Naunyn-Schmiedeberg's Naunyn-Schmiedeberg's Arch. Pharmacol. 306, 203-207 (1979)
Archivesof
Pharmacology 9 by Springer-Verlag 1979
Comparison of Sodium Nitroprusside and Isoprenaline Aerosols in Histamine-Induced Bronchial Asthma of the Guinea Pig* Volker A. W. Kreye and Erich Marquard II. Physiologisches Institut and Anatomisches Institut III, Universifftt Heidelberg, Im Neuenheimer Feld 326, D-6900 Heidelberg, Federal Republic of Germany
Summary. 1. Sodium nitroprusside (NP) is a potent
relaxant of the guinea pig's tracheal smooth muscle contracted by carbachol or histamine. 2. At high doses, aerosols of NP delay the occurrence of symptoms of bronchial asthma in guinea pigs subjected to a histamine aerosol. However, the fladrenergic bronchodilator isoprenaline is at least 2,600 times more potent as an anti-asthmatic than NP on a molar basis. 3. NP and isoprenaline both reduce the blood pressure in rats when given either as infusion or as aerosol. The aerosol/infusion dosage ratio for comparable reductions of blood pressure were 1 for isoprenaline, and 10 for NP. After cessation of the application of the NP aerosol, the blood pressure rose more than twice as fast (t~/2 of blood pressure recovery) than after cessation of the application of the isoprenaline aerosol. 4. The poor antiasthmatic effect of NP aerosols may result from low permeability of the bronchial epithelium to the NP anion, from partial destruction in the airways or during tissue permeation, and from short half life of NP in the circulating blood. 5. Because of its inferiority to known bronchodilators, aerosols of NP cannot be recommended for the treatment of acute bronchial asthma. Key words: Antiasthmatics - Bronchodilators Nitroprusside - Isoprenaline - Pulmonary drug resorption - Blood pressure effects.
Introduction
Sodium nitroprusside [Na2Fe(CN)sNO-2HzO; Nipride ~; Nipruss "~; NP) is an extremely potent reSend offpr&t requests to V. A. W. Kreye at the above address * This work was supported by the German Research Foundation (DFG) within SFB 90 "Cardiovascular System". The experiments were performed at the Pharmacological Institute, University of Heidelberg
laxant of isolated vascular smooth muscle. In an investigation on the sensitivity to the relaxant action of NP of various different types of smooth muscle from rats and guinea pigs, only the tracheal chain of the guinea pig was found to respond similarly well to NP (Kreye et al., 1975). This finding suggested that NP might be a useful relaxant of the bronchiolar tree. Therefore, we have investigated in guinea pigs the anti-asthmatic effect of NP aerosols on histamineinduced asthma. As a reference substance, the known bronchodilator drug, isoprenaline, was included in the study. In preceding in vitro experiments, the relaxant potency (ICs0) of both drugs on histamine- and carbachol-contracted tracheal smooth muscle was studied. Finally, the effect of inhaled NP and isoprenaline aerosols on the time-course of blood pressure changes was studied in rats. From these latter experiments, some information on the invasion of the drugs from the bronchiolar/alveolar lumen into the blood compartment was expected. Methods a) In vitro Studies on Isolated Tracheal Smooth Muscle. Male guinea pigs weighing 250 - 300 g were killed by a blow on the head, and their tracheae were excised. Under chilled physiological salt solution, tracheal rings were prepared according to Castillo and de Beer (1947). For each tracheal chain, 3 rings were tied together with thread. In organ baths (volume 10 ml) containing physiological salt solution (37~C; gassed with 95 % O z - 5 % CO2 ; pH 7.2), the tracheal chains were suspended at an initial tension of 12 raN. The composition of the physiological salt solution, and the procedures for measuring isometric tension changes have been described in detail elsewhere (Kreye et al., 1975). The tracheal chains were contracted by the addition of histamine or carbachol to reach bath concentrations of 5 • I 0 - ~ M or 10 -7 M, respectively. When this stimulation had resulted in a constant muscular tone, NP or isoprenaline (Aludrin ,"~; Boehringer, Ingelheim) were added in increasing doses in a cumulative fashion. From the resulting dose-response curves, single ICs0 values were obtained graphically, and used for the calculation of geometrical means of ICs0. b) Induction of Asthma by Histamine Aerosol, and Effect of NP or [soprenaline Aerosols. For the production of aerosols, a modification
0028-1298/79/0306/0203/$01.00
204 of the method described by Halpern (1942) was used. Two streams of compressed air were passed through bottles filled with distilled water for humidification, and then led via two atomizers (Palmer and Kingsbury, 1952; aerosol particle size: 1 - 2 p ) into an animal chamber made ofacryl glass and having a volume of 6.51. Opposite to the inlets coming from the atomizers, two holes in the wall of the animal chamber permitted the escape of the aerosol vapors into the open air via an exhaust pipe. To control the compressed air stream, a manometer an(i a volumeter to measure air pressure and flow, respectively, and a three way valve for immediate interruption of the aerosols were placed between the humidifyer bottles and the atomizers. During the experiments, the air pressure in the system was kept at 72.57 kPa above atmospheric pressure, and the flow rate at 4.5 l/rain; this resulted in the vaporisation of 0.15ml/min of the fluid in the atomizers. One of the atomizers contained a solution of I mg histamine dihydrochloride/ml distilled water to produce the asthmatogenic histamine aerosol. The other atomizer either contained pure distilled water (Regimen A) or solutions of the bronchodilator drugs in distilled water at various concentrations: isoprenaline sulphate 10 gg/ml (B), 33 pg/ml (C), and 100 gg/ml (D); NP - 3.3 mg/ml (E), 10mg/ml (F), and 30mg/mI (G). According to a randomisation scheme, 21 guinea pigs were subdivided into 7 groups of 3 animals each. Per experimental day, one of the groups was submitted to one of the regimens A to G. For the selection of animal groups and regimens, a cross-over design had been elaborated so that at the end of the experiments, all the animals had been subjected to all of the regimens. Single guinea pigs were placed into the center of the animal chamber, and observed by the experimentator. According to Preuner (1939), the following stages of asthma development can be distinguished in the guinea pig: 0 - no reaction 1 - slight reaction, i.e. slight coughs 2 - increased breathing frequency with involvement of auxiliary respiratory muscles 3 - moderate orthopnoea at reduced breathing frequency 4 - severe orthopnoea with the head elevated and the extremities extended 5 - collapse, and start of clonic seizures 6 - death. The time from the start of the histamine aerosol to reaching stage 5 is identical with the "preconvulsion time" as defined by Herxheimer (1952). In our experiments, the time to reach stages 1 to 5 was used as the criterion to judge the anti-asthmatic effects of the bronchodilator drugs. When the animals had reached stage 5, the aerosol was stopped immediately, and the chamber was aerated. The maximal duration of the animals' exposures to histamine and drug aerosols was restricted to 15 rain (i.e. 900 s). With the higher doses of bronchodilator drugs, several animals did not reach all stages of asthma development; in these cases, for the evaluation a time ofg00 s was assumed arbitrarily.
