Annie Delaunois

Modulation of acetylcholine, capsaicin and substance P effects by histamine H3 receptors in isolated perfused rabbit lungs.

The modulatory role of histamine H3 receptors in pulmonary oedema induced by acetylcholine, capsaicin and by exogenous substance P was investigated in isolated, ventilated rabbit lungs. Endothelial permeability was evaluated by measuring the capillary filtration coefficient (Kf,c). Acetylcholine (10(-8) to 10(-4) M), substance P (10(-10) to 10(-6) M), capsaicin (10(-4) M) and 5-hydroxytryptamine (5-HT) (10(-4) M) induced an increase in the Kf,c. Carboperamide, a novel histamine H3 receptor antagonist, induced a significant leftward shift of the concentration-response curve to acetylcholine and also enhanced the effect of capsaicin on the Kf,c, while it had no significant effect on the response to substance P and 5-HT. Imetit, a new histamine H3 receptor agonist, strongly inhibited the effects of acetylcholine and capsaicin. Imetit also strongly protected the lung against substance P effects but did not prevent the 5-HT-induced increase in the Kf,c. Carboperamide completely blocked the inhibitory effect of Imetit on the acetylcholine response. (R)-alpha-Methylhistamine, an other histamine H3 receptor agonist, had the same protective effect against acetylcholine response as Imetit. We conclude that histamine H3 receptors could protect the lung against acetylcholine- and capsaicin-induced oedema via a prejunctional modulatory effect on the C-fibres. However, since the response to exogenous substance P was also inhibited by histamine H3 receptor stimulation, the presence of such receptors at a postsynaptic level, probably on mast cells, was also suggested.

Modulatory effect of imetit, a histamine H3 receptor agonist, on C-fibers, cholinergic fibers and mast cells in rabbit lungs in vitro

The pharmacological mechanisms involved in the interactions between C-fibers, cholinergic fibers and mast cells were investigated in tracheally perfused rabbit lungs by measuring the simultaneous release of substance P and histamine in lung effluents. The amounts of substance P and histamine released in lung superfusates were measured by radioimmunoassay (RIA) after administration of capsaicin and carbachol. Capsaicin (10(-4) M) induced a simultaneous increase in substance P (273 +/- 56% of baseline) and histamine (460 +/- 138%) release. Similarly, carbachol (10(-4) M) caused an increase in the release of both substance P (367 +/- 111%) and histamine (1379 +/- 351%). The effect of capsaicin was prevented by pretreating the lungs with the tachykinin NK1 receptor antagonist SR 140333 (10(-7) M), and atropine (10(-6) M). SR 140333 prevented the carbachol-induced release of substance P but not of histamine. Exogenous substance P induced an increase in histamine release (136 +/- 7%) which was significantly greater in lungs perfused with the neutral endopeptidase inhibitor, thiorphan (10(-5) M) (272 +/- 35%). This effect was prevented by atropine (10(-6) M). Pretreatment of lungs with imetit (5 x 10(-8) M), a selective H3 receptor agonist, prevented the capsaicin-induced release of both mediators. Imetit also blocked the carbachol-induced release of substance P but not of histamine. Exogenous substance P-evoked histamine release was inhibited by imetit. Therefore, it can be concluded that substance P released through the action of capsaicin can activate cholinergic fibers, leading to cholinoceptor stimulation with subsequent activation of C-fibers and mast cells. While the presence of presynaptic H3 receptors modulating substance P-induced acetylcholine release was only surmised, the existence of modulating histamine H3 receptors on C-fibers was confirmed.

Altered capillary filtration coefficient in parathion- and paraoxon-induced edema in isolated and perfused rabbit lungs

