Crystal structure of human macrophage elastase (MMP-12) in complex with a hydroxamic acid inhibitor.

Human macrophage elastase (MMP-12) is a member of the family of matrix metalloproteinases (MMPs) that plays, like other members of the family, an important role in inflammatory processes contributing to tissue remodelling and destruction. In particular, a prominent role of MMP-12 in the destruction of elastin in the lung alveolar wall and the pathogenesis of emphysema has been suggested. It is therefore an attractive therapeutic target. We describe here the crystal structure of the catalytic domain of MMP-12 in complex with a hydroxamic acid inhibitor, CGS27023A. MMP-12 adopts the typical MMP fold and binds a structural zinc ion and three calcium ions in addition to the catalytic zinc ion. The enzyme structure shows an ordered N terminus close to the active site that is identical in conformation with the superactivated form of MMP-8. The S1'-specificity pocket is large and extends into a channel through the protein, which puts MMP-12 into the class of MMPs 3, 8 and 13 with large and open specificity pockets. The two crystallographically independent molecules adopt different conformations of the S1'-loop and its neighbouring loop due to differing crystal packing environments, suggesting that flexibility or the possibility of structural adjustments of these loop segments are intrinsic features of the MMP-12 structure and probably a common feature for all MMPs. The inhibitor binds in a bidentate fashion to the catalytic zinc ion. Its polar groups form hydrogen bonds in a substrate-like manner with beta-strand sIV of the enzyme, while the hydrophobic substituents are either positioned on the protein surface and are solvent-exposed or fill the upper part of the specificity pocket. The present structure enables us to aid the design of potent and selective inhibitors for MMP-12.

Multiple-conformation and protonation-state representation in 4D-QSAR: the neurokinin-1 receptor system.

Using a 4D-QSAR approach (software Quasar) allowing for multiple-conformation, orientation, and protonation-state ligand representation as well as for the simulation of local induced-fit phenomena, we have validated a family of receptor surrogates for the neurokinin-1 (NK-1) receptor system. The evolution was based on a population of 500 receptor models and simulated during 40 000 crossover steps, corresponding to 80 generations. It yielded a cross-validated r(2) of 0.887 for the 50 ligands of the training set (represented by a total of 218 conformers and protomers) and a predictive r(2) of 0.834 for the 15 ligands of the test set (70 conformers and protomers). A series of five "scramble tests" (with an average predictive r(2) of -0.438) demonstrates the sensitivity of the surrogate toward the biological data, for which it should establish a QSAR. On the basis of this model, the activities of 12 new compounds - four of which have been synthesized and tested in the meantime - are predicted. For most of the NK-1 antagonists, the genetic algorithm selected a single entity - out of the up to 12 conformers or protomers - to preferably bind to the receptor surrogate. Moreover, the evolution converged at an identical protonation scheme for all NK-1 antagonists. This indicates that 4D-QSAR techniques may, indeed, reduce the bias associated with the choice of the bioactive conformation as each ligand molecule can be represented by an ensemble of conformations, orientations, and protonation states.

BIIE0246: a selective and high affinity neuropeptide Y Y(2) receptor antagonist.

The in vitro biological characterisation of the first potent and selective non-peptide neuropeptide Y Y(2) receptor antagonist, (S)-N(2)-[[1-[2-[4-[(R,S)-5,11-dihydro-6(6h)-oxodibenz[b, e]azepin-11-yl]-1-piperazinyl]-2-oxoethyl] cylopentyl] acetyl]-N-[2-[1,2-dihydro-3,5(4H)-dioxo-1,2-diphenyl-3H-1,2, 4-triazol-4-yl]ethyl]-argininamid (BIIE0246) is reported. BIIE0246 displaced [125I]neuropeptide Y with high affinity (IC(50)=3.3 nM) from the human neuropeptide Y Y(2) receptor and proved to be highly selective. BIIE0246 displayed antagonistic properties and thus represents the first selective non-peptide neuropeptide Y Y(2) receptor antagonist.

