Cloning, expression, and characterization of a chitinase gene from the Antarctic psychrotolerant bacterium Vibrio sp. strain Fi:7.

A marine psychrotolerant bacterium from the Antarctic Ocean showing high chitinolytic activity on chitin agar at 5 degrees C was isolated. The sequencing of the 16S rRNA indicates taxonomic affiliation of the isolate Fi:7 to the genus Vibrio. By chitinase activity screening of a genomic DNA library of Vibrio sp. strain Fi:7 in Escherichia coli, three chitinolytic clones could be isolated. Sequencing revealed, for two of these clones, the same open reading frame of 2,189 nt corresponding to a protein of 79.4 kDa. The deduced amino acid sequence of the open reading frame showed homology of 82% to the chitinase ChiA from Vibrio harveyi. The chitinase of isolate Fi:7 contains a signal peptide of 26 amino acids. Sequence alignment with known chitinases showed that the enzyme has a chitin-binding domain and a catalytic domain typical of other bacterial chitinases. The chitinase ChiA of isolate Fi:7 was overexpressed in E. coli BL21(DE3) and purified by anion-exchange and hydrophobic interaction chromatography. Maximal enzymatic activity was observed at a temperature of 35 degrees C and pH 8. Activity of the chitinase at 5 degrees C was 40% of that observed at 35 degrees C. Among the main cations contained in seawater, i.e., Na+, K+, Ca2+, and Mg2+, the enzymatic activity of ChiA could be enhanced twofold by the addition of Ca2+.

[Results of conversion from conventional immunosuppressive therapy to cyclosporin A in complications following kidney transplantation]. / Ergebnisse der Konversion von konventioneller immunsuppressiver Therapie auf Cyclosporin A bei Komplikationen nach Nierentransplantation.

In 41 patients suffering from complications late after kidney transplantation the conventional immunosuppression was converted to Cyclosporin A. A complete normalization was obtained in the case of acquired immunoglobulin G deficiency and in persistent leukocytopenia. 6 patients suffering from life-threatening bacterial infections did survive after conversion with functioning allograft and 8 out of 9 late rejections were reversible. Conversion may be necessary in chronic hepatopathy and chronic rejection. A conversion to cyclosporin A is recommended because of control of complications in 78% (46/59) of the recipients.

The teaching and organisation of clinical pharmacology in European medical schools (W.H.O. Working Group on Clinical Pharmacology).

A World Health Organisation (European Regional Office) working party has been established to review the progress of clinical pharmacology in European countries. As part of this review a questionnaire on the teaching of clinical pharmacology was sent to the Deans of all 350 medical schools in the region. Very few replies were received from U.S.S.R., Greece and Portugal and these countries' returns were not analysed further. The overall compliance rate (excluding these countries) was 82% with a figure of 84% from Western Europe and 74% from Eastern Europe. An average time of 96 h (range 0-320) was devoted to pharmacology teaching in the medical curriculum in Western Europe with 124 (0-240) h in Eastern Europe. In contrast 28 h (0-210) was devoted to clinical pharmacology teaching in Western Europe and 27 h (0-90) in Eastern Europe. On average in Western Europe each medical school had 2 individuals trained in clinical pharmacology with 1.3 posts in the subject and the figures for Eastern Europe were 2.3 and 1.1 respectively. However these figures hide a wide variance in the teaching of clinical pharmacology. Particularly in Western Europe there are a number of medical schools in Italy, Spain and the Federal Republic of Germany (FRG) where clinical pharmacology is not taught and there is a dearth of individuals trained in the subject. Every effort to encourage clinical pharmacology and its teaching should be made, particularly in these countries.

Effect of methotrexate on the pharmacokinetics and renal excretion of cisplatin.

The kinetics of platinum in plasma and erythrocytes, and its renal excretion, have been examined in five patients with non-small cell carcinoma of the lung, after treatment with cisplatin 50 mg/m2 (Platidiame) and, three weeks later, a combination of 50 mg/m2 cisplatin and 40 mg/m2 methotrexate. The patients were given 0.9% saline 1 l 1 h prior to drug application. Plasma platinum elimination was biphasic with a short initial phase (t1/2 alpha 10-31 min) and a long beta-phase (t1/2 beta 65-91 h). With the exception of increased AUC values in all five patients 0-8 h after the injection, no significant change in the kinetics of platinum in plasma was found after coadministration of methotrexate. In four of the five patients renal platinum excretion was reduced in the first 6 h after administration of methotrexate. The renal clearance of platinum was 50% lower in those four patients 0-3 h after the injection. With the exception of one patient, no signs of nephrotoxicity were observed after combined drug administration. Other toxic effects were mild and showed no increase after the initial administration of methotrexate.

Capillary blood, dried on filter paper, as sample for monitoring cyclosporin A concentrations.

For monitoring the immunosuppressive drug cyclosporin A by radioimmunoassay (Sandoz Ltd.) we propose a simple method of sampling in which 20 microL of capillary blood is dried on filter paper. Patients can collect their own samples and mail or bring them to the laboratory. Results for such samples, and their variability, correspond to those for conventional methods of sampling (collection of venous or capillary blood into buffer). Capillary blood can be stored on paper at room temperature for more than four weeks with no effect on assay results.

[Pharmacokinetics of dihydralazine following intravenous administration in laboratory animals]. / Pharmakokinetik von Dihydralazin nach intravenöser Gabe bei Versuchstieren.

