Agents and Actions vol. 8/3 (1978) Birkh~iuser Verlag, Basel
199
The Effect of Sodium 5,6-Dimethyl-2-Nitroindanedione on Anaphylactic reactions in vitro b y T 3 . SHARPE, JANET W . ROSS a n d BARBARA A . SPICER Beecham Pharmaceuticals, Research Division, Brockham Park, Betchworth, Surrey, England
Abstract A uitroinflaneflione (BRL 10833) inhibited the antigen induced release of histamine and slow reacting substance of anaphylaxis (SRS-A) from passively sensitized human lung at similar concentrations to those required for the inhibition of histamine release by disodium eromoglyeate (DSCG). BRL 10833 was more potent than DSCG as an inhibitor of histamine release by antigen from actively and passively sensitized rat peritoneal cells and rat skin fragments.
Introduction BRL 10833 (the s o d i u m salt o f 5,6d i m e t h y l - 2 - n i t r o i n d a n e d i o n e ) is m o r e p o t e n t t h a n d i s o d i u m c r o m o g l y c a t e ( D S C G ) in vivo in t h e r a t as an i n h i b i t o r o f p a s s i v e c u t a n e o u s a n d p e r i t o n eal a n a p h y l a x i s [1, 2]. T h e d a t a p r e s e n t e d h e r e c o m p a r e s t h e activities o f B R L 10833 a n d D S C G in h u m a n a n d r a t t i s s u e s in vitro.
Materials and methods Animals. Male Wistar rats, 250-300 g. Antigens. Freeze dried extracts of mixed grass pollen (12 grasses) or Dermatophagoidespteronyssinus prepared in our laboratories were used to challenge the human tissues and chicken egg albumin (ovalbumin, crystallized and lyophilized, Sigma Grade III) was used for the rat studies. Compounds. Theophylline and isoprenaline sulphate were from Sigma Chemical Co., England. Disodium cromoglycate was from Fisons Pharmaceuticals, Loughborough, England. BRL 10833 was the sodium salt of 5,6-dimethyl-2nitroindane- 1,3 -dione monohydrate [ 3]. Sensitization of rats. The rats were given intraperitoneal injections of 0.5 ml Bordetella pertussis vaccine (4 x 101~organisms/ml) and subcutaneous injections of 0.5 ml of an emulsion of 100 mg ovalbumin in 2 ml isotonic saline and 3 ml of Freund's incomplete adjuvant. Eighteen days later the rats were killed and either the skin samples, peritoneal ceils or sera were collected. Histamine assay. All samples were deproteinized with 0.4 N perchloric acid before the histamine was assayed using an automated spectrofluorimeter [4]. In the human leucocyte experiments at concentrations of histamine below 50
ng/ml, BRL 10833 at concentrations above 10-4 M interfered with the assay. Allowance was made for this by the use of a calibration curve of histamine standards containing the appropriate concentration of BRL 10833. SRS-A assay. Solutions to be assayed for their SRS-A content were heated immediately in a boiling water bath for 5 minutes and stored at - 2 0 ~ until assayed on the guinea pig ileum [2]. Calculation of the results. The individual observations were determined as: Percent histamine released = Histamine in diffusate
x 100
Histamine in diffusate + histamine in tissue residue Each observation was expressed as a percentage of the mean control histamine release within that experiment. Results are expressed as the mean + S.E.M. with the number of observations. Human lung. Macroscopically normal human lung tissue was obtained at the time of surgery for carcinoma. The lung parenchyma was sliced, thoroughly washed with Tyrode's solution, replicated into 200 mg or 4 • 50 mg samples and left overnight at room temperature in 1 ml of diluted human serum. The sensitizing serum was obtained from a donor who gave a positive skin test in response to the mixed grass extract and was used at I in 4 to 1 in 20 dilutions. After removal of the serum and washing the lung pieces twice with Tyrode's solution, 2 ml Tyrode's solution were added to each tube. The tubes were put in a water bath at 37~ and exactly 15 minutes later the compound in ! ml Tyrode's solution, or Tyrode's solution alone at 37~ was added. After 1 or 3 minutes, 25 gg mixed grass extract in 50 pl Tyrode's solution was added and the tubes were incubated for a further 15 minutes. The solution from each tube (diffusate) was then removed and retained for the determination of amounts of histamine and slow reacting substance of anaphylaxis (SRS-A) released. 3 ml Tyrode's solution were added to the residual lung tissue and the tubes were placed in a boiling water bath for 8 minutes. The solutions from these boiled samples were assayed for the residual histamine. Triplicate lung samples were processed for each of the drug treatments and in each experiment there
