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Effects of Heparin on the Inhibitory Activities of Human Urinary Trypsin Inhibitor (Ulinastatin) on Trypsin, Chymotrypsin and Leukocyte Elastase
Inhibitory
Effects
Tadayuki
of Emedastine
Difumarate
Saito', Asako Hagihara', Naoko Igarashi', Naomi Matsuda', Keizo Ito', Mitsunobu Mio2 and Kenji Tasaka2 'New Drug Research Laboratories
2Department
of Pharmacology
inhibitory
January
13, 1993
effects of emedastine
Release
Akira Yamashita',
, Kanebo, Ltd., Osaka 534, Japan
, Faculty of Pharmaceutical Received
ABSTRACT-The
on Histamine
Sciences, Okayama Accepted
March
difumarate
University, Okayama
700, Japan
6, 1993
on histamine release were studied in rat
peritoneal mast cells. Emedastine significantly inhibited substance P (SP)-induced histamine release at con centrations above 10-9 M in the presence of extracellular Ca2+ and at concentrations above 10-" M in its absence. At concentrations of 10-8 M or higher, emedastine significantly inhibited SP-induced Ca" release from intracellular Ca stores and SP-induced 45Ca uptake into mast cells. Emedastine also inhibited passive peritoneal anaphylaxis in rats and guinea pigs. We conclude that the clinical antiallergic effects of emedastine involve the inhibition of histamine release and that this inhibition is mediated by the inhibition of Ca" release from intracellular Ca stores and the inhibition of Ca" influx into mast cells. Keywords:
Emedastine
difumarate,
Histamine
release,
Histamine release from mast cells is induced by various stimuli apart from IgE-mediated antigen-antibody reac tions. In particular, histamine release induced by neuro peptides such as substance P (SP) has been discussed ex tensively in recent years (1-6). Intracutaneous injection of histamine produces circumscribed edema, wheals and flares; an axon reflex model has been suggested for the spread of flares. Some histamine-induced orthodromic im pulses from sensory nerve endings spread as antidromic impulses through terminal arborizations. These anti dromic impulses cause the release of SP which stimu lates neighboring mast cells to release histamine (1). Emedastine difumarate is a new antiallergic drug which possesses an antihistaminic effect (7) and an inhibitory effect on IgE-mediated histamine release from rat peritoneal mast cells (8). In the present study, we exam ined the effects of emedastine on SP-induced histamine re lease from rat peritoneal mast cells. To elucidate the mechanism of histamine release inhibition induced by emedastine, we studied its effects on SP-induced Ca 21 release from intracellular Ca stores and 45Ca influx into mast cells. In addition, we also studied the effects of emedastine on passive peritoneal anaphylaxis (PPA) in rats and guinea pigs to clarify the effectiveness of this drug in preventing in vivo anaphylaxis in relation to the inhibition of histamine release.
Substance
P, Mast cell, Intracellular
MATERIALS
Ca 21
AND METHODS
Animals Male Wistar rats weighing 210 420 g and male or fe male Hartley guinea pigs weighing 300-400 g were used. The animals were housed in a temperature and humidity controlled room with free access to food and water. Drugs Emedastine and ketotifen were synthesized by Kanebo, Ltd. Disodium cromoglycate (DSCG) was extracted from Intal (Fujisawa Pharmaceutical Co., Ltd., Osaka) by Kanebo, Ltd. Verapamil was purchased from Sigma Chemical Co. (St. Louis, MO, USA). To study the effects of the test compounds in the presence of extracellular Ca2+ in vitro, emedastine and ketotifen were dissolved in physiological buffered solution (PBS; 154 mM NaCI, 2.7 mM KCI, 0.9 mM CaCl2i 5.6 mM glucose, 0.1076 bovine serum albumin, 5 mM N-2-hydroxyethylpiperazine-N'-2 ethanesulfonic acid, pH 7.4). Verapamil was dissolved in distilled water and diluted with PBS. To study the effects of test compounds in a Ca-free medium, Ca-free PBS con taining 0.1 mM ethylenediaminetetraacetic acid (EDTA) was prepared and substituted for PBS. For the in vivo stud ies, emedastine and ketotifen were dissolved in distilled water, and DSCG was dissolved in saline.
Histamine release from rat peritoneal mast cells Rats were exsanguinated, 25 ml of glucose-free PBS was injected into the abdominal cavity, and the abdomi nal wall was gently massaged. Fluid from the abdominal cavity was collected and centrifuged at 80 x g for 5 min at 4'C. The pellet was then washed with fresh glucose-free PBS and suspended in PBS. The cell suspensions (5 x 105 cells/ml) and test compounds were preincubated at 371C for 10 min, after which 0.1 ml of cell suspension was ad ded to 0.8 ml of each test compound solution or PBS as a control. After a 60-min incubation, 0.1 ml of SP was ad ded, and incubation was continued for another 10 min. The reaction was terminated by the addition of 1 ml of ice-cold PBS. After centrifugation, histamine in the super natant and residual histamine in the cell pellet were deter mined by spectrofluorimetric assay, according to the method of Shore et al. (9). In the study performed in the absence of extracellular Ca", Ca-free PBS containing 0.1 mM EDTA was substituted for PBS. Histamine re leased in the supernatant was expressed as a percentage of the total histamine content in the specimen. The spon taneous histamine release was 4.3-L0.8% (n =11) or 3.7±0.7% in the presence or absence of extracellular Ca", respectively. The percent inhibition was calculated as follows: Inhibition (%)=(C-D)/(C-S) x 100 C: SP-induced histamine release without drugs D: SP-induced histamine release with drugs S: spontaneous histamine release Fluorescence measurement of mast cells loaded with quin 2 Fluorescence measurement of mast cells loaded with quin 2 was carried out according to the method of Tasaka et al. (2). Rat peritoneal mast cells were harvested from the abdominal cavity and purified to a level higher than 87% by density gradient centrifugation with Percoll (Pharmacia Biotechnology AB, Uppsala, Sweden). The mast cells were suspended in PBS containing 10-5 M of quin 2/AM (Dojindo Laboratories, Kumamoto) and incu bated for 60 min at 37 1C, followed by washing with Ca free PBS. Mast cell suspension (0.4 ml) was then added to the microchamber, and test compounds were added; after 10 min of incubation, SP was added to the medium. Fluorescent changes derived from quin 2 chelated with in tracellular Ca' were measured as integrated values every 2 sec under a fluorescence microscope (XF, Nikon Corpo ration, Tokyo) connected to a video-intensified micros copy system (ARGUS-100, Hamamatsu Photonics, Hamamatsu). Results were expressed as a percentage of the fluorescence intensity determined before the addition of SP, and the maximum values detected in the control and drug-treated groups were compared. Fluorescence in
