ACTIONS OF MOGUISTEINE ON COUGH AND PULMONARY RAPIDLY ADAPTING RECEPTOR ACTIVITY IN THE GUINEA PIG

Pharmacological Research, Vol. 35, No. 2, 1997

ACTIONS OF MOGUISTEINE ON COUGH AND PULMONARY RAPIDLY ADAPTING RECEPTOR ACTIVITY IN THE GUINEA PIG TSUGUO MORIKAWA*, LICIA GALLICO† and JOHN WIDDICOMBE *Yatsu 7-7-25, 1-602, Narashino, Chiba 275, Japan and †Boehringer Mannheim Italia SpA, Viale G. B. Stucchi 110, 1-20052 Monza, Italy Accepted 29 November 1996 With anaesthetized guinea pigs, the actions of moguisteine were tested on the cough reflex, the resting discharge of lung rapidly adapting receptors (RARs), RAR activity induced by aerosols of capsaicin, stimulation of RARs due to i.v. injection of capsaicin, and on the reflex responses to i.v. capsaicin. I.v. moguisteine (20 µg kg −1), compared with vehicle, decreased the spontaneous firing of RARs. Intragastric (i.g.) moguisteine (200 mg kg−1) had no effect on resting discharge. I.g. moguisteine depressed the cough response due to capsaicin aerosol (0.01–1 mg ml−1) and significantly reduced the increased discharge of the RARs due to the aerosol. I.v. and i.g. moguisteine reduced the proportionate increase in RAR discharge due to i.v. capsaicin (50 µg kg−1). It did not appreciably affect the cardiovascular and respiratory responses to i.v. capsaicin, which presumably activated lung C-fibre receptors. We conclude that the antitussive action of moguisteine is mediated at least in part by a decrease in the excitatory response of RARs to tussive stimuli. 1997 The Italian Pharmacological Society KEY WORDS: cough, capsaicin, moguisteine, lung receptors.

INTRODUCTION Moguisteine [(R,S)-2-(2-methoxyphenoxy)-methyl-3ethoxycarbonyl-acetyl-1,3 thiazolidine] is a novel, non-narcotic antitussive agent. In dogs it inhibits cough due to electrical stimulation of the trachea, and in guinea-pigs it depresses cough due to inhalation of citric acid or capsaicin aerosols, or due to mechanical or electrical stimulation of the trachea [1, 2]. Moguisteine suppresses clinical cough in patients with lung or airway disease [3–6]. The observation that moguisteine, unlike codeine [7] is inactive in suppressing cough when it was administered into the cerebral ventricles and that it does not appreciably change respiratory and lung mechanic variables after oral dosing suggests that the mode of action of the agent may be peripheral rather than central [2]. Other antitussive agents suppress cough by an action on lung sensory receptors [8, 9]. This conclusion is supported by the observation that moguisteine in guinea-pigs suppresses the airway hyperactivity and vascular plasma extravasation due to cigarette smoke [10]. To establish convincingly a peripheral action for an antitussive agent it is necessary to show that it inhibits activity in airway receptors responsible for cough.

Address for correspondence: Professor J. G. Widdicombe, Sherrington School of Physiology, UMDS, St Thomas’ Hospital, Lambeth Palace Road, London, SE1 7EH. 1043–6618/97/020113–06/$25.00/0/fr960122

Most evidence points to the airway rapidly adapting receptors (RARs) as the main source of cough in the lower airways, although C-fibre receptors may also be involved (see Discussion). We have therefore studied the action of moguisteine on RARs to test the site of action of the drug. In addition the effect of moguisteine on lung reflexes set up by intravenous capsaicin, which stimulates pulmonary C-fibre receptors, and by inhaled capsaicin, which causes coughing, was tested.

