Kallikrein inhibitors limit kinin B(2) antagonist-induced progression of oedematous to haemorrhagic pancreatitis in rats.

BACKGROUND AND PURPOSE: Exocrine hyperstimulation with caerulein is an established model for oedematous acute pancreatitis. Prevention of oedema formation by bradykinin B(2) receptor antagonists induces a progression to a haemorrhagic course in this model. We have investigated whether increased kallikrein activity in the pancreas is responsible for vascular damage and whether this could be prevented by selective kallikrein inhibitors. EXPERIMENTAL APPROACH: Caerulein was infused i.v. and vascular damage was assessed by histological evaluation and determination of haemoglobin accumulation in the tissue. In addition, oedema formation, tissue and plasma kallikrein (PK) activities and the endogenous kallikrein inhibitors alpha(1)-antitrypsin (alpha(1)-AT) and alpha(2)-macroglobulin (alpha(2)-M) were measured. KEY RESULTS: Haemorrhagic lesions induced by icatibant in caerulein-induced pancreatitis were associated with a reduction in alpha(1)-AT and alpha(2)-M in the pancreas and a concomitant augmentation of tissue kallikrein (TK) activity. The TK inhibitor VA999024 (previously FE999024), or its combination with the PK inhibitor VA999026 (previously FE999026), inhibited oedema formation to the same extent but did not induce vascular damage. Furthermore, VA999024 inhibited TK activity. When icatibant was combined with VA999024 and VA999026, progression from oedematous to haemorrhagic pancreatitis was abolished. CONCLUSIONS AND IMPLICATIONS: Reduced oedema formation by B(2) antagonists prevented influx of endogenous kallikrein inhibitors and led to an excessive activity of kallikrein in the pancreas leading to vascular damage. This can be prevented by a combined inhibition of both tissue-type and plasma-type kallikrein. Kallikrein inhibitors thus should be further evaluated for their therapeutic potential in preventing haemorrhagic lesions in acute pancreatitis.

Effects of the non-peptide B2 receptor antagonist FR173657 in models of visceral and cutaneous inflammation.

OBJECTIVE: The non-peptide B2 receptor antagonist (E)-3-(6-acetamido-3-pyridyl)-N-[N-[2,4-dichloro-3-[(2-methyl-8-quinolin yl)oxymethyl]phenyl]-N-methylaminocarbonylmethyl]acrylamide (FR173657) was compared to the peptide antagonist icatibant in models of visceral and cutaneous inflammation. METHODS: Pancreatitis was induced by caerulein in anaesthetized Sprague-Dawley rats. Acute cystitis was induced by intravesical instillation of xylene or i.p. cyclophosphamide injection. Cutaneous inflammation was induced in anaesthetized guinea-pigs by s.c. injection of collagenase from Clostridium histolyticum. RESULTS: FR173657 inhibited oedema formation and tissue enzyme retention during pancreatitis at 500 nmol/kg and above after peroral administration, and from 30 nmol/kg after s.c. injection; icatibant was effective at 3 nmol/kg s. c. Protein extravasation in both cystitis models was abolished by s.c. FR173657 at 300 nmol/kg. Collagenase-induced oedema was attenuated equieffectively by FR173657 and icatibant at doses of 10 micomol/kg and 300 nmol/kg s.c., respectively. CONCLUSIONS: FR173657 inhibits kinin-mediated effects in visceral and cutaneous inflammation at doses that are about 10 times higher than those of icatibant. However, FR173657 is also active following oral administration.

Kallikrein-kinin system in acute pancreatitis: potential of B(2)-bradykinin antagonists and kallikrein inhibitors.

The development of selective antagonists for bradykinin B(2) receptors has greatly advanced research on the role of the kallikrein-kinin system in acute pancreatitis. Kinins released during the course of the inflammatory injury are the major cause of the vascular symptoms, i.e. pancreatic oedema formation and its consequences, such as haemoconcentration, hypovolaemia and hypotension. Kinins are also involved in the accumulation of potentially cytotoxic factors in the pancreatic tissue. However, treatment with B(2) antagonists must begin prior to the formation of pancreatic oedema in order to inhibit or attenuate the vascular effects. Visceral pain as a possible target symptom for treatment with B(2) antagonists at later time points is suggested by the B(2) receptor-mediated activation of nociceptive afferents.

5-hydroxytryptamine release from skin mast cells in vivo induced by peptide but not by nonpeptide ligands for bradykinin receptors.