tensities were converted into pseudo-color black according to pixel intensity. 45Ca uptake into rat peritoneal
from white to
mast cells
The measurement of 45Ca uptake into rat peritoneal mast cells was carried out according to a modification of the method of Spataro and Bosmann (10). Rat peritoneal mast cells purified with Percoll density gradient centrifu gation were suspended in PBS. Mast cell suspension (0.35 ml, 2.86 x 105cells/ml) was preincubated at 371C for 10 min, after which 0.05 ml of 45CaC12(1 MBq/ml) was added; the suspension was then incubated for 10 min prior to the addition of 0.05 ml of test compound solu tion or PBS as a control. After a 10-min incubation, 0.05 ml of SP was added, and the suspension was incubated for another 10 min. The reaction was terminated by the addition of 0.5 ml of ice-cold PBS. The cells, collected by centrifugation at 340 x g for 5 min at 41C, were washed twice with ice-cold PBS. After solubilization of the cell pellet in 0.2 ml of PBS containing 10% Triton X-100, 2 ml of liquid scintillator (Scintisol EX-H, Wako Pure Chemical Industries, Ltd., Osaka) was added to 0.15 ml of the specimen. The radioactivity was measured by a liquid scintillation counter (LSC-1050, Aloka Co., Ltd., Mitaka). The percent inhibition was calculated as fol lows: Inhibition (%) _ (C D)/(C S) x 100 C: SP-induced 45Ca uptake without drugs D: SP-induced 45Ca uptake with drugs S: spontaneous 45Ca uptake Preparation of anti-egg albumin (EA) rat serum Anti-EA rat serum was prepared according to a modifi cation of the method of Stotland and Share (11). Rats were immunized by intracutaneous injection of 0.6 ml of saline containing 1 mg of EA, 2 mg of aluminum hydroxide gel, and 1010 killed Bordetella pertussis (Tohama strain phase I, Chiba Serum Institute, Ichi kawa) into the footpads. Five days after immunization, the animals were boosted with an intramuscular injection of 0.5 ml of 1 mg/ml EA. Sera were isolated from blood collected at 15 days after the first immunization. The IgE titer of the serum was examined by inducing 48-hour homologous passive cutaneous anaphylaxis, and the sera with levels of 1:256 or above were pooled and kept at 80'C until immediately before the experiment. PPA in rats Rats were passively sensitized by the intraperitoneal
in
jection of 3 ml anti-EA rat serum (1/12 dilution). After 48 hours, 1 ml of 5 mg/ml EA was intravenously injected. Immediately after that, 5 ml of Tyrode's solution (137 mM NaCl, 2.7 mM KCI, 1.8 mM CaC12i 1.0 MM MgC12,
12.0 mM NaHCO3, 0.4 mM NaH2PO4, and 5.6 mM glu cose) was intraperitoneally injected. The animals were killed 5 min after the antigen injection, and the peritoneal fluid was collected. After centrifugation at 400 x g for 5 min at 41C, histamine in the supernatant was determined by a spectrofluorimetric assay according to the method of Shore et al. (9). The test compounds were administered orally 60 min before the antigen injection. Preparation
of anti-benzylpenicilloyl
Effects on histamine release in the absence of extracellular Ca 2+ When rat peritoneal mast cells were exposed to 2 x 10-6 M of SP in the absence of extracellular Ca", histamine was released at 22.2 ± 2.9% (n = 9) of the total histamine content. Emedastine, at 10-11 M or higher, significantly inhibited histamine release (Fig. 2), while ketotifen produced significant inhibition only at 10-6 M or higher.
bovine r-globulin
(BPO •BGG) guinea pig serum Anti-BPO . BGG guinea pig serum was prepared accord ing to the method of Levine et al. (12). Female guinea pigs were immunized intraperitoneally by repeated injec tions (twice a month, a total of 15-21 times) of 1 ml of 0.1070aluminum hydroxide gel which contained 2 pg/ml of BPO • BGG as antigen. Sera were isolated from blood collected at 10 days after the last immunization. The IgE titer of the serum was examined by inducing 7-day homologous passive cutaneous anaphylaxis; sera with levels of 1:512 or above were pooled and kept at 801C until immediately before the experiment. PPA in guinea pigs Male guinea pigs were passively sensitized with intra peritoneal injection of 3 ml anti-BPO • BGG serum (1/ 100 dilution). After 7 days, 0.3 mg/kg of pyrilamine was in jected intravenously, followed 5 min later by the in travenous injection of 1 ml of BPO. BSA (protein concen tration, 0.2 mg/ml). Immediately after that, 10 ml of Tyrode's solution was intraperitoneally injected. The animals were killed 5 min after the antigen injection, and peritoneal 400 x g for determined method of ministered
Fig. 1. Effects of emedastine and ketotifen histamine release from rat peritoneal mast extracellular Cat+. Each point represents experiments. Vertical bars indicate S.E. significantly different from the control. ketotifen.
on substance P-induced cells in the presence of the mean of 5 or 6 *P<0.05, **P<0.01, 0: emedastine, •:
Fig. 2. Effects of emedastine and ketotifen histamine release from rat peritoneal mast extracellular Ca 21. Each point represents experiments. Vertical bars indicate S.E. significantly different from the control. ketotifen.
on substance P-induced cells in the absence of the mean of 4 or 5 *P<0.05, **P<0.01, 0: emedastine, •:
fluid was collected. After centrifugation at 5 min at 41C, histamine in the supernatant was by spectrofluorimetric assay according to the Shore et al. (9). The test compounds were ad orally 60 min before the antigen injection.
