Thromboembolic diseases are widespread in modern society because, as it is known, a number of factors increase the risk of their development. The number of patients gradually increases due to the aging of population, extending of treatment duration, and expansion of
indications. According to that, both anticoagulation therapy and prophylaxis represent an important part of the health care system and huge pharmaceutical market worldwide. Heparin is the oldest anticoagulant medicine that is still largerly involved in clinical practice. This is a large mucopolysaccharide molecule with a strong negative charge, and therefore its application is limited to the parenteral, intravenous (i.v.) or subcutaneous (s.c.) injections. Since the elimination half-life of heparin is short, it should be administered several times a day, which could be very uncomfortable for the patients. The main objective of the doctoral thesis is the development of an advanced delivery system for prolonged subcutaneous release of heparin, which would enable less frequent application, as well as an improved therapeutic outcome of the treatment - prolonged anticoagulant effect and reduced side effects. In our research, the design, preparation and in vitro and in vivo evaluation of the subcutaneous delivery systems for heparin based on poloxamers’ temperature-responsive hydrogels and pH-responsive chitosan nanocomplexes are presented. In the initial studies, we worked on the optimization of the preparation process and physico-chemical evaluation of polyelectrolyte complexes (PEC) from chitosan and heparin because their formation and stability are affected by many factors. PECs were prepared by spontaneous complexation of heparin and chitosan in various mass ratios. The influence of the dispersion medium pH on the formation and dissociation of complexes between countercharged polymers was thorougly examined. Our results indicate that both complexes’ size and zeta potential certainly depend on dispersion pH, and that pH can be controlled by the concentration and the ratio between polyelectrolytes. We found that mass ratio of 1:1 between heparin and chitosan at pH 5.2 is the most appropriate for the formation of small,
homogeneous and stable PEC. We also prepared and evaluated PEC between low molecular weight heparin (LMWH) and chitosan, and showed that 1:2 LMWH:chitosan mass ratio is required for the formation of small and stable complexes. In parallel with research on the PEC
we developed thermoresponsive poloxamer-based hydrogels, which exhibit sol to gel transition upon an increase in temperature, and are suitable for various ways of applications, especially for subcutaneous administration.. Thermoresponsive hydrogels were prepared from poloxamer 407 (P407), poloxamer 188 (P188), and hydroxypropyl methylcellulose (HPMC) in various ratios. In in vitro tests, the impact of each component on the decisive properties of a hydrogel was determined. It was demonstrated that with appropriate combination of polymers we are able to prepare a low viscous liquid at ambient temperature which transforms into hydrogel after subcutaneous application. We found that P188 increased gelation temperature and improved gel dissolution, but the addition of HPMC to basic P407 formulations lowered gelation temperature and prolonged gel dissolution. Heparin/chitosan PEC were incorporated into thermoresponsive gelling systems and therefore, heparin release was prolonged (18 days), while other properties of thermoresponsive system remain unchanged. In further studies our in vitro results were upgraded with cytotoxicity testing of selected formulations in keratinocyte cell line, followed by a central part of the doctoral research, focused on their in vivo evaluation in animals. Cytotoxicity tests demonstrated good cell proliferation after 24 h and 48 h long exposure to heparin/chitosan PEC and thermoresponsive hydrogels. In vivo test was performed in healthy rats, to which heparin plasma concnetrationtime profiles were determined after subcutaneous administration of tested formulations. The results demonstrated longer duration of heparin in blood (5 days) after thermoresponsive hydrogel with nanocomplexes was administered. We have also shown that heparin absorption rate constant from thermoresponsive hydrogel with nanocomplexes was the lowest. The results of in vivo study have proved that a delivery system consisting of thermoresponsive hydrogel with heparin/chitosan nanocomplexes’ dispersion can enable less frequent drug
administration, which is of great importance especially during the longlasting prophylactic treatment of thromboembolic diseases. In the last part of the doctoral dissertation, we successfully developed and validated a novel size-exclusion chromatographic method for the determination of heparin in pharmaceuticals. The research work undoubtly showed that the combination of thermoresponsive hydrogels and pH-responsive nanocomplexes represented a promising subcutaneous delivery system for heparin and LMWH. We proved that biocompatible and injectable dually-responsive system can be prepared by proper polymers’ selection and their ratios. We believe that the results of our research contribute new knowledge in the field of in situ gelling, smart delivery systems, and that their use would possibly enable more comfortable treatment to chronic patients.
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