Evžen Kasafírek

Effect of alaptide, its analogues and oxiracetam on memory for an elevated plus-maze in mice

In the present study, the elevated plus-maze was used to evaluate memory in female mice. In Experiment 1, the mice retested on day 1, 4 or 7 after the initial session escaped from the open arm into the enclosed arm in a significantly shorter time than those retested on day 10 or 14. Thus, a 10-day inter-session interval was chosen for testing drugs which were expected to enhance memory. In Experiment 2, in the retest performed on day 10, both alaptide (cyclo(L-alanyl-1-amino-1- cyclopentanecarbonyl)) and oxiracetam, given immediately after the 1st session, reduced the transfer latency from the open arm into the enclosed arm as compared with that of the controls. In Experiment 3, a similar effect, i.e., the retention of spatial information, was facilitated by post-session injections of 5 out of 21 alaptide analogues. The new compounds represent the 2,5-piperazinedione derivatives which contain 1-amino-1-cyclo-alkanecarboxylic acid (C3 to C7 ring). The cyclopentane- and cyclohexane-ring was substituted by an alkyl group. In the series with the cycloalkane ring, the importance of the structure of alaptide was confirmed again, which underlines the importance of the cyclopentane ring; the active structures had L-alanine instead of glycine as the second amino acid. Isomers of the cyclohexane series which contained methyl or tert-butyl were most active when the substitution was at position 3. Our results demonstrate that the model of long-term memory can be used to discriminate between closely related chemical structures.

Effective peritoneal therapy of acute pancreatitis in the rat with glutaryl-trialanin-ethylamide: a novel inhibitor of pancreatic elastase.

The six hour peritoneal lavage with glutaryl-trialanin-ethylamide, a low molecular competitive inhibitor of pancreatic elastase (IC50-8 mumol/l), effectively suppresses the evolution of taurocholate induced acute pancreatitis in the rat. The lavage alone is followed by a marked decrease of fat necrosis and amylase and lipase activity in serum. The area of pancreatic haemorrhage was significantly reduced only after the lavage solution was supplemented with Glt-Ala3-NHEt. The effect was not enhanced by a bolus injection of the inhibitor before starting the lavage. The combination of Glt-Ala3-NHEt with aprotinin or nafamstate mesilate produced only marginal greater benefit. The effect of Glt-Ala3-NHEt on pancreatic haemorrhage is time and dose related even with delayed onset of the lavage. Animals treated with peritoneal lavage without Get-Ala3-NHEt lived longer than controls (p less than 0.05), but by 60 hours the survival rate of both groups was almost the same (76 v 74%). All animals lavaged with Glt-Ala3-NHEt survived 120 hours and the difference in the survival rate between this and both remaining groups was significant (100% v 76% v 74% - p less than 0.05). The results were considered favourable and preliminary clinical trials of Glt-Ala3-NHEt in subjects with acute pancreatitis justified.

Elastolytic activity of human duodenal contents

Elastolytic activity of human duodenal contents was determined using the new chromogenic substrate succinyl-trialanine-p-nitroanilide (Suc-Ala3-NAp). The mean output values after pancreatic stimulation with pancreozymin and secretin were significantly higher in controls than in subjects with impairment of other secretory values (volume, bicarbonate, amylase, lipase). Agar gel electrophoresis and chromatography on DEAE-Sephadex revealed one to two fractions which differed in mobility (cathodic and anodic fraction), elution with different NaCl concentrations (0.15 M, cathodic fraction; 0.3 M, anodic fraction), and in behaviour towards synthetic and natural substrate (Suc-Ala3-NAp) and elastin-Congo Red). The cathodic fraction cleaved both substrates, whereas the anodic fraction cleaved only Suc-Ala3-NAp. After trypsin and enterokinase treatment the anodic fraction behaved as the cathodic fraction on DEAE-Sephadex chromatography. The molecular weights (Sephadex G-100) and the Michaelis constants (Suc-Ala3-NAp) of both fractions were identical (24 500; 0.45 X 10(-3) M). These fractions represent probably diffenent activation forms of pancreatic elastase.

