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Peripheral site of action of levodropropizine in experimentally-induced cough: Role of sensory neuropeptides
Pulmonary Pharmacology (1992) 5, 143-147
PULMONARY PHARMACOLOGY
Peripheral Site of Action of Levodropropizine in Experimentally-induced Cough: Role of Sensory Neuropeptides A. Lavezzo, G. Melillo, G . Clavenna, C . Omini Dompe Research and Development Laboratories, Dompe Farmaceutici S .p .A ., Milan, Italy
SUMMARY : The mechanism of action of levodropropizine has been investigated in different models of experimentally-induced cough in guinea-pigs . In particular it has been demonstrated that the antitussive drug has a peripheral site of action by injecting the drug intracerebroventricularly (i .c.v.). In these experiments levodropropizine (40 µg/50 gI i .c .v .) did not prevent electrically-induced cough . On the other hand, codeine (5 µg/50 µl i.c.v.) markedly prevented coughing . A difference in the potency ratio of levodropropizine and codeine has been demonstrated in capsaicin-induced cough ; after oral administration, codeine was about two to three times more potent than levodropropizine . However, after aerosol administration the two compounds were equipotent . These data might suggest a peripheral site of action for levodropropizine which is related to sensory neuropeptides . Further support for the role of sensory neuropeptides in the mechanism of action of levodropropizine comes from the results obtained in capsaicin-desensitized animals . In this experimental model levodropropizine failed to prevent the vagally elicited cough in neuropeptide-depleted animals, whereas codeine did not differentiate between control and capsaicintreated animals. In conclusion, our results support the suggestion that levodropropizine has a peripheral site of action . In addition, the interference with the sensory neuropeptide system may explain, at least in part, its activity in experimentally-induced cough .
the same compound, renamed 443C81, given by aerosol in human was not active in preventing the respiratory responses of sub-tussive doses of capsaicin . 10 Levodropropizine is an antitussive drug in current clinical use which does not interact with opioid receptors ." In addition, it has already been demonstrated that levodropropizine is active against cough induced by different experimental methods" and in clinical trials ." In particular, levodropropizine, in animal experiments, is active against cough induced by irritants such as citric acid, NH4OH and H2S04 and by electrical stimulation of the tracheal mucosa and vagus nerve." Levodropropizine both in human and animal studies showed only minor side-effects, particularly at the CNS level ." In order to further characterize the mechanism of action of levodropropizine we performed some experiments with the aim of confirming the peripheral site of action of the compound and possible involvement with the sensory neuropeptide system . In fact, it has been shown that sensory neuropeptides, and particularly substance P, may be involved in the cough reflex ." Nadel's group' 1,16 has shown that neutral endopeptidase inhibitors such as phosphoramidon potentiate substance P- and capsaicin-induced cough in the guinea-pig . These data,
INTRODUCTION Cough is a common feature of respiratory infection, chronic bronchitis and asthma, this protective reflex helps to clear the airways of excessive mucus production and inspired noxious agents .' Cough may often be a reflection of airway hyperreactivity which typically accompanies the asthmatic syndrome' and in some patients it may occur as the main symptom of asthma.' Until recently the cough-centre in the CNS was believed to be the only site of action of the classical antitussive drugs such as codeine .' ,' Today a peripheral effect has been suggested for codeine, in particular acting on peripheral opioid receptors . These receptors have been localized in the peripheral nerves' including vagal sensory neurons' even if the functional significance of these receptors has to be completely investigated .' In addition to this, Adcock et al' reported that BW443C, a pentapeptide opioid agonist, at doses consistent with non-penetration into the CNS, inhibited citric acid-induced cough in guinea-pig, indicating the presence of peripheral opioid receptors in the lung . More recently, however, it has been shown that 0952-0600/92/020143 + 05 $03 .00/0
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together with that obtained by other groups, 14,17 strongly support an important role for sensory neuropeptides in the cough reflex .
MATERIALS AND METHODS Cough induced by electrical stimulation of the guinea-pig trachea Male Dunkin-Hartley guinea-pigs (350-450 g ; Rodentia, Italy) fasted for 24 h were anaesthetized with urethane (1 .2 g/kg i .p .) and the trachea exposed . Two stainless steel electrodes (0 .2 mm diameter) were wrapped around the trachea and threaded under the skin out through the back of the animal ." Cough was induced by electrical stimulation (20 s) with the following characteristics : 0 .6 ms, 40 Hz, 2-8 V . The compounds (levodropropizine and codeine, dissolved in saline) and saline (controls) were administered intracerebroventricularly (i .c.v .) in a constant volume of 50 gl through a cannula (PEI 0) inserted into the right ventricle . The number of coughs induced by electrical stimuli was recorded before and 1, 3 and 5 min after i .c .v . drug administrations .