c) Effects of NP and Isoprenaline Given by Aerosol or Infusion on Blood Pressure. These experiments were performed in rats as this species is a standard preparation for the recording of blood pressure in our laboratory~ For anaesthesia, Sprague-Dawley rats weighing about 300 g received 1.4 g urethane/kg body weight s.c. One carotid artery and one jugular vein were dissected, and polyethylene catheters (internal diameter: 0.5ram; external diameter: 1.0mm) were introduced into the vessels. The arterial catheter was connected to a Statham pressure transducer P23Db; after amplification, the blood pressure dependent electrical signals were recorded on a Siemens Kompensograph III servo chart recorder. By the jugular catheter, aqueous solutions of drugs in the concentrations stated below could be infused with an infusion pump at a rate of 0.1 ml/min. To record the effects ot~NP and isoprenaline aerosols on blood-pressure, the
Naunyn-Schmiedeberg's Arch. Pharmacol. 306 (1979) anaesthetized animals were placed into the animal chamber, and the aerosols were produced as described above. The aim of the study was to compare the time courses of quantitatively similar blood-pressure changes. In preliminary experiments, the following concentrations of aqueous NP and isoprenaline solutions for aerosol production and infusion, respectively, had been found to be roughly equidepressor, and were used for the experiments: solutions for aerosols - 6rag NP/ml, 0.3 mg isoprenaline/ml; solutions for infusions - 20 gg NP/ ml, 10 pg isoprenaline/ml. Taking into account the vaporization rate of the atomizers, the flow rate of the aerosols, and the tidal respiratory volume of rats (103 ml/min ; H6bel et al., 1971), an intrapulmonary administration of 20.6 ng NP/min and of 1 ng isoprenaline/min was calculated, whereas the rates of i.v. infusions were 2 ng NP/min and 1 ng isoprenaline/min, respectively.
d) StatisticalEvaluations. By using the R/s-ratio method described by David et al. (1954), normal distribution of the results could not be established. Therefore, the Wilcoxon test was used for the control of significance of the results where necessary.
Results A. In vitro Studies on Isolated Tracheal Smooth Muscle T r a c h e a l c h a i n s o f the g u i n e a p i g s t i m u l a t e d e i t h e r by 1 0 - T M c a r b a c h o l o r 5 • 1 0 - T M h i s t a m i n e w e r e rel a x e d b y t h e c u m u l a t i v e a d d i t i o n o f N P in a c o n c e n t r a t i o n r a n g e o f 10 - 9 to ] 0 - 6 M , a n d by i s o p r e naline in a c o n c e n t r a t i o n r a n g e o f 10 - 1 ~ to 10 - 7 M, r e @ e c t i v e l y . T h e c o n c e n t r a t i o n s for h a l f - m a x i m a l rel a x a t i o n (ICso) o f the p r e p a r a t i o n s w e r e d e t e r m i n e d g r a p h i c a l l y f r o m the single d o s e - r e s p o n s e curves, and u s e d for t h e c a l c u l a t i o n o f g e o m e t r i c a l m e a n s + S.E. (n = 4) o f I C s o - v a l u e s . W i t h the c a r b a c h o l - c o n t r a c t e d tissues, the I C s o for N P w a s f o u n d to be 1.04 ( + 0 . 5 5 ; - 0 . 3 6 ) • 1 0 - T M a n d f o r i s o p r e n a l i n e 3.61 ( + 0 . 5 0 ; - 0 . 4 4 ) • l 0 - 9 M . W i t h the h i s t a m i n e - c o n t r a c t e d tissues, t h e I C s o f o r N P was 3.05 ( + 0 . 9 3 ; -0.66) • 10 - 8 M , a n d for i s o p r e n a l i n c 1.62 ( + 0 . 2 3 ; - 0 . 2 0 ) x 10 . 9 M . T h u s , N P is a p o t e n t r e l a x a n t o f t r a c h e a l s m o t h m u s c l e , a l t h o u g h in this tissue i s o p r e n a l i n e is o f g r e a t e r p o t e n c y ; as c o m p a r e d to i s o p r e n a l i n e , a 2 9 - f o l d h i g h e r m o l a r c o n c e n t r a t i o n o f N P was n e e d e d f o r halfmaximal relaxation of carbachol-stimulated tracheal s m o o t h m u s c l e , a n d a 19-fold h i g h e r c o n c e n t r a t i o n in histamine-stimulated preparations.
