Stability of Betamethasone Esters in Some Topical Dosage Forms and Its Impact on Their Biological Potential
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]. Glucocorticoids play their role by decreasing circulating lymphocytes (including T cells), eosinophils, basophils, monocytes and macrophages, whereas on the other hand they increase the number of circulating neutrophils, hemoglobin and erythrocytes. The anti-inflammatory effects of glucocorticoids are due to decreased production of prostaglandins and leukotrienes [4].
Glucocorticoids used in therapy, are mainly produced synthetically and can be divided into oral, inhalational, injectable and topical corticosteroids according to their type of administration. Systemic use of their synthetic derivatives is indicated mainly for the treatment of rheumatoid arthritis [5] and allergic manifestations [6], while topically they are effectively utilized in dermatoses and other dermatological disorders [7, 8].
Topical corticosteroids can be classified into different classes according to their vasoconstrictor assay and/or clinical efficacy in mitigating signs and symptoms of inflammatory dermatoses [9, 10]. The clinical assessment of different types of topical corticosteroids is shown in Table 1.
3
Out of a vast variety of compounds of the cortisone series’ betamethasone derivatives such as betamethasone valerate and betamethasone dipropionate are most commonly used in contact dermatitis, atopic dermatitis, pruritis with lichenification, allergic eczema and psoriasis [11] in the form of creams, ointments, gels, lotions or solutions. Their wide application, highly potent and photolabile nature and formulation in multiple dosage forms make them important candidates for advance research both from chemical, pharmaceutical and biological point of view. It is the objective of this study to conduct research on the stability aspects of these drugs and their formulations and also to explore their toxic/phototoxic potential on cells and biological molecules via various in vitro phototoxicity tests.
4
CH2OH CH2OH
C=O C=O CH3 CH3 O HO OH OH
CH3 CH3
O O CORTISONE HYDROCORTISONE
CH OH CH2OH CH2OH 2
C=O C=O C=O CH CH3 CH3 3 O HO HO OH OH OH CH CH3 CH3 3
F
O O O PREDNISONE PREDNISOLONE FLUDROCORTISONE
CH OH CH2OH CH2OH 2
C=O C=O C=O CH CH3 CH3 3 HO HO HO OH OH OH CH CH3 CH3 3 OH CH3
F F
O O O
CH3
METHYLPREDNISOLONE DEXAMETHASONE TRIAMCINOLONE
Figure 1. Evolutionary development of compounds of cortisone series.
5
Table 1. Clinical assessment of different types of topical corticosteroids.
Very potent Potent
Clobetasol propionate 0.05% Amcinonide 0.1% Diflucortolone valerate 0.3% Beclomethasone dipropionate 0.025% Fluocinolone valerate 0.2% Betamethasone benzoate 0.025% Halcinonide 0.1% Betamethasone dipropionate 0.05% Ulobetasol propionate 0.05% Betamethasone valerate 0.1% Diflorasone diacetate 0.05% Fluolorolone acetonide 0.025% Triamcinolone acetonide 0.1%
Moderately potent Mildly potent
Alclometasone dipropionate 0.05% Fluocinolone acetonide 0.0025% Betamethasone valerate 0.025% Hydrocortisone 0.5% & 1% Fludroxycortide 0.0125% Hydrocortisone acetate 1% Flumetasone pivalate 0.002% Methylprednisolone acetate 0.25% Prednicarbate 0.25%
6
1.2 Physicochemical Characteristics
The physicochemical characteristics of selected betamethasone derivatives [12-14] are shown in Table 2.
Table 2. The Physicochemical characteristics of selected betamethasone derivatives.
Physicochemical Betamethasone Betamethasone Valerate characteristics Dipropionate
Molecular formula C27 H37 FO 6 C28 H37 FO 7
Molecular weight 476.6 504.6
White or creamy-white Almost white crystalline Appearance crystalline powder powder Practically insoluble in Practically insoluble in water, water, sparingly soluble in soluble in alcohol, freely Solubility alcohol, freely soluble in soluble in acetone and in acetone and in dichloromethane dichloromethane
About 190 0C with About 170 to 179 0C with Melting point decomposition decomposition
[]25 0 = + 65.7 0 Dioxane []25 0 = + 77 0 Optical rotation []27 0 = + 89.4 0 Dioxane Methanol
UV max (nm) 238 ( 1.57 x 10 4) 239 ( 1.592 x 10 4)
7
1.3 Chemical Structure
Betamethasone Dipropionate Chemically betamethasone dipropionate is 9-floro- 11 , 17, 21-trihydoxy-16 - methyl pregna-1, 4-diene-3, 20-dione-17, 21-dipropionate.The empirical formula of
the compound is C 28 H37 FO 7 and it has the following chemical structure [12].
O
H C 3 O O H H3C O HO CH3 H H3C H O
CH3
F H
O
BETAMETHASONE DIPROPIONATE
Betamethasone Valerate Chemically betamethasone valerate is 9-floro-11 , 17, 21-trihydoxy-16 -methyl pregna-1, 4-diene-3, 20-dione-17-valerate. The empirical formula of the compound
is C 27 H37 FO 6 and it has the following chemical structure [12].
O
H C 3 O O H H3C OH HO H H H3C CH3 F H
O
BETAMETHASONE-17-VALERATE
8
1.4 Synthesis
Betamethasone Dipropionate Betamethasone dipropionate is synthesized by reacting betamethasone alcohol with ethyl orthopropionate and toluene-p-sulphonic acid to yield betamethasone 17, 21- ethylorthopropionate [15]. This compound is then reacted with acetic acid to yield betamethasone-17-propionate, which upon further treatment with propionyl chloride at 0 oC, for 1 hour, dilution with water and acidification with dilute hydrochloric acid gives the crude diester. The crude diester yields the final pure form of betamethasone dipropionate upon recrystallization from acetone-petroleum ether [16]. Betamethasone Valerate Betamethasone alcohol is suspended in ethyl acetate with stirring. Toluene-p- sulphonic acid monohydrate and methyl orthovalerate are then added. The mixture is warmed to form a complete solution. The solution is then treated with 2N-aqeous solution of sulphuric acid at room temperature for 15 minutes before washing with saturated sodium bicarbonate solution and water. The organic phase is dried over anhydrous magnesium sulphate, filtered and evaporated to dryness under reduced pressure. The crude betamethasone-17-valerate is then dissolved by stirring at reflux temperature in acetone followed by a slow addition of petroleum ether to the mixture. The mixture is then allowed to cool to room temperature and the product is collected by filtration. The product is then washed by displacement with 10% acetone-petrol and dried in vacuo at 40 oC to yield white crystalline solid [16].
1.5 Stability
Stability of betamethasone derivatives and other glucocorticoids has long been the subject of investigation by many workers. Several reviews have been published on the stability and related aspects of glucocorticoids [17-22]. Different workers have evaluated the chemical stability [23-32], physical stability [33, 34] and even the stability in the presence of micro-organisms (biodegradation) [35] of the pure drugs and their formulations. Principles of chemical kinetics [36-47] have been applied during these studies. 9
1.5.1 Chemical Stability
Glucocorticoids possessing a dihydroxyacetone side-chain at C-17 have been shown to degrade mainly by hydrolysis [48, 49], oxidation [50] or photolysis [51].
1.5.1.1 Hydrolysis
Hydrolysis has been shown to be the most common degradation pathway of C-17 and/ or C-21 esterified corticosteroids in aqueous and biological media [52]. Reversible ester migration and subsequent hydrolysis (Figure 2) has been reported for a number of corticosteroids in aqueous solutions and in various pharmaceutical preparations.
Ester group migration
C-17 Steroidal ester C-21 Steroidal ester
Hydrolysis Hydrolysis
Steroid base
Figure 2. Ester group migration and hydrolysis in C-17 and C-21 esterified corticosteroids.
The hydrolytic degradation and subsequent stabilization of glucocorticoids was studied in aerosol solution formulations [53]. Addition of an acid to the aerosol solution formulation has been shown to provide stability against hydrolytic degradation. Wurthwein and Rohdewald [54] studied the hydrolysis of beclomethasone dipropionate in simulated intestinal fluid. They reported that 10 beclomethasone dipropionate is hydrolyzed rapidly to beclomethasone-17- monopropionate initially with subsequent slow hydrolysis to beclomethasone. In addition steroidal esters’ hydrocortisone butyrate [55], hydrocortisone hemisuccinate [56] and hydrocortisone-21-lysinate [57] have shown pH dependent hydrolysis and / or reversible ester migration between C-21 and C-17-hydroxy groups in aqueous media. Foe et al . [58] have shown a reversible ester migration between beclomethasone-17-propionate and beclomethasone-21-propionate in human plasma. The C-21 ester isomer then degrades to the corresponding alcohol through hydrolysis. The stability of an aqueous suspension of betamethasone dipropionate was also evaluated [59]. The compound showed maximum stability at pH 4. It also showed high stability as compared to other corticosteroids. Hydrolysis of the compound resulted in the formation of betamethasone alcohol. Reversible ester migration and further hydrolytic degradation has been shown for betamethasone valerate in a number of references [60, 61]. The compound has been shown to convert to betamethasone-21-valerate in neutral and alkaline solutions while the maximum stability of the compound in an aqueous solution was found to be at pH 5 [62]. Bundgaard et al . [63] have communicated a valuable work on the kinetic of the rearrangement of betamethasone-17-valerate to the 21-valerate in aqueous solution. Yip et al . [64] investigated the stability of betamethasone-17- valerate in various ointment bases and reported the degradation of betamethasone-17-valerate to betamethasone-21-valerate and betamethasone alcohol. Quantification of the degradation was determined by direct densitometry on thin layer chromatographic plates. The degradation was found and assessed to be an apparent first-order process and to depend on the diluent used and its concentrations. Temperature effect on the degradation rate was also evaluated. Results also indicated that the degradation of the drug was base-catalyzed. Storage of the acidified solutions at room temperature showed that the drug was also subject to acid-catalyzed hydrolysis, but at a rate much smaller than the base-catalyzed hydrolysis. Mehtha et al . [65] have studied the stability of betamethasone-17- valerate (Betnovate Ointment) in emulsifying ointment. The degradation of the drug was quantified by HPLC. More than 60% of the drug degraded within 6 hours. 11
Continuous increase in the concentration of betamethasone-21-valerate was observed which peaked within 2 days followed by a slow degradation (half-life 8 days) to betamethasone alcohol. The Stability of betamethasone valerate has also been evaluated in culture medium in the presence of artificial living skin equivalent (LSE) by Kubota et al . [66]. Degradation profile (%) of betamethasone-17-valerate in the culture medium with skin homogenate did not differ from those without homogenate, however, the conversion of betamethasone-21-valerate to betamethasone was accelerated by skin homogenate.
1.5.1.2 Oxidation
Substituents at ring D make 17-ketol steroids sensitive to oxidation [67]. Oxidative alteration of the side chain at C-17 could be affected both in aerobic and anaerobic conditions. The ketol (1) undergoes rearrangement via the enediol (2) to the aldol (3) which is broken down by a retro-aldol reaction to give the ketone (4).
OH OH
H2C O C O OHC OH
C H C CH OH OH OH
(1) (2) (3)
O CHO
+ CH2 OH
GLYCOLALDEHYDE (4)
In alkaline medium, under the effect of oxygen, the dihydroxyacetone group at C-17 breaks oxidatively in a form of glycol cleavage to a hydroxyaldehyde which, by intramolecular rearrangement, changes to the carboxylic acid anion (5). 12
OH
H2C O
C COOH OH H
O2
HO-
(1) (5)
Oxidative attack at C-21 can result in glyoxal derivative (6) which rearranges in acid medium and with the addition of water produces the hydroxy acid (7).
OH
H2C O CHC O HOOC O
C C CH OH OH OH
O2 H2O
HO-
(1) (6) (7)
Guttman and Meister [68] reported the base-catalyzed oxidative degradation of prednisolone in aqeous solutions. The rate of prednisolone degradation increased with increase in hydroxyl ion concentration under both aerobic and anaerobic conditions. However, more rapid degradation of the drug was found under aerobic conditions. The oxidative degradation of prednisolone with trace metal impurities in buffer salts and inhibition by ethtylene diamine tetra-acetate was explored in alkaline solutions [69]. Metal ions catalyzed oxidation of hydrocortisone to its 21- dehydro derivative and inhibition by ethyline diamine tetra-acetate was also reported [70]. Oxidation of corticosteroids was also observed in polyethylene glycol 300 [71, 72]. In addition, air oxidation of betamethasone dipropionate in solid state was also assessed [73]. The compound was shown to be stable towards air 13
oxidation. Heating at 75°C for 6 months in the presence of air, displayed no change in colour or in the thin layer chromatogram.