METHODS Guinea-pigs (Duncan Hartley, weight 0.4–0.75 kg, of either sex) were anaesthetized with urethane (1.5 g kg−1) intraperitoneally. A tracheal cannula below the larynx, a femoral arterial catheter for blood pressure recording (P23 Db, Gould) and an external jugular venous catheter for injections of drugs were inserted. A peroral-oesophageal gastric catheter was inserted. Breathing was recorded via a Fleisch pneumotachograph attached to the tracheal cannula. Intratracheal pressure was also measured. All variables were recorded on a Gould chart recorder. Both cervical vagus nerves were isolated. Initially the left vagus was cut, placed in a paraffin-filled dissecting trough, and single fibres were teased out. Action potentials were amplified (Tektronik 122), displayed on an oscilloscope and recorded on the Gould chart recorder. If a satisfactory single fibre from an 1997 The Italian Pharmacological Society

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RAR was not obtained in the left vagus, the right vagus was then cut and explored. The majority of single fibres identified were from slowly adapting pulmonary stretch receptors. These were identified by their large action potentials, their regular and slowly-adapting discharge on lung inflation, their near-linear correlation of discharge with the size of lung inflation, and their inhibition by (or occasionally a slow regular discharge) on lung deflation [11]. RARs were identified by their smaller action potentials, irregular firing pattern, vigorous response to both lung inflations and deflations with the latter usually being the more powerful stimulus, rapid adaptation to the inflations and deflations, and non-linear correlation of discharge with the size of volume changes [11]. In early experiments some were also identified by their positive response to i.v. injections of histamine, but this agent was not used for any of the results described here since it would interfere with airway function. In practice the distinction between SARs and RARs was easy, and independent observers looking at the records of firing always agreed with the definition of the receptor. Once the recording of the RAR fibre activity had been achieved, one of two protocols was pursued.

Protocol 1 After a control baseline recording of at least 2 min, capsaicin (50 µg kg−1) was injected i.v. The changes in fibre activity, airflow, intratracheal pressure and blood pressure were continuously recorded until baseline conditions or a steady state had been reached. After an interval of at least 5–10 min, DMSO (the vehicle, 0.4%, 0.2 ml kg−1) was injected i.v. while recording variables. After another interval of 5–10 min, the capsaicin injection was repeated. Five to ten mins later, moguisteine (20 µg kg−1, as 0.2 ml kg−1 solution) in DMSO was injected i.v. Five to ten min later the capsaicin administration was repeated. If the RAR remained viable, 5–10 min later moguisteine (200 mg kg−1) was administered through the gastric tube (i.g.). DMSO was not included. After 30 min the capsaicin i.v. injection was repeated.

Protocol 2 After control recording of variables, a stream of capsaicin aerosol was passed through a T-tube connected to the pneumotachograph and tracheal cannula, the gas flowing at 1 l min−1 and the concentration strong enough to induce coughing, as tested before fibre recording. In practice the concentration was usually 10 µg ml−1, but on one occasion was as high as 1 mg ml−1. The aerosol was generated from a modified DeVilbiss ultrasonic nebulizer, that gave an aerodynamic mass median diameter of 7–8 µm. Aerosol administration was continued for 2 min with continuous recording of variables. After a recovery period (15–30 min), moguisteine (200 mg kg−1) was administered i.g. After a further 30 min, administration of the

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capsaicin aerosol was repeated. Five control experiments were done to test for tachyphylaxis of cough to capsaicin aerosol. The animals had both vagus nerves intact and capsaicin aerosol was given at times 0, 30 and 60 min, without administration of moguisteine.

Analysis of results For each intervention (capsaicin, moguisteine and DMSO) variables were analysed of 15–60 s periods before and after the intervention, and at intervals subsequently. When the intervention caused marked changes in respiration (eg. apnoea or coughing) these were noted but were not usually quantitated because of difficulties in interpretation. (For example, the change between apnoea and rapid shallow breathing is difficult to mark precisely; the distinction between a cough and a deep inspiration/expiration is likewise problematical—see below; even if respiratory frequency and peak flow are identical, the pattern of flow may have changed and is difficult to quantitate simply.) We therefore report: (1) fibre discharge; (2) respiratory frequency; (3) peak inspiratory-expiratory flow difference as an index of respiratory effort; (4) blood pressure, the mean of systolic and diastolic pressure; and (5) heart rate. Unless otherwise stated, these have all been averaged over 15-s periods. Since the animals were not breathing through their larynges, they could not produce a true cough. However, after capsaicin aerosol (but not with other interventions), the guinea-pigs often made respiratory efforts that resembled coughing, i.e. a deep breath followed by what seemed to be a forced expiration. These were apparent on the flow record, and will be referred to as coughs. They were only seen when one vagus was intact or, in the absence of fibre recording, when both vagi were intact.