B. Effect o f N P and Isoprenaline on Histamine-Induced Asthma M o s t o f t h e g u i n e a pigs s u b j e c t e d to the h i s t a m i n e a e r o s o l d e v e l o p e d t h e t y p i c a l signs o f b r o n c h i a l a s t h m a a c c o r d i n g to t h e c r i t e r i a g i v e n u n d e r M e t h o d s . O f these criteria, s t a g e 2 ( i n c r e a s e d b r e a t h i n g f r e q u e n c y ) a n d s t a g e 5 (collapse) c o u l d be o b s e r v e d w i t h the l e a s t s u b j e c t i v e e r r o r since these s y m p t o m s a p p e a r e d abruptly. T h e t i m e c o u r s e o f a s t h m a d e v e l o p m e n t is g i v e n
V. A. W. Kreye and E. Marquard: Nitroprusside Aerosols in Bronchial Asthma x 100 9
l
8 84
iii ii 1 --TIT
IS
100 p g l m l
IS
33 ,ug I ml
IS
10 p g / m l
NP
3 0 mg I ml
NP
10 mglml
NP
3.3 mg I ml
Control
s
0 1
2
3
4
, S t a g e of A s t h m a 5
Fig. 1. Development of bronchial asthma in guinea pigs (n = 17) subjectedto aerosolsof histaminedihydrochloride(1 mg/mlsolution) in the absence(Control)and presenceof sodiumnitroprusside(NP)or isoprenaline sulphate (IS) aerosols. The concentrationsof the drug solutions used for the aerosolsare given.Abscissa : stage of asthmatic reaction according to Preuner (1939) (see text); ordinate; time to reach stage of asthmaticreactionin s (~ + S.E.).Maximalduration of experiments was 900 s. When animals did not reach an appropriate stage of reaction, for evaluation a time of 900s was assumed arbitrarily
in Fig. 1. For unknown reasons, 4 of the 21 animals did not react with bronchial asthma to the histamine aerosol during 900 s, i.e. t h e maximal duration of the experiments. Although these animals were also submitted to all other regimens because of the random• sat• scheme, they were eliminated from the final evaluation of the study resulting in a number of animals of only 17. Both bronchodilator drugs as aerosols prolonged the time course of asthma development (Fig. 1) or even prevented asthma development, but considerable differences in their protective effects were found. Whereas NP aerosols of solutions containing 3.3mg/ml, 10mg/ml, and 30mg/ml abolished the histamineinduced collapse (stage 5) in 2, 3, and 3 animals, respectively, isoprenaline in the concentrations of 10 lag/ml, 33 ktg/ml, and 100 ~tg/ml protected 10, 15, and 16 of the 17 animals from the asthmatic collapse. F r o m these results and the time courses given in Fig. 1 it is clear that the lowest dose of isoprenaline (10 pg/ml) had a greater antiasthmatic effect than the highest dose of N P (30 mg/ml). With these doses, the
205
time to reach asthma stage 2 was similar for both drugs (10~tg/ml isoprenaline: 483.3 +_ 96.7s; 30mg/ml NP: 471.3 • 63.3 s). This permits a rough estimate of the ant• efficacy of the two drugs : for a comparable ant• effect, the dose of N P needed was 3,000fold higher than that of isoprenaline on a weight basis, and 2,600-fold higher on a molar basis.
C. Effects of Infused and Inhaled N P or lsoprenaline on Blood Pressure At the concentrations given, both N P and isoprenaline produced comparable reductions of the blood pressure in rats when administered by infusion or as an aerosol. The following changes in blood pressure were found (~? • S.E., n = 8; percentage figures in brackets represent percentage changes from initial blood pressures): infusion of 2ng NP/min, - 3 6 . 3 • 4 . 5 m m H g ( - 4 1 . 4 + 1.8 ~o); aerosol of N P (approx. 20.6 rig/rain), - 33.0 • (-39.9• infusion of l n g isoprenaline/min, - 3 0 . 9 • 2.7ram Hg ( - 4 1 . 7 • 1.2 ~ ) ; aerosol of isoprenaline (approx. 1 ng/min), - 2 6 . 9 + 4.3 m m H g ( - 3 4 . 5 • 3.2 ~). These values did not differ significantly ( P > 0.1). If one compares the doses of drugs needed to induce these blood pressure changes, it is obvious that isoprenaline was approximately equieffective irrespective of whether given by the i.v. or by the intrapulmonary route of administration, whereas in the case of NP, the intrapulmonary dose had to be 10-fold higher than the i.v. dose in order to produce a comparable reduction of the blood pressure. The process of invasion of the drugs from the pulmonary compartment as judged from the timecourse of the blood pressure reduction was unexpectedly fast; the time from the onset of aerosol production to reach 50 ~ of the maximal blood pressure reduction (tl/2) was found to be 117.0_+ 15.0s ( 2 + S.E., n = 8 ) with the NP-aerosol, and 142.3 • 31.3 s (n = 8) with the isoprenaline-aerosol (difference not significant; P > 0.I). On infusion of the drugs, the half-time of the blood pressure reduction was 40.0 _+ 2.5 s (n = 8) with NP, and 44.6 _+ 3.8 s (n = 8) with isoprenaline (difference not significant; P > 0.1). Both, aerosols and infusions were stopped after 10rain, and the blood pressure was recorded.continuously until control blood pressures were reestablished in the animals. The half-time of recovery from the reduced blood pressure (ff + S.E., n = 8) was found to be 32.0 + 3.1 s after infusion of NP, 289.5 _+ 33.8safter N P as aerosol, 135.4• 17.8s after infusion of isoprenaline, and 649.7 • 53.1 s after isoprenaline as aerosol. These findings permit the conclusion that after cessation of the aerosol administration, the resorption
206 of both drugs from the pulmonary compartment continued, and that NP was probably more rapidly eliminated from the blood than isoprenaline. However, because of the complexity of the pharmacokinetic and pharmacodynamic processes involved, no sound decision can be made to what degree the invasion and/or the elimination process may have accounted for the longer duration of blood pressure effects after isoprenaline-aerosols than after NP-aerosols.
