Effectiveness of anthracycline against experimental prion disease in Syrian hamsters.

Prion diseases are transmissible neurodegenerative conditions characterized by the accumulation of protease-resistant forms of the prion protein (PrP), termed PrPres, in the brain. Insoluble PrPres tends to aggregate into amyloid fibrils. The anthracycline 4'-iodo-4'-deoxy-doxorubicin (IDX) binds to amyloid fibrils and induces amyloid resorption in patients with systemic amyloidosis. To test IDX in an experimental model of prion disease, Syrian hamsters were inoculated intracerebrally either with scrapie-infected brain homogenate or with infected homogenate coincubated with IDX. In IDX-treated hamsters, clinical signs of disease were delayed and survival time was prolonged. Neuropathological examination showed a parallel delay in the appearance of brain changes and in the accumulation of PrPres and PrP amyloid.

Prolonged half-life in the circulation of a chemical conjugate between a pro-urokinase derivative and human serum albumin.

Pro-urokinase is a natural plasminogen activator that displays a clot-lysis activity through a fibrin-dependent mechanism. It seems to be a promising agent for the treatment of coronary thrombosis. Like tissue-type plasminogen activator and two-chain urokinase-type plasminogen activator, pro-urokinase has a very short half-life in circulation. It has been described that conjugation of serum albumin with pro-urokinase in plasma may occur that could protect this protein from degradation. In this study we describe the insertion of an extra cysteine residue in the N-terminal end of des-(C11-K135)-pro-urokinase (delta 125-proUK), a pro-urokinase deletion mutant lacking amino acids 11-135. We have expressed and purified the new mutein [H5K, S9C, N10T] des-(C11-K135)-pro-urokinase (Cys-delta 125-pro-urokinase) and chemically conjugated it with serum albumin via the extra cysteine of Cys-delta-pro-urokinase. The purified conjugate obtained has a lower specific amidolytic activity (72,000 U/mg) than unconjugated Cys-delta 125-pro-urikinase (240,000 U/mg) due to its higher molecular mass and has a similar fibrinolytic activity in a clot lysis test to that of delta 125-pro-urokinase. We established an ELISA to measure the concentration of the conjugate in plasma and to follow the pharmacokinetics of the conjugate in monkeys after bolus injection. The conjugate displays significant lysis of human plasma clots in vivo and a dramatic increase of the half-life in the circulation, with respect to pro-urokinase and delta 125-pro-urokinase. Therefore, preliminary biological characterisation of this conjugate indicates that it could be a good candidate to inject as a bolus, compared with the infusion regimen needed with pro-urokinase.

Thrombomodulin is a cofactor for thrombin degradation of recombinant single-chain urokinase plasminogen activator "in vitro" and in a perfused rabbit heart model.

Thrombin cleaves single-chain urokinase-type plasminogen activator (scu-PA) to a two-chain derivative (tcu-PA) fibrinolytically inactive. This reaction was accelerated in vitro by purified rabbit lung thrombomodulin in equimolar complex with thrombin. Polyclonal antibodies to rabbit thrombomodulin prevented this effect. We also observed that heparin and other sulfated polysaccharides had an accelerating effect on thrombin cleavage of recombinant scu-PA. Their effect was concentration-dependent and then reversed at high levels. The effect of heparin and heparan sulfate was independent and synergic with respect to thrombomodulin. All observations except the effect of heparin, could be confirmed in a Langendorff isolated rabbit heart model. From competition experiments carried out with scu-PA derivatives and mutants, we postulate that the amino-terminal sequence of rscu-PA, containing the epidermal growth factor (EGF)-like and the kringle domains is involved in the cofactor effect of thrombomodulin on scu-PA inactivation by thrombin. We conclude that a regulatory mechanism of scu-PA inactivation is present at the cell surface.

Psycho-educative group psychotherapy for Jewish child-survivors of the Holocaust and non-Jewish child-survivors of Japanese concentration camps.

