Background. Electrochemotherapy is a tumour ablation modality, based on electroporation of the cell membrane, allowing non-permeant anticancer drugs to enter the cell, thus augmenting their ...cytotoxicity by orders of magnitude. In preclinical studies, bleomycin and cisplatin proved to be the most suitable for clinical use. Intravenous administration of cisplatin for electrochemotherapy is still not widely accepted in the clinics, presumably due to its lower antitumor effectiveness, but adjuvant therapy by immunomodulatory or vascular-targeting agents could provide a way for its potentiation. Hence, the aim of the present study was to explore the possibility of adjuvant tumour necrosis factor α (TNF-α) therapy to potentiate antitumor effectiveness of electrochemotherapy with intravenous cisplatin administration in murine sarcoma.
Materials and methods. In vivo study was designed to evaluate the effect of TNF-α applied before or after the electrochemotherapy and to evaluate the effect of adjuvant TNF-α on electrochemotherapy with different cisplatin doses.
Results. A synergistic interaction between TNF-α and electrochemotherapy was observed. Administration of TNF-α before the electrochemotherapy resulted in longer tumour growth delay and increased tumour curability, and was significantly more effective than TNF-α administration after the electrochemotherapy. Tumour analysis revealed increased platinum content in tumours, TNF-α induced blood vessel damage and increased tumour necrosis after combination of TNF-α and electrochemotherapy, indicating an anti-vascular action of TNF-α. In addition, immunomodulatory effect might have contributed to curability rate of the tumours.
Conclusion. Adjuvant intratumoural TNF-α therapy synergistically contributes to electrochemotherapy with intravenous cisplatin administration. Due to its potentiation at all doses of cisplatin, the combined treatment is predicted to be effective also in tumours, where the drug concentration is suboptimal or in bigger tumours, where electrochemotherapy with intravenous cisplatin is not expected to be sufficiently effective.
Skeletal muscle is an attractive target tissue for delivery of therapeutic genes, since it is well vascularized, easily accessible, and has a high capacity for protein synthesis. For efficient ...transfection in skeletal muscle, several protocols have been described, including delivery of low voltage electric pulses and a combination of high and low voltage electric pulses. The aim of this study was to determine the influence of different parameters of electrotransfection on short-term and long-term transfection efficiency in murine skeletal muscle, and to evaluate histological changes in the treated tissue. Different parameters of electric pulses, different time lags between plasmid DNA injection and application of electric pulses, and different doses of plasmid DNA were tested for electrotransfection of tibialis cranialis muscle of C57Bl/6 mice using DNA plasmid encoding green fluorescent protein (GFP). Transfection efficiency was assessed on frozen tissue sections one week after electrotransfection using a fluorescence microscope and also noninvasively, followed by an in vivo imaging system using a fluorescence stereo microscope over a period of several months. Histological changes in muscle were evaluated immediately or several months after electrotransfection by determining infiltration of inflammatory mononuclear cells and presence of necrotic muscle fibers. The most efficient electrotransfection into skeletal muscle of C57Bl/6 mice in our experiments was achieved when one high voltage (HV) and four low voltage (LV) electric pulses were applied 5 seconds after the injection of 30 μg of plasmid DNA. This protocol resulted in the highest short-term as well as long-term transfection. The fluorescence intensity of the transfected area declined after 2–3 weeks, but GFP fluorescence was still detectable 18 months after electrotransfection. Extensive inflammatory mononuclear cell infiltration was observed immediately after the electrotransfection procedure using the described parameters, but no necrosis or late tissue damage was observed. This study showed that electric pulse parameters, time lag between the injection of DNA and application of electric pulses, and dose of plasmid DNA affected the duration of transgene expression in murine skeletal muscle. Therefore, transgene expression in muscle can be controlled by appropriate selection of electrotransfection protocol.
Background. Electrochemotherapy is a tumor ablation modality, based on electroporation of the cell membrane, allowing non-permeant anticancer drugs to enter the cell, thus augmenting their ...cytotoxicity by orders of magnitude. In preclinical studies, bleomycin and cisplatin proved to be the most suitable for clinical use. Intravenous administration of cisplatin for electrochemotherapy is still not widely accepted in the clinics, presumably due to its lower antitumor effectiveness, but adjuvant therapy by immunomodulatory or vascular-targeting agents could provide a way for its potentiation. Hence, the aim of the present study was to explore the possibility of adjuvant tumor necrosis factor α (TNF-α) therapy to potentiate antitumor effectiveness of electrochemotherapy with intravenous cisplatin administration in murine sarcoma. Materials and methods. In vivo study was designed to evaluate the effect of TNF-α applied before or after the electrochemotherapy and to evaluate the effect of adjuvant TNF-α on electrochemotherapy with different cisplatin doses. Results. A synergistic interaction between TNF-α and electrochemotherapy was observed. Administration of TNF-α before the electrochemotherapy resulted in longer tumor growth delay and increased tumor curability, and was significantly more effective than TNF-α administration after the electrochemotherapy. tumor analysis revealed increased platinum content in tumors, TNF-α induced blood vessel damage and increased tumor necrosis after combination of TNF-α and electrochemotherapy, indicating an anti-vascular action of TNF-α. In addition, immunomodulatory effect might have contributed to curability rate of the tumors. Conclusion. Adjuvant intratumoural TNF-α therapy synergistically contributes to electrochemotherapy with intravenous cisplatin administration. Due to its potentiation at all doses of cisplatin, the combined treatment is predicted to be effective also in tumors, where the drug concentration is suboptimal or in bigger tumors, where electrochemotherapy with intravenous cisplatin is not expected to be sufficiently effective.