Discussion
A strong relaxant effect of NP on isolated tracheal smooth muscle of the guinea pig was found in this study confirming the results of a preceding investigation (Kreye et al., 1975). Because of this finding, and in spite of the fact that isoprenaline proved to be the more potent relaxant of the tracheal smooth muscle preparation in vitro, we felt encouraged to study a possible antiasthmatic effect of NP aerosols; a directly acting bronchodilator without the known cardiac side-effects of fl-adrenergic antiasthmatics would offer many advantages in the treatment of acute asthmatic attacks. In comparison to the controls, NP as an aerosol had a definite protective effect on the development of asthmatic symptoms in guinea pigs subjected to an aerosol of histamine. For instance, the time to reach asthma reaction stage 2 (increasing breathing frequency with involvement of accessory respiratory muscles) and asthma reaction stage 5 (collapse, start of clonic seizures) was doubled at the lowest dose of NP used, and increased 4-fold at the highest dose. However, in comparison to the antiasthmatic effect of isoprenaline aerosols, NP proved to be definitely inferior; a dose of NP at least 3,000 times higher than that of isoprenaline had to be given as an aerosol for a comparable antiasthmatic effect. With respect to the results from the organ bath experiments, the dose of NP effective as an aerosol in the asthma studies was two orders of magnitude higher than expected. Several reasons may account for this low effectiveness of inhaled NP aerosols. The most plausible explanation would be poor penetration of NP through the bronchoepithelial layer and the lamina propria of the bronchioli to the smooth muscle layer; NP, as a highly dissociable salt, should be present in the bronchial tree as a bulky anion with neglible lipophilicity, and accordingly little ability to pass cell membranes. In contrast, the good antiasthmatic effect of isoprenaline could be explained by the fact that this drug with its pKa of 8.64 will at least in part be present in the bronchiolar tree in the undissociated form. A second explanation for the weak antiasthmatic effect of the NP aerosol can be derived from our
Naunyn-Schmiedeberg'sArch. Pharmacol. 306 (1979) experiments regarding its effect on blood pressure. In contrast to the asthma studies, they were performed in the rat which is a standard preparation for this purpose. We have no reasons to assume, however, that this difference in the species used is a principal shortcoming of this study. Based on a rough estimate of the intrapulmonarily applied doses of the drugs as aerosols, about the same amount of isoprenaline either given by aerosol or by infusion produced the same reduction of blood pressure, whereas a 10-fold higher intrapulmonary dose of NP than by the i.v. route had to be given for comparable effects on blood pressure. This finding suggests that only one tenth of the intrapulmonary NP may have entered the general circulation, and that the major part may have been destroyed by contact with the tissues of the airways, or during passage through the cellular layers. It has been known for long that NP is destroyed on contact with tissue homogenates (Hill, 1942; Casinelli, 1956; Smith and Kruszina, 1974), and we have shown recently that NP is inactivated by about 30% during a single passage through peripheral vascular beds of anaesthetized rats (Kreye et al., 1977). It has been proposed that at least in part, the antiasthmatic effect of bronchodilator drugs given as aerosols may, after their resorption from the airways, be produced by the substance acting from the vascular compartment (Dautrebande at el., 1941). With respect to this assumption, isoprenaline would have another advantage over NP as an antiasthmatic; in our experiments, the survival of isoprenaline in the circulation as derived from the half-life of recovery from the blood pressure reduction after cessation of the aerosol administration lasted more than twice as long as that of NP. Finally, it cannot be excluded that bronchiolar smooth muscle cells differ from tracheal smooth muscle in their sensitivity to drugs, and that the high susceptibility of the isolated tracheal preparation to the relaxant action of NP is not necessarily shared by the bronchial smooth muscle. As a conclusion from our experiments, we cannot recommend NP as an aerosol for the treatment of bronchial asthma; although at high doses effective in prolonging the time course of asthma development in guinea pigs, NP proved to be definitively inferior to the known bronchodilator, isoprenaline.
References
Casinelli,C. M. : La actionhipotensoradelnitroprusiatoaplicationes therapeuticas. Anales de la Facultad de MedicinaUniversidad nacionalmayorde San Marcosde Lima39, 1330-1331 (1956) Castillo,J. C., de Beer,E. J. : The trachealchain.I. A preparationfor the study of antispasmodicswith particular referenceto bronchodilatordrugs.J. Pharmacol.Exp. Ther. 90, 104-109 (1947)
V. A. W. Kreye and E. Marquard: Nitroprusside Aerosols in Bronchial Asthma Dautrebande, L., Philipot, E., Nogarode, F., Charlier, R. : Aerosols madicamenteux. I. P~n6tration dans l'organisme et action ~i distance de substances m6dicamenteuses introduites par voie transpulmonaire. Arch. Int. Pharmacodyn. 66, 138 - 167 (1941) David, H. A., Hartley, H. O., Pearson, E. S. : The distribution of the ratio, in a single normal sample, of range to standard deviation. Biometrika 41, 482-493 (1954) Halpern, B. N.: Les antihistaminiques de synth&e. Essais de chimith6rapy des 6tats allergiques. Arch. Int. Pharmacodyn. 68, 3 3 9 - 408 (1942) Herxheimer, H. : Repeatable microshocks of constant strength in guinea-pig anaphylaxis. J. Physiol. (Lond.) 117, 251 - 255 (1952) Hill, H. E. : A contribution to the toxicology of sodium nitroprusside. 1. The decomposition and determination of sodium nitroprusside. Austral. Chem. Inst. J. Proc. 9, 8 9 - 9 3 (1942) H6bel, M., Maroske, D., Eichler, O. : Eine einfache Methode zur Bestimmung des Atemminutenvolumens yon Ratten und Meerschweinchen. Arch. Int. Pharmacodyn. 194, 3 7 1 - 374 (1971)