Statistics The results were expressed as the mean±S.E. A one way analysis of variance with Dunnett's test and a paired t-test were used to determine statistical significance. RESULTS Effects on histamine release in the presence of extracellu lar Ca" When rat peritoneal mast cells were exposed to 3 x 10-6 M of SP in the presence of extracellular Ca", histamine was released at 27.3 ±2.1076 (n=11) of the total histamine content. Emedastine, at 10-9 M or higher, produced sig nificant inhibition of histamine release (Fig. 1), while ketotifen did not have any significant effect.
Fig. 3. Pseudo-color conversion of the fluorescence images of quin 2-loaded mast cells before (a and c) and 2 sec after (b and d) exposure to substance P. (a) and (b): control mast cells. (c) and (d): mast cells pretreated with emedastine.
Effects on increase of intracellular Ca" concentration When mast cells loaded with quin 2 were exposed to 2 x 10-6 M of SP in the absence of extracellular Cat+, the
fluorescence intensity of these cells was increased transient ly at 2 4 sec after SP stimulation and returned to the basal level by 10 sec after SP stimulation, indicating Ca" release from intracellular Ca stores. Emedastine, at 10-8 M, significantly inhibited the fluorescence increase at 2-4 sec after SP stimulation (Figs. 3 and 4), while ketotifen did not show any significant effect at this concentration. Effects on 45Ca uptake into rat peritoneal mast cells When mast cells were exposed to 3 x 10-6 M of SP, 45Ca uptake into mast cells was increased in a time -de pendent manner (Fig. 5). The increase of 45Ca uptake was continued for at least 30 min. In the following study, mast cells were incubated for 10 min after SP stimulation. SP (3 x 10-6 M) increased 45Ca uptake by 1272 ± 236 cpm (n = 5) compared to the value of 139 ± 34 cpm (n = 5) in the control group. Emedastine inhibited the SP-induced increase of 45Ca uptake in a concentration-dependent manner at concentrations ranging from 10-8 to 10-5 M
Fig.
4.
Effects
increases Each dicate
of
of emedastine
intracellular
column represents S.E. **P<0.01,
and Ca'-'
ketotifen
levels
the mean significantly
in rat
of 7-20 different
on substance peritoneal cells. from
P-induced mast
Vertical bars the control.
cells. in
(Fig. 6), while ketotifen did not have any significant effect. Verapamil, a calcium channel blocker, significantly inhibited 45Ca uptake at concentrations of 10-5 M or higher.
Fig. 5. Time course of substance P-induced 45Ca uptake into rat peritoneal mast cells. Each point represents the mean of 3 or 4 ex periments. Vertical bars indicate S.E. D: substance P-free, /: substance P, 3 x 10-6 M.
Fig. 6. Effects of emedastine, ketotifen and verapamil on substance P-induced 45Cauptake into rat peritoneal mast cells. Each point represents the mean of 5 experiments. Vertical bars indicate S.E. *P<0.05, **P<0.01, significantly different from the control. 0: emedastine, •: ketotifen, A: verapamil.
Effects on PPA in rats When antigen was intravenously injected to passively sensitized rats, the histamine concentration in the abdomi nal fluid was increased to 1.17 ± 0.25 pg/ml (n = 10) com pared to the concentration of 0.01 ± 0.01 ppg/ml (n = 8) in the group that had not received antigen injections, indicat ing that histamine was released into the abdominal cavity from peritoneal mast cells. Emedastine and DSCG slight ly inhibited PPA in rats at 30 mg/kg, p.o. and 10 mg/kg, i.v., respectively (Fig. 7).
Fig.
7.
Effects
anaphylaxis
of
emedastine
animals.
in rats.
Each
Vertical
different
from
Fig.
Effects
8.
anaphylaxis represents *P<0 .05,
bars
the
indicate
of emedastine mean
**P<0.01,
DSCG
on
represents S.E.
passive
the
peritoneal
mean
of
**P<0.01,
8 or
10
significantly
control.
in guinea the
and
column
pigs of
5-15
and treated
ketotifen with
animals.
significantly
on
passive
pyrilamine. Vertical
different
from
peritoneal
Each
bars
indicate
column S.E.
the control.
