Making progress and gaining momentum in global 3Rs efforts: how the European pharmaceutical industry is contributing.

Animal research together with other investigational methods (computer modeling, in vitro tests, etc) remains an indispensable part of the pharmaceutical research and development process. The European pharmaceutical industry recognizes the responsibilities inherent in animal research and is committed to applying and enhancing 3Rs principles. New nonsentient, ex vivo, and in vitro methods are developed every day and contribute to reducing and, in some instances, replacing in vivo studies.

Discovery of further pyrrolidine trans-lactams as inhibitors of human neutrophil elastase (HNE) with potential as development candidates and the crystal structure of HNE complexed with an inhibitor (GW475151).

Described herein is a modern approach to the rapid preparation and evaluation of compounds as potential back-up drug candidates. GW311616A, 1, a derivative of pyrrolidine trans-lactams, has previously been described as a potent, orally active inhibitor of human neutrophil elastase (HNE) for the treatment of respiratory disease. These properties made it a suitable candidate for development.

The discovery of a potent, intracellular, orally bioavailable, long duration inhibitor of human neutrophil elastase--GW311616A a development candidate.

The discovery of a potent intracellular inhibitor of human neutrophil elastase which is orally active and has a long duration of action is described. The pharmacodynamic and pharmacokinetic properties of a trans-lactam development candidate, GW311616A, are described.

Intracellular inhibition of human neutrophil elastase by orally active pyrrolidine-trans-lactams.

Described are the acylation binding of trans-lactam 1 to porcine pancreatic elastase, the selection of the SO2Me activating group for the lactam N which also confers metabolic stability in hamster liver microsomes, the introduction of aqueous solubility through the piperidine salt 9, the in vivo oral activity of 9 and its bioavailability, and the introduction of 9 as an intracellular neutrophil elastase inhibitor.

The resistance of low density lipoprotein to oxidation promoted by copper and its use as an index of antioxidant therapy.

The measurement ex vivo of the resistance of low density lipoprotein (LDL) to oxidation promoted by copper is now being used in surveys of human populations at risk of developing atherosclerosis. However, it is not known whether a relationship between LDL oxidisability measured in this way and the development of atherosclerotic lesions exists. Using Watanabe rabbits as a model of the disease, we have found that dietary supplementation with the antioxidants, probucol and alpha-tocopherol, increased the resistance of LDL isolated from small volumes of plasma to oxidation.

Changes in the caldesmon isoform content and intimal thickening in the rabbit carotid artery induced by a silicone elastomer collar.

The presence of a silicone elastomer collar around one carotid artery of a rabbit induces thickening of the tunica intima. We used immunoblotting to study quantitatively changes in the isoforms of caldesmon, a protein implicated in the regulation of contractility in smooth muscle, while also monitoring the histological changes during 28 days after collaring. Control rabbit carotid arteries (n = 28) contained 245 +/- 6.

The adventitia and atherogenesis: removal initiates intimal proliferation in the rabbit which regresses on generation of a 'neoadventitia'.

Removal of the carotid artery adventitia from rabbits induced the formation of an intimal hyperplastic lesion. In rabbits fed a normal diet, the lesion (measured as the intimal:medial ratio) was maximal by day 14 (0.456 +/- 0.

Involvement of the renin-angiotensin system in ischemic damage and reperfusion arrhythmias in the isolated perfused rat heart.

We have investigated the contribution of the renin-angiotensin system to the damage caused by 40-min global ischemia in the isolated rat heart. A converting enzyme inhibitor, enalaprilat (70 nM), an angiotensin II receptor antagonist, compound 89 (2 microM), and an inhibitor of rat renin, CGP 44099A (20 nM), given before ischemia reduced the median duration of ventricular fibrillation on reperfusion to a similar extent (5.53, 5.

The effects of radiographic contrast media on myocardial contractility and coronary resistance: osmolality, ionic concentration, and viscosity.

With the ongoing development of new contrast agents, questions develop concerning the cardiac effects of these drugs. We used the perfused rat heart model to investigate the effects on cardiac and coronary function of hypertonic ionic (sodium chloride) and nonionic (glucose) solutions and conventional and low osmolality radiographic contrast media (RCM). We also evaluated the concurrent effects of RCM on prostacyclin and adenine nucleotide/nucleoside release.

Kinetics of adenine nucleotide catabolism in coronary circulation of rats.

We have used the rat isolated, perfused heart to study the metabolism of adenine nucleotides on a single passage through the coronary circulation. Low doses (3-30 nmol) of ATP, ADP, or AMP injected as a bolus were extensively catabolized by ectoenzymes. Increasing doses of each nucleotide demonstrated saturability of catabolism that occurred at significantly lower doses of AMP than of ADP or ATP.

Purinoceptors in the rat heart.

The effects of an intracoronary bolus of adenosine triphosphate (ATP), alpha, beta-methylene ATP (APCPP), beta, gamma-methylene ATP (APPCP), adenosine diphosphate (ADP), adenosine monophosphate (AMP) and adenosine on coronary tone and ventricular myocardial contraction were investigated in the perfused rat heart. Adenine nucleotides, given by bolus injection were negatively inotropic in amounts greater than 3 X 10(-7) mol. The potency order was ATP greater than ADP greater than AMP.

Diastolic coronary resistance in the isolated rabbit heart during and after ischaemia: contribution of extravascular forces.

Coronary blood flow is influenced by contraction of the myocardium. The contribution of extravascular forces to coronary resistance during and after a period of low pressure perfusion or hypoxia and after a period of global ischaemia was studied in experiments on rabbit hearts perfused by the Langendorff method. End diastolic flow, perfusion pressure, developed tension, and resting tension were measured.

Diastolic coronary resistance after ischaemia in the isolated rabbit heart: effect of nifedipine.

The effect of nifedipine on tension, coronary flow and perfusion pressure was studied in the Langendorff rabbit heart after 15 and 60 min global ischaemia. Nifedipine (1.44 X 10(-8) M) added to the perfusate before 15 min ischaemia prevented the increase of diastolic coronary resistance which occurred on reperfusion in the absence of the drug.

Purines and prostaglandins as regulators in the coronary circulation.

In the isolated rat heart perfused at constant flow a bolus injection of an adenine nucleotide reduced coronary perfusion pressure and was negatively inotropic (ATP greater than ADP greater than AMP). Adenosine had no effect on perfusion pressure or contractile force. ATP and ADP stimulated production of PGI2 from the coronary bed.

Absence of a relationship between extracellular potassium accumulation and contractile failure in the ischemic or hypoxic rabbit heart.

Ischemia and hypoxia both cause a rapid loss of potassium from myocardial cells. We have investigated the relationship between the accumulation of potassium in the extracellular fluid and the early loss of contractility. Experiments were performed on the isolated rabbit heart perfused with physiological saline at 36 degrees C, paced at 3 Hz.

Mechanical response of rabbit myocardium and coronary arteries to leukotriene D4. Failure to demonstrate a role in the pathophysiology of hypoxia.

We have studied the effect of leukotriene D4 (LTD4) on rabbit and rat myocardial contractility and on rabbit coronary arteries. A concentration of 2 X 10(-7) M caused only a small reduction of myocardial contractility, but caused a contraction of smooth muscle in coronary arteries similar to that obtained with potassium (30 mmoles/liter). LTD4 (2 X 10(-7) M) added to the perfusate 10 min before or at the time of reoxygenation after a period of 30 min of hypoxia did not alter contractility or resting tension.