STABILITY OF BETAMETHASONE ESTERS IN SOME TOPICAL DOSAGE FORMS AND ITS IMPACT ON THEIR BIOLOGICAL POTENTIAL Thesis submitted in partial fulfillment of the requirement for the degree of DOCTOR OF PHILOSOPHY by Saif-ur-Rehman Khattak B.Pharm, M.Pharm SUPERVISOR: PROF. DR. DILNAWAZ SHEIKH CO-SUPERVISOR: PROF. DR. USMAN GHANI KHAN Faculty of Pharmacy HAMDARD UNIVERSITY Karachi – 74600 March 2010 iii ABSTRACT The present work involves an investigation of the thermal and photochemical degradation of betamethasone esters i.e. betamethasone valerate and betamethasone dipropionate under various conditions and the evaluation of the photoxicity of these compounds. The thermal degradation (40 oC) of betamethasone-17-valerate leads to the formation of betamethasone-21-valerate and betamethasone alcohol whereas betamethasone dipropionate gives rise to betamethasone-17-propionate, betamethasone-21-propionate and betamethasone alcohol at pH 2.5-7.5, betamethasone-21-propionate being an intermediate in this reaction. The betamethasone esters on photodegradation, using a UV radiation source (300-400nm), yield two major unknown products in aqueous and organic solvents. The detection of the photodegradation products of betamethasone valerate and betamethasone dipropionate has been carried out by HPLC and the t R values of the unknown products have been reported. The USP HPLC method, after proper validation, has been used for the assay of betamethasone esters and their thermal and photodegradation products. The analytical data have been used to evaluate the kinetics of thermal and photochemical reactions. In both reactions the betamethasone esters have been found to follow the first-order kinetics under the conditions employed. The apparent first-order rate constants for the thermal degradation of betamethasone valerate and betamethasone dipropionate in various media lie in the range of 0.339-9.07x10 -3 hr -1 and 0.239-1.87x10 -3 hr -1, respectively. The values of these rate constants for the photodegradation of betamethasone valerate and betamethasone dipropionate are in the range of 1.617-11.303x10 -3 min -1 and 1.101- 7.657x10 -3 min -1, respectively. The buffer and ionic strength effects on the rate of thermal and photodegradation have also been studied. It has been found that phosphate buffer inhibits the rate of degradation of both esters at pH 7.5. This could be due to deactivation of the thermal and photo-excited species involved in the reaction .An increase in the ionic strength of the phosphate buffer also leads to a decrease in the rate of reaction. iv Attempts on photostabilization of betamethasone esters in cream and gel formulations using compounds causing spectral overlay (vanillin and butyl hydroxytoluene) and light scattering agent (titanium dioxide) show promising results. However, the use of titanium dioxide was most effective in the photostabilization of the esters, causing 39.62-42.56 % and 33.84-35.70 % greater protection in cream and gel formulations compared to the control formulations of betamethasone valerate and betamethasone dipropionate, respectively. An important aspect of this work has been the evaluation of in vitro phototoxicity of betamethasone esters. This involved the application of the tests including photohemolysis, lipid photoperoxidation and protein photodamage. The results indicate that betamethasone esters and their photodegradation products are toxic to mouse red blood cells under UV irradiation. Photodegradation products of the esters are toxic in the dark also, therefore, appropriate precautions may be taken in their clinical applications to avoid any adverse effects. v ACKNOWLEDGEMENTS First of all I am extremely thankful to Allah Subhana-hu-Taala, the merciful and mighty, for giving me the courage to conduct the research work presented in this thesis. I also pay thousands of Salams to the Holy prophet Muhammad (peace be upon him) whose sunna provided me the guidance to live in this world. I express my deep sense of gratitude to my supervisor Prof. Dr. Dilnawaz Sheikh and Co-supervisor Prof. Dr. Usman Ghani Khan for their keen interest, guidance and encouragement throughout the course of this investigation. I extend my grateful thanks to Prof. Dr. Iqbal Ahmed of the Institute of Pharmaceutical Sciences, Baqai Medical University Karachi, for his continuous guidance and encouragement. I would like to thank Mrs. Sadia Rashid, President Hamdard Foundation Pakistan, Prof. Dr. Naseem A.Khan, Vice Chancellor, Hamdard University and Prof. Dr. Javaid Iqbal, Dean, Faculty of Pharmacy, Hamdard University, for providing an excellent environment and encouragement during my research work. My thanks are due to Prof. Dr. Mustafa Kamal, Chairman Biotechnology Department, University of Karachi, Mr. Saleem Qazi, PCSIR Complex, Karachi, Dr. Muhammad Ashraf, Mr. Ross Mamen, Mr. Shakeel Ahmed Ansari, Mr. Irfan Ahmed and Mr. Tanveer Akhter for their technical assistance. Mr. Mubeen