Amphotericin B Nano-Formulations: Development, characterisation and suitability for oral administration Dissertation zur Erlangung des Grades "Doktor der Naturwissenschaften" im Promotionsfach Pharmazie am Fachbereich Chemie, Pharmazie und Geowissenschaften der Johannes Gutenberg-Universität in Mainz Raphael Johnson geb. in Obuasi, Ghana Mainz, 2015 DEKAN: 1. Gutachter: 2. Gutachter: Tag der mündlichen Prüfung: 11/12/2015 Dedication i Acknowledgement ACKNOWLEDGEMENT ii Table of Contents TABLE OF CONTENTS Abbreviations viii 1.0 Introduction…………………………………………………………………. 1 1.1 Oral drug absorption………………………………………………………….. 1 1.2 Factors affecting oral drug absorption………………………………………... 1 1.2.1 Physiological factors………………………………………………………….. 1 1.2.2 Physicochemical factors………………………………………………………. 2 1.2.3 Biopharmaceutical factors……………………………………………………. 2 1.3 Oral drug delivery systems of poorly water soluble drugs…………………… 3 1.3.1 Liposomes…………………………………………………………………….. 4 1.3.1.1 Classification of liposomes…………………………………………………... 4 1.3.1.2 Methods of liposome preparation……………………………………………. 5 1.3.1.3 Multilamellar vesicles (MLV)………………………………………………... 5 1.3.1.4 Unilamellar vesicles (SUV and LUV)……………………………………….. 5 1.3.1.5 Applications of liposomes in drug delivery………………………………….. 5 1.3.1.6 Limitations of liposome technology………………………………………….. 6 1.4 Self-emulsifying drug delivery systems (SEDDS)…………………………… 6 1.4.1 Components of SEDDS Formulation…………………………………………. 7 1.4.1.1 Oil phase……………………………………………………………………… 7 1.4.1.2 Surfactants…………………………………………………………………….. 8 1.4.1.2 Co-surfactants/Co-solvents…………………………………………………… 8 1.5 Amphotericin B……………………………………………………………….. 9 1.5.1 Therapeutic indication, therapeutic index and toxicity……………………….. 9 1.5.2 Chemical properties…………………………………………………………... 10 1.5.3 Solubility……………………………………………………………………… 10 1.5.4 Aggregation…………………………………………………………………… 12 1.5.5 pKa……………………………………………………………………………. 13 1.5.6 Partition coefficient…………………………………………………………… 13 1.5.7 Pharmacokinetic properties…………………………………………………… 13 1.5.7.1 Absorption and permeability………………………………………………….. 13 1.5.7.2 Distribution…………………………………………………………………… 13 1.5.7.3 Metabolism and excretion…………………………………………………….. 13 1.5.7.4 Commercial formulations…………………………………………………….. 13 iii Table of Contents 1.6 Scattering techniques for structural investigations…………………………… 15 1.6.1 Dynamic light scattering……………………………………………………… 15 1.6.1.1 Theory of dynamic light scattering…………………………………………… 15 1.6.1.2 Small angle scattering (SAS)…………………………………………………. 16 1.6.1.3 The scattering vector………………………………………………………….. 17 1.6.1.4 The contrast term……………………………………………………………... 18 2.0 Aim of thesis…………………………………………………………………. 20 3.0 Materials and methods……………………………………………………… 21 3.1 Materials………………………………………………………………………. 21 3.1.1 Materials for HST preparation………………………………………………... 21 3.1.2 Materials for liposome preparation…………………………………………… 21 3.1.3 Materials for Self-emulsifying system (SEDDS)……………………………... 21 3.1.4 Cell culture……………………………………………………………………. 22 3.1.5 Chemicals and solvents……………………………………………………….. 23 3.1.6 General equipment and disposables…………………………………………... 24 3.1.7 Data processing……………………………………………………………….. 26 3.2 Methods……………………………………………………………………….. 26 3.2.1 Formulation of AmB-HST……………………………………………………. 26 3.2.2. Solubility of Amphotericin B in intestinal model media FaSSIF-C………….. 26 3.2.3 Manufacture of liposomes…………………………………………………….. 27 3.2.3.1 Preparation of multilamellar liposomes………………………………………. 27 3.2.3.2 Preparation of small unilamellar liposomes for contrast variation experiment. 27 3.2.4 Preparation of self-emulsifying drug delivery system………………………... 28 3.2.4.1 Solubility of Amphotericin B in excipients…………………………………... 28 3.2.4.2 Preparation of ternary phase diagram………………………………………… 28 3.2.4.3 Preparation of drug containing pre-concentrate………………………………. 28 3.2.5 Freeze drying of samples……………………………………………………... 28 3.2.6 Characterization of formulations……………………………………………... 29 3.2.6.1 Drug content…………………………………………………………………... 29 3.2.6.2 Drug entrapment in liposomes………………………………………………... 29 3.2.6.3 Size measurements……………………………………………………………. 29 3.2.6.4 Emulsification properties of nanoemulsion…………………………………... 30 3.2.6.5 Precipitation study of nanoemulsion………………………………………….. 30 3.2.6.6 Microscopy……………………………………………………………………. 30 iv Table of Contents 3.2.6.7 UV-Visible spectrum…………………………………………………………. 30 3.2.6.8 Fluorescence spectrum of Amphotericin B…………………………………… 31 3.2.7 Differential scanning calorimetry (DSC)……………………………………... 31 3.2.8 Fourier transform infrared spectroscopy (ATR-FTIR)……………………….. 31 3.2.9 Density gradient centrifugation……………………………………………….. 31 3.2.10 Culture of Caco-2 cells……………………………………………………….. 31 3.2.10.1 Cytotoxicity assay…………………………………………………………….. 