Nalpha-tosyl-L-arginine-p-nitroanilide as substrate in color test and polarographic test of trypsin.

Abstract A practical procedure is described for a sensitive photometric and polarographic determination of trypsin, using N α - tosyl- l -arginine -p- nitroanilide ( l -TAPA) as substrate. The test is suitable for routine analysis of series of clinical samples. The enzymatic activity is determined from the quantity of p-nitroaniline liberated hydrolytically and established photometrically or polarographically. The polarographic determination is suitable for nonhomogeneous or colored media. The test can be carried out even if the sample concentration of trypsin is lower than 1 μg/ml. A method is presented for calculation of the enzyme units directly from the polarographic wave height of p-nitroaniline liberated enzymatically.

Isolation and analysis of peptidic fragments of α-gliadin using reversed-phase high-performance liquid chromatography

Summary Peptidic fragments of α-gliadin were obtained by peptic-tryptic-pancreatic (PTP) digestion of the α-gliadin fraction isolated by ion-exchange chromatography on a sulphopropyl-Sephadex C-50 column. The proteolytic digest was fractionated by ultrafiltration into three subfractions, PTPa 1 –PTPa 3 . The subfraction PTPa 2 was then analysed and individual peaks were separated using reversed-phase high-performance liquid chromatography (RP-HPLC) using a gradient of acetonitrile in 0.1% trifluoroacetic acid and a Separon SGX-C 18 sorbent. A 100-mg amount of the PTPa 2 subfraction was separated in a single analysis by preparative RP-HPLC and twenty peaks were obtained for further characterization. The molecular mass in range 300–3000 was established for individual peptidic fragments by gel-permeation chromatography on a TSK-G2000 SW column.

Two-step generation of spirocyclic dipeptides from linear peptide ethyl ester precursors

Linear tri- and tetrapeptide precursors of 2,5-piperazinedione were prepared and their conversion to spirocyclic dipeptidase enzymes, the spirocyclic dipeptides (SpDp) were generated from the precursors by a two-step mechanism consisting in the proteolytic release of the C-terminal dipeptide ethyl ester and its subsequent spontaneous cyclization. After intraperitoneal administration of urokinase and Ac-Leu-Lys-Gly-Acp-OEt, a SpDp precursor targeted to endogenous plasmin, or the administration of the activated Hageman factor fragment and Ac-Leu-Arg-Ala-Acp-OEt, a SpDp precursor, targeted to endogenous kallikrein, the generated corresponding C-terminal dipeptide ethylester intermediates and SpDp, cyclo(Gly-Acp) and cyclo (Ala-Acp), respectively, were detected in the blood serum of C57B1 mice. Suppression of partial amnesia induced by sodium nitrite was observed in rats where it was subcutaneously administered with H-Leu-Ala-Acp-OEt, a peptide precursor of alaptide, the active SpDp, i.e. cyclo(1-amino-1-cyclopentanecarbonyl-L-alanyl).

New low-molecular inhibitors of pancreatic elastase with possible in vivo application: Alkylamides of N-acylated tripeptides

Out of a series of alkylamides of N-acylated tripeptides, Glt-(Ala)2-Pro-NH-Et and Glt-(Ala)3-NH-Pr were found to be potent inhibitors of porcine and human pancreatic elastase, and because they are free of toxic groups they might be considered for in vivo application.

Cleavage of p-nitroanilides of N-acylated tri- and tetrapeptides by alanine endopeptidase from the brush border membranes of rat enterocytes

The activity of the alanine endopeptidase from the intestinal brush border was studied using chromogenic substrates of the general fomula Sc-Ala2-X-pNA, Sc-Y-Z-Ala-pNA and W-Ala3-pNA respectively. Substrates with C-terminal Leu or Nle are hydrolyzed more readily than Ala-analogues. At least one Ala-residue in one of the positions adjacent to the C-terminus is necessary for the enzyme activity. An Na-substituent has no effect on the activity.