The authors describe the application of a form of group psychotherapy for middle aged patients with a past history of being child-survivors of the Second World War. In a group of Jewish survivors, the group members initially expressed anger at the perpetrators, while a group of Dutch-Indonesian survivors of Japanese concentration camps were less able to do so. Therapy was helpful in working through these and other stages of progression until the group members were able to look at their own present lives and think of their futures. Overall results of the group psychotherapy are presented. Despite the patients' age and past traumatic experiences, psycho-educative group psychotherapy was helpful for both groups.

Efficient renaturation and fibrinolytic properties of prourokinase and a deletion mutant expressed in Escherichia coli as inclusion bodies.

Prourokinase is a plasminogen activator of 411 amino acids which displays a clot-lysis activity through a fibrin-dependent mechanism, and which seems to be a promising agent for the treatment of acute myocardial infarction. The preparation of recombinant prourokinase in bacteria has been hampered by its insolubility and by difficulty in refolding the polypeptide chain. In this paper we describe the renaturation process of two recombinant proteins expressed in Escherichia coli as inclusion bodies: prourokinase and a deletion derivative (delta 125-prourokinase) in which 125 amino acids of the N-terminal region have been removed. Deletion of this sequence brings to higher refolding yields and faster kinetics (first-order rate constant of renaturation of 0.57 h-1 for delta 125-prourokinase and 0.25 h-1 for prourokinase). Our process involves sequential steps of denaturation, reduction and controlled refolding of the polypeptide chain. When applied to pure, non-glycosylated and active prourokinase, it gives a refolding yield of about 80%, demonstrating the efficiency of the renaturation procedure. Lower yields (15% and 30%, respectively, for prourokinase and delta 125-prourokinase) were obtained when the same refolding protocol was applied to inclusion bodies from bacteria. After purification to homogeneity (as shown by HPLC and SDS/PAGE) specific activities were 160,000 and 250,000 IU/mg protein, respectively, for prourokinase and delta 125-prourokinase. As with prourokinase, the deletion mutant delta 125-prourokinase displays a zymogenic nature, being activated by plasmin to the active two-chain form; however, this mutant is approximately fourfold more resistant than prourokinase to plasmin activation, and consequently shows a different fibrinolytic profile.

"Supersulfated" heparin fragments, a new type of low-molecular weight heparin. Physico-chemical and pharmacological properties.

A new type of low-molecular-weight heparin (ss-LMW-H) was prepared (by controlled depolymerization and concurrent sulfation of heparin with a mixture of sulfuric and chlorosulfonic acid), to test the influence of extra-sulfate groups on biological properties of heparin fragments. The fragments had an average molecular weight ranging from 5000 to 10,000, a sulfate-to-carboxyl molar ratio of 2.8-3.1, and electrophoretic mobilities and NMR spectra distinctly different from those of the parent heparins. Depolymerization with oversulfation reduced the anticoagulant activity of heparin (ex vivo, in rats) much more than depolymerization alone, to about 10% of the original APTT and 25-30% of the original a.Xa units. By contrast, the antithrombotic activity (venous stasis model, in rats) was still comparable to that of heparin, and bleeding times were not significantly increased. The lipasemic (lipoprotein-lipase-releasing) activity of ss-LMW-H fragments was more than twice that of heparin. Results are discussed in terms of contribution of charge-density effects to different activities and to different mechanisms for the same activity of heparin.

Inhibition of vascular smooth muscle cell proliferation in culture by pentosan polysulphate and related compounds.

Pentosan polysulphate (PPS; SP 54; Hemoclar), a highly sulphated semi-synthetic polysaccharide of MW 4.7 kD, was tested in vitro as an inhibitor of rabbit aortic smooth muscle cell (SMC) proliferation and its effects were compared with those of dextran sulphate, laminarin sulphate and heparin fractions. When added to the cell cultures simultaneously with foetal calf serum, all sulphated polysaccharides inhibited cell growth. PPS was five fold more active than heparin and its low molecular weight fractions. A high molecular weight fraction of PPS was the most active inhibitor (IC50: 2.7 micrograms/ml vs 45 micrograms/ml for PPS, and 241 micrograms/ml for heparin). When PPS and heparin were pre-incubated with the cells for three days, then removed before foetal calf serum was added to stimulate growth, their inhibitory effects were comparable with those seen when the compounds were present throughout the growth phase, suggesting that they exert their effect by binding to the cell surface, and not by interacting with the growth factors in serum.