1.5.1.3 Photolysis
Photolytic degradation of glucocorticoids has been studied extensively and cited by different workers both in solutions [74, 75] and in the solid state [76, 77].The photochemical behavior of glucocorticoids was preliminarily investigated by Barton and Taylor [78, 79] who focused attention on prednisone acetate. It was found sensitive to light and converted into a range of novel molecules depending upon the reaction conditions. Hamlin et al. [80] studied the photolysis of alcoholic solutions of hydrocortisone, prednisolone and methylprednisolone under ordinary fluorescent light. They observed that the degradation follows first-order kinetics and the rate of degradation of prednisolone and methylprednisolone was alike, whereas hydrocortisone degraded at about 1/7 the rate of the other two steroids. A more systematic work on prednisone and its 21-acetate was performed by Williams et al. [81]. They reported that irradiation of prednisone (1a) or prednisone acetate (1b) in dry dioxane with 254 nm light produced lumiprednisone (2a) and (2b), respectively, in 65% yield.
CH2OR CH2OR
CO CO
O O OH H OH
O hν O
1a , R=H 2a , R=H 1b , R=COCH3 2b , R=COCH3
14
The general scheme for the lumi rearrangements is given as below.
O
hν neutral O O
CH3 OH
CH3 H H3O H O H2O 2 βββ − attack ααα − attack O HO O
The same rearrangement pattern has been observed for prednisolone and its acetate [82], dexamethasone and its acetate [83, 84], betamethasone [85, 86], diflorasone, triamcinolone acetonide and fluocinolone acetonide [87], as shown below.
COCH OR COCH OR 2 2 OH OH X X R2 R2 O hν R1 R Solid 1 O
R3 R3
15
Compound X R R1 R2 R3
OH
Pridnisolone / Acetate C H, Ac F H H
H
OH
Dexamethasone / Acetate C H F -CH3 H
H
OH
Betamethasone C H F -CH3 H
H
OH
Diflorasone C H F –CH3 H
H
CH2OH CH2OH O O O O HO HO
H O H O O
F H F H hν O
X X
TRIAMCINOLONE ACETONIDE.. X=H
FLUOCINOLONE ACETONIDE.. X=F
16
Degradation in the ring A has also been observed in hydrocortisone in polyethylene glycol ointment base. [88]. The primary photoproducts may undergo further transformation with cleavage of the three-membered ring, resulting in rearomatisation or cleavage of ring A or in the expansion of ring B according to conditions as shown in prednisolone and dexamethasone [89] (Scheme 1,2,3a,3b).
CH2OH O
HO HO HO
hν
O O
PREDNISOLONE
HO HO HO
O HO
H2O HO
O HO HO O
O OH OH
Scheme 1
CH2OH CH2OH O O HO HO HO HO
hν O H2O O OH PREDNISOLONE
Scheme 2
17
CH2OH O O O HO HO HO O
O H2O hν
O PREDNISOLONE O Scheme 3a
CH OH CH2OH 2 O CH OH O O 2 HO OH HO HO OH O H CH3 CH3 CH3 O HO hν F H H2O O
DEXAMETHASONE Scheme 3b
Photo-oxidation of glucocorticoids e.g. hydrocortisone, cortisone and their acetates was also explored in the solid state [90]. The main process involves the loss of side chain at C-17 to give androstendione and trione derivatives as shown below.
18
COCH2R O
OH X X
hν, N2 Solid O O
XR OH H, COCH3
HYDROCORTISONE/ ACETATE C H CORTISONE/ ACETATE CO H, COCH3
Solid state photochemistry of halomethasone and prednicarbate has been evaluated by Reish et al. [91]. The observed processes involve the C-17 side chain, however, with a different pathway than that seen in photo-oxidation of hydrocortisone and cortisone as shown below.
COCH2OH COCH2OH O O O OH OH HO HO HO H H H CH3 CH3 CH3 Cl Cl Cl
F hν F F Solid O O O F F F F
O
F Cl
CHOH CH3 HALOMETHASONE CO HO OH HO HOCH2CO HO H CH3 Cl
F O
19
COCH2OCOCH2CH3 COCH2OR HO OCO CH CH 2 2 HO OR1
hν Solid O O
PREDNICARBATE R=COCH2CH3,R1=H R=H,R1=COOCH2CH3
Takacks et al. [92] have reported 45-51% photodegradation in hydrocortisone, prednisolone and betamethasone, 20-31% in desoxycortone acetate, hydrocortisone acetate, methylprednisolone, dexamethasone and triamcinolone acetonide and less than15% in fluocinolone acetonide, prednisolone and cortisone acetate, after 48 hours irradiation in the solid state. Photodegradation of betamethasone-17-valerate was also monitored in isopropanolic hydrogel [93]. After 20 minutes of irradiation with novasol test and in sunlight, 17% more loss of the drug content was observed than the tests performed in dark.
1.5.2 Physical Stability
Unlike chemical stability, very little information is available in the literature on the physical stability of steroids and steroidal preparations. Polymorphism has been shown to be the main physical degradative route [94]. Haleblian et al. [95] have studied the intercoversion of fluprednisolone polymorphs. Some work has also been carried out on the polymorphism of cortisone acetate [96]. When a more soluble crystal form (form II) of cortisone acetate is formulated into an aqueous suspension, it converts to a less soluble form (form V). This phase change leads to caking of the cortisone acetate suspension. Phase separation has also been observed in topical corticosteroid formulations upon mixing with commercially available ointments and/or creams [97].
20
1.6 Chromatographic Methods for Identification and Determination of Betamethasone Valerate, Betamethasone Dipropionate and their Thermal Degradation Products
1.6.1 Thin Layer Chromatography
The details of thin layer chromatography used for the identification and determination of betamethasone valerate, betamethasone dipropionate and their degradation products are given in Table 3.
Table 3. Rf values of betamethasone valerate, betamethasone dipropionate and degradation products.
Pure drug/ Rf Values of the Solvent Substance Dosage Adsorbent parent compound and Reference System form its thermal degrades
Semisolid Chloroform Bet-17-valerate = 0.219 Betamethasone ointment Silica gel 60 ethylacetate Bet-21-valerate = 0.454 [64] valerate bases (1:1, v/v) Bet = 0.129
Silica gel with Water: fluorescent methanol indicator ether:dichloro- Bet-17-valerate = --- // Pure drug having an [98,99] methane Bet- 21-valerate = --- optimal (1.2:8:15:77, intensity of v/v) 254nm Silica gel with fluorescent indicator Chloroform : Bet-17-valerate = 0.246 Present // Pure drug having an ethylacetate Bet- 21-valerate = 0.513 work optimal (1:1, v/v) Bet =0.12 intensity of 254nm. Silica gel with Bet-17-propionate = fluorescent 0.18 indicator Chloroform : Betamethasone Bet-21-propionate = 0.4 Present Pure drug having an ethylacetate dipropionate Bet- dipropionate = work optimal (1:1, v/v) 0.48 intensity of Bet = 0.12 254nm.
Bet = Betamethasone
21
1.6.2 High Performance Liquid Chromatography
The details of high performance liquid chromatography applied for the separation and determination of betamethasone valerate, betamethasone dipropionate and their degradation products by some workers are given in Table 4.
Table 4. HPLC conditions for the separation and determination of betamethasone valerate, betamethasone dipropionate and their degradation products.
Flow Retention time of the Pure drug/ Substance Column Mobile phase rate Detector parent compound and its Reference Dosage form (ml/min) thermal degrades Stainless Steel Column Water: Bet- Bet-17-valerate=7min Pure drug 250mm x 4.6mm i.d. acetonitrile 1.0 UV (254nm) [98,99] valerate Bet-21-valerate=9min packed with ODS (60: 40, v/v) Water: 25cm x 4.6mm i.d. Bet-17-valerate= --- // Ointment acetonitrile 1.5 UV (239nm) [100] packed with 10 µ ODS Bet-21-valerate= --- (45:55, v/v) Pre-column (RP18) = 3cm x 4.6mm i.d. packed with Water: Diode-array Bet-17-valerate=5.7min // Cream/lotion lichrosorb. Analytical acetonitrile 1.5 [101] (239nm) Bet-21-valerate=6.8min column = 25cm x (45: 55, v/v) 4.6mm i.d. packed with 10µ ODS Pre-column (RP18) = 3cm x 4.6mm i.d. Variable- packed with Water: Wavelength Isopropyl Bet-17-valerate=5.6min // lichrosorb. Analytical acetonitrile 1.5 /diode-array [102] Myristate Bet-21-valerate=6.5min column = 25cm x (45: 55, v/v) UV detectors 4.6mm i.d. packed (239nm) with 10µ ODS
250mm x 4.6mm i.d. Water: Bet-17-valerate =5.67min Pure drug/ Present // (µBondapak, C18) acetonitrile 1.2 UV (254nm) Bet-21-valerat =7.26min Cream/ gel work packed with 5µ ODS (40:60, v/v) Bet=2.48min
Stainless Steel column Water: Bet (Permaphase) 1m x Bet-monopropionate=5min Pure Drug acetonitrile 0.5 UV (254nm) [59] dipropionate 2mm i.d. packed with Bet-dipropionate=7min (3:1, v/v) ODS
Bet-17-propionate=3.72min 250mm x 4.6mm i.d. Water: Pure drug/ Bet-21-propionate=4.34min Present // (µBondapak, C18) acetonitrile 1.2 UV (254nm) Cream/ gel Bet-dipropionate=8.20min work packed with 5µ ODS (40:60, v/v) Bet=2.34min
Bet = Betamethasone
22
1.7 Photostabilization of Topical Preparations
Generally topical preparations have long been protected from light through specific packaging material like other dosage forms, however, protection with suitable excipients has also been effectively used [103]. Protection with light absorbers (spectral overlay) is achieved by adding an excipient to a formulation which absorbs light in the region of absorption maximum of the substance to be protected. [104-106]. The principle of photostabilization through spectral overlay with absorbing excipients is shown in Figure 3. Another approach utilizes substances which block light radiations through reflection and scattering [107]. A number of substances are found in the literature used for this purpose. Some of the commonly used substances that bring about photoprotection are curcumin, vanillin, quinosol, titanium dioxide, colors/pigments, flavonoids, para-aminobenzoic acid and sulfonates, etc [108-109]. Photostabilization of an isopropanolic polyacrylate hydrogel containing betamethasone-17-valerate has been investigated [93]. The investigation proved that by the addition of 4% 2- phenylbenz-imidazole-5-sulfonic acid to the hydrogel, UV irradiation has no effect on the drug content. Light protection can also be achieved by the addition of quenchers to the formulation if photo reactions proceed through a type I (reactions which occur via the formation of free radicals) or type II (reactions which occur via the formation of singlet oxygen) photosensitization mechanisms [110]. Substances such as ascorbic acid, -tocopherol, butyl hydroxytoluene and butyl hydroxyanisole which are capable of acting as free radical scavengers as well as weak singlet oxygen quenchers, can be used effectively.
1.8 Phototoxicity
Phototoxicity is an acute toxic response that is elicited after the first exposure of skin to certain chemicals and subsequent exposure to light, or that is induced by skin irradiation after the systemic administration of a chemical. Phototoxic reactions in humans occur exclusively on skin exposed to light.
23
absorption region of the rediation
ultraviolet visible
200 300 400 500 600
λ (nm) vanillin
yellow colourants
red colourants
blue colourants
Figure 3. Photostabilization through spectral overlay with absorbing excipients
24
Their morphology and clinical symptoms may vary. In some cases a burning and painful sensation is felt during light exposure, while in other cases reactions such as erythema, oedema and vesiculation occur at later stages with time. It is believed that phototoxic reaction causes damage of cells by direct modification of certain targets such as DNA, lipids and/or amino acids, proteins, lysosomes, mitochondria and plasma membrane [111]. Phototoxic reactions may be oxygen dependent (photodynamic) or oxygen independent (non-photodynamic). In general, it is the capacity of the drug to generate free radicals that have been regarded as the most potentially damaging characteristic, because of the possibility of chain reactions that occur subsequently [112]. In addition phototoxicity may be produced by toxic photoproducts that may be produced by the action of sunlight on the drug in the epidermal layers of the skin of patients. The adverse photosensitivity effects produced by these toxic photoproducts may either be due to their undesirable physiological properties or because they can easily transfer energy to body compounds [113]. Most phototoxic reactions occur in the wavelength range from 300 – 400 nm. Most drug-induced phototoxic reactions are acute, occurring within a few minutes to several hours after exposure. They reach a peak from several hours to several days later, and usually disappear within a short time period after stopping either the drug or the exposure to radiation [114]. But it definitely brings an adverse drug reaction that could be viable in its intensity. The list of phototoxic drugs includes several common antibiotics, sulfonamides, quinolines, diuretics, tranquilizers, oral diabetes medication and anti-neuplastic drugs [115]. There are also some dermatologic drugs both topical and oral that can sensitize skin and exhibit phototoxicity. Some information is available in the literature on the phototoxicity of glucocorticoids [116-120]. The phototoxicity of prednisolone and dexamethasone has also been shown in aquatic organism C. dubia [89].Various in vitro phototoxicity test models have been designed to evaluate a drug for phototoxicity [121-124]. In this work betamethasone valerate and betamethasone dipropionate are objectively evaluated for their phototoxic potentiatial on erythrocytes, lipids (linoleic acid) and proteins.