Statistical analysis Whether or not the intervention caused a response was tested by Student’s paired t-test comparing measurements before with measurements after the intervention. Whether or not moguisteine or DMSO changed the responses to capsaicin was similarly tested, comparing the size of the response to capsaicin before the injection with that after. Both types of analysis were therefore with paired data. Results and changes are expressed as absolute values. However, to show more clearly the size of the effect, in the Tables percentage changes are also given. These are percentage changes based upon means rather than percentage changes in individual tests re-expressed as mean percentages. The reason is that for some variables, action potentials occasionally and coughing always, the control values were zero so that any percentage increase would be infinity which would invalidate averaging the percentages.

Drugs Moguisteine was from Boehringer Mannheim Italia;

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it was dissolved in 0.4% DMSO for i.v. injection, and given as a suspension in 0.15 M NaCl for i.g. administration. Capsaicin was obtained from Sigma; it was dissolved in 10% ethanol and 10% Tween 80, and diluted with 0.15 M NaCl solution.

RESULTS

Effects of moguisteine and DMSO (Table I) Before assessment of the effects of moguisteine on responses to capsaicin it was important to see whether the former had any action on the variables being studied which might affect the results. In particular, we wished to see whether moguisteine changed the spontaneous discharge of RARs; such an action might explain any antitussive effects. Since i.v. moguisteine was given as a solution with DMSO, the latter as vehicle was also tested. Table I summarizes the results. Moguisteine (20 µg kg−1 i.v.) in DMSO was tested on four RARs (Table I). Initial hypotensions and depressions of breathing recovered quickly (within 1–2 min), and after recovery there were no significant differences between respiratory rate, heart rate and blood pressure compared with controls before moguisteine. Of the four RARs studied, three showed a decrease in firing; the fourth, silent during the control, responded with a few impulses. The mean decrease in firing frequency was not statistically significant. DMSO, alone (0.2 ml kg−1 i.v.) transiently decreased respiratory frequency and blood pressure in four tests, with recovery usually within 1–2 min. After recovery there were no significant changes in respiratory rate, heart rate and blood pressure. Of the four RARS studied, three showed moderate increases in frequency of firing after DMSO; the fourth had an usually high resting frequency (10.4 impulses s−1) which considerably decreased after DMSO. The mean change (−1.63 impulses s−1, −45%) was not statistically significant.

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The results with RARs and i.v. DMSO and moguisteine were analysed by subtracting the changes due to DMSO from those due to moguisteine. Moguisteine caused a statistically significant (−16.5±4.1%, n=4, P <0.05) decrease in RAR firing frequency. When moguisteine (200 mg kg−1) was given i.g. on seven preparations, an interval of 30 min was left to allow gastric absorption. At the end of this time there were no significant changes in any of the four variables measured compared with control values before moguisteine (Table I).

Effect of i.g. moguisteine on responses to capsaicin aerosols (Table II) Capsaicin aerosol caused several coughs (see Discussion) in most of the animals, followed by a moderate but significant increase in respiratory frequency (Table II). The size of peak flow difference was little affected. Heart rate and blood pressure remained unchanged. The most conspicuous response was an increase in the firing of RARs, seen in all five instances (Fig. 1). The increase of firing was far out of proportion to the changes in breathing, and is unlikely to have been caused by them; this conclusion was supported by examination of control records comparing the pattern of breathing with RAR firing frequency. After i.g. moguisteine, capsaicin aerosol still had no effect on heart rate or blood pressure (Table II). Respiratory frequency and peak flow difference due to capsaicin were not statistically significant from those before moguisteine. However the capsaicin caused fewer coughs and this difference was statistically significant. Capsaicin still significantly increased the discharge of all the RARs tested but the size of the increase was significantly reduced to about half the control increase (Fig. 1). This reduction in activity is unlikely to be explained by changes in respiration due to capsaicin aerosol which were if anything larger after moguisteine. To test whether the cough responses to capsaicin aerosol showed tachyphylaxis, in five experiments cough was induced by capsaicin

