Clin. exp. Immunol. (1975) 21, 419-429.
INHIBITION OF IMMEDIATE HYPERSENSITIVITY REACTIONS IN THE RAT BY DISODIUM CROMOGLYCATE AND A NITROINDANEDIONE BARBARA A. SPICER, JANET W. ROSS AND H. SMITH Beecham Pharmaceuticals, Research Ditvision, Chemotherapeutic Research Centre, Brockham Park, Betchworth, Surrey (Received 24 March 1975) SUMMARY
A nitroindanedione (BRL 10833) and disodium cromoglycate (DSCG) showed similar activities as inhibitors of IgE-mediated passive cutaneous anaphylaxis (PCA) and passive peritoneal anaphylaxis (PPA) reactions in the rat. BRL 10833 was more active than DSCG when given parenterally and unlike DSCG it inhibited the PCA reaction in the rat after oral administration. In the PCA test both compounds produced a state of refractoriness both to themselves and to each other. Isoprenaline also inhibited the PCA reaction but its activity was not reduced when the rats were refractory to DSCG.
INTRODUCTION Disodium cromoglycate (DSCG) is effective in the treatment of some types of asthma. The reasons for its effectiveness are not fully known and it is therefore difficult to assess the reliability of screening procedures designed to detect other compounds which might give similar protection to asthmatics. Screens using primates or primate tissues, especially human tissues, would be desirable and DSCG has been reported to protect passively or spontaneously sensitized monkeys against antigen provoked bronchoconstriction (Cox et al., 1970; Patterson, Talbot & Brandfonbreuer, 1971) but the procedures involved are expensive and technically difficult. DSCG will inhibit the antigen-induced release of spasmogens from fragments of passively sensitized human lung (Assem & Schild, 1969) and monkey lung (Assem & Mongar, 1970), but when compared with other drugs, such as the sympathomimetic amines, DSCG is not very active in these systems. For example, in the human lung system DSCG was a million times less potent than isoprenaline in inhibiting histamine release (Assem & Schild, 1969), and the inhibition given by DSCG was not dose-dependent (Orange & Austen, 1971b). It has been reported that DSCG does not inhibit the release of histamine from actively sensitized human leucocytes by challenge with antigen (Lichtenstein & Atkinson, 1969). Correspondence: Mr H. Smith, Beecham Pharmaceuticals, Research Division, Chemotherapeutic Research Centre, Brockham Park, Betchworth, Surrey RH3 7AJ.
419
420
Barbara A. Spicer, Janet W. Ross and H. Smith
DSCG seems to be most reliably active in test systems which make use of the rat or of rat tissue. Passive cutaneous anaphylaxis (PCA) reactions in the rat mediated by rat IgE are inhibited by DSCG and the system has been used to screen compounds for similar activities (Cairns et al., 1972). Unfortunately, the test is not very specific and again some sympathomimetic amines, including isoprenaline, showed greater activity than DSCG (Ankier, 1971). However, when rat IgE was used to passively sensitize the peritoneal cavity of the rat DSCG was the only drug, amongst a series studied including isoprenaline, which inhibited the, antigen-induced release of histamine (Orange & Austen, 1971 a). DSCG inhibited the release of histamine from passively sensitized rat peritoneal mast cells and was most active if added simultaneously with antigen. If added before antigen a refractory state was produced so that DSCG added subsequently with antigen showed reduced activity (Kusner, Rubnick & Herzig, 1973). A similar state of refractoriness was produced in it'oil in the rat PCA test (Thomson & Evans, 1973). We wish to show that a nitroindanedione, the sodium salt of 5,6-dimethyl-2-nitroindane1,3-dione, BRL 10833 (Fig. 1) and DSCG show similar activities in the rat PCA and the rat passive peritoneal anaphylaxis (PPA) systems and that in the rat PCA test both compounds can produce a state of refractoriness not only to themselves but also to each other. We have also attempted to see if isoprenaline is active in the PCA test when the rats are refractory to DSCG.
] 1l 3
F~~N02Na
H/
FIG. 1. The sodium salt of 5,6-dimethyl-2-nitroindane-1,3-dione, BRL 10833.
MATERIALS AND METHODS Antigen The antigen used was chicken egg albumin (ovalbumin, crystallized and lyophilized Sigma Grade III).
Compounds tested DSCG (Fisons Pharmaceuticals) was administered to rats dissolved in an isotonic solution of saline buffered with 0 05 M, pH 7-2, Sorenson buffer (PBS) (Bacto Hemagglutination buffer, Difco Laboratories). Isoprenaline sulphate (Burroughs Wellcome) was dissolved in PBS and used immediately. The sodium salt of 5,6-dimethyl-2-nitroindane-1,3-dione (BRL 10833) (Fig. 1) was prepared in our laboratories by Dr D. R. Buckle (Buckle et al., 1973). BRL 10833 was administered in PBS except that at concentrations above 2 mg/ml it was suspended in a 1: 1 mixture of PBS and 10% methyl cellulose in distilled water.