200 were 9 positive and 6 negative controls. At the concentrations of BRL 10833 used there was no interference in the SRS-A assay. Human leucocytes. The method was similar to that of MAY et al. [5]. Briefly, 40 ml blood were taken from each of 4 donors, two of whom gave positive skin tests to the mixed grass extract and two to the extract of D. pteronyssinus. The erythrocytes were sedimented with 6% dextran in saline for 1 hour. The leucocyte rich upper layer was collected and the cells were washed twice with Tris A prior to suspension in 30 ml Tris A C M , preheated to 37~ 0.8 ml of this leucocyte suspension was added to tubes containing 0.2 ml Tris A C M in which the antigen and the drugs were dissolved. The antigen concentration of 0.1 /~g/ml and the drug concentrations quoted in the results are the concentrations in this 1 ml of solution. Each treatment was carried out in 2 or 4 replicate tubes within each experiment. After incubation at 3 7 ~ for 1 hour, the tubes were centrifuged at 150 g for 5 minutes. The histamine contents of the supernatant and the cell residue for each tube were assayed. Rat peritoneal cells. The buffer solutions used were Dulbecco's pH 7.2 phosphate buffered saline modified to contain 1 m M CaCI2, 1 m M MgC12 and 0.5 m M glucose or a solution based on RPM1 1640 tissue culture medium modified according to BACH, BLOCH and AUSTEN [6]. Similar results were obtained with the two buffers. Passively sensitized cells. Experiments were performed on mixed peritoneal cells (1-10% mast cells) collected from freshly killed rats, in 5 ml ice cold buffer. The cells were washed twice, in buffer, resuspended in 0.2 ml buffer and added to 0.2 ml serum from sensitized rats. After 2 hours at 3 7 ~ the peritoneal cells were washed twice with buffer and then resuspended in a modified buffer at 37~ 0.8 ml aliquots of this cell suspension were added to 0.2 ml of the modified buffer at 3 7 ~ containing the compounds and antigen. The tubes were incubated at 37~ for 5 minutes and then centrifuged at 150 g for 5 minutes. The histamine contents of the supernatants and the cell residues were determined. Each treatment was carried out in 2 or 4 replicate tubes in each of 12 experiments. Actively sensitized cells. The experiments were carried out on mixed peritoneal cells collected from freshly killed actively sensitized rats in 5 ml ice cold buffer. The cells were washed twice and resuspended in buffer. Subsequent incubation with antigen in the presence of 10833 or D S C G was carried out using the procedures described under passively sensitized cells.
Rat skin Passively sensitized skin. 10 mg pieces of shaved, freshly obtained rat abdominal skin were incubated for 2 hours at 37~ with a 1 in 4 dilution, in Tyrode's solution, of a serum obtained from sensitized rats. The supernatant was removed and the skin samples were washed once with 5 ml Tyrode's solution. The skin samples were preincubated at 3 7 ~ in 1 ml Tyrode's solution before the addition of 1 ml compound, also at 37~ and after 3 minutes 100 pl Tyrode's solution containing 200 pg ovalbumin. After a further 15 minutes incubation at 3 7 ~ the diffusate was removed and assayed for its histamine content. 2 ml Tyrode's solution were added to the skin, and residual histamine was determined in the solution obtained after 8 minutes heating in a boiling water bath. Actively sensitized skin. Pieces of shaved abdominal skin were removed from actively sensitized rats; after 15
A Nitroindanedione and in vitro Anaphylaxis
minutes at 37~ in 1 ml Tyrode's solution, drugs and antigen were added and the tubes processed as with passively sensitized skin.