Table I Effects of i.v. and i.g. moguisteine on measured variables Drug

n

Variable

Before Drug

After drug Absolute

% Change

Moguisteine i.v. (20 µm kg−1)

4

Action potential (s−1) Respiratory rate (min−1) Peak flow (arb) HR (min−1) BP (mm Hg)

5.00±2.88 26.3±4.1 25.2±3.7 213±8.9 60.8±5.2

2.35±0.82 24.1±2.8 26.4±4.5 210±8.5 66.0±3.7

−53 −8 +10 −1 +9

Moguisteine i.g. (200 mg kg−1)

7

Action potential (s−1) Respiratory rate (min−1) Peak flow (arb) HR (min−1) BP (mm Hg)

1.23±0.36 28.1±3.9 18.0±2.6 241±16.4 54.7±1.7

1.13±0.47 27.0±4.1 20.3±4.2 236±16.7 57.0±2.3

−8 −4 +11 −2 +4

Means±SEM . No changes were statistically significant. arb=arbitrary units.

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Effect of moguisteine on responses to i.v. capsaicin (Table III)

Action potentials (imps s–1)

20

15

10 * 5

0

Control

Caps

Control Moguist.

Caps

Fig. 1. Mean action potential discharges from RARs in response to capsaicin aerosols. The first column shows control discharge; the second column the response to inhalation of capsaicin aerosol; the third column the resting discharge 15–30 min later; the fourth column the resting discharge 30 min after moguisteine 200 mg kg−1 i.g; the last column the subsequent response to capsaicin aerosol. *P< 0.05 for before and after moguisteine.

(2 min) at times of 0, 30 and 60 min, without moguisteine administration. The three stimuli caused 7.2±3.1, 5.8±2.0 and 8.2±4.6 coughs respectively, suggesting that tachyphylaxis had not occurred.

Intravenous capsaicin decreased blood pressure and heart rate significantly, although the change in the latter was small (Table III). After moguisteine (i.v., n=3; i.g., n=2), i.v. capsaicin caused responses of similar size. Intravenous capsaicin caused apnoea and rapid shallow breathing of variable pattern. Coughing was never seen. Although these changes in breathing are difficult to quantitate, the records indicated that similar respiratory changes were seen when capsaicin was administered before and after moguisteine. All five RARs were strongly stimulated by i.v. capsaicin (Table III). It seems unlikely that these responses were secondary to respiratory changes since apnoea and shallow breathing would decrease rather than increase the firing of RARs. After moguisteine (i.v. and i.g.) capsaicin stimulated the receptors to a similar mean maximum firing compared with controls. However the baseline discharge was greater, presumably due to the i.v. injections of capsaicin and, when expressed as proportionate changes in firing frequency, the increase in discharge was significantly smaller. For the three RAR’s tested with i.v. moguisteine, controls with DMSO vehicle showed no significant effect on the RAR response to capsaicin. Before DMSO, capsaicin increased the activity of 19.7±6.21 impulses s−1, and after DMSO by 22.5±4.30

Table II Responses to capsaicin aerosols before and after i.g. moguisteine Variable

Before moguisteine Before capsaicin

Action potentials (s−1) Respiratory rate (min−1) Peak flow (arb) Coughs (absolute) HR (min−1) BP (mm Hg)

1.29±0.58 30.8±3.3 21.8±3.3 0 254±13 57±1.4

After moguisteine

After capsaicin Absolute

% change

12.06±5.34* 37.7±3.5* 23.3±3.4 2.6±0.7* 253±14 57±1.6

+835* +22* +7 — 0 0

Before capsaicin

After capsaicin Absolute

1.53±0.74 28.8±4.6 23.5±3.3 0 246±16 53±2.8

6.56±2.70*† 43.7±9.7* 25.5±4.9 0.8±0.4† 243±14 57±2.1

% change +329*† +52* +9 — −1 0

Means±SEM, n=5; *P<0.05 for response to capsaicin; †P<0.05 for after moguisteine (200 mg kg −1, i.g.) compared with before. arb=arbitrary units.

