Pharmacology 7 7: 323 329 (1978)
Evaluation of the Antiasthmatic Potential of Oxarbazole in Guinea Pigs Z.E. Mietern Department of Pharmacology, Sterling-Winthrop Research Institute, Rensselaer, N.Y.
Key Words. Antiasthmatic • Bradykinin • Bronchoconstriction ■Oxarbazole • SRS-A Abstract. Oxarbazole, 9-benzoyl-2,3,4,9,-tetrahydro-6-methoxy-l H-carbazole-3-carboxylic acid, inhibited bronchoconstriction induced by a crude SRS-A preparation or by bradykinin in dose-re lated manner in anesthetized guinea pigs. Oxarbazole was ineffective against bronchoconstriction induced by histamine, carbachol, serotonin, or PGF2cr Inhibition o f bronchoconstriction induced by the crude SRS-A preparation by oxarbazole was unimpeded by the presence of |3-adrenergic- or cholinergic-blocking agents, but it could be overcome by increased doses of the crude SRS-A preparation.
Agents capable of antagonizing SRS-A have been reported before. Berry and Collier (4) reported antagonism o f SRS-A and kinins by nonsteroidal anti-inflammatory drugs. Selective antagonism of SRS-A on guinea pig ileum in vitro has been reported for FPL 55712 (1, 2) and on guinea pig tracheal chains for hydratropic acids (11).
In our search for antagonists of SRS-A, we found that members o f a series of 9-benzoyl-2,3, 4.9-tetrahydro-l H-carbazole-3-carboxylic acids, synthesized by Dr. E.J. Alexander, inhibited bronchoconstriction induced by crude SRS-A preparation in guinea pigs. Oxarbazole, a 6-methoxy derivative o f this basic structure, was found to be the most promising member of this series. This report presents the antiasthmat ic properties of oxarbazole in guinea pigs.
Methods SRS-A Crude SRS-A was prepared from rat peritoneal cavities according to the method of Orange et al. (17). The crude SRS-A preparation (henceforth designated SRS-A) was stored at - 20 °C, and it was thawed
Downloaded by: King's College London 137.73.144.138 - 1/16/2019 9:00:48 AM
Slow-reacting substance o f anaphylaxis (SRS-A) has been found to be a mediator of anaphylaxis in man (3, 12, 18), monkeys (5, 15. 16). guinea pigs (5, 15), and calves (8). Since it has been assumed that SRS-A plays a significant role in human allergic asthma (6, 10), there is belief that pharmacological agents capable o f inhibiting the bronchoconstrictor ef fects of SRS-A should be effective against asthma (7).
shortly before each experiment. Control and medicat ed animals were injected with the same lot of the SRS-A preparation in each experiment. Although the SRS-A preparation has been found to contain little or no histamine, an antihistaminic drug was incorporated in each experiment in order to exclude possible histaminic effects. At the beginning of these studies it was deemed unnecessary to purify SRS-A in order to de fine pharmacological activity of antiasthmatic com pounds. Animals Hartley-strain guinea pigs, weighing from 250 to 350 g each were used throughout these studies. The guinea pigs were fasted overnight, anesthetized with 1.5 g of urethane/kg i.p., and the tracheas and jugular veins were cannulated. The guinea pigs were respired by a Harvard rodent respiratory pump at 40 strokes/ min. The intratracheal pressure (ITP) was recorded continuously via a Statham P23A transducer and a polygraph. Bronchoconstrictor responsiveness follow ing the start of artificial respiration of each guinea pig was monitored by repeated intravenous injections of 5 Mg of histamine(base)/kg, and studies with a spasmogen were commenced when maximum bron choconstrictor responsiveness to histamine was ob tained. Bronchoconstriction Induced by SRS-A. Each guinea pig was injected intravenously with 0.1 mg/kg of propranolol and thenyldiamine each, and, 5 min later, a predetermined optimal vol ume of SRS-A that would result in increases in ITP in control guinea pigs from 15 to 40 mm Hg was injected intravenously. This optimal volume ranged from 0.1 to 0.3 ml/lOOg body weight in most experiments. SRSA-induced bronchoconstriction was studied in 2 guinea pigs at a time: the first guinea pig was medicat ed, whereas the second guinea pig received the vehicle or solvent of the medication (control). Both guinea pigs received SRS-A at the same time. The effect of the administered compound upon SRS-A-induced bronchoconstriction was expressed as percent in hibition of the peak increase in ITP as compared to the peak increase in the control guinea pig. In an experiment on the reversibility of the in hibitory activity of oxarbazole, guinea pigs were used in units of 3 where 1 guinea pig was injected with a low dose of SRS-A, the second with a low dose of SRS-A and oxarbazole, and a third one with a multiple