Hypersensitivity reactions in the rat
421
Antiserum Serum containing heat-labile homocytotropic antibody was raised in rats to ovalbumin by a method similar to that described by Mota (1964). Male Wistar rats of 250-300 g were given intraperitoneal injections of 0 5 ml of Bordetella pertussis vaccine (4 x 1010 organisms/ ml; Burroughs Wellcome, London) and subcutaneous injections of 0 5 ml of an emulsion of 100 mg of ovalbumin in 2 ml of isotonic saline and 3 ml of Freund's incomplete adjuvant. -The rqts were bled by cardiac puncture on day 18, the blood was pooled and the serum separated, stored at -20'C and thawed only once before use. The serum produced PCA activity in recipient rats down to a dilution of 1:32 to 1:64 after 72 hr and persisted for several days. This activity was lost to a dilution of 1:2 by heating at 560 for 4 hr even when the sensitizing period was reduced to 4 hr. Passive cutaneous anaphylaxis This was carried out by a method based on that of Ovary & Bier (1952) as modified by
Goose & Blair (1969). Male Wistar rats of 250-300 g were given 0-1 ml of each of six two-fold serial dilutions of the antiserum in 0.900 saline, injected intradermally into separate sites on their shaved backs. Seventy-two hours later the animals were challenged by intravenous injection of 0 3 ml of a 100 solution of ovalbumin in PBS mixed with 0-2 ml of a 500 solution of pontamine sky-blue (6 BX. C.I. 24410, Raymond A. Lamb, London) in isotonic saline. The rats were killed after 20 min and the diameter of the blue wheals at the antibody injection sites were measured on the outer surface of the skin. The starting dilution of the serum was adjusted so that there was no response, after challenge, at the injection site of the highest dilution and a maximum response at the two lowest dilutions. Typically, six two-fold serial dilutions of the serum from 1:4 to 1: 128 were used.
Inhibition of PCA Compounds were tested for their ability to reduce the diameter of the wheals at the three intradermal sites which in control animals gave less than maximum response. Wheals were usually symmetrical, but when not, the mean of the largest and smallest diameters was used. Each dose of the compound was administered to an animal at a measured time prior to intravenous challenge with ovalbumin. Control groups of six animals were given the same volume of carrier fluid at the same time prior to challenge. Rats were starved for 15 hr prior to giving compounds or carrier fluid orally. The results were calculated as follows: percentage inhibition of PCA = 100 (1 - a/b), where a = the sum of the diameters of the wheals produced in the test animal at the three sites of antibody dilutions which in control animals gave less than maximum response, and b = the mean sum of the diameters of the wheals produced in the control group of animals at the antibody sites which gave less than maximum response. A typical variation in a control group of six animals gave an s.e.m. of +6% Passive peritoneal anaphylaxis The method used was based on that of Orange, Stechschulte & Austen (1970) with the difference that blue dye was injected intravenously into the rats just prior to the intraperitoneal challenge with antigen. The subsequent leakage of blue dye into the peritoneal cavity was used as a measure of extravasation.
422
Barbara A. Spicer, Janet W. Ross and H. Smith
Male Wistar rats of 250-300 g were given intraperitoneal injections of 2 ml of a 1:5 dilution of the rat antiserum containing rat IgE. Two hours later 0 3 ml of PBS saturated with Evans Blue (G. T. Gurr, London) was injected intravenously followed by intraperitoneal injection of 1 ml of a solution of a test compound in saline or of saline. Thirty seconds later the animals were challenged with an intraperitoneal injection of 5 ml of Tyrode's solution containing 0 4 mg/ml of ovalbumin and 50 pg/ml of heparin. Five minutes after challenge the rats were stunned and bled. The peritoneal fluid was collected by opening the peritoneal, cavity over a funnel into polycarbonate tubes in ice. The supernatants were separated from the cellular residue by centrifuging at 150 g for 5 min, and retained for estimation of the amounts of dye, histamine and SRS-A. Dye assay The optical densities (OD) of the supernatants which were not contaminated with blood were read immediately at 615 nm. The supernatants were then placed in a boiling water bath for 5 min and stored frozen at - 20'C until it was convenient to assay for histamine and SRS-A. The optical density was taken as a measure of the concentration of blue dye leaked into the peritoneal cavity. Histamine assay Histamine was assayed by bio-assay or spectrofluorimetrically. In the bio-assay histamine was assayed on the isolated guinea-pig ileum preparation in the presence of 5 x 10-7 M atropine. Two concentrations of the unknown were bracketed between three concentrations of standard. For the spectrofluorimetric assay an automated system was used similar to that described by Evans, Lewis & Thomson (1973) using the Technicon Autoanalyser system. At the concentrations used the compounds tested did not interfere with the assays.