Changes in pulmonary endothelium permeability and in microvascular hemodynamics induced by parathion (Pth) and paraoxon (Pox), its active metabolite, were investigated in isolated, perfused rabbit lungs. Blood-free perfusate was recirculated through isolated and ventilated lungs in an isogravimetric state and in zone III conditions. The arterial/venous/double occlusion technique was used to divide the total vascular resistance (Rt) into four components: arterial, precapillary, postcapillary, and venous. The capillary filtration coefficient (Kfc) was evaluated by measuring the amount of fluid filtering through the endothelium when the arterial and venous pressures were suddenly increased. Pth and Pox induced pulmonary edema by increasing endothelium permeability without changing the hemodynamic parameters at any level of the vascular bed. The Kfc value increased progressively, reaching a maximum (Emax) 60 min after administration of organophosphate (558 +/- 65% (n = 5) and 707 +/- 109% (n = 5) of baseline values, for Pth and Pox, respectively). During the next 60 min, it decreased. The time course of Pox-induced changes in Kfc was similar to that obtained with Pth. The concentration-response curve (Emax) expressed as a percentage of the baseline value versus the logarithm of the molar Pth concentration, ranging from 2 x 10(-5) to 4 x 10(-4) M) was linear (y = 1661.1 + 327.3x, r = 0.89, p < 0.001, n = 14). Piperonyl butoxide (4 x 10(-4) M), an inhibitor of cytochrome P450, had a strong protective effect against Pth (4 x 10(-4) M)-induced alterations of endothelium permeability (n = 5, p < 0.001). The effects of Pox (4 x 10(-4) M) on Kfc were completely abolished by pretreatment with 10(-5) M atropine, as shown by the significantly lower Emax value recorded in atropine-pretreated lungs (129 +/- 33%, n = 4) than in Pox-treated lungs (707 +/- 109%, n = 5, p < 0.001). The effects of Pth, on the other hand, were only partially inhibited, since the Emax value recorded in atropine-pretreated lungs (196 +/- 20%, n = 4) remained significantly higher than that recorded for control lungs (129 +/- 15%; n = 5; p < 0.05). These results show that isolated and perfused rabbit lungs constitute an appropriate model for studying the direct pulmonary effects of organophosphates. The edema-inducing action of Pth depends on its activation by conversion to Pox in the lung tissue.(ABSTRACT TRUNCATED AT 400 WORDS)

Permeability of the endothelium and partitioning of the pulmonary blood flow resistance in isolated perfused pig lungs: effects of breed and age.

The right and left lungs of 5 healthy Minipigs and of 13 healthy Landrace piglets were isolated, perfused at constant pressure and maintained in an isogravimetric state under zone III conditions (pulmonary venous pressure>alveolar pressure). By applying the double, arterial and venous, occlusion technique, the total blood flow resistance (Rt) was partitioned into four components: arterial (Ra), pre-(Ra′) and post-capillary (Rv′) and venous (Rv). The capillary filtration coefficient (Kf,c) was evaluated by measuring the weight gained by the lungs when the arterial and venous pressures were suddenly increased. In the youngest Landrace piglets (5 weeks old), there was an uncontrolled vasoconstriction which sometimes prevented perfusion of the lungs and induced a large increase inRt. These high values ofRt were decreased by tolazoline administration. The values ofRt recorded in older pigs (12–13 weeks old) were lower in Minipigs (33.66±3.77 cmH2O min L−1 per 100 g of lungs;n=5) than in Landrace piglets (55.20±6.18 cmH2O min L−1 per 100 g;n=5). This breed difference was due to the differences inRa′ andRv′. The mean values ofKf,c were 0.193±0.015 and 0.202±0.029 ml min (cmH2O)−1 per 100 g of the lungs in Minipigs and Landrace piglets respectively. All these parameters were stable for the 3 hours following the equilibrium period. It was concluded that: (1) There is an age-related maturation of the control of the vasomotor tone in porcine lungs. (2) Pulmonary microvascular haemodynamics are influenced by the breed of the pigs. (3) There was no difference in theKf,c values between both the breeds. (4) A comparison of the values reported for dogs and rabbits with our data shows that the pre- and post-capillary resistances and, to a lesser extent, the arterial and venous resistances are relatively high in pigs.

Interactions between acetylcholine and substance P effects on lung mechanics in the rabbit.

Summary— The pharmacological mechanisms involved in the acetylcholine (ACh)‐ and substance P (SP)‐induced changes in pulmonary mechanics were studied in isolated perfused rabbit lungs. Tracheal pressure (Ptr) and airflow were measured by a Fleisch pneumotachograph and pressure transducers. Air volume, lung resistance (RL) and dynamic compliance (Cdyn) were calculated. ACh induced a dose‐dependent increase in Ptr and RL, and a decrease in Cdyn. These effects were strongly prevented by atropine, and partly by SR140333, a neurokinin NK1 receptor antagonist; SR48968, a neurokinin NK2 receptor antagonist; indomethacin and antihistaminics. Ketanserin had no significant protective effect against ACh. SP also induced concentration‐dependent increases in RL and decreases in Cdyn. SR140333 and atropine strongly inhibited the effects of SP, while ketanserin, SR48968, antihistaminics and indomethacin did not protect the lungs against this drug. 5‐hydroxytryptamine induced no significant change in lung mechanic parameters. Cumulative concentrations of histamine increased RL and decreased Cdyn. We conclude that ACh‐induced changes in lung resistance and compliance are in part mediated by a direct effect on airway smooth muscle and in part by the stimulation of C fibers, by the release of histamine from mast cells and by the synthesis of arachidonic acid metabolites. In turn, the effects of SP on lung mechanics are partly due to cholinergic activation.