25
Aims and Objectives of Present Study
Betamethasone is a synthetic corticosteroid used widely in the treatment of various diseases. Systemic use of this compound is mainly indicated in the treatment of rheumatoid arthritis and allergic manifestations while topically it is effectively used in dermatoses and other dermatological disorders. Mono and diesters of the compound are mainly meant for topical purpose and are formulated as ointments, creams, gels and topical solutions. These esters are unstable and may undergo hydrolytic and oxidative degradation in the presence of acids and / or bases. The resultant products are generally less active as compared to the parent compound e.g. Betamethasone-21-valerate has been found to possess one fifteenth of the activity of the 17-valerate. These ester are also sensitive to light and may decompose to various photodegrades. These degrades may not only be of low activity but may have enhanced toxicity to cells and other biological molecules. The degradative processes may occur individually or simultaneously depending upon the reaction conditions (pH, oxygen content, solvent, buffer type and concentration, ionic strength, intensity of light, wavelength of light, etc). Degradation of the compounds in the formulated products upon extemporaneous dilution or exposing to sunlight, when applied to the skin, could be of clinical and toxicological significance. Therefore, it is necessary to undertake detailed work on the thermal/photostability and phototoxicity of these compounds. The present study is an attempt to explore some of the stability aspects regarding the pure drugs and their topical formulations (creams and gels) along with screening for phototoxicity using some basic in vitro phototoxicity test models. The various aspects involved in this investigation may be summarized as below. 1. Preparation of cream and gel formulations containing betamethasone valerate and betamethasone dipropionate. 2. Thermolysis of betamethasone valerate and betamethasone dipropionate in pure solutions and in cream and gel formulations. 3. Photolysis of betamethasone valerate and betamethasone dipropionate in pure solutions and in cream and gel formulations in the presence and absence of photoprotective additives as stablizers. 26
4. To develop and validate high performance liquid chromatographic methods for the determination of betamethasone valerate, betamethasone dipropionate and their thermal and photodegrades. 5. To evaluate kinetics of aerobic thermolysis and photolysis of betamethasone valerate and betamethasone dipropionate in different solvents. 6. To determine the pH of maximum stability of betamethasone valerate and betamethasone dipropionate in water-acetonitrile mixture using pH-rate profile. 7. To study the effects of ionic strength, buffer concentration and solvent dielectric constant on the degradation kinetics of the esters at constant pH. 8. To evaluate phototoxicity of the esters to cells and other biological molecules like lipids and proteins using in vitro phototoxicity tests.
CHAPTER TWO
EXPERIMENTAL WORK
28
2.1 Materials and Equipments
Acrylamide (Serva chemicals, Germany) Agarose (Sigma Chemicals, Germany) Betamethasone-17- valerate USP (Glaxo,Pakistan) Betamethasone-21-valerate (Glaxo, Pakistan) Betamethasone-17-propionate (Crystal, Malaysia) Betamethasone-21-propionate (Crystal, Malaysia) Betamethasone dipropionate USP (Crystal, Malaysia) Betamethasone (Glaxo,Pakistan) The purity of the aforementioned material was checked using Thin Layer Chromatography and High Performance Liquid Chromatography. Bovine serum albumin (Sigma Chemicals, Germany) Butyl hydroxyanisole (Sigma Chemicals, Germany) Butyl hydroxytoluene (Sigma Chemicals, Germany) Carbomer 940 (North Chemicals, Colombia) Cetostearyl alcohol (Croda, Japan) Coomassie Brilliant Blue R-250 (Fluka, Germany) Hydroxyethyl cellulose (Spectrum,USA) Linoleic acid (Spectrum, USA) Methylene blue (Merck, Germany) N, N-Methylene bis acrylamide (Serva Chemicals, Germany) N, N, N, N-tetramethylethylenediamine (TEMED) (Merck, Germany) Sodium dodecyl sulphate (Merck, Germany) Titanium dioxide (Merck, Germany) Tris-(hydroxymethyl) –amino methane (Fluka, Germany) Tween 20 (Sigma, Germany) Vanillin (Merck, Germany) -mercaptoethanol (Merck, Germany) All the reagents used were analytical grade and the solvents were spectroscopic grade. Freshly prepared deionized/ distilled water was used throughout the work. HPLC (Agilent, 1100 Series, USA) 29
HPLC (Shimadzu, LC-20A, Japan) Spectrophotometer (Shimadzu, UV-1601 PC, Japan) Bio-Rad power pac 300 electrophoresis apparatus (Bio-Rad, Italy) Slab gel apparatus (Bio-Rad, Italy) Densitometer (Hewlett Packard Scanjet Scanner 8300, USA) Centrifuge Machine (Damon/ IEC, B-20A, USA) pH meter (WTW, 702, Germany) Radiation chamber (Local) Silver san mixer (Local) TLC precoated plates (Merck, Germany) UV illuminator (Upland, USA) Illuminance meter (TES-1332A, TES Electrical Corporation, Taiwan)
2.2 Methods
2.2.1 Thin Layer Chromatography (TLC)
An appropriate volume of the sample solution was applied to silica gel 254 precoated plates and subjected to ascending chromatography using chloroform: ethyl acetate (1:1, v/v) as the developing solvent. Pure compounds were dissolved in acetonitrile and then applied to the TLC plates. The solvent was allowed to ascend the plate upto a distance of 15cm. The plate was then air dried and viewed under ultraviolet light at 254 and 366 nm to locate the parent compounds and their degradation products. The plate was alternatively sprayed with a mixture of sulfuric acid, methanol and nitric acid (10:10:1, v/v/v) and heated at 105 oC for 15 minutes.
2.2.2 High Performance Liquid Chromatography (HPLC)
An HPLC (Agilent, 1100 Series, USA) system consisted of a solvent delivery system, a syringe loading, six-port sample injector equipped with a diode-array UV detector and a 250mm x 4.6mm column, packed with 5µ octadecylsilane, was used in all development and methods validation studies while separation and determination of betamethasone valerate , betamethasone dipropionate and their thermal and photodegrades was performed on a Shimadzu class -20 A HPLC 30
(Kyoto, Japan) system that consisted of an LC-20AT pump, an SPD-20A UV-visible detector and an inbuilt CBM-20A lite communication bus module. Data collection and integration were achieved using Shimadzu LC solution computer software version 1.2 (Kyoto, Japan). All separations were carried out isocratically at room temperature (20 ± 1oC).
2.2.3 Ultraviolet and Visible Spectroscopy
All absorbance measurements and spectral determinations were made on a Shimadzu UV-visible recording spectrophotometer using matched silica cells of 10mm pathlength. The cells were employed always in the same orientation using appropriate control solutions in the reference beam .The baseline was automatically corrected by the built-in baseline memory at the initializing period . Auto-zero adjustment was made with zero adjustment key. Data collection and spectral determinations were achieved by Shimadzu personal spectroscopy computer software version 3.7. The instrument was periodically checked using the following calibration standards. Wavelength scale: Holmium Oxide Filter (NIST SRM 2034)
Absorbance scale: 50 mg /l of K2 Cr 2 O 7 in 0.01N H 2SO 4 Absorbance at 257 nm =0.725 350 nm= 0.539± 0.005 [125].
2.2.3 pH Measurements
All pH measurements were carried out with a pH meter (WTW-Germany, model 702, Sensitivity ± 0.01 pH units). The electrode was standardized with buffer solutions (pH 2.0, 4.0 and 7.0, Merck) at 25 0C. For determination of pH of the formulated products (cream/gel) a 2 g sample was mixed thoroughly with 30 ml of double distilled water in a beaker and pH of the mixture was determined.
2.2.4 Electrophoresis
The polyacrylamide gel electrophoresis was carried out using a Bio-Rad power pac 300 electrophoresis apparatus (Bio-Rad, Italy). Quantification of the bands was 31 achieved by the gel densitometry using scanjet scanner photo and imaging software scanplot version 2.0.
2.2.4.1 Preparation of solutions
Solution A {Acrylamide-bis Acrylamide (30:0.8)}
30g acrylamide and 0.8g bis- acrylamide were dissolved in distilled water and made up the volume upto 100 ml. The solution was filtered to remove any suspended particles and stored at 4 oC in dark bottle.
Solution B {3M Tris-HCl (pH 8.8)}
36.3g Tris and 48 ml 1M HCl were mixed and the pH adjusted to 8.8 using 0.1M HCl if required. The volume was made upto 100 ml with distilled water and stored at 4 oC.
Solution C {0.5M Tris-HCl (pH 6.8)}
6.05 g Tris was dissolved in 40 ml of distilled water and the pH adjusted to 6.8 with 0.1M HCl. The volume was made upto 100 ml with distilled water and stored at 4 oC.
Solution D {1% Sodium Dodecyl Sulphate (SDS)}
1g SDS was dissolved in distilled water and made the volume upto 100 ml and stored at room temperature.
Solution E {1.5% Ammonium per Sulphate (APS)}
0.15g APS was dissolved in 5 ml distilled water and made the volume upto 10 ml. It was prepared fresh before use. 32
Reservoir Buffer {0.124M Tris, 1mM Glycine, 0.5% SDS (pH 8.3)}
15 g Tris, 0.075g glycine and 5 g SDS were dissolved in 500 ml distilled water and made upto 1 liter and stored at 4 oC.
Sample Diluting Buffer {0.0625 M Tris-HCl (pH 6.8), 2% SDS, 2% 2-Mercaptoethanol, 10% Glycerol or Sucrose}
12.5 ml solution C, 2g SDS, 5 ml 2-mercaptoetnanol and 10 ml glycerol were mixed together and made upto 100 ml and stored at 4 oC.
Staining Solution {0.0025% Coomassie Brilliant Blue R-250, 45.5% Acetic Acid, 4.6% Methanol}
2.5 mg Coomassie brilliant blue R-250 was dissolved in 454 ml acetic acid and 46 ml methanol and made upto 1 liter with distilled water. The solution was filtered and stored at room temperature.
Destaining Solution {7.5% Acetic Acid, 5% Methanol}
150 ml acetic acid and 100 ml methanol were mixed together and made upto 2 liters and stored at room temperature.
TEMED
TEMED was used as supplied.
2.2.4.2 Procedure
1. The resolving and stacking gels were prepared using Table 5. 2. The stacking gel was poured into the plates after polymerization of the resolving gel and wells were formed with well forming comb. 3. 40µl samples were loaded in sample wells and voltage was applied until complete separation achieved. 4. The gel was removed for staining with staining solution. 33
5. The gel was destained by repeated washing with destaining solution. 6. Quantification of the bands was achieved densitometrically by the scanjet scanner photo and imaging software scanplot version 2.0.
Table 5. Standard composition of stacking and resolving gel
Stacking Gel (ml) Resolving Gel (ml) Stock
Solution 2.5% 5% 7.5% 10% 12.5% 15% 17% 20%
A 1.25 2.5 3.75 5.0 6.25 7.5 8.75 10.0 B - 2.0 2.0 2.0 2.0 2.0 20. 2.0 C 2.5 ------
D 1.0 1.5 1.5 1.5 1.5 1.5 1.5 1.5
E 0.5 0.75 0.75 0.75 0.75 0.75 0.75 0.75
TEMED 0.005 0.005 0.005 0.005 0.005 0.005 0.005 0.005
Water 5.5 8.5 7.75 6.5 5.25 4.0 2.75 1.5
34
2.2.5 Thermal/Photodegradation of Betamethasone Valerate and Betamethasone Dipropionate in Aqueous and Organic Media
2x10-4M solutions of the compounds were prepared in phosphate buffer (pH 7.5) and organic solvents e.g. methanol and acetonitrile. Zero time samples were withdrawn immediately while the remainder solutions were divided into 100 ml aliquots in 100 ml plastic capped glass bottles. The number of samples was so that a separate sample could be used for each analysis. The samples bottles were wrapped with aluminum foil for light protection and then placed in an oven at 40 oC. In the case of photodegradation the samples where stirred and flushed with purified oxygen gas for 30 minutes and then irradiated in the radiation chamber under controlled temperature (25±1 oC). The solutions were removed after regular time intervals and then subjected to HPLC analysis of the parent compounds and their major thermal or photodegrades as described in section 2.2.8. In the case of thermal degradation the samples were brought to room temperature before HPLC analysis. The effect of pH on the thermal stability of betamethasone esters was studied with citrophosphate buffers of different pH. 2x10-3M solutions (500 ml) of the esters were prepared by dissolving the exactly weighed quantities of the compounds in acetonitrile (100 ml) and then mixed with buffers (400 ml) of different pH. The pH of the final solutions was adjusted with 20% orthophosphoric acid or 1N sodium hydroxide to 2.5, 3.5, 4.5, 5.5, 6.5 and 7.5, respectively. The ionic strength (µ) was kept constant at 0.15M. Zero time samples were taken immediately. The remainder of the solution was divided into aliquots of 50 ml each in clean 100 ml volumetric flasks. All the flasks were wrapped with aluminum foil and then kept in an oven at 40 oC. The samples were removed at regular time intervals and the reaction was immediately terminated by adding 20% orthophosphoric acid or 1N sodium hydroxide solution to adjust pH of the samples to approximately 4.0. The volume of the samples was made upto 100 ml with acetonitrile after bringing the temperature of the samples to room temperature. Analysis of the samples was performed by a validated high performance liquid chromatographic assay method as described in section 2.2.8. A similar method was 35
used to study the effect of ionic strength and buffer concentration on the thermal/ photodegradation of betamethasone esters.