SRS-A assay
Slow-reacting substance of anaphylaxis was assayed on the isolated guinea-pig ileum preparation in the presence of atropine (5 x 1O -M) and mepyramine maleate (10-6 M), the latter to abolish the histamine response (Brocklehurst, 1960). The term SRS-A was used to designate the material which gave a slow contraction of the guinea-pig ileum under the conditions described. Samples from test animals receiving a drug and from control animals given no drug were assayed on the same piece of prepared guinea-pig ileum and the mean of the SRS-A concentration of the samples from the control group was taken as 100% and the SRS-A concentrations were expressed relative to this. At the concentrations used the compounds tested did not interfere with the assay. Statistics
Probability values throughout were assessed by the Student's t-test. RESULTS Inhibition of Rat PCA We routinely determine the activities of compounds in the rat PCA test by injecting them subcutaneously prior to challenge with antigen. The time between administration of the compound and challenge with antigen is critical. DSCG, BRL 10833 and isoprenaline
423
Hypersensitivity reactions in the rat
a_ c (
.2 D .r_
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I 0
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2
I
I
30 40 30 0 10 20 30 0 10 20 20 10 Time (min) between s.c. administration of the drug and i.v. antigen challenge
Fig 2
c
.
100 a1)
90 80 70
60
(12)
50
1(12)
40 30
20
(6)
10 -
0-001
0-01
1.0
0-1
10
100
Dose (mg / kg) Fig. 3
FIG. 2. Inhibition of rat PCA by drugs given subcutaneously at various times before i.v. antigen challenge: (a) DSCG (20 mg/kg); (b) BRL 10833 (0 5 mg/kg); (c) isoprenaline (0-02 mg/kg). The results are expressed as the mean ± s.e.m. The numbers in parentheses refer to the number
of rats used at that dose. FIG. 3. Inhibition of rat PCA by different compounds. Isoprenaline (0), BRL 10833 (0) and DSCG (-) were given subcutaneously 10 min before antigen challenge and BRL 10833 was also given orally 15 min before challenge (L). The results are expressed as the mean ± s.e.m. The numbers in parentheses refer to the number of rats used at that dose.
showed highest activity if given subcutaneously 10 min before antigen challenge (Fig. 2), and the rank order of potency of the compounds was isoprenaline > BRL 10833 > DSCG. BRL 10833 inhibited the rat PCA test when given orally and showed more activity by this
424
Barbara A. Spicer, Janet W. Ross and H. Smith
route than did DSCG when the latter was given subcutaneously (Fig. 3). No inhibition of the test was found after giving DSCG orally at doses up to 100 mg/kg and at times up to 30 min before antigen challenge. Cross-reacting tachyphylaxis It has been shown that a large intravenous dose of DSCG (40 mg/kg) produces a marked inhibition in the rat PCA test if given with antigen but that this activity was reduced with, increasing time between drug administration and challenge (Thomson & Evans, 1973). We have confirmed this and have found that a similar effect was obtained with BRL 10833 (Table 1). TABLE 1. Inhibition of rat PCA after a large dose of drugs given at various times before antigen challenge
Drug BRL 10833 (2 mg/kgi.v.)
DSCG (40 mg/kg i.v.)
Time (min) between dose and antigen challenge
Percentage inhibition of rat PCA (Mean (six rats) ± s.e.m.)
0 15 30 60 0 20 30 40 60
78± 5-3 38±11 1 13± 52 14± 4-6 100± 0 31± 10-8 8± 8-3 4± 2-3 2+ 7-4
Thomson & Evans (1973) also found that, although a dose of DSCG of 40 mg/kg given intravenously 30 min before challenge produced little inhibition of the rat PCA test, it reduced the inhibitory activity of the same dose of DSCG given just before challenge. We investigated the effects of pre-dosing intravenously with this dose of DSCG or with a dose of 2 mg/kg of BRL 10833 given 30 min before challenge on the inhibition produced by intravenous dosing with DSCG and BRL 10833 given just before challenge using those doses which normally give good inhibition (Figs 4 and 5). The amount of inhibition produced by those doses of DSCG which normally produce well over 50% inhibition was markedly reduced not only by pre-dosing with DSCG but also by pre-dosing with BRL 10833 (Fig. 4). A similar reduction in activity normally shown by BRL 10833 was obtained by pre-dosing with BRL 10833 or with DSCG (Fig. 5). Predosing with DSCG produced little effect on the dose response curve of isoprenaline (Table 2). Rat passive peritoneal anaphylaxis (PPA)
Negative controls. Under the conditions of the test there was very little leakage of histamine, SRS-A or of blue dye into the peritoneal cavity of rats either sensitized with serum containing rat IgE and injected with carrier fluid instead of antigen or treated with normal rat serum and challenged with antigen.