used . This sequence was repeated every 10 min . The mean value of three sequences obtained before the delivery of drugs or saline was considered to be the basal value . The antitussive effect of the compounds was calculated 5, 15 and 25 min after the drug injection, as percent inhibition over the basal mean . Capsaicin desensitization in guinea-pigs Capsaicin desensitization was performed in guineapigs according to the method of Gamse et al 19 as modified by Del Monte et al .2' Briefly, increasing doses of capsaicin (0 .3, 0 .6, 1 .2 and 2 .4 mg/kg s .c . on the first day and 2 .5, 10, 15 and 20 mg/kg s .c . on the second day) were injected at 2-h intervals . Fifteen minutes before each dose of capsaicin, the animals received (i .p .) a cocktail of agonists and antagonists with the following composition : atropine (1 .25 mg/ kg) ; pyrilamine (2 .5 mg/kg) ; aminophylline (10 mg/ kg) ; and salbutamol (0 .1 mg/kg) . Pentobarbitone sodium (10-20 mg/kg i .p .) was also injected 15 min before the first four doses of capsaicin to minimize its irritating effect . The guinea-pigs which survived (80%) this treatment were used 5-15 days later . Statistical analysis
Capsaicin-induced cough in the awake guinea-pig Guinea-pigs (details as before) were placed (one at the time) into a perspex box (20 x 14 x 12 cm) with two holes (1 cm diameter), to provide fresh air, and allowed to relax . An aerosol of capsaicin (0 .0045% w/v in ethanol/H 2O 10/90 solution) was generated for 4 min using an ultrasonic nebuliser (DeVilbiss, Pulmosonic, DeVilbiss Co ., Somerset, PA, USA) connected to a Palmer pump adjusted to 64 strokes/min and 12 ml per stroke . The IN and OUT limbs of the ventilator pump were connected to the two holes of the box . The number of coughs within 4 min and the time of the first cough were recorded blind; the drugs were administered by os and by aerosol 60 min and 30 s respectively, before capsaicin . Cough induced by vagal stimulation in the guinea-pig Guinea-pigs (details as before) were anaesthetized with urethane (1 .2 g/kg) and tracheotomized . The changes in tracheal pressure were recorded on a sidearm of a tracheal cannula via a pressure transducer (Bentley Trantec, Basile, Italy) connected to a pen recorder (Gemini, Basile, Italy) . The vagus nerve was isolated and electrically stimulated (0 .1-0 .5 ms, 2-5 V, 25 Hz) with bipolar electrodes connected to a Grass Stimulator (Grass Instruments Co ., Quincy, MA, USA) . A stimulation period of 10 s followed by a second 10-s stimulation period after a 1-min interval was
The comparison of the different treatments and groups was performed according to the Friedman analysis of variance for non-parametric data ." Drugs The drugs used were as follows : levodropropizine (Dompe, Farmaceutici S . p . a, Milan, Italy) ; codeine (SIFAC, Milan, Italy); capsaicin, pyrilamine, aminophylline, atropine, salbutamol, and urethane (Sigma, St Louis, MO, USA) ; pentobarbitone (CFM, Milan, Italy) ; ethanol (Carlo Erba, Milan, Italy) .
RESULTS Levodropropizine (40 gg/50 µl i .c .v .) did not modify the degree of coughing induced by electrical stimulation of the trachea in the guinea-pig at any of the times considered (Fig . 1) . On the other hand, codeine (5 gg/50 gl i .c .v .) markedly inhibited cough in the same experimental conditions (Fig . 1) . In the capsaicin aerosol experiments both levodropropizine and codeine markedly inhibited cough induced by the irritating compound . In particular, the two drugs showed a different reciprocal potency depending on the route of administration used (Table 1) . In fact, after oral administration the ratio of the ED 50 calculated for the two compounds was 1 .74, indicating that levodropropizine was almost two times less potent than codeine . Conversely, after aerosol
Action of Levodropropizine in Experimental Cough
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Time (min) Fig. 1 Effects of intracerebroventricular (i.c .v .) administration of levodropropizine (40 µg; ∎) and codeine (5 µg ; 0) on cough evoked by tracheal stimulation in guinea-pig . Each column represents the mean± SE of ten different experiments . The analysis of variance for non-parametric data gave the following statistical differences between pre- and post-treatments : levodropropizine P>0 .05 ; codeine P<0.05 .
administration the relative potency of the two compounds was inverted, levodropropizine being equiactive to or more active than codeine (Table 1) . In order to substantiate the possible role of sensory neuropeptides in the mechanism of action of levodropropizine we investigated the capacity of this compound to prevent cough induced by electrical stimulation of the vagus nerve in the capsaicin-desensitized guinea-pig . In this experimental model capsaicin pretreatment slightly, but statistically significantly (P < 0 .05) increased the number of electrically-evoked coughs when compared to controls, ranging from 7.7 ± 0 .8 (n =10) to 10 .3 ± 0 .6 (n =10) in control and capsaicin-desensitized animals, respectively . Levodropropizine (10 mg/kg i.v .) inhibited the vagus-mediated cough in control animals (Fig . 2) . However, in the capsaicin-desensitized animals, levodropropizine lost its capacity to prevent the onset of cough almost completely. Codeine (1 .5 mg/kg i.v .) did not discriminate between normal and desensitized animals . In fact, in both groups the protective activity of codeine was evident and the degree of activity was similar . Table 1 Effects of levodropropizine and codeine on capsaicin aerosol-induced cough in conscious guinea-pigs . ED, Compounds
Oral (mg/kg)
Aerosol (w/v %)
Levodropropizine
129 (85-196) 74 (52-107)
1 .19 (0 .98-1 .45) 1 .37 (1 .09-1 .73)
Codeine
Each ED, was calculated from ten different experiments . Values in parentheses are the 95% confidence limits ; P<0 .05 .
