Introduction

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Kinins are a family of proinflammatory peptides generated in plasma and tissues through the enzymatic cleavage of precursors (kininogens) (Bhoola et al., 1992). In mammals, many effects of kinins are mediated through bradykinin B₂ receptors (Regoli and Barabé, 1980; Hess et al., 1992), which are constitutively expressed by a number of target cells. Another bradykinin receptor is present in mammals (bradykinin B₁ receptor), which has a low level of expression in normal tissues but is de novo expressed during inflammation or injury (Marceau et al., 1998).

Among natural kinins, bradykinin (BK) and kallidin are the natural agonists for B₂ receptors, whereas their des-Arg metabolites are the natural agonists for B₁ receptors, with negligible degree of cross talk between different peptides and receptors (Regoli and Barabé, 1980; Hall, 1992). In guinea pigs, there is molecular and pharmacological evidence for the presence of B₂ receptors (Hall, 1992; Farmer et al., 1998), whereas there are no conclusive data documenting the expression of B₁ receptor, even following inflammation.

BK produces a number of effects in the airways that may be relevant for the pathophysiology of asthma, including bronchoconstriction (Fuller et al., 1987), increased mucus secretion (Davis et al., 1982), induction of an increase in microvascular permeability and edema formation (Saria et al., 1983), stimulation of afferent nerves (Fox et al., 1993), and recruitment and stimulation of inflammatory cells (Sato et al., 1996). Part of these effects is indirect, mediated through the release of other mediators such as cytokines and chemokines (Koyama et al., 1995; Pagelow et al., 1995), sensory neuropeptides (Geppetti, 1993), prostanoids (Greenberg et al., 1982), and nitric oxide (Ricciardolo et al., 1994).

Because of its potent proinflammatory and bronchoconstrictor effects, it has been hypothesized that BK could play a role in the pathophysiology of airway irritation/allergy, contributing to the symptoms and pathophysiology of airway diseases, including asthma (Proud, 1998). To date, a clinical phase II study has been reported in asthmatic patients following 4 weeks of aerosol treatment with Icatibant, a peptide B₂ receptor antagonist (Akbary et al., 1996).

On the other hand, kinins, especially via the B₂ receptor, seem to have a protective role on cardiovascular function. In particular, there is accumulating evidence indicating that part of the beneficial effect exerted by angiotensin-converting enzyme inhibitors on the cardiovascular system could be indirectly produced by preventing the degradation of kinins, which are inactivated by angiotensin-converting enzyme (Gohlke et al., 1997).

Thus, when considering a putative beneficial role of B₂ receptor antagonists in asthma or other airway diseases, it remains to be established whether this effect could be accompanied (or not) by a detrimental effect on cardiovascular function due to a concomitant blockade of cardiovascular B₂ receptors. To date there is no evidence suggesting a heterogeneity of B₂ receptors in the airways versus the cardiovascular system, which could be an amenable starting point to develop "airway-selective" B₂ receptor antagonists. On the other hand, it could be hypothesized that a topical (e.g., aerosol) administration of these drugs may allow the desired effect on the airways with a relatively less efficient blockade of B₂ receptors in the cardiovascular system.

In the present study, we have characterized the in vivo effects of two peptide B₂ receptor antagonists, Icatibant (Wirth et al., 1991) and MEN 11270 (Meini et al., 1999) on bronchoconstriction and hypotension induced by BK in anesthetized guinea pigs compared with the nonpeptide antagonist FR 173657 (Asano et al., 1997). The three antagonists were administered by the intravenous and intratracheal route.

To further investigate the differences in potency and duration of action of these compounds in vivo, we also determined their affinity for B₂ receptors in guinea pig lungs by radioligand binding and have evaluated the metabolic stability of Icatibant and MEN 11270 in guinea pig plasma and homogenates of guinea pig liver and lung.