425
Hypersensitivity. reactions in the rat 80
70 U 0-
.5 60 0 c 0
50
z
40
.rQ) ol
2 c
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p
30 20
(6)
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10
1*0 Dose of DSCG (mg/kg) Fig. 4
0.1
90
(I1)
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70
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I)
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Fig.5 FIG. 4. Inhibition of rat PCA by DSCG when given intravenously just prior to antigen challenge and 30 min after i.v. administration of saline (0), DSCG (40 mg/kg) (0) and BRL 10833 (2 mg/kg) (A). The results are given as the mean ± s.e.m. The numbers in parentheses refer to the number of rats used at that dose. FIG. 5. Inhibition of rat PCA by BRL 10833 when given intravenously just prior to antigen challenge and 30 min after i.v. administration of saline (o), DSCG (40 mg/kg) (A) and BRL 10833 (2 mg/kg) (A). The results are given as the mean+ s.e.m. The numbers in parentheses refer to the number of rats used at that dose.
Inhibition of ratPPA. BRL 10833 at a concentration of 2 10-6 M in the peritoneal cavity showed a similar type and order of activity to those shown by DSCG at a concentration of 2 x 10- 5 M. At these concentrations both compounds produced more than a 9000 inhibition of histamine release, but were less effective at the inhibition of SRS-A release. Isoprenaline at 2 x 10- 6 M produced no significant inhibition of histamine release. Peritoneal fluids collected from non-challenged rats treated with isoprenaline at 2 10-6 M interfered with the assay for SRS-A. All three compounds produced a similar inhibition of blue dye leakage. x
x
426
Barbara A. Spicer, Janet W. Ross and H. Smith TABLE 2. Inhibition of rat PCA by isoprenaline when given just prior to antigen challenge and 30 min after administration of saline or DSCG
Percentage inhibition of rat PCA mean (five or six rats) + s.e.m. Dose of isoprenaline (mg/kg i.v.) 0 00025 0005 001 002
Predose saline (i.v.)
Predose DSCG (40 mg/kg i.v.) -
22 + 74 39+59 62+6 2 63+68
1*+8-4 45 + 90
50±67 69±5-8 72+45
* Enhanced histamine release, not significant. TABLE 3. Rat passive peritoneal anaphylaxis Rats sensitized with serum containing antigen-specific rat IgE (no challenge)*
Rats treated with normal rat serum (challenged with antigen)
Histamine
SRS-A
Blue dye
Histamine
SRS-A
Blue dye
2-7+0 5
0 4±0 1
9-2+ 1-3
34±06
05+0 1
14-1+1-6
The results are expressed as the mean ± s.e.m. for groups of six rats of the concentrations in peritoneal fluid as percentages of that in sensitized and challenged controls. * Injected with carrier fluid.
These were not significantly different from each other but were significantly different from controls. A concentration of isoprenaline of 2 x 10-3 M in the peritoneal cavity produced a significant inhibition of histamine release. DISCUSSION
DSCG is not absorbed to any extent after oral administration to the rat (Moss et al., 1970) or to man (Walker et al., 1972) and for therapeutic use it has to be given by inhalation. Relatively large amounts have to be inhaled, which can only be achieved by the insufflation of a powder and this limits the dose. After insufflation it is rapidly cleared both from the lungs and the blood (Walker et al., 1972). A compound with greater activity than DSCG and with more favourable absorption characteristics might be more effective clinically. It is possible that the clinical effectiveness of DSCG is connected with its ability to reduce the release of spasmogens induced by reaction between antigen and reagin. BRL 10833 was
Hypersensitivity reactions in the rat
427
TABLE 4. Rat passive peritoneal anaphylaxis. Effects of DSCG BRL 10833 and isoprenaline
Concentration of drug
(M)* Control rats No drug DSCG (2x 10-5) BRL 10833 (2x 10-6) Isoprenaline (2x 10-6) 2x10-5 2x 10-4 2 x 10-3
Concentrations in peritoneal fluids collected 5 min after antigen challenge, as percentage of mean concentration in fluids from control rats ± s.e.m. (number of rats)t
Histamine
SRS-A
OD 625 nm
100±12-2 (13)
100±15-5 (11)
100± 5 3 (13)
6-2+ 1 4 (14) P0.05 93 4+ 6-2 (7) P>0-05 50 0+ 6 0 (6) P
INHIBITION OF IMMEDIATE HYPERSENSITIVITY REACTIONS IN THE RAT BY DISODIUM CROMOGLYCATE AND A NITROINDANEDIONE BARBARA A. SPICER, JANET W. ROSS AND H. SMITH Beecham Pharmaceuticals, Research Ditvision, Chemotherapeutic Research Centre, Brockham Park, Betchworth, Surrey (Received 24 March 1975) SUMMARY
A nitroindanedione (BRL 10833) and disodium cromoglycate (DSCG) showed similar activities as inhibitors of IgE-mediated passive cutaneous anaphylaxis (PCA) and passive peritoneal anaphylaxis (PPA) reactions in the rat. BRL 10833 was more active than DSCG when given parenterally and unlike DSCG it inhibited the PCA reaction in the rat after oral administration. In the PCA test both compounds produced a state of refractoriness both to themselves and to each other. Isoprenaline also inhibited the PCA reaction but its activity was not reduced when the rats were refractory to DSCG.