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20
25
30
Fig. 2 Effects of levodropropizine (10 mg/kg i .v .) and codeine (1 .5 mg/kg i.v.) on vagally (electrically-stimulated) evoked cough reflex in control and capsaicin-desensitized guinea-pig . Each column represents the mean ± SE of five different experiments . The analysis of variance for non-parametric data gave the following statistical differences between pre- and post-treatments : Control animals: levodropropizine P<0 .02 ; codeine P<0.02. Capsaicin-desensitized animals : levodropropizine P>0.05; codeine P<0 .02. (0) Levodropropizine control ; (0) codeine control ; (ii) levodropropizine capsaicin-desensitized ; (0) codeine capsaicin-desensitized .
DISCUSSION Levodropropizine is a well-known antitussive drug currently used in therapy, with a peripheral site of action that does not involve interaction with opioid receptors." The results presented here confirm and further support the evidence of a peripheral site of action for levodropropizine . The comparison with codeine has shown clearly that the two compounds behave differently in all the experimental procedures used . In particular, after i .c .v . injection codeine, but not levodropropizine, is able to antagonize electrically-induced cough . We used a single dose of the compound due to the technical difficulties involved in using this experimental procedure . However, the dose used (40 tg/50 µl) was very high and it has already been demonstrated that given by os levodropropizine was one-fifth as active as codeine in the same experimental model of electrically-induced cough .' When higher dose-ratio (eight times) was used, the antitussive activity of codeine was very evident, whereas levodropropizine was completely inactive after i .c .v . injection . These results may indicate that levodropropizine has no capacity to interact with CNS structures related to the cough-centre. The involvement of sensory neuropeptides in the cough reflex is well documented, in fact the exogenous administration of substance P induces cough"" and it has been suggested that its mechanism of action is
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related to a direct stimulation of sensory nerves ." The differences in potency ratio between levodropropizine and codeine, when given by os or by aerosol, in the inhibition of capsaicin-induced cough may suggest that levodropropizine is more selective for the peripheral sensory nerves related to the cough reflex . On the other hand, the different rate of metabolism of the two compounds after oral administration should be taken into account; levodropropizine is rapidly metabolized in humans22 even if the therapeutic effect of the compound is present for at least 6-8 h . 23 The possible involvement of sensory neuropeptides in the mechanism/s of action of levodropropizine is suggested by the results obtained in capsaicindesensitized guinea-pig . In this model of cough the capacity of levodropropizine is markedly reduced by the depletion of sensory neuropeptides by capsaicin, 19 whereas the effect of codeine is not affected by capsaicin pretreatment . Apart from the fact that these results further emphasize the different sites of action of the two compounds, they support the existence of an inhibitory effect of levodropropizine on the release or activity of sensory neuropeptides, particularly against substance P . In this regard, preliminary data' suggest that the levodropropizine aerosol is active in inhibiting the airway hyperreactivity induced by cigarette smoke inhalation, an effect which is dependent on the release of sensory neuropeptides . 25 However, an inhibition of levodropropizine on the sensory function of airway afferent nerves could not be ruled out. Therefore, substance P and related peptides may not be the only targets of levodropropizine since we found a small but statistically significant increase in the number of coughs induced by vagal stimulation in capsaicin-desensitized guinea-pigs as compared to controls . These data may indicate that after neuropeptide depletion, some adaptative phenomena might occur in order to maintain the cough reflex efficient . In addition, it is possible to speculate that vagal stimulation, besides excitatory neuropeptides (substance P), also releases inhibitory peptides which could modulate the vagal-induced cough . Hence, an alternative explanation for the lack of activity of levodropropizine in sensory neuropeptide depleted animals is that levodropropizine may release an inhibitory peptide which may also be depleted by capsaicin treatment . In conclusion, our results indicate that levodropropizine is a potent peripherally acting antitussive drug and that its mechanism of action may be, at least in part, interference with the capsaicin-sensitive sensory neuropeptide system . References I . Freedman S. Mechanism of ventilation . In : Brewis R A L, Gibson G J, Geddes D M, eds . Respiratory medicine . London : Bailliere Tindall 1990 : 114-130.
