No Effect of the Oral Neutral Endopeptidase Inhibitor Candoxatril, on Bronchomotor Tone and Histamine Reactivity in Asthma

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No effect of the oral neutral endopeptidase inhibitor candoxatril, on bronchomotor tone and histamine reactivity in asthma

R.M. Angus, M.J.A. McCallum, J.E. Nally, N.C. Thomson

No effect of the oral neutral endopeptidase inhibitor, candoxatril, on bronchomotor tone Dept of Respiratory Medicine, Western and histamine in asthma. RM. Angus, M.J.A. McCallum, J.E. Nally, N.C. Thomson. Infirmary, Glasgow, UK. ERS Journals Ltd 1994. ABSTRACT: Neutral endopeptidase (NEP) is found in many tissues in man, in- Correspondence: N.C. Thomson cluding the lung. Metabolism by NEP is one of the main mechanisms for the clear- Dept of Respiratory Medicine Western Infirmary ance of atrial natriuretic peptide (ANP), a hormone that causes bronchodilation Glasgow G11 6NT and reduces nonspecific bronchial reactivity in man. Candoxatril, an oral NEP UK. inhibitor has been shown to elevate circulating ANP levels. We have sought to determine whether the administration of candoxatril will alter bronchomotor tone Keywords: Atrial natriuretic peptide (forced expiratory volume in one second (FEV1)) and histamine reactivity. bronchial reactivity Ten male asthmatic patients with stable asthma were enrolled (mean (SD) age bronchomotor tone candoxatril 32 (10) yrs; FEV1 92 (11) % predicted) in a randomized, double-blind, placebo- controlled study. On each study day, after baseline spirometry, patients rec- histamine eived 200 mg of candoxatril or placebo. Spirometry was repeated at half hourly neutral endopeptidase inhibitor intervals. After 2 h a histamine inhalation test was performed. Received: July 7 1993 There was no significant difference in FEV1 values at baseline or at 2 h post- Accepted after revision January 30 1994 dosing between active and placebo study days, with mean (SEM) FEV1 at baseline and 2 h of 3.71(0.29) l and 3.85(0.29) l on the placebo day, and 3.89(0.27) l and 4.05(0.82) l on the active day, respectively. The geometric mean (range) pro- vocative concentration of histamine producing a 20% fall in FEV1 (PC20) on the placebo day and active day did not differ significantly, being 1.17(0.25Ð25.8) and 0.93(0.13Ð32) mgáml-1, respectively. ANP levels rose significantly on the active day, from a mean (SEM) baseline value of 12.1 (2.1) pgáml-1 to 29.1(4.8) pgáml-1 at 2 h, but did not change on the placebo day (baseline 11.54(2) pgáml-1 and 2 h level 10.8(1.9) pgáml-1. Plasma drug levels were maximally elevated between 2 and 3 h mean (SEM) plasma candoxatril at 1 h 161(53), 2 h 846(136), 3 h 896(68) and 4 h 626(60) ngáml-1. No side-effects were observed or noted by the patients. We conclude that the acute administration of the oral NEP inhibitor, candoxatril, does not alter bronchomotor tone or bronchial reactivity to histamine in stable asthmatic patients. This neutral effect of candoxatril is reassuring, and permits further exploration of NEP inhibitors in the treatment of e.g. cardiac failure. Eur Respir J., 1994, 7, 1084Ð1089.