INTRODUCTION Disodium cromoglycate (DSCG) is effective in the treatment of some types of asthma. The reasons for its effectiveness are not fully known and it is therefore difficult to assess the reliability of screening procedures designed to detect other compounds which might give similar protection to asthmatics. Screens using primates or primate tissues, especially human tissues, would be desirable and DSCG has been reported to protect passively or spontaneously sensitized monkeys against antigen provoked bronchoconstriction (Cox et al., 1970; Patterson, Talbot & Brandfonbreuer, 1971) but the procedures involved are expensive and technically difficult. DSCG will inhibit the antigen-induced release of spasmogens from fragments of passively sensitized human lung (Assem & Schild, 1969) and monkey lung (Assem & Mongar, 1970), but when compared with other drugs, such as the sympathomimetic amines, DSCG is not very active in these systems. For example, in the human lung system DSCG was a million times less potent than isoprenaline in inhibiting histamine release (Assem & Schild, 1969), and the inhibition given by DSCG was not dose-dependent (Orange & Austen, 1971b). It has been reported that DSCG does not inhibit the release of histamine from actively sensitized human leucocytes by challenge with antigen (Lichtenstein & Atkinson, 1969). Correspondence: Mr H. Smith, Beecham Pharmaceuticals, Research Division, Chemotherapeutic Research Centre, Brockham Park, Betchworth, Surrey RH3 7AJ.
419
420
Barbara A. Spicer, Janet W. Ross and H. Smith
DSCG seems to be most reliably active in test systems which make use of the rat or of rat tissue. Passive cutaneous anaphylaxis (PCA) reactions in the rat mediated by rat IgE are inhibited by DSCG and the system has been used to screen compounds for similar activities (Cairns et al., 1972). Unfortunately, the test is not very specific and again some sympathomimetic amines, including isoprenaline, showed greater activity than DSCG (Ankier, 1971). However, when rat IgE was used to passively sensitize the peritoneal cavity of the rat DSCG was the only drug, amongst a series studied including isoprenaline, which inhibited the, antigen-induced release of histamine (Orange & Austen, 1971 a). DSCG inhibited the release of histamine from passively sensitized rat peritoneal mast cells and was most active if added simultaneously with antigen. If added before antigen a refractory state was produced so that DSCG added subsequently with antigen showed reduced activity (Kusner, Rubnick & Herzig, 1973). A similar state of refractoriness was produced in it'oil in the rat PCA test (Thomson & Evans, 1973). We wish to show that a nitroindanedione, the sodium salt of 5,6-dimethyl-2-nitroindane1,3-dione, BRL 10833 (Fig. 1) and DSCG show similar activities in the rat PCA and the rat passive peritoneal anaphylaxis (PPA) systems and that in the rat PCA test both compounds can produce a state of refractoriness not only to themselves but also to each other. We have also attempted to see if isoprenaline is active in the PCA test when the rats are refractory to DSCG.
] 1l 3
F~~N02Na
H/
FIG. 1. The sodium salt of 5,6-dimethyl-2-nitroindane-1,3-dione, BRL 10833.
MATERIALS AND METHODS Antigen The antigen used was chicken egg albumin (ovalbumin, crystallized and lyophilized Sigma Grade III).