In vitro short-term study of ammonium-nitrogen production from cattle urine : influence of ampicillin, hydroquinone and animal litter materials

The production of NH + 4 -N following in vitro incubation of cattle urine was monitored for 24 h in the presence of ampicillin sodium salt (0, 32, 64, 128 mg/l), hydroquinone (0, 16·7, 33·4, 66·8 mg/l), wheat straw (0, 3·3, 6·6, 13·2 g/l) or spruce sawdust (0, 3·3, 6·6, 13·2 g/l) with (20 IU) or without urease. Each concentration of ampicillin, hydroquinone, wheat straw or spruce sawdust was tested in triplicate. The equipment consisted of Woulff flasks containing 300 ml of a buffered solution (0·02 M ; pH 7·50) with 1 ml of cattle urine. The cattle urine was characterized by measuring the main nitrogen contents, which were 6·52 mg total-N/ml, 5·96 mg urea-N/ml and 0·026 mg ammonium-N/ml. The initial pH of urine was 7·84. Ammonium and nitrate concentrations, and pH were monitored at zero- time and after 3, 6 and 24 h of incubation with the cattle urine. The addition of urease to the flasks containing urine induced a significant increase in the production of ammonium-N, from 1·83 to 6·32 mg NH + 4 -N/flask after 24 h of incubation. In the presence of urease, an inhibitory effect was recorded in NH + 4 -N production with ampicillin and spruce sawdust. In contrast, hydroquinone inhibited urease activity and wheat straw adsorbed the NH + 4 -N produced, both causing a dose-dependent relationship. In the absence of urease, ampicillin, hydroquinone, wheat straw or spruce sawdust caused a dose- related decrease in NH + 4 -N production. However, the highest amounts of wheat straw (6·6 and 13·2 g/flask) exhibited a temporary increase in NH + 4 -N production during the first 6 h. This is probably due to a generation of extra NH + 4 -N as compared to the control flasks without straw. However, at 24 h, the situation paralleled the other materials. Although pH increased approximately from 7·50 to 7·65 during the hydrolysis of urea in the presence of urease, no gaseous ammonia was volatilized into a 0·1 M HCl flask. Moreover, no increase in nitrate concentration was found during the incubation. The present results suggest that ampicillin, hydroquinone and spruce sawdust could be used in order to reduce NH + 4 -N production from cattle urine. The NH + 4 -N already produced could probably be absorbed on wheat straw.

RADIOIMMUNOASSAY OF SUBSTANCE P IN LUNG PERFUSATE

A radioimmunoassay of the undecapeptide substance P (SP) and its application in rabbit lung superfusate has been developed. The assay was based on the use of a radioactive tracer, 125I-SP-Tyr8, which showed, with an excess of antibodies, a specific binding greater than 90% and a nonspecific binding lower than 2%. This tracer was stable for 4 weeks at -20 degrees C. Its specific radioactivity was 384 Ci/mmol. The assays lower limit of detection was 10 pg/ml (0.7 fmol). The determination of SP in rabbit lung perfusate required extraction and concentration using octadecylsilane cartridges. Under these conditions, the recovery of SP from experiments on lung perfusates (n=4) was 75.0+/-4.5%, and the intraassay and interassay coefficients of variation were 7.3+/-1.9% and 10.7+/-1.3%, respectively. Amounts of SP released in rabbit lung perfusates were determined following stimulation of pulmonary C fibers with capsaicin (10(-4) M). During the stimulation period, the values of SP from lung perfusates (n=6) increased significantly when compared with baseline values.

