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Bronchodilator effects of sympathomimetic amines given singly and in combination
EUROPEAN JOURNAL OF PHARMACOLOGY14 (1971) 140-149. NORTH-HOLLANDPUBLISHINGCOMPANY
BRONCHODILATOR EFFECTS OF SYMPATHOMIMETIC AMINES GIVEN SINGLY AND IN COMBINATION L-Q PUN, M.W. McCULLOCH and M.J. RAND Department of Pharmacology, University of Melbourne, Parkville, Victoria 3052, Australia
Accepted 28 December 1970
Received 9 March 1970
L-Q PUN, M.W. McCULLOCHand M.J. RAND, Bronchodilator effects of sympathomimetic amines given singly and in combination, European J. Pharmacol. 14 (1971) 140-149. The hronchodilator effects of a number of sympathomimetic amines were assessed in terms of reduction of histamine-induced bronchospasm. Their order of potency when given by injection 20 sec before injection of histamine was: isoprenaline ~ adrenaline > > orciprenaline > noradrenaline ~ phenylephrine; when given by infusion it was: isoprenaline ~ adrenaline ~>noradrenaline :> orciprenaline. No tachyphylaxis to the bronchodilator effect occurred during infusions of up to 2.5 hr. Combinations of the sympathomimetics, one given by injection the other by infusion, were synergistic in bronchodilator activity except for phenylephfine with orciprenaline. Sympathomimetic drugs Bronchodilator drugs
Synergistic action of sympathomimetics Histamine bronchospasm
1. INTRODUCTION Recently attention has been drawn to the increasing incidence of mortality in asthmatics in the U.K., Australia, Japan, Western Europe and the U.S.A. According to Speizer, Doll and Heaf (1968) there was no evidence to suggest that there has been any change in diagnostic habits or methods of certification of death which would account for the increase. Since 1952, there has been increasing use in the treatment of asthma of corticosteroid preparations and of pressurized aerosols containing sympathomimetic bronchodilator drugs: suspicion has been thrown on these drugs as a possible cause of the increased mortality. There has been much correspondence in medical journals suggesting that sympathomimetic bronchodilator drugs may be responsible for sudden death in asthmatics and for the increasing mortality among asthmatics (Van Metre, 1969; Kieghley, 1969; Inman and Adelstein, 1969). The hazards of bronchodilator drugs are attributed to their excessive use or to their use in combination.
Experiments on the cardiovascular effects in cats of the commonly used bronchodilator drugs isoprenaline, adrenaline and orciprenaline fared to provide evidence of cardiac toxicity when they were given by infusion to mimic excessive use, or when they were given by intravenous injections during infusions to mimic their use in combination (Atkinson and Rand, 1968). There was, however, tachyphylaxis to the effects of these drugs: thus, the responses to injections were reduced during the infusion of any of them. Tachyphylaxis developed only to the effects exerted through fl-adrenoreceptors; namely, tachycardia and vasodilation. The pressor response to an injection of adrenaline, which involves vascular ct.adrenoreceptors, was not diminished during infusions of isoprenaline or orciprenaline. These observations led us to consider that a possible hazard of excessive use of sympathomimetics in the treatment of asthma may be the development of tachyphylaxis to their bronchodilator effects, since this involves /3-receptors of bronchial smooth muscle which might be expected to react similarly to the/3-receptors of
L.Q Pun et al., Sympathomimetic bronchodilators vascular smooth muscle and of the heart. No previous experimental observations appear to have been made on the bronchodilator effects of sympathomimetics given in combination, therefore, we were prompted to study this, and also to determine if tachyphylaxis developed to the bronchodilator action of the drugs used singly.
2. METHODS Guinea-pigs of either sex weighing 250 to 700 g were used. They were anaesthetised by intraperitoneal injection of 4 ml/kg of a solution containing 250 mg/ml of urethane and 15 mg/ml of chloralose. Blood pressure was measured from the right common carotid artery by means of a cannula connected to a Statham transducer (Model P23Db) which was coupled to an Offner Dynograph pen-writer. Intravenous injections of drugs were made through a cannula inserted into the left jugular vein. Intravenous infusions were given through a cannula inserted into the right jugular vein using a Palmer slow infusion apparatus. Concentrations of drugs given by infusion were adjusted to accord with a constant volume rate of about 0.02 ml/min. The guinea-pigs were artificially ventilated with a Palmer constant volume respiration pump (25 ml capacity) through a tracheal cannula. The rate was 72 strokes/min and the volume was 2 to 6 ml/stroke, being approximately 0.8 ml per 100 g of guinea-pig body weight. Intratracheal pressure was measured by connecting a T-junction on the tracheal cannula to a Statham transducer (Model P 23Db), the output of which was coupled to the Offner Dynograph. An increase in intratracheal pressure was taken to denote bronchoconstriction (McCulloch, Proctor and Rand, 1967). Intravenous injections of histamine were given at 8 to 10 rain intervals until constant bronchoconstrictor responses to a given dose were obtained. In some experiments, constant control responses were obtained for two or three doses of histamine. The bronchodilator effects of sympathomimetic amines were determined by reduction of the bronchoconstrictor response to histamine. Intravenous injections were given 20 sec before an injection of histamine: infusions of sympathomimetics were given for at least 15 min before testing their effect on response to
141
histamine. The increases in intratracheal pressure produced by histamine injections have been expressed as a multiple of the basal intratracheal pressure which was taken as unity. The effect of the sympathomimetics was expressed as a percentage change in the histamine-induced bronchoconstriction. Each experiment was identified by a different symbol. The following drugs were used: histamine acid phosphate; dl-isoprenaline hydrochloride (Isuprel, Winthrop Laboratories); 1-adrenaline tartrate (Adrenate, Burroughs Wellcome and Co. Ltd.); 1-noradrenaline bitartrate (Levophed, Winthrop Laboratories); orciprenaline sulphate (Alupent, Boehringer Ingelheim Pty. Ltd.); and 1-phenylephrine hydrochloride (Neosynephrine, Winthrop Laboratories).