Compounds tested DSCG (Fisons Pharmaceuticals) was administered to rats dissolved in an isotonic solution of saline buffered with 0 05 M, pH 7-2, Sorenson buffer (PBS) (Bacto Hemagglutination buffer, Difco Laboratories). Isoprenaline sulphate (Burroughs Wellcome) was dissolved in PBS and used immediately. The sodium salt of 5,6-dimethyl-2-nitroindane-1,3-dione (BRL 10833) (Fig. 1) was prepared in our laboratories by Dr D. R. Buckle (Buckle et al., 1973). BRL 10833 was administered in PBS except that at concentrations above 2 mg/ml it was suspended in a 1: 1 mixture of PBS and 10% methyl cellulose in distilled water.
Hypersensitivity reactions in the rat
421
Antiserum Serum containing heat-labile homocytotropic antibody was raised in rats to ovalbumin by a method similar to that described by Mota (1964). Male Wistar rats of 250-300 g were given intraperitoneal injections of 0 5 ml of Bordetella pertussis vaccine (4 x 1010 organisms/ ml; Burroughs Wellcome, London) and subcutaneous injections of 0 5 ml of an emulsion of 100 mg of ovalbumin in 2 ml of isotonic saline and 3 ml of Freund's incomplete adjuvant. -The rqts were bled by cardiac puncture on day 18, the blood was pooled and the serum separated, stored at -20'C and thawed only once before use. The serum produced PCA activity in recipient rats down to a dilution of 1:32 to 1:64 after 72 hr and persisted for several days. This activity was lost to a dilution of 1:2 by heating at 560 for 4 hr even when the sensitizing period was reduced to 4 hr. Passive cutaneous anaphylaxis This was carried out by a method based on that of Ovary & Bier (1952) as modified by
Goose & Blair (1969). Male Wistar rats of 250-300 g were given 0-1 ml of each of six two-fold serial dilutions of the antiserum in 0.900 saline, injected intradermally into separate sites on their shaved backs. Seventy-two hours later the animals were challenged by intravenous injection of 0 3 ml of a 100 solution of ovalbumin in PBS mixed with 0-2 ml of a 500 solution of pontamine sky-blue (6 BX. C.I. 24410, Raymond A. Lamb, London) in isotonic saline. The rats were killed after 20 min and the diameter of the blue wheals at the antibody injection sites were measured on the outer surface of the skin. The starting dilution of the serum was adjusted so that there was no response, after challenge, at the injection site of the highest dilution and a maximum response at the two lowest dilutions. Typically, six two-fold serial dilutions of the serum from 1:4 to 1: 128 were used.
Inhibition of PCA Compounds were tested for their ability to reduce the diameter of the wheals at the three intradermal sites which in control animals gave less than maximum response. Wheals were usually symmetrical, but when not, the mean of the largest and smallest diameters was used. Each dose of the compound was administered to an animal at a measured time prior to intravenous challenge with ovalbumin. Control groups of six animals were given the same volume of carrier fluid at the same time prior to challenge. Rats were starved for 15 hr prior to giving compounds or carrier fluid orally. The results were calculated as follows: percentage inhibition of PCA = 100 (1 - a/b), where a = the sum of the diameters of the wheals produced in the test animal at the three sites of antibody dilutions which in control animals gave less than maximum response, and b = the mean sum of the diameters of the wheals produced in the control group of animals at the antibody sites which gave less than maximum response. A typical variation in a control group of six animals gave an s.e.m. of +6% Passive peritoneal anaphylaxis The method used was based on that of Orange, Stechschulte & Austen (1970) with the difference that blue dye was injected intravenously into the rats just prior to the intraperitoneal challenge with antigen. The subsequent leakage of blue dye into the peritoneal cavity was used as a measure of extravasation.
422
Barbara A. Spicer, Janet W. Ross and H. Smith
Male Wistar rats of 250-300 g were given intraperitoneal injections of 2 ml of a 1:5 dilution of the rat antiserum containing rat IgE. Two hours later 0 3 ml of PBS saturated with Evans Blue (G. T. Gurr, London) was injected intravenously followed by intraperitoneal injection of 1 ml of a solution of a test compound in saline or of saline. Thirty seconds later the animals were challenged with an intraperitoneal injection of 5 ml of Tyrode's solution containing 0 4 mg/ml of ovalbumin and 50 pg/ml of heparin. Five minutes after challenge the rats were stunned and bled. The peritoneal fluid was collected by opening the peritoneal, cavity over a funnel into polycarbonate tubes in ice. The supernatants were separated from the cellular residue by centrifuging at 150 g for 5 min, and retained for estimation of the amounts of dye, histamine and SRS-A. Dye assay The optical densities (OD) of the supernatants which were not contaminated with blood were read immediately at 615 nm. The supernatants were then placed in a boiling water bath for 5 min and stored frozen at - 20'C until it was convenient to assay for histamine and SRS-A. The optical density was taken as a measure of the concentration of blue dye leaked into the peritoneal cavity. Histamine assay Histamine was assayed by bio-assay or spectrofluorimetrically. In the bio-assay histamine was assayed on the isolated guinea-pig ileum preparation in the presence of 5 x 10-7 M atropine. Two concentrations of the unknown were bracketed between three concentrations of standard. For the spectrofluorimetric assay an automated system was used similar to that described by Evans, Lewis & Thomson (1973) using the Technicon Autoanalyser system. At the concentrations used the compounds tested did not interfere with the assays.
