart, Ind.; rabbit antihuman IgE, Dako I mmunoglobulins, Accurate Chemicals, Westbury, N.Y.; RIA kit for LTC,, LTD,, and LTE,, Amersham Corp., Arlington Heights, Ill.; RIA kit for TXB2, New England Nuclear-Dupont, Boston, Mass.; and A23187, Behring Diagnostics, La Jolla, Calif.

"

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FIG. 1. Structure of CI-949.

Preparation of drugs CI-949, cromolyn sodium, and nedocromil were synthesized in the Department of Chemistry, Parke-Davis Pharmaceutical Research Division, Warner-Lambert Co., Ann Arbor, Mich. Ketotifen was obtained from Sandoz Pharmaceuticals Corp., East Hanover, N.J. Drugs were first solubilized in 0.5% dimethylsulfoxide and water (or only water) to which concentrated HEPES buffer was added to bring the solution to physiologic pH and ionic strength. The buffer used throughout the basophil experiments and for mediator release from human lung consisted of HEPES (25 mmol/L), NaC1 (125 mmol/L), KC1 (5 mmol/L), human serum albumin (0.03%), CaC12 (1 mmol/L), and MgC12 (1 mmol/L), pH 7.4. Neither the drugs themselves nor the diluent interfered with the mediator assays.

Preparation of HRF and C5a Cell-free supematants from Con A-stimulated mononuclear cell cultures were used as a source of HRF. 16 Briefly, washed mononuclear cells from a Hypaque-Ficoll gradient 17 were incubated with Con A for 3 hours at 37 ~ C in a 5% CO2 atmosphere. After cells were washed, the cells were cultured for 48 hours. The supematants containing HRF were then harvested and dialyzed in the cold. The samples were additionally treated with 0.01 mol/L of ct-methyl-Dmannoside, a competitive inhibitor of Con A. C5a was prepared from normal human serum according to the procedure of Femandez and Hugli. ~8

Preparation and challenge of human lung Portions of grossly normal lung were obtained from the Department of Surgery, University of Michigan School of Medicine, Ann Arbor, Mich., after surgery for carcinoma. The lung was placed in Tyrode's buffer (containing, in grams per liter, NaC1 [8.0], NaHCO3 [1.0], CaC12 [0.2], KC1 [0.2], MgCI2 [0.1], NaH2PO4 [0.05], and glucose [1.0], pH 7.4), dissected free of larger bronchioles and b l o o d vessels, and then minced with scissors into 25 to 75 mg fragments. The fragments were washed with approximately 1 L of Tyrode's buffer and then stored overnight in buffer at room temperature. Before use, the tissue was washed with HEPES buffer, and approximately 400 mg samples of lung tissue were placed in plastic vials containing buffer.

904

C o n r o y et al.

J. ALLERGY CLIN. IMMUNOL. DECEMBER 1990

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quantitation of allergic mediators. Mediator release and inhibition of mediator release by CI-949 was comparable in ceils prepared by either method.

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After a 10-minute incubation at 37~ C in HEPES buffer, drug or buffer was added to the appropriate vials. Anti-IgE (final dilution, 3: 1000) was added to the vials, and the incubation was continued with shaking for 30 minutes. Supernatant solutions were removed for assay and processed as described below.

Preparation and challenge of human basophilic leukocytes Basophilic leukocytes were prepared from anticoagulanttreated venous blood from allergic volunteers. For studies of stimulus effects on histamine release, blood was mixed with hydroxyethyl starch. The leukocytes collected after sedimentationwere washed with calcium-free HEPES buffer and resuspended in calcium-containing HEPES buffer at a concentration of approximately 107 cells per milliliter. In studies of eicosanoid metabolism, the mononuclear fraction, containing basophils and lymphocytes isolated according to the method of Ferrante and Thong) 7 was used as a source of cells. The cells were preincubated with the test drug or buffer (to measure spontaneous release) for 10 minutes at 37~ C. The cells were then challenged with appropriate stimulus in a concentration to obtain histamine release from the ascending portion of the dose-response curve from each donor (or with buffer) and incubated for 50 minutes at 37 ~ C. The reaction was stopped by centrifugation, and the resulting supernatant solutions were decanted and saved for