Effects on PPA in guinea pigs In a preliminary study, when antigen was intravenously injected into passively sensitized guinea pigs, all of the animals died of anaphylactic shock within 5 min. There fore, in the present study, guinea pigs were pretreated with pyrilamine to avoid anaphylactic death and to obtain the control value for the evaluation of the emedastine effect. This treatment prevented anaphylactic death of the animals, and inhibited the increase of histamine concen tration in the abdominal fluid; histamine concentrations of pyrilamine-nontreated group and pyrilamine-treated group were 92.0 -1:16.3 ng/ml (n =12) and 60.5 ± 12.6 ng/ml (n= 12), respectively. However, this effect was not
significant. When antigen was intravenously
injected into
guinea pigs treated with pyrilamine, histamine concentra tion in the abdominal fluid was increased to 58.6±9.0 ng/ml (n=:15) compared to the level of 4.3 ± 0.6 ng/ml (n= 15) in the group that had not received antigen injec tions. Emedastine produced significant inhibition of PPA at 0.03 mg/kg, p.o. (Fig. 8), while ketotifen did not in hibit it, even at 0.3 mg/kg, p.o. DISCUSSION Many factors, including allergic reactions, induce hista mine release from mast cells. Although type I hypersensi tive reactions mediated by IgE are thought to be the main reason for histamine release, proteins and peptides secret ed by activated eosinophils and neutrophils during the anaphylactic reaction are also responsible for the hista mine release from mast cells (13, 14). When the spreading of inflammation beyond the point of stimulation takes place via the axon reflex, neuropeptides, such as SP, secreted from nerve endings, induce histamine release from mast cells (1). In this study, we selected SP as a hista mine releaser, and we examined the effects of emedastine on histamine release. It has been reported that SP-induced histamine release was mediated by Ca2+ release from the intracellular Ca store in the absence of extracellular Ca 21 (2, 3). In fact, as shown in Figs. 2 and 3, SP elicits both histamine release and increase in cytoplasmic Ca 21 concentrations. On the other hand, in the presence of extracellular Ca2+, SP in creased 45Ca uptake into mast cells. These findings were consistent with data showing that SP-induced histamine release was mediated not only by Ca 2+ release from in tracellular Ca stores, but also by Ca 21 influx into mast cells (3). SP-induced Ca 2+ release from intracellular Ca stores occurred immediately after SP stimulation, and it was transient. On the other hand, the increase of SP-in duced 45Ca uptake into mast cells continued for 30 min. These results suggest that the early phase of SP-induced histamine release from mast cells was mediated by Ca" release from intracellular Ca stores, and the late phase was mediated by Ca 21 influx of extracellular Ca 21 into mast cells. Emedastine inhibited the SP-induced hista mine release and the increase of fluorescence intensity de rived from quin 2 chelated with intracellular Ca 2+ as well as SP-induced 45Ca uptake into mast cells. These results indicate that the inhibitory effects of emedastine on SP induced histamine release are related to the inhibition of Ca 2+ release from intracellular Ca stores as well as to the inhibition of Ca 21 influx into mast cells. Emedastine inhibited SP-induced histamine release at concentrations as low as 10-9 M in the presence of ex tracellular Ca 2+. The IC50 value of emedastine for anti
histaminic
activity is 6.1 x 10-9 M in vitro (7), and the
plasma level of emedastine was about 10-9 g/ml (1.9 X 10-9 M) when 2 mg of emedastine was administered repeatedly in humans (15). This indicates that emedastine elicits the inhibition of histamine release and shows anti histaminic effects at clinical dose levels. Hence, we exam ined PPA models in rats and guinea pigs to determine whether the same results for histamine release obtained in vitro could be obtained in vivo. In rats, emedastine showed slight inhibition of PPA at 30 mg/kg, p.o. To pre vent anaphylactic death, guinea pigs were pretreated with an antihistamine, pyrilamine, since pyrilamine, among many other antihistamines, is reported to have the least inhibitory effect on histamine release (16, 17). Pyrilamine at 0.3 mg/kg, i.v. prevented anaphylactic death in these animals due to its antihistaminic effects, and it gave the control value that is necessary for the evaluation of the emedastine effect. Pyrilamine was effective in inhibiting histamine release into the abdominal cavity. Emedastine exhibited an additive effect with pyrilamine in inhibiting the increase of histamine concentration, and this inhibi tory effect of emedastine became significant at 0.03 mg/kg, p.o. This was equivalent to the dose at which other type I allergic reactions were inhibited in guinea pigs (8, 18). Therefore, it was concluded that orally ad ministered emedastine was effective in preventing hista mine release in vivo. The effective dose of emedastine in rats was different from that in guinea pigs. Sakai et al. (19) reported that there were great species differences in the Cmax/dose and AUC/dose of emedastine, and they were due to the species difference in the first pass effect between rats and guinea pigs. Therefore, it was con sidered that the species difference in bioavailabilities of emedastine was one of the reasons for the difference in effective dose between rats and guinea pigs. From these results, we concluded that the clinical effi cacy of emedastine, an antiallergic agent, can be attribut ed to its inhibition of histamine release in addition to its antihistaminic effect. The mechanisms through which it in hibits histamine release are related to its significant inhibi tion of both Ca2+ release from intracellular Ca stores and Ca" influx into mast cells.
REFERENCES
1 Foreman, J.C. and Jordan, C.C.: Histamine release and vas cular changes induced by neuropeptides. Agents Actions 13, 105-116 (1983) 2 Tasaka, K., Mio, M. and Okamoto, M.: Intracellular calcium release induced by histamine releasers and its inhibition by some antiallergic drugs. Ann. Allergy 56, 464-469 (1986) 3 Fewtrell, C.M.S., Foreman, J.C., Jordan, C.C., Oehme, P., Renner, H. and Stewart, J.M.: The effects of substance P on histamine and 5-hydroxytryptamine release in the rat. J.