A number of biologically active peptides including bradykinin (BK) are known to elicit an unspecific, non-receptor-mediated release of mediators from mast cells. We have investigated whether novel, nonpeptide BK B2 receptor ligands, i.e., the antagonists N-[N-[3-[(3-bromo-2-methylimidazo(1,2-a]pyridin-8-yl)oxymethyl]-2, 4-dichlorophenyl]-N-methylaminocarbonylmethyl]-4-(dimethylamino carbonyl)cinnamylamide hydrochloride (FR167344) and (E)-3-(6-acetamido-3-pyridyl)-N-[N-(2,4-dichloro-3-¿(2-methyl-8-quinolin yl)oxymethyl¿ phenyl]-N-methylaminocarbonylmethyl]acryamide (FR173657), and the agonist 8-[2,6-dichloro-3-[N-[(E)-4-(N-methylcarbamoyl)cinnamidoacetyl+ ++]-N-methylamino] benzyloxy]-2-methyl-4-(2-pyridylmethoxy)quinoline (FR190997), would be devoid of this unspecific action. Paw oedema in anaesthetized rats was observed following subplantar injection of BK, FR190997, the B2 antagonist D-Arg-[Hyp3, Thi5, D-Tic7, Oic8]-BK (icatibant, previously Hoe-140), the B1 agonist des-Arg9-BK (DABK) and the B1 antagonist des-Arg9 [Leu8]-BK (DALBK). The effect of BK was inhibited by systemic pretreatments with icatibant and methysergide in an additive manner, whereas the local effect of icatibant was only sensitive to methysergide. Oedema induced by DABK and DALBK was also attenuated by methysergide. Mepyramine pretreatment was ineffective for all oedema-producing agents. Effects of FR190997 were abolished by pretreatment with icatibant. FR167344 and FR173657 did not induce oedema formation. Depletion of mast cells by compound 48/80 mimicked the effects of methysergide on BK and icatibant and had no effect on FR190997. It is concluded that the novel nonpeptide receptor ligands are devoid of the unspecific mast cell-degranulating action observed with the peptide ligands.

Inhibition of kinin action and kinin generation compared to dexamethasone pretreatment with respect to vascular effects and pancreatic enzymes in experimental acute pancreatitis.

It was determined earlier that inhibition of the action of endogenous kinins by the bradykinin B2 antagonist, icatibant (Hoe-140; D-Arg-[Hyp3, Thi5, D-Tic7, Oic8]-bradykinin), prevents pancreatic oedema formation during caerulein-induced acute pancreatitis, and simultaneously improves the egress of activated pancreatic enzymes from the pancreas. We have now investigated whether inhibition of increases in vascular permeability by another approach, i.e., pretreatment with dexamethasone, would have comparable effects. In addition, preliminary data are presented on the effects of the selective low molecular weight inhibitor of tissue kallikrein, H-(4-Cl)-D-Phe-1Nal-(3-aminopropyl)-guanidine (CH-2856). Icatibant abolished plasma extravasation into the pancreatic tissue and prevented the development of hypovolaemia. Caerulein-induced increases of amylase activity in the pancreas were significantly reduced by icatibant, while amylase activity in blood was augmented. Inhibition of kinin generation by CH-2856 had similar effects, as oedema formation was inhibited and enzyme activities were reduced in the pancreas and augmented in the blood serum. Dexamethasone completely abolished oedema formation, but only partially inhibited the development of hypovolaemia and haemoconcentration. Amylase activities in the pancreas and in blood remained completely unaffected by dexamethasone. The results suggest that retention of activated enzymes in the pancreatic tissue during acute pancreatitis involves a B2 receptor-mediated, but glucocorticoid-insensitive mechanism.

Vespula vulgaris venom: role of kinins and release of 5-hydroxytryptamine from skin mast cells.

Wasp venoms contain several active components, among them kinin-related peptides. Like bradykinin and [Thr6]bradykinin, Vespula vulgaris venom caused paw oedema following subplantar injection in anaesthetized rats. The oedema was partly inhibited by the bradykinin B2 receptor antagonist icatibant (Hoe 140); the remaining part was abolished by additional pretreatment with 5-hydroxytryptamine (5-HT) receptor antagonists or mast cell depletion. Histamine receptor antagonists were ineffective. Capsaicin pretreatment attenuated oedema formation indicating a neurogenic sensory component. Nociceptive behavioural responses induced by the venom in unanaesthetized rats were abolished by icatibant. It is concluded that kinins, either contained in the venom or released from the tissue, play the predominant role in the inflammatory and algesic effects. The inflammatory effects only partly rely on direct, bradykinin receptor-mediated mechanisms while the remaining part depends on the release of 5-HT from skin mast cells. The algesic effects of the venom are entirely due to direct B2 receptor activation.

The nonpeptide B2 receptor antagonist FR173657: inhibition of effects of bradykinin related to its role in nociception.