3. RESULTS 3.1. Bronchodilator effects o f sympathomimetic amines given singly 3.1.1. Bronchodilator effects of injections of sym. pathomimetic amines In the control period, constant bronchoconstrictor responses were obtained to each of three doses of histamine chosen to produce graded increases in intratracheal pressure. Fig. 1 shows the effect of the three doses of histamine in producing bronchoconstriction in 16 experiments and the effect of 2 doses of histamine in 3 experiments. The guinea-pigs varied in their bronchoconstrictor responses; the mean doses of histamine producing 50, 150 and 200 percent increase in intratracheal pressure were 2.9 (n = 19), 4.3 (n = 19) and 7.5 (n = 16)/ag/kg respectively, where n is equal to the number of observations used to calculate the mean doses. Injections of sympathomimetic amines were given 20 sec before a histamine injection; the doses were chosen to produce a decrease in the response to the middle dose of histamine of about one third. The effect of the sympathomimetic amines is shown in fig. 2. The chosen dose of each sympathomimetic amine was in general more effective in counteracting the bronchoconstrictor responses to lower than to higher doses of histamine. The relative potency of the sympathomimetic amines tested by single intravenous injection was isoprenaline ~ adrenaline > > orciprenaline > noradrenaline > phenylephrine. Records illustrating the reduction in histamine-
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induced bronchoconstriction produced by injections of isoprenaline (50ng/kg) and of phenylephrine (6/ag/kg) are shown in figs. 3 and 4 respectively, in each of which the two left hand panels should be compared. The injection of a sympathomimetic amine was not repeated until the bronchoconstriction induced by histamine had returned to control levels. The effects of adrenaline, isoprenaline and noradrenaline were no longer apparent when the next injection of histamine was given, indicating that the effect persisted for less than 8 min. However, orciprenaline and phenylephrine had more persistent effects lasting for longer than 10 rain but for less than 30 rain. 3.1.2. Bronchodilator effects of infusions of sympathomimetic amines The effects of infusions were studied in two ways: firstly, by determining the percentage reduction in histamine-induced bronchoconstriction produced by an infusion of a sympathomimetic amine; secondly,
by determining the increase in dose of histamine necessary to maintain a constant bronchoconstrictor response with increasing rates of infusion of each sympathomimetie amine. Firstly to study the effects of infusions in decreasing bronchoconstrictor responses, three doses of histamine were given. Control observations were obtained as described above, and then a rate of infusion of each sympathomimetic amine was selected such that the response to the middle dose of histamine was reduced by approximately 50%. Fig. 5 shows the effect of the infusion of four sympathomimetic amines in nineteen experiments. The percentage decrease in histamine-induced bronchospasm was greater with lower than with higher doses of histamine. The relative potency of the sympathomimetics given by infusion was isoprenaline > adrenaline :> noradrenaline > orciprenaline. The relative differences in potencies are less when the drugs Were given by infusion than when they were given by injection. Representative records showing the reductions in
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Fig. 2. The effect of injections of sympathomimetic amines in reducing bronchoconstriction induced by histamine. Each graph represents a different sympathomimetic amine, isoprenaline (ISO); adrenaline (ADR); orciprenaline (ORCI); noradrenaline (NA); phenylephrine (PE). Each experiment is identified by a different symbol, injected doses of sympat;lomimetic amines are written alongside the symbols. The sympathomimetic amines were more effective in counteracting the bronchoconstrictor responses to lwer than to higher doses of histamine.
