The in vitro influence of sulfated bis-lactobionic acid amides on O6-alkylguanine-DNA alkyltransferase, DNase I, nucleic acid synthesis and chromatin structure.

The influence of four sulfated bis-lactobionic acid amides (BLAA) of molecular weights between 2388 and 2514 on O6-alkylguanine-DNA alkyltransferase (AT), DNase I and nucleic acid synthesis as well as on nucleoid sedimentation and the viscosity of alkaline lysates of chicken embryo cells was studied in vitro. The activities of AT and DNase I were inhibited by BLAA in a dose-dependent manner. Depending on the polyanion used, concentrations depleting AT activity by 50% ranged between 3.5 and 7.0 microM, whereas BLAA concentrations of almost 250-320 microM were needed to halve DNase I activity. At concentrations above 8 microM, BLAA decreased scheduled DNA synthesis in a dose-dependent fashion whereas RNA synthesis remained unchanged even at the highest BLAA concentrations used (2 mM). In chicken embryo brain cells BLAA exerted a biphasic effect on the nucleoid sedimentation and the viscosity of alkaline cell lysates reflecting a decrease in chromatin compactness at lower BLAA concentrations (10-100 microM) and an increase in chromatin compactness at higher polyanion concentrations (> or = 200 microM). The remarkably high sensitivity of the nuclear enzyme AT deserves further investigation in regard to the fate of the polyanions within cells and tissues.

Anticoagulant and antithrombotic properties of synthetic sulfated bis-lactobionic acid amides.

Sulfated bis-lactobionic acid amides, a new class of polyanions with heparin-like properties, were synthesized and their antithrombotic and anticoagulant activities were determined. Compared to heparin and Fragmin, a low molecular weight heparin, the substances exhibited moderate to low anticoagulant activities in aPTT, thrombin clotting time and Heptest assays. In amidolytic assays no anti-IIa activity and only exceedingly low anti-Xa activity was observed. The antithrombotic activity of the bis-lactobionic acid amides was determined using two thrombosis models. In rabbit and rat models thrombi were induced by a combination of endothelial damage and reduction of blood flow or only by endothelial damage. At least one of the bis-lactobionic acid amides (LW 10082) exhibited a considerable antithrombotic activity which was similar to low molecular weight heparin.

Endogenous release of tissue factor pathway inhibitor by topical application of an ointment containing mucopolysaccharide polysulfate to nonhuman primates.

Several studies have shown that tissue factor pathway inhibitor (TFPI) is released after the intravenous and subcutaneous administration of heparin and heparin-related drugs. Mucopolysaccharide polysulfate (MPS) is a preparation of glycosaminoglycans (GAGS) derived from mammalian cartilage, which has several structural and functional properties similar to heparin. Previous reports have shown that MPS is capable of releasing TFPI after intravenous administration. Therefore, this investigation was performed to determine the ability of topically administered MPS to release TFPI in a nonhuman primate model. A group of four monkeys were administered 3% MPS ointment in a dosage of 0.5 g/kg corresponding to 15 mg MPS/kg; another four monkeys were administered placebo ointment at a dosage of 0.5 g/kg once a day for 5 days in a period of 10 days. No effect of MPS was observed on the coagulation assays activated partial thromboplastin time (APTT), thrombin time (TT) and Heptest or on the platelet count. However, both the total and free TFPI levels were significantly and progressively elevated over the 10-day period in comparison to the placebo control group (P<.05). It is proposed that the ability of the topically administered MPS to increase the free and total TFPI levels may be one of the modes of action that contributes to the anticoagulant and anti-inflammatory actions of this agent.

[Treatment of superficial thrombophlebitis]. / Behandlung der oberflächlichen Thrombophlebitis.

Superficial thrombophlebitis is a frequently occurring disease which, because of risk factors, affects women to a greater extent than men. The presence of deep venous thrombosis should be excluded by imaging procedures. Heparin or low-molecular weight heparins are indicated for treatment, if deep venous thrombosis is present or threatening. In the acute stage non-steroidal antiinflammatory drugs can be taken orally in order to alleviate pain and inflammation. In the case of concomitant chronic venous insufficiency, edema protective drugs (e. g. preparations containing horse chestnut extract and flavonoids) can support the therapy. For the treatment of inflammatory conditions in relatively short superficial vein segments without involvement of the deep venous system, a limitation to topical treatment is possible. In addition to compression therapy, the application of creams or gels containing heparin or heparinoids are the usual treatment approach. The use of topical products containing mucopolysaccharide polysulfate (MPS) has been proven to be beneficial. The problem of providing evidence of the efficacy of topical products in the therapy for superficial thrombophlebitis under the current strict demands of our regulatory authorities and of developing new products under these conditions is discussed.