Intravenous atrial natriuretic peptide (ANP) has been orally bioavailable and inhalation studies have, so far, shown to cause bronchodilation and reduce bronchial demonstrated only a modest effect. It is likely, there- reactivity in man [1Ð6]. When given by inhalation to fore, that any clinical application in asthma would be asthmatic patients, ANP has been shown to attenuate limited by the need for continuous intravenous infusion histamine- and methacholine-induced contraction, and and its relatively short duration of action in high doses it has a small bronchodilator effect [7Ð9]. ANP is cleared from the plasma by two main path- In vitro we have shown that ANP relaxes human bron- ways: degradation by the enzyme NEP [11] and binding chial smooth muscle preconstricted by methacholine, to a non-guanylyl cyclase clearance receptor [12]. Neutral and that preincubation with ANP attentuates methacho- endopeptidase 3.4. 24.11 (NEP) is a zinc-containing line-induced contraction [10]. Both of these effects are metalloendopeptidase, first localized to the brush bor- significantly enhanced by the addition of the neutral der of the kidney, in 1974 [13]. Since then, NEP has endopeptidase (NEP) inhibitor, phosphoramidon [10]. been found in many sites within the body and in a ANP would appear, therefore, to have therapeutic poten- variety of species other than man [11]. In the lung, NEP tial in asthma. However, as it is a peptide, ANP is not appears to be present in high concentrations in airway NEUTRAL ENDOPEPTIDASE INHIBITOR IN ASTHMA 1085 epithelium, although it is also found in submucosal glands, and patients attended at the same time of day on both β airway smooth muscle and nerves [14]. An alternative occasions. Inhaled 2-agonists were discontinued for approach to harnessing the therapeutic potential of ANP 8 h prior to attendance on each study day but inhaled on the airway might be to systemically inhibit NEP, corticosteroids were continued unchanged. Patients were thereby allowing elevation of endogenous hormone asked to fast, except for water, for 2 h prior to the study. levels. Candoxatril (Pfizer Central Research, Sandwich, If the FEV1 was within 10% of the baseline on the screen- Kent, UK) is a potent specific inhibitor of NEP, which ing day, the study proceeded on that day. Patients were has been shown to enhance plasma ANP concentra- asked to lie semirecumbent and an intravenous cannula tions [15, 16]. The present study is based on the ratio- (Venflon; Viggo AB, Helsingborg, Sweden) was insert- nale that the systemic inhibition of NEP by candoxatril ed for blood-sampling in the ante-cubital vein. Patients might elevate airway ANP levels sufficiently to favour- remained in a semirecumbent position, except for per- ably affect bronchomotor tone and bronchial reactivity forming spirometry. Prior to dosing, heart rate and and to overcome the potential effects of bronchocon- blood pressure were measured. Blood pressure and pulse strictor tachykinins, which are also metabolized by were monitored every 15 min using a semi-automatic NEP. sphygmomamometer. Blood (20 ml) was sampled for drug and hormonal (ANP, cyclic guanosine monphos- Methods phate (cGMP) and catecholamines) assay at times 0, 30, 60, 90, 120, 180, 240 min. Spirometry was checked every 30 min, and at 2 h histamine inhalation test was Patients performed using the method of COCKCROFT et al. [18]. After completion of the challenge, the patient received Ten adult male, atopic asthmatic patients were re- 2.5 mg of salbutamol via a nebulizer and spirometry SD) age 32 cruited from the out-patient clinic (mean ( was repeated after 20 min. After 4 h, the patient was (10) yrs, forced expiratory volume in one second (FEV ) 1 allowed home following completion of a physical exam- 92(11) % predicted) (table 1). Asthma was diagnosed ination. in accordance with American Thoracic Society guide- lines [17]. As a condition of enrolment, the patient's ≥ FEV1 had to be 70% of predicted despite withholding β Measurements 2-agonists. Each patient's asthma had been stable (de- fined as no respiratory tract infection or deterioration of FEV1. This was measured using a dry wedge spiro- asthma control requiring an alteration in anti-asthma meter (Vitalograph S, Vitalograph, Buckingham, UK), treatment) for at least 2 months prior to recruitment. All β the best of three readings being taken at each time-point. were taking short-acting 2-agonists, and 7 of the 10 inhaled corticosteroids. These were measured using The study had the approval of the Glasgow West Ethics Pulse and blood pressure. a semi-automatic sphygmomanometer (Critikon; Dina- Committee and informed written consent was obtained map vital signs monitor 1846 FX, Berkshire, UK). Three from each patient. readings were taken at each time-point from the non- dominant arm, the mean being recorded. Study design