Physiol. (Lond.) 330, 393-411 (1982) Devillier, P., Renoux, M., Giroud, J.-P. and Regoli, D.: Pep tides and histamine release from rat peritoneal mast cells. Eur. J. Pharmacol. 117, 89-96 (1985) 5 Erjavec, F., Lembeck, F., Florjanc-Irman, T., Skofitsch, G., Donnerer, J., Saria, A. and Holzer, P.: Release of histamine by substance P. Naunyn Schmiedebergs Arch. Pharmacol. 317, 67-70 (1981) 6 Pearce, F.L., Kassessinoff, T.A. and Liu, W.L.: Characteris tics of histamine secretion induced by neuropeptides: Implica tions for the relevance of peptide-mast cell interactions in al lergy and inflammation. Int. Arch. Allergy Appl. Immunol. 88, 129-131 (1989) 7 Fukuda, T., Morimoto, Y., lemura, R., Kawashima, T., Tsukamoto, G. and Ito, K: Effect of 1-(2-ethoxyethyl)-2-(4 methyl-1-homopiperazinyl)benzimidazole difumarate (KB 2413), a new antiallergic, on chemical mediators. Arzneimittel forschung 34, 801-805 (1984) 8 Fukuda, T., Saito, T., Tajima, S., Shimohara, K. and Ito, K.: Antiallergic effect of 1-(2-ethoxyethyl)-2-(4-methyl-l-homo piperazinyl)benzimidazole difumarate (KB-2413). Arzneimittel forschung 34, 805-810 (1984) 9 Shore, P.A., Burkhalter, A. and Cohn, V.H.: A method for the fluorimetric assay of histamine in tissues. J. Pharmacol. Exp. Ther. 127, 182-186 (1959) 10 Spataro, A.C. and Bosmann, H.B.: Mechanisms of action of disodium cromoglycate-mast cell calcium ion influx after a his tamine-releasing stimulus. Biochem. Pharmacol. 25, 505-510 (1976) 11 Stotland, L.M. and Share, N.N.: Active bronchial anaphylaxis in the rat. Can. J. Physiol. Pharmacol. 52, 1114-1118 (1974) 12 Levine, B.B., Chang, H., Jr. and Vaz, N.M.: The production 4
13
14
15
16
17
18
of hapten-specific reaginic antibodies in guinea pig. J. Im munol. 106, 29-33 (1971) Zheutlin, L.M., Ackerman, S.J., Gleich, G.J. and Thomas, L.L.: Stimulation of basophil and rat mast cell histamine release by eosinophil granule-derived cationic proteins. J. Immunol. 133, 2180-2185 (1984) Henderson, W.R., Chi, E.Y. and Klevanoff, S.J.: Eosinophil peroxidase-induced mast cell secretion. J. Exp. Med. 152, 265-279 (1980) Nakajima, S., Tsutani, Y., Ohishi, M., Doi, Y., Yamazaki, K. and Ohba, Y.: Phase I study of emedastine difumarate (KG 2413). II. Single and consecutive administration studies using capsules containing sustained release granules of KG-2413. Clin. Rep. 23, 5431-5440 (1989) (in Japanese) Lichtenstein, L.M. and Gillespie, E.: The effects of the H, and H2 antihistamines on "allergic" histamine release and its inhibi tion by histamine. J. Pharmacol. Exp. Ther. 192, 441-450 (1975) Church, M.K. and Gradidge, C.F.: Inhibition of histamine release from human lung in vitro by antihistamines and related drugs. Br. J. Pharmacol. 69, 663-667 (1980) Saito, T., Nishimura, N., Tajima, S., Fukuda, T. and Ito, K.: Anti-allergic effects of 1-(2-ethoxyethyl)-2-(4-methyl-l-homo
piperazinyl)benzimidazole difumarate (KB-2413). Folia Pharma col. Japon. 89, 55-62 (1987) (Abs. in English) 19 Sakai, T., Hamada, T., Awata, N. and Watanabe, J.: Pharmacokinetics of an antiallergic agent, 1-(2-ethoxyethyl)-2 (hexahydro-4-methyl-lH--1,4-diazepin-l-yl)-1H-benzimidazole difumarate (KG-2413) after oral administration: Interspecies differences in rats, guinea pigs and dogs. J. Pharmacobiodyn. 12, 530-536 (1989)
Effects
Tadayuki
of Emedastine
Difumarate
Saito', Asako Hagihara', Naoko Igarashi', Naomi Matsuda', Keizo Ito', Mitsunobu Mio2 and Kenji Tasaka2 'New Drug Research Laboratories
2Department
of Pharmacology
inhibitory
January
13, 1993
effects of emedastine
Release
Akira Yamashita',
, Kanebo, Ltd., Osaka 534, Japan
, Faculty of Pharmaceutical Received
ABSTRACT-The
on Histamine
Sciences, Okayama Accepted
March
difumarate
University, Okayama
700, Japan
6, 1993
on histamine release were studied in rat
peritoneal mast cells. Emedastine significantly inhibited substance P (SP)-induced histamine release at con centrations above 10-9 M in the presence of extracellular Ca2+ and at concentrations above 10-" M in its absence. At concentrations of 10-8 M or higher, emedastine significantly inhibited SP-induced Ca" release from intracellular Ca stores and SP-induced 45Ca uptake into mast cells. Emedastine also inhibited passive peritoneal anaphylaxis in rats and guinea pigs. We conclude that the clinical antiallergic effects of emedastine involve the inhibition of histamine release and that this inhibition is mediated by the inhibition of Ca" release from intracellular Ca stores and the inhibition of Ca" influx into mast cells. Keywords:
Emedastine
difumarate,
Histamine
release,
Histamine release from mast cells is induced by various stimuli apart from IgE-mediated antigen-antibody reac tions. In particular, histamine release induced by neuro peptides such as substance P (SP) has been discussed ex tensively in recent years (1-6). Intracutaneous injection of histamine produces circumscribed edema, wheals and flares; an axon reflex model has been suggested for the spread of flares. Some histamine-induced orthodromic im pulses from sensory nerve endings spread as antidromic impulses through terminal arborizations. These anti dromic impulses cause the release of SP which stimu lates neighboring mast cells to release histamine (1). Emedastine difumarate is a new antiallergic drug which possesses an antihistaminic effect (7) and an inhibitory effect on IgE-mediated histamine release from rat peritoneal mast cells (8). In the present study, we exam ined the effects of emedastine on SP-induced histamine re lease from rat peritoneal mast cells. To elucidate the mechanism of histamine release inhibition induced by emedastine, we studied its effects on SP-induced Ca 21 release from intracellular Ca stores and 45Ca influx into mast cells. In addition, we also studied the effects of emedastine on passive peritoneal anaphylaxis (PPA) in rats and guinea pigs to clarify the effectiveness of this drug in preventing in vivo anaphylaxis in relation to the inhibition of histamine release.
Substance
P, Mast cell, Intracellular
MATERIALS
Ca 21
AND METHODS
Animals Male Wistar rats weighing 210 420 g and male or fe male Hartley guinea pigs weighing 300-400 g were used. The animals were housed in a temperature and humidity controlled room with free access to food and water. Drugs Emedastine and ketotifen were synthesized by Kanebo, Ltd. Disodium cromoglycate (DSCG) was extracted from Intal (Fujisawa Pharmaceutical Co., Ltd., Osaka) by Kanebo, Ltd. Verapamil was purchased from Sigma Chemical Co. (St. Louis, MO, USA). To study the effects of the test compounds in the presence of extracellular Ca2+ in vitro, emedastine and ketotifen were dissolved in physiological buffered solution (PBS; 154 mM NaCI, 2.7 mM KCI, 0.9 mM CaCl2i 5.6 mM glucose, 0.1076 bovine serum albumin, 5 mM N-2-hydroxyethylpiperazine-N'-2 ethanesulfonic acid, pH 7.4). Verapamil was dissolved in distilled water and diluted with PBS. To study the effects of test compounds in a Ca-free medium, Ca-free PBS con taining 0.1 mM ethylenediaminetetraacetic acid (EDTA) was prepared and substituted for PBS. For the in vivo stud ies, emedastine and ketotifen were dissolved in distilled water, and DSCG was dissolved in saline.