Results
Human lung. BRL 10833 and D S C G inhibited the antigen induced release of histamine from passively sensitized human lung at concentrations between 1 x 10 -5 M and 3 • 1 0 - 4 M, the levels reaching significance in 13 of 15 lungs in which BRL 10833 was tested and 8 of 10 lungs in which D S C G was tested. The combined results from all the experiments are shown in Figure 1.
'2~1 100-
.., T 33
=~.
L- 8
T Contro, l* 18
0~
DSCG 7- 60-6
36
~BRLI0833 6
8 40N 20-
Non-specific Z 0'
125
"|
10-6
10-5
I 0"4
10-3
Molarity of compound Figure 1 Antigen induced histamine release from passively sensitized h u m a n lung in the presence of BRL 10833 or D S C G . Results are the mean + S.E.M. with the number of observations given. *Mean control histamine release was 17.7% of the total histamine content.
Isoprenaline at 1 • 10 -7 M caused significant inhibition of histamine release in all of the 8 lungs in which it was tested. We attempted to assay SRS-A in 9 lung experiments but in 4 lungs the amounts of SRS-A were too low to measure. In the other 5 lungs the mean SRS-A release by sensitization and challenge was 4.7 units/ml (SEM 0.71, 36 observations) and the SRS-A release in the absence of sensitization or challenge was less than 0.9 units/ml. BRL 10833 significantly inhibited SRSA release in these five lung samples at concentrations of 1 x 10 -5 to 1 x 10 -4 M (Table 1).
201
A Nitroindanedione and in vitro Anaphylaxis
120-
Table 1 Inhibition of SRS-A release from h u m a n lung by B R L 10833. Mean percent inhibition of SRS-A release by BRL 10833
Concentration of BRL 10833
*~ I00
,,,/
,"t'L"~,,
x',
.
I
'
Control* 32
801X 3x 1x 3 x
10 -4 M lO-SM 10 -s M 10 -6 M
Mean
S.E.M
P
59.4 52.1 48.0 --9.9
_+5.5 (9) +7.8 (13) +11.2 (11) -+24.6 (3)
199
The Effect of Sodium 5,6-Dimethyl-2-Nitroindanedione on Anaphylactic reactions in vitro b y T 3 . SHARPE, JANET W . ROSS a n d BARBARA A . SPICER Beecham Pharmaceuticals, Research Division, Brockham Park, Betchworth, Surrey, England
Abstract A uitroinflaneflione (BRL 10833) inhibited the antigen induced release of histamine and slow reacting substance of anaphylaxis (SRS-A) from passively sensitized human lung at similar concentrations to those required for the inhibition of histamine release by disodium eromoglyeate (DSCG). BRL 10833 was more potent than DSCG as an inhibitor of histamine release by antigen from actively and passively sensitized rat peritoneal cells and rat skin fragments.
Introduction BRL 10833 (the s o d i u m salt o f 5,6d i m e t h y l - 2 - n i t r o i n d a n e d i o n e ) is m o r e p o t e n t t h a n d i s o d i u m c r o m o g l y c a t e ( D S C G ) in vivo in t h e r a t as an i n h i b i t o r o f p a s s i v e c u t a n e o u s a n d p e r i t o n eal a n a p h y l a x i s [1, 2]. T h e d a t a p r e s e n t e d h e r e c o m p a r e s t h e activities o f B R L 10833 a n d D S C G in h u m a n a n d r a t t i s s u e s in vitro.