1. The nonpeptide bradykinin B2 receptor antagonist, FR173657 ((E)-3-(6-acetamido-3-pyridyl)-N-[N-(2, 4-dichloro-3-[(2-methyl-8-quinolinyl) oxymethyl] phenyl]-N-methylaminocarbonylmethyl] acrylamide), was tested in models involving bradykinin-induced activation of primary afferent neurones in vitro and in vivo. 2. Bradykinin-induced contractions of the rabbit isolated iris sphincter muscle mediated by tachykinin release from trigeminal afferent neurones were inhibited in a non-competitive manner by FR173657. A pKB value of 7.9 was calculated. Effects of substance P were unaffected by FR173657. 3. Nociceptive behavioural responses following intraplantar injection of bradykinin in unanaesthetized rats were reduced by 0.3 micromol kg(-1) FR173657 s.c. (P < 0.05), and completely abolished by 3 micromol kg(-1) (P < 0.05). Peroral administration of 5 micromol kg(-1) FR173657 abolished the bradykinin effects (P < 0.05); lower doses had no significant effect. 4. Shortening by intraplantar injection of bradykinin of the paw withdrawal latency in response to radiant heat was abolished by 3 micromol kg(-1) FR173657 s.c. (P < 0.05), while 300 nmol kg(-1) had an intermediate effect. Hyperalgesia induced by prostaglandin E2 remained unaffected by FR173657. 5. Blood pressure reflexes following i.p. instillation of bradykinin in anaesthetized rats were inhibited by FR173657 s.c. with an ID50 of 1.1 micromol kg(-1), while the peptidic B2 antagonist icatibant (Hoe-140; D-Arg0-[Hyp3, Thi5, D-Tic7, Oic8]-bradykinin) caused inhibition at significantly lower doses (ID50 8.5 nmol kg(-1) P < 0.001). Responses to hydrochloric acid i.p. remained unaffected by FR173657. 6. FR173657 or similar nonpeptide compounds may be useful for the development of drugs for diseases involving pain induced by the release of endogenous kinins, i.e. especially in acute inflammatory conditions.

Effects of the non-peptide B2 antagonist FR173657 on kinin-induced smooth muscle contraction and relaxation, vasoconstriction and prostaglandin release.

1. The non-peptide bradykinin (BK) antagonist (E)-3-(6-acetamido-3-pyridyl)-N-[N-[2,4-dichloro-3-[(2-methyl-8-quinolin yl) oxymethyl]phenyl]-N-methylaminocarbonylmethyl]acrylamide (FR173657) was tested in intestinal, uterine, tracheal and vascular in vitro preparations. The investigation aimed at determining the antagonistic potency, duration of action, specificity for BK receptors and apparent mode of antagonistic action of FR173657. 2. Contractions of the isolated ileum of the guinea-pig in response to BK were inhibited by FR173657 (10-300 nM) in a concentration-dependent manner. The inhibition lasted for up to 90 min after wash-out of FR173657. Cumulative concentration-response curves to BK were shifted to the right with a concomitant decrease in the maximum effect. A pKB value of 8.7 was determined. FR173657 had no effect on contractions induced by acetylcholine, histamine, 5-hydroxytryptamine, substance P, angiotensin II or caerulein. 3. The concentration-response curves for B2 receptor-mediated relaxations of the rat isolated duodenum induced by BK were shifted to the right together with a concomitant reduction of the maximum BK effect in the presence of FR173657 (10-300 nM). A pKB of 9.0 +/- 0.2 was calculated. FR173657 had no effect on B1 receptor-mediated relaxations in response to des-Arg9-BK. 4. The concentration-response curves for BK-induced contractions of the rat isolated uterus were shifted to the right by FR173657 (3-300 nM) in a concentration-dependent and parallel manner. The Schild plot for the inhibition caused by FR173657 had a slope of -0.98 indicating a competitive mode of antagonism. A pA2 value of 9.1 was determined. 5. Contractions of the circular smooth muscles of the guinea-pig isolated trachea in response to BK were concentration-dependently inhibited by FR173657 (10-100 nM). An affinity estimate of 9.3 was calculated for FR173657. Contractions induced by acetylcholine and relaxations in response to isoprenaline remained completely unaffected by FR173657. 6. In the rabbit isolated perfused ear, BK (0.01-10 nmol) produced a dose-dependent vasoconstriction. In the presence of 30 nM FR173657, the effects of BK were reduced by at least 60%, while FR173657 completely abolished the effects of all BK doses at 300 nM. FR173657 did not affect vasoconstriction induced by noradrenaline or angiotensin II. 7. The arterial injection of BK (10 nmol) into the rabbit isolated perfused ear caused an approximately three fold increase in the release of the prostaglandins E2 and I2 into the venous effluent. The BK-stimulated prostaglandin release was completely abolished in the presence of FR173657 (300 nM) while the basal prostaglandin release was unchanged. 8. In summary, FR173657 was shown to be a highly potent and selective BK antagonist which was active on B2, but not B1, receptors. FR173657 was a competitive antagonist in the rat uterus but showed a deviation from competitive inhibition in the other preparations studied similar to other second generation peptide antagonists. The inhibitory action in vitro was long-lasting, but was fully reversible.

Effects of FR173657, a non-peptide B2 antagonist, on kinin-induced hypotension, visceral and peripheral oedema formation and bronchoconstriction.