Mielens
of the low dose of SRS-A and oxarbazole. Activity was expressed as ratios between SRS-A-induccd peak increases in ITP of guinea pigs 1 and 2, and 3. Bradykinin. Guinea pigs received 0.1 mg of propranolol/kg i.v.. followed by 30 Mg of bradykinin/kg i.v. 5 min later. The peak increases in ITP were re corded. After the tracheal pressure had returned to normal level, the determinations of the peak responses to bradykinin were repeated. Histamine, carbachol, and serotonin. These were injected sequentially into the same guinea pigs. Hista mine was injected intravenously four times in 5-min intervals at 5 Mg>basc)/kg: the last two peak increases in ITP were averaged and recorded. Carbachol was injected intravenously at 6 Mg/kg, 5 min after the last injection of histamine, and serotonin was injected intravenously at 5 Mg (base) of serotonin-creatininesulfatc complex/kg, 5 min after the injection of carbachol. The peak increases in ITP after the intra venous injections of carbachol and serotonin were recorded. PGF^a- This was injected into another set of guinea pigs at 1.0 mg/kg i.v. The guinea pigs received 0.1 mg of propranolol/kg i.v. before PGF-2Q. Peak in creases in ITP after the intravenous injections of PGl' 2cr were recorded. Propranolol was used in the studies with SRS-A, bradykinin and PGF'2a in order to enhance the uni formity and magnitude of bronchoconstriction in guinea pigs following intravenous injections of these spasmogens. Drugs Phenylbutazone was used as a reference drug for anti-SRS-A and antibradykinin activity because of a lack of a specific anti-SRS-A drug at the time of these studies. Aminophylline was used as a reference drug for bronchodilator activity. Oxarbazole and the reference drugs were adminis tered either per os or intravenously. Orally, the com pounds were administered via an esophageal tube 1.0 ml/100g body weight 1 h before the injections of the spasmogens (30 min before anesthesia). Control guinea pigs received the vehicle of these medications, 0.5% gum tragacanth. For intravenous administration, oxarbazole and phenylbutazone w>ere dissolved in 10% N-methylglucamine and N/5 NaOH. respectively. Final dilutions were made in saline, and the volume of injections was 0.1 ml/100g body weight. Both com pounds were injected 1 min before the injections of
Downloaded by: King's College London 137.73.144.138 - 1/16/2019 9:00:48 AM
324
325
Antiasthmatic Evaluation of Oxaibazole
propranolol in one study (SRS-A), and 5 min before the injections of bradykinin in a second study. Control guinea pigs received the solvents of the medications. In another study. 1.0 mg/kg of atropine (base) was injected intravenously 2 min before the injections of oxarbazole.
Results Intravenous injections o f SRS-A into control guinea pigs resulted in sustained increases in U P, following a lag phase of approximately half a minute. The peak increase in ITP was observed within 1 min in most cases, and the increases in ITP declined slowly over the next 10 min. Intravenously or orally administered ox arbazole resulted in dose-related inhibition of SRS-A-induced increases in ITP (table I). Intra venously administered oxarbazole was 3.8 times more potent than phenylbutazone (95% con-
Drug-Induced Increases in ITP Oxarbazole was studied for its effects upon ITP in anesthetized, 500-600 g guinea pigs following slow intravenous injections at the relatively high dose of 100 mg/kg. The guinea pigs were pre-treatcd with 1.0 mg of propranolol/kg i.v. (16), and oxarbazole and its solvent were administered to 5 guinea pigs each. The peak increases in ITP were recorded.
Table I. Inhibition of SRS-A-induced increase in intra-trachcal pressure (ITP) in anesthetized guinea pigs
Oxarbazole
mg/kg
Route
Peak increase in ITP due to SRS-A control guinea pigs mm Hg ± SE 23 27 28 27
3.3 10.0
p.o.
5 5
23 i 2 28 ± 4
36 ± 13* 87 ± 4**
1.0
i.v.
8 6 7 5
22 23 22 32
-7 59 72 87
-
8
27 ± 5
4t 6
i.v.
5
30 ± 4
91 t 6
i.v.
5
31 + 2
84 t 10
i.v.