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Kreye, V. A. W., Baron, G. D., Liith, J. B., Schmidt-Gayk, H. : Mode of action of sodium nitroprusside on vacular smooth muscle. Naunyn-Schmiedeberg's Arch. Pharmacol. 288, 381 - 402 (1975) Kreye, V. A. W., Reske, S. N., Schultz, K. D. : Vasodilatatorisch wirkende Antihypertensiva: Modellsubstanz Natrium-Nitroprussid. Verh. Dtsch. Ges. Kreislaufforsch. 43, 8 7 - 9 6 (1977) Palmer, F., Kingsbury, St. S. : Particle size in nebulized aerosols. Am. J. Pharmacy 124, 112-124 (1952) Preuner, R. : Allergiestudien. I. Die Wirkung der Witterung auf das experimentelle Asthma bronchiale. Z. Hyg. 121, 559-568 (1939) Smith, R. P., Kruszina, H. : Nitroprusside produces cyanide poisoning via a reaction with hemoglobin. J. Pharmacol. Exp. Ther. 191, 557-563 (1974)
Received November 3, 1978/Accepted January 24, 1979
Archivesof
Pharmacology 9 by Springer-Verlag 1979
Comparison of Sodium Nitroprusside and Isoprenaline Aerosols in Histamine-Induced Bronchial Asthma of the Guinea Pig* Volker A. W. Kreye and Erich Marquard II. Physiologisches Institut and Anatomisches Institut III, Universifftt Heidelberg, Im Neuenheimer Feld 326, D-6900 Heidelberg, Federal Republic of Germany
Summary. 1. Sodium nitroprusside (NP) is a potent
relaxant of the guinea pig's tracheal smooth muscle contracted by carbachol or histamine. 2. At high doses, aerosols of NP delay the occurrence of symptoms of bronchial asthma in guinea pigs subjected to a histamine aerosol. However, the fladrenergic bronchodilator isoprenaline is at least 2,600 times more potent as an anti-asthmatic than NP on a molar basis. 3. NP and isoprenaline both reduce the blood pressure in rats when given either as infusion or as aerosol. The aerosol/infusion dosage ratio for comparable reductions of blood pressure were 1 for isoprenaline, and 10 for NP. After cessation of the application of the NP aerosol, the blood pressure rose more than twice as fast (t~/2 of blood pressure recovery) than after cessation of the application of the isoprenaline aerosol. 4. The poor antiasthmatic effect of NP aerosols may result from low permeability of the bronchial epithelium to the NP anion, from partial destruction in the airways or during tissue permeation, and from short half life of NP in the circulating blood. 5. Because of its inferiority to known bronchodilators, aerosols of NP cannot be recommended for the treatment of acute bronchial asthma. Key words: Antiasthmatics - Bronchodilators Nitroprusside - Isoprenaline - Pulmonary drug resorption - Blood pressure effects.
Introduction
Sodium nitroprusside [Na2Fe(CN)sNO-2HzO; Nipride ~; Nipruss "~; NP) is an extremely potent reSend offpr&t requests to V. A. W. Kreye at the above address * This work was supported by the German Research Foundation (DFG) within SFB 90 "Cardiovascular System". The experiments were performed at the Pharmacological Institute, University of Heidelberg
laxant of isolated vascular smooth muscle. In an investigation on the sensitivity to the relaxant action of NP of various different types of smooth muscle from rats and guinea pigs, only the tracheal chain of the guinea pig was found to respond similarly well to NP (Kreye et al., 1975). This finding suggested that NP might be a useful relaxant of the bronchiolar tree. Therefore, we have investigated in guinea pigs the anti-asthmatic effect of NP aerosols on histamineinduced asthma. As a reference substance, the known bronchodilator drug, isoprenaline, was included in the study. In preceding in vitro experiments, the relaxant potency (ICs0) of both drugs on histamine- and carbachol-contracted tracheal smooth muscle was studied. Finally, the effect of inhaled NP and isoprenaline aerosols on the time-course of blood pressure changes was studied in rats. From these latter experiments, some information on the invasion of the drugs from the bronchiolar/alveolar lumen into the blood compartment was expected. Methods a) In vitro Studies on Isolated Tracheal Smooth Muscle. Male guinea pigs weighing 250 - 300 g were killed by a blow on the head, and their tracheae were excised. Under chilled physiological salt solution, tracheal rings were prepared according to Castillo and de Beer (1947). For each tracheal chain, 3 rings were tied together with thread. In organ baths (volume 10 ml) containing physiological salt solution (37~C; gassed with 95 % O z - 5 % CO2 ; pH 7.2), the tracheal chains were suspended at an initial tension of 12 raN. The composition of the physiological salt solution, and the procedures for measuring isometric tension changes have been described in detail elsewhere (Kreye et al., 1975). The tracheal chains were contracted by the addition of histamine or carbachol to reach bath concentrations of 5 • I 0 - ~ M or 10 -7 M, respectively. When this stimulation had resulted in a constant muscular tone, NP or isoprenaline (Aludrin ,"~; Boehringer, Ingelheim) were added in increasing doses in a cumulative fashion. From the resulting dose-response curves, single ICs0 values were obtained graphically, and used for the calculation of geometrical means of ICs0. b) Induction of Asthma by Histamine Aerosol, and Effect of NP or [soprenaline Aerosols. For the production of aerosols, a modification
0028-1298/79/0306/0203/$01.00
