Development of an in vitro release test for in situ gelling ophthalmic formulation

Cilj ovog diplomskog rada bio je razviti diskriminativnu metodu za ispitivanje oslobađanja lijeka iz inovativnog in situ gelirajućeg pripravka za oftalmičku primjenu. Kao aparatura za ispitivanje oslobađanja in vitro odabrana je vertikalna difuzijska ćelija. Za potrebe rada pripremljene su i četiri formulacije s timolol maleatom koje se temelje na poloksamerima P407 i P188 kao pomoćnim tvarima koje određuju temperaturu geliranja te kitozanu koji poboljšava mehaničku čvrstoću nastalog gela. Kako bi se odabrao medij za ispitivanje brzine oslobađanja, ispitana je topljivost timolol maleata koristeći tri medija, simulirani suzni fluid, Krebs-Ringerov pufer i fosfatni pufer. Nije bilo značajne razlike u topljivosti timolol maleata u ispitivanim medijima, pa je kao medij odabran simulirani suzni fluid, zbog najbolje simulacije uvjeta in vivo. Za odabir membrane za ispitivanje brzine oslobađanja ispitana je adsorpcija timolola i otpor difuziji koristeći pet membrana koje su se razlikovale po svom sastavu i veličini pora. Optimalnom se pokazala membrana izrađena od celuloza acetata, veličine pora 0,8 μm. Kao analitička metoda korištena je ultravisoko-učinkovita tekućinska kromatografija. U sklopu validacije analitičke metode potvrđena je njena specifičnost, linearnost i točnost za timolol maleat u rasponu koncentracija od 0,5 μg/mL do 516,9 μg/mL. Za provjeru diskriminatornosti razvijene metode oslobađanja lijeka in vitro analizirane su četiri formulacije koje se razlikuju po količini pomoćnih tvari ili količini djelatne tvari. Provedene analize pokazale su da je razvijena metoda sposobna detektirati razlike u formulaciji, pa može biti koristan alat u razvoju formulacije inovativnog in situ gelirajućeg pripravka timolol maleata za oftalmičku primjenu.The aim of this Diploma thesis was to develop a discriminative method for testing drug release from the innovative in situ gelling formulation for ophthalmic administration. The vertical diffusion cell was selected as the apparatus for in vitro release assay. Four formulations with timolol maleate based on poloxamers P407 and P188, as excipients that control the temperature of gelation, and chitosan, which improves the mechanical strength of the resulting gel. In order to choose a release medium, the solubility of timolol maleate was tested using three media, a simulated tear fluid, Krebs-Ringer buffer and phosphate buffer. There was no significant difference in the solubility of timolol maleate in tested media, so the simulated tear fluid was selected because of the best simulation of in vivo conditions. To select a membrane for in vitro release assay, we tested the timolol adsorption and diffusional resistance using five membranes which varied in their composition and pore size. The optimal mebrane was shown to be the one made of cellulose acetate with pore size of 0.8 μm. The ultra-performance liquid chromatography was used as an analytical method. The validation of the analytical method was confirmed in terms of its specificity, linearity and accuracy for timolol maleate in a concentration range of 0.5 mg/mL to 516.9 mg/mL. To check the discriminatory of developed method for in vitro drug release, we analyzed four formulations differing in the amount of excipients or active ingredient. These analyses showed that the developed method is able to detect differences in the formulation, so it may be a useful tool in the development of formulation of innovative in situ gelling formulation containing timolol maleate for ophthalmic administration