[Mineral metabolism and aluminum burden with hydrotalcit. A placebo-controlled randomized double-blind study]. / Mineralhaushalt und Aluminiumbelastung unter Hydrotalcit. Eine Plazebo-kontrollierte randomisierte Doppelblindstudie.

In a randomized, double-blind study, the influence of hydrotalcit (aluminium-magnesium hydroxide carbonate hydrate) on the mineral metabolism was investigated. No significant and clinically relevant changes in calcium, phosphate, magnesium or aluminium concentrations in the serum were observed. Nor were these substances eliminated in greater amounts in the urine. Merely the elimination of magnesium was higher in the group receiving the test substance than in the placebo group; after discontinuation of the medication, no difference in the elimination rates was found. Thus, provided that renal function is intact, the use of hydrotalcit does not lead to any disturbance in the mineral balance, or, in particular, to any additional aluminium burden.

Gastrin release and gastric secretion induced by dimethylxanthine in man.

Gastrin release and gastric secretion in response to i.v. dimethylxanthine (2 mg/kg for 5 min followed by a 55-min infusion of 2.5 mg/kg) were studied in 25 normal subjects. Gastrin release was significantly stimulated, whereas gastric acid and pepsin secretion was inhibited. The adenylcyclase activation may, therefore, play a role in the mechanism of gastrin release. Moreover, dimethylxanthine seems to be a useful aid in studies on G-cell function in man.

Effects of secretin and cholecystokinin-pancreocymin on water, electrolyte and glucose absorption in human jejunum. A comparison between different hormone preparations.

The effects of natural secretin (90%) and synthetic secretin as well as impure (10%) and pure (99%) cholecystokinin-pancreozymin (CCK) on net absorption of water, electrolytes, and glucose in human jejunum were studied in 31 normal subjects. An intestinal perfusion technique with a triple-lumen tube was used. Net absorption of water and solute was significantly inhibited by both hormones only with larger doses, pure CCK being less active than impure CCK. A dose-dependent response of water and electrolyte absorption to graded doses of pure CCK was observed, without concomitant inhibition of glucose absorption with lower doses. The findings suggest that secretin and CCK may not be of physiologic importance regarding intestinal absorption in man. The definite changes in intestinal motility and transit rate caused by these hormones seem more likely to result in a reduction of intestinal absorption and an increase in the secretion of water and electrolytes along the proximal small bowel.

Synthetic human gastrin I and gastrin-like pentapeptide in studies of intestinal absorption in man. Role of gastrin in human intestinal absorption.

The effects of synthetic human gastric I (SHG I) and gastrin-like pentapeptide (PG) on jejunal water, electrolyte, and glucose absorption were studied in 11 normal subjects. The i.v. administration of graded doses of SHG I increased plasma gastrin levels similar to those after food intake and in the Zollinger-Ellison syndrome. SHG I and PG caused no significant changes in the net movement of water and solute. The findings indicate that gastrin has no direct effect on intestinal absorption in normal man, and does not account for the mechanism of diarrhea in the Zollinger-Ellison syndrome.

[Absorption in the human small intestine relative to the intraluminal milieu]. / Das Absorptionsverhalten des menschlichen Dünndarms in Abhängigkeit von dem intraluminalen Milieu.

The bulk of water and electrolyte absorption takes place in the human jejunum from isotonic solutions, and is determined largely by special transport mechanisms for different monosaccharides, amino acids and dipeptides. This is of considerable significance for regaining the large volumes of fluid delivered to the small intestine during the digestion of food. Small changes in intraluminal pH do not significantly influence the absorptive function of the jejunum and are rapidly compensated by the buffering capacity of the gut. The maintenance of an isotonic as well as neutral intraluminal milieu seems to be essential to the physiological processes of intestinal absorption.