2.2.6 Thermal Degradation of Betamethasone Esters in Cream and Gel Formulations 2.2.6.1 Preparation of Cream and Gel Formulations Simple formulations (cream and gel) of the compounds were prepared in a laboratory scale silver san mixer. The formulae and manufacturing procedures of the formulations are as under:
2.2.6.1.1 Formulae
Cream Material % of the total formula Betamethasone ester 0.1 Carbomer (940) 1.5 Propylene glycol 8.0 Cetostearyl alcohol 7.0 Isopropyl alcohol 2.0 Ethyl paraben 0.2 Deionized water 81.0 1N Sodium Hydroxide solution q.s Gel Material % of the total formula Betamethasone ester 0.1 Carbomer (940) 0.7 Hydroxy ethyl cellulose 0.5 Propylene glycol 20 Di-isopropenolamine 0.5 Isopropyl alcohol 2.0 Ethyl paraben 0.2 Deionized water 75.9 4N Hydrochloric acid solution q.s 36
2.2.6.1.2 Manufacturing procedures
Cream
1. Carbomer 940 was soaked overnight in water 2. Betamethasone ester and ethyl paraben were dissolved in isopropyl alcohol 3. Propylene glycol, cetostearyl alcohol and remaining water were mixed together 4. Steps1, 2 and 3 were mixed together thoroughly in silver san mixer 5. pH was maintained with 1N sodium hydroxide solution under gentle mixing.
Gel
1. Carbomer 940 and Hydroxy ethyl cellulose were soaked overnight in water 2. Betamethasone ester and ethyl paraben were dissolved in isopropyl alcohol 3. Propylene glycol and remaining water were mixed together 4. Steps1, 2 and 3 were mixed together thoroughly in silver san mixer 5. Di-isopropanolamine was added to the mixture under vigorous mixing 6. pH was maintained with 4N hydrochloric acid solution under gentle mixing.
2.2.6.2 Method
Exactly weighed samples (cream or gel) were spreaded evenly (approx. 2mm thickness) in petri dishes and then placed in an oven at 40 oC. The samples were withdrawn at regular intervals for analysis. The whole content of the petri dish was dissolved in acetonitrile and then filtered through 0.45µ filter paper. The filtrate was diluted with acetonitrile to make the solution 0.1 mg/ ml. Assay of the parent compounds and their major thermal degrades was performed as described in section 2.2.8.
37
2.2.7 Photodegradation of Betamethasone Esters
2.2.7.1 Radiation chamber
The irradiation of betamethasone esters solutions, cream and gel formulations was carried out in a 2 x1.5 x1.75 feet (lxwxh) wooden chamber fitted with a wooden cover. Two small chambers, provided with arrangements for the fixation of the radiation source and a powerful exhaust fan for the temperature control, were fitted to the main chamber, one on the sidewall and one on the top of the chamber. Adjustable wooden supports were also provided inside the chamber for the placement of samples containers at particular distance i.e. 30 cm, from the radiation source. The temperature was maintained at 25± 10C throughout the course of irradiation. The intensity of light was measured with a digital illuminance meter (TES-1332A, TES.Electrical Corporation, Taiwan).
2.2.7.2 Radiation source
A 300 watt UV bulb (Ultra-vitalux, Osram, Germany) emitting in the region of 300- 400 nm was used in all photolytic studies. The technical data of the bulb is as under: Construction wattage =300 Construction voltage =230 Dimensions (h x w x l) = 203mm x134mm x131mm Base (standard designation) = E27 Illumination (at sample location) = approx 16,500 lux
2.2.7.3 Method a. Photodegradation in aqueous and organic solutions
Solutions of the compounds were irradiated in glass flasks/ glass bottles with horizontal beam of UV radiations ( 300-400 nm) for increasing time intervals in the radiation chamber. The samples were removed at regular intervals for the analysis of the parent compounds and their photodegradation products via HPLC method as mentioned in section 2.2.8.
38
b. Photodegradation in cream and gel formulations
Exactly weighed samples (cream or gel) were spreaded evenly (approx. 2mm thickness) in Petri dishes and then placed at a distance of 30 cm from the radiation source in the radiation chamber. The samples were irradiated with vertical beam of UV radiation and then removed at regular intervals for analysis. The whole content of the Petri dish was dissolved in acetonitrile and then filtered through 0.45µ filter paper. The filtrate was diluted with acetonitrile to make the solution 0.1 mg/ ml. Assay of the parent compounds and their major photodegrades was performed as described in section 2.2.8. The photodegradation of betamethasone esters in cream and gel formulations in the presence of photoprotectors was carried out by dissolving/ suspending 0.1% each of the photoprotector such as titanium dioxide, vanillin and butyl hydroxytoluene in isopropyl alcohol/ water and then mixing thoroughly with the cream or gel formulation in the laboratory scale silver san mixer. The samples were irradiated and analyzed accordingly by HPLC.
2.2.8 Assay of Betamethasone Valerate, Betamethasone Dipropionate and Their Major Thermal and Photodegrades
2.2.8.1 Preparation of calibration standard solutions
Standard stock solutions (12.5, 25, 50, 75 and 100 µg/ ml) of betamethasone-17- valerate were prepared in acetonitrile each containing 25µg beclomethasone dipropionate as an internal standard. For quantification of the thermal degradation products, betamethasone-21-valerate and betamethasone alcohol, solutions (12.5, 25, 50, 75 and 100 µg/ ml) of the degradation products were prepared in acetonitrile each containing 25µg beclomethasone dipropionate. Similarly, stock solutions of betamethasone-17, 21-dipropionate and its degradation products, betamethasone- 17- propionate, betamethasone-21-propionate and betamethasone alcohol, were prepared in acetonitrile in the same concentrations and with the same internal standard. 39
2.2.8.2 Sample preparation
An exactly weighed quantity of the formulation equivalent to 0.5 mg of betamethasone esters was mixed with 5 ml of acetonitrile and then made up the volume to 10 ml with the mobile phase. In case of liquid sample the volume of the sample containing 0.5 mg betamethasone ester was mixed with the volume of the mobile phase to make the final volume upto 10 ml. The mixture was filtered through 0.45µ filter paper prior to injection into the HPLC system.
2.2.8.3 Chromatographic procedure
A 20µl of sample or calibration standard solution was injected into the chromatographic system equipped with a 250mm x 4.6mm column that contained packing 5µ octadecylsilane and a 254 nm detector. The mobile phase was a filtered and degassed mixture of actonitrile and water (60:40, v/v) and the flow rate was about 1 ml/ minute. Injections of samples were alternated with calibration standard solutions until each sample had been injected at least three times. Peak height ratios of injected samples were compared with calibration standard solutions for the determination of the amount of the parent compounds and their major thermal degradation products. In case of photostability studies a mixture of acetonitrile and water (50:50, v/v) was used as a mobile phase. The photodegrades were detected at 210 nm while their estimation was made as percentage of the principal peak.
2.2.9 Photohemolysis
The whole blood of a healthy and untreated albino mouse, using heparin as anticoagulant, was obtained. The blood was washed with phosphate buffer saline (0.01M phosphate buffer, 0.135M NaCl, pH7.4) in centrifuge machine (2500rpm for 15min), and the supernatant was removed carefully. The procedure was repeated until the supernatant was colorless. Red blood cells were resuspended in phosphate buffer saline so that the resultant suspension had an optical density of 0.6-0.7 at 650 nm (corresponding to 10 6 cells/ ml). For photohemolysis experiments, small 40
volumes (less than 1% ) of pre-irradiated and untreated concentrated ethanol solutions of the compounds were added to RBC suspension (final concentration 50µM). The suspension was then irradiated with ultraviolet light (300-400 nm) under gentle shaking in a controlled temperature (25±1 oC) chamber for increasing time intervals. Samples containing scavengers like butyl hydroxyanisole (50µM) and sodium azide (50µM) were also irradiated similarly. Hemolysis was determined by measuring the decreasing optical density of the samples at 650 nm [126]. Control samples were (1) untreated RBC (2) RBC in the presence of untreated compounds and kept in the dark (3) RBC in the presence of pre-irradiated compounds and kept in the dark and (4) RBC irradiated without compounds.
2.2.10 Photoperoxidation of Linoleic Acid
Linoleic acid (1x10 -3M) in phosphate buffer saline (0.01M phosphate buffer, 0.135M NaCl, pH 7.4) containing 0.05% Tween 20 as emulsifying agent was irradiated with UV light (300-400 nm) in the presence of the compounds (1x10 -5M) for increasing time intervals. Peroxidaton of linoleic acid was determined by measuring the increasing absorbance at 233 nm corresponding to the conjugated dienic hydroperoxides formed during irradiation [127]. The process was repeated with pre-irradiated compounds also.
2.2.11 Protein Photodamage
For determination of protein photocross linking white membranes (ghosts) were irradiated with the untreated and pre-irradiated compounds and then subjected to polyacrylamide gel electrophoresis as described as under.
2.2.11.1 Preparation of white membranes (ghosts)
White membranes (ghosts) were prepared by gradual osmotic lysis method [128]. Whole blood collected from untreated albino mouse using heparin as anticoagulant, was centrifuged at 2500rpm for 15minutes for the separation of RBCs. The RBCs were washed three times with saline (0.9%NaCl) at 4 oC and subsequently lysed 1:40 with 50mM phosphate buffer (pH 8.3) at 4 oC. The membranes (ghosts) were 41
washed with the same lysis buffer at least five times at 25000g at 4 oC until a colorless solution of ghosts was obtained .The ghosts were aliquoted and stored at -70 oC . Ghosts were resuspended in phosphate buffer saline before use.
2.2.11.2 Determination of membranes protein contents
Membrane protein contents were determined by Bradford protein assay method using bovine serum albumin (BSA) as a standard [129]. Water and proteins were added into ten colorimetric tubes (10x100mm) according to the top three rows of table 6. Tube1 was used as a blank while tube 2 to tube 6 were for construction of a standard calibration curve. Tubes 7 to tube 10 were duplicates of two different concentrations of the ghost’s solution. 5 ml of dilute Bradford dye reagent was added to each tube and mixed well by gentle inversion .After a period of at least 5minutes absorbance of each tube was taken at 595 nm. Concentration of protein (mg/ ml) in the membrane ghosts was calculated from the standard calibration curve.
Table 6. Procedure for Bradford protein assay
Reagents T-1 T-2 T-3 T-4 T-5 T-6 T-7 T-8 T-9 T-10
Water 1.0 0.9 0.8 0.6 0.4 0.2 0.7 0.7 0.4 0.4
Standard - 0.1 0.2 0.4 0.6 0.8 - - - - BSA
Membrane ------0.3 0.3 0.6 0.6 protein
Dye solution 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0
42
2.2.11.3 Irradiation of ghosts/ compound suspension and polyacrylamide gel electrophoretic analysis
Pre-irradiated and untreated compounds dissolved in small volume of ethanol were added to the membrane suspension (3 mg/ ml protein concentration) and incubated in the dark for 15minutes before UV irradiation. 500µl samples were irradiated in 1mm quartz cuvettes in a controlled temperature (25± 1 oC) chamber for increasing time intervals. 40µl of the irradiated membrane samples were reduced and denatured by addition of -mercaptoethanol and sodium dodecyl sulphate (SDS) at 90 0C for 3minutes and bromophenol blue (BPB) was added before plyacrylamide gel electrophoretic analysis (8% running gel, 4% stacking gel). The electrophoresis was carried out at 100V for 4hrs in a Bio- rad power pac 300 apparatus, using 0.124 M Tris, 1mM glycine and 0.5% SDS as running buffer. The gel was stained with Coomassie brilliant blue R-250 solution and then washed with a mixture of methanol, acetic acid and water (5:7.5:87.5, v/v/v). The gel was then submitted to densitometric analysis by the photo and imaging software scanplot version 2.0. Controls used were (i) untreated ghosts (ii) ghosts irradiated without the compounds (iii) ghosts with the irradiated compounds and kept in the dark and (iv) ghosts with untreated compounds and kept in the dark.