204 of the method described by Halpern (1942) was used. Two streams of compressed air were passed through bottles filled with distilled water for humidification, and then led via two atomizers (Palmer and Kingsbury, 1952; aerosol particle size: 1 - 2 p ) into an animal chamber made ofacryl glass and having a volume of 6.51. Opposite to the inlets coming from the atomizers, two holes in the wall of the animal chamber permitted the escape of the aerosol vapors into the open air via an exhaust pipe. To control the compressed air stream, a manometer an(i a volumeter to measure air pressure and flow, respectively, and a three way valve for immediate interruption of the aerosols were placed between the humidifyer bottles and the atomizers. During the experiments, the air pressure in the system was kept at 72.57 kPa above atmospheric pressure, and the flow rate at 4.5 l/rain; this resulted in the vaporisation of 0.15ml/min of the fluid in the atomizers. One of the atomizers contained a solution of I mg histamine dihydrochloride/ml distilled water to produce the asthmatogenic histamine aerosol. The other atomizer either contained pure distilled water (Regimen A) or solutions of the bronchodilator drugs in distilled water at various concentrations: isoprenaline sulphate 10 gg/ml (B), 33 pg/ml (C), and 100 gg/ml (D); NP - 3.3 mg/ml (E), 10mg/ml (F), and 30mg/mI (G). According to a randomisation scheme, 21 guinea pigs were subdivided into 7 groups of 3 animals each. Per experimental day, one of the groups was submitted to one of the regimens A to G. For the selection of animal groups and regimens, a cross-over design had been elaborated so that at the end of the experiments, all the animals had been subjected to all of the regimens. Single guinea pigs were placed into the center of the animal chamber, and observed by the experimentator. According to Preuner (1939), the following stages of asthma development can be distinguished in the guinea pig: 0 - no reaction 1 - slight reaction, i.e. slight coughs 2 - increased breathing frequency with involvement of auxiliary respiratory muscles 3 - moderate orthopnoea at reduced breathing frequency 4 - severe orthopnoea with the head elevated and the extremities extended 5 - collapse, and start of clonic seizures 6 - death. The time from the start of the histamine aerosol to reaching stage 5 is identical with the "preconvulsion time" as defined by Herxheimer (1952). In our experiments, the time to reach stages 1 to 5 was used as the criterion to judge the anti-asthmatic effects of the bronchodilator drugs. When the animals had reached stage 5, the aerosol was stopped immediately, and the chamber was aerated. The maximal duration of the animals' exposures to histamine and drug aerosols was restricted to 15 rain (i.e. 900 s). With the higher doses of bronchodilator drugs, several animals did not reach all stages of asthma development; in these cases, for the evaluation a time ofg00 s was assumed arbitrarily.
c) Effects of NP and Isoprenaline Given by Aerosol or Infusion on Blood Pressure. These experiments were performed in rats as this species is a standard preparation for the recording of blood pressure in our laboratory~ For anaesthesia, Sprague-Dawley rats weighing about 300 g received 1.4 g urethane/kg body weight s.c. One carotid artery and one jugular vein were dissected, and polyethylene catheters (internal diameter: 0.5ram; external diameter: 1.0mm) were introduced into the vessels. The arterial catheter was connected to a Statham pressure transducer P23Db; after amplification, the blood pressure dependent electrical signals were recorded on a Siemens Kompensograph III servo chart recorder. By the jugular catheter, aqueous solutions of drugs in the concentrations stated below could be infused with an infusion pump at a rate of 0.1 ml/min. To record the effects ot~NP and isoprenaline aerosols on blood-pressure, the
Naunyn-Schmiedeberg's Arch. Pharmacol. 306 (1979) anaesthetized animals were placed into the animal chamber, and the aerosols were produced as described above. The aim of the study was to compare the time courses of quantitatively similar blood-pressure changes. In preliminary experiments, the following concentrations of aqueous NP and isoprenaline solutions for aerosol production and infusion, respectively, had been found to be roughly equidepressor, and were used for the experiments: solutions for aerosols - 6rag NP/ml, 0.3 mg isoprenaline/ml; solutions for infusions - 20 gg NP/ ml, 10 pg isoprenaline/ml. Taking into account the vaporization rate of the atomizers, the flow rate of the aerosols, and the tidal respiratory volume of rats (103 ml/min ; H6bel et al., 1971), an intrapulmonary administration of 20.6 ng NP/min and of 1 ng isoprenaline/min was calculated, whereas the rates of i.v. infusions were 2 ng NP/min and 1 ng isoprenaline/min, respectively.
d) StatisticalEvaluations. By using the R/s-ratio method described by David et al. (1954), normal distribution of the results could not be established. Therefore, the Wilcoxon test was used for the control of significance of the results where necessary.
Results A. In vitro Studies on Isolated Tracheal Smooth Muscle T r a c h e a l c h a i n s o f the g u i n e a p i g s t i m u l a t e d e i t h e r by 1 0 - T M c a r b a c h o l o r 5 • 1 0 - T M h i s t a m i n e w e r e rel a x e d b y t h e c u m u l a t i v e a d d i t i o n o f N P in a c o n c e n t r a t i o n r a n g e o f 10 - 9 to ] 0 - 6 M , a n d by i s o p r e naline in a c o n c e n t r a t i o n r a n g e o f 10 - 1 ~ to 10 - 7 M, r e @ e c t i v e l y . T h e c o n c e n t r a t i o n s for h a l f - m a x i m a l rel a x a t i o n (ICso) o f the p r e p a r a t i o n s w e r e d e t e r m i n e d g r a p h i c a l l y f r o m the single d o s e - r e s p o n s e curves, and u s e d for t h e c a l c u l a t i o n o f g e o m e t r i c a l m e a n s + S.E. (n = 4) o f I C s o - v a l u e s . W i t h the c a r b a c h o l - c o n t r a c t e d tissues, the I C s o for N P w a s f o u n d to be 1.04 ( + 0 . 5 5 ; - 0 . 3 6 ) • 1 0 - T M a n d f o r i s o p r e n a l i n e 3.61 ( + 0 . 5 0 ; - 0 . 4 4 ) • l 0 - 9 M . W i t h the h i s t a m i n e - c o n t r a c t e d tissues, t h e I C s o f o r N P was 3.05 ( + 0 . 9 3 ; -0.66) • 10 - 8 M , a n d for i s o p r e n a l i n c 1.62 ( + 0 . 2 3 ; - 0 . 2 0 ) x 10 . 9 M . T h u s , N P is a p o t e n t r e l a x a n t o f t r a c h e a l s m o t h m u s c l e , a l t h o u g h in this tissue i s o p r e n a l i n e is o f g r e a t e r p o t e n c y ; as c o m p a r e d to i s o p r e n a l i n e , a 2 9 - f o l d h i g h e r m o l a r c o n c e n t r a t i o n o f N P was n e e d e d f o r halfmaximal relaxation of carbachol-stimulated tracheal s m o o t h m u s c l e , a n d a 19-fold h i g h e r c o n c e n t r a t i o n in histamine-stimulated preparations.
