| Popis: |
Tromboembolicne bolezni so v sodobni družbi zelo razširjene, kajti znano je, da številni dejavniki vecajo tveganje za nastanek le-teh. Število pacientov iz leta v leto narašca zaradi staranja prebivalstva, podaljševanja trajanja zdravljenja in širjenja indikacij. Heparin je najstarejše antikoagulantno zdravilo, ki pa je še danes v veliki meri vkljuceno v klinicno prakso. To je velika mukopolisaharidna molekula z mocnim negativnim nabojem, zato je njegova aplikacija omejena na parenteralno, v obliki intravenskih (i.v.) ali subkutanih (s.c.) injekcij. Ker je razpolovni cas eliminacije heparina kratek, ga je potrebno dajati veckrat na dan, kar je s stališca pacienta zelo neugodno. Osrednji cilj doktorskega dela predstavlja razvoj naprednega dostavnega sistema za subkutano podaljšano sprošcanje heparina, ki bi omogocil manj pogosto aplikacijo, kot tudi izboljšan terapevtski izid zdravljenja. V naših raziskavah predstavljamo nacrtovanje, izdelavo ter in vitro in in vivo vrednotenje subkutanih dostavnih sistemov heparina, ki temeljijo na temperaturno-odzivnih hidrogelih na osnovi poloksamerov in pH-odzivnih hitosanskih nanokompleksih. V zacetnih raziskavah smo se ukvarjali z optimiziranjem postopka izdelave in s fizikalnokemijskim vrednotenjem polielektrolitnih kompleksov (PEK) heparina in hitosana, kajti na njihov nastanek in stabilnost vplivajo številni dejavniki. PEK smo izdelali s spontanim združevanjem heparina in hitosana v razlicnih masnih razmerjih. Natancno smo proucili vpliv pH vrednosti disperznega medija na tvorbo in razgradnjo kompleksov med nasprotno nabitima polimeroma. Naši rezultati kažejo, da sta tako velikost nastalih kompleksov kot zeta potencial nedvomno odvisna od pH disperznega medija, ter da nanj lahko vplivamo s spremembo koncentracije in razmerja med polielektrolitoma. Ugotovili smo, da je za tvorbo majhnih, homogenih in stabilnih PEK najbolj primerno masno razmerje 1:1 med nefrakcioniranim heparinom in hitosanom pri pH 5,2. Prav tako smo izdelali in ovrednotili PEK med nizkomolekularnim heparinom (NMH) in hitosanom ter pokazali, da je za tvorbo majhnih in stabilnih kompleksov potrebno masno razmerje NMH:hitosan 1:2. Vzporedno z raziskavami na PEK smo razvijali termoodzivne hidrogele na osnovi poloksamerov, ki izkazujejo prehod iz sol v gel stanje pri povišanju temperature in so primerni za razlicne nacine aplikacij, predvsem pa za subkutano dajanje. Termoodzivne hidrogele smo razvili iz poloksamera 407 (P407), oloksamera 188 (P188) in hidroksipropilmetilceluloze (HPMC) v razlicnih razmerjih. Z in vitro testi smo opredelili vpliv posameznih sestavin na odlocilne lastnosti hidrogelov ter pokazali, da je z ustrezno izbiro polimerov mogoce doseci formulacijo, ki bo na sobni temperaturi še tekoca in se bo lahko injicirala, po subkutani aplikaciji pa bo pod kožo nastal hidrogel. Ugotovili smo, da P188 zvišuje tocko geliranja in pospešuje raztapljanje gela, ce pa osnovnim P407 formulacijam dodamo HPMC, le-te gelirajo pri nižjih temperaturah, njihovo raztapljanje pa traja dalj casa. Najbolj primerne PEK heparina in hitosana s stališca velikosti in stabilnosti smo nato vgradili v termoodzivne gelirajoce sisteme in dokazali, da lahko bistveno podaljšamo cas sprošcanja heparina (18 dni), medtem ko druge lastnosti termoodzivnega sistema ostanejo nespremenjene. V nadaljevanju doktorskega dela smo naše ugotovitve in vitro raziskav nadgradili s proucevanjem citotoksicnosti izbranih formulacij na celicni liniji keratinocitov, temu pa je sledil osrednji del naših raziskav, usmerjen v in vivo testiranje na živalih. Testi citotoksicnosti so pokazali, da disperzije nanokompleksov nefrakcioniranega heparina ali NMH in hitosana ter formulacije termoodzivnih hidrogelov ne zavirajo celicne proliferacije po 24 h in 48 h izpostavitvi. In vivo študijo smo izvedli na štirih skupinah podgan, katerim smo dolocili potek plazemskih koncentracij heparina skozi cas po subkutani aplikaciji testiranih formulacij. Rezultati poskusov na živalih so pokazali daljšo prisotnost heparina v krvi (5 dni) po dajanju termoodzivnega hidrogela z hitosanskimi nanokompleksi. Absorpcija heparina iz termoodzivnega hidrogela z nanokompleksi je bila najpocasnejša med testiranimi formulacijami. Na ta nacin smo dokazali, da dostavni sistem, sestavljen iz poloksamernega termoodzivnega hidrogela in disperzije nanokompleksov heparina in hitosana lahko omogoci manj pogosto aplikacijo zdravila, kar je izrednega pomena predvsem pri dolgotrajnem profilakticnem zdravljenju tromboembolicnih bolezni. V zadnjem delu doktorske disertacije smo predstavili izkljucitveno kromatografsko metodo za kvantitativno dolocanje heparina v farmacevtskih izdelkih, ki smo jo kot prvi uspešno razvili in validirali. Raziskovalno delo v okviru doktorske disertacije je nedvomno pokazalo, da kombinacija termoodzivnih hidrogelov in pH-odzivnih nanokompleksov predstavlja obetaven subkutani dostavni sistem za heparin in NMH. Dokazali smo, da lahko z ustrezno izbiro sestavin in njihovih deležev pripravimo kombiniran dvojno-odzivni dostavni sistem, ki je varen in primeren za enostavno subkutano aplikacijo. Menimo, da rezultati naših raziskav prispevajo nova znanja na podrocju in situ gelirajocih, pametnih dostavnih sistemov, ter da bi z uporabo le-teh lahko omogocili kronicnim bolnikom prijaznejše zdravljenje. 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. |