2 . Widdicombe J G . Respiratory reflex and defense . In : Brain D J, Proctor D F, Reid L M, eds . Respiratory defense mechanism. New York : M . Dekker Inc . 1977 : 593-630 . 3 . Corrao W M, Braman S S, Irwin R S . Chronic cough as the sole presenting manifestation of bronchial asthma . N Engl J Med 1979 ; 300 : 633 . 4. Eddy N B, Friebel H, Hahn K J, Halbach H . Codeine and its alternates for pain and cough relief. 3 . The antitussive action of codeine-mechanisms, methodology and evaluation . Bull WHO 1969 ; 40 : 425-454 . 5 . Salem H, Aviado D M . Antitussive drugs with special reference to a new theory for the initiation of the cough reflex and the influence of bronchodilators. Am J Med Sci 1964 ; 247 : 585-600 . 6 . Atwen S F, Murrin L C, Kuhar M J . Presynaptic localisation of opiate receptors in the vagal and accessory optic system : an autoradiographic study . Neuropharmacology 1978 ; 17 : 65-71 . 7 . Young W S, Wamsley J K, Zarbin M A, Kuhar M J . Opioid receptors undergo axonal flow . Science 1980 ; 210: 76-78 . 8 . Adcock J J . Peripheral opioid receptors and the cough reflex . Respir Med 1991 ; 85 (suppl . A) : 43-46 . 9 . Adcock J J, Schneider C, Smith T W . Effects of codeine, morphine and a novel opioid pentapeptide BW443C, on cough, nociception and ventilation in the unanaesthetized guinea-pig. Br J Pharmacol 1988 ; 93 : 93-100 . 10 . Choudry N B, Gray S J, Callaghan J M, Posner J M, Fuller R W . Effect of 443C81 on the respiratory responses to capsaicin in humans . Eur J Pharm 1990; 183 : 1095 . 11 . Melillo G, Malandrino S, Rossoni G, et al . General pharmacology of the new antitussive levodropropizine . Arzneim Forsch/Drug Res 1988 ; 38 : 1144-1150. 12 . Malandrino S, Melillo G, Bestetti A, Borsa M, Giuliani P, Tonon G C . Ar4itussive properties of levodropropizine . Arzneim Forsch/Drug Res 1988 ; 38 : 1141-1143 . 13 . Allegra L, Bossi R . Clinical trials with the new antitussive levodropropizine in adult bronchitic patients . Arzneim Forsch/Drug Res 1988; 38 : 1163-1166 . 14 . Forsberg K, Karlsson J A . Cough induced by stimulation of capsaicin-sensitive sensory neurons in conscious guinea-pigs . Acta Physiol Scand 1986 ; 128: 319-320 . 15 . Kohrogi H, Graf P D, Sekizawa K, Borson D B, Nadel J A . Neutral endopeptidase inhibitors potentiate substance P- and capsaicin-induced cough in awake guinea pigs . J Clin Invest 1988 ; 82 : 2063-2068 . 16 . Kohrogi H, Nadel J A, Malfroy B, et al . Recombinant human enkephalinase (neutral endopeptidase) prevents cough induced by tachykinins in awake guinea pigs . J Clin Invest 1989; 84 : 781-786 . 17 . Karlsson J A, Sant'Ambrogio G, Widdicombe J . Afferent neural pathways in cough and reflex bronchoconstriction . J Appl Physiol 1988; 65 : 1007-1023 . 18 . Cavanagh R L, Gylys J A, Bierwagen M E . Antitussive properties of butorphanol . Arch Int Pharmacodyn 1976 ; 220 : 258-268 . 19. Gamse R, Wax A, Zigmond R E, Leeman S E . Immunoreactive substance P in sympathetic ganglia : distribution and sensitivity towards capsaicin . Neuroscience 1981 ; 6 : 437 . 20 . Del Monte M, Omini C, Subissi A . Ganglion-blocking agents enhance neurally mediated bronchoconstriction in the guinea-pig : possible role of sensory neuropeptides . Eur J Pharmacol 1990; 180 : 239-245 . 21 . Friedman M . The use of ranks to avoid the assumption of normality implicit in the analysis of variance . J Am Stat Assoc 1937; 32 : 675-701 . 22 . Zaratin P, De Angelis L, Cattabeni F . Gas chromatographic-mass spectrometric determination of levodropropizine plasma levels in healthy volunteers . Arzneim Forsch/Drug Res 1988 ; 38 : 1156-1158 . 23 . Bossi R, Braga P C, Centanni S, Legnani D, Moavero N E, Allegra L . Antitussive activity and respiratory system effects of levodropropizine in man . Arzneim Forsch/Drug Res 1988 ; 38: 1159-1162 . 24 . Daffonchio L, Hernandez A, Melillo G, Omini C . The antitussive drug levodropropizine prevents airway
Action of Levodropropizine in Experimental Cough hyperreactivity induced by exposure to cigarette smoke in guinea-pig . Eur Respir J 1991 ; 4 (suppl . 14) : 228s. 25. Daffonchio L, Hernandez A, Gallico L, Omini C . Airways hyperreactivity induced by active cigarette smoke exposure in guinea pig : possible role of sensory neuropeptides . Pulmon Pharmacol 1990; 3 : 1-6 .
