[Online patent searching--rapid responses to critical questions]. / Online-Patentrecherchen--schnelle Antworten auf kritische Fragen.

Online searching in publically available patent files opens up interesting possibilities to provide a rapid response to critical questions. A computerized analysis of all patents of leading German pharmaceutical companies over the last decade in important indication areas is described. Supported by subsequent manual processing of individual patents it is shown that duplicate experiments on animals practically never occur.

Influence of muzolimine on arterial wall elastin.

Muzolimine, 3-amino-1-(3,4-dichloro-alpha-methylbenzyl)-2- pyrazolin -5-one, an antihypertensive and diuretic drug, accumulates in the arterial tissue of rats and dogs after oral administration. Two weeks after the administration of 3 mg [14C]muzolimine, the aorta of rats contained 60-300 times more 14C-radioactivity/weight unit than the skin or tail tendon. The 14C-radioactivity was exclusively bound to the isolated aortic elastin and corresponded to 0.04% of the applied muzolimine dose. Up to ca 250 ng bound muzolimine/mg elastin was found in the aorta of dogs treated with non-labelled muzolimine for 52 weeks. The elastin-bound [14C]muzolimine was not extractable by organic solvents or by weak acids or bases but was released in a soluble form by pancreatic elastase and extracted from the elastase digest by dichloromethane. In the dichloromethane extract muzolimine was detected by HPLC and HPTLC, and was identified by mass spectrometry. Muzolimine pretreatment of rats for 2 months did not influence the elastin content of arterial tissue or [3H]glycine incorporation into aortic elastin under organ culture conditions, but after labelling the elastin with [4,5-3H]lysine, the [3H]desmosine and [3H]-isodesmosine isolated from the elastin of muzolimine-pretreated rats and incorporated under organ culture conditions was lower than that of control animals. In addition, aortic elastin of rats pretreated for 2 months with 800 ppm muzolimine in the diet was more resistant to elastase degradation. This effect might give some implications for muzolimine in the therapy of cardiovascular disorders with impaired arterial elastin metabolism.

In vivo evidence for the chemotactic activity of cyclooxygenase- and lipoxygenase-dependent compounds using a corneal implantation technique.

Cyclooxygenase- and lipoxygenase-dependent compounds of arachidonic acid were studied in a corneal implantation technique concerning their chemotactic activity for polymorphonuclear leukocytes. Arachidonic acid, prostaglandins A1, A2, B2, I2, thromboxane A2 and leukotriene D4 showed no activity, whereas leukotriene B4 was the most potent chemoattractant substance for polymorphonuclear leukocytes, followed by 5-HETE, prostaglandins E2 and E1. This gives evidence for the decisive role of some prostaglandins and leukotrienes in inflammation in general, and in corneal inflammatory processes in special.

[Experimental studies on the effect of prostaglandin and leukotriene synthesis inhibitors on epithelial regeneration of the cornea]. / Experimentelle Untersuchungen zum Einfluss von Prostaglandin- und Leukotrien-Synthesehemmern auf die Epithelregeneration der Hornhaut.

Total abrasion of the corneal epithelium was performed in New Zealand white rabbits; then the influence of nonsteroidal anti-inflammatory drugs was studied macro- and microscopically. The cyclooxygenase inhibitor indomethacin had no influence on reepithelialization, but increased the number of polymorphonuclear leukocytes in the epithelium and stroma. The lipoxygenase inhibitor Bay 08276 accelerated reepithelialization significantly by inhibiting the chemotaxis and degranulation of polymorphonuclear leukocytes. From our results it is possible to produce a flowchart on the pathophysiology of corneal regeneration, including the different mechanisms of prostaglandins and leukotrienes.

The antithrombotic activity of BAY g 6575.

The activity of 1-[2-(beta-naphthyloxy)ethyl]-3-methyl-2-pyrazolin-5-one (= BAY g 6575) was evaluated in models of experimental thrombosis caused by traumatically induced damage of vessel segments. After prophylactic administration of BAY g 6575 (0.3 mg/kg p.o.) to rats the thrombus formation was significantly reduced in the carotid artery as well as in the jugular vein. The thrombus formation in the femoral arteries of rabbits is inhibited at a minimal effective dose of 1 mg/kg p.o. The incidence of occlusive thrombi is not influenced. BAY g 6575 is 10 times more potent than acetylsalicylic acid (ASA). In the arterial system the thrombus formation is frequently completely abolished.

Influence on thromboxane and malondialdehyde synthesis in human thrombocytes by various inhibitors of platelet function.