	Experimental Procedures

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Radioligand Binding Experiments. Male albino guinea pigs (400-450 g) were stunned and bled. Whole lungs were removed, dissected free of connective and vascular tissue, and placed in TES (10 mM, pH 7.4, at 4°C) with a cocktail of peptidase inhibitors: 1,10 phenanthroline (1 mM), EGTA (1 mM), captopril, leupeptin, soybean trypsin inhibitor, DL-2-mercaptomethyl-3-guanidoethylthiopropanoic acid (1 µM each), chymostatin (3.3 µM), phenylmethylsulfonyl fluoride (0.1 mM), and bacitracin (140 µg/ml). The lungs were chopped (McIlwain tissue chopper; The Mickle Laboratory Engineering, Gomshall, Surrey, UK) and homogenized with a Polytron (PT 3000; Kinematica, Lucerne, Switzerland), set at 15,000 rpm for 30 s in 10 ml/g of the above-mentioned buffer. The homogenate was centrifuged at 2500g for 10 min to remove cellular debris. The supernatant was homogenized and centrifuged at 20,000 rpm (4°C) for 30 min. The pellet was resuspended in binding buffer to obtain 10 mg/ml membrane protein and frozen immediately in 3-ml aliquots by immersion in liquid nitrogen, and then stored at 80°C until use.

In Vivo Experiments. Male albino guinea pigs (Charles River, Calco, Italy) weighing 350 to 400 g were anesthetized with urethane (1.5 g/kg s.c.). A polyethylene catheter was inserted into the jugular vein, for drug administration. The blood pressure was recorded through a polyethylene catheter inserted into the carotid artery and connected by a pressure transducer (Hewlett Packard 1240) to MacLab/8s ML 780.

Metabolic Stability. Dunkin-Hartley guinea pig (300-350 g) were used. The animals were fasted for approximately 16 h before the experiments but water was freely available. Blood was collected in tubes containing EDTA and the plasma obtained by centrifugation (2000g for 10 min) was stored at 25°C until used.

Evaluation of Data. Competition experiments were processed by Prism 2.0 (GraphPad, San Diego, CA) to determine the IC₅₀. All values are given as mean ± S.E. of the mean from the stated number of experiments (n) performed in duplicate.

Materials. [³H]BK (specific activity 114 mCi/nmol) was provided by DuPont NEN (Hertfordshire, UK). BK was obtained from Peninsula (St. Helens, UK). All B₂ receptor antagonists used, MEN 11270, Icatibant, and FR 173657 were synthesized at Menarini Ricerche (Florence, Italy). Leupeptin was obtained from Boehringer-Mannheim (Germany) and DL-2-mercaptomethyl-3-guanidoethylthiopropanoic acid from Calbiochem (La Jolla, CA). GF/B glass fiber filtermats were provided by Brandel (Semat, St. Albans, Hertfordshire, UK). Phenanthroline, EGTA, EDTA, captopril, chymostatin, bacitracin, and propranolol were purchased from Sigma (St. Louis, MO). Atropine was purchased from Serva (Heidelberg, Germany), 2-hydroxypropil--cyclodextrin from Research Biochemicals International (Natick, MA), and [Hyp³,Tyr(Me)⁸]-bradykinin from Calbiochem-Novabiochem (Laüfelfingen, Switzerland). Dimethyl sulfoxide was purchased from Merck (Darmstad, Germany).

	Results

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Binding of [³H]BK to Guinea Pig Lung Membranes

BK (1 pM-1000 nM) displaced [³H]BK binding (0.2 nM) from guinea pig lung membranes with an IC₅₀ of 0.97 ± 0.53 nM (n = 3) (Hill slope of 0.78 ± 0.091). All the B₂ receptor antagonists were tested in the same concentration range.

Icatibant displaced the [³H]BK binding with an IC₅₀ of 0.24 ± 0.06 nM (n = 3) (Hill slope of 1.33 ± 0.12). MEN 11270 showed a comparable affinity value, its IC₅₀ being 0.43 ± 0.13 nM (n = 3) (not statistically different from BK or Icatibant) (Hill slope of 0.87 ± 0.026). As shown in Fig. 1 the nonpeptide B₂ receptor antagonist FR 173657 showed a lower affinity in inhibiting the [³H]BK binding, its IC₅₀ amounting to 15.3 ± 10 nM (P < 0.05 versus BK, Icatibant, and MEN 11270; Fisher's least-significant difference test) (Hill slope of 0.72 ± 0.04).

View larger version (21K): [in this window] [in a new window]   Fig. 1.   Concentration-inhibition curves for BK, MEN 11270, Icatibant, and FR 173657 on [3H]BK (0.2 nM)-specific binding in guinea pig lung membranes. Each point and bar represents the mean ± S.E.M of three experiments performed in duplicate.