6
35 ± 2
5 ± 18
3.3 10.0 33.0 Controls
-
Oxarbazole Oxarbazole Atropine Atropine
3.3
3.3 ..0 1.0
n
J
* Significant at p
Evaluation of the Antiasthmatic Potential of Oxarbazole in Guinea Pigs Z.E. Mietern Department of Pharmacology, Sterling-Winthrop Research Institute, Rensselaer, N.Y.
Key Words. Antiasthmatic • Bradykinin • Bronchoconstriction ■Oxarbazole • SRS-A Abstract. Oxarbazole, 9-benzoyl-2,3,4,9,-tetrahydro-6-methoxy-l H-carbazole-3-carboxylic acid, inhibited bronchoconstriction induced by a crude SRS-A preparation or by bradykinin in dose-re lated manner in anesthetized guinea pigs. Oxarbazole was ineffective against bronchoconstriction induced by histamine, carbachol, serotonin, or PGF2cr Inhibition o f bronchoconstriction induced by the crude SRS-A preparation by oxarbazole was unimpeded by the presence of |3-adrenergic- or cholinergic-blocking agents, but it could be overcome by increased doses of the crude SRS-A preparation.
Agents capable of antagonizing SRS-A have been reported before. Berry and Collier (4) reported antagonism o f SRS-A and kinins by nonsteroidal anti-inflammatory drugs. Selective antagonism of SRS-A on guinea pig ileum in vitro has been reported for FPL 55712 (1, 2) and on guinea pig tracheal chains for hydratropic acids (11).
In our search for antagonists of SRS-A, we found that members o f a series of 9-benzoyl-2,3, 4.9-tetrahydro-l H-carbazole-3-carboxylic acids, synthesized by Dr. E.J. Alexander, inhibited bronchoconstriction induced by crude SRS-A preparation in guinea pigs. Oxarbazole, a 6-methoxy derivative o f this basic structure, was found to be the most promising member of this series. This report presents the antiasthmat ic properties of oxarbazole in guinea pigs.
Methods SRS-A Crude SRS-A was prepared from rat peritoneal cavities according to the method of Orange et al. (17). The crude SRS-A preparation (henceforth designated SRS-A) was stored at - 20 °C, and it was thawed
Downloaded by: King's College London 137.73.144.138 - 1/16/2019 9:00:48 AM
Slow-reacting substance o f anaphylaxis (SRS-A) has been found to be a mediator of anaphylaxis in man (3, 12, 18), monkeys (5, 15. 16). guinea pigs (5, 15), and calves (8). Since it has been assumed that SRS-A plays a significant role in human allergic asthma (6, 10), there is belief that pharmacological agents capable o f inhibiting the bronchoconstrictor ef fects of SRS-A should be effective against asthma (7).
shortly before each experiment. Control and medicat ed animals were injected with the same lot of the SRS-A preparation in each experiment. Although the SRS-A preparation has been found to contain little or no histamine, an antihistaminic drug was incorporated in each experiment in order to exclude possible histaminic effects. At the beginning of these studies it was deemed unnecessary to purify SRS-A in order to de fine pharmacological activity of antiasthmatic com pounds. Animals Hartley-strain guinea pigs, weighing from 250 to 350 g each were used throughout these studies. The guinea pigs were fasted overnight, anesthetized with 1.5 g of urethane/kg i.p., and the tracheas and jugular veins were cannulated. The guinea pigs were respired by a Harvard rodent respiratory pump at 40 strokes/ min. The intratracheal pressure (ITP) was recorded continuously via a Statham P23A transducer and a polygraph. Bronchoconstrictor responsiveness follow ing the start of artificial respiration of each guinea pig was monitored by repeated intravenous injections of 5 Mg of histamine(base)/kg, and studies with a spasmogen were commenced when maximum bron choconstrictor responsiveness to histamine was ob tained. Bronchoconstriction Induced by SRS-A. Each guinea pig was injected intravenously with 0.1 mg/kg of propranolol and thenyldiamine each, and, 5 min later, a predetermined optimal vol ume of SRS-A that would result in increases in ITP in control guinea pigs from 15 to 40 mm Hg was injected intravenously. This optimal volume ranged from 0.1 to 0.3 ml/lOOg body weight in most experiments. SRSA-induced bronchoconstriction was studied in 2 guinea pigs at a time: the first guinea pig was medicat ed, whereas the second guinea pig received the vehicle or solvent of the medication (control). Both guinea pigs received SRS-A at the same time. The effect of the administered compound upon SRS-A-induced bronchoconstriction was expressed as percent in hibition of the peak increase in ITP as compared to the peak increase in the control guinea pig. In an experiment on the reversibility of the in hibitory activity of oxarbazole, guinea pigs were used in units of 3 where 1 guinea pig was injected with a low dose of SRS-A, the second with a low dose of SRS-A and oxarbazole, and a third one with a multiple