Histamine release from rat peritoneal mast cells Rats were exsanguinated, 25 ml of glucose-free PBS was injected into the abdominal cavity, and the abdomi nal wall was gently massaged. Fluid from the abdominal cavity was collected and centrifuged at 80 x g for 5 min at 4'C. The pellet was then washed with fresh glucose-free PBS and suspended in PBS. The cell suspensions (5 x 105 cells/ml) and test compounds were preincubated at 371C for 10 min, after which 0.1 ml of cell suspension was ad ded to 0.8 ml of each test compound solution or PBS as a control. After a 60-min incubation, 0.1 ml of SP was ad ded, and incubation was continued for another 10 min. The reaction was terminated by the addition of 1 ml of ice-cold PBS. After centrifugation, histamine in the super natant and residual histamine in the cell pellet were deter mined by spectrofluorimetric assay, according to the method of Shore et al. (9). In the study performed in the absence of extracellular Ca", Ca-free PBS containing 0.1 mM EDTA was substituted for PBS. Histamine re leased in the supernatant was expressed as a percentage of the total histamine content in the specimen. The spon taneous histamine release was 4.3-L0.8% (n =11) or 3.7±0.7% in the presence or absence of extracellular Ca", respectively. The percent inhibition was calculated as follows: Inhibition (%)=(C-D)/(C-S) x 100 C: SP-induced histamine release without drugs D: SP-induced histamine release with drugs S: spontaneous histamine release Fluorescence measurement of mast cells loaded with quin 2 Fluorescence measurement of mast cells loaded with quin 2 was carried out according to the method of Tasaka et al. (2). Rat peritoneal mast cells were harvested from the abdominal cavity and purified to a level higher than 87% by density gradient centrifugation with Percoll (Pharmacia Biotechnology AB, Uppsala, Sweden). The mast cells were suspended in PBS containing 10-5 M of quin 2/AM (Dojindo Laboratories, Kumamoto) and incu bated for 60 min at 37 1C, followed by washing with Ca free PBS. Mast cell suspension (0.4 ml) was then added to the microchamber, and test compounds were added; after 10 min of incubation, SP was added to the medium. Fluorescent changes derived from quin 2 chelated with in tracellular Ca' were measured as integrated values every 2 sec under a fluorescence microscope (XF, Nikon Corpo ration, Tokyo) connected to a video-intensified micros copy system (ARGUS-100, Hamamatsu Photonics, Hamamatsu). Results were expressed as a percentage of the fluorescence intensity determined before the addition of SP, and the maximum values detected in the control and drug-treated groups were compared. Fluorescence in
tensities were converted into pseudo-color black according to pixel intensity. 45Ca uptake into rat peritoneal
from white to
mast cells
The measurement of 45Ca uptake into rat peritoneal mast cells was carried out according to a modification of the method of Spataro and Bosmann (10). Rat peritoneal mast cells purified with Percoll density gradient centrifu gation were suspended in PBS. Mast cell suspension (0.35 ml, 2.86 x 105cells/ml) was preincubated at 371C for 10 min, after which 0.05 ml of 45CaC12(1 MBq/ml) was added; the suspension was then incubated for 10 min prior to the addition of 0.05 ml of test compound solu tion or PBS as a control. After a 10-min incubation, 0.05 ml of SP was added, and the suspension was incubated for another 10 min. The reaction was terminated by the addition of 0.5 ml of ice-cold PBS. The cells, collected by centrifugation at 340 x g for 5 min at 41C, were washed twice with ice-cold PBS. After solubilization of the cell pellet in 0.2 ml of PBS containing 10% Triton X-100, 2 ml of liquid scintillator (Scintisol EX-H, Wako Pure Chemical Industries, Ltd., Osaka) was added to 0.15 ml of the specimen. The radioactivity was measured by a liquid scintillation counter (LSC-1050, Aloka Co., Ltd., Mitaka). The percent inhibition was calculated as fol lows: Inhibition (%) _ (C D)/(C S) x 100 C: SP-induced 45Ca uptake without drugs D: SP-induced 45Ca uptake with drugs S: spontaneous 45Ca uptake Preparation of anti-egg albumin (EA) rat serum Anti-EA rat serum was prepared according to a modifi cation of the method of Stotland and Share (11). Rats were immunized by intracutaneous injection of 0.6 ml of saline containing 1 mg of EA, 2 mg of aluminum hydroxide gel, and 1010 killed Bordetella pertussis (Tohama strain phase I, Chiba Serum Institute, Ichi kawa) into the footpads. Five days after immunization, the animals were boosted with an intramuscular injection of 0.5 ml of 1 mg/ml EA. Sera were isolated from blood collected at 15 days after the first immunization. The IgE titer of the serum was examined by inducing 48-hour homologous passive cutaneous anaphylaxis, and the sera with levels of 1:256 or above were pooled and kept at 80'C until immediately before the experiment. PPA in rats Rats were passively sensitized by the intraperitoneal
in
jection of 3 ml anti-EA rat serum (1/12 dilution). After 48 hours, 1 ml of 5 mg/ml EA was intravenously injected. Immediately after that, 5 ml of Tyrode's solution (137 mM NaCl, 2.7 mM KCI, 1.8 mM CaC12i 1.0 MM MgC12,
12.0 mM NaHCO3, 0.4 mM NaH2PO4, and 5.6 mM glu cose) was intraperitoneally injected. The animals were killed 5 min after the antigen injection, and the peritoneal fluid was collected. After centrifugation at 400 x g for 5 min at 41C, histamine in the supernatant was determined by a spectrofluorimetric assay according to the method of Shore et al. (9). The test compounds were administered orally 60 min before the antigen injection. Preparation
of anti-benzylpenicilloyl
Effects on histamine release in the absence of extracellular Ca 2+ When rat peritoneal mast cells were exposed to 2 x 10-6 M of SP in the absence of extracellular Ca", histamine was released at 22.2 ± 2.9% (n = 9) of the total histamine content. Emedastine, at 10-11 M or higher, significantly inhibited histamine release (Fig. 2), while ketotifen produced significant inhibition only at 10-6 M or higher.