Mechanisms of substance P‐induced pulmonary oedema in the rabbit: interactions between parasympathetic and excitatory NANC nerves

Summary— The pharmacological mechanisms involved in the substance P (SP)‐induced pulmonary oedema were studied in isolated perfused rabbit lungs. Substance P induced a dose‐dependent increase in the capillary filtration coefficient (Kf,c), responsible for oedema. Atropine, hemicholinium‐3 and ruthenium red pretreatment partly protected the lungs against SP effects, while tetrodotoxin and hexamethonium did not significantly modify them. (±)CP96,345, a NK1 receptor antagonist, completely inhibited the SP‐induced increase in the Kf,c. Like SP, acetylcholine (ACh) and capsaicin also increased the Kf,c. Atropine and (±)CP96,345 completely blocked the oedema induced by both drugs. Tetrodotoxin and ruthenium red strongly inhibited the response to capsaicin and acetylcholine. It was concluded that SP‐induced pulmonary oedema is in part mediated by a stimulating action on cholinergic efferent nerves, with subsequent release of endogenous acetylcholine. Acetylcholine can, in turn, stimulate the release of SP from excitatory non adrenergic, non cholinergic nerves. The effects induced by capsaicin and exogenous acetylcholine, thus endogenous SP, involve tetrodotoxin‐sensitive mechanisms, while those produced by exogenous SP are tetrodotoxin‐resistant.

Investigative safety strategies to improve success in drug development

Understanding and reducing attrition rate remains a key challenge in drug development. Preclinical and clinical safety issues still represent about 40% of drug discontinuation, of which cardiac and liver toxicities are the leading reasons. Reducing attrition rate can be achieved by various means, starting with a comprehensive evaluation of the potential safety issues associated to the primary target followed by an evaluation of undesirable secondary targets. To address these risks, a risk mitigation plan should be built at very early development stages, using a panel of in silico , in vitro , and in vivo models. While most pharmaceutical companies have developed robust safety strategies to de-risk genotoxicity and cardiotoxicity issues, partly driven by regulatory requirements; safety issues affecting other organs or systems, such as the central nervous system, liver, kidney, or gastro-intestinal system are less commonly addressed during early drug development. This paper proposes some de-risking strategies that can be applied to these target organ systems, including the use of novel biomarkers that can be easily integrated in both preclinical and clinical studies. Experiments to understand the mechanisms’ underlying toxicity are also important. Two examples are provided to demonstrate how such mechanistic studies can impact drug development. Novel trends in investigative safety are reviewed, such as computational modeling, mitochondrial toxicity assessment, and imaging technologies. Ultimately, understanding the predictive value of non-clinical safety testing and its translatability to humans will enable to optimize assays in order to address the key objectives of the drug discovery process, i.e., hazard identification, risk assessment, and mitigation.

INTERACTIONS BETWEEN CYTOCHROME P-450 ACTIVITIES AND OZONE-INDUCED MODULATORY EFFECTS ON ENDOTHELIAL PERMEABILITY IN RABBIT LUNGS: Influence of Gender

The effects of rabbit exposure to ozone (O(3)) (0.4 ppm for 4 h) on two different cytochrome P-450 (CYP450)-dependent activities were investigated. In turn, the role of CYP450 in the inhibitory effect of O(3) on acetylcholine (ACh)-evoked increase in endothelial permeability was also assessed. Immediately after the period of exposure, rabbits of both sexes were sacrificed and their lungs were extracted. Some lungs were used for preparation of microsomes and measurement of 7-ethoxyresorufin O-deethylase (EROD) and parathion oxidase activities. Other rabbit lungs were isolated and recirculatingly blood-free perfused. Arterial, venous pressures, and lung weight were continuously recorded. Capillary pressure was measured by applying the double occlusion method. Capillary filtration coefficient (K(f,c)) was evaluated by measuring the amount of fluid filtering through the endothelium when vascular pressures were suddenly increased. Dose-response curves to ACh were constructed in air- or O(3)-exposed rabbits. Some animals were pretreated with piperonyl butoxide (PBO), a well-known inhibitor of CYP450. O(3) significantly reduced both EROD and parathion oxidase lung microsomal activities in females, while it had no effect in males. Exposure to O(3) strongly inhibited the ACh-induced increase in K(f,c). Pretreatment with PBO reversed the modulatory effect of O(3) on endothelial permeability in male rabbits, but not in females. It was concluded (1) that inhibition of 2 different CYP450-dependent activities after exposure to 0.4 ppm O(3) for 4 h appears to be a gender-dependent phenomenon, and (2) that CYP450 is probably involved in the O(3)-evoked inhibitory mechanism against ACh-induced increase in endothelial permeability, but only in males.