RESULTS AND DISCUSSION
CHAPTER THREE
THERMAL DEGRADATION REACTIONS
45
3.1 Introduction
Betamethasone derivatives including the betamethasone valerate and betamethasone dipropionate are sensitive to heat [11, 64] and undergo degradation to form a number of products. Spectrophotometeric and chromatographic methods have been used for the identification of betamethasone esters and their degradation products. In the present work high performance liquid chromatography (HPLC) has been used for the identification of the thermal degradation products of betamethasone esters formed under the present conditions in organic solvents, phosphate buffer, cream and gel preparations.
3.2 Identification of the Thermal Degradation Products of Betamethasone Esters
The degradation products of betamethasone esters obtained during the present
reactions were identified by comparison of their tR values with those of the reference standards and are reported in Table 7. A typical chromatogram showing betamethasone valerate and its degradation products (betamethasone-21-valerate and betamethasone alcohol) formed in methanol is shown in Fig 4. In all the media (organic solvents, phosphate buffer, cream and gel) only two thermal products were identified (Table 7). These products are formed at a relatively low temperature (40 0C) and are produced by the ester group migration from C17 to C21, and further hydrolysis as proposed by yip et al . [64] (Fig 5). In the case of betamethasone dipropionate three degradation products (betamethasone-17-propionate, betamethasone -21-propionate and betamethasone alcohol) were identified by HPLC in all the media studied. A typical chromatogram of betamethasone dipropionate and its degradation products formed in methanol is shown in Fig 6. The degradation of betamethasone dipropionate with the products formed is shown in Fig 7.The reaction involves deacylation (C17 and C21), interconversion of 17 to 21-propionate and further hydrolysis to betamethasone alcohol. Some minor products were also identified in all the media.
46
Table 7. Thermal degradation products of betamethasone esters (40 oC).
Compound Medium Degradation Products
Betamethasone valerate Acetonitrile, methanol, Betamethsone-21- valerate phosphate buffer ( pH 7.5), Betamethasone alcohol cream, gel
Betamethasone dipropionate Acetonitrile, methanol, Betamethasone-17-propionate phosphate buffer ( pH 7.5), Betamethasone-21-propionate cream, gel Betamethasone alcohol
47
[mV] Betamethasone alcohol
Betamethasone 5.0 -17-valerate Betamethasone -21-valerate
Beclomethasone 2.5 dipropionate
0
0.0 2.5 5.0 7.5 10.0 12.5 min
Figure 4. HPLC chromatogram showing betamethasone-17-valerate and its thermal degradation products, betamethasone-21-valerate and betamethasone alcohol with internal standard beclomethasone dipropionate.
48
O O
H3C O H3C O OH O H C O OH H 3 H H3C HO HO
H Ester group migration H H3C H H3C H
CH3 CH3
F H F H
O O BETAMETHASONE-21-VALERATE
H BETAMETHASONE-17-VALERATE y d r o l y s O i s H H3C HO OH OH H H3C H
CH3
F H
O
BETAMETHASONE ALCOHOL
Figure 5. Degradation pathway for the thermal transformation of betamethasone-17- valerate into betamethasone-21-valerate and betamethasone alcohol.
49
[mV] Betamethasone-17-propionate
20
10 Betamethasone alcohol Betamethasone-21-propionate
Betamethasone Beclomethasone dipropionate dipropionate
0
0.0 2.5 5.0 7.5 10.0 12.5 min
Figure 6. HPLC chromatogram showing betamethasone dipropionate and its thermal degradation products betamethasone-17-propionate, betamethasone-21- propionate and betamethasone alcohol with internal standard beclomethasone dipropionate.
50
O
H3C O
O O H H3C HO
CH3 H H3C H O
CH3
F H
O
BETAMETHASONE DIPROPIONATE Deacylation Deacylation O
H3C O O
O OH H H3C O HO OH H H3C HO
OH CH3 H H H3C H H3C H O CH3 CH3 Ester group migration F H F H
O O
BETAMETHASONE-17-PROPIONATE BETAMETHASONE-21-PROPIONATE
H y d r o l y s i s O
OH H H3C HO OH
H3C H H
CH3
F H
O BETAMETHASONE ALCOHOL
Figure 7. Proposed degradation pathways of betamethasone dipropionate to give betamethasone-17-propionate, betamethasone-21-propionate and betamethasone alcohol. Thick arrows indicate major pathways.
51
3.3 Assay of Betamethasone Esters and Degradation Products
Various spectrophotometeric and chromatographic methods have been used for the assay of betamethasone esters and their thermal degradation products [59, 98-101]. In the present case the United States Pharmacopeia (USP) method based on HPLC has been used for the assay of betamethasone esters and their thermal degradation products. The USP method is mainly used for the determination of the purity of betamethasone esters, however, it has been found that this method could be used for the assay of betamethasone esters as well as their degradation products because there
is sufficient difference in their t R values . The method was validated under the present experimental conditions before its application to the assay of betamethasone esters and their degradation products formed in various media.
3.3.1 Validation
3.3.1.1 Specificity
In order to ensure that the excipients of the formulations do not contribute to the peaks of betamethasone esters and their degradation products, reference standards, cream, gel and placebo cream and placebo gel were separately dissolved in methanol and then analyzed by HPLC method as described in section 2.2.9. No interference was found from the excipients.
3.3.1.2 Linearity
Linearity was determined by constructing calibration curves of betamethasone esters and their degradation products. Calibration curves constructed on the basis of peaks height ratios of the reference standards / internal standard versus reference standards concentrations were linear over the concentration range studied. The linearity data is shown in Table 8.
52
3.3.1.3 Precision (Repeatability)
Repeatability was determined by carrying out six replicate assays on a sample of betamethasone esters and the overall RSD was found to be within 2%.
3.3.1.4 Accuracy (Recovery)
Accuracy studies were performed on cream and gel formulations only. This was performed by adding known amounts of the esters to the formulations followed by the normal assay procedure. The results in Table 9-10 indicate that accuracy of the method is acceptable since overall mean of the recovery is within 97-103%.
The assay data on betamethasone esters and their degradation products in organic solvents, phosphate buffer (pH 7.5), cream and gel preparations is given in Table 11-20.
53
Table 8. Linearity data of betamethasone esters and their thermal degradation products.
Compound Slope Corr. Coefficient
Betamethasone-17-valerate 0.0192 0.994
Betamethasone-21-valerate 0.0165 0.995
Betamethasone alcohol 0.0625 0.994
Betamethasone dipropionate 0.0135 0.998
Betamethasone-21-propionte 0.0241 0.999
Betamethasone-17-propionate 0.0218 0.994
54
Table 9. Recoveries of betamethasone valerate from spiked samples.
Dosage form µg added µg found % Recovery
250.3 249.05 99.50 249.9 250.65 100.30 250.2 254.2 101.59 Cream 251.5 247.23 98.30 247.8 250.03 100.89 Mean: 100.116 RSD: 1.27%
249.8 250.78 100.39 250.9 249.15 99.30 248.5 251.3 101.12 Gel 251.3 247.92 98.65 255.1 255.35 100.09 Mean: 99.91 RSD: 0.96%
Table 10. Recoveries of betamethasone dipropionate from spiked samples.
Dosage form µg added µg found % Recovery
253.7 259.0 102.08 254.1 257.3 101.26 250.8 252.52 100.67 Cream 250.2 246.2 98.40 246.5 244.51 99.18 Mean: 100.32 RSD: 1.5%
250.7 247.85 98.86 251.9 259.45 103.00 245.2 249.49 101.75 Gel 248.2 249.19 100.40 253.1 248.62 98.23 Mean: 100.45 RSD: 1.97% 55
Table 11. Assay of betamethasone-17-valerate and degradation products formed in methanol (40 0C).
Degradation products Betamethasone Time (Hour) -17-valerate Betamethasone-21- Betamethasone (M x 10 5) valerate alcohol (M x 10 5) (M x 105) 0 10.04 0.00 0.00 24 8.69 1.06 0.34 48 7.40 2.00 1.16 72 6.00 2.43 2.19 96 4.75 2.90 3.04 120 3.65 3.35 4.10 144 2.72 3.73 4.97
Table 12. Assay of betamethasone-17-valerate and degradation products formed in acetonitrile (40 0C).
Degradation products Betamethasone Time (Hour) -17-valerate Betamethasone-21- Betamethasone (M x 10 5) valerate alcohol (M x 10 5) (M x 10 5) 0 9.97 0.00 0.00 24 8.80 0.91 0.00 48 7.62 1.84 0.32 72 6.51 2.35 1.12 96 5.40 2.73 2.06 120 4.23 2.92 3.23 144 3.24 3.18 3.97
56
Table 13. Assay of betamethasone-17-valerate and degradation products formed in phosphate buffer (pH 7.5) at 40 0C.
Degradation products Betamethasone Time (Hour) -17-valerate Betamethasone-21- Betamethasone (M x 10 5) valerate alcohol (M x 10 5) (M x 10 5) 0 10.14 0.00 0.00 24 9.20 0.79 0.12 48 8.29 1.58 0.87 72 7.31 2.19 1.45 96 6.45 2.73 2.30 120 5.48 2.90 3.65 144 4.60 3.38 4.50
Table 14. Assay of betamethasone-17-valerate and degradation products formed in cream (40 0C).
Degradation products Betamethasone Time (Hour) -17-valerate Betamethasone-21- Betamethasone (M x 10 5) valerate alcohol (M x 10 5) (M x 10 5) 0 10.26 0.00 0.00 24 10.08 0.21 0.00 48 9.91 0.20 0.06 72 9.75 0.24 0.15 96 9.60 0.33 0.20 120 9.43 0.31 0.30 144 9.29 0.32 0.59
57
Table 15. Assay of betamethasone-17-valerate and degradation products formed in gel (40 0C).
Degradation products Betamethasone Time (Hour) -17-valerate Betamethasone-21- Betamethasone (M x 10 5) valerate alcohol (M x 10 5) (M x 10 5) 0 10.02 0.00 0.00 24 9.92 0.13 0.00 48 9.83 0.21 0.00 72 9.75 0.24 0.11 96 9.63 0.20 0.18 120 9.56 0.29 0.25 144 9.45 0.31 0.38
Table 16. Assay of betamethasone dipropionate and degradation products formed in methanol (40 0C).
Degradation products Time Betamethasone (Hour) dipropionate Betamethasone Betamethasone Betamethasone (M x 10 5) -17-propionate -21-propionate alcohol (M x 10 5) (M x 10 5) (M x 10 5) 0 9.98 0.00 0.00 0.00 24 9.55 0.00 0.18 0.00 48 9.19 0.00 0.43 0.00 72 8.78 0.15 0.60 0.00 96 8.42 0.29 0.77 0.00 120 8.00 0.38 0.94 0.13 144 7.63 0. 43 1.13 0.28
58
Table 17. Assay of betamethasone dipropionate and degradation products formed in acetonitrile (40 0C).
Degradation products Time Betamethasone (Hour) dipropionate Betamethasone- Betamethasone- Betamethasone (M x 10 5) 17-propionate 21-propionate alcohol (M x 10 5) (M x 10 5) (M x 10 5) 0 10.01 0.00 0.00 0.00 24 9.69 0.00 0.11 0.00 48 9.36 0.00 0.20 0.00 72 9.00 0.00 0.39 0.00 96 8.70 0.09 0.45 0.00 120 8.41 0.16 0.52 0.00 144 8.10 0.21 0.63 0.04
Table 18. Assay of betamethasone dipropionate and degradation products formed in phosphate buffer (pH 7.5) at 40 0C.
Degradation products Time Betamethasone (Hour) dipropionate Betamethasone- Betamethasone- Betamethasone (M x 10 5) 17-propionate 21-propionate alcohol (M x 10 5) (M x 10 5) (M x 10 5) 0 9.93 0.00 0.00 0.00 24 9.78 0.00 0.15 0.00 48 9.65 0.14 0.24 0.10 72 9.49 0.23 0.40 0.18 96 9.37 0.25 0.51 0.34 120 9.20 0.28 0.67 0.52 144 9.09 0.28 0.69 0.74
59
Table 19. Assay of betamethasone dipropionate and degradation products formed in cream (40 0C).