SRS-A assay
Slow-reacting substance of anaphylaxis was assayed on the isolated guinea-pig ileum preparation in the presence of atropine (5 x 1O -M) and mepyramine maleate (10-6 M), the latter to abolish the histamine response (Brocklehurst, 1960). The term SRS-A was used to designate the material which gave a slow contraction of the guinea-pig ileum under the conditions described. Samples from test animals receiving a drug and from control animals given no drug were assayed on the same piece of prepared guinea-pig ileum and the mean of the SRS-A concentration of the samples from the control group was taken as 100% and the SRS-A concentrations were expressed relative to this. At the concentrations used the compounds tested did not interfere with the assay. Statistics
Probability values throughout were assessed by the Student's t-test. RESULTS Inhibition of Rat PCA We routinely determine the activities of compounds in the rat PCA test by injecting them subcutaneously prior to challenge with antigen. The time between administration of the compound and challenge with antigen is critical. DSCG, BRL 10833 and isoprenaline
423
Hypersensitivity reactions in the rat
a_ c (
.2 D .r_
a1)
I 0
t7
2
I
I
30 40 30 0 10 20 30 0 10 20 20 10 Time (min) between s.c. administration of the drug and i.v. antigen challenge
Fig 2
c
.
100 a1)
90 80 70
60
(12)
50
1(12)
40 30
20
(6)
10 -
0-001
0-01
1.0
0-1
10
100
Dose (mg / kg) Fig. 3
FIG. 2. Inhibition of rat PCA by drugs given subcutaneously at various times before i.v. antigen challenge: (a) DSCG (20 mg/kg); (b) BRL 10833 (0 5 mg/kg); (c) isoprenaline (0-02 mg/kg). The results are expressed as the mean ± s.e.m. The numbers in parentheses refer to the number
of rats used at that dose. FIG. 3. Inhibition of rat PCA by different compounds. Isoprenaline (0), BRL 10833 (0) and DSCG (-) were given subcutaneously 10 min before antigen challenge and BRL 10833 was also given orally 15 min before challenge (L). The results are expressed as the mean ± s.e.m. The numbers in parentheses refer to the number of rats used at that dose.
showed highest activity if given subcutaneously 10 min before antigen challenge (Fig. 2), and the rank order of potency of the compounds was isoprenaline > BRL 10833 > DSCG. BRL 10833 inhibited the rat PCA test when given orally and showed more activity by this
424
Barbara A. Spicer, Janet W. Ross and H. Smith
route than did DSCG when the latter was given subcutaneously (Fig. 3). No inhibition of the test was found after giving DSCG orally at doses up to 100 mg/kg and at times up to 30 min before antigen challenge. Cross-reacting tachyphylaxis It has been shown that a large intravenous dose of DSCG (40 mg/kg) produces a marked inhibition in the rat PCA test if given with antigen but that this activity was reduced with, increasing time between drug administration and challenge (Thomson & Evans, 1973). We have confirmed this and have found that a similar effect was obtained with BRL 10833 (Table 1). TABLE 1. Inhibition of rat PCA after a large dose of drugs given at various times before antigen challenge
Drug BRL 10833 (2 mg/kgi.v.)
DSCG (40 mg/kg i.v.)
Time (min) between dose and antigen challenge
Percentage inhibition of rat PCA (Mean (six rats) ± s.e.m.)
0 15 30 60 0 20 30 40 60
78± 5-3 38±11 1 13± 52 14± 4-6 100± 0 31± 10-8 8± 8-3 4± 2-3 2+ 7-4
Thomson & Evans (1973) also found that, although a dose of DSCG of 40 mg/kg given intravenously 30 min before challenge produced little inhibition of the rat PCA test, it reduced the inhibitory activity of the same dose of DSCG given just before challenge. We investigated the effects of pre-dosing intravenously with this dose of DSCG or with a dose of 2 mg/kg of BRL 10833 given 30 min before challenge on the inhibition produced by intravenous dosing with DSCG and BRL 10833 given just before challenge using those doses which normally give good inhibition (Figs 4 and 5). The amount of inhibition produced by those doses of DSCG which normally produce well over 50% inhibition was markedly reduced not only by pre-dosing with DSCG but also by pre-dosing with BRL 10833 (Fig. 4). A similar reduction in activity normally shown by BRL 10833 was obtained by pre-dosing with BRL 10833 or with DSCG (Fig. 5). Predosing with DSCG produced little effect on the dose response curve of isoprenaline (Table 2). Rat passive peritoneal anaphylaxis (PPA)
Negative controls. Under the conditions of the test there was very little leakage of histamine, SRS-A or of blue dye into the peritoneal cavity of rats either sensitized with serum containing rat IgE and injected with carrier fluid instead of antigen or treated with normal rat serum and challenged with antigen.