B. Effect o f N P and Isoprenaline on Histamine-Induced Asthma M o s t o f t h e g u i n e a pigs s u b j e c t e d to the h i s t a m i n e a e r o s o l d e v e l o p e d t h e t y p i c a l signs o f b r o n c h i a l a s t h m a a c c o r d i n g to t h e c r i t e r i a g i v e n u n d e r M e t h o d s . O f these criteria, s t a g e 2 ( i n c r e a s e d b r e a t h i n g f r e q u e n c y ) a n d s t a g e 5 (collapse) c o u l d be o b s e r v e d w i t h the l e a s t s u b j e c t i v e e r r o r since these s y m p t o m s a p p e a r e d abruptly. T h e t i m e c o u r s e o f a s t h m a d e v e l o p m e n t is g i v e n
V. A. W. Kreye and E. Marquard: Nitroprusside Aerosols in Bronchial Asthma x 100 9
l
8 84
iii ii 1 --TIT
IS
100 p g l m l
IS
33 ,ug I ml
IS
10 p g / m l
NP
3 0 mg I ml
NP
10 mglml
NP
3.3 mg I ml
Control
s
0 1
2
3
4
, S t a g e of A s t h m a 5
Fig. 1. Development of bronchial asthma in guinea pigs (n = 17) subjectedto aerosolsof histaminedihydrochloride(1 mg/mlsolution) in the absence(Control)and presenceof sodiumnitroprusside(NP)or isoprenaline sulphate (IS) aerosols. The concentrationsof the drug solutions used for the aerosolsare given.Abscissa : stage of asthmatic reaction according to Preuner (1939) (see text); ordinate; time to reach stage of asthmaticreactionin s (~ + S.E.).Maximalduration of experiments was 900 s. When animals did not reach an appropriate stage of reaction, for evaluation a time of 900s was assumed arbitrarily
in Fig. 1. For unknown reasons, 4 of the 21 animals did not react with bronchial asthma to the histamine aerosol during 900 s, i.e. t h e maximal duration of the experiments. Although these animals were also submitted to all other regimens because of the random• sat• scheme, they were eliminated from the final evaluation of the study resulting in a number of animals of only 17. Both bronchodilator drugs as aerosols prolonged the time course of asthma development (Fig. 1) or even prevented asthma development, but considerable differences in their protective effects were found. Whereas NP aerosols of solutions containing 3.3mg/ml, 10mg/ml, and 30mg/ml abolished the histamineinduced collapse (stage 5) in 2, 3, and 3 animals, respectively, isoprenaline in the concentrations of 10 lag/ml, 33 ktg/ml, and 100 ~tg/ml protected 10, 15, and 16 of the 17 animals from the asthmatic collapse. F r o m these results and the time courses given in Fig. 1 it is clear that the lowest dose of isoprenaline (10 pg/ml) had a greater antiasthmatic effect than the highest dose of N P (30 mg/ml). With these doses, the
205
time to reach asthma stage 2 was similar for both drugs (10~tg/ml isoprenaline: 483.3 +_ 96.7s; 30mg/ml NP: 471.3 • 63.3 s). This permits a rough estimate of the ant• efficacy of the two drugs : for a comparable ant• effect, the dose of N P needed was 3,000fold higher than that of isoprenaline on a weight basis, and 2,600-fold higher on a molar basis.
C. Effects of Infused and Inhaled N P or lsoprenaline on Blood Pressure At the concentrations given, both N P and isoprenaline produced comparable reductions of the blood pressure in rats when administered by infusion or as an aerosol. The following changes in blood pressure were found (~? • S.E., n = 8; percentage figures in brackets represent percentage changes from initial blood pressures): infusion of 2ng NP/min, - 3 6 . 3 • 4 . 5 m m H g ( - 4 1 . 4 + 1.8 ~o); aerosol of N P (approx. 20.6 rig/rain), - 33.0 • (-39.9• infusion of l n g isoprenaline/min, - 3 0 . 9 • 2.7ram Hg ( - 4 1 . 7 • 1.2 ~ ) ; aerosol of isoprenaline (approx. 1 ng/min), - 2 6 . 9 + 4.3 m m H g ( - 3 4 . 5 • 3.2 ~). These values did not differ significantly ( P > 0.1). If one compares the doses of drugs needed to induce these blood pressure changes, it is obvious that isoprenaline was approximately equieffective irrespective of whether given by the i.v. or by the intrapulmonary route of administration, whereas in the case of NP, the intrapulmonary dose had to be 10-fold higher than the i.v. dose in order to produce a comparable reduction of the blood pressure. The process of invasion of the drugs from the pulmonary compartment as judged from the timecourse of the blood pressure reduction was unexpectedly fast; the time from the onset of aerosol production to reach 50 ~ of the maximal blood pressure reduction (tl/2) was found to be 117.0_+ 15.0s ( 2 + S.E., n = 8 ) with the NP-aerosol, and 142.3 • 31.3 s (n = 8) with the isoprenaline-aerosol (difference not significant; P > 0.I). On infusion of the drugs, the half-time of the blood pressure reduction was 40.0 _+ 2.5 s (n = 8) with NP, and 44.6 _+ 3.8 s (n = 8) with isoprenaline (difference not significant; P > 0.1). Both, aerosols and infusions were stopped after 10rain, and the blood pressure was recorded.continuously until control blood pressures were reestablished in the animals. The half-time of recovery from the reduced blood pressure (ff + S.E., n = 8) was found to be 32.0 + 3.1 s after infusion of NP, 289.5 _+ 33.8safter N P as aerosol, 135.4• 17.8s after infusion of isoprenaline, and 649.7 • 53.1 s after isoprenaline as aerosol. These findings permit the conclusion that after cessation of the aerosol administration, the resorption
206 of both drugs from the pulmonary compartment continued, and that NP was probably more rapidly eliminated from the blood than isoprenaline. However, because of the complexity of the pharmacokinetic and pharmacodynamic processes involved, no sound decision can be made to what degree the invasion and/or the elimination process may have accounted for the longer duration of blood pressure effects after isoprenaline-aerosols than after NP-aerosols.