Recommendations on biosimilar low-molecular-weight heparins.

Based on the results of large clinical trials, several low-molecular-weight heparins (LMWHs) have been approved for prophylaxis and the treatment of venous and arterial thromboembolism. As a result of expiration or pending expiration of patent protection of the originator LMWHs, many generic or biosimilar LMWHs have been approved in some countries and more are likely to be approved elsewhere. Their greater availability may reduce the treatment costs. The Working Party on Requirements for Development of Biosimilar LMWHs of the Subcommittee on Control of Anticoagulation, Scientific and Standardization Committee of the International Society on Thrombosis and Haemostasis has reached a consensus on recommendations to ensure the quality of biosimilar LMWHs as compared with the originator LMWHs.

[Comparison of the antiphlogistic effect of mucopolysaccharide-polysulfate ointments with heparin-containing ointments in the UV erythema test]. / Vergleich der antiphlogistischen Wirkung von Mucopolysaccharid-polyschwefelsäureester-Salben mit heparinhaltigen Salben im UV-Erythem-Test.

It could be demonstrated by means of the UV erythema model that four different ointment preparations containing mucopolysaccharide polysulfate (MPS) have a marked antiinflammatory effect and inhibit the formation of erythemas to a large extent. In this trial arrangement the different preparations containing MPS were significantly superior to altogether eight commercial heparin ointments, which were tested, too. With an inhibition of 69% of the erythema induced by UV rays, Hirudoid 40000 ointment is the most effective of all preparations tested.

Antithrombotic effect of a mucopolysaccharide polysulfate after systemic, topical and percutaneous application.

Antithrombotic activity of mucopolysaccharide polysulfuric acid ester (MPS) was investigated in vitro as well as in animal models of thrombosis. In vitro studies were performed by using native human blood and the haemostatometer. MPS (greater than or equal to 25 micrograms/ml) caused concentration dependent inhibition of the in vitro haemostasis (platelet reactivity). Antihaemostatic activity of epoprostenol (prostacyclin) was synergized by MPS. In vivo platelet thrombus formation was induced by laser irradiation in the microvasculature of hamster cheek pouch. The time until occlusion of the irradiated small vein and the duration of occlusion were measured. MPS administered intravenously at a dose range of 1-10 mg/kg b.w. inhibited thrombus formation and prolonged the time until occlusion of the irradiated vessel. Upon topical administration of a solution of 1, 5 or 25% MPS the occlusion time was also prolonged. Topical application of MPS as a 0.3% cream (Hirudoid) increased the occlusion time significantly. 24 h percutaneous application of 0.3% MPS cream but not the placebo on the back skin of rats resulted in a significant (p less than 0.001) inhibition of thrombus formation in the microvessels of the mesoappendix. A common finding at all modes of administration of MPS was that reperfusion of the occluded vessel occurred much earlier in MPS treated animals than in the controls.

[Percutaneous penetration of mucopolysaccharide-polysulfate ester from a combination preparation]. / Zur perkutanen Penetration von Mucopolysaccharid-polyschwefelsäureester aus einem Kombinationspräparat.

The cutaneous penetration of mucopolysaccharide polysulfate (MPS) from a commercial available combination drug (Mobilat) could be shown by means of histochemical and immunohistological methods. Deposition of MPS from ointment and gel in corium and subcutis of different animal species has been improved by the keratolytic activity of the salicylic acid component. There are differences in the intensity of absorption of MPS not only in the various animal species, but also caused by the particular galenic preparations investigated.

[Effects of 6-methyl-uracil upon the phagocytic activity in mice following whole-body X-irradiation OR 2,4,6-triethyleneimino-s-triazine treatment (author's transl)]. / Zur Wirkung von 6-Methyluracil auf die Phagozytoseaktivität von Mäusen nach Einwirkung von Ganzkörper-Röntgenbestrahlung oder 2,4,6-Triäthylenimino-s-triazin.