Comparative turn-over of heparin cofactor II and antithrombin III in baboons. Influence of heparin and pentosan polysulfate administration.

Heparin and pentosan polysulfate (PPS) interact in plasma with antithrombin III (AT III) and Heparin cofactor II (HC II) respectively. To assess the influence of heparin or PPS treatment on the metabolism of their respective cofactors, we performed a double tracer study in baboons receiving heparin or PPS. Purified AT III and HC II from human plasma were labelled with 131I and 125I respectively by the lactoperoxidase-glucose oxidase technique. The tracers had unchanged biological activities, were homogeneous in SDS-PAGE, migrated as native proteins by crossed immunoelectrophoresis in the presence of heparin or PPS and virtually coeluted with endogenous baboon proteins from heparin-agarose. Nine animals were randomly allocated to receive, during the metabolic study, heparin (500 IU/kg/d, n = 3), PPS (5 mg/kg,d, n = 3) or a placebo (n = 3) given in 2 daily subcutaneous injections. Heparin levels and anticoagulant effects were similar in extent and duration to those usually achieved in man. The plasma concentrations of AT III and HC II did not vary under treatment. The half-life of the elimination phase in the placebo group ranged from 1.95 to 2.33 d for AT III and from 1.96 to 2.21 d for HC II. There was no significant difference in the half-lives of the 2 inhibitors between the placebo group and the animals receiving heparin or PPS. This suggest that clinical conditions associated with heparin treatment may be important for the effect of heparin on AT III metabolism previously reported in patients.(ABSTRACT TRUNCATED AT 250 WORDS)

Tolerance and biological activity of pentosan polysulfate after intramuscular or subcutaneous administration for ten days in human volunteers.

This study reports on the tolerance and the pharmacological activity of pentosan polysulfate (PPS) administered to healthy volunteers for 10 days. Three groups of 10 subjects received either one daily injections of 100 mg of PPS by I. M. route (group I), or two daily injection of 50 mg of PPS by I. M. or S. C. route (groups II and III, respectively). In each group two random subjects received a placebo for the 10 days; on day 0, each subject was injected by a placebo. Clinical tolerance was checked by a daily physical examination; biological tolerance was assessed comparing the results of the main biochemical and haematological constants measured before starting the treatment (day 0) and 12 or 24 h after the end of the treatment (day 11). The pharmacological activity was measured on serial samples taken before treatment and between 1 and 6 h after the drug injection on days 1, 3 and 10; the results were compared to those obtained on day 0. Clinical tolerance was good. The biological side effects concern the transaminase levels and the platelet counts. An increase above the upper normal limit was observed in 18/24 and 3/24 for alanine and aspartic transaminase respectively. The mean platelet reduction ranged between 24 and 34% according to the groups. The drug injection induced a slight Quick time (PT) prolongation, no significant alteration of factors II, VII-X, V levels and of thrombin clotting time. The activated partial thromboplastin time (APTT) was significantly prolonged and there was a weak but significant circulating anti-Xa activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of long-term treatment with suloctidil on blood viscosity, erythrocyte deformability and total fibrinogen plasma levels in diabetic patients.

1-(4-Isopropylthiophenyl)-2-n-octylaminopropanol (suloctidil, Sulocton), a drug introduced into clinical practice for the treatment of cerebral and peripheral vascular insufficiency and its complications, has been shown to decrease blood hyperviscosity after long-term treatment in diabetic patients. Suloctidil was particularly effective in lowering measured blood viscosity at high shear rate (230 s-1). At low shear rate (0.77 s-1) a decreasing effect was also observed but reached a statistically significant level only after one month. Since neither total plasma fibrinogen levels nor red blood cell deformability were modified by suloctidil treatment, its lowering effect on blood hyperviscosity could be due to a hitherto unrecognized mechansim of action.