A randomized, double-blind, placebo-controlled study Histamine inhalation test [18]. Baseline FEV1 was design was employed. Two study days were required, measured, taking the best of three readings. Thereafter, a saline inhalation was administered and then doubling Table 1. Ð Patient characteristics doses of nebulized histamine were administered at 5 min intervals, beginning with 0.0625 mgáml-1. Each concen- Pt Age FEV1 Therapy No. yrs l % pred tration was given for 2 min via a Wright's gas nebulizer driven by compressed air, with an output of 0.13 1 31 2.44 74 S, B* mlámin-1 The FEV was measured at 0.5, 1.5 and 3 min 2 57 2.89 85 S 1 3 29 4.79 112 S, B* after each inhalation, until a fall of at least 20% was 4 27 4.12 91 S achieved. The result was then expressed as the hista- 5 39 3.69 103 S, B* mine provocation concentration producing a 20% fall 6 28 4.62 87 T, Bu in FEV1 ( PC20). 7 29 3.2 97 S, B** 8 21 4.62 97 S, B* ANP. Venous blood (l0 ml) was collected into po- 9 29 3.81 84 A, B* tassium ethylenediamine tetra-aceticacid (EDTA) tubes 10 26 4.52 96 A, B* containing 1,000 IU aprotinin (Bayer, Newbury, UK), Mean (SD) 31.6 (10) 3.87 (0.8) 92 (11) stored on ice and spun within 2 h. Plasma was stored at -20¡C, and ANP was later measured by radioimmuno- Pt: patient; A: antihistamine; Bu: inhaled budesonide; B: inhaled beclomethasone; S: inhaled salbutamol; T: inhaled terbutaline; assay following pre-extraction with C18 reverse phase µ columns (Sep-Pak; Waters, Milford, MA, USA). The FEV1: forced expiratory volume in one second. *: <1,000 g beclomethasone or <800 µg budesonide; **: ≥1,000 µg assay has previously been described in detail [19], both beclomethasone. inter- and intra-assay variation ≤8%. 1086 R.M. ANGUS ET AL.

cGMP. Venous blood (5 ml) was collected into lith- 4.4 ium heparin tubes, stored on ice and spun within 2 h. Cyclic GMP was later measured by radio-immunoassay 4.2 after pre-extraction of plasma onto Amprep minicolumns (Amersham International plc., Aylesbury, Buckingham- 4.0

shire, UK) (RPN 1918) [20]. The assay has previously l

been described in detail [20]. 1 3.8 Candoxatrilat (active metabolite of candoxatril). Ven- FEV ous blood (5 ml) was collected into plain tubes, stored 3.6 on ice and spun within 2 h. Plasma was stored at -20¡C and candoxatrilat, the active metabolite of candoxatril, 3.4 was later estimated using an assay dependent on the abil- ity of NEP to hydrolyse 14C-hippuryl-phenylalanyl- 3.2 arginine to hippuric acid and phenylyl-arginine. The amount of radiolabelled hippuric acid is inversely re- 3.0 0.0 0.5 1.0 1.5 2.0 lated to log concentration of candoxatrilat [21]. Time h Drugs Fig. 1. Ð Forced expiratory volume in one second (FEV1) after treat- Candoxatril (Pfizer Central Research, Sandwich, UK). ment with placebo or oral candoxatril (200 mg) in 10 asthmatic patients. Data are presented as mean and SEM. ❏ : placebo; ❍ : can- Capsules of 200 mg were provided and administered doxatril. with 200 ml of water. Placebo capsules (Pfizer Central Research, Sandwich, UK) identical to the above were provided and administered in the same way. Histamine 100 acid phosphate (Sigma Chemical Co., Poole, Dorset, UK) was made up in phosphate buffered saline. Salbutamol (Allen & Hanbury's, Greenford, UK) was made up in 2.5 mgáml-1 aliquots by the pharmacy.