bovine r-globulin
(BPO •BGG) guinea pig serum Anti-BPO . BGG guinea pig serum was prepared accord ing to the method of Levine et al. (12). Female guinea pigs were immunized intraperitoneally by repeated injec tions (twice a month, a total of 15-21 times) of 1 ml of 0.1070aluminum hydroxide gel which contained 2 pg/ml of BPO • BGG as antigen. Sera were isolated from blood collected at 10 days after the last immunization. The IgE titer of the serum was examined by inducing 7-day homologous passive cutaneous anaphylaxis; sera with levels of 1:512 or above were pooled and kept at 801C until immediately before the experiment. PPA in guinea pigs Male guinea pigs were passively sensitized with intra peritoneal injection of 3 ml anti-BPO • BGG serum (1/ 100 dilution). After 7 days, 0.3 mg/kg of pyrilamine was in jected intravenously, followed 5 min later by the in travenous injection of 1 ml of BPO. BSA (protein concen tration, 0.2 mg/ml). Immediately after that, 10 ml of Tyrode's solution was intraperitoneally injected. The animals were killed 5 min after the antigen injection, and peritoneal 400 x g for determined method of ministered
Fig. 1. Effects of emedastine and ketotifen histamine release from rat peritoneal mast extracellular Cat+. Each point represents experiments. Vertical bars indicate S.E. significantly different from the control. ketotifen.
on substance P-induced cells in the presence of the mean of 5 or 6 *P<0.05, **P<0.01, 0: emedastine, •:
Fig. 2. Effects of emedastine and ketotifen histamine release from rat peritoneal mast extracellular Ca 21. Each point represents experiments. Vertical bars indicate S.E. significantly different from the control. ketotifen.
on substance P-induced cells in the absence of the mean of 4 or 5 *P<0.05, **P<0.01, 0: emedastine, •:
fluid was collected. After centrifugation at 5 min at 41C, histamine in the supernatant was by spectrofluorimetric assay according to the Shore et al. (9). The test compounds were ad orally 60 min before the antigen injection.
Statistics The results were expressed as the mean±S.E. A one way analysis of variance with Dunnett's test and a paired t-test were used to determine statistical significance. RESULTS Effects on histamine release in the presence of extracellu lar Ca" When rat peritoneal mast cells were exposed to 3 x 10-6 M of SP in the presence of extracellular Ca", histamine was released at 27.3 ±2.1076 (n=11) of the total histamine content. Emedastine, at 10-9 M or higher, produced sig nificant inhibition of histamine release (Fig. 1), while ketotifen did not have any significant effect.
Fig. 3. Pseudo-color conversion of the fluorescence images of quin 2-loaded mast cells before (a and c) and 2 sec after (b and d) exposure to substance P. (a) and (b): control mast cells. (c) and (d): mast cells pretreated with emedastine.
Effects on increase of intracellular Ca" concentration When mast cells loaded with quin 2 were exposed to 2 x 10-6 M of SP in the absence of extracellular Cat+, the
fluorescence intensity of these cells was increased transient ly at 2 4 sec after SP stimulation and returned to the basal level by 10 sec after SP stimulation, indicating Ca" release from intracellular Ca stores. Emedastine, at 10-8 M, significantly inhibited the fluorescence increase at 2-4 sec after SP stimulation (Figs. 3 and 4), while ketotifen did not show any significant effect at this concentration. Effects on 45Ca uptake into rat peritoneal mast cells When mast cells were exposed to 3 x 10-6 M of SP, 45Ca uptake into mast cells was increased in a time -de pendent manner (Fig. 5). The increase of 45Ca uptake was continued for at least 30 min. In the following study, mast cells were incubated for 10 min after SP stimulation. SP (3 x 10-6 M) increased 45Ca uptake by 1272 ± 236 cpm (n = 5) compared to the value of 139 ± 34 cpm (n = 5) in the control group. Emedastine inhibited the SP-induced increase of 45Ca uptake in a concentration-dependent manner at concentrations ranging from 10-8 to 10-5 M
Fig.
4.
Effects
increases Each dicate
of
of emedastine
intracellular
column represents S.E. **P<0.01,
and Ca'-'
ketotifen
levels
the mean significantly
in rat
of 7-20 different
on substance peritoneal cells. from
P-induced mast
Vertical bars the control.
cells. in
(Fig. 6), while ketotifen did not have any significant effect. Verapamil, a calcium channel blocker, significantly inhibited 45Ca uptake at concentrations of 10-5 M or higher.
Fig. 5. Time course of substance P-induced 45Ca uptake into rat peritoneal mast cells. Each point represents the mean of 3 or 4 ex periments. Vertical bars indicate S.E. D: substance P-free, /: substance P, 3 x 10-6 M.
Fig. 6. Effects of emedastine, ketotifen and verapamil on substance P-induced 45Cauptake into rat peritoneal mast cells. Each point represents the mean of 5 experiments. Vertical bars indicate S.E. *P<0.05, **P<0.01, significantly different from the control. 0: emedastine, •: ketotifen, A: verapamil.
Effects on PPA in rats When antigen was intravenously injected to passively sensitized rats, the histamine concentration in the abdomi nal fluid was increased to 1.17 ± 0.25 pg/ml (n = 10) com pared to the concentration of 0.01 ± 0.01 ppg/ml (n = 8) in the group that had not received antigen injections, indicat ing that histamine was released into the abdominal cavity from peritoneal mast cells. Emedastine and DSCG slight ly inhibited PPA in rats at 30 mg/kg, p.o. and 10 mg/kg, i.v., respectively (Fig. 7).