Date received : 6 August 1991 Date revised : 11 September 1991 Date accepted: 19 November 1991
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PULMONARY PHARMACOLOGY
Peripheral Site of Action of Levodropropizine in Experimentally-induced Cough: Role of Sensory Neuropeptides A. Lavezzo, G. Melillo, G . Clavenna, C . Omini Dompe Research and Development Laboratories, Dompe Farmaceutici S .p .A ., Milan, Italy
SUMMARY : The mechanism of action of levodropropizine has been investigated in different models of experimentally-induced cough in guinea-pigs . In particular it has been demonstrated that the antitussive drug has a peripheral site of action by injecting the drug intracerebroventricularly (i .c.v.). In these experiments levodropropizine (40 µg/50 gI i .c .v .) did not prevent electrically-induced cough . On the other hand, codeine (5 µg/50 µl i.c.v.) markedly prevented coughing . A difference in the potency ratio of levodropropizine and codeine has been demonstrated in capsaicin-induced cough ; after oral administration, codeine was about two to three times more potent than levodropropizine . However, after aerosol administration the two compounds were equipotent . These data might suggest a peripheral site of action for levodropropizine which is related to sensory neuropeptides . Further support for the role of sensory neuropeptides in the mechanism of action of levodropropizine comes from the results obtained in capsaicin-desensitized animals . In this experimental model levodropropizine failed to prevent the vagally elicited cough in neuropeptide-depleted animals, whereas codeine did not differentiate between control and capsaicintreated animals. In conclusion, our results support the suggestion that levodropropizine has a peripheral site of action . In addition, the interference with the sensory neuropeptide system may explain, at least in part, its activity in experimentally-induced cough .
the same compound, renamed 443C81, given by aerosol in human was not active in preventing the respiratory responses of sub-tussive doses of capsaicin . 10 Levodropropizine is an antitussive drug in current clinical use which does not interact with opioid receptors ." In addition, it has already been demonstrated that levodropropizine is active against cough induced by different experimental methods" and in clinical trials ." In particular, levodropropizine, in animal experiments, is active against cough induced by irritants such as citric acid, NH4OH and H2S04 and by electrical stimulation of the tracheal mucosa and vagus nerve." Levodropropizine both in human and animal studies showed only minor side-effects, particularly at the CNS level ." In order to further characterize the mechanism of action of levodropropizine we performed some experiments with the aim of confirming the peripheral site of action of the compound and possible involvement with the sensory neuropeptide system . In fact, it has been shown that sensory neuropeptides, and particularly substance P, may be involved in the cough reflex ." Nadel's group' 1,16 has shown that neutral endopeptidase inhibitors such as phosphoramidon potentiate substance P- and capsaicin-induced cough in the guinea-pig . These data,
INTRODUCTION Cough is a common feature of respiratory infection, chronic bronchitis and asthma, this protective reflex helps to clear the airways of excessive mucus production and inspired noxious agents .' Cough may often be a reflection of airway hyperreactivity which typically accompanies the asthmatic syndrome' and in some patients it may occur as the main symptom of asthma.' Until recently the cough-centre in the CNS was believed to be the only site of action of the classical antitussive drugs such as codeine .' ,' Today a peripheral effect has been suggested for codeine, in particular acting on peripheral opioid receptors . These receptors have been localized in the peripheral nerves' including vagal sensory neurons' even if the functional significance of these receptors has to be completely investigated .' In addition to this, Adcock et al' reported that BW443C, a pentapeptide opioid agonist, at doses consistent with non-penetration into the CNS, inhibited citric acid-induced cough in guinea-pig, indicating the presence of peripheral opioid receptors in the lung . More recently, however, it has been shown that 0952-0600/92/020143 + 05 $03 .00/0
143
© 1992 Academic Press Limited
144
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together with that obtained by other groups, 14,17 strongly support an important role for sensory neuropeptides in the cough reflex .
MATERIALS AND METHODS Cough induced by electrical stimulation of the guinea-pig trachea Male Dunkin-Hartley guinea-pigs (350-450 g ; Rodentia, Italy) fasted for 24 h were anaesthetized with urethane (1 .2 g/kg i .p .) and the trachea exposed . Two stainless steel electrodes (0 .2 mm diameter) were wrapped around the trachea and threaded under the skin out through the back of the animal ." Cough was induced by electrical stimulation (20 s) with the following characteristics : 0 .6 ms, 40 Hz, 2-8 V . The compounds (levodropropizine and codeine, dissolved in saline) and saline (controls) were administered intracerebroventricularly (i .c.v .) in a constant volume of 50 gl through a cannula (PEI 0) inserted into the right ventricle . The number of coughs induced by electrical stimuli was recorded before and 1, 3 and 5 min after i .c .v . drug administrations .