Table III Responses to i.v. capsaicin before and after moguisteine Variable

Before moguisteine Before capsaicin −1

Action potentials (s ) HR (min−1) BP (mm Hg)

0.86±2.11 198±17.8 60±4.7

After moguisteine

After capsaicin Absolute

% change

24.46±3.73* 190±18.5* 48±6.6*

+2737* −4* −20*

Before capsaicin 2.13±0.69 192±13.8 62±4.4

After capsaicin Absolute

% change

24.82±4.39* 185±1.88* 48±5.6*

+106*† −4* −23*

Means±SEM, n=5 (n=3 for moguisteine 20 µg kg−1 i.v.; n=2 for moguisteine 200 mg kg−1 i.g.); *P<0.05 for response to capsaicin; †P<0.05 for after moguisteine compared with before.

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impulses s−1. When the DMSO values were subtracted from those for moguisteine, capsaicin caused smaller absolute (+23.6±5.72 impulses s−1 before, +20.5±7.13 impulses s−1 after) and percentage (+1804% before, +590% after) responses after moguisteine.

DISCUSSION I.v. moguisteine inhibited the spontaneous discharge of RARs more than did the carrier DMSO. Given i.g., it significantly inhibited the coughs and the stimulation of RAR’s due to capsaicin aerosol. And given i.g. and i.v. it significantly inhibited the stimulation of RAR’s due to i.v. capsaicin. In experiments of this type it is desirable to stabilize the RAR preparation as much as possible by paralysis and artificial ventilation of the animal, and to study prompt responses to i.v. moguisteine to avoid long-term shifts in baseline. With our experimental preparation it proved impossible to administer enough capsaicin aerosol through a respiratory pump with paralysed animals to cause fictive cough; this may be due to the small size of the apparatus and connecting tube. Therefore RAR activity was recorded during spontaneous breathing and changes in breathing pattern might affect the RARs. The results, however, did not include activities during large changes in respiration such as coughing or apnoeas, and the respiratory responses to capsaicin before and after administration of moguisteine were always closely similar, apart from the suppression of cough, and seem most unlikely to account for the large changes in RAR firing seen with capsaicin and depressed by moguisteine. Even if paralysed artificially-ventilated animals had been used it is still difficult to determine whether an effect is ‘direct’ or ‘indirect’. For example, capsaicin might induce changes in bronchomotor tone, particularly conspicuous in the guinea-pig [12] and possibly changes in mucus secretion, blood pressure and haemodynamic variables in the lungs [13]. These reflex changes are due to activation by capsaicin of both lung C-fibre receptors [13] and lung RARs [14, 15]. One cannot therefore be certain under any experimental circumstances that the response of an RAR to a chemical is ‘direct’. We had hoped to test moguisteine i.v. since the short time-scale of its actions would have removed the possible complication of changing baselines over a longer period. However we could only give i.v. moguisteine in solution in DMSO, and initial tests showed that there were powerful and immediate strong respiratory and cardiovascular responses, especially with doses larger than 20 µg kg−1. Even with this particular dose, the response to the vehicle (DMSO) was closely similar to that of DMSO plus moguisteine, suggesting that the DMSO was causing the pronounced effects. Since i.v. injections of these small doses of moguisteine would probably not test adequately the antituss-