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Fig. 3. The effect of injections of isoprenaline (50 ng/kg) ('~) in modifying histamine-induced bronchoconstriction (t), before and during the infusion of orciprenaline (100 ng/kg/min) in the anaesthetised guinea-pig. Both isoprenaline injection and orciprenaline infusion were effective in reducing the histamine-induced bronchoconstriction. The combined effect of isoprenaline injection during orciprenaline infusion was greater than when either drug was used alone. bronchoconstrictor responses to three doses of histamine by infusions of orciprenaline (100 and 50 ng/kg/min) are shown in figs. 3 and 4 in each of which the upper two panels should be compared. No observations are reported for infusions of phenylephrine, because guinea-pigs did not survive infusions at a rate necessary to cause a 50% reduction in bronchoconstrictor responses to the middle dose of histamine. Secondly to assess the bronchodilator potency o f sympathomimetic amines, the following procedure was adopted. Constant responses were obtained to a
dose of histamine that produced an increase in intratracheal pressure approximately equal to the pressure produced by the inflation stroke of the respiration pump. The infusion o f a sympathomimetic amine was then started at a low rate which was chosen to produce a just detectable decrease in the bronchoconstricter response to histamine. During the infusion, the dose of histamine was increased until it produced the same bronchoconstrictor response as before the infusion. This procedure was repeated several times, the rates of infusion being increased, and a larger equi-effective dose of histamine being found for each --ORCIPRENALINE INFUSION 5 0 n g / k g / m i n - -
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rate. The ratio of equi-effective doses of histamine during an infusion and in the control period was termed the 'histamine ratio'. The results are shown graphically in fig. 6 from which it may be seen that the relative potencies of the sympathomimetics tested were isoprenaline > adrenaline > orciprenaline > noradrenaline ~ phenylephrine. Experiments were carried out to determine whether tachyphylaxis to the bronchodilator effects of the
sympathomimetics developed after prolonged infusion. A rate of infusion was chosen for each such that the histamine ratio was about 1.5. The rates of infusion were as follows: isoprenaline, 0.01/ag/ kg/min; adrenaline, 0.04 /ag/kg/min; orciprenaline, 0.10 tag/kg/min; and noradrenaline, 0.65/~g/kg/min. During infusions of these drugs for periods of 30 rain to 2.5 hr, the histamine ratio stayed constant throughout; that is to say, tachyphylaxis was not
L-Q Pun et al., Sympathomimetic bronchodilators
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produced. Typical experiments with orciprenaline and isoprenaline are shown in fig. 7. Bronchoconstrictor responses to histamine returned to control levels within 20 to 50 min after ceasing infusions of sympathomimetic amines. Prolonged infusion of phenylephrine at a rate such that the histamine ratio was greater than 1.5 was not possible since the guinea-pigs did not survive these infusions ( > 1/~g/kg/min).
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Having established separately the effects of injections and infusions of sympathomimetic amines in reducing the bronchoconstrictor responses to three doses of histamine, the effects of injections of one sympathomimetic amine were determined during the infusion of another. Each amine was either injected or infused, except phenylephrine which was given by injection only. The doses given by injection were those shown in fig. 2, and the rates of infusion were as shown in fig. 5. The effects of the combination of one injected and one infused sympathomimetic amine are shown in fig. 8, where the percentage reduction produced by the infusion of different sympathomimetic amines were plotted against the percentage reduction produced by injections during infusions of either isoprenaline, adrenaline, noradrenaline or orciprenaline. With most combinations, the injection plus the infusion had a greater effect than either the injection or the infusion given singly, the only exception observed was with injections of phenylephrine during infusions of orciprenaline.
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Fig. 8. The effects of the combination of infused and injected sympathomimetic amines on histamine-induced bronchoconstriction. Isoprenaline (ISO); noradrenaline (NA); orciprenaline (ORCI); phenylephrine (PE); adrenaline (ADR). Points to the right of the line show synergism.
As with injections and infusions given singly, the percentage reductions in histamine-induced bronchoconstriction produced by combinations of injection and infusion were generally greater with lower than with higher doses of histamine. The effect of isoprenaline injections during an infusion of orciprenaline is illustrated in fig. 3; in this, the bronchoconstrictor responses to histamine when the drugs were given in combination are shown in the bottom right panel, and are clearly smaller than those
elicited during the infusion alone (top right panel) or after injection alone (bottom left panel). A similar experiment with injections of phenylephrine during an infusion of orciprenaline is shown in fig. 4 although phenylephrine was effective in reducing histamine-induced bronchoconstriction when injected alone (bottom left versus top left panel), it produced no further reduction of histamine-induced bronchoconstrictor responses that had already been reduced by infusion of orciprenaline (bottom right panel).
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L-Q Pun et aL, Sympathomimetic bronchodilators
3.3. Observations on cardiovascular effects o f histamine and sympathomimetic amines
4. DISCUSSION
As mentioned in the Introduction, it has been previously observed that the cardiovascular effects of injections of sympathomimetics that were mediated through 13-receptors in the cat were reduced or abolished during infusions of sympathomimetics. Since the bronchodilator effects of sympathomimetics are mediated through /3-receptors, but the effects of injections and infusions were generally synergistic in the guinea-pig, it is worth commenting on the cardiovascular effects of the sympathomimetics in the guinea-pig. The usual response to an injection of histamine in the guinea-pig was a slight fall of blood pressure which was followed by a slight rise and then a longer lasting fall. Injections of sympathomimetic drugs were given 20 sec before an injection of histamine, hence the responses to them were complicated by their interaction with histamine. With isoprenaline and orciprenaline, the depressor response was greater than with histamine alone. With noradrenaline, adrenaline and phenylephrine, the depressor response to histamine was reduced, abolished or replaced by a rise of pressure. Infusions of isoprenaline or orciprenaline at the rates used in these experiments did not affect the blood pressure of the guinea-pigs. Infusions of noradrenaline, adrenaline and phenylephrine produced an increase in blood pressure of 10 to 20 mm Hg and an increase in pulse pressure which was most marked with adrenaline infusions: the responses to histamine during these infusions consisted of either a small fall or a small rise in blood pressure, the effect being of only a few millimeters Hg either way. The injection of sympathomimetic amines (20 sec before a histamine injection) during an infusion produced a rise in blood pressure. The heart rate of the guinea-pigs was only slightly increased by injections or infusions of sympathomimetic amines. No arrhythmias occurred despite administration of injections and infusions of sympathomimetic amines, singly or in combination, for periods of up to 4 hr.