1. Phagocytic activity measured by means of the intravasal clearance of a soot dispersion in male NMRI-mice was increased six to ten days after whole-body X-irradiation (640 R) and decreased during the same period after i.v. administration of 2,4,6-triethyleneimino-s-triazine (TEM 2.0 mg/kg). 2. By means of 6-methyl-uracil food admixtures (200 to 400 ppm during 2 or 3 weeks) or by repeated intravenous injections of a N-methyl-D-glucosamine-6-methyluracil complex (62.5 to 250 mg/kg daily during five days), a significant augmentation of the phagocytic index being related to time and dosage was obtained in otherwise untreated mice. Comparable results were seen using cytidine and cytidine-s'-phosphate, whereas guanosine-5'-phosphate remained ineffective. 3. Whilst stimulating effects of 6-methyl-uracil or its N-methyl-D-glucosamine complex on X-irradiated mice were suspended, an increase up to supernormal values of the phagocytic index was produced by the pyrimidine base in animals treated with TEM. In accordance to this the survival rate of lethally X-irradiated mice (960 R) could not be increased; with animals given lethal TEM-doses, however, a significantly increased survival rate was obtained. 4. The present investigations as well as former biochemical analyses confirm the assumption that 6-methyluracil produces its regeneration effects, to some extent at least, by specific pathways influencing the reticuloendothelium. Different results from X-irradiated and TEM-treated mice are referring to the different points of attack of the two noxa.

[Effects of 6-methyluracil and exogenous nucleic acids on DNA-synthesis of bone-marrow cells of rats following X-irradiation and tretamine or cyclophosphamide treatment (author's transl)]. / Wirkung von 6-Methyluracil und exogenen Nukleinsäuren auf die DNS-Synthese von Knochenmarkszellen der Ratte nach Einwirkung von Röntgenstrahlen, Tretamin oder Cyclophosphamid.

The in vitro incorporation of 14C-thymidine into DNA of bone-marrow cells as well as DNA content of spleen and thymus of male Wistar rats were increased significantly by 6-methyluracil (within an N-methyl-D-glucosamine-6-methyluracil complex) and/or heterologous, low-molecular nucleic acids, when given therapeutically at doses of 100--250 mg/kg b.w. following i.v. injection of tretamine. Maximum effects were reached as early as two days after the onset of experiment. When tretamine was replaced by X-irradiation or cyclophosphamide, the influence of the 6-methyluracil complex and nucleic acids was less significant. Whereas normal bone-marrow cells and cells damaged in vitro by X-irradiation, tretamine and/or inhibitors of protein synthesis could not be influenced by the 6-methyluracil complex, incorporation of 14C-L-phenylalanine into proteins of bone-marrow cells was enhanced by exogenous DNA or RNA (10--100 micrograms/ml). The present investigations underline the previously drawn conclusions that certain biological alkylating agents, nucleic acids and pyrimidines may interfere to some extent through the reticuloendothelial system.

E1On the effect of 6-methyluracil on mice damaged by 2,4,6-triethylene-imino-1,3,5-triazine or by X-irradiation (author's transl). / Zur Wirkung von 6-Methyluracil auf Mäuse nach Schädigung durch 2,4,6-Triäthylenimino-1,3,5-triazin oder Röntgenbestrahlung

Survival time of mice after i.v. injections of 2,4.6-triethylene-imino-1,3,5-trazine (TEM) or total body-X-irradiation (TBI) was increased by 6-methyluraclil (6-MU) when given in food (200 ppm). Under the same conditions, 6-MU decreased the involution of spleen and thymus (as measured by DNA-content and DNase II activity) under the infuence of TEM and enhanced the regeneration of the spleen after TBI. Elevation of DNase I- and protein content of the kidneys and a (short-dated) increase of incorporation of 14C-phenylalamine into microsomes of liver of 14C-orotic acid into RNA of liver and kidney suggest that the influence of 6-MU is mediated at least partly by a specfically anabolic effect.

[The antiexudative and anti-edematous action of sympathomimetics]. / Zur antiexsudativen und antiödematösen Wirkung von Sympathikomimetika.

The anti-exudative and anti-edematous effects and the dose-effect relationship of a-sympathomimetics, especially of l-phenylephrine-HCl (PE), have been demonstrated using as model the rat pad-carrageenan edema and the histamine liberator test, the dosage being administered cutaneously, orally, and intraperitoneally. The beta-receptor activating compound bamethane sulfate was not effective when used on the same models. Evidence of percutaneous penetration of PE was provided both on isolated skin and in viro. PE, when injected intravenously or subcutaneously, produced a rise of blood pressure in experimental animals. When PE was administered orally and cutaneously, it did not, however, alter the blood pressure. The anti-inflammatory and anti-exudative effects remianed unchanged.

Antithrombotic action of recombinant hirudin in a venous thrombosis model.