1. Kinins are believed to play a key role in many inflammatory conditions. Therefore, bradykinin antagonists are being developed for potential therapeutic applications. In the present investigation we describe the pharmacology, in vivo, of (E)-3-(6-acetamido-3-pyridyl)-N-[N-[2,4-dichloro-3-[2-methyl-8-quinoliny l) oxymethyl]phenyl]-N-methylaminocarbonylmethyl]acrylamide (FR173657), a novel, non-peptide bradykinin antagonist. 2. The hypotensive effects of i.v. injections of bradykinin (50 pmol) in captopril-pre-treated anaesthetized rats were significantly inhibited by 100 nmol kg-1 FR173657 s.c., and completely abolished by 300 nmol kg-1. The full inhibitory effect developed within 60 min and remained unchanged for at least 4 h. However, the effect was reversible, since 24 h after an injection of 300 nmol kg-1 FR173657 no inhibitory effect could be observed. 3. The plasma protein extravasation into the pancreas and duodenum induced by an i.v. infusion of bradykinin (11 nmol kg-1 within 20 min) in captopril-treated anaesthetized rats was completely abolished by FR173657 at doses of 30 nmol kg-1 s.c. and above, given 60 min before bradykinin. FR173657 3 nmol kg-1 was ineffective, while a dose of 10 nmol kg-1 produced an intermediate effect. 4. The paw oedema induced by the subplantar injection of bradykinin (30 nmol) in anaesthetized rats was inhibited slightly by s.c. injection of FR173657 0.3 mumol kg-1, whereas 1 and 3 mumol kg-1 produced significant inhibition of the bradykinin-induced oedema. The maximum inhibition amounted to about 50% and could not be increased even when the dose of FR173657 was increased to 30 mumol kg-1. FR173657 did not effect the oedema caused by histamine or 5-hydroxytryptamine. 5. Bradykinin (20 nmol kg-1, i.v.) caused increases in pulmonary inflation pressure by 300-600 Pa in anaesthetized, respirated guinea-pigs. The effect was reduced to 58 +/- 9% of the initial value 60 min after the s.c. injection of FR173657 1 mumol kg-1, whereas only 9 +/- 7% remained after 10 mumol kg-1. The bronchoconstrictor actions of histamine remained unaffected by FR173657. 6. In summary, FR173657 is a highly potent and selective bradykinin antagonist. The inhibitory action in vivo lasts for longer than 4 h but is fully reversible. FR173657, or similar compounds, will be a useful tool for the pharmacological investigation of pathophysiological states and may possess a therapeutic potential in diseases involving the endogenous release of kinins.

Pathophysiological and possible physiological roles of kinins in the pancreas.

The i.v. infusion of a low dose of the cholecystokinin agonist caerulein elicited a sustained secretion of amylase into the biliopancreatic duct of rats. Pretreatment with the bradykinin antagonist icatibant (Hoe-140) had no effect on unstimulated amylase release and did not affect caerulein-induced amylase secretion. An i.v. infusion of bradykinin in doses not producing a pancreatic oedema elicited an increase in pancreatic juice production lasting 20-40 min after the end of the infusion. This pro-secretory effect was also visible at higher doses in captopril-pretreated rats producing an oedema similar to that observed in caerulein-induced pancreatitis. Using the Monastral blue method, it was found that the kininase II blocker captopril induced an opening of endothelial gaps in pancreatic capillaries. This effect was blocked by icatibant suggesting that kinins are formed in the pancreas under basal conditions. Thus, kinins appear not to be involved in the regulation of the production of digestive enzymes. However, kinins may have a modulatory role in the production of pancreatic juice and in the microcirculatory regulation in the pancreas.

Evidence for the presence of NK1 and NK3 receptors on cholinergic neurones in the guinea-pig ileum.

In a guinea-pig ileum longitudinal muscle preparation, substance P (SP) (> or = 6 nM) caused an initial contraction followed by a sustained plateau contraction of about 20-50% of the initial response. This plateau contraction is caused by the SP-induced activation of cholinergic motoneurones which contract the smooth muscles by the released acetylcholine (ACh). We investigated the contribution of neurokinin NK1 and NK3 receptors during this 'plateau phase' of contraction. The plateau contraction induced by SP (60 nM) was significantly reduced by the NK1 receptor antagonist CP-96,345 (200 nM) added 5 min after SP, but was not affected by its inactive enantiomer CP-96,344 (200 nM). The NK1 receptor antagonist CP-99,994 (100 nM) significantly reduced the plateau contraction induced by SP (60 nM and 600 nM) and that induced by the NK1 receptor agonist substance P-O-methylester (SPOMe; 100 nM). CP-99,994 (100 nM), however did not affect the plateau contraction induced by the NK3 receptor agonist [Asp5,6, MePhe8]-SP(5-11) (100 nM). The plateau contraction induced by SP (600 nM) was not affected by the NK3 receptor antagonist SR-142,801 (100 nM), added 5 min after SP. Pre-incubation of the ileum with SR-142,801 (100 nM) 30 min prior to the addition of SP (600 nM) also had no significant effect on the plateau contraction. However, it significantly reduced the ileal contraction in the first minutes after the initial spasmogenic contraction. We suggest that SP induces the plateau contraction of the guinea-pig ileum longitudinal muscle mainly by the activation of NK1 receptors on cholinergic neurones.