Ahmed deserves special thanks for preparing this manuscript. I am also thankful to M/S. GSK Pakistan (Pvt) Ltd. M/S. Nabi Qasim Pharmaceutical (Pvt) Ltd. M/S. Tabros Pharma (Pvt) Ltd. PCSIR complex, Karachi and Biotechnology Department, University of Karachi, for providing their technical facilities to enable me to complete this work. I also acknowledge M/S. GSK Pakistan (Pvt) Ltd. and M/S. Crystal Pharma (Malysia) for providing reference standards of betamethasone valerate, betamethasone dipropionate and their thermal degradation products. I also record my special thanks to all my colleagues for their valuable suggestions and support. Finally, I would like to acknowledge my wife and children for their support and deep understanding. SAIF-UR-REHMAN KHATTAK vi DEDICATED TO MY BELOVED MOTHER (LATE) JEHAN BIBI vii CONTENTS Abstract iii Acknowledgements v CHAPTER Page 1. INTRODUCTION AND LITERATURE SURVEY 1 1.1 Introduction 2 1.2 Physicochemical Characteristics 6 1.3 Chemical Structure 7 1.4 Synthesis 8 1.5 Stability 8 1.5.1 Chemical Stability 9 1.5.1.1 Hydrolysis 9 1.5.1.2 Oxidation 11 1.5.1.3 Photolysis 13 1.5.2 Physical Stability 19 1.6 Chromatographic Methods for Identification and 20 Determination of Betamethasone Valerate, Betamethasone Dipropionate and Their Degradation Products 1.6.1 Thin Layer Chromatography 20 1.6.2 High Performance Liquid Chromatography 21 1.7 Photostabilization of Topical Preparations 22 1.8 Phototoxicity 22 AIMS AND OBJECTIVES OF PRESENT STUDY 25 2. EXPERIMENTAL WORK 27 2.1 Materials and Equipments 28 2.2 Methods 29 2.2.1 Thin Layer Chromatography (TLC) 29 viii 2.2.2 High Performance Liquid Chromatography (HPLC) 29 2.2.3 Ultraviolet and Visible Spectroscopy 30 2.2.3 pH Measurements 30 2.2.4 Electrophoresis 30 2.2.4.1 Preparation of solutions 31 2.2.4.2 Procedure 32 2.2.5 Thermal/Photodegradation of Betamethasone Valerate and 34 Betamethasone Dipropionate in Aqueous and Organic Media 2.2.6 Thermal Degradation of Betamethasone Esters in Cream 35 and Gel Formulations 2.2.6.1 Preparation of Cream and Gel Formulations 35 2.2.6.1.1 Formulae 35 2.2.6.1.2 Manufacturing procedures 36 2.2.6.2 Method 36 2.2.7 Photodegradation of Betamethasone Esters 37 2.2.7.1 Radiation chamber 37 2.2.7.2 Radiation source 37 2.2.7.3 Method 37 2.2.8 Assay of Betamethasone Valerate, Betamethasone 38 Dipropionate and Their Major Thermal and Photodegrades 2.2.8.1 Preparation of calibration standard solutions 38 2.2.8.2 Sample preparation 39 2.2.8.3 Chromatographic procedure 39 2.2.9 Photohemolysis 39 2.2.10 Photoperoxidation of Linoleic Acid 40 2.2.11 Protein Photodamage 40 2.2.11.1 Preparation of white membranes (ghosts) 40 2.2.11.2 Determination of membranes protein contents 41 2.2.11.3 Irradiation of ghosts/ compound suspension and 42 polyacrylamide gel electrophoretic analysis ix RESULTS AND DISCUSSION 43 3. THERMAL DEGRADATION REACTIONS 44 3.1 Introduction 45 3.2 Identification of the Thermal Degradation Products 45 of Betamethasone Esters 3.3 Assay of Betamethasone Esters and Degradation Products 51 3.3.1 Validation 51 3.3.1.1 Specificity 51 3.3.1.2 Linearity 51 3.3.1.3 Precision (Repeatability) 52 3.3.1.4 Accuracy (Recovery) 52 3.4 Kinetics of Thermal Degradation 61 3.5 Solvent Effect 68 3.6 pH Effect 68 3.6.1 pH-Rate Profile 68 3.6.2 Product Distribution 72 3.7 Buffer Effect 74 3.8 Ionic Strength Effect 82 4. PHOTOCHEMICAL DEGRADATION REACTIONS 92 4.1 Introduction 93 4.2 Identification of the Photodegradation Products of 93 Betamethasone Esters 4.3 Assay of Betamethasone Esters and Photodegradation Products 96 4.4 Product Distribution 96 4.5 Kinetics of Photolysis 96 4.5.1 Solvent Effect 105 4.5.2 Buffer Effect 105 4.5.3 Ionic Strength Effect 108 4.6 Photostabilization of Betamethasone Esters in Cream 108 x and Gel Formulations 5. IN VITRO PHOTOTOXICITY TESTING 121 5.1 Introduction 122 5.2 Photohemolysis 122 5.3 Lipid Photoperoxidation 123 5.4 Protein Photodamage 123 CONCLUSIONS 130 REFERENCES 134 CHAPTER ONE INTRODUCTION AND LITERATURE SURVEY 2 1.1 Introduction Glucocorticoids are naturally produced adrenal cortical steroid hormones or synthetic compounds that are used in a variety of disorders for their metabolic, anti-inflammatory and anti-allergic actions [1]. The first member of these compounds “Cortisone” was introduced into therapy in 1949, following its first clinical trial to determine its efficacy against rheumatoid arthritis by Hench and associates at Mayo clinic in Rochester in 1948 [2]. Since then a large number of valuable members of the cortisone series have been developed synthetically and progressively prescribed in the treatment of different diseases. The evolutionary development of these compounds is shown in Figure 1. The physiologic effects of glucocorticoids are known to be diverse. These agents regulate the metabolism of proteins, carbohydrates and lipids. They are involved in gluconeugenesis in the liver which leads to increased blood glucose levels [3].