Development of biomimetic phospholipid vesicle-based permeation assays (PVPA) as screening tool in drug development

Although several routes of administration can be utilized to bring a drug to the desired site of action, oral administration is still the most important and prevalent route of administration, due to its cost efficiency, convenience and patient compliance. A prerequisite for successful oral therapy is the ability of a drug to cross the gastrointestinal barrier. Over the past two decades, the number of new biological active chemical entities has increased due to the modern discovery programs, often based on combinatorial chemistry and high-throughput screening. Consequently, appropriate and reliable high-throughput in vitro models to assess the permeability of new drug candidates and drug formulations are required to increase the success rate and to reduce the time and cost for development. The phospholipid vesicle-based permeation assay (PVPA) is an in vitro permeability model consisting of a tight layer of liposomes immobilized on a filter that successfully has been used to test novel active substances and formulations. The first part of this thesis was to employ the PVPA for the first time as a screening tool to assess and improve the permeability of acyclovir (ACV), a poorly permeable model drug, by designing mucoadhesive liposomal formulations. The incorporation of ACV into liposomes resulted in a significant increase in the in vitro permeability of ACV, and mucoadhesive coating further enhanced the permeability for some of the formulations. The next part of this thesis was to develop a more robust, biomimetic PVPA with a lipid composition mimicking that of the intestinal barrier. The permeability values obtained showed that the positively charged basic compounds showed increased permeability through the negatively charged biomimetic PVPA compared to the original PVPA. The results from the model drugs also correlated well with in vivo on fractions absorbed in humans. Further, the charge in lipid composition resulted in a tremendously increase in barrier robustness in the presence of tensides compared with the original PVPA as well as improved storage stability for up to 6 months at -70oC. The biorelevance of the model was further improved by using biorelevant media. The biomimetic barrier was found to be compatible with fasted state and fed state simulated intestinal fluids (FaSSIF and FeSSIF). Four model drugs exhibited changes in permeability in the presence of the different simulated intestinal fluids in agreement with previous reports. Collectively, these findings moved the biomimetic PVPA an important step forward toward use as a better in vitro permeability model in drug development

Investigation and optimization of liposome formulation for use as drug carrier for the anticancer agent Camptothecin

In this thesis, the method development and investigation of different liposomal formulations to incorporate and retain Camptothecin (CPT) is described. CPT is a potent anticancer drug that has shown to be active against a broad spectrum of cancers. However, due to its challenging physicochemical properties, like poor water solubility, severe toxic effects to normal tissues and instability, its clinical development has been limited for nearly 40 years. A strategy to overcome CPT’s challenging properties is to use liposome-based carrier system. By taking advantage of this carrier system, we may solubilise CPT in the phospholipid bilayer of liposomes, protect it from blood proteins and achieve a selective drug accumulation in tumor tissues or tumor-associated cells by enhanced permeability and retention effect (EPR). A good liposome formulation of clinical utility must fulfil two important criteria. The liposomal drug carrier must incorporate CPT in the liposomal bilayer in a relevant therapeutic concentration and be able to retain the drug within the liposome to make it bioavailable at the target site after i.v. administration. The focus of this thesis was to study different liposomal formulations and their ability to incorporate and retain CPT. Screening of eight different liposome formulations with respect to association with CPT was performed. The 1,2-di-oleyl-3 trimethyl-ammonium-propane (DOTAP) containing formulations showed superior incorporation capacity, giving an CPT incorporation of 250 µg/130 µmoles lipid. The DOTAP containing formulations exhibited as well a trend toward higher retention ability in serum compared to the other formulations. Although they showed better retention ability, only 25 % of the drug was associated with the liposomes, which is far from being optimal. One of the important criteria mentioned above for liposomes as drug delivery systems is their ability to remain stable in blood circulation for prolonged time in order to reach the specific target and to avoid rapid clearance by RES after i.v. injection. To achieve this, PEG decoration on the liposome surface can be employed. We chose to PEGylate DOTAP formulations in order to get a better understanding of this system. PEGylation lead, as expected, to increased stability of the liposomes, however a reduced incorporation capacity was observed. The presence of 1 % and 10 % PEG gave better retention and slower leakage from the liposomes. We conclude that DOTAP inclusion in our liposomes increased the incorporation of CPT into the lipid bilayer, that liposomal retention in our current formulations must be improved, and while PEGylation is necessary in order to prevent rapid in vivo clearance, the inclusion of PEG reduces incorporation, and therefore further studies are needed in order to improve incorporation of CPT in PEGylated liposomes

Biomimetic PVPA in vitro model for estimation of the intestinal drug permeability using fasted and fed state simulated intestinal fluids

A prerequisite for successful oral drug therapy is the drug’s ability to cross the gastrointestinal barrier. Considering the increasing number of new chemical entities in modern drug discovery, reliable and fast in vitro models are required for early and efficient prediction of intestinal permeability. To mimic the intestinal environment, use of biorelevant media may provide valuable information on in vivo drug permeation. The present study aims at improving the novel biomimetic phospholipid vesicle-based permeation assay’s (PVPAbiomimetic) biorelevance by investigating the applicability of the biorelevant media; fasted state simulated intestinal fluid (FaSSIF) and fed state simulated intestinal fluid (FeSSIF). The FaSSIF and FeSSIF’s influence on the permeability of the model drugs acyclovir, indomethacin, griseofulvin and nadolol was then assessed. The barriers’ robustness in terms of storage stability was also evaluated. The barriers were found to maintain their integrity in presence of FaSSIF and FeSSIF. The model drugs showed changes in permeability in presence of the different simulated intestinal fluids that was in agreement with previous reports. Moreover, the barriers showed improved storage stability by maintaining its integrity for 6 months. Altogether, this study moves the PVPAbiomimetic an important step towards a better in vitro permeability model for use in drug development