Effects of the bradykinin antagonist, icatibant (Hoe 140), on pancreas and liver functions during and after caerulein-induced pancreatitis in rats.

It has been found earlier that the bradykinin antagonist, icatibant (Hoe 140), prevents the pancreatic oedema and the ensuing hypotension and haemoconcentration, and facilitates the removal of activated enzymes form the tissue during caerulein-induced acute pancreatitis. For a potential therapeutic use of the compound in clinical situations it is essential to investigate whether the associated increase in enzyme activities in the blood serum has any adverse effects on the pancreas itself or on other organs. Normal amylase secretion into the biliopancreatic duct stimulated by a low dose of caerulein (0.4 nmol kg-1 h-1, i.v.) was not affect by icatibant (100 nmol kg-1, s.c.) Acute pancreatitis, induced by a high dose of caerulein (4 nmol kg-1 h-1 for 2 h, i.v.), resulted in elevations in the activities of amylase and lipase in the pancreatic tissue and in the blood serum lasting for at least 4 h after the end of the caerulein infusion. While the rise in enzyme activities in the blood serum was augmented in icatibant-treated rats only at the end of the caerulein-infusion, the enzyme accumulation in the pancreas was significantly reduced by icatibant for at least 4 h after the end of the caerulein infusion. The secretion of amylase and lipase into the biliopancreatic duct was significantly increased only during the first 20 min of acute pancreatitis; in rats pre-treated with icatibant, no significant increase could be observed. Twenty-four hours after induction of pancreatitis, a low-dose caerulein stimulation of the exocrine function of the pancreas led to a reduced but sustained secretion of amylase regardless of whether the animals had received icatibant or not. During the first 45 min of pancreatitis, blood glucose concentrations were significantly reduced, but returned to values not different from those obtained in saline-infused controls. This effect was not affected by icatibant. No changes in the response to an i.v. glucose tolerance test were found on the day after induction of acute pancreatitis. The serum activities of glutamic pyruvic transaminase and gamma-glutamyl transpeptidase determined up to 24 h after induction of pancreatitis were not different from saline controls. icatibant had no effect on the activities of these enzymes. It is concluded that during caerulein-induced acute pancreatitis normal exocrine secretion of pancreatic enzymes into the pancreatic duct ceases almost immediately. Pre-treatment with icatibant significantly reduces the accumulation of activated enzymes in the pancreatic tissue for several hours after induction of pancreatitis while a concomitant augmentation in enzyme activities in the blood serum lasts much shorter. There is no indication of adverse effects on the function of the endocrine or exocrine pancreas and that of the liver, either during the acute stages of pancreatitis or during the recovery period.

Anti-inflammatory and analgesic activity of the bradykinin antagonist, icatibant (Hoe 140), against an extract from Porphyromonas gingivalis.

1. Porphyromonas gingivalis is one of the bacteria likely to be related to pain in periodontitis. Several enzymes isolated from P. gingivalis have been reported to have kininogenase activity. Since kinin release could be held responsible for inflammatory symptoms and pain in periodontitis, we investigated whether the inflammatory and algesic effects of a sonic extract from P. gingivalis (PGSE) could be inhibited by the potent bradykinin B2 receptor antagonist, icatibant (Hoe 140). 2. In anaesthetized rats, the subplantar injection of PGSE (0.1 and 1.0 mg) caused a dose-dependent oedema of the hind paws. The net increase of the paw volume 60 min after the injection was 23 +/- 5% and 77 +/- 12%, respectively. The oedema was rich in plasma proteins as determined by the Evans blue method. Pretreatment with icatibant (300 nmol kg-1, s.c.) significantly reduced the effect of 1.0 mg of PGSE whereas the effects of 0.1 mg of PGSE remained unaffected. 3. The subplantar injection of 1.0 mg of PGSE in unanaesthetized rats caused nociceptive behavioural responses which started about 5 min after the injection and lasted for about 10-15 min. These responses were completely prevented by pretreatment with icatibant (300 nmol kg-1, s.c.). 4. The present results show that the plasma extravasation induced by non-algesic doses of a sonic extract from P. gingivalis are caused by mechanisms other than B2 kinin receptor activation whereas inflammatory effects of algesic doses are due to the action of kinins. The pain elicited by the extract is solely mediated by kinins and can be prevented by icatibant. The bradykinin antagonist could thus have a potential for a clinical use against pain associated with periodontal inflammation.

Mediation by bradykinin of rat paw oedema induced by collagenase from Clostridium histolyticum.