-1 10 Analysis mg·ml

Changes in FEV1, pulse, blood pressure, hormonal 20

and drug levels were compared using analysis of vari- PC ance, Fisher's exact test and the Scheffe F-test were used 1 to correct for multiple comparisons. This was performed on an Apple Macintosh SE personal computer, using the Statview software package. 0.1 Results Placebo Candoxatril There was no significant difference in FEV and 1 Fig. 2. Ð Provocation concentration of histamine producing a 20% PC20 values between active and placebo study days. fall in forced expiratory volume in one second (PC20) after pretreat- The mean(SEM) FEV1 at baseline and 2 h was 3.71(0.29) ment with placebo or oral candoxatril (200 mg) in 10 asthmatic and 3.85(0.29) l, respectively, on the placebo day; and patients.

Table 2. Ð Pulse systolic and diastolic blood pressure (BP) following placebo or oral candoxatril (200 mg) in 10 asthmatic patients Time Pulse beatsámin-1 Systolic BP mmHg Diastolic BP mmHg

h Placebo Active Placebo Active Placebo Active 0 72.9 (6.2) 66.3 (3.6) 132 (5.8) 131 (5.5) 78 (3.7) 76 (2.9) 0.5 64.5 (3.8) 63.8 (3.7) 134 (5.4) 126 (4.5) 79 (4.0) 77 (2.0) 1 63.8 (3.4) 64.3 (3.2) 137 (4.6) 123 (4.1)* 81 (3.9) 74 (2.5) 1.5 63.4 (3.3) 63.1 (2.8) 133 (4.9) 123 (4.2)* 78 (3.8) 75 (3.0) 2 63.1 (3.4) 65 (3.7) 133 (5.0) 128 (3.8) 77 (3.4) 75 (2.3) 2.5 68.5 (3.5) 72.8 (3.6) 131 (5.0) 128 (4.1) 80 (2.8) 80 (3.2) 3 68.0 (3.2) 70.6 (2.7) 131 (6.1) 134 (4.2) 76 (3.4) 79 (3.2) 3.5 67.3 (3.4) 73.0 (3.9) 135 (5.1) 136 (3.3) 75 (3.9) 75 (2.4)

Data are presented as mean, and SEM in parenthesis. *: p<0.05. NEUTRAL ENDOPEPTIDASE INHIBITOR IN ASTHMA 1087

3 BP was noted at 1 and 1.5 h on the treatment day, when * compared with placebo (table 2). * Plasma drug levels were maximally elevated between 2 and 3 h (mean(SEM) plasma candoxatrilat at 1 h 161(53), 2 h 846(136), 3 h 896(68), and 4 h 62(60) -1 2 ngáml-1) (fig. 3). * ANP levels rose significantly on the active day from a mean(SEM) baseline value of 12.1(2.1) pgáml-1 to 29.1 * (4.8) pgáml-1 at 2 h, (p<0.005); but did not change on 1 the placebo day, baseline 11.5 (2) pgáml-1, 2 h level 10.8(1.9) pgáml-1 (fig. 3). Cyclic GMP levels rose on the active day from mean cGMP levels pmol·ml cGMP (SEM) baseline value of 0.69(0.28) pmolál-1 to be signif- 0 icantly elevated at 1.40(0.75) pmolál-1 at 2 h, and to be further elevated at 2.57(0.94) pmolál-1 at 4 h. On the placebo day there was no change with time (fig. 3). 70 60 Discussion * -1 50 * Our results demonstrate that the acute administration of the oral NEP inhibitor, candoxatril, does not alter 40 bronchomotor tone or bronchial reactivity to histamine 30 * in stable asthmatic patients. Candoxatril is an orally * active indanyl ester prodrug of candoxatrilat [15], which 20 is a potent specific inhibitor of neutral endopeptidase ANP levels pg·ml ANP [21, 22]. The oral administration of candoxatril has been 10 shown to elevate circulating ANP levels in normal [15] 0 and mild hypertensive patients [16]. Given by oral administration, peak plasma candoxatrilat levels are achieved at around 1.5 h, with peak ANP levels at 2Ð3 h, although they remain elevated at 4 h [16]. The oral 1000 administration of candoxatril (200 mg) is also associated with a significant natriuresis and diuresis [15, 16]; 800 higher doses are not associated with higher plasma ANP levels or increased natriuresis or diuresis. NEP is ubiq- -1 uitous and is plentiful in the lung, particularly in the air- 600 way [14]. The pulmonary uptake of ANP is approximately 20% [23], but it is not clear whether this is due to binding to clearance receptors or degradation by NEP with- 400 in the lung. The exact contribution of lung NEP in determining circulating levels has not been quantified; 200 however, the circulating levels of ANP doubtless reflect a general inhibition of NEP within the body. The systemic elevation of ANP does imply a significant NEP