Degradation products Betamethasone Time (Hour) dipropionate Betamethasone-17- Betamethasone-21- (M x 10 5) propionate propionate (M x 10 5) (M x 10 5) 0 10.04 0.00 0.00 24 9.98 0.00 0.04 48 9.88 0.00 0.04 72 9.79 0.00 0.09 96 9.71 0.00 0.13 120 9.65 0.00 0.19 144 9.58 0.03 0.26
Table 20. Assay of betamethasone dipropionate and degradation products formed in gel (40 0C).
Degradation products Betamethasone Time (Hour) dipropionate Betamethasone-17- Betamethasone-21- (M x 10 5) propionate propionate (M x 10 5) (M x 10 5) 0 10.26 0.00 0.00 24 10.19 0.00 0.00 48 10.14 0.00 0.05 72 10.07 0.00 0.13 96 10.00 0.00 0.17 120 9.96 0.12 0.21 144 9.91 0.08 0.25
60
3.4 Kinetics of Thermal Degradation
The thermal degradation of betamethasone valerate and betamethasone dipropionate involves complex reactions as shown in Figure 5 and 7, respectively. The molecules are quite stable and in cream and gel formulations undergo less than 10% degradation at 40 oC in 144 hours. The HPLC determination of these esters is accurate (Section 3.3.1) and the analytical data represent the residual amount of betamethasone valerate and betamethasone dipropionate during the degradation reactions.
In order to evaluate the rate of degradation of these compounds the analytical data obtained on betamethasone valerate and betamethasone dipropionate (Table 11-20) were subjected to kinetic treatment. The thermal degradation of betamethasone esters has been shown to follow first-order kinetics. The first-order plots for the reactions carried out in various media are shown in Fig 8-17 and the apparent first- o order rate constants, kobs , for the degradation reactions at 40 C are reported in Table 21. The correlation coefficients for the rate constants are in the range of 0.990-0.999.
It appears that the rate of degradation of betamethasone esters decreases generally in the order of the medium. Organic solvents > phosphate buffer > cream > gel
Thus betamethasone esters are most stable in semisolid preparations. The evaluation of the kinetics of the thermal degradation reactions of betamethasone esters on the basis of first-order kinetics is a simplified treatment of these reactions. As shown in Figure 5 the degradation of betamethasone valerate is a consecutive first-order reaction involving betamethasone-21-valerate as an intermediate. However, the reaction may be considered as an overall first-order degradation for which the rate constants have been reported. The degradation of betamethasone dipropionate involves the formation of betamethasone-21-propionate and betamethasone-17- propionate. In these reactions betamethasone-21-propionate is the major reaction 61 product which is further degraded to betamethasone alcohol and betamethasone-17- propionate is the minor degradation product which is converted to betamethasone- 21-propionate during the reaction. Therefore, the overall degradation of betamethasone dipropionate could be considered to follow first-order kinetics. This has been observed in the treatment of the analytical data for betamethasone dipropionate and on the basis the values of apparent first-order rate constants for the overall degradation have been reported.
62
1.2
1
0.8
0.6
0.4
0.2
0 0 24 48 72 96 120 144 168
Time (Hour)
Figure 8. First-order plot for the degradation of betamethasone valerate in methanol (40 oC).
1.2
1
0.8
0.6
0.4
0.2
0 0 24 48 72 96 120 144 168
Time (Hour)
63
Figure 9. First-order plot for the degradation of betamethasone valerate in acetonitrile (40 oC).
1.2
1
0.8
0.6
0.4
0.2
0 0 24 48 72 96 120 144 168 Time (Hour)
Figure 10. First-order plot for the degradation of betamethasone valerate in phosphate buffer (pH 7.5) at 40 oC.
1.01
1.005
1
0.995
0.99
0.985
0.98
0.975
0.97 0 24 48 72 96 120 144 168 Time (Hour)
Figure 11. First-order plot for the degradation of betamethasone valerate in cream (40 oC). 64
1.005
1
0.995
0.99
0.985
0.98
0.975
0.97 0 24 48 72 96 120 144 168 Time (Hour)
Figure 12. First-order plot for the degradation of betamethasone valerate in gel (40 oC).
1.02
1
0.98
0.96
0.94
0.92
0.9
0.88
0.86 0 24 48 72 96 120 144 168 Time (Hour)
Figure 13. First-order plot for the degradation of betamethasone dipropionate in methanol (40 oC).
65
1.02
1
0.98
0.96
0.94
0.92
0.9 0 24 48 72 96 120 144 168 Time (Hour)
Figure 14. First-order plot for the degradation of betamethasone dipropionate in acetonitrile (40 oC).
1 0.995 0.99 0.985 0.98 0.975 0.97 0.965 0.96 0.955 0 24 48 72 96 120 144 168 Time (Hour)
Figure 15. First-order plot for the degradation of betamethasone dipropionate in phosphate buffer (pH 7.5) at 40 oC.
66
1.005
1
0.995
0.99
0.985
0.98
0.975 0 24 48 72 96 120 144 168 Time (Hour)
Figure 16. First-order plot for the degradation of betamethasone dipropionate in cream (40 oC).
1.012 1.01 1.008 1.006 1.004 1.002 1 0.998 0.996 0.994 0 24 48 72 96 120 144 168 Time (Hour)
Figure 17. First-order plot for the degradation of betamethasone dipropionate in gel (40 oC).
67
Table 21. Apparent first-order rate constants ( kobs ) for the thermal degradation of betamethasone-17-valerate and betamethasone dipropionate (40 oC).
Betamethasone-17-valerte Betamethasone dipropionate
3 -1 3 -1 Medium Dielectric Constant kobs x10 , hr Corr. kobs x10 , hr Corr. 25 oC Coefficient Coefficient
Methanol 32.6 9.07 0.992 1.87 0.999
Acetonitrile 40.1 7.78 0.990 1.46 0.999 pH 7.5 78.5 5.48 0.994 0.59 0.997
Cream --- 0.479 0.994 0.30 0.993
Gel --- 0.399 0.998 0.239 0.998
68
3.5 Solvent Effect
In the present work organic and aqueous solvents have been used to study the thermal degradation of betamethasone valerate and betamethasone dipropionate and the rate constants (Table 21) in these solvents have been determined. Organic solvents are known to influence the rate of degradation of drugs and the formulator may take the advantage of this fact in the preparation and formulation development of sensitive drugs. The degradation of pharmaceutical compounds in a medium depends on solvent characteristics including the dielectric constant which is a measure of the polarity of a medium [36, 130]. To find out a relation between the rate of degradation of betamethasone valerate and betamethasone dipropionate and
the dielectric constant of the medium, plots of kobs versus dielectric constants of the medium were prepared (Figures 18-19). It has been observed that the rate of thermal degradation for both the compounds decreases with an increase in the dielectric constant. This indicates the participation of a non-polar intermediate in the thermal degradation reaction. The activity of the intermediate is increased in the solvents of decreased polarity which is due to the existence of a non-polar intermediate in the reaction.
3.6 pH Effect
Thermal degradation reaction on betamethasone esters were carried out in the pH range 2.5-7.5. The relationship between the rate of degradation and pH is discussed below.
3.6.1 pH- Rate Profile
The pH-rate profile for the thermal degradation of betamethasone dipropionate (Fig 20) represents the break down of the ester side chain followed by hydrolysis. The molecule may undergo specific acid-base catalysis resulting in an increase in the rate with a decrease in pH in the acid region and with an increase in rate in pH in the alkaline region. 69
10 9 8 7 6
. 5 4 3 2 1 0 0 10 20 30 40 50 60 70 80 90 Dielectric constant.
( ) Methanol ( ) Acetonitrile ( ) Water (Phosphate buffer (pH 7.5) Figure 18. Dependence of the rate constant of thermal degradation of betamethasone valerate on the solvent dielectric constant.
2 1.8 1.6 1.4 1.2
. 1 0.8 0.6 0.4 0.2 0 0 10 20 30 40 50 60 70 80 90 Dielectric constant.
( ) Methanol ( ) Acetonitrile ( ) Water (Phosphate buffer (pH 7.5) Figure 19. Dependence of the rate constant of thermal degradation of betamethasone dipropionate on the solvent dielectric constant. 70
The very slow rate around pH 4.5 appears to be due to the solvent catalytic effect, that is, the un-ionized water-catalyzed reaction of the molecule. The rate law for the acid-base catalyzed reaction may be written as:
+ - kobs = ko + k1 [H ] + k2 [OH ]
+ At low pH the term k 1 [H ] is greater and specific hydrogen ion catalysis is observed. Similarly, at high pH, the concentration of [OH -] is greater and specific hydroxyl ion catalysis is observed. This explains the v-shaped pH-rate profile for the thermal degradation of betamethasone dipropionate. The pH-rate profile for the thermal degradation of betamethasone velarate (Fig 21) represents ester hydrolysis over the pH range 2.5-7.5 and probably involves an intermediate in the reaction as observed in the case of the hydrolysis of hydrochlorothiazide [131]. The profile indicates an increase in the rate in the pH range 2.5-3.5 due to H + ion catalysis. This is followed by a relatively pH independent region extending over the range of pH 3.5-4.5. On increasing the pH there is a gradual increase in the rate above pH 4.5. This appears to be due to the water / hydroxyl ion-catalyzed hydrolysis of the molecule in the neutral and alkaline region. The hydrolysis of betamethasone valerate represents v-shaped curve and is a case of specific acid-base catalyzed degradation.
71
-1.1
-1.6
-2.1
-2.6
-3.1
-3.6
-4.1
-4.6 2 3 4 5 6 7 8 pH
Figure 20. pH-rate profile for the degradation of betamethasone dipropionate (40 oC).
-1
-1.5
-2
-2.5
-3
-3.5
-4 2 3 4 5 6 7 8 pH
Figure 21. pH-rate profile for the degradation of betamethasone-17-valerate (40 oC).
72
3.6.2 Product Distribution
The product distribution (% ratio) at 10% thermal degradation of betamethasone- 17-valerate in the pH range 2.5 to 5.5 is given in Table 22. It appears that the thermal degradation of betamethasone-17-valerate increases as a function of pH in the range of 2.5-5.5 leading to the formation of betamethasone-21-valerate (8.33- 9.65%) and betamethasone alcohol (0.17-0.9%). The degradation of betamethasone- 17-valerate leads to the formation of betamethasone alcohol through betamethasone-21-valerate as an intermediate in the reaction. Some minor unknown products were also found during the degradation reaction.
The product distribution (% ratio) at 10% thermal degradation of betamethasone dipropionate is reported in Table 23. Betamethasone dipropionate leads to the formation of betamethasone-17-propionate, betamethasone-21-propionate and betamethasone alcohol. However, betamethasone-21-propionate is the only product formed at pH 2.5. Betamethasone-21-propionate and betamethasone alcohol are the only products formed at pH 3.5 and 4.5. Betamethasone-17-propionate and betamethasone-21-propionate are the only products formed at pH 7.5. Betamethasone-17-propionate, betamethasone-21-propionate and betamethasone alcohol are all formed at pH 5.5 and 6.5. The formation of betamethasone-17- propionate increases with pH whereas the formation of betamethasone-21- propionate and betamethasone alcohol decreases with pH in the pH range 2.5-7.5. It appears that in the pH range 2.5-4.5 any betamethasone-17-propionate formed is unstable and is converted to betamethasone-21-propionate. Since betamethasone alcohol is formed through betamethasone-21-propionate, its decreased formation with pH is in accordance with the decreased formation of betamethasone-21- propionate with pH. It also indicates that betamethasone alcohol is a product of betamethasone-21-propionate.
73
Table 22. Product distribution at 10% thermal degradation of betamethasone-17-valerate (40 oC).
Betamethasone-21- Betamethasone pH valerate alcohol 2.5 8.33 0.17 3.5 9.10 0.90 4.5 9.55 0.45 5.5 9.65 0.35
Table 23. Product distribution at 10% thermal degradation of betamethasone dipropionate (40 oC).
Betamethasone-17- Betamethasone-21- Betamethasone pH propionate propionate alcohol 2.5 - 10.00 - 3.5 - 9.20 0.80 4.5 - 6.80 3.20 5.5 0.48 8.68 0.83 6.5 3.18 6.69 0.13 7.5 5.39 4.61 -
74
3.7 Buffer Effect
In order to observe the effect of phosphate buffer (pH 7.5) on the rate of thermal degradation of betamethasone esters, reactions were carried out in the presence of 0.05-0.2 M buffer. The concentrations of betamethasone esters determined during the reactions at various time intervals are given in Table 24-25. The apparent first- order rate constants (Table 26) were determined from the slopes of the log concentration versus time plots (Fig 22-29). The second-order rate constants
determined from the slopes of the plots of kobs versus phosphate concentration are reported as 3.02x10 -6 M-1s-1 and 1.305x10 -6 M-1s-1 for betamethasone valerate and betamethasone dipropionate degradation, respectively. The plots show that buffer -3 -1 causes inhibition of the reaction. This is evident from the values of k0 {(5.5x10 hr (betamethasone valerate) and 1.22x10 -3 hr -1 (betamethasone dipropionate)} which are higher than those in the presence of the buffer. This observation is in agreement with the effect of phosphate buffer on the degradation of mometasone furoate [52]. This may be due to the interaction of phosphate with thermally activated species leading to the inhibition of the reaction.
