Polymer-conjugated bovine pancreatic and seminal ribonucleases inhibit growth of human tumors in nude mice.

The hydrophilic poly[N-(2-hydroxypropyl)methacrylamide] (PHPMA) was used for RNase A or BS-RNase modification to prevent their degradation in bloodstream or fast elimination. Two PHPMA chains (classic and star-like) were synthesized and their conjugates with both enzymes were tested on the CD-1 nude mice bearing various human tumors. These RNase conjugates injected intravenously or intraperitoneally into the mice bearing melanoma, neuroblastoma or ovarian tumor caused significant reduction of transplanted tumors following ten daily doses of 2.5 and/or 1 mg/kg, respectively, while free RNase A or BS-RNase injected in doses of 10 mg/kg exerted only negligible antitumor activity. Histological examination confirmed potent cytotoxic effect of RNase A conjugates in ovarian tumor. Despite the antitumor activity observed in vivo, the in vitro cytotoxic activity of RNase A conjugates was not pronounced and did not differ from that caused by the free RNase A. The in vitro experiments with 125I-labeled preparations demonstrated that polymer conjugates were internalized by tumor cells very poorly in contrast to the dose-dependent internalization of the wild enzyme preparation. Surprisingly, mice injected with EL-4 leukemic cells, which were preincubated for 4 h with BS-RNase conjugates, exerted significantly prolonged survival compared with the control non-treated mice. It may be supposed that both BS-RNase and RNase A conjugates with PHPMA act after administration in vivo by a mechanism different from that or those occurring under in vitro conditions because in vivo they exert an antitumor action, whereas in vitro, they are ineffective. The experiments proved that RNase A, when conjugated to PHPMA, produced identical aspermatogenic and antitumor effects as BS-RNase conjugated to this polymer and that this preparation may be regarded as a potential anticancer drug.

Poly[N-(2-hydroxypropyl)methacrylamide] conjugates of bovine pancreatic ribonuclease (RNase A) inhibit growth of human melanoma in nude mice.

Recently hydrophilic poly[N-(2-hydroxypropyl)methacrylamide] (PHPMA) was used for BS-RNase modification to prevent its degradation in bloodstream or fast elimination. Polymer-conjugated BS-RNase preparations proved to be cytotoxic after intravenous or intraperitoneal application, whereas native BS-RNase was ineffective. Here RNase A unimer was conjugated with two HPMA polymers (classic and star) and their antitumor effects both in vitro and in vivo were compared with those of BS-RNase polymers. Surprisingly, the antitumor effect of RNase A conjugates was also pronounced. The RNase A conjugates (classic and star) injected intravenously to mice bearing melanoma tumor caused a significant reduction in tumor volume following ten doses of 5 and 1 mg/kg, respectively. Despite the antitumor activity observed in vivo, the in vitro tested cytotoxic activity of RNase A did not differ from that caused by native RNase A while native BS-RNase (50 microg/ml) totally inhibited DNA synthesis in treated cells. The experiments with 125I-labeled preparations demonstrated concentration-dependent internalization of native BS-RNase by tumor cells within an hour, whereas the polymer conjugate (S-BS) was not internalized. On the contrary, the in vivo experiments showed that whereas 40% of S-BS conjugate persisted in bloodstream for 24h after administration, 98% of the native BS-RNase was already eliminated. Improved antitumor activities of PHPMA-modified RNases in vivo might be ascribed to their prolonged retention in bloodstream, better proteolytic stability and resistance to the action of the ribonuclease inhibitor.

Polymer conjugated bovine seminal ribonuclease inhibits growth of solid tumors and development of metastases in mice.

Bovine seminal ribonuclease (BS-RNase) exerts a potent cytotoxic activity when administered intratumorally (i.t.) to the nude mice bearing human tumors. The ineffective treatment with intravenous (i.v.) or intraperitoneal (i.p.) administration led us to the synthesis of polymeric conjugates with BS-RNase to prevent it from degradation in the blood vessel. Hydrophilic poly[N-(2-hydroxypropyl)methacrylamide] (PHPMA) was used for BS-RNase modification and a PHPMA-BS-RNase conjugates were prepared. Classic conjugate (P-BS) with BS-RNase bound to the polymer by its oligopeptide site chains was prepared by aminolytic reaction of the polymer precursor bearing reactive ester groups situated in the side chains of polymer, while star-like conjugate (S-BS) was synthesized by the reaction of PHPMA containing end-chain reactive group with BS-RNase in aqueous buffer solution at pH 8. In contrast to the total ineffectiveness of free BS-RNase administered i.v. at a daily dose 10 mg/kg, application of P-BS and S-BS conjugates at doses 2 mg/kg and 0.5 mg/kg caused significant inhibition of the growth of human melanoma in nude mice. On the base of these results the effect of i.v. administered S-BS on the metastatic process and the survival of C57Bl/6 inbred mice inoculated with B16 melanoma cells was investigated. Sixty per cent of mice treated with S-BS (0.5 mg/kg/day) survived 100 days without metastatic foci when the experiment terminated. The average survival time of the treated groups was 75.5 days compared to 32.7 days in the control group. BS-RNase conjugated to water soluble polymers appears to be the first BS RNase preparation which exerts anticancer and antimetastatic activity following its intravenous administration.