Comparative pharmacokinetic studies with high i.v. doses of dihydralazine (1, 5 and 7.5 mg/kg-1, respectively) were performed in rats, rabbits, and dogs. 1 was distributed in rabbits and dogs in two phases with half lives of 0.25-0.4 min and 1.0-2.0 min. The terminal slope was species dependent. The relative clearance values decreased in the order rabbit, rat, dog. High tissue concentrations were observed in rabbits and dogs in parenchymatous organs and endothelium (aorta). Skeletal and heart muscles belonged to the central compartment. The biliary excretion was low in rabbits. Dogs accumulated the drug in bile (bile/serum-ratios = 19-390). Maximally 5% of the given dose were excreted in rabbits with the 12-h-urine.

Pharmacokinetics of adriamycin, adriamycinol, and antipyrine in patients with moderate tumor involvement of the liver.

The pharmacokinetics of adriamycin, its metabolite adriamycinol, and antipyrine were studied in 17 patients with moderate tumor involvement of the liver and compared to that of 19 tumor patients with normal liver function (Preiss et al. 1985). The individual liver function parameters deviated from normal by a factor ranging from 2.5 to 12.2. The t1/2 alpha and t1/2 beta, the AUC (corrected for body weight and dose) and the total body clearance (CL, corrected for body weight) of adriamycin did not differ significantly between the two groups of patients. Likewise, there was no difference in the kinetic parameters of antipyrine between the two groups. Unlike adriamycin and antipyrine, adriamycinol was found to have a significantly longer t1/2term (60.5 vs 28.3 h, P less than 0.001), an increased AUC (3.00 vs 1.43 h/ug per ml, P less than 0.02), and a higher AUCadriamycinol/AUCadriamycin ratio (0.94 vs 0.52, P less than 0.02) in patients with moderate tumor involvement of the liver. The CL, the AUC, and t1/2 beta of adriamycin correlated significantly (P less than 0.001 and P less than 0.01) with the corresponding kinetic parameters of antipyrine, but not with the usual liver function parameters. No correlation could be found between the kinetic parameters of adriamycinol and those of antipyrine.

[Quantitative determination and kinetics of dihydralazine in hypertension patients]. / Quantitative Bestimmung und Kinetik von Dihydralazin bei Hypertoniepatienten.

For the quantitative determination of dihydralazine (1) a derivative with acetylacetone in biological material was formed at pH = 4.9, extracted with n-hexane, and measured gaschromatographically with N-P-FID. Acid labile 1 was hydrolyzed with HCl (1 mol/l) for 24 h. The detection limit was 25 nmol/l plasma. Kinetic studies were performed in 16 patients with essential hypertension under steady-state conditions after the oral application of 50 mg 1. The acetylator phenotype was determined with sulfamethazine. Complete dihydralazine plasma level-time courses were found in only 5 cases. The concentrations were below the detection limit in 4 patients for the whole period. Only single values could be registered in the remaining patients. Maximal plasma levels of the free (58-314 nmol/l) and acid labile 1 (147-367 nmol/l) were reached 20-40 min after the application. The elimination half life was 23-47 min for the free 1, 55-92 min for the acid labile 1. Less than 0.5% of the applied drug were excreted into the 24 h urine in its free form, about 0.4% as acid labile derivatives. No correlation could be found between the acetylator phenotype of the patients and the kinetic behaviour of the drug. Preliminary studies concerning the biliary excretion of 1 after i. m. application in two patients with T-drain showed an accumulation of the free compound with bile/plasma ratios up to 7.4.

[The pharmacokinetics of bendamustine (Cytostasane) in humans]. / Untersuchungen zur Pharmakokinetik von Bendamustin (Cytostasan) am Menschen.

The pharmacokinetics of bendamustine (Cytostasane) was determined in plasma on seven patients after its intravenously and oral application, respectively. Cytostasane was given in a dosis of 4.2-5.5 mg . kg-1 as an intravenous infusion over 3 min and as gelatine capsules in a 7-d intervall. Its elimination from the plasma is fast, monoexponentially and two-phasic after intravenous application (t1/2 alpha = 9.6 min, t1/2 beta = 36.1 min). The AUC was 11.17 micrograms . ml-1 . h, the central distribution volume 11.15 l and the distribution volume in steady state 20.51 l. The mean total clearance was 528.9 ml . min-1. After oral application maximal plasma levels of Cytostasane were detectable before 1 h. The mean oral bioavailability was 0.57, ranged from 0.25 to 0.94. Cytostasane undergoes metabolism. Its hydrolysis in plasma is slow (t1/2 = 1.67 h). After Cytostasane the depression of leucocytes was mild.

[Blood level oriented ambulatory cyclosporin A therapy following kidney transplantation]. / Blutspiegelorientierte ambulante Cyclosporin-A-Therapie nach Nierentransplantation.

It is reported on the blood level-oriented oral long-term therapy with cyclosporin-A (Sandimmun) in 20 patients after kidney transplantation. The Cy-A-concentrations were measured in the whole blood by means of a radioimmunoassay (Sandoz) under steady state conditions. The mathematical analysis of the steady state daily minimum concentrations in the whole blood depending upon the dosage rate allows individual calculations of the dosage for the stabilisation of the therapeutic concentration desirable for the individual patient in each case. In the majority of the patients a non-linear relation between the steady state blood concentration and the dosage rate was present. The observation of a defined therapeutic area under the conditions of the ambulatory treatment could be achieved without any problems. The control intervals were 4-6 weeks. The long-term stability of the individual pharmacokinetic parameters can at present not be judged reliably, since the average observation time is still too short. In 6 patients who are controlled on the way demonstrated already for several months no systemic changes in the behaviour of the blood level were to be seen.