Histamine content in the supernatants from either human leukocyte or human lung preparations was assayed by an automated fluorometric method described by Siraganian. 19 Total histamine was determined by perchloric acid lysis of a n aliquot of leukocytes or by heating the lung fragment samples to 80~ C with perchloric acid. Histamine release was expressed as a percentage of the total histamine content less the spontaneous release. Mean ---SEM percent histamine release by anti-IgE was 44% -+ 2.6% (n = 45) from leukocytes, and 10% • 2.2% (n = 10) from lung. Histamine release from leukocyte preparations by FMLP was 52% • 2.8% (n = 35); by HRF, 35.3% _+ 4.2% ( n - - 9 ) ; by Con A, 36% • 5.6% ( n - - 7 ) ; by C5a, 42% _ 5.6% (n = 12); by A23187, 76% • 6.6% (n = 10); and by PMA, 67% --- 6.2% (n = 8). Assays for leukotriene and thromboxane were performed with commercially available RIA kits. The amount of released mediator in each sample was calculated from the standard curve. The activated lung cells released 13.5 • 4.7 pg of immunoreactive LTC4/D4, n = 8; and 12.9 • 5.4 pg of immunoreactive TXB2, n = 6, per milligram of wet tissue. These values were normalized with the total histamine content of the sample, since histamine content of the lung is quite uniform. This step adjusted raw leukotriene and TXB2 data for differences in the mass of tissue used in each sample. The normalization step was not required in the leukocyte system because sampling error is very small ( • 1.5%). The amount of immunoreactive leukotriene released from human leukocytes, calculated according to the method of MacGlashan et al. ~~was 44.9 pmol/ixg of histamine for antiIgE challenge (n = 5) and 119 pmol/Ixg of histamine for FMLP challenge (n = 6). Thromboxane release from human leukocytes was 4 pmol/p~g of histamine for anti-IgE challenge (n = 6) and 10 pmol/Fg of histamine for FMLP challenge (n = 5). Percent inhibition of release was calculated by comparing release in the presence of drug to the control release.

RESULTS CI-949 inhibited, in a dose-dependent manner, the release of histamine, leukotriene, and thromboxane from human basophilic leukocytes challenged with anti-IgE (Fig. 2). The IC50 for inhibition of histamine release was 11.4 p~mol/L. Virtually complete inhibition of histamine release occurred at 100 txmol/L, with negligible inhibition of release < 3 ixmol / L. Both LTC4/LTD4 and TXB2 release were inhibited at lower concentrations (IC50, 0.5 and 0.1 txmol/L, respectively). Complete inhibition of leukotriene and thromboxane synthesis/release was obtained with 10 and 1 Ixmol/L of CI-949, respectively. Because there is information suggesting that addi-

CI-949 i n h i b i t s m e d i a t o r release

VOLUME 86 NUMBER 6, PART 1

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CI-949 (/~M) FIG. 3. Effect of CI-949 on histamine release from human basophilic leukocytes challenged with anti-lgE (o) (n = 45), Con A (e) (n = 7), and HRF (A) (n = 9). Cells were preincubated with CI-949 for 10 minutes before the addition of stimulus.

0 tional stimuli might also precipitate allergic or asthmatic responses, we tested the ability of CI-949 to inhibit the release of histamine, leukotriene, and thromboxane from leukocytes challenged with FMLP. CI-949 was an effective inhibitor of release of all three mediators in response to this stimulus. The ICs0s for inhibition of histamine, leukotriene, and thromboxane were 6.3, 2, and 0.1 ixmol/L for FMLP challenge (data not presented). To characterize further the inhibitory effect of the compound, we compared its effect on histamine release from basophils challenged with those stimuli studied above plus Con A, HRF, C5a, A23187, and PMA. CI-949 inhibited release by all stimuli but demonstrated differential potencies. ICs0s for anti-IgE, Con A and HRF (Fig. 3), as well as for FMLP (data not presented), were essentially identical at 10 to 15 txmol/L each, whereas C5a-induced release was considerably more resistant to inhibition by CI-949 (IC5o, 60 I~mol/L). The percent of histamine release from control cells (FMLP, 52% +- 2.8%, n = 35 and C5a, 42% __. 5.6%, n = 12) did not appear to be a factor in the differential potency of inhibition by CI949, since release was comparable to the release in Fig. 2. A23187 and PMA were used to induce receptorindependent release of histamine by basophils. The dose response for inhibition by CI-949 was similar to the response noted for receptor-mediated stimulation. Nearly complete inhibition of A23187-induced release and 70% maximal inhibition of PMA-induced release occurred with 100 ~xmol/L of CI-949. Again the differential potency of CI-949 for these stimuli was evident, with ICsos of 54 ~mol/L for PMA and 30 p~mol/L for A23187 (data not presented). The mean control release for these stimuili was higher than re-

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FIG. 4. Inhibition by c1-g49 of LTC4/D4 (o), TXB2 (e), and histamine (zx) release from human lung. Chopped lung fragments were incubated with CI-949 at 37 ~ C for 10 minutes before the addition of anti-lgE. Vertical bars represent the standard errors from 4 to 10 experiments (except for the lowest CI-949 concentration in the TXB2 measurements, where n = 2) run in triplicate.

lease induced by the other stimuli reported above: A23187, 76% - 6.6% (n = 10) and PMA, 67% _ 6.2% (n = 8). We have also examined the effect of CI-949 on mediator release from anti-IgE-challenged human chopped lung preparations (Fig. 4). In this model, CI-949 was approximately equally effective in inhibiting release of histamine (IC50, 16.6 izmol/L) leukotriene (IC5o, 7,1 ixmol/L), and thromboxane (ICso, 6.2 p,mol/L). Virtually complete inhibition of all three mediators was observed at the 75 txmol/L concentration of CI-949. Clinical agents that have been reported to act via inhibition of mediator release were also tested in the leukocyte and lung models to compare the profiles of inhibition with that of CI-949. Neither cromolyn, nedocromil, nor ketotifen inhibited histamine release from human leukocytes challenged with anti-IgE (