In Vivo Experiments

Effect of Intravenous Administration of Bradykinin or [Hyp³,Tyr(Me)⁸]-Bradykinin on Bronchoconstriction and Blood Pressure. BK (0.1 nmol, 1 µmol/kg i.v., n = 6) induced a dose-dependent increase of insufflation pressure (bronchoconstriction) and concomitant reduction in both systolic and diastolic blood pressure in anesthetized, propranolol (3.4 µmol/kg)-pretreated guinea pigs.

View larger version (16K): [in this window] [in a new window]   Fig. 2.   Traces showing effect of intravenous administration of bradykinin (10 nmol/kg i.v.) on systemic pressure (A and C) and bronchoconstriction (B and D) before and after vehicle (top) or MEN 11270 (100 nmol/kg i.v.) (bottom) in urethane-anesthetized guinea pigs.

Effect of MEN 11270, Icatibant, and FR 173657 on Bronchoconstriction and Hypotension Induced by Intravenous Administration of BK. MEN 11270, Icatibant, and FR 173657 at the highest dose tested (100 nmol/kg i.v.) did not affect the basal values of blood pressure and insufflation pressure (data not shown).

View larger version (36K): [in this window] [in a new window]   Fig. 3.   Effect of intravenous administration of MEN 11270, Icatibant, and FR 173657 on bronchoconstriction (A, C, and E) and hypotension (diastolic pressure) (B, D, and F) induced by intravenous administration of bradykinin (10 nmol/kg) in anesthetized guinea pigs. In all experiments the animals were treated with propranolol (3.4 µmol/kg i.v.) and gallamine (3.4 µmol/kg i.v.) as a bolus (1 ml/kg) followed by continuous infusion of the same solution for the duration of the experiment. In each panel results are expressed as a percentage of the control response to bradykinin recorded before treatment. Each value is the mean ± S.E.M. of five to seven experiments. *P < 0.05, significantly different from the respective value in the vehicle group.

View larger version (35K): [in this window] [in a new window]   Fig. 4.   Effect of intratracheal administration of MEN 11270, Icatibant, and FR 173657 on bronchoconstriction (A, C, and E) and hypotension (diastolic pressure) (B, D, and F) induced by intravenous administration of bradykinin (10 nmol/kg) in anesthetized guinea pigs. In all experiments the animals were treated with propranolol (3.4 µmol/kg i.v.) and gallamine (3.4 µmol/kg i.v.) as a bolus (1 ml/kg) followed by continuous infusion of the same solution for the duration of the experiment. In each panel results are expressed as a percentage of the control response to bradykinin recorded before treatment. Each value is the mean ± S.E.M. of five to eight experiments. *P < 0.05, significantly different from the respective value in the vehicle group.

Metabolic Stability of MEN 11270 and Icatibant

Both MEN 11270 and Icatibant incubated in guinea pig plasma at a concentration of 10 µM (for 24 h at 37°C) were stable, since their recovery was greater than 95%. As shown in Table 1, both peptide antagonists (at 10, 20, or 50 µM) were metabolized in guinea pig liver homogenates. Striking differences between the metabolic stability of MEN 11270 and Icatibant were observed in guinea pig lung homogenates where MEN 11270 (at 10 or 50 µM) was stable for 24 h, whereas Icatibant underwent extensive time- and concentration-dependent degradation.

	Discussion

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BK, under the action of carboxypeptidases can generate its des-Arg metabolite, which is a selective ligand for bradykinin B₁ receptors (Marceau et al., 1998). Although we have used BK as agonist in most of our in vivo experiments, we are confident that the effects reported in this study are exclusively dependent on stimulation of bradykinin B₂ receptors for the following reasons: 1) although B₁ receptors have been cloned from various species there is little evidence, to date, for their expression in guinea pigs (Perron et al., 1999); 2) both bronchoconstriction and hypotension induced by BK were effectively blocked by Icatibant, MEN 11270, and FR 173657, which are potent B₂ receptor antagonists with a high degree of selectivity for B₂ versus B₁ receptors (Wirth et al., 1991; Asano et al., 1997; Meini et al., 1999); 3) the effects of BK were reproduced by [Hyp³,Tyr(Me)⁸]-BK, a selective kinin B₂ receptor agonist, and the responses to this synthetic ligand were blocked by Icatibant (dose 30 nmol/kg i.v.) with similar efficacy and duration of action compared with those of BK.