32 3.2.10.2 Uptake study of liposomes……………………………………………………. 32 3.3 Hemolysis study………………………………………………………………. 32 3.4 In vitro release of Amphotericin B from liposomes………………………...... 33 3.4.1 In vitro release study of Amphotericin B from AmB-HST…………………... 34 3.5 Rheological properties of nanoemulsion pre-concentrates…………………… 34 3.6 Stability study of nanoemulsion pre-concentrates……………………………. 34 3.7 Small angle neutron scattering (SANS) experiments………………………… 35 3.7.1 Contrast variation method for estimation of core-shell structure of AmB-HST 35 3.7.2 Contrast variation for evaluation of equivalent scattering length density……. 35 3.7.3 Determination of internal structure of freeze-dried (final product) liposome…. 36 3.7.4 Estimation of the structure of the nanoemulsions by SANS………………….. 37 3.7.5 Statistical analysis…………………………………………………………….. 37 4.0 Results………………………………………………………………………... 38 4.1 AmB-HST formulation……………………………………………………….. 38 4.1.1 Formulation of AmB-HST……………………………………………………. 38 4.1.2 UV-Visible spectra of Amphotericin B………………………………………. 39 4.1.3 Fluorescence spectrum of Amphotericin B…………………………………… 39 4.1.4 Microscopy of AmB-HST…………………………………………………….. 40 4.1.5 Particle size of formulation…………………………………………………… 41 4.1.6 Small angle scattering of AmB-HST…………………………………………. 42 4.1.7 In vitro release of Amphotericin B from AmB-HST…………………………. 43 4.2 Results of solubility of Amphotericin B in FaSSIF-C………………………... 44 4.3 Results of multilamellar liposomes formulation……………………………… 45 4.3.1 Drug content, entrapment and particle size of liposomes…………………….. 45 4.3.2 Microscopy of liposomes……………………………………………………... 48 4.3.3 Spectrum of Amphotericin B in liposomes…………………………………… 48 4.3.4 DSC of Amphotericin B, liposomes and blanks……………………………… 49 v Table of Contents 4.3.5 FTIR of liposomes and their physical mixtures………………………………. 50 4.3.6 Density gradient centrifugation of liposomes………………………………… 51 4.3.7 Cytotoxicity test of liposomes………………………………………………… 52 4.3.8 Liposomal Amphotericin B uptake study…………………………………….. 53 4.3.9 In vitro release study of amphotericin B from liposomes…………………….. 54 4.3.10 Hemolysis test of liposomes………………………………………………….. 55 4.4 Small angle neutron scattering SANS………………………………………… 55 4.4.1 Scattering profiles of DOPC-Amphotericin B small unilamellar liposomes in 100% D2O…………………………………………………………………….. 56 4.4.2 Analysis of the scattering profiles…………………………………………….. 57 4.4.3 Scattering profile of DOPC-Cholesterol-Amphotericin B liposomes (SUV)… 58 4.4.4 Scattering profile of DOPC liposomes (SUV) in H2O buffer………………… 59 4.4.5 Scattering profile of DOPC-Cholesterol (SUV) in H2O buffer………………. 60 4.4.6 Scattering profiles of AmB-HST……………………………………………... 61 4.4.7 Evaluation of particle size of samples measured after SANS contrast variation 63 4.4.7.1 Particle size for DOPC-Amphotericin B (SUV)……………………………… 63 4.4.7.2 Particle size for DOPC-Cholesterol-Amphotericin B (SUV)………………… 64 4.4.7.3 Particle size for AmB-HST in D2O buffers…………………………………... 65 4.4.7.4 Structural parameters of SANS in comparison with DLS……………………. 66 4.4.7.4.1 Structural parameters for DOPC-Amphotericin B (SUV)…………………… 66 4.4.7.4.2 Structural parameters for DOPC-Cholesterol-Amphotericin B (SUV)……… 66 4.4.7.4.2 Structural parameters for AmB-HST………………………………………… 66 4.4.8 Determination of equivalent scattering length density by SANS contrast Variation……………………………………………………………………… 67 4.4.9 Internal structure evaluation of the freeze-dried liposomes………………….. 73 4.5 Results of self-nanoemulsifying system……………………………………... 84 4.5.1 Solubility of Amphotericin B in excipients………………………………….. 84 4.5.2 Ternary phase diagrams……………………………………………………… 85 4.5.3 Emulsification properties of blanks………………………………………….. 86 4.5.4 Emulsification properties of pre-concentrates……………………………….. 88 4.5.5 Effect of surfactant and co-surfactant on drug content………………………. 91 4.5.6 Effect of surfactant and co-surfactants on droplet size……………………….. 92 4.5.7 Precipitation study of nanoemulsions………………………………………… 93 4.5.8 UV-Visible spectrum of Amphotericin B in nanoemulsion…………………... 93 vi Table of Contents 4.5.9 Optimisation of formulations…………………………………………………. 94 4.5.10 Results of cytotoxicity test of nanoemulsion…………………………………. 97 4.5.11 Hemolysis test of nanoemulsions……………………………………………... 98 4.5.12 Rheology of pre-concentrates………………………………………………… 99 4.5.13 Structure determination of nanoemulsion by small angle neutron scattering… 99 4.5.13 Stability study of nanoemulsions……………………………………………... 102 5.0 Discussions…………………………………………………………………… 104 5.1 Development and structural evaluation of Amphotericin B core-shell microparticles………………………………………………………………… 105 5.2 Development of self-microemulsifying drug delivery system………………... 108 5.3 Solubility of Amphotericin B in FaSSIF-C…………………………………… 114 5.4 Development of Amphotericin