Fig.
7.
Effects
anaphylaxis
of
emedastine
animals.
in rats.
Each
Vertical
different
from
Fig.
Effects
8.
anaphylaxis represents *P<0 .05,
bars
the
indicate
of emedastine mean
**P<0.01,
DSCG
on
represents S.E.
passive
the
peritoneal
mean
of
**P<0.01,
8 or
10
significantly
control.
in guinea the
and
column
pigs of
5-15
and treated
ketotifen with
animals.
significantly
on
passive
pyrilamine. Vertical
different
from
peritoneal
Each
bars
indicate
column S.E.
the control.
Effects on PPA in guinea pigs In a preliminary study, when antigen was intravenously injected into passively sensitized guinea pigs, all of the animals died of anaphylactic shock within 5 min. There fore, in the present study, guinea pigs were pretreated with pyrilamine to avoid anaphylactic death and to obtain the control value for the evaluation of the emedastine effect. This treatment prevented anaphylactic death of the animals, and inhibited the increase of histamine concen tration in the abdominal fluid; histamine concentrations of pyrilamine-nontreated group and pyrilamine-treated group were 92.0 -1:16.3 ng/ml (n =12) and 60.5 ± 12.6 ng/ml (n= 12), respectively. However, this effect was not
significant. When antigen was intravenously
injected into
guinea pigs treated with pyrilamine, histamine concentra tion in the abdominal fluid was increased to 58.6±9.0 ng/ml (n=:15) compared to the level of 4.3 ± 0.6 ng/ml (n= 15) in the group that had not received antigen injec tions. Emedastine produced significant inhibition of PPA at 0.03 mg/kg, p.o. (Fig. 8), while ketotifen did not in hibit it, even at 0.3 mg/kg, p.o. DISCUSSION Many factors, including allergic reactions, induce hista mine release from mast cells. Although type I hypersensi tive reactions mediated by IgE are thought to be the main reason for histamine release, proteins and peptides secret ed by activated eosinophils and neutrophils during the anaphylactic reaction are also responsible for the hista mine release from mast cells (13, 14). When the spreading of inflammation beyond the point of stimulation takes place via the axon reflex, neuropeptides, such as SP, secreted from nerve endings, induce histamine release from mast cells (1). In this study, we selected SP as a hista mine releaser, and we examined the effects of emedastine on histamine release. It has been reported that SP-induced histamine release was mediated by Ca2+ release from the intracellular Ca store in the absence of extracellular Ca 21 (2, 3). In fact, as shown in Figs. 2 and 3, SP elicits both histamine release and increase in cytoplasmic Ca 21 concentrations. On the other hand, in the presence of extracellular Ca2+, SP in creased 45Ca uptake into mast cells. These findings were consistent with data showing that SP-induced histamine release was mediated not only by Ca 2+ release from in tracellular Ca stores, but also by Ca 21 influx into mast cells (3). SP-induced Ca 2+ release from intracellular Ca stores occurred immediately after SP stimulation, and it was transient. On the other hand, the increase of SP-in duced 45Ca uptake into mast cells continued for 30 min. These results suggest that the early phase of SP-induced histamine release from mast cells was mediated by Ca" release from intracellular Ca stores, and the late phase was mediated by Ca 21 influx of extracellular Ca 21 into mast cells. Emedastine inhibited the SP-induced hista mine release and the increase of fluorescence intensity de rived from quin 2 chelated with intracellular Ca 2+ as well as SP-induced 45Ca uptake into mast cells. These results indicate that the inhibitory effects of emedastine on SP induced histamine release are related to the inhibition of Ca 2+ release from intracellular Ca stores as well as to the inhibition of Ca 21 influx into mast cells. Emedastine inhibited SP-induced histamine release at concentrations as low as 10-9 M in the presence of ex tracellular Ca 2+. The IC50 value of emedastine for anti
histaminic
activity is 6.1 x 10-9 M in vitro (7), and the
plasma level of emedastine was about 10-9 g/ml (1.9 X 10-9 M) when 2 mg of emedastine was administered repeatedly in humans (15). This indicates that emedastine elicits the inhibition of histamine release and shows anti histaminic effects at clinical dose levels. Hence, we exam ined PPA models in rats and guinea pigs to determine whether the same results for histamine release obtained in vitro could be obtained in vivo. In rats, emedastine showed slight inhibition of PPA at 30 mg/kg, p.o. To pre vent anaphylactic death, guinea pigs were pretreated with an antihistamine, pyrilamine, since pyrilamine, among many other antihistamines, is reported to have the least inhibitory effect on histamine release (16, 17). Pyrilamine at 0.3 mg/kg, i.v. prevented anaphylactic death in these animals due to its antihistaminic effects, and it gave the control value that is necessary for the evaluation of the emedastine effect. Pyrilamine was effective in inhibiting histamine release into the abdominal cavity. Emedastine exhibited an additive effect with pyrilamine in inhibiting the increase of histamine concentration, and this inhibi tory effect of emedastine became significant at 0.03 mg/kg, p.o. This was equivalent to the dose at which other type I allergic reactions were inhibited in guinea pigs (8, 18). Therefore, it was concluded that orally ad ministered emedastine was effective in preventing hista mine release in vivo. The effective dose of emedastine in rats was different from that in guinea pigs. Sakai et al. (19) reported that there were great species differences in the Cmax/dose and AUC/dose of emedastine, and they were due to the species difference in the first pass effect between rats and guinea pigs. Therefore, it was con sidered that the species difference in bioavailabilities of emedastine was one of the reasons for the difference in effective dose between rats and guinea pigs. From these results, we concluded that the clinical effi cacy of emedastine, an antiallergic agent, can be attribut ed to its inhibition of histamine release in addition to its antihistaminic effect. The mechanisms through which it in hibits histamine release are related to its significant inhibi tion of both Ca2+ release from intracellular Ca stores and Ca" influx into mast cells.
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