425
Hypersensitivity. reactions in the rat 80
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Fig.5 FIG. 4. Inhibition of rat PCA by DSCG when given intravenously just prior to antigen challenge and 30 min after i.v. administration of saline (0), DSCG (40 mg/kg) (0) and BRL 10833 (2 mg/kg) (A). The results are given as the mean ± s.e.m. The numbers in parentheses refer to the number of rats used at that dose. FIG. 5. Inhibition of rat PCA by BRL 10833 when given intravenously just prior to antigen challenge and 30 min after i.v. administration of saline (o), DSCG (40 mg/kg) (A) and BRL 10833 (2 mg/kg) (A). The results are given as the mean+ s.e.m. The numbers in parentheses refer to the number of rats used at that dose.
Inhibition of ratPPA. BRL 10833 at a concentration of 2 10-6 M in the peritoneal cavity showed a similar type and order of activity to those shown by DSCG at a concentration of 2 x 10- 5 M. At these concentrations both compounds produced more than a 9000 inhibition of histamine release, but were less effective at the inhibition of SRS-A release. Isoprenaline at 2 x 10- 6 M produced no significant inhibition of histamine release. Peritoneal fluids collected from non-challenged rats treated with isoprenaline at 2 10-6 M interfered with the assay for SRS-A. All three compounds produced a similar inhibition of blue dye leakage. x
x
426
Barbara A. Spicer, Janet W. Ross and H. Smith TABLE 2. Inhibition of rat PCA by isoprenaline when given just prior to antigen challenge and 30 min after administration of saline or DSCG
Percentage inhibition of rat PCA mean (five or six rats) + s.e.m. Dose of isoprenaline (mg/kg i.v.) 0 00025 0005 001 002
Predose saline (i.v.)
Predose DSCG (40 mg/kg i.v.) -
22 + 74 39+59 62+6 2 63+68
1*+8-4 45 + 90
50±67 69±5-8 72+45
* Enhanced histamine release, not significant. TABLE 3. Rat passive peritoneal anaphylaxis Rats sensitized with serum containing antigen-specific rat IgE (no challenge)*
Rats treated with normal rat serum (challenged with antigen)
Histamine
SRS-A
Blue dye
Histamine
SRS-A
Blue dye
2-7+0 5
0 4±0 1
9-2+ 1-3
34±06
05+0 1
14-1+1-6
The results are expressed as the mean ± s.e.m. for groups of six rats of the concentrations in peritoneal fluid as percentages of that in sensitized and challenged controls. * Injected with carrier fluid.
These were not significantly different from each other but were significantly different from controls. A concentration of isoprenaline of 2 x 10-3 M in the peritoneal cavity produced a significant inhibition of histamine release. DISCUSSION
DSCG is not absorbed to any extent after oral administration to the rat (Moss et al., 1970) or to man (Walker et al., 1972) and for therapeutic use it has to be given by inhalation. Relatively large amounts have to be inhaled, which can only be achieved by the insufflation of a powder and this limits the dose. After insufflation it is rapidly cleared both from the lungs and the blood (Walker et al., 1972). A compound with greater activity than DSCG and with more favourable absorption characteristics might be more effective clinically. It is possible that the clinical effectiveness of DSCG is connected with its ability to reduce the release of spasmogens induced by reaction between antigen and reagin. BRL 10833 was
Hypersensitivity reactions in the rat
427
TABLE 4. Rat passive peritoneal anaphylaxis. Effects of DSCG BRL 10833 and isoprenaline
Concentration of drug
(M)* Control rats No drug DSCG (2x 10-5) BRL 10833 (2x 10-6) Isoprenaline (2x 10-6) 2x10-5 2x 10-4 2 x 10-3
Concentrations in peritoneal fluids collected 5 min after antigen challenge, as percentage of mean concentration in fluids from control rats ± s.e.m. (number of rats)t
Histamine
SRS-A
OD 625 nm
100±12-2 (13)
100±15-5 (11)
100± 5 3 (13)
6-2+ 1 4 (14) P0.05 93 4+ 6-2 (7) P>0-05 50 0+ 6 0 (6) P