1. Collagenases are thought to play a major role in the pathology of gas gangrene caused by Clostridium histolyticum, because they can destroy the connective tissue barriers. We investigated possible mediators involved in the oedema formation and plasma protein extravasation which follow the injection of a collagenase (EC 3.4.24.3) from Clostridium histolyticum into one hind paw of anaesthetized rats. 2. The magnitude of the oedema following a subplantar injection was dependent on the dose of collagenase (30, 100 and 300 micrograms) injected. It reached its maximum within 30 min and remained unchanged for at least 5 h. Plasma protein extravasation into the paw was most pronounced within 20 min of the injection. Heat-inactivated collagenase was ineffective. 3. The B2 bradykinin (BK) antagonist icatibant (D-Arg-[Hyp3-Thi5-D-Tic7- Oic8] bradykinin, formerly named Hoe-140) reduced oedema formation in a dose-dependent manner with a maximal reduction of around 65% at a dose of 100 nmol kg-1 (s.c.). A significant effect could already be observed at a dose of 10 nmol kg-1. The duration of the effect of icatibant (100 nmol kg-1) was found to be at least 3 h. These results demonstrate the high potency and long duration of action of icatibant. Pretreatment of rats with the bradykinin B1 antagonist, des-Arg9-[Leu8]-BK did not affect collagenase-induced paw oedema. Thus, the observed collagenase-induced effects are mainly mediated by BK through activation of B2 receptors. 4. Pretreatment of adult rats with capsaicin (125 mg kg-1, s.c.) three weeks before the collagenase injection caused a significant attenuation of the paw oedema and of plasma extravasation but was significantly less effective than icatibant (100 nmol kg-1, s.c.). The non-peptide substance P antagonist,CP-96,345 (l0 micromol kg-1, i.v.) significantly reduced collagenase-induced oedema formation to a degree comparable with that seen after capsaicin pretreatment. The inhibition by the substance P antagonist was significantly smaller than that seen after icatibant. The inhibitory effect of icatibant in capsaicin pretreated rats, or of icatibant together with CP-96,345 in untreated rats, was not greater than that oficatibant alone in rats treated with the vehicle for either capsaicin or CP-96,345. CP-96,344(10 micromol kg-1, i.v.), the inactive enantiomer of CP-96,345, did not affect collagenase-induced paw oedema. In capsaicin-pretreated rats, CP-96,345 (10 micromol kg-1, i.v.) did not reduce collagenase-induced paw oedema.The subplantar injection of bradykinin (30 nmol) induced a paw oedema comparable with that induced by collagenase (100 microg). CP-96,345 (10 micromol kg-1, i.v.), but not CP-96,344 (1O micromol kg-1, i.v.),significantly reduced the bradykinin-induced paw oedema. These findings indicate that collagenase leads to the release of bradykinin; bradykinin then stimulates afferent C-fibre terminals and causes the release of substance P and probably also neurokinin A, which augment the oedema-inducing effect of bradykinin.5. Indomethacin or mepyramine plus cimetidine failed to inhibit collagenase-induced paw oedema.Thus, prostaglandins and histamine do not seem to be involved in collagenase-induced paw oedema.6. After subplantar injection of collagenase, the sensitivity scores in a modified formalin-test rapidly increased during the first 10 min. This increase was abolished by pretreatment with icatibant(100 nmol kg-1, s.c.) indicating that the stimulation of nociceptive afferent neurones following injection of collagenase is due to the action of released kinins.7. In conclusion, bradykinin appears to be the main mediator of inflammation induced by a collagenase from Clostridium histolyticum. As well as having direct relevance to a known pathological condition,collagenase-induced paw oedema could prove to be a useful model in inflammation research and in the investigation of bradykinin antagonists. The present results might provide an experimental basis for clinical investigations of the effects of icatibant in infectious diseases where the release of collagenases from bacteria causes rapid spreading of inflammation.

Blood pressure reflexes following activation of capsaicin-sensitive afferent neurones in the biliopancreatic duct of rats.