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ive actions of the drug, we resorted to administration of moguisteine i.g. This had the disadvantage that we had to wait 30 min, to allow maximal drug absorption [16]. It had the advantages that i.g. moguisteine seemed to cause no significant shifts to respiratory, cardiovascular and RAR baselines, that presumably the blood concentrations of moguisteine were more stable for repeated testing with capsaicin, and that DMSO did not need to be used. We tried to measure ‘coughs’ after capsaicin aerosol. Since the animals were not breathing through their larynges, strictly speaking they could not cough. However direct observation and examination of the flow record showed that they made pronounced respiratory efforts consisting of a deep inspiration followed by a forced expiration. These efforts we have assumed to be coughs. Moguisteine i.g. inhibited the cough response to inhaled capsaicin, reducing it to about one-third. Since only one RAR could be studied in each guinea pig, and dose-response relationships for moguisteine could not be established, we deliberately chose a high dose of moguisteine compared with other studies [2]. In our experiments deep anaesthesia was needed for successful recording of nerve action potentials, whereas Gallico et al. [2] used unanesthetized or lightly unanesthetized guinea-pigs. Deep anaesthesia may depress the responsiveness to moguisteine. The drug also decreased the RAR response to capsaicin aerosol, more than halving it. In view of the evidence that RARs are the sensory endings responsible for cough [17–19] these two results are presumably causally related. We did not attempt to obtain doseresponse curves with moguisteine, or try to follow the time-course of inhibition of cough and RAR activity after i.g. administration of the drug, since the duration of such experiments would have been much greater than the life-span of RAR recordings. The depression of RAR response to capsaicin aerosol after moguisteine is unlikely to be due to tachyphylaxis to capsaicin, since the latter provoked similar cough and respiratory changes over the same time course without moguisteine. Intravenous moguisteine may have diminished the sensitivity of RARs stimulated by capsaicin i.v. This conclusion is bound to be tentative because i.v. capsaicin causes pronounced cardiovascular and respiratory changes, the latter being difficult to quantitate. In addition, depression of the RAR responses to i.v. capsaicin by moguisteine was shown not from absolute values of impulse frequency change but from proportional changes in receptor activity. Thus i.v. capsaicin increased the spontaneous discharge of RAR’s, possibly by secretion of mucus or by causing neurogenic inflammatory changes [20]. The relative decrease in response to RARs to i.v. capsaicin after moguisteine is unlikely to be due to tachyphylaxis to capsaicin, since the latter produced similar respiratory and cardiovascular changes after moguisteine.

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Moguisteine itself, given i.v., inhibited spontaneous RAR activity, although the absolute change was not statistically significant, possibly due to the small N-values and one aberrant result. When the changes due to the vehicle (DMSO) were subtracted from those due to moguisteine plus DMSO then the response to moguisteine became statistically significant. This observation is consistent with the results of moguisteine on responses of coughing and RAR activity to capsaicin. Moguisteine suppresses the bronchial hyperactivity and vascular plasma extravasation due to cigarette smoke in guinea-pigs [10]. These responses are thought to be due to stimulation of RARs in this species [21], a view consistent with the belief that moguisteine inhibits RAR activity. Moguisteine, at least when given i.v., does not seem to affect pulmonary C-fibre reflexes, assessed from cardiovascular changes activated by i.v. capsaicin. Examination of the experimental records suggests that the respiratory changes due to i.v. capsaicin are also not influenced by i.v. moguisteine, but this point may need further testing. Capsaicin-induced vascular plasma extravasation is not blocked by moguisteine, a result consistent with the view that the agent is not acting primarily on C-fibre receptors. Capsaicin i.v. never caused cough, which has never been described in many experiments on anaesthetized and unanesthetized animals of many species with i.v. drug stimulation of pulmonary C-fibres [18, 19]. Indeed their stimulation inhibits cough in the cat [22]. The fact that i.v. capsaicin stimulates RARs but does not cause cough may be because the tussive response is inhibited by simultaneous activation of C-fibre receptors. We conclude that moguisteine can depress the resting discharge of RAR’s when given i.v., and inhibits the cough reflex and excitation of RAR activity due to capsaicin. Its antitussive action is presumably by inhibition of RAR activity rather than via C-fibre receptors.

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4.

5.

6.

7. 8. 9. 10.

11. 12.

13.

14. 15.

16.

17.

ACKNOWLEDGEMENTS TM was supported by Boehringer Mannheim Italia, to whom we are also grateful for the supply of moguisteine.

18. 19.

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