The effects of various inhibitors of platelet function have been compared with respect to the minimal effective concentration required to inhibit platelet aggregation and the thromboxane (TXA2) and malondialdehyde (MDA) synthesis in washed platelets. The potencies of the agents in the different platelet function tests showed no consistent correlation. The most striking difference was found between the prostaglandins and the adenine nucleotides. The prostaglandins PGE1 and PGI2 inhibited all platelet reactions (10(-7) - 10(-8) g/ml) which may be explained by their stimulation of adenylcyclase. In contrast, the adenine nucleotides adenosine and AMP which are supposed to raise cAMP platelets via the same mechanism and strongly inhibited primary and secondary platelet aggregation (3 x 10(-6) - 3 x 10(-7) g/ml) had no effect on TXA2 and MDA synthesis. The results suggest that the pharmacological inhibition of platelet function may not act via a uniform mechanism controlled by the arachidonate pathway.

Arachidonic acid induced mortality in animals - an appropriate model for the evaluation of antithrombotic drugs?

Sodium arachidonate (1.1 mg/kg b.w.) was injected into a marginal ear vein of rabbits according to SILVER M.J. et al. (1). 94% of the animals died within a few minutes. We investigated to what extent these lethal effects of arachidonic acid (possibly caused by embolizing platelet thrombi) can be prevented by potential antithrombotic drugs. As indicated in the tables the animals are protected by some compounds, especially the nonsteroidal anti-inflammatory agents, in a dose-dependent manner. However, the results of this in vivo model showed no correlation with the potency of the drugs for inhibiting platelet aggregation and inhibition of experimental thrombosis. It is therefore reasonable to assume that the drug effects in this model reflect an overall inhibition of prostaglandins including thromboxanes.

Cyanogen bromide treatment of methionine-containing compounds.

The preparation of a series of X-Met-Gly-OEt and X-Met-Phe-OMe and their treatment with CNBr in either 70% or 97-100% formic acid at 25 degrees C are described where X is methanesulfonyl (mesyl), p-nitrobenzyloxycarbonyl, phthaloyl, trifluoroacetyl, acetyl, formyl, or tert-butyloxycarbonyl. Total cleavage of the peptide esters was found with mesyl-, p-nitrobenzyloxycarbonyl-, phthaloyl-, and trifluoroacetylmethionyl derivatives which indicated the suitability of these derivatives as amino protecting groups in peptide synthesis. Treatment of the acetylmethionyl peptide esters with CNBr in 70 and 97-100% formic acid resulted in 92 and 98% cleavage, respectively. With formylmethionyl peptide esters, about 85-95% cleavage was estimated when either 70 or 97-100% formic acid was used as the solvent. With the tert-butyloxycarbonylmethionyl derivatives, CNBr treatment in 70% formic acid resulted in about 93% cleavage of peptides, while treatment in 97-100% formic acid led to only 30-33% release of C-terminal amino acid esters. Quantitative cleavage of the carbonylbis(methionyl peptide esters) was observed. The reaction of CNBr with N-terminal methionyl derivatives containing free alpha-amino groups revealed that free methionine was quantitatively converted to homoserine lactone, whereas methionine ethyl ester and methionyl peptides (Met-Gly and Met-Phe) disappeared from the reaction mixture in 70% formic acid with only partial splitting of the ester (16%) or peptide bond (45%).

Synthesis and properties of carbonylbis(methionyl)insulin, a proinsulin analogue which is convertible to insulin by cyanogen bromide cleavage.

The preparation and use of carbonylbis (L-methionine p-nitrophenyl ester) as a reversible cross-linking reagent for insulin are described. The reaction of 1 equiv of reagent with zinc insulin in dimethylformamide in the presence of triethylamine yields as one of the products NalphaA1, NepsilonB29-carbonylbis(methionyl)insulin, (CBM-insulin). The CBM-insulin was characterized by end group analysis and by the products formed on tryptic and chymotryptic cleavage. It possessed 91% of the immunological and 6.5% of the hormonal activity of insulin. Treatment of CBM-insulin with cyanogen bromide (CNBr) in 70% formic acid for 1 h resulted in nearly complete removal of the methionine bridge to yield insulin. A small amount of a side product was removed on DEAE-cellulose at pH 7.2 to give an overall recovery of insulin of 70-80%. Oxidative sulfitolyses of CBM-insulin gave the hexa(S-sulfonate) which was reduced with dithiothreitol to yield reduced CBM-insulin. The latter compound, containing 6 sulfhydryls, exhibited a pH-dependent circular dichroic spectrum. The form at pH 10 exhibited a spectrum typical of random coil which was converted to a form at pH 7.8 which was characterized by a negative extremum at 213 nm. The change in the spectrum at 213 nm with pH was characterized by an apparent pKa of 8.5. Studies on the reoxidation of reduced CBM-insulin were performed at pH values between 7.8 and 10 and at protein concentrations of 0.01-1 mg/ml. The best yields (ca. 85%) of the correctly paired disulfide bonds were obtained in reoxidations at pH 9.5-10 at protein concentration of 0.01-0.1 mg/ml. CBM-insulin, which had been isolated from reoxidation at high pH of the reduced CBM-insulin, was cleaved by CNBr to yield a fully active insulin in an overall yield of 60% from the reduced CBM-insulin.