Materials and methods Animals. Male Wistar rats, 250-300 g. Antigens. Freeze dried extracts of mixed grass pollen (12 grasses) or Dermatophagoidespteronyssinus prepared in our laboratories were used to challenge the human tissues and chicken egg albumin (ovalbumin, crystallized and lyophilized, Sigma Grade III) was used for the rat studies. Compounds. Theophylline and isoprenaline sulphate were from Sigma Chemical Co., England. Disodium cromoglycate was from Fisons Pharmaceuticals, Loughborough, England. BRL 10833 was the sodium salt of 5,6-dimethyl-2nitroindane- 1,3 -dione monohydrate [ 3]. Sensitization of rats. The rats were given intraperitoneal injections of 0.5 ml Bordetella pertussis vaccine (4 x 101~organisms/ml) and subcutaneous injections of 0.5 ml of an emulsion of 100 mg ovalbumin in 2 ml isotonic saline and 3 ml of Freund's incomplete adjuvant. Eighteen days later the rats were killed and either the skin samples, peritoneal ceils or sera were collected. Histamine assay. All samples were deproteinized with 0.4 N perchloric acid before the histamine was assayed using an automated spectrofluorimeter [4]. In the human leucocyte experiments at concentrations of histamine below 50
ng/ml, BRL 10833 at concentrations above 10-4 M interfered with the assay. Allowance was made for this by the use of a calibration curve of histamine standards containing the appropriate concentration of BRL 10833. SRS-A assay. Solutions to be assayed for their SRS-A content were heated immediately in a boiling water bath for 5 minutes and stored at - 2 0 ~ until assayed on the guinea pig ileum [2]. Calculation of the results. The individual observations were determined as: Percent histamine released = Histamine in diffusate
x 100
Histamine in diffusate + histamine in tissue residue Each observation was expressed as a percentage of the mean control histamine release within that experiment. Results are expressed as the mean + S.E.M. with the number of observations. Human lung. Macroscopically normal human lung tissue was obtained at the time of surgery for carcinoma. The lung parenchyma was sliced, thoroughly washed with Tyrode's solution, replicated into 200 mg or 4 • 50 mg samples and left overnight at room temperature in 1 ml of diluted human serum. The sensitizing serum was obtained from a donor who gave a positive skin test in response to the mixed grass extract and was used at I in 4 to 1 in 20 dilutions. After removal of the serum and washing the lung pieces twice with Tyrode's solution, 2 ml Tyrode's solution were added to each tube. The tubes were put in a water bath at 37~ and exactly 15 minutes later the compound in ! ml Tyrode's solution, or Tyrode's solution alone at 37~ was added. After 1 or 3 minutes, 25 gg mixed grass extract in 50 pl Tyrode's solution was added and the tubes were incubated for a further 15 minutes. The solution from each tube (diffusate) was then removed and retained for the determination of amounts of histamine and slow reacting substance of anaphylaxis (SRS-A) released. 3 ml Tyrode's solution were added to the residual lung tissue and the tubes were placed in a boiling water bath for 8 minutes. The solutions from these boiled samples were assayed for the residual histamine. Triplicate lung samples were processed for each of the drug treatments and in each experiment there
200 were 9 positive and 6 negative controls. At the concentrations of BRL 10833 used there was no interference in the SRS-A assay. Human leucocytes. The method was similar to that of MAY et al. [5]. Briefly, 40 ml blood were taken from each of 4 donors, two of whom gave positive skin tests to the mixed grass extract and two to the extract of D. pteronyssinus. The erythrocytes were sedimented with 6% dextran in saline for 1 hour. The leucocyte rich upper layer was collected and the cells were washed twice with Tris A prior to suspension in 30 ml Tris A C M , preheated to 37~ 0.8 ml of this leucocyte suspension was added to tubes containing 0.2 ml Tris A C M in which the antigen and the drugs were dissolved. The antigen concentration of 0.1 /~g/ml and the drug concentrations quoted in the results are the concentrations in this 1 ml of solution. Each treatment was carried out in 2 or 4 replicate tubes within each experiment. After incubation at 3 7 ~ for 1 hour, the tubes were centrifuged at 150 g for 5 minutes. The histamine contents of the supernatant and the cell residue for each tube were assayed. Rat peritoneal cells. The buffer solutions used were Dulbecco's pH 7.2 phosphate buffered saline modified to contain 1 m M CaCI2, 1 m M MgC12 and 0.5 m M glucose or a solution based on RPM1 1640 tissue culture medium modified according to BACH, BLOCH and AUSTEN [6]. Similar results were obtained with the two buffers. Passively sensitized cells. Experiments were performed on mixed peritoneal cells (1-10% mast cells) collected from freshly killed rats, in 5 ml ice cold buffer. The cells were washed twice, in buffer, resuspended in 0.2 ml buffer and added to 0.2 ml serum from sensitized rats. After 2 hours at 3 7 ~ the peritoneal cells were washed twice with buffer and then resuspended in a modified buffer at 37~ 0.8 ml aliquots of this cell suspension were added to 0.2 ml of the modified buffer at 3 7 ~ containing the compounds and antigen. The tubes were incubated at 37~ for 5 minutes and then centrifuged at 150 g for 5 minutes. The histamine contents of the supernatants and the cell residues were determined. Each treatment was carried out in 2 or 4 replicate tubes in each of 12 experiments. Actively sensitized cells. The experiments were carried out on mixed peritoneal cells collected from freshly killed actively sensitized rats in 5 ml ice cold buffer. The cells were washed twice and resuspended in buffer. Subsequent incubation with antigen in the presence of 10833 or D S C G was carried out using the procedures described under passively sensitized cells.