used . This sequence was repeated every 10 min . The mean value of three sequences obtained before the delivery of drugs or saline was considered to be the basal value . The antitussive effect of the compounds was calculated 5, 15 and 25 min after the drug injection, as percent inhibition over the basal mean . Capsaicin desensitization in guinea-pigs Capsaicin desensitization was performed in guineapigs according to the method of Gamse et al 19 as modified by Del Monte et al .2' Briefly, increasing doses of capsaicin (0 .3, 0 .6, 1 .2 and 2 .4 mg/kg s .c . on the first day and 2 .5, 10, 15 and 20 mg/kg s .c . on the second day) were injected at 2-h intervals . Fifteen minutes before each dose of capsaicin, the animals received (i .p .) a cocktail of agonists and antagonists with the following composition : atropine (1 .25 mg/ kg) ; pyrilamine (2 .5 mg/kg) ; aminophylline (10 mg/ kg) ; and salbutamol (0 .1 mg/kg) . Pentobarbitone sodium (10-20 mg/kg i .p .) was also injected 15 min before the first four doses of capsaicin to minimize its irritating effect . The guinea-pigs which survived (80%) this treatment were used 5-15 days later . Statistical analysis
Capsaicin-induced cough in the awake guinea-pig Guinea-pigs (details as before) were placed (one at the time) into a perspex box (20 x 14 x 12 cm) with two holes (1 cm diameter), to provide fresh air, and allowed to relax . An aerosol of capsaicin (0 .0045% w/v in ethanol/H 2O 10/90 solution) was generated for 4 min using an ultrasonic nebuliser (DeVilbiss, Pulmosonic, DeVilbiss Co ., Somerset, PA, USA) connected to a Palmer pump adjusted to 64 strokes/min and 12 ml per stroke . The IN and OUT limbs of the ventilator pump were connected to the two holes of the box . The number of coughs within 4 min and the time of the first cough were recorded blind; the drugs were administered by os and by aerosol 60 min and 30 s respectively, before capsaicin . Cough induced by vagal stimulation in the guinea-pig Guinea-pigs (details as before) were anaesthetized with urethane (1 .2 g/kg) and tracheotomized . The changes in tracheal pressure were recorded on a sidearm of a tracheal cannula via a pressure transducer (Bentley Trantec, Basile, Italy) connected to a pen recorder (Gemini, Basile, Italy) . The vagus nerve was isolated and electrically stimulated (0 .1-0 .5 ms, 2-5 V, 25 Hz) with bipolar electrodes connected to a Grass Stimulator (Grass Instruments Co ., Quincy, MA, USA) . A stimulation period of 10 s followed by a second 10-s stimulation period after a 1-min interval was
The comparison of the different treatments and groups was performed according to the Friedman analysis of variance for non-parametric data ." Drugs The drugs used were as follows : levodropropizine (Dompe, Farmaceutici S . p . a, Milan, Italy) ; codeine (SIFAC, Milan, Italy); capsaicin, pyrilamine, aminophylline, atropine, salbutamol, and urethane (Sigma, St Louis, MO, USA) ; pentobarbitone (CFM, Milan, Italy) ; ethanol (Carlo Erba, Milan, Italy) .
RESULTS Levodropropizine (40 gg/50 µl i .c .v .) did not modify the degree of coughing induced by electrical stimulation of the trachea in the guinea-pig at any of the times considered (Fig . 1) . On the other hand, codeine (5 gg/50 gl i .c .v .) markedly inhibited cough in the same experimental conditions (Fig . 1) . In the capsaicin aerosol experiments both levodropropizine and codeine markedly inhibited cough induced by the irritating compound . In particular, the two drugs showed a different reciprocal potency depending on the route of administration used (Table 1) . In fact, after oral administration the ratio of the ED 50 calculated for the two compounds was 1 .74, indicating that levodropropizine was almost two times less potent than codeine . Conversely, after aerosol
Action of Levodropropizine in Experimental Cough
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Time (min) Fig. 1 Effects of intracerebroventricular (i.c .v .) administration of levodropropizine (40 µg; ∎) and codeine (5 µg ; 0) on cough evoked by tracheal stimulation in guinea-pig . Each column represents the mean± SE of ten different experiments . The analysis of variance for non-parametric data gave the following statistical differences between pre- and post-treatments : levodropropizine P>0 .05 ; codeine P<0.05 .
administration the relative potency of the two compounds was inverted, levodropropizine being equiactive to or more active than codeine (Table 1) . In order to substantiate the possible role of sensory neuropeptides in the mechanism of action of levodropropizine we investigated the capacity of this compound to prevent cough induced by electrical stimulation of the vagus nerve in the capsaicin-desensitized guinea-pig . In this experimental model capsaicin pretreatment slightly, but statistically significantly (P < 0 .05) increased the number of electrically-evoked coughs when compared to controls, ranging from 7.7 ± 0 .8 (n =10) to 10 .3 ± 0 .6 (n =10) in control and capsaicin-desensitized animals, respectively . Levodropropizine (10 mg/kg i.v .) inhibited the vagus-mediated cough in control animals (Fig . 2) . However, in the capsaicin-desensitized animals, levodropropizine lost its capacity to prevent the onset of cough almost completely. Codeine (1 .5 mg/kg i.v .) did not discriminate between normal and desensitized animals . In fact, in both groups the protective activity of codeine was evident and the degree of activity was similar . Table 1 Effects of levodropropizine and codeine on capsaicin aerosol-induced cough in conscious guinea-pigs . ED, Compounds
Oral (mg/kg)
Aerosol (w/v %)
Levodropropizine
129 (85-196) 74 (52-107)
1 .19 (0 .98-1 .45) 1 .37 (1 .09-1 .73)
Codeine
Each ED, was calculated from ten different experiments . Values in parentheses are the 95% confidence limits ; P<0 .05 .