75
Table 24. Concentration of betamethasone valerate (Mx10 5) at various buffer (Phosphate) concentration (0.05-0.2M) at 40 oC.
Phosphate Time (Hour) Concentration 0 24 48 72 96 (M)
0.05 10.00 9.18 8.23 7.2 6.22
0.10 10.00 9.24 8.30 7.38 6.54
0.15 10.00 9.32 8.51 7.72 6.89
0.20 10.00 9.40 8.78 8.00 7.27
Table 25. Concentration of betamethasone dipropionate (Mx10 5) at various buffer (Phosphate) concentration (0.05-0.2M) at 40 oC.
Phosphate Time (Hour) Concentration 0 24 48 72 96 (M)
0.05 10.00 9.80 9.59 9.36 9.14
0.10 10.00 9.83 9.64 9.47 9.31
0.15 10.00 9.89 9.76 9.62 9.50
0.20 10.00 9.95 9.89 9.83 9.78
76
1.1
1
0.9
0.8
0.7
0.6
0.5
0.4 0 24 48 72 96 Time (Hour)
Figure 22. First-order plot of the thermal degradation of betamethasone valerate (40 oC) at pH 7.5 (0.05M phosphate buffer).
1.1
1
0.9
0.8
0.7
0.6
0.5
0.4 0 24 48 72 96 Time (Hour)
Figure 23. First-order plot of the thermal degradation of betamethasone valerate (40 oC) at pH 7.5 (0.1M phosphate buffer). 77
1.1
1
0.9
0.8
0.7
0.6
0.5
0.4 0 24 48 72 96 Time (Hour)
Figure 24. First-order plot of the thermal degradation of betamethasone valerate (40 oC) at pH 7.5 (0.15M phosphate buffer).
1.1
1
0.9
0.8
0.7
0.6
0.5
0.4 0 24 48 72 96 Time (Hour)
Figure 25. First-order plot of the thermal degradation of betamethasone valerate (40 oC) at pH 7.5 (0.2M phosphate buffer).
78
1.005 1 0.995 0.99 0.985 0.98 0.975 0.97 0.965 0.96 0.955 0 24 48 72 96 Time (Hour)
Figure 26. First-order plot of the thermal degradation of betamethasone dipropionate (40 oC) at pH 7.5 (0.05M phosphate buffer).
1.005
1
0.995
0.99
0.985
0.98
0.975
0.97
0.965 0 24 48 72 96 Time (Hour)
Figure 27. First-order plot of the thermal degradation of betamethasone dipropionate (40 oC) at pH 7.5 (0.1M phosphate buffer).
79
1.005
1
0.995
0.99
0.985
0.98
0.975 0 24 48 72 96 Time (Hour)
Figure 28. First-order plot of the thermal degradation of betamethasone dipropionate (40 oC) at pH 7.5 (0.15M phosphate buffer).
1.002
1
0.998
0.996
0.994
0.992
0.99
0.988 0 24 48 72 96 Time (Hour)
Figure 29. First-order plot of the thermal degradation of betamethasone dipropionate (40 oC) at pH 7.5 (0.2M phosphate buffer).
80
6
5.5
5
4.5
4
3.5
3
2.5
2 0 0.05 0.1 0.15 0.2 0.25
Phosphate concentration (M)
Figure 30. Plot of kobs vs phosphate concentration of thermal degradation of betamethasone valerate (40 oC) at pH 7.5.
1.4
1.2
1
0.8
0.6
0.4
0.2
0 0 0.05 0.1 0.15 0.2 0.25 Phosphate concentration (M)
Figure 31. Plot of kobs vs phosphate concentration of thermal degradation of betamethasone dipropionate (40 oC) at pH 7.5.
81
Table 26. Apparent first-order rate constants ( kobs ) for the thermal degradation of betamethasone-17-valerate and betamethasone dipropionate at various o phosphate concentrations (40 C).
Betamethasone-17-valerte Betamethasone dipropionate
3 -1 3 -1 Phosphate kobs x10 , hr Corr. Coefficient kobs x10 , hr Corr. Coefficient concentration (M)
0.05 4.965 0.995 0.938 0.999
0.10 4.438 0.997 0.746 0.999
0.15 3.886 0.996 0.534 0.999
0.20 3.334 0.995 0.233 0.999
82
3.8 Ionic Strength Effect
The effect of ionic strength on the thermal degradation of betamethasone valerate and betamethasone dipropionate was also studied in sodium phosphate buffers (pH 7.5) of ionic strength 0.3, 0.6, 0.9, 1.2 and 1.5M. The ionic strength was adjusted with KCl. The concentrations of betamethasone esters determined during the reactions at various time intervals are given in Table 27-28. The observed rate of degradation of both compounds followed first-order kinetics over the ionic strength tested. The log concentration versus time plots for both compounds are shown in Fig 32-41. The first-order rate constants determined from the slopes of the lines are reported in Table 29. Plots of the values of the first-order rate constants against the ionic strength (Fig 42-43) showed that the rate of degradation decreased with increasing ionic strength implying that the degradation is influenced by the ionic
strength of phosphate buffer. The value of k0 determined by extrapolation to zero ionic strength is 4.80x10 -3 hr -1 and 0.85x10 -3 hr -1 for betamethasone valerate and betamethasone dipropionate, respectively.
Plots of log k/k0 against the square root of ionic strength (Fig 44-45) were found to be linear (Corr. coefficient, 0.992 and 0.991) suggesting that the relationship is obeyed for the values of ionic strength investigated (0.3-1.5M). The number of unit
charges Z A Z B, calculated from the slopes of the plots using the Debye-Huckel
equation (log k/k0 = 1.02Z √ u) were found to be 0.386 and 0.612 for betamethasone valerate and betamethasone dipropionate, respectively. These results do not support the applicability of Debye-Huckel limiting law as the values obtained are much lower than the values expected from the Debye-Huckel equation.This may be due to the high ionic strength and temperature used in this study, whereas Debye-Huckel equation assumes for a reaction involving ions in a diluted aqueous solution (u < 0.01) at 25 ºC.
83
Table 27. Concentration of betamethasone valerate (Mx10 5) at different ionic strength (0.3-1.5M) at 40 oC.
Ionic Strength Time (Hour) (M) 0 24 48 72 96
0.3 10.0 9.21 8.3 7.44 6.55
0.6 10.0 9.3 8.4 7.58 6.82
0.9 10.0 9.37 8.58 7.94 7.18
1.2 10.0 9.36 8.74 8.14 7.45
1.5 10.0 9.41 8.86 8.29 7.70
Table 28. Concentration of betamethasone dipropionate (Mx10 5) at different ionic strength (0.3-1.5M) at 40 oC.
Ionic Strength Time (Hour) (M) 0 24 48 72 96
0.3 10.0 9.81 9.64 9.45 9.28
0.6 10.0 9.85 9.69 9.55 9.40
0.9 10.0 9.88 9.75 9.63 9.51
1.2 10.0 9.90 9.79 9.70 9.59
1.5 10.0 9.92 9.86 9.77 9.68
84
1.1
1
0.9
0.8
0.7
0.6
0.5
0.4 0 24 48 72 96 Time (Hour)
Figure 32. First-order plot of the thermal degradation of betamethasone valerate (40 oC) at pH 7.5 (µ = 0.3M).
1.1
1
0.9
0.8
0.7
0.6
0.5
0.4 0 24 48 72 96 Time (Hour)
Figure 33. First-order plot of the thermal degradation of betamethasone valerate (40 oC) at pH 7.5 (µ = 0.6M).
85
1.1
1
0.9
0.8
0.7
0.6
0.5
0.4 0 24 48 72 96 Time (Hour)
Figure 34. First-order plot of the thermal degradation of betamethasone valerate (40 oC) at pH 7.5 (µ = 0.9M).
1.1
1
0.9
0.8
0.7
0.6
0.5
0.4 0 24 48 72 96 Time (Hour)
Figure 35. First-order plot of the thermal degradation of betamethasone valerate (40 oC) at pH 7.5 (µ = 1.2M).
86
1.1
1
0.9
0.8
0.7
0.6
0.5
0.4 0 24 48 72 96 Time (Hour)
Figure 36. First-order plot of the thermal degradation of betamethasone valerate (40 oC) at pH 7.5 (µ = 1.5M).
1.02
1
0.98
0.96
0.94
0.92
0.9
0.88 0 24 48 72 96 Time (Hour)
Figure 37. First-order plot of the thermal degradation of betamethasone dipropionate (40 oC) at pH 7.5 (µ = 0.3M).
87
1.02
1
0.98
0.96
0.94
0.92
0.9
0.88 0 24 48 72 96 Time (Hour)
Figure 38. First-order plot of the thermal degradation of betamethasone dipropionate (40 oC) at pH 7.5 (µ = 0.6M).
1.02
1
0.98
0.96
0.94
0.92
0.9
0.88 0 24 48 72 96 Time (Hour)
Figure 39. First-order plot of the thermal degradation of betamethasone dipropionate (40 oC) at pH 7.5 (µ = 0.9M).
88
1.02
1
0.98
0.96
0.94
0.92
0.9
0.88 0 24 48 72 96 Time (Hour)
Figure 40. First-order plot of the thermal degradation of betamethasone dipropionate (40 oC) at pH 7.5 (µ = 1.2M).
1.02
1
0.98
0.96
0.94
0.92
0.9
0.88 0 24 48 72 96 Time (Hour)
Figure 41. First-order plot of the thermal degradation of betamethasone dipropionate (40 oC) at pH 7.5 (µ = 1.5M).
89
Table 29. Apparent first-order rate constants ( kobs ) for the thermal degradation of betamethasone-17-valerate and betamethasone dipropionate at various ionic strength ( 40 oC ).
Betamethasone-17-valerte Betamethasone dipropionate
3 -1 3 -1 Ionic strength kobs x10 , hr Corr. Coefficient kobs x10 , hr Corr. Coefficient (M)
0.3 4.414 0.997 0.779 0.999
0.6 3.989 0.998 0.645 0.999
0.9 3.452 0.998 0.525 0.999
1.2 3.070 0.998 0.455 0.999
1.5 2.734 0.999 0.359 0.998
90
6
5
4
3
2
1
0 0 0.3 0.6 0.9 1.2 1.5 1.8
Ionic strength (µ)
Figure 42. Plot of kobs vs ionic strength (µ) of thermal degradation of betamethasone valerate (40 oC) at pH 7.5.
1
0.8
0.6
0.4
0.2
0 0 0.3 0.6 0.9 1.2 1.5 1.8 Ionic strength (µ)
Figure 43. Plot of kobs vs ionic strength (µ) of thermal degradation of betamethasone dipropionate (40 oC) at pH 7.5.
91
0.2
0.1
0
-0.1
-0.2
-0.3
-0.4 0 0.2 0.4 0.6 0.8 1 1.2 1.4
µ
Figure 44. Plot of log (kobs /ko) vs µ of thermal degradation of betamethasone valerate (40 oC) at pH 7.5.
0.4
0.2
0
-0.2
-0.4
-0.6
-0.8
-1
-1.2 0 0.2 0.4 0.6 0.8 1 1.2 1.4
µ
Figure 45. Plot of log ( kobs /ko) vs µ of thermal degradation of betamethasone dipropionate (40 oC) at pH 7.5.
CHAPTER FOUR
PHOTOCHEMICAL DEGRADATION REACTIONS
93
4.1 Introduction
It is well established that betamethasone esters are sensitive to light and undergo photodegradation on UV irradiation (Section 1.5.1.3). Therefore the degradation of betamethasone esters was also studied on exposure to UV light. The photochemical degradation of betamethasone valerate and betamethasone dipropionate led to the formation of different products which were separated by HPLC as in the case of the thermal degradation products (Section 3.2) for the reactions carried out in organic solvents, phosphate buffer, gels and creams. The details of photodegradation reactions are given in the following section.