From these experiments two main points emerge; (1) tachyphylaxis to the bronchodilator effects of sympathomimetic amines did not develop on prolonged infusion; (2)the bronchodilator effects of sympathomimetic amines were generally synergistic when any two were used in combination, except when phenylephrine injections were given during orciprenaline infusion. A possible explanation is that orciprenaline being a relatively weak/3-receptor agonist had to practically saturate the /3-receptor sites before any reduction in the histamine-induced bronchoconstriction was observed. Hence further reduction by phenylephrine, another weak /3-receptor agonist was impossible. This situation would also tend to unmask the action of phenylephrine on o~-receptors. Everitt and Cairncross (1969) reported contraction in the isolated trachea of the guinea pig produced by adrenaline and noradrenaline in the presence of proprano1ol. Our findings on bronchial smooth muscle are in contrast to the observations of Atkinson and Rand (1968) on the cardiovascular effects of sympathomimetic amines involving /3-receptors, namely, that tachyphylaxis developed to the depressor effect and the tachycardia during infusions, and synergism was not observed. That there should be a difference between cardiovascular and bronchial/3-receptors is not entirely unexpected since it has been reported by Brittain, Farmer, Jack, Martin and Simpson (1968) and by Kelman and Palmer (1969) that there are ~-receptor stimulating drugs having a selective action on the bronchial masculature with relatively weak cardiovascular actions. Also, /3-receptor antagonists may not act equally on bronchial and cardiovascular /3-receptors since Levy and Wilkenfeld (1969) and Wale, Pun and Rand (1969) have reported the cardioselective /3-receptor blocking action of ICI 50172. Lands, Arnold, McAuliff, Luduena and Brown (1967) proposed that/3-receptors may be divided into two types: t31-receptors associated with cardiac tissue and /32-receptors associated with bronchial smooth muscle. The tachyphylaxis and mutual suppression of effects of/3-receptor agonists observed in the cardiovascular system by Atkinson and Rand (1968) may therefore involve /31-receptors. The lack of tachyphylaxis and the synergistic effect of 13-receptor
L-Q Pun et al., Sympathomimetic bronchodilators agonists on bronchial smooth muscle may involve ~32-receptors. In any event, our findings are further evidence for a difference between fl-receptors in different tissues. It was observed that the relative bronchodilator potency of the sympathomimetics depended on whether they were injected (fig. 2) or infused (fig. 5). Furthermore, for noradrenaline and orciprenaline the order of potency differed. A possible explanation is that for injected noradrenaline, uptake into adrenergic nerve terminals plays an important part in diminishing its effect, hence injected noradrenaline was less potent than orciprenaline, but it was more potent than orciprenaline when it was given by an infusion during which uptake mechanisms may have been saturated. These experiments show that the administration of large doses of bronchodilator sympathomimetic drugs singly or in combination over some hours did not lead to tolerance and loss of bronchodilator efficacy; on the other hand tolerance developed to their effects on the heart (Atkinson and Rand, 1968). If the use of sympathomimetics in the treatment of asthma is responsible for the increase in mortality among asthmatics, our findings suggest that the explanation is not to be found in increased cardiac toxicity or to loss of bronchodilator efficacy, at least during shortterm administration.
ACKNOWLEDGEMENTS The expenses of this research were defrayed by grants from the National Health and Medical Research Council and the National Heart Foundation of Australia. One of us (L-Q.P.) was supported by grants from the Asthma Founda-
149
tion of Victoria while part of this research was in progress. We are grateful for further support and gifts of drugs from Winthrop Sterling (Aust.), and Boehringer Ingelheim (Aust.) Pry. Ltd., REFERENCES Atkinson, J.M. and M.J. Rand, 1968, Mutual suppression of cardiovascular effects of some 0-adrenoreceptor agonists in the cat, J. Pharm. Pharmacol. 20,916. Brittain, R.T., J.B. Farmer, D. Jack, L.E. Martin and W.T. Simpson, 1968, a-[t-butylamino-methyl] -4-hydroxym-xylene-c~1,a3-diol (AH.3365): a selective 0-adrenergic stimulant, Nature 219, 862. Everitt, B.J. and K.D. Cairncross, 1969, Adrenergic receptors in the guinea-pig trachea, J. Pharm. Pharmacol. 21, 97. Inman, W.H.W. and A.H. Adelstein, 1969, Rise and fall of asthma mortality in England and Wales in relation to use of pressurized aerosols, Lancet 2, 279. Keighley, G.R., 1969, Refractory asthma and adrenergic aerosols, N.Y. State J. Med. 69,662. Kelman, G.R., K.N.V. Palmer and H.R. Cross, 1969, Cardiovascular effects of AH 3365 (Salbutamol), Nature 221, 1251. Lands, A.H., A. Arnold, J.P. McAuliff, F.P. Luduena and T.G. Brown, 1967, Differentiation of receptor systems activated by sympathomimetic amines, Nature 214, 597. Levy, B. and B.E. Wilkenfeld, 1969, An analysis of selective /3-receptor blockade, European J. Pharmacol. 5, .227. McCulloch, M.W., C. Proctor and M.J. Rand, 1967, Evidence for an adrenergic homeostatic bronchodilator reflex mechanism, European J. Pharmacol. 2, 214. Metre, Van T.E., Jr., 1969, Adverse effects of inhalation of excessive amounts of nebulised isoproterenol (isoprenaline) in status asthmaticus, J. Allergy 43, 101. Speizer, F.E., R. Doll and P. Heaf, 1968, Observations on recent increase in mortality from asthma, Brit. Med. J. 1, 335. Wale, J., L-Q. Pun and M.J. Rand, 1969, The effects of ICI 50172, propranolol, pronethalol and MJ 1999 on 0-receptors, European J. Pharmacol. 8, 25.