Mielens
of the low dose of SRS-A and oxarbazole. Activity was expressed as ratios between SRS-A-induccd peak increases in ITP of guinea pigs 1 and 2, and 3. Bradykinin. Guinea pigs received 0.1 mg of propranolol/kg i.v.. followed by 30 Mg of bradykinin/kg i.v. 5 min later. The peak increases in ITP were re corded. After the tracheal pressure had returned to normal level, the determinations of the peak responses to bradykinin were repeated. Histamine, carbachol, and serotonin. These were injected sequentially into the same guinea pigs. Hista mine was injected intravenously four times in 5-min intervals at 5 Mg>basc)/kg: the last two peak increases in ITP were averaged and recorded. Carbachol was injected intravenously at 6 Mg/kg, 5 min after the last injection of histamine, and serotonin was injected intravenously at 5 Mg (base) of serotonin-creatininesulfatc complex/kg, 5 min after the injection of carbachol. The peak increases in ITP after the intra venous injections of carbachol and serotonin were recorded. PGF^a- This was injected into another set of guinea pigs at 1.0 mg/kg i.v. The guinea pigs received 0.1 mg of propranolol/kg i.v. before PGF-2Q. Peak in creases in ITP after the intravenous injections of PGl' 2cr were recorded. Propranolol was used in the studies with SRS-A, bradykinin and PGF'2a in order to enhance the uni formity and magnitude of bronchoconstriction in guinea pigs following intravenous injections of these spasmogens. Drugs Phenylbutazone was used as a reference drug for anti-SRS-A and antibradykinin activity because of a lack of a specific anti-SRS-A drug at the time of these studies. Aminophylline was used as a reference drug for bronchodilator activity. Oxarbazole and the reference drugs were adminis tered either per os or intravenously. Orally, the com pounds were administered via an esophageal tube 1.0 ml/100g body weight 1 h before the injections of the spasmogens (30 min before anesthesia). Control guinea pigs received the vehicle of these medications, 0.5% gum tragacanth. For intravenous administration, oxarbazole and phenylbutazone w>ere dissolved in 10% N-methylglucamine and N/5 NaOH. respectively. Final dilutions were made in saline, and the volume of injections was 0.1 ml/100g body weight. Both com pounds were injected 1 min before the injections of
Downloaded by: King's College London 137.73.144.138 - 1/16/2019 9:00:48 AM
324
325
Antiasthmatic Evaluation of Oxaibazole
propranolol in one study (SRS-A), and 5 min before the injections of bradykinin in a second study. Control guinea pigs received the solvents of the medications. In another study. 1.0 mg/kg of atropine (base) was injected intravenously 2 min before the injections of oxarbazole.
Results Intravenous injections o f SRS-A into control guinea pigs resulted in sustained increases in U P, following a lag phase of approximately half a minute. The peak increase in ITP was observed within 1 min in most cases, and the increases in ITP declined slowly over the next 10 min. Intravenously or orally administered ox arbazole resulted in dose-related inhibition of SRS-A-induced increases in ITP (table I). Intra venously administered oxarbazole was 3.8 times more potent than phenylbutazone (95% con-
Drug-Induced Increases in ITP Oxarbazole was studied for its effects upon ITP in anesthetized, 500-600 g guinea pigs following slow intravenous injections at the relatively high dose of 100 mg/kg. The guinea pigs were pre-treatcd with 1.0 mg of propranolol/kg i.v. (16), and oxarbazole and its solvent were administered to 5 guinea pigs each. The peak increases in ITP were recorded.
Table I. Inhibition of SRS-A-induced increase in intra-trachcal pressure (ITP) in anesthetized guinea pigs
Oxarbazole
mg/kg
Route
Peak increase in ITP due to SRS-A control guinea pigs mm Hg ± SE 23 27 28 27
3.3 10.0
p.o.
5 5
23 i 2 28 ± 4
36 ± 13* 87 ± 4**
1.0
i.v.
8 6 7 5
22 23 22 32
-7 59 72 87
-
8
27 ± 5
4t 6
i.v.
5
30 ± 4
91 t 6
i.v.
5
31 + 2
84 t 10
i.v.
6
35 ± 2
5 ± 18
3.3 10.0 33.0 Controls
-
Oxarbazole Oxarbazole Atropine Atropine
3.3
3.3 ..0 1.0
n
J
* Significant at p