1. Inflammatory diseases of the pancreas or diseases which cause obstruction within the biliary or within the biliary or pancreatic duct system are associated with severe pain. Although neuropeptides such as substance P are present in the biliary tree, only few capsaicin-sensitive, substance P-positive nerve fibres have been found in the ducts. In order to obtain functional evidence whether capsaicin-sensitive afferent neurones transmit nociceptive information arising from the biliopancreatic duct, blood pressure reflexes following electrical stimulation of the duct or increases in intraductal pressure were determined in barbiturate-anaesthetized rats. 2. Electrical stimulation of neurones in the biliopancreatic duct was carried out at 30 V, 3 ms, 50 Hz for 20s. In untreated animals the electrical stimulation resulted in rises in blood pressure by up to 25 mmHg, but in about a quarter of all animals tested this response was absent. Following the administration of phentolamine (7 mumol kg-1, i.p.) the blood pressure responses were changed to pronounced and reproducible depressor reflexes of -5 to -30 mmHg. Retrograde injections into the biliopancreatic duct of 300 microliters of a 154 mM sodium chloride solution produced increases in intraductal pressure of approximately 10 mmHg. This elicited shortlasting falls in blood pressure of 3-15 mmHg. Phentolamine significantly augmented the fall in blood pressure to 8-30 mmHg. 3. The depressor reflexes observed in both models after the administration of phentolamine were abolished by morphine (1 mumol kg-1, i.v.). The inhibition by morphine was reversed by naloxone (3 mumol kg-1, i.v.). Naloxone given before morphine did not affect the depressor reflex but prevented the inhibitory action of subsequently injected morphine.4. Acute s.c. injection of capsaicin (30 mg kg-1) abolished the depressor reflexes in response to both types of nociceptive stimulation in phentolamine-treated rats. The initial pressor effects of electrical stimulation were only partly inhibited by capsaicin whereas the basal depressor reflexes in response to elevation of intraductal pressure were abolished. In rats which had received capsaicin on the day before the experiment or had been treated with capsaicin as neonates, only minor rises in blood pressure were induced by electrical stimulation at the beginning of the experiment and no changes in blood pressure occurred after the administration of phentolamine. After adult or neonatal pretreatment with capsaicin the depressor reflexes in response to increased intraductal pressure were only small and were unchanged by phentolamine.5. The depressor reflexes following either electrical stimulation or increases in intraductal pressure were abolished by the unselective Beta-blocker, (-)-propranolol (3 micromol kg-1, i.p.), and greatly reduced by the Beta 1-blocker, metoprolol (6 micromol kg- 1, i.p.). The Beta2-preferring adrenoceptor antagonist, butoxamine(3 micromol kg-1, i.p.), had no effect on the depressor responses. The reflex falls in blood pressure were also abolished by hexamethonium (10 micromol kg-1, i.p.) but not by atropine (3 micromol kg-1, i.p.).6. Both models of stimulation of nociceptive afferents caused identical patterns of blood pressure responses following adrenalectomy or chemical sympathectomy. In adrenalectomized rats, the initial responses consisted of depressor reflexes which were not augmented but significantly reduced by phentolamine and further inhibited by metoprolol. In rats that had been pretreated with 6-hydroxydopamine(total dose 0.6 mmol kg-1) to accomplish chemical sympathectomy, nociceptive stimulation caused rises in blood pressure. Phentolamine treatment abolished these pressor effects but revealed only minor, if any, depressor responses that were unaffected by metoprolol.7. In summary, the hypotensive effects in both models constitute nociceptive reflexes since they are abolished by morphine and restored by naloxone. The afferent part of the reflex is mediated by nerve fibres sensitive to capsaicin. Both experimental procedures seem to elicit two, presumably separate, reflex mechanisms. Firstly, catecholamines released from the adrenal medulla elevate blood pressure or limit hypotensive responses via activation of vascular alpha receptors. Secondly, the reflex inhibition of the sympathetic nerve activity in the heart and the vasculature causes the nociceptive depressor reflexes.

Pathological events in experimental acute pancreatitis prevented by the bradykinin antagonist, Hoe 140.

1. In a previous investigation, Hoe 140, a specific and potent bradykinin B2 receptor antagonist, prevented the pancreatic oedema and the hypotension observed during acute experimental pancreatitis; however, it augmented the associated rises in the serum activities of pancreatic enzymes. Therefore, we have now investigated the consequences of the pancreatic oedema for the fate of activated enzymes released into the tissue during the course of acute pancreatitis. 2. Acute oedematous pancreatitis was induced in rats, pretreated with captopril (50 mumol kg-1, i.p.), by hyperstimulation of the exocrine function of the pancreas with the cholecystokinin analogue, caerulein (4 nmol kg-1 h-1, i.v.), for up to 120 min. 3. Pancreatic oedema began to develop 10 min after the start of the caerulein infusion, reached a maximum within about 45 min, and then declined slightly. The development of the oedema parallelled the second phase of the caerulein-induced fall in blood pressure found in earlier experiments. No further extravasation of plasma proteins occurred during the 2nd hour of the caerulein infusion. The oedema formation was completely blocked in animals pretreated with the bradykinin receptor antagonist, Hoe 140 (100 nmol kg-1, s.c.). Pretreatment with aprotinin or soy bean trypsin inhibitor did not result in a significant inhibition of the oedema. 4. The haematocrit of animals with experimental pancreatitis showed a pronounced increase which started 10 min after the start of the caerulein infusion and reached maximal values at 60 min. The changes in haematocrit showed a reduction in total blood volume of 28% due to a 48% loss of plasma. This effect was completely blocked by Hoe 140. 5. In rats with caerulein-induced pancreatitis, there was a time-dependent increase in the activities of amylase and lipase in blood serum as well as in the pancreas. Pretreatment with Hoe 140 greatly augmented the caerulein-induced rise in enzyme activities in blood serum but potently attenuated it in the pancreas. The activities of trypsin in both the blood serum and the pancreas were below or near the limit of detection in all experimental groups.6. It is concluded that the second phase of hypotension in this model of acute pancreatitis is due to the liberation of kinins which cause a massive loss of blood plasma into the pancreas and into the retroperitoneal space. Activated enzymes are trapped in the pancreas, at least in part, by the oedema of the gland. Treatment with Hoe 140 prevents the oedema formation and greatly facilitates the egress of activated enzymes from the pancreas.