BRONCHODILATOR EFFECTS OF SYMPATHOMIMETIC AMINES GIVEN SINGLY AND IN COMBINATION L-Q PUN, M.W. McCULLOCH and M.J. RAND Department of Pharmacology, University of Melbourne, Parkville, Victoria 3052, Australia
Accepted 28 December 1970
Received 9 March 1970
L-Q PUN, M.W. McCULLOCHand M.J. RAND, Bronchodilator effects of sympathomimetic amines given singly and in combination, European J. Pharmacol. 14 (1971) 140-149. The hronchodilator effects of a number of sympathomimetic amines were assessed in terms of reduction of histamine-induced bronchospasm. Their order of potency when given by injection 20 sec before injection of histamine was: isoprenaline ~ adrenaline > > orciprenaline > noradrenaline ~ phenylephrine; when given by infusion it was: isoprenaline ~ adrenaline ~>noradrenaline :> orciprenaline. No tachyphylaxis to the bronchodilator effect occurred during infusions of up to 2.5 hr. Combinations of the sympathomimetics, one given by injection the other by infusion, were synergistic in bronchodilator activity except for phenylephfine with orciprenaline. Sympathomimetic drugs Bronchodilator drugs
Synergistic action of sympathomimetics Histamine bronchospasm
1. INTRODUCTION Recently attention has been drawn to the increasing incidence of mortality in asthmatics in the U.K., Australia, Japan, Western Europe and the U.S.A. According to Speizer, Doll and Heaf (1968) there was no evidence to suggest that there has been any change in diagnostic habits or methods of certification of death which would account for the increase. Since 1952, there has been increasing use in the treatment of asthma of corticosteroid preparations and of pressurized aerosols containing sympathomimetic bronchodilator drugs: suspicion has been thrown on these drugs as a possible cause of the increased mortality. There has been much correspondence in medical journals suggesting that sympathomimetic bronchodilator drugs may be responsible for sudden death in asthmatics and for the increasing mortality among asthmatics (Van Metre, 1969; Kieghley, 1969; Inman and Adelstein, 1969). The hazards of bronchodilator drugs are attributed to their excessive use or to their use in combination.
Experiments on the cardiovascular effects in cats of the commonly used bronchodilator drugs isoprenaline, adrenaline and orciprenaline fared to provide evidence of cardiac toxicity when they were given by infusion to mimic excessive use, or when they were given by intravenous injections during infusions to mimic their use in combination (Atkinson and Rand, 1968). There was, however, tachyphylaxis to the effects of these drugs: thus, the responses to injections were reduced during the infusion of any of them. Tachyphylaxis developed only to the effects exerted through fl-adrenoreceptors; namely, tachycardia and vasodilation. The pressor response to an injection of adrenaline, which involves vascular ct.adrenoreceptors, was not diminished during infusions of isoprenaline or orciprenaline. These observations led us to consider that a possible hazard of excessive use of sympathomimetics in the treatment of asthma may be the development of tachyphylaxis to their bronchodilator effects, since this involves /3-receptors of bronchial smooth muscle which might be expected to react similarly to the/3-receptors of
L.Q Pun et al., Sympathomimetic bronchodilators vascular smooth muscle and of the heart. No previous experimental observations appear to have been made on the bronchodilator effects of sympathomimetics given in combination, therefore, we were prompted to study this, and also to determine if tachyphylaxis developed to the bronchodilator action of the drugs used singly.
2. METHODS Guinea-pigs of either sex weighing 250 to 700 g were used. They were anaesthetised by intraperitoneal injection of 4 ml/kg of a solution containing 250 mg/ml of urethane and 15 mg/ml of chloralose. Blood pressure was measured from the right common carotid artery by means of a cannula connected to a Statham transducer (Model P23Db) which was coupled to an Offner Dynograph pen-writer. Intravenous injections of drugs were made through a cannula inserted into the left jugular vein. Intravenous infusions were given through a cannula inserted into the right jugular vein using a Palmer slow infusion apparatus. Concentrations of drugs given by infusion were adjusted to accord with a constant volume rate of about 0.02 ml/min. The guinea-pigs were artificially ventilated with a Palmer constant volume respiration pump (25 ml capacity) through a tracheal cannula. The rate was 72 strokes/min and the volume was 2 to 6 ml/stroke, being approximately 0.8 ml per 100 g of guinea-pig body weight. Intratracheal pressure was measured by connecting a T-junction on the tracheal cannula to a Statham transducer (Model P 23Db), the output of which was coupled to the Offner Dynograph. An increase in intratracheal pressure was taken to denote bronchoconstriction (McCulloch, Proctor and Rand, 1967). Intravenous injections of histamine were given at 8 to 10 rain intervals until constant bronchoconstrictor responses to a given dose were obtained. In some experiments, constant control responses were obtained for two or three doses of histamine. The bronchodilator effects of sympathomimetic amines were determined by reduction of the bronchoconstrictor response to histamine. Intravenous injections were given 20 sec before an injection of histamine: infusions of sympathomimetics were given for at least 15 min before testing their effect on response to
141
histamine. The increases in intratracheal pressure produced by histamine injections have been expressed as a multiple of the basal intratracheal pressure which was taken as unity. The effect of the sympathomimetics was expressed as a percentage change in the histamine-induced bronchoconstriction. Each experiment was identified by a different symbol. The following drugs were used: histamine acid phosphate; dl-isoprenaline hydrochloride (Isuprel, Winthrop Laboratories); 1-adrenaline tartrate (Adrenate, Burroughs Wellcome and Co. Ltd.); 1-noradrenaline bitartrate (Levophed, Winthrop Laboratories); orciprenaline sulphate (Alupent, Boehringer Ingelheim Pty. Ltd.); and 1-phenylephrine hydrochloride (Neosynephrine, Winthrop Laboratories).