Discussion
A strong relaxant effect of NP on isolated tracheal smooth muscle of the guinea pig was found in this study confirming the results of a preceding investigation (Kreye et al., 1975). Because of this finding, and in spite of the fact that isoprenaline proved to be the more potent relaxant of the tracheal smooth muscle preparation in vitro, we felt encouraged to study a possible antiasthmatic effect of NP aerosols; a directly acting bronchodilator without the known cardiac side-effects of fl-adrenergic antiasthmatics would offer many advantages in the treatment of acute asthmatic attacks. In comparison to the controls, NP as an aerosol had a definite protective effect on the development of asthmatic symptoms in guinea pigs subjected to an aerosol of histamine. For instance, the time to reach asthma reaction stage 2 (increasing breathing frequency with involvement of accessory respiratory muscles) and asthma reaction stage 5 (collapse, start of clonic seizures) was doubled at the lowest dose of NP used, and increased 4-fold at the highest dose. However, in comparison to the antiasthmatic effect of isoprenaline aerosols, NP proved to be definitely inferior; a dose of NP at least 3,000 times higher than that of isoprenaline had to be given as an aerosol for a comparable antiasthmatic effect. With respect to the results from the organ bath experiments, the dose of NP effective as an aerosol in the asthma studies was two orders of magnitude higher than expected. Several reasons may account for this low effectiveness of inhaled NP aerosols. The most plausible explanation would be poor penetration of NP through the bronchoepithelial layer and the lamina propria of the bronchioli to the smooth muscle layer; NP, as a highly dissociable salt, should be present in the bronchial tree as a bulky anion with neglible lipophilicity, and accordingly little ability to pass cell membranes. In contrast, the good antiasthmatic effect of isoprenaline could be explained by the fact that this drug with its pKa of 8.64 will at least in part be present in the bronchiolar tree in the undissociated form. A second explanation for the weak antiasthmatic effect of the NP aerosol can be derived from our
Naunyn-Schmiedeberg'sArch. Pharmacol. 306 (1979) experiments regarding its effect on blood pressure. In contrast to the asthma studies, they were performed in the rat which is a standard preparation for this purpose. We have no reasons to assume, however, that this difference in the species used is a principal shortcoming of this study. Based on a rough estimate of the intrapulmonarily applied doses of the drugs as aerosols, about the same amount of isoprenaline either given by aerosol or by infusion produced the same reduction of blood pressure, whereas a 10-fold higher intrapulmonary dose of NP than by the i.v. route had to be given for comparable effects on blood pressure. This finding suggests that only one tenth of the intrapulmonary NP may have entered the general circulation, and that the major part may have been destroyed by contact with the tissues of the airways, or during passage through the cellular layers. It has been known for long that NP is destroyed on contact with tissue homogenates (Hill, 1942; Casinelli, 1956; Smith and Kruszina, 1974), and we have shown recently that NP is inactivated by about 30% during a single passage through peripheral vascular beds of anaesthetized rats (Kreye et al., 1977). It has been proposed that at least in part, the antiasthmatic effect of bronchodilator drugs given as aerosols may, after their resorption from the airways, be produced by the substance acting from the vascular compartment (Dautrebande at el., 1941). With respect to this assumption, isoprenaline would have another advantage over NP as an antiasthmatic; in our experiments, the survival of isoprenaline in the circulation as derived from the half-life of recovery from the blood pressure reduction after cessation of the aerosol administration lasted more than twice as long as that of NP. Finally, it cannot be excluded that bronchiolar smooth muscle cells differ from tracheal smooth muscle in their sensitivity to drugs, and that the high susceptibility of the isolated tracheal preparation to the relaxant action of NP is not necessarily shared by the bronchial smooth muscle. As a conclusion from our experiments, we cannot recommend NP as an aerosol for the treatment of bronchial asthma; although at high doses effective in prolonging the time course of asthma development in guinea pigs, NP proved to be definitively inferior to the known bronchodilator, isoprenaline.
References
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Kreye, V. A. W., Baron, G. D., Liith, J. B., Schmidt-Gayk, H. : Mode of action of sodium nitroprusside on vacular smooth muscle. Naunyn-Schmiedeberg's Arch. Pharmacol. 288, 381 - 402 (1975) Kreye, V. A. W., Reske, S. N., Schultz, K. D. : Vasodilatatorisch wirkende Antihypertensiva: Modellsubstanz Natrium-Nitroprussid. Verh. Dtsch. Ges. Kreislaufforsch. 43, 8 7 - 9 6 (1977) Palmer, F., Kingsbury, St. S. : Particle size in nebulized aerosols. Am. J. Pharmacy 124, 112-124 (1952) Preuner, R. : Allergiestudien. I. Die Wirkung der Witterung auf das experimentelle Asthma bronchiale. Z. Hyg. 121, 559-568 (1939) Smith, R. P., Kruszina, H. : Nitroprusside produces cyanide poisoning via a reaction with hemoglobin. J. Pharmacol. Exp. Ther. 191, 557-563 (1974)
Received November 3, 1978/Accepted January 24, 1979