4.2 Identification of the Photodegradation Products of Betamethasone Esters
The HPLC chromatograms showing the peaks of betamethasone valerate, betamethasone dipropionate and their photoproducts are presented in Figure 46 and 47, respectively. In the case of batamethasone valerate two major products
(A and B) were detected with t R values of 14.1 and 20.9min, respectively. In the case of betamethasone dipropionate two major products (C and D) were detected
with t R values of 19.28 and 31.2min, respectively. It appears from the t R values of the photoproducts of the two compounds that the products formed from these compounds are not similar. The comparison of UV spectra of the degradation products with those of the parent compounds (Figure 48-49) indicating a similarity in the structural features of the degradation products. The absorption maxima of the two photoproducts of betamethasone valerate (A and B) occur at 204 and 214nm and 198 and 223nm which are different from those of the absorption maxima of betamethasone valerate i.e. 198 and 241nm. Similarly the two photoproducts of betamethasone dipropionate (C and D) exhibit absorption maxima at 201nm and 204 and 215nm, respectively. Since the absorption maxima of betamethasone dipropionate appear at 198 and 241nm, the two photoproducts of this compound may be considered as having different chemical structures. This is supported by the
fact that the photoproducts of the two compounds have different t R values and consequently different chemical structures. In view of the absence of reference
94
mAU
200 Betamethasone -17-valerate 150
Photoproduct B 100 Photoproduct A
50
0
0 10 20 30
Time (min) Figure 46. HPLC chromatogram showing betamethasone-17-valerate and its photoproducts A and B.
mAU
Betamethasone 200 dipropionate 150
Photoproduct D 100 Photoproduct C
50
0
0 10 20 30
Time (min) Figure 47. HPLC chromatogram showing betamethasone dipropionate and its photoproducts C and D.
95
1 . 0 0 4 . 0 0 0 . 1 0
0 . 9 0 3 . 6 0
0 . 8 0 3 . 2 0 0 . 0 8
0 . 7 0 2 . 8 0
0 . 6 0 2 . 4 0 0 . 0 6
0 . 5 0 2 . 0 0
0 . 4 0 1 . 6 0 0 . 0 4
0 . 3 0 1 . 2 0
0 . 2 0 0 . 8 0 0 . 0 2
0 . 1 0 0 . 4 0
0 . 0 0 0 . 0 0 0 . 0 0 2 0 0 2 5 0 3 0 0 3 5 0 4 0 0 2 0 0 2 5 0 3 0 0 3 5 0 4 0 0 2 0 0 2 5 0 3 0 0 3 5 0 4 0 0 Wavelength (nm) Wavelength (nm) Wavelength (nm) ( 1 ) ( A ) ( B )
Figure 48. UV spectra of betamethasone-17-valerate (1) and its photoproducts A and B.
1 . 0 0 2 . 0 2 . 0
0 . 9 0 1 . 8 1 . 8
0 . 8 0 1 . 6 1 . 6
0 . 7 0 1 . 4 1 . 4
0 . 6 0 1 . 2 1 . 2
0 . 5 0 1 . 0 1 . 0
0 . 4 0 0 . 8 0 . 8
0 . 3 0 0 . 6 0 . 6
0 . 2 0 0 . 4 0 . 4
0 . 1 0 0 . 2 0 . 2
0 . 0 0 0 . 0 0 . 0 2 0 0 2 5 0 3 0 0 3 5 0 4 0 0 2 0 0 2 5 0 3 0 0 3 5 0 4 0 0 2 0 0 2 5 0 3 0 0 3 5 0 4 0 0 Wavelength (nm) Wavelength (nm) Wavelength (nm) ( 2 ) ( C ) ( D )
Figure 49. UV spectra of betamethasone dipropionate (2) and its photoproducts C and D. 96
standards a complete identification of the photodegradation products of betamethasone esters could not be achieved.
4.3 Assay of Betamethasone Esters and Photodegradation Products
In order to quantify betamethasone valerate, betamethasone dipropionate and their photodegradation products, all the compounds were assayed by HPLC method (USP 2009). Since the nature of photodegradation products is not known, it was assumed that these products give a similar detector response as that of betamethasone valerate and betamethasone dipropionate. Therefore, the degradation products were assayed with reference to the peak height of the parent compounds, respectively. The values of assay data on the photodegradation of betamethasone valerate and betamethasone dipropionate are given in Table 30-31.
4.4 Product Distribution In order to observe the composition of the photoproducts in 10% degraded sample of the betamethasone esters the % ratios were determined by normalization. The ratios for the major unknown products (A and B) and the minor unknown products of betamethasone valerate are given in Table 32 while for betamethasone dipropionate and its major unknown products (C and D) and minor unknown products in Table 33. The formation of product A, B and minor are in the range of 2.60-7.90 %, 1.46-6.30 % and 0.64-1.10 %, respectively. The formation of product C, D and minor are in the range of 3.30-9.40 %, 1.66-5.70 % and 0.60-1.00 %, respectively.
4.5 Kinetics of Photolysis
Photolysis of betamethasone esters was carried out in different media i.e. methanol, acetonitrile, phosphate buffer (pH 7.5), cream and gel formulations. Kinetic treatment of the assay data (Table 30-31) of photolysis of betamethasone esters in different media has been shown to follow first-order kinetics. The first- order plots for the photolytic reactions carried out in different media are shown in Fig 48-57
and the apparent first-order rate constants ( kobs ) are reported in Table 34. 97
Table 30. Assay of betamethasone-17-valerate on photodegradation in different media.
Time Acetonitrile Methanol Phosphate buffer, pH Cream Gel (min) (M x 10 5) (M x 10 5) 7.5 (M x 10 5) (M x 10 5) (M x 10 5)
0 10.05 9.98 9.95 10.14 9.92
30 7.30 7.10 7.53 9.57 9.45
60 5.33 5.00 5.70 9.05 9.02
90 3.85 3.56 4.31 8.52 8.63
120 2.80 2.57 3.26 7.97 8.17
Table 31. Assay of betamethasone dipropionate on photodegradation in different media.
Time Acetonitrile Methanol Phosphate buffer, Cream Gel (min) (M x 10 5) (M x 10 5) pH 7.5 (M x 10 5) (M x 10 5) (M x 10 5)
0 10.0 9.96 10.20 9.89 10.09
30 8.04 7.88 8.45 9.40 9.75
60 6.45 6.25 7.00 8.94 9.41
90 5.27 5.06 5.82 8.48 9.10
120 4.19 3.98 4.78 8.10 8.84
98
Table 32. Product distribution at 10% photodegradation of betamethasone-17-valerate in different media.
Photoproduct Photoproduct Minor Medium A B Photoproducts
Acetonitrile 7.90 1.46 0.64
Methanol 2.80 6.30 0.90
Phosphate 2.60 6.30 1.10 buffer (pH7.5)
Cream 6.60 3.40 --
Gel 5.70 4.30 --
Table 33. Product distribution at 10% photodegradation of betamethasone dipropionate in different media.
Photoproduct Photoproduct Minor Medium C D Photoproducts
Acetonitrile 4.95 4.20 0.85
Methanol 3.30 5.70 1.00
Phosphate 9.40 -- 0.60 buffer (pH7.5)
Cream 7.30 2.70 --
Gel 8.34 1.66 --
99
1.2
1
0.8
0.6
0.4
0.2
0 0 30 60 90 120 150 Time (min)
Figure 50. First-order plot for the photodegradation of betamethasone valerate in methanol.
1.1 1 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0 30 60 90 120 150
Time (min)
Figure 51. First-order plot for the photodegradation of betamethasone valerate in acetonitrile.
100
1.1 1 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 30 60 90 120 150
Time (min)
Figure 52. First-order plot for the photodegradation of betamethasone valerate in phosphate buffer (pH 7.5).
1
0.98
0.96
0.94
0.92
0.9
0.88
0.86 0 30 60 90 120 150
Time (min)
Figure 53. First-order plot for the photodegradation of betamethasone valerate in cream.
101
1
0.98
0.96
0.94
0.92
0.9
0.88 0 30 60 90 120 150 Time (min)
Figure 54. First-order plot for the photodegradation of betamethasone valerate in gel.
1.1 1 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 30 60 90 120 150
Time (min)
Figure 55. First-order plot for the photodegradation of betamethasone dipropionate in methanol.
102
1.1 1
0.9 0.8 0.7 0.6
0.5 0.4 0.3 0 30 60 90 120 150
Time (min)
Figure 56. First-order plot for the photodegradation of betamethasone dipropionate in acetonitrile.
1.2 1.1 1 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 0 30 60 90 120 150 Time (min)
Figure 57. First-order plot for the photodegradation of betamethasone dipropionate in phosphate buffer (pH 7.5).
103
1.1
1.05
1
0.95
0.9
0.85
0.8 0 30 60 90 120 150
Time (min)
Figure 58. First-order plot for the photodegradation of betamethasone dipropionate in cream.
1.1
1.05
1
0.95
0.9
0.85
0.8 0 30 60 90 120 150
Time (min)
Figure 59. First-order plot for the photodegradation of betamethasone dipropionate in gel.
104
Table 34. Apparent first-order rate constants ( kobs ) for the photodegradation of betamethasone-17-valerate and betamethasone dipropionate.
Betamethasone-17-valerte Betamethasone dipropionate
3 -1 3 -1 Medium Dielectric kobs x10 , min Corr. kobs x10 , min Corr. Constant Coefficient Coefficient (25 0C)
Methanol 32.6 11.303 0.999 7.657 0.999
Acetonitrile 40.1 10.651 0.999 7.254 0.999
Phosphate 78.5 9.288 0.999 6.314 0.999 buffer (pH 7.5)
Cream --- 2.007 0.999 1.663 0.999
Gel --- 1.617 0.999 1.101 0.999
105
The correlation coefficients for the rate constants are in the range of 0.998-0.999. It appears that photodegradation generally decreases in the order of the medium as: organic solvents > phosphate buffer > cream > gel
The mode of photodegradation of betamethasone valerate and betamethasone dipropionate is not known. In the present work to major photoproducts of each compound have been detected. However, the route of their formation can not be speculated on the basis of the analytical data for more than 50 % degradation. It may be concluded that these esters undergo photodegradation by first-order kinetics. The rate constants indicate that betamethasone valerate degrades faster than betamethasone dipropionate, suggesting that betamethasone valerate is more susceptible to photodegradation compared to that of the betamethasone dipropionate.
4.5.1 Solvent Effect
It has been observed that solvent dielectric constant plays an important role in the thermal degradation of betamethasone valerate and betamethasone dipropionate (Section 3.5). In order to observe the role of solvent on the rate of photodegradation
of betamethasone valerate and betamethasone dipropionate, plots of kobs versus the solvent dielectric constant were prepared (Figure 60-61) and a behavior similar to that observed in the case of thermal degradation was indicated. Thus the thermal and photodegradation of betamethasone valerate and betamethasone dipropionate are influenced by the solvent dielectric constant and the rate is increased with a decrease in the solvent dielectric constant suggesting the presence of a non-polar intermediate in the reaction.
4.5.2 Buffer Effect
Photodegradation of betamethasone esters was carried out in varying concentration of phosphate buffer as in the case of thermal degradation (Section 3.6). Plots of the
kobs versus buffer concentration are shown in Figures 62-63. Similar to the behavior of thermal degradation a decrease in the rate is observed in photodegradation with 106
12
10
8
6
4
2
0 0 10 20 30 40 50 60 70 80 90 Dielectric constant
Figure 60. Dependence of the rate constant of photodegradation of betamethasone valerate on the solvent dielectric constant. ( ) Methanol ( ) Acetonitrile ( ) Water (Phosphate buffer (pH 7.5)
9 8 7 6 5 4 3 2 1 0 0 10 20 30 40 50 60 70 80 90 Dielectric constant
Figure 61. Dependence of the rate constant of photodegradation of betamethasone dipropionate on the solvent dielectric constant. ( ) Methanol ( ) Acetonitrile ( ) Water (Phosphate buffer (pH 7.5) 107
16
14
12
10
8
6
4
2
0 0 0.05 0.1 0.15 0.2 Phosphate concentration (M)
Figure 62. Plot of kobs vs phosphate concentration of photodegradation of betamethasone valerate at pH 7.5.
12
10
8
6
4
2
0 0 0.05 0.1 0.15 0.2 Phosphate concentration (M)
Figure 63. Plot of kobs vs phosphate concentration of photodegradation of betamethasone dipropionate at pH 7.5.
108
an increase in the buffer concentration in both cases. Therefore, the buffer causes an inhibition in the rate of reaction. This may be due to deactivation of the excited species with an increase in buffer concentration. A decrease in the rate of degradation of such compounds has been observed with an increase in phosphate buffer [52]. It may be concluded that phosphate buffer has a significant effect on the photodegradation kinetics of betamethasone ester.
4.5.3 Ionic Strength Effect
The rate of photodegradation of both esters decreases with an increase in ionic strength of phosphate buffer (Figure 64-65) as observed in the case of thermal degradation. The explanation of the effect of ionic strength on the rate of photodegradation has been presented in section 3.7.
4.6 Photostabