1 FORMULATION AND EVALUATION OF SOME IN VITRO AND IN VIVO PROPERTIES OF ARTEMETHER-LOADED SELF EMULSIFYING DRUG DELIVERY SYSTEM BY UGWU CALISTER ELOCHUKWU PG/M.PHARM/11/59496 DEPARTMENT OF PHARMACEUTICAL TECHNOLOGY AND INDUSTRIAL PHARMACY UNIVERSITY OF NIGERIA, NSUKKA FEBRUARY, 2014 2 TITLE PAGE FORMULATION AND EVALUATION OF SOME IN VITRO AND IN VIVO PROPERTIES OF ARTEMETHER-LOADED SELF EMULSIFYING DRUG DELIVERY SYSTEM APPROVAL PAGE 3 THIS DISSERTATION WAS APPROVED BY THE DEPARTMENT OF PHARMACEUTICAL TECHNOLOGY AND INDUSTRIAL PHARMARCY, FACULTY OF PHARMACEUTICAL SCIENCES UNIVERSITY OF NIGERIA, NSUKKA …………………………… .......…………………. PROF. G. C ONUNKWO DR. N. C OBITTE (SUPERVISOR) (SUPERVISOR) ……………………. DR. I. V ONYISHI (HEAD OF DEPARTMENT) …………………….. ……………………. EXTERNAL EXAMINER DEAN OF FACULTY DEDICATION 4 This work is dedicated to my darling, Gabriel and lovely daughters, Ugwu Gabriela and Sonia. 5 DECLARATION We certify that Ugwu, Calister Elochukwu, a postgraduate student in the Department of Pharmaceutical Technology and Industrial Pharmacy, University of Nigeria, Nsukka, has completed the requirements for the award of the degree of Master of Pharmacy in Pharmaceutical Technology and Industrial Pharmacy. The research work reported in this dissertation is original and has not been submitted in support of an application for another degree or qualification of this or any other university. …………………………… .......…………………. PROF. G. C ONUNKWO DR. N. C OBITTE (SUPERVISOR) (SUPERVISOR) ……………………. DR. I. V ONYISHI (HEAD OF DEPARTMENT) 6 ACKNOWLEDGEMENTS I will start by thanking the Almighty God who has been by my side throughout the period of this work. To Him be the glory. My thanks to Step B management for their support in this research work, may God bless you. I wish to thank my first supervisor, Professor G. C. Onunkwo, who has always given me his support and assistance. I would like to express my warmest gratitude to my second supervisor Dr N. C. Obitte for his constant patience and practical support. You were always present when needed. I enjoyed working under your expert supervision and wished everybody would have a supervisor like you! My gratitude to our honourable HOD, Dr. I. V. Onyishi, who has provided excellent working facilities and environment for research work to progress, may the good Lord continue to shower more wisdom on you, thank you for the endless support. I appreciate our able Professors; Prof A. Ckukwu, Prof S. I Ofoefule and Dr. J. Onyechi, for providing professorial advice and even accommodation to facilitate this work. My thanks to the staff of our sister department such as Prof A. A. Attama, Prof E. Ibezim, Prof K. C Ofokansi, Dr.E. Onuigbo, Dr. P. Nnamani, Dr. M. Mommoh, Pharm. John and Pharm Kene. My thanks to Pharm. Agubata, Pharm. S. A Chime, and Pharm. N. O. Umeh. To my classmates and co- researchers; Pharm. Valentine, Reginald, Kenneth, and Nduka. To our staff Mr Nwodo, Mrs. Ezugwu, Anti Mau-Mau, and labouratory workers Mr. Odoh, Mrs okeh and Mrs. Oledinma, G.O.D, Emeka and KC, they were all helpful. I appreciate my friends and well-wishers; Pharm. (Mrs) Uwaoma Nwanneka, Pharm. (Mrs) Ezeani Chiamaka, Pharm. (Mrs) Peace (Nee