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10 15 Time (min)
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Fig. 2 Effects of levodropropizine (10 mg/kg i .v .) and codeine (1 .5 mg/kg i.v.) on vagally (electrically-stimulated) evoked cough reflex in control and capsaicin-desensitized guinea-pig . Each column represents the mean ± SE of five different experiments . The analysis of variance for non-parametric data gave the following statistical differences between pre- and post-treatments : Control animals: levodropropizine P<0 .02 ; codeine P<0.02. Capsaicin-desensitized animals : levodropropizine P>0.05; codeine P<0 .02. (0) Levodropropizine control ; (0) codeine control ; (ii) levodropropizine capsaicin-desensitized ; (0) codeine capsaicin-desensitized .
DISCUSSION Levodropropizine is a well-known antitussive drug currently used in therapy, with a peripheral site of action that does not involve interaction with opioid receptors." The results presented here confirm and further support the evidence of a peripheral site of action for levodropropizine . The comparison with codeine has shown clearly that the two compounds behave differently in all the experimental procedures used . In particular, after i .c .v . injection codeine, but not levodropropizine, is able to antagonize electrically-induced cough . We used a single dose of the compound due to the technical difficulties involved in using this experimental procedure . However, the dose used (40 tg/50 µl) was very high and it has already been demonstrated that given by os levodropropizine was one-fifth as active as codeine in the same experimental model of electrically-induced cough .' When higher dose-ratio (eight times) was used, the antitussive activity of codeine was very evident, whereas levodropropizine was completely inactive after i .c .v . injection . These results may indicate that levodropropizine has no capacity to interact with CNS structures related to the cough-centre. The involvement of sensory neuropeptides in the cough reflex is well documented, in fact the exogenous administration of substance P induces cough"" and it has been suggested that its mechanism of action is
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related to a direct stimulation of sensory nerves ." The differences in potency ratio between levodropropizine and codeine, when given by os or by aerosol, in the inhibition of capsaicin-induced cough may suggest that levodropropizine is more selective for the peripheral sensory nerves related to the cough reflex . On the other hand, the different rate of metabolism of the two compounds after oral administration should be taken into account; levodropropizine is rapidly metabolized in humans22 even if the therapeutic effect of the compound is present for at least 6-8 h . 23 The possible involvement of sensory neuropeptides in the mechanism/s of action of levodropropizine is suggested by the results obtained in capsaicindesensitized guinea-pig . In this model of cough the capacity of levodropropizine is markedly reduced by the depletion of sensory neuropeptides by capsaicin, 19 whereas the effect of codeine is not affected by capsaicin pretreatment . Apart from the fact that these results further emphasize the different sites of action of the two compounds, they support the existence of an inhibitory effect of levodropropizine on the release or activity of sensory neuropeptides, particularly against substance P . In this regard, preliminary data' suggest that the levodropropizine aerosol is active in inhibiting the airway hyperreactivity induced by cigarette smoke inhalation, an effect which is dependent on the release of sensory neuropeptides . 25 However, an inhibition of levodropropizine on the sensory function of airway afferent nerves could not be ruled out. Therefore, substance P and related peptides may not be the only targets of levodropropizine since we found a small but statistically significant increase in the number of coughs induced by vagal stimulation in capsaicin-desensitized guinea-pigs as compared to controls . These data may indicate that after neuropeptide depletion, some adaptative phenomena might occur in order to maintain the cough reflex efficient . In addition, it is possible to speculate that vagal stimulation, besides excitatory neuropeptides (substance P), also releases inhibitory peptides which could modulate the vagal-induced cough . Hence, an alternative explanation for the lack of activity of levodropropizine in sensory neuropeptide depleted animals is that levodropropizine may release an inhibitory peptide which may also be depleted by capsaicin treatment . In conclusion, our results indicate that levodropropizine is a potent peripherally acting antitussive drug and that its mechanism of action may be, at least in part, interference with the capsaicin-sensitive sensory neuropeptide system . References I . Freedman S. Mechanism of ventilation . In : Brewis R A L, Gibson G J, Geddes D M, eds . Respiratory medicine . London : Bailliere Tindall 1990 : 114-130.