Plasma candoxatril at ng·ml 0 inhibitory effect and, as we have shown that NEP is 01234important in regulating the in vitro effects of exogen- ous ANP on the airway, it is possible that inhibition of Time h NEP within the lung may add to this and facilitate local Fig. 3. Ð Plasma candoxatril at levels plasma ANP levels and plas- ANP effects. Overall, we believe it is unlikely, there- ma cGMP levels following placebo or oral candoxatril (200 mg) fore, that the absence of an effect of candoxatril on air- in 10 asthmatic patients. Data are presented as mean ±SEM. way function was due to a failure to inhibit NEP, to *: p<0.01 candoxatril vs placebo; ANP: atrial natriuretic peptide; the administered dose of the drug being too low, or to cGMP: cyclic guanylyl monophosphate. ❏ :placebo; ❍ : candoxatril. inappropriate timing of the bronchial challenge after dosing. 3.89(0.27) and 4.05(0.82) l, respectively, on the ac- The absence of a bronchodilator response may reflect tive day (fig. 1). The geometric mean (range) PC20 the fact that the patients had well-controlled asthma and -1 was 1.17(0.25Ð25.8) and 0.93(0.13Ð32) mgáml on the had baseline FEV1 values close to their predicted best. placebo and active day, respectively, (fig. 2). There However, the plasma ANP and cGMP levels achieved were no measured changes in pulse or blood pressure were much lower than those associated with broncho- (BP) at 2 h, although a small significant fall in systolic dilation in our previously reported infused study [4]. At 1088 R.M. ANGUS ET AL.

the higher rates of infused ANP associated with bron- erties, including tachykinins, which may be involved in chodilation, we also witnessed mild haemodynamic neurogenic inflammation and the pathogenesis of asth- changes, with a fall in systolic BP and a rise in pulse ma [2, 29]. It might be predicted, therefore, that inhi- [4]. In this study, there were no haemodynamic changes bition of NEP would increase bronchial reactivity. In observed, except for a slight reduction in systolic blood vitro work on human tissue has demonstrated that inhibi- pressure at 1 and 1.5 h. It seems likely that the levels tion of NEP potentiates substance P and capsaicin- of ANP achieved by inhibiting NEP are inadequate induced bronchial smooth muscle contraction [30]. to exert a significant bronchodilator action on the Thiorphan, an NEP inhibitor, has been given by in- airway in asthma. However, in patients with chronic ob- halation to normal subjects and asthmatic patients; no structive pulmonary disease, particularly with associated effect on airway tone was noted, and there was no pulmonary hypertension, resting ANP levels are ele- effect on a subsequent bronchial challenge by meth- vated [25]. In this setting, further elevation of circu- acholine [31, 32]. However, there was an enhanced lating ANP levels by the inhibition of NEP may result bronchoconstrictor response to the neuropeptide, neu- in pharmacological levels of ANP being achieved, al- rokinin A [31, 32]. One other study looked at respon-