The present results indicate that both MEN 11270 and Icatibant are potent and long-acting inhibitors of the B₂ receptor-induced bronchoconstriction in guinea pigs following i.v. administration. When given by the i.v. route, FR 173657 consistently showed a good potency in inhibiting bronchoconstriction at a short time after administration, but this effect was short lived, at least compared with the peptide antagonists. This difference in the in vivo activity between peptide and nonpeptide (FR 173657) B₂ receptor antagonists was even more evident when considering BK-induced hypotension, and consistent with the results of other in vivo studies (Meini et al., 2000). However, in a previous study, following s.c. administration (Griesbacher and Legat, 1997) FR 173657 showed a long-duration antagonist effect against BK-induced hypotension. This discrepancy is probably due to the different routes of administration of FR 173657 (i.v. versus s.c.) between their study and ours. It is likely that following s.c. administration absorption is slower, allowing for a longer duration of effect compared with i.v. injection.

It could be speculated that the poor in vivo activity of FR 173657 compared with peptide antagonists simply reflects the lower binding affinity of FR 173657 for guinea pig B₂ receptors (Inamura et al., 1997; present study). However, in vitro functional studies on guinea pig tissues (trachea, ileum) indicate that peptide and the nonpeptide (FR 173657) compounds antagonize B₂ receptor-mediated responses with a similar potency (Griesbacher et al., 1997; Inamura et al., 1997). Since in binding studies the affinity of FR 173657 for B₂ receptors is temperature-dependent and that of peptide antagonists is not (Meini et al., 1999), it is possible that the different temperatures at which binding and functional studies have been carried out (room temperature and 37°C, respectively) are responsible for the underestimation of apparent affinity of FR 173657 in binding studies (Inamura et al., 1997; present study). It appears likely that factors related to distribution, degradation, or elimination greatly limit the efficacy of this antagonist after systemic administration.

All the antagonists tested were more effective in inhibiting BK-induced bronchoconstriction than BK-induced hypotension after i.v. administration. The dose of BK selected as a challenge was clearly more effective in producing hypotension (about 80% of E_max) than bronchoconstriction (about 50% of E_max); therefore, the observed difference in antagonist efficacy in inhibiting BK-induced bronchoconstriction versus BK-induced hypotension after i.v. administration can be partly ascribed to these different effects of BK. Despite this limitation it is interesting to observe that the relative efficacy of different antagonists in inhibiting BK-induced bronchoconstriction versus BK-induced hypotension was increased after i.t. administration. In this respect, the most striking gain of airway selectivity was observed for FR 173657, which was practically ineffective in producing a significant block of BK-induced hypotension after administration by the i.t. route. This may indicate that the systemic bioavailability of this compound is limited following i.t. administration, preventing a blockade of the vascular effect of BK. The data obtained after i.v. administration of FR 173657, as discussed above, are in favor of this second hypothesis.

An increase in airway selectivity after i.t. administration was also observed for MEN 11270 and Icatibant. Although the profile of action of these two peptide antagonists was virtually the same after i.v. administration, the degree of blockade of BK-induced bronchoconstriction after topical delivery to the airways seems more intense/prolonged with MEN 11270 than for Icatibant. Our data indicate that both drugs are substantially resistant to degradation in guinea pig plasma and liver, indicating that chemical modification of these structures (both derived from BK) has produced a substantial reduction in degradation by peptidases. Although Icatibant was relatively resistant to degradation for short periods of incubation in homogenates of guinea pig lung, a substantial degradation of this compound was also observed after 4 and 24 h, whereas MEN 11270 was not degraded at all for up to 24 h of incubation. Therefore, the present data indicate the existence of an enzymatic activity in homogenates of guinea pig lung, which slowly but efficiently degrades Icatibant but not MEN 11270. It appears conceivable that, within the time frame of the in vivo experiments, this enzymatic activity may have limited the activity of Icatibant in preventing BK-induced bronchoconstriction. Therefore, as already described (Whalley et al., 1997), the duration of the in vivo action of Icatibant can be improved by structurally modified analogs, and the present data suggest that this goal can be achieved by improving metabolic stability.

In conclusion, the present findings indicate that B₂ receptor antagonists either of peptide or nonpeptide origin are effective in blocking B₂ receptors in guinea pig airways, and that after topical delivery of these drugs it is possible to reduce their ability to block B₂ receptors in the cardiovascular system. It is possible that compounds derived from these prototypes may represent a novel class of drugs for treatment of airway diseases in which BK has a pathogenic role.