2 . Widdicombe J G . Respiratory reflex and defense . In : Brain D J, Proctor D F, Reid L M, eds . Respiratory defense mechanism. New York : M . Dekker Inc . 1977 : 593-630 . 3 . Corrao W M, Braman S S, Irwin R S . Chronic cough as the sole presenting manifestation of bronchial asthma . N Engl J Med 1979 ; 300 : 633 . 4. Eddy N B, Friebel H, Hahn K J, Halbach H . Codeine and its alternates for pain and cough relief. 3 . The antitussive action of codeine-mechanisms, methodology and evaluation . Bull WHO 1969 ; 40 : 425-454 . 5 . Salem H, Aviado D M . Antitussive drugs with special reference to a new theory for the initiation of the cough reflex and the influence of bronchodilators. Am J Med Sci 1964 ; 247 : 585-600 . 6 . Atwen S F, Murrin L C, Kuhar M J . Presynaptic localisation of opiate receptors in the vagal and accessory optic system : an autoradiographic study . Neuropharmacology 1978 ; 17 : 65-71 . 7 . Young W S, Wamsley J K, Zarbin M A, Kuhar M J . Opioid receptors undergo axonal flow . Science 1980 ; 210: 76-78 . 8 . Adcock J J . Peripheral opioid receptors and the cough reflex . Respir Med 1991 ; 85 (suppl . A) : 43-46 . 9 . Adcock J J, Schneider C, Smith T W . Effects of codeine, morphine and a novel opioid pentapeptide BW443C, on cough, nociception and ventilation in the unanaesthetized guinea-pig. Br J Pharmacol 1988 ; 93 : 93-100 . 10 . Choudry N B, Gray S J, Callaghan J M, Posner J M, Fuller R W . Effect of 443C81 on the respiratory responses to capsaicin in humans . Eur J Pharm 1990; 183 : 1095 . 11 . Melillo G, Malandrino S, Rossoni G, et al . General pharmacology of the new antitussive levodropropizine . Arzneim Forsch/Drug Res 1988 ; 38 : 1144-1150. 12 . Malandrino S, Melillo G, Bestetti A, Borsa M, Giuliani P, Tonon G C . Ar4itussive properties of levodropropizine . Arzneim Forsch/Drug Res 1988 ; 38 : 1141-1143 . 13 . Allegra L, Bossi R . Clinical trials with the new antitussive levodropropizine in adult bronchitic patients . Arzneim Forsch/Drug Res 1988; 38 : 1163-1166 . 14 . Forsberg K, Karlsson J A . Cough induced by stimulation of capsaicin-sensitive sensory neurons in conscious guinea-pigs . Acta Physiol Scand 1986 ; 128: 319-320 . 15 . Kohrogi H, Graf P D, Sekizawa K, Borson D B, Nadel J A . Neutral endopeptidase inhibitors potentiate substance P- and capsaicin-induced cough in awake guinea pigs . J Clin Invest 1988 ; 82 : 2063-2068 . 16 . Kohrogi H, Nadel J A, Malfroy B, et al . Recombinant human enkephalinase (neutral endopeptidase) prevents cough induced by tachykinins in awake guinea pigs . J Clin Invest 1989; 84 : 781-786 . 17 . Karlsson J A, Sant'Ambrogio G, Widdicombe J . Afferent neural pathways in cough and reflex bronchoconstriction . J Appl Physiol 1988; 65 : 1007-1023 . 18 . Cavanagh R L, Gylys J A, Bierwagen M E . Antitussive properties of butorphanol . Arch Int Pharmacodyn 1976 ; 220 : 258-268 . 19. Gamse R, Wax A, Zigmond R E, Leeman S E . Immunoreactive substance P in sympathetic ganglia : distribution and sensitivity towards capsaicin . Neuroscience 1981 ; 6 : 437 . 20 . Del Monte M, Omini C, Subissi A . Ganglion-blocking agents enhance neurally mediated bronchoconstriction in the guinea-pig : possible role of sensory neuropeptides . Eur J Pharmacol 1990; 180 : 239-245 . 21 . Friedman M . The use of ranks to avoid the assumption of normality implicit in the analysis of variance . J Am Stat Assoc 1937; 32 : 675-701 . 22 . Zaratin P, De Angelis L, Cattabeni F . Gas chromatographic-mass spectrometric determination of levodropropizine plasma levels in healthy volunteers . Arzneim Forsch/Drug Res 1988 ; 38 : 1156-1158 . 23 . Bossi R, Braga P C, Centanni S, Legnani D, Moavero N E, Allegra L . Antitussive activity and respiratory system effects of levodropropizine in man . Arzneim Forsch/Drug Res 1988 ; 38: 1159-1162 . 24 . Daffonchio L, Hernandez A, Melillo G, Omini C . The antitussive drug levodropropizine prevents airway
Action of Levodropropizine in Experimental Cough hyperreactivity induced by exposure to cigarette smoke in guinea-pig . Eur Respir J 1991 ; 4 (suppl . 14) : 228s. 25. Daffonchio L, Hernandez A, Gallico L, Omini C . Airways hyperreactivity induced by active cigarette smoke exposure in guinea pig : possible role of sensory neuropeptides . Pulmon Pharmacol 1990; 3 : 1-6 .
Date received : 6 August 1991 Date revised : 11 September 1991 Date accepted: 19 November 1991
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