The design, synthesis and biological activity of new thrombin inhibitors with a pyridinone or pyrazinone core and different heterobicyclic P
1 arginine side-chain mimetics are described. The arginine ...side-chain mimetics used in this study are (±)-4,5,6,7-tetrahydro-2
H-indazol-5-ylmethanamine and both enantiomers thereof, (±)-4,5,6,7-tetrahydro-1,3-benzothiazole-2,6-diamine and the corresponding
R enantiomer. Compound
25, the most potent in the series of pyrazinone inhibitors, exhibited a K
i of 41 nM
in vitro and high selectivity against trypsin and factor Xa.
The optimization of 3-amino-2-pyridinone acetamide thrombin inhibitors incorporating weakly basic heterobicyclic P
1-arginine side-chain mimetics is described.
Optimization of lead compounds
1 and
2 ...resulted in novel, selective, and potent thrombin inhibitors incorporating weakly basic heterobicyclic P
1-arginine mimetics. The design, synthesis, and biological activity of racemic thrombin inhibitors
17–
29 and enantiomerically pure thrombin inhibitors
30–
33 are described. The arginine side-chain mimetics used in this study are 4,5,6,7-tetrahydro-1,3-benzothiazol-2-amine, 4,5,6,7-tetrahydro-2
H-indazole, and 2-imino-4,5,6,7-tetrahydro-1,3-benzothiazol-3(2
H)-ylamine.
Endoglin is a transforming growth factor- beta (TGF- beta ) co-receptor that participates in the activation of a signaling pathway that mediates endothelial cell proliferation and migration in ...angiogenic tumor vasculature. Therefore, silencing of endoglin expression is an attractive approach for antiangiogenic therapy of tumors. The aim of our study was to evaluate the therapeutic potential of small interfering RNA (siRNA) molecules against endoglin in vitro and in vivo. Therapeutic potential in vitro was assessed in human and murine endothelial cells (HMEC-1, 2H11) by determining endoglin expression level, cell proliferation and tube formation. In vivo, the therapeutic potential of siRNA molecules was evaluated in TS/A mammary adenocarcinoma growing in BALB/c mice. Results of our study showed that siRNA molecules against endoglin have a good antiangiogenic therapeutic potential in vitro, as expression of endoglin mRNA and protein levels in mouse and human microvascular endothelial cells after lipofection were efficiently reduced, which resulted in the inhibition of endothelial cell proliferation and tube formation. In vivo, silencing of endoglin with triple electrotransfer of siRNA molecules into TS/A mammary adenocarcinoma also significantly reduced the mRNA levels, number of tumor blood vessels and the growth of tumors. The obtained results demonstrate that silencing of endoglin is a promising antiangiogenic therapy of tumors that could not be used as single treatment, but as an adjunct to the established cytotoxic treatment approaches.
The design, synthesis and biological activity of new thrombin inhibitors with a pyridinone or pyrazinone core and different heterobicyclic P(1) arginine side-chain mimetics are described. The ...arginine side-chain mimetics used in this study are (+/-)-4,5,6,7-tetrahydro-2H-indazol-5-ylmethanamine and both enantiomers thereof, (+/-)-4,5,6,7-tetrahydro-1,3-benzothiazole-2,6-diamine and the corresponding R enantiomer. Compound 25, the most potent in the series of pyrazinone inhibitors, exhibited a K(i) of 41 nM in vitro and high selectivity against trypsin and factor Xa.
Optimization of lead compounds 1 and 2 resulted in novel, selective, and potent thrombin inhibitors incorporating weakly basic heterobicyclic P(1)-arginine mimetics. The design, synthesis, and ...biological activity of racemic thrombin inhibitors 17-29 and enantiomerically pure thrombin inhibitors 30-33 are described. The arginine side-chain mimetics used in this study are 4,5,6,7-tetrahydro-1,3-benzothiazol-2-amine, 4,5,6,7-tetrahydro-2H-indazole, and 2-imino-4,5,6,7-tetrahydro-1,3-benzothiazol-3(2H)-ylamine.
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