Production of TGF-beta1 in lungs after low-dose whole-body radiation exposure in fibrosing (C57BL/6) and non-fibrosing (C3H/J) mouse strain.

The aim of the presented study was to observe acute and subacute discrete TGF-beta1 production after a low-dose whole-body radiation stimulus, known to induce thrombocytopenia. TGF-beta1 mRNA production and the number of thrombocytes was followed up in two mouse strains with different tendencies to the origination of fibroses. Mice of the C57BL/6 and C3H/J strains were exposed to a whole-body dose of 7 Gy. Non-irradiated mice of both strains were used as negative controls. The relative number of thrombocytes recorded in lung capillaries was significantly lower in both strains on day 9 after irradiation in comparison with controls. This finding was in accordance with a decrease in the number of thrombocytes in the peripheral blood in irradiated animals of both strains. On day 56 relative platelet counts reached physiological numbers in comparison to controls. On the other hand, TGF-beta1 mRNA production was higher in the C57BL/6 strain (on day 9) contrary to minimal production in the C3H/J strain (on day 9) or no production in both groups on day 56 and in controls. Thus, TGF-beta1 production without increased thrombocyte trapping in lung vessels in acute stage suggests that an additional mechanism is involved in low-dose radiation-induced cytokine synthesis in lung tissue besides the release of growth factors from thrombocytes.

Antitumor action of bovine seminal ribonuclease.

Unlike the bovine pancreatic ribonuclease (RNAase A), bovine seminal ribonuclease (BS RNAase) displays various biological activities including antitumor cytotoxicity. To learn more about its antitumor activity, we investigated BS RNAase effect on athymic nude mice bearing various tumors. BS RNAase (250 micrograms per mouse per day) was administered to the mice with prostate carcinoma for three weeks by three different routes (intraperitoneally--i.p., subcutaneously--s.c., and intratumorally-i.t.). Administration i.p. was ineffective, while s.c. administration reduced significantly size of tumors and i.t. administration abolished half of the tumors in treated mice. The i.t. administration of BS RNase to nude mice bearing melanoma showed even better results. Eighty % of mice were without tumors and in the other mice the tumors were significantly diminished. The best antitumor effect was obtained in case of seminoma. All mice bearing this tumor were cured after ten doses of BS RNAase.

Antitumor action of bovine seminal ribonuclease. Cytostatic effect on human melanoma and mouse seminoma.

This paper reports on the antitumor activity of BS RNase on human melanoma and mouse seminoma. Human melanoma cells established in culture were extremely susceptible to BS RNase, administered in concentrations ranging from 1-100 microg/ml. Concentrations of BS RNase over 10 microg/ml caused complete inhibition of cell growth. Bovine pancreatic ribonuclease (RNase A), a prototype of the ribonuclease superfamily, did not exert any effect under these conditions. Based on our previous results, athymic mice bearing human melanoma or mouse seminoma were treated with intratumoral administration of BS RNase (12.5 mg/kg b.w.). This dose was injected for five consecutive days excluding weekends. The intratumoral administration of BS RNase to nude mice bearing human melanoma showed a significant antitumor effect. There were no tumors seen in eighty percent of mice treated for three weeks, and tumors in the other mice diminished significantly. After some delay the tumors started to regrow. Prolonging of the treatment to five weeks had a similar effect. The effect of BS RNase on mouse seminoma was well pronounced. Five to seven doses of BS RNase were sufficient to eliminate tumors in all treated mice. However, as in the previous experiment, the growth of tumor tissue later reappeared.

A comparison of haemocoagulation tests in the experimental endotoxin model DIC and in rats whole-body irradiated by 250 Gy.