Ibezim), Rev. Fr. Charles Ozioko and Mr Clement Okwor for their prayers. They are wonderful too. I cannot thank enough my darling husband, Gabriel, for his financial and moral support. To my dear lovely daughter Anulinna, I appreciate her for her patience always tolerating mummy’s absence. Many thanks to my dear brother, Engr. Emeka, and wife, all my sisters, my niece-Obinwa and my 7 beloved mother, Roseline (always available to assist) for their prayers, financial support and assistance. Thank you all and God bless. TABLE OF CONTENTS TITLE PAGES i APPROVAL ii DEDICATION iii DECLARATION iv ACKNOWLEDGEMENT v TABLE OF CONTENTS vi LIST OF TABLES ix LIST OF FIGURES x ABSTRACT xi CHAPTER ONE 1.0 GENERAL INTRODUCTION 1 1.1 BIOPHARMACEUTICAL CLASSIFICATION SYSTEM (BCS) OF DRUGS 3 1.2 DISPERSED SYSTEM 6 1.2.1 COLLOIDAL DISPERSION 6 1.2.2 MACROEMULSION OR CONVENTIONAL EMULSION 7 1.2.3 GEL 8 1.2.4 SUSPENSION 9 1.2.5 MICROEMULSION 10 1.2.6 FOAMS 11 1.3 FORMULATION TECHNIQUES OF HYDROPHOBIC DRUGS 12 1.3.1 SALT FORMATION 13 1.3.2 SOLID DISPERSION 13 1.3.3 COMPLEXATION WITH CYCLODEXTRIN 14 1.3.4 SOLID LIPID NANOPARTICLES 15 1.3.5 LIPOSOMES 16 1.3.6 NIOSOMES 17 1.3.7 DENDRIMERS 17 1.3.8 ANTISOLVENT 19 1.3.9 CO-GRINDING 20 1.3.10 SELF EMULSIFYING OIL FORMULATION 22 1.3.10.1 LIQUID SELF EMULSIFYING DRUG DELIVERY SYSTEM 34 8 1.3.10.2 SOLID SELF EMULSIFYING DRUG DELIVERY SYSTEM 34 1.3.10.3 SUPERSATURABLE /GELLED SELF EMULSIFYING DRUG DELIVERY SYSTEM 44 1.4 LIPID FORMULATION CLASSIFICATION SYSTEM (LFCS) 50 1.5 COMPOSITION OF SELF EMULSIFYING OIL FORMULATION 52 1.5.1 LIPOPHILIC BASE 53 1.5.2 SURFACTANT 56 1.5.3 CO-SURFACTANT/CO-SOLVENT 58 1.6 LITERATURE REVIEW OF EXCIPIENTS 60 ® 1.6.1 LABRASOL 60 1.6.2 TRIACETIN ® 60 1.6.3 TRANSCUTOL P® 61 1.6.4 HYDROXYPROPYLMETHYLCELLULOSE 62 1.7 MALARIA 63 1.8 ARTEMETHER (ARM) 67 1.8.1ASSAY OF ARTEMETHER 71 1.8.2 PHARMACOLOGICAL USES OF ARTEMETHER 73 1.8.2.1 ANTIMALARIAL EFFECT OF ARTEMETHER 73 1.8.2.2 ANTISCHITOSOMIASIS EFFECT OF ARTEMETHER 74 1.8.2.3 ANTITUMOR EFFECT OF ARTEMETHER 75 1.9 SELF EMULSIFYING DRUG DELIVERY SYSTEM (SEDDS) 77 1.9.1 PREPARATION OF SEDDS 77 1.9.2 MECHANISMS OF SEDDS AND ITS APPLICATIONS 78 1.9.3 CHARACTERIZATION OF SEDDS 81 1.9.3.1 DROPLET SIZE 81 1.9.3.2 EQUILIBRIUM PHASE DIAGRAM 81 1.9.3.3 ZETA POTENTIAL MEASUREMENT 82 1.9.3.4 DETERMINATION OF EMULSIFICATION TIME 82 1.9.3.5 LIQUEFACTION TIME 82 1.9.3.6 RELEASE STUDIES 83 1.9.3.7 THERMODYNAMIC STABILITY STUDIES 83 1.10 OBJECTIVES OF THE STUDY 84 CHAPTER TWO 2.0 MATERIAL AND METHODS 85 2.1 MATERIALS AND APPARATUS 85 2.2 METHODS 86 2.2.1 MELTING POINT DETERMINATION 86 2.2.2 STANDARD BEER-LAMBERT PLOT 86 2.2.3 SOLUBILITY OF DRUG IN DIFFERENT VEHICLES 87 2.2.4 CONSTRUCTION OF PHASE DIAGRAMS 87 9 2.2.6 SOLUBILITY STUDIES OF THE OPTIMIZED SEDDS BATCHES 88 2.2.7 PREFORMULATION ISOTROPICITY TESTS 88 2.2.8 FORMULATION OF ARM-LOADED SEDDS 88 2.2.9 FORMULATION OF ARM-LOADED SUPERSATURABLE SEDDS (S-SEDDS) 89 2.3 EVALUATION OF THE SEDDS AND S-SEDDS 89 2.3.1 DROPLET SIZE, POLYDISPERSITY INDEX AND ZETA POTENTIAL 90 2.3.2 POSTFORMULATION ISOTROPICITY TESTS/STABILITY TEST 90 2.3.3 EMULSIFICATION TIME TESTS 90 2.3.4 REFRIGERATION CYCLE TEST 90 2.3.5 CENTRIFUGATION 90 