Concentrations of prostaglandin A-, E- and F-like substances in gastric mucosa of normal subjects and of patients with various gastric diseases.

1. Prostaglandin A-, prostaglandin E- and prostaglandin F-like substances were determined radioimmunologically in antral biopsy material obtained by endoscopy. 2. In patients with gastritis, the concentrations of prostaglandin (E+A)-like substances were six times as high and of prostaglandin F-like substances twice as high as in normal subjects. In chronic atrophic gastritis, the concentrations of prostaglandin (E+A)-like material was four times as high as in normal subjects whereas prostaglandin-F like material remained unchanged. In acute gastric ulcer, prostaglandin (E+A)-like material reached concentrations four times times higher than in normal subjects, accompanied by a fivefold increase of prostglandin F-like substances. After healing of the gastric ulcer, prostaglandins returned to normal values. 3. There was no correlation between gastrin and prostaglandins in all biopsy specimens.

Hypergastrinemia induced by glucocorticoid and corticotropin treatment in man.

To elucidate further the pathogenesis of steroid-induced ulceration, plasma gastrin levels, both basal and after a test meal, were studied in normal volunteers and patients treated with glucocorticoids or corticotropin. In normal subjects the acute intravenous administration of 100 mg prednisolone had no effect on plasma gastrin levels. After oral administration of prednisolone (40 mg daily, for four days) a significant increase of the basal, the reactive, and the over 90-min integrated gastrin release was observed. In this group, the glucocorticoid treatment had a slight, but significant influence on gastric acid and pepsin secretion, while acidity and pepsin output stimulated by pentagastrin was not affected. In patients treated with prednisolone for more than 24 weeks, the oral administration of this hormone failed to alter basal gastrin values but affected significantly secretion after the test meal. In patients with multiple sclerosis, after intramuscular administration of corticotropin (60 IU daily, for 12 days), an increase of the basal, the reactive, and the integrated gastrin release also was found. Glucocorticoid-induced hypergastrinemia provides information on the pathogenesis of steroid-induced ulceration.

Effects of somatostatin on exocrine and endocrine pancreatic function stimulated by intestinal hormones in man.

To evaluate the action of somatostatin on exocrine and endocrine pancreatic function, synthetic somatostatin (GIF) was administered (intravenous bolus of 300 mug followed by a constant 60-minute infusion, 5 mug/min) to 17 normal subjects. The secretin-induced volume and total bicarbonate contents of the duodenal aspirate were not affected whereas the bicarbonate concentration was significantly diminished. GIF reduced decisively the pancreatic enzyme secretion stimulated by pure (99%) cholecystokinin-pancreozymin. After the GIF infusion was stopped, a significant rise in enzyme secretion was observed. The secretin-induced insulin release was almost completely suppressed. Because GIF can be extracted in large quantities from pancreas, these data suggest that somatostatin may play a physiological role in the regulation of the secretory processes of this organ. Furthermore, GIF may be a useful adjunct in the treatment of acute pancreatitis.

Effect of lipids on insulin, growth hormone and exocrine pancreatic secretion in man.

Influences of fat on release of insulin, growth hormone and pancreatic enzyme secretion were studied in 35 metabolically healthy subjects. A fat solution containing 40 g of soy bean oil was administered, I.V., orally and intraduodenally. In all cases there was a similar increase of insulin but the rise in serum insulin after oral or intraduodenal fat administration was not related to the changes in plasma free fatty acids, free glycerol and triglyceride levels. Blood surgar responded according to insulin secretion. The route of fat administration may possibly influence growth hormone secretion. Following intraduodenal fat administration volume and bicarbonate contents of the duodenal juice rose slightly whereas trypsin and bilirubin content increased considerably. These results suggest that insulin secretion after oral or intraduodenal administration of fat is influenced by intestinal factors. Cholecystokinin-pancroezymin and gastric inhibitory polypeptide are qualified to serve as such factors.