lowing the manifestation of beneficial airway effects. siveness to the inflammatory mediator leukotriene D4 That ANP levels achieved are inadequate to signifi- in normal subjects, and found that preinhalation with cantly affect airway function is supported by the absence thiorphan enhances its effect [33]. In a study of an of a protective effect against histamine-induced bron- orally-administered NEP inhibitor, acetorphan, in a choconstriction. In concentrations lower than those dose sufficient to enhance skin responses to substance required to cause bronchodilation, infused ANP has P, no effect on airway function or responsiveness to been shown to modify the airway response to inhaled inhaled metabisulphide, which is thought to act in part histamine and to ultrasonically nebulized distilled water by the release of sensory neuropeptides, was noted in in asthmatics [5, 6]. However, the plasma ANP con- mildly asthmatic subjects [34]. The role of NEP in the centrations produced by these infusion studies were still lung is unclear, but is a balance between the potential higher than those achieved by the administration of can- beneficial effects of inhibiting ANP metabolism against doxatril; at the time of the challenge the mean (SEM) ANP the deleterious effects of inhibiting the breakdown of level was 29.1(4.8) after candoxatril compared with 41(4.3) other agents, such as tachykinins. No measure of sub- pgáml-1 with infused ANP (1 pmolákg-1ámin-1). Inhaled stance P or neurokinin A has been made, and we can- ANP (1 mg) has also been shown to attenuate histamine- not exclude that a challenge with these tachykinins might and methacholine-induced [7, 8] bronchoconstriction. have resulted in bronchoconstriction. Nevertheless, our The plasma levels associated with this dose of inhaled results suggest that in mild stable asthmatics the acute ANP are similar to those seen in this study (personal systemic inhibition of NEP does not alter the bronchial observation), however, it can be speculated that airway response to histamine. levels may be much higher when given by inhalation. Overall, the effect of the acute inhibition of NEP on Local concentrations are likely to be the most impor- airway tone and nonspecific reactivity appears to be tant consideration when examining the effects of ANP on neutral in these mild stable asthmatics. It is reassuring the airway, as it is probable that ANP achieves its effects that no deleterious effects on airway function have in vivo by acting directly on smooth muscle in the air- been observed, particularly if NEP inhibitors prove use- way. ANP is thought to act on particulate guanylyl ful in the treatment of other conditions, such as cardiac cyclase to cause the generation of cGMP, which is believed failure. A cautionary note is required, however, as this to be the second messenger for ANP in the cell [25]. study has only examined the acute effects of inhibiting Evidence for this includes several studies in animal mod- NEP in patients with mild stable asthma, and does not els, in which a direct dose-relaxant effect of ANP in air- address changes in NEP function or levels which may way smooth muscle has been demonstrated, and work occur in patients with more severe asthma. Our find- on isolated bovine trachea suggesting that this mecha- ings would suggest that the systemic inhibition of NEP nism is likely to be mediated by cGMP [26, 27]. ANP will not allow the beneficial effects of ANP on the receptors have also been demonstrated in the rat lung, airway to be harnessed in patients with asthma. One and these are located in the bronchial and bronchiolar possible intervention that could be envisaged, however, muscle [28], although there are no studies, so far, which is the administration of an NEP inhibitor concomit- have sought ANP receptors on the human airway. Further antly with ANP by inhalation. Here it could be specu- evidence for a direct action is the observation that ANP lated that the local inhibition of airway NEP would confers protection against methacholine-induced con- promote the effects of ANP. Alternatively, if the guan- traction in isolated human and bovine bronchial tissue [10]. ylyl cyclase pathway acted upon by ANP is to be ex- Whilst no beneficial effects were noted with the ad- ploited therapeutically in asthma, it seems likely that ministration of candoxatril, neither were any side-effects ANP analogues stable to NEP would hold more promise. observed or reported. Likewise, there was no increase Acknowledgements: The authors thank Pfizer Central in airway responsiveness to histamine. NEP cleaves a Research (UK) for providing candoxatril. They thank H. variety of active peptides in addition to ANP, including Johnson and J. Allen of Pfizer for their guidance with study encephalins, tachykinins (such as neurokinin A and design and help with the logistics of the study. They also which to thank J.J. Morton for his help with the hormone substance P), bradykinin, and chemotactic peptide [11]. assays. RMA is supported by a British Lung Foundation A number of substances have bronchoconstrictor prop- Research Fellowship. NEUTRAL ENDOPEPTIDASE INHIBITOR IN ASTHMA 1089

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