Effects of the bradykinin antagonist, HOE 140, in experimental acute pancreatitis.

1. The novel bradykinin antagonist, HOE 140, completely blocked the fall in rabbit blood pressure caused, not only by i.v. bradykinin, but also by i.v. kallikrein. This shows that both the effects of exogenously administered bradykinin and those of endogenously released kinins are antagonized by HOE 140. 2. Acute pancreatitis was induced in rats by i.v. infusion of the cholecystokinin analogue, caerulein. This treatment resulted in massive oedema of the pancreas, increased activities of amylase and lipase in serum and a characteristic, biphasic fall in blood pressure. 3. HOE 140 prevented the caerulein-induced pancreatic oedema and the second phase of hypotension whereas NPC 349, a widely used, but short-acting, bradykinin antagonist did not show a significant inhibition. HOE 140, in contrast to its inhibitory effects on caerulein-induced pancreatic oedema and hypotension, significantly augmented the increases in amylase and lipase activities in serum. 4. It is concluded that in this model of acute pancreatitis, the release of kinins induces pancreatic oedema and hypotension. Prevention by HOE 140 of the kinin-induced oedema allows the pancreatic enzymes to leave the tissue without hindrance and thus will diminish subsequent pathological events. It is suggested that the results obtained with the highly potent and long-acting bradykinin antagonist, HOE 140, provide a pharmacological basis for a clinical trial in acute pancreatitis.

CP-96,345, a non-peptide antagonist of substance P: II. Actions on substance P-induced hypotension and bronchoconstriction, and on depressor reflexes in mammals.

In order to extend the in vivo pharmacological profile of CP-96,345 ((2S,3S)-cis-2-(diphenylmethyl)-N-((2-methoxyphenyl)- methyl)-1-azabicyclo[2.2.2]octan-3-amine dihydrochloride), a non-peptide antagonist of substance P, the compound was tested against substance P-induced effects on blood pressure in rabbits and on respiration pressure in guinea-pigs. In addition, CP-96,345, and its inactive (2R,3R)-enantiomer, CP-96,344, were also tested in 2 models involving presumably substance P-mediated reflexes in the rat. The fall in blood pressure evoked by i.v. injections of substance P in the rabbit was inhibited by 0.3 mumol kg-1 CP-96,345 i.v. for at least 30 min, and almost abolished, for at least 2 h, by 3 mumol kg-1. In guinea-pigs, the bronchoconstriction induced by i.v. injections of substance P, but not that in response to histamine or bradykinin, was inhibited by CP-96,345 (0.6 and 6.0 mumol kg-1, i.v.) in a dose-dependent manner and this inhibition lasted for 40 min and 70 min, respectively. CP-96,345 (3-20 mumol kg-1, i.v.) reduced depressor reflexes in response to stimulation of peripheral capsaicin-sensitive neurones in the rat. However, the inhibition was not dose-dependent and was also seen with the inactive enantiomer, CP-96,344. This effect can hardly be attributed to receptor-specific effects of CP-96,345 and may be related to unspecific actions such as those involved in the cardiac depression exhibited by both enantiomers. The present results show that CP-96,345 is a potent antagonist of substance P in vivo.(ABSTRACT TRUNCATED AT 250 WORDS)

CP-96,345, a non-peptide antagonist of substance P: I. Effects on the actions mediated by substance P and related tachykinins on the guinea-pig ileum and rabbit jejunum.

The effects of a non-peptide antagonist of substance P, CP-96,345, were investigated, in vitro, on the guinea-pig ileum and the rabbit jejunum. Contractions of the guinea-pig ileum, induced by substance P and neurokinin A, were specifically inhibited by the racemate (+/-)CP-96,345 (pIC50 7.8 and 7.3, respectively). The inhibition by (+/-)CP-96,345 of contractions evoked by neurokinin B and by bradykinin (pIC50 6.1 and 4.9, respectively) was attributed to unspecific effects of the antagonist. The inhibition of substance P-induced contractions of the rabbit jejunum required a 10 times higher concentration of (+/-)CP-96,345 (pIC50 = 6.8) than was required with the guinea-pig ileum. The plateau phase of contraction of the guinea-pig ileum induced by high concentrations of substance P, neurokinin A or neurokinin B, which is known to be mediated through tachykinin receptors on intrinsic cholinergic neurones, was inhibited by 200 nM (+/-)CP-96,345 but not by the inactive enantiomer, CP-96,344. This indicates a specific inhibition of these neuronal tachykinin receptors by (+/-)CP-96,345. Contractions known to be mediated by the release of substance P, such as those evoked by capsaicin and by mesenteric nerve or field stimulation, were partially inhibited by (+/-)CP-96,345 at concentrations of 200 to 600 nM. Unspecific inhibitory effects of CP-96,345, in concentrations of 1 microM or higher, were observed on histamine-induced contractions, and on the cholinergic twitch response to electrical stimulation, of the guinea-pig ileum.(ABSTRACT TRUNCATED AT 250 WORDS)