Rat skin Passively sensitized skin. 10 mg pieces of shaved, freshly obtained rat abdominal skin were incubated for 2 hours at 37~ with a 1 in 4 dilution, in Tyrode's solution, of a serum obtained from sensitized rats. The supernatant was removed and the skin samples were washed once with 5 ml Tyrode's solution. The skin samples were preincubated at 3 7 ~ in 1 ml Tyrode's solution before the addition of 1 ml compound, also at 37~ and after 3 minutes 100 pl Tyrode's solution containing 200 pg ovalbumin. After a further 15 minutes incubation at 3 7 ~ the diffusate was removed and assayed for its histamine content. 2 ml Tyrode's solution were added to the skin, and residual histamine was determined in the solution obtained after 8 minutes heating in a boiling water bath. Actively sensitized skin. Pieces of shaved abdominal skin were removed from actively sensitized rats; after 15
A Nitroindanedione and in vitro Anaphylaxis
minutes at 37~ in 1 ml Tyrode's solution, drugs and antigen were added and the tubes processed as with passively sensitized skin.
Results
Human lung. BRL 10833 and D S C G inhibited the antigen induced release of histamine from passively sensitized human lung at concentrations between 1 x 10 -5 M and 3 • 1 0 - 4 M, the levels reaching significance in 13 of 15 lungs in which BRL 10833 was tested and 8 of 10 lungs in which D S C G was tested. The combined results from all the experiments are shown in Figure 1.
'2~1 100-
.., T 33
=~.
L- 8
T Contro, l* 18
0~
DSCG 7- 60-6
36
~BRLI0833 6
8 40N 20-
Non-specific Z 0'
125
"|
10-6
10-5
I 0"4
10-3
Molarity of compound Figure 1 Antigen induced histamine release from passively sensitized h u m a n lung in the presence of BRL 10833 or D S C G . Results are the mean + S.E.M. with the number of observations given. *Mean control histamine release was 17.7% of the total histamine content.
Isoprenaline at 1 • 10 -7 M caused significant inhibition of histamine release in all of the 8 lungs in which it was tested. We attempted to assay SRS-A in 9 lung experiments but in 4 lungs the amounts of SRS-A were too low to measure. In the other 5 lungs the mean SRS-A release by sensitization and challenge was 4.7 units/ml (SEM 0.71, 36 observations) and the SRS-A release in the absence of sensitization or challenge was less than 0.9 units/ml. BRL 10833 significantly inhibited SRSA release in these five lung samples at concentrations of 1 x 10 -5 to 1 x 10 -4 M (Table 1).
201
A Nitroindanedione and in vitro Anaphylaxis
120-
Table 1 Inhibition of SRS-A release from h u m a n lung by B R L 10833. Mean percent inhibition of SRS-A release by BRL 10833
Concentration of BRL 10833
*~ I00
,,,/
,"t'L"~,,
x',
.
I
'
Control* 32
801X 3x 1x 3 x
10 -4 M lO-SM 10 -s M 10 -6 M
Mean
S.E.M
P
59.4 52.1 48.0 --9.9
_+5.5 (9) +7.8 (13) +11.2 (11) -+24.6 (3)