The dose- and time-dependent antithrombotic activity of recombinant hirudin (r-hirudin) was examined in a new animal model of venous thrombosis. For the evaluation of the antithrombotic activity of r-hirudin, an occluding thrombus was produced by repeated clamping of the rat jugular vein. The number of clampings necessary to induce thrombosis served as a measure of antithrombotic activity. The gradual reduction of blood flow was registered by Doppler sonography. In order to estimate the relative antithrombotic activity of r-hirudin, its potency in this model was compared with other antithrombotic substances. r-Hirudin proved to be less potent than heparin after intravenous administration. Its half-life after subcutaneous administration was relatively short; however, the antithrombotic potency after subcutaneous administration was comparable to that of heparin.

Antithrombotic and anticoagulant properties of synthetic polyanions: sulfated bis-aldonic acid amides.

Fifteen polyanionic bis-aldonic acid amides were synthesized by condensation of the corresponding aldonic acids and alpha,omega-diamines and by subsequent sulfation of the intermediates. The compounds were completely sulfated and exhibited molecular weights between 1450 and 2600 daltons. The antithrombotic activity of these synthetic heparin analogs was evaluated in a rat jugular vein clamping induced thrombosis model. Several compounds pounds were potent antithrombotic substances comparable in potency to the low molecular weight heparin Fraxiparin. Some structure activity relationships on their pharmacologic action could be deduced. The antithrombotic activity of a representative agent was confirmed using the Wessler stasis model in rabbits. In the in vitro tests the bis-aldonic acid amides exhibited moderate to low anticoagulant effects depending on the assay used. In the activated partial thromboplastin time assay, the anticoagulant potency of these compounds was between 2- and 30-fold lower than that of heparin. The anti-factor Xa activity of the experimental substances was at least 50 times lower than that of heparin, whereas the compounds were devoid of anti-factor IIa activity when measured in an amidolytic assay. The anticoagulant effects of these agents were dependent on the length of the spacing and on the type of aldonic acid moieties. These bisaldonic acid amides represent a novel class of potent antithrombotic substances without any anti-factor IIa activities and only very low anti-factor Xa activities, suggesting that these antiprotease actions are not a prerequisite for the antithrombotic action of polyanionic substances.

Biochemical studies on sulfated lactobionic acid amides.

A series of sulfated bis-lactobionic acid amides was prepared. The compounds comprise highly charged poly-anions of very low molecular weight. Usually, the compounds contain 16 sulfate groups per molecule and are homogeneous, monodisperse substances. The molecular weights range from 2388 to 2514. The compounds were evaluated for anticoagulant activity using a number of standard tests. The compounds exhibited moderate to good APTT activity. Interestingly, the anti-Xa and anti-IIa activities were very low, particularly in amidolytic assays. Using prothrombin and Factor X activation assays, it was demonstrated that these compounds were more potent in these experimental settings than in standard anti-Xa or anti-IIa assays. Compared with heparin and LMW heparins, the bis-lactobionic acid amides were particularly active as inhibitors of the intrinsic system activation of Factor X. The bis-lactobionic acid amides exhibited potent anticomplement activity, being clearly superior to heparin and LMW heparins. For the most interesting substance, LW 10082, experimental results suggest that its anticoagulant activity is independent of antithrombin II. The anticoagulant activity of LW 10082 was readily neutralized by protamine sulfate. Platelet factor 4, however, did not reduce its anticoagulant activity. Because of these favorable properties, it is hoped that LW 10082 might prove to be an interesting alternative to heparin or LMW heparin in clinical use.

Derivatives of bis-aldonic acid amides and their anticoagulant and antithrombotic properties.

Aprosulate sodium was the first representative of a new class of synthetic polyanions showing antithrombotic efficacy in different animal models. In several clinical trails (Phase I) in human volunteers aprosulate demonstrated anticoagulant properties, too. Searching for other active substances of similar structure, a series of bis-aldonic acid amides were synthesized. These compounds exhibited interesting antithrombotic and anticoagulant activities. The pharmacodynamic activities of the compounds LW 10121, LW 10125, LW 10114, 10244, and LW 10168 are summarized in this article. These substances prolonged the APTT to 150% of the blank values at concentrations of 1.5 to 13.5 micrograms/mL. The thrombin time and anti-Xa test were only slightly influenced by concentrations up to 100 micrograms/mL. All compounds were investigated in a jugular vein hemostasis model in rats to examine their antithrombotic potential. They all had an antithrombotic activity lower than aprosulate, except compound LW 10121, which seemed to be a little more active. The subcutaneous injection of 10 mg/kg LW 10121 resulted in a longer duration of action than aprosulate and heparin. On the basis of the chemical structure and the profile of action, it is assumed that the new compounds may possess the same mode of action as aprosulate, but the mechanism of action may be different from heparin and low molecular weight heparins.