Based on comparing results of tests followed in the present work between the endotoxin model of the disseminated intravascular coagulation (DIC) and irradiated groups of rats we consider that after the whole-body irradiation by a high dose of 250 Gy, the DIC occurs, in spite of the fact that the first stage--hypercoagulation condition, can be hardly observed. In the experimental endotoxin model, an increase of activated partial thromboplastin test (APTT) values and prolongation of the thrombin time was observed up to 24 hours in both endotoxin doses. After both endotoxin doses, the fibrinogen level was transiently decreased with its subsequent increase. The fibrin monomers correspond to decreasing the fibrinogen level. After the first dose, they were positive between the 3rd and 12th hours and after the second dose, the positivity was observed 6 hours after the application. The antithrombin III level was decreased after 12 hours in both endotoxin doses. The thrombocyte count was considerably reduced already from the 6th hour after administering endotoxin to the end of the experiment. Considerable changes of the thrombocyte aggregation were observed only 3 hours after administering the second dose. When comparing the resulting values of these tests with values observed in irradiated animals, then we can see a certain agreement in the nature of the changes after the exposure to 250 Gy. The fibrinogen level was transiently decreased 3 hours after irradiation, when considerable changes in the thrombocyte aggregation also occurred.

[Changes in platelet aggregation and cAMP levels in isolated blood platelets in rats after 5.0 Gy and 10.0 Gy whole-body irradiation doses]. / Zmeny agregace krevních desticek a hladiny cAMP v izolovaných krevních destickách u celotelove ozárených potkanu dávkami 5.0 Gy a 10.0 Gy.

Rats were subjected to whole-body irradiation with doses of 5.0 Gy and 10.0 Gy. After intervals of 1.5 hours, 3, 6, 12 and 24 hours following irradiation the authors tested the aggregation capacity of thrombocytes after induction with adenosine--5--diphosphate and the cAMP level in isolated thrombocytes. Statistically significant differences between experimental and control groups were found particularly at some time intervals in rats irradiated with 10.0 Gy. The authors found a significant acceleration of the time of maximal aggregation at time intervals of 3.6 and 24 hours following irradiation with 10.0 Gy. Only at the time interval of 3 hours after irradiation with 10 Gy a statistically significant reduction of the maximum aggregation, as compared with the control group, occurred, and similarly the tang alpha value indicates that also the initial rate of aggregation was lower. The cAMP level in isolated thrombocytes was markedly reduced in particular after 5.0 Gy already during the early intervals after irradiation. A temporary increase of the cAMP levels was recorded 3 hours after irradiation with 10.0 Gy which may explain the reduced aggregation capacity of thrombocytes found during the same time interval. The results of both tests are not sufficient evidence of a hypercoagulation state after irradiation with the mentioned doses within 24 hours after irradiation.

[Review: heparin. 2. Effect on thrombocytes, vascular walls and fibrinolysis]. / Ubersicht: Das Heparin. 2. Beeinflussung der Thrombozyten, Gefässwand und Fibrinolyse.

In a brief survey the impact on thrombocyte functions and thrombocyte values caused by heparin and its fractions is reported, with the acute and immuno-allergic form of heparin-induced thrombopenia being widely dealt with. Furthermore, the relationship between heparin and vessel wall, particularly then its endothelial layer, is discussed and finally, its impact on the mechanisms of fibrinolysis is reported.

[Review: heparin. 1. Methods of determination and inhibitory mechanisms of some coagulation factors]. / Ubersicht: Das Heparin. 1. Bestimmungsverfahren und Hemmechanisme einzelner Gerinnungsfaktoren.

In a brief survey four groups of determining methods are reported for the purpose of monitoring the heparin therapy, with their advantages and disadvantages being assessed simultaneously. Emphasis is placed on the heterogeneity of heparin with approximately 120 fractions which are the cause of different manners of response and an impediment to the development of a rapid, reliable and economically advantageous method. Finally, all heparin factors in the plasma are enumerated and their mechanisms of action on individual coagulation factors are referred to.

[Hemostatic mechanisms and malignant tumors]. / Ubersicht. Blutstillungsmechanismen und maligne Geschwülste.

The mutual relationships between malignant tumours and mechanisms of blood coagulation are presented in a brief survey. In this connection, the mechanisms of a tumour cell entering the circulation through the vessel well and its leaving into the tissues are discussed, the theory of microtrauma being used for explaining these processes. Subsequently, the alterations to be found in the count and function of thrombocytes after contact with a malignant cell and the impact on this cell by blood platelets are represented. As a third factor the activation of blood coagulation which is exercised by substances with a procoagulatory effect produced by the malignant tissue and the frequently observed thrombosis in the course of neoplastic diseases are dealt with in connection with blood level changes of some coagulation factors. In a fourth section the significance of fibrinolysis, its activation and inhibition as well as the production of fibrinolytic activators by neoplasms are discussed.