3. RESULTS 3.1. Bronchodilator effects o f sympathomimetic amines given singly 3.1.1. Bronchodilator effects of injections of sym. pathomimetic amines In the control period, constant bronchoconstrictor responses were obtained to each of three doses of histamine chosen to produce graded increases in intratracheal pressure. Fig. 1 shows the effect of the three doses of histamine in producing bronchoconstriction in 16 experiments and the effect of 2 doses of histamine in 3 experiments. The guinea-pigs varied in their bronchoconstrictor responses; the mean doses of histamine producing 50, 150 and 200 percent increase in intratracheal pressure were 2.9 (n = 19), 4.3 (n = 19) and 7.5 (n = 16)/ag/kg respectively, where n is equal to the number of observations used to calculate the mean doses. Injections of sympathomimetic amines were given 20 sec before a histamine injection; the doses were chosen to produce a decrease in the response to the middle dose of histamine of about one third. The effect of the sympathomimetic amines is shown in fig. 2. The chosen dose of each sympathomimetic amine was in general more effective in counteracting the bronchoconstrictor responses to lower than to higher doses of histamine. The relative potency of the sympathomimetic amines tested by single intravenous injection was isoprenaline ~ adrenaline > > orciprenaline > noradrenaline > phenylephrine. Records illustrating the reduction in histamine-
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induced bronchoconstriction produced by injections of isoprenaline (50ng/kg) and of phenylephrine (6/ag/kg) are shown in figs. 3 and 4 respectively, in each of which the two left hand panels should be compared. The injection of a sympathomimetic amine was not repeated until the bronchoconstriction induced by histamine had returned to control levels. The effects of adrenaline, isoprenaline and noradrenaline were no longer apparent when the next injection of histamine was given, indicating that the effect persisted for less than 8 min. However, orciprenaline and phenylephrine had more persistent effects lasting for longer than 10 rain but for less than 30 rain. 3.1.2. Bronchodilator effects of infusions of sympathomimetic amines The effects of infusions were studied in two ways: firstly, by determining the percentage reduction in histamine-induced bronchoconstriction produced by an infusion of a sympathomimetic amine; secondly,
by determining the increase in dose of histamine necessary to maintain a constant bronchoconstrictor response with increasing rates of infusion of each sympathomimetie amine. Firstly to study the effects of infusions in decreasing bronchoconstrictor responses, three doses of histamine were given. Control observations were obtained as described above, and then a rate of infusion of each sympathomimetic amine was selected such that the response to the middle dose of histamine was reduced by approximately 50%. Fig. 5 shows the effect of the infusion of four sympathomimetic amines in nineteen experiments. The percentage decrease in histamine-induced bronchospasm was greater with lower than with higher doses of histamine. The relative potency of the sympathomimetics given by infusion was isoprenaline > adrenaline :> noradrenaline > orciprenaline. The relative differences in potencies are less when the drugs Were given by infusion than when they were given by injection. Representative records showing the reductions in
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Fig. 2. The effect of injections of sympathomimetic amines in reducing bronchoconstriction induced by histamine. Each graph represents a different sympathomimetic amine, isoprenaline (ISO); adrenaline (ADR); orciprenaline (ORCI); noradrenaline (NA); phenylephrine (PE). Each experiment is identified by a different symbol, injected doses of sympat;lomimetic amines are written alongside the symbols. The sympathomimetic amines were more effective in counteracting the bronchoconstrictor responses to lwer than to higher doses of histamine.
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Fig. 3. The effect of injections of isoprenaline (50 ng/kg) ('~) in modifying histamine-induced bronchoconstriction (t), before and during the infusion of orciprenaline (100 ng/kg/min) in the anaesthetised guinea-pig. Both isoprenaline injection and orciprenaline infusion were effective in reducing the histamine-induced bronchoconstriction. The combined effect of isoprenaline injection during orciprenaline infusion was greater than when either drug was used alone. bronchoconstrictor responses to three doses of histamine by infusions of orciprenaline (100 and 50 ng/kg/min) are shown in figs. 3 and 4 in each of which the upper two panels should be compared. No observations are reported for infusions of phenylephrine, because guinea-pigs did not survive infusions at a rate necessary to cause a 50% reduction in bronchoconstrictor responses to the middle dose of histamine. Secondly to assess the bronchodilator potency o f sympathomimetic amines, the following procedure was adopted. Constant responses were obtained to a
dose of histamine that produced an increase in intratracheal pressure approximately equal to the pressure produced by the inflation stroke of the respiration pump. The infusion o f a sympathomimetic amine was then started at a low rate which was chosen to produce a just detectable decrease in the bronchoconstricter response to histamine. During the infusion, the dose of histamine was increased until it produced the same bronchoconstrictor response as before the infusion. This procedure was repeated several times, the rates of infusion being increased, and a larger equi-effective dose of histamine being found for each --ORCIPRENALINE INFUSION 5 0 n g / k g / m i n - -
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Fig. 5. The effects of infusion of sympathomimetic amines in reducing bronchoconstriction induced by histamine. Each graph represents a different sympathomimetic amine, isoprenaline (ISO); adrenaline (ADR); noradrenaline (NA), orciprenaline (ORCI). Each experiment is identified by a different symbol, infused doses of sympathomimetic amines are written along side the symbols. The percentage decrease in histamine-induced bronchospasm was greater with lower than with higher doses of histamine, as with injected sympathomimetic amines (fig. 2).