2.3.6 AQUEOUS DILUTION TEST 90 2.3.7 VISCOSITY OF ARM-LOADED SEDDS 91 2.3.8 DETERMINATION OF PH 91 2.3.9 LOADING EFFICIENCY 91 2.3.10 CRYSTALLIZATION/PRECIPITATION STUDIES 91 2.3.10.1 PHOTOMICR. OF STAND. DISPERSION OF ARM-SEDDS AND S-SEDDS 91 2.3.11 DRUG RELEASE STUDIES 92 2.3.12 IN VIVO STUDIES 92 CHAPTER THREE 3.0 RESULTS AND DISCUSSIONS 94 3.1 MELTING POINT 94 3.2 SOLUBILITY OF ARM IN DIFFERENT VEHICLES 94 3.3 PSEUDOTERNARY PHASE DIAGRAM 96 3.4 SOLUBILITY STUDIES OF OPTIMIZED BATCHES 105 3.5 PREFORMULATION ISOTROPICITY TESTS 106 3.6 DROPLET SIZES, POLYDISPERSITY INDEX AND ZETA POTENTIAL 108 3.7 POSTFORMULATION ISOTROPICITY TESTS 110 3.8 EMULSIFICATION TIME 110 3.9 REFRIGERATION CYCLE TEST 111 3.10 CENTRIFUGATION 111 3.11 AQUEOUS DILUTION TEST 112 3.12 VISCOSITY STUDIES 112 3.13 PH STUDIES 119 3.14 LOADING EFFICIENCY OF ARM-LOADED SEDDS AND S-SEDDS 126 3.15 CRYSTALLIZATION/PRECIPITATION STUDIES 132 3.16 RELEASE STUDIES ARM-LOADED SEDDS AND S-SEDDS 135 3.17 RELEASE MECHANISMS OF HPMC BATCHES IN SGF AND SIF 141 3.18 THE RESULTS OF IN VIVO STUDIES OF ARM-LOADED SEDDS 150 CHAPTER FOUR 10 CONCLUSION 153 RECOMMENDATION 154 REFERENCES 155 APPENDICES 177 LIST OF TABLES Table 1: Lipid formulation classification system 50 Table 2: The percent composition ratios of the SEDDS and S-SEDDS 89 Table 3: Droplet size, polydispersity and zeta potential of the SEDDS 108 Table 4: The loading efficiency of 40 mg ARM-loaded SEDDS of non-HPMC 126 Table 5: The loading efficiency of 50 mg ARM-loaded SEDDS of non-HPMC 127 Table 6: The loading efficiency of 55 mg ARM-loaded SEDDS of non-HPMC 127 Table 7: The loading efficiency of 40 mg ARM–loaded S-SEDDS of HPMC 128 Table 8: The loading efficiency of 50 mg ARM-loaded S-SEDDS of HPMC 128 Table 9: The loading efficiency of 55 mg ARM-loaded S-SEDDS of HPMC 129 11 LIST OF FIGURES Fig 1: Formulation techniques of hydrophobic drugs 12 Fig 2: The role of HPMC as PPI to inhibit the formation of crystalline materials 47 Fig 3: The structural formular of artemether 68 Fig 4: Synthesis of artemisinin derivative 69 Fig 5: Structure of α-β unsaturated decalone 72 Fig 6: Solubility (mg/ml) profile of ARM in different vehicles 94 Fig 7: Pseudoternary phase diagram plot of Smix(1:0) 96 Fig 8: The phase diagram of the Smix ratio 1:0.5 97 Fig 9: The phase diagram of the Smix ratio 1:1 98 Fig 10: The phase diagram of the Smix ratio 1:2 99 Fig 11: The phase diagram of the Smix ratio 1:3 100 Fig 12: The phase diagram of the Smix ratio 3: 1 101 Fig 13: The phase diagram of the Smix ratio 3:2 102 Fig 14: A typical aqueous of SEDDS dispersion 104 Fig 15: Solubility of ARM in the optimized SEDDS batches 105 Fig 16: Capsules of ARM-loaded SEDDS and S-SEDDS 107 Fig 17: Viscosity of non-HPMC batches of 40 mg ARM SEDDS 113 Fig 18: Viscosity of non-HPMC batches of 50 mg ARM SEDDS 114 Fig 19: Viscosity of non-HPMC batches of 55 mg ARM SEDDS 115 Fig 20: Viscosity of HPMC batches of 40 mg ARM SEDDS 116 Fig 21: Viscosity of HPMC batches of 50 mg ARM SEDDS 117 Fig 22: Viscosity of HPMC batches of 55 mg ARM SEDDS 118 Fig 23: pH versus time (month) for 40 mg ARM-loaded SEDDS of non-HPMC batches 120 Fig 24: pH versus time (month) for 50 mg ARM-loaded SEDDS