In vitro lipolysis assay as a prognostic tool for the development of lipid based drug delivery systems D i s s e r t a t i o n zur Erlangung des akademischen Grades Doctor rerum naturalium (Dr. rer. nat.) vorgelegt der Naturwissenschaftlichen Fakultät I Biowissenschaften der Martin-Luther-Universität Halle-Wittenberg von Frau Sandra Klein geb. am 24.02.1982 in Halle (Saale) Gutachter 1. Prof. Dr. rer. nat. Karsten Mäder 2. Prof. Dr. rer. nat. Markus Pietzsch 3. Prof. Dr. rer. nat. Thomas Rades Datum der öffentlichen Verteidigung: 30.01.2013 I Content I Introduction.................................................................................................................... 1 II Scientific background..................................................................................................... 4 1 Formulation strategies for poorly soluble drugs.......................................................... 4 1.1 Salt formation..................................................................................................... 4 1.2 Inclusion in cyclodextrins.................................................................................... 5 1.3 Micronisation...................................................................................................... 5 1.4 Nanosuspensions............................................................................................... 6 1.5 Solid dispersions................................................................................................ 8 1.6 Lipid formulations............................................................................................... 9 2 Lipid digestion and its impact on drug absorption......................................................12 2.1 Biochemistry of fat digestion .............................................................................12 2.2 Influence of fat digestion on drug absorption.....................................................18 3 Assessment of lipid formulations...............................................................................19 3.1 Dispersion testing..............................................................................................19 3.2 Simulating fat digestion: In vitro lipolysis assay .................................................20 III Materials and Methods .................................................................................................25 1 Materials...................................................................................................................25 2 Methods....................................................................................................................26 2.1 Dispersion experiments.....................................................................................26 2.2 In vitro digestion experiments............................................................................27 2.3 pH-stat titration..................................................................................................29 2.4 Lipid analyses by high performance thin layer chromatography (HPTLC) .........30 IV Results and discussion.................................................................................................33 1 Influence of digestion on lipid based formulations: Impact of formulation type and triglyceride source ............................................................................................................33 1.1 Introduction.......................................................................................................33 1.2 Materials and methods......................................................................................36 1.3 Results and Discussion.....................................................................................38 1.4 Conclusion ........................................................................................................51 2 Susceptibility of surfactants towards pancreatic digestion.........................................53 2.1 Introduction.......................................................................................................53 2.2 Materials and Methods......................................................................................54 2.3 Results and discussion......................................................................................59 2.4 Conclusion ........................................................................................................70 3 Susceptibility of sucrose ester formulations towards pancreatic enzyme-mediated digestion...........................................................................................................................71 II 3.1 Introduction.......................................................................................................71 3.2 Materials and methods......................................................................................73 3.3 In vitro digestion experiments............................................................................74 3.4 Dissolution of Ibuprofen sustained release tablets in biorelevant media............75 3.5 Results and Discussion.....................................................................................77 3.6 Conclusion ........................................................................................................85 4 Modelling gastric fat digestion: Development of a gastric lipolysis assay ..................86 4.1 Introduction.......................................................................................................86 4.2 Scientific background........................................................................................87 4.3 Materials and methods......................................................................................91 4.4 Results and Discussion.....................................................................................96 4.5 Conclusion ......................................................................................................103 V Summary and perspectives ........................................................................................105 III List of abbreviations API active pharmaceutical ingredient BCS biopharmaceutical classification system BA bile acid BS bile salt CEH carboxyl ester hydrolase CMC critical micellar concentration DAG(s) diacylglyceride(s) DG(s) diglyceride(s) ER endoplasmatic reticulum EQ. equation FA(s) fatty acid(s) FaSSIF fasted state simulated intestinal fluid FaSSGF fasted state simulated gastric fluid FeSSIF fed state simulated intestinal fluid FeSSGF fed state simulated gastric fluid GC gas chromatography GI gastrointestinal GRAS generally regarded as safe HGL human gastric lipase HLB hydrophilic-lipophilic balance HPL human pancreatic lipase HTS high throughput screening HPLC high performance liquid chromatography HPTLC high performance thin-layer chromatography LC long chain LCS lipid classification system LCT long chain triglyceride LD laser diffraction LFCS lipid formulation classification system LP lipoprotein MAG(s) monoacylglyceride(s) MC medium chain MCT medium chain triglyceride MG(s) monoglyceride(s) PCS photon correlation spectroscopy IV PDI polydispersity index PEG polyethylene glycol P-gp P-glycoprotein PLRP2 pancreatic lipase-related protein 2 PL phospholipids PPL porcine pancreatic lipase PSD particle size distribution rDGL recombinant dog gastric lipase SD standard derivation SE sucrose ester SEDDS self-emulsifying drug delivery systems SIF simulated intestinal fluid SLS sodium lauryl sulphate SEDDS self-emulsifying drug delivery systems SMEDDS self-microemulsifying drug delivery systems SMEDDS self-nanoemulsifying drug delivery systems TAG(s) triacylglyceride(s) TBU tributyrin unit TIM TNO intestinal model THL Tetrahydrolipstatin (Orlistat) TG(s) triglyceride(s) USP United States Pharmacopeia V Introduction III Introduction The advances in automated synthesis, combinatorial chemistry and innovative high- throughput screening have led to an increasing number of drug candidates which are characterised by high molecular weight, unchanged H-bonding properties, higher lipophilicity and, hence, poor aqueous solubility 1. The proportion of new chemical entities (NCEs) with poor solubility in water is estimated to be more than 40% as these drug candidates exert their pharmacological action in or at biological membranes or membrane associated proteins 2. This challenges drug delivery institutions in industry or academia to develop carrier systems for the optimal oral and parenteral administration of these drugs. According to the Biopharmaceutical Classification system (BCS), drugs are classified into four categories based on their aqueous solubility and ability to permeate biological membranes (Figure 1)3. A drug is considered as ‘highly soluble’ when the highest dose strength is soluble in < 250 ml water over a pH range of 1-7.5. A compound with 90% oral bioavailability is considered as ‘highly permeable’. Drugs with reasonable membrane permeability but poor aqueous solubility belong to class II of the BCS. For these drugs, the dissolution is often the rate-limiting step of absorption. The bioavailability from conventional drug delivery systems like tablets might be unacceptable but an appropriate formulation design can help to improve the dissolution step and, thereby, enhance their oral bioavailability. For drugs with poor solubility and poor permeability (belonging to class IV of the BCS), the best option to improve