rate. The ratio of equi-effective doses of histamine during an infusion and in the control period was termed the 'histamine ratio'. The results are shown graphically in fig. 6 from which it may be seen that the relative potencies of the sympathomimetics tested were isoprenaline > adrenaline > orciprenaline > noradrenaline ~ phenylephrine. Experiments were carried out to determine whether tachyphylaxis to the bronchodilator effects of the
sympathomimetics developed after prolonged infusion. A rate of infusion was chosen for each such that the histamine ratio was about 1.5. The rates of infusion were as follows: isoprenaline, 0.01/ag/ kg/min; adrenaline, 0.04 /ag/kg/min; orciprenaline, 0.10 tag/kg/min; and noradrenaline, 0.65/~g/kg/min. During infusions of these drugs for periods of 30 rain to 2.5 hr, the histamine ratio stayed constant throughout; that is to say, tachyphylaxis was not
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produced. Typical experiments with orciprenaline and isoprenaline are shown in fig. 7. Bronchoconstrictor responses to histamine returned to control levels within 20 to 50 min after ceasing infusions of sympathomimetic amines. Prolonged infusion of phenylephrine at a rate such that the histamine ratio was greater than 1.5 was not possible since the guinea-pigs did not survive these infusions ( > 1/~g/kg/min).
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Having established separately the effects of injections and infusions of sympathomimetic amines in reducing the bronchoconstrictor responses to three doses of histamine, the effects of injections of one sympathomimetic amine were determined during the infusion of another. Each amine was either injected or infused, except phenylephrine which was given by injection only. The doses given by injection were those shown in fig. 2, and the rates of infusion were as shown in fig. 5. The effects of the combination of one injected and one infused sympathomimetic amine are shown in fig. 8, where the percentage reduction produced by the infusion of different sympathomimetic amines were plotted against the percentage reduction produced by injections during infusions of either isoprenaline, adrenaline, noradrenaline or orciprenaline. With most combinations, the injection plus the infusion had a greater effect than either the injection or the infusion given singly, the only exception observed was with injections of phenylephrine during infusions of orciprenaline.
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As with injections and infusions given singly, the percentage reductions in histamine-induced bronchoconstriction produced by combinations of injection and infusion were generally greater with lower than with higher doses of histamine. The effect of isoprenaline injections during an infusion of orciprenaline is illustrated in fig. 3; in this, the bronchoconstrictor responses to histamine when the drugs were given in combination are shown in the bottom right panel, and are clearly smaller than those
elicited during the infusion alone (top right panel) or after injection alone (bottom left panel). A similar experiment with injections of phenylephrine during an infusion of orciprenaline is shown in fig. 4 although phenylephrine was effective in reducing histamine-induced bronchoconstriction when injected alone (bottom left versus top left panel), it produced no further reduction of histamine-induced bronchoconstrictor responses that had already been reduced by infusion of orciprenaline (bottom right panel).
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3.3. Observations on cardiovascular effects o f histamine and sympathomimetic amines
4. DISCUSSION
As mentioned in the Introduction, it has been previously observed that the cardiovascular effects of injections of sympathomimetics that were mediated through 13-receptors in the cat were reduced or abolished during infusions of sympathomimetics. Since the bronchodilator effects of sympathomimetics are mediated through /3-receptors, but the effects of injections and infusions were generally synergistic in the guinea-pig, it is worth commenting on the cardiovascular effects of the sympathomimetics in the guinea-pig. The usual response to an injection of histamine in the guinea-pig was a slight fall of blood pressure which was followed by a slight rise and then a longer lasting fall. Injections of sympathomimetic drugs were given 20 sec before an injection of histamine, hence the responses to them were complicated by their interaction with histamine. With isoprenaline and orciprenaline, the depressor response was greater than with histamine alone. With noradrenaline, adrenaline and phenylephrine, the depressor response to histamine was reduced, abolished or replaced by a rise of pressure. Infusions of isoprenaline or orciprenaline at the rates used in these experiments did not affect the blood pressure of the guinea-pigs. Infusions of noradrenaline, adrenaline and phenylephrine produced an increase in blood pressure of 10 to 20 mm Hg and an increase in pulse pressure which was most marked with adrenaline infusions: the responses to histamine during these infusions consisted of either a small fall or a small rise in blood pressure, the effect being of only a few millimeters Hg either way. The injection of sympathomimetic amines (20 sec before a histamine injection) during an infusion produced a rise in blood pressure. The heart rate of the guinea-pigs was only slightly increased by injections or infusions of sympathomimetic amines. No arrhythmias occurred despite administration of injections and infusions of sympathomimetic amines, singly or in combination, for periods of up to 4 hr.
From these experiments two main points emerge; (1) tachyphylaxis to the bronchodilator effects of sympathomimetic amines did not develop on prolonged infusion; (2)the bronchodilator effects of sympathomimetic amines were generally synergistic when any two were used in combination, except when phenylephrine injections were given during orciprenaline infusion. A possible explanation is that orciprenaline being a relatively weak/3-receptor agonist had to practically saturate the /3-receptor sites before any reduction in the histamine-induced bronchoconstriction was observed. Hence further reduction by phenylephrine, another weak /3-receptor agonist was impossible. This situation would also tend to unmask the action of phenylephrine on o~-receptors. Everitt and Cairncross (1969) reported contraction in the isolated trachea of the guinea pig produced by adrenaline and noradrenaline in the presence of proprano1ol. Our findings on bronchial smooth muscle are in contrast to the observations of Atkinson and Rand (1968) on the cardiovascular effects of sympathomimetic amines involving /3-receptors, namely, that tachyphylaxis developed to the depressor effect and the tachycardia during infusions, and synergism was not observed. That there should be a difference between cardiovascular and bronchial/3-receptors is not entirely unexpected since it has been reported by Brittain, Farmer, Jack, Martin and Simpson (1968) and by Kelman and Palmer (1969) that there are ~-receptor stimulating drugs having a selective action on the bronchial masculature with relatively weak cardiovascular actions. Also, /3-receptor antagonists may not act equally on bronchial and cardiovascular /3-receptors since Levy and Wilkenfeld (1969) and Wale, Pun and Rand (1969) have reported the cardioselective /3-receptor blocking action of ICI 50172. Lands, Arnold, McAuliff, Luduena and Brown (1967) proposed that/3-receptors may be divided into two types: t31-receptors associated with cardiac tissue and /32-receptors associated with bronchial smooth muscle. The tachyphylaxis and mutual suppression of effects of/3-receptor agonists observed in the cardiovascular system by Atkinson and Rand (1968) may therefore involve /31-receptors. The lack of tachyphylaxis and the synergistic effect of 13-receptor
L-Q Pun et al., Sympathomimetic bronchodilators agonists on bronchial smooth muscle may involve ~32-receptors. In any event, our findings are further evidence for a difference between fl-receptors in different tissues. It was observed that the relative bronchodilator potency of the sympathomimetics depended on whether they were injected (fig. 2) or infused (fig. 5). Furthermore, for noradrenaline and orciprenaline the order of potency differed. A possible explanation is that for injected noradrenaline, uptake into adrenergic nerve terminals plays an important part in diminishing its effect, hence injected noradrenaline was less potent than orciprenaline, but it was more potent than orciprenaline when it was given by an infusion during which uptake mechanisms may have been saturated. These experiments show that the administration of large doses of bronchodilator sympathomimetic drugs singly or in combination over some hours did not lead to tolerance and loss of bronchodilator efficacy; on the other hand tolerance developed to their effects on the heart (Atkinson and Rand, 1968). If the use of sympathomimetics in the treatment of asthma is responsible for the increase in mortality among asthmatics, our findings suggest that the explanation is not to be found in increased cardiac toxicity or to loss of bronchodilator efficacy, at least during shortterm administration.
ACKNOWLEDGEMENTS The expenses of this research were defrayed by grants from the National Health and Medical Research Council and the National Heart Foundation of Australia. One of us (L-Q.P.) was supported by grants from the Asthma Founda-
149
tion of Victoria while part of this research was in progress. We are grateful for further support and gifts of drugs from Winthrop Sterling (Aust.), and Boehringer Ingelheim (Aust.) Pry. Ltd., REFERENCES Atkinson, J.M. and M.J. Rand, 1968, Mutual suppression of cardiovascular effects of some 0-adrenoreceptor agonists in the cat, J. Pharm. Pharmacol. 20,916. Brittain, R.T., J.B. Farmer, D. Jack, L.E. Martin and W.T. Simpson, 1968, a-[t-butylamino-methyl] -4-hydroxym-xylene-c~1,a3-diol (AH.3365): a selective 0-adrenergic stimulant, Nature 219, 862. Everitt, B.J. and K.D. Cairncross, 1969, Adrenergic receptors in the guinea-pig trachea, J. Pharm. Pharmacol. 21, 97. Inman, W.H.W. and A.H. Adelstein, 1969, Rise and fall of asthma mortality in England and Wales in relation to use of pressurized aerosols, Lancet 2, 279. Keighley, G.R., 1969, Refractory asthma and adrenergic aerosols, N.Y. State J. Med. 69,662. Kelman, G.R., K.N.V. Palmer and H.R. Cross, 1969, Cardiovascular effects of AH 3365 (Salbutamol), Nature 221, 1251. Lands, A.H., A. Arnold, J.P. McAuliff, F.P. Luduena and T.G. Brown, 1967, Differentiation of receptor systems activated by sympathomimetic amines, Nature 214, 597. Levy, B. and B.E. Wilkenfeld, 1969, An analysis of selective /3-receptor blockade, European J. Pharmacol. 5, .227. McCulloch, M.W., C. Proctor and M.J. Rand, 1967, Evidence for an adrenergic homeostatic bronchodilator reflex mechanism, European J. Pharmacol. 2, 214. Metre, Van T.E., Jr., 1969, Adverse effects of inhalation of excessive amounts of nebulised isoproterenol (isoprenaline) in status asthmaticus, J. Allergy 43, 101. Speizer, F.E., R. Doll and P. Heaf, 1968, Observations on recent increase in mortality from asthma, Brit. Med. J. 1, 335. Wale, J., L-Q. Pun and M.J. Rand, 1969, The effects of ICI 50172, propranolol, pronethalol and MJ 1999 on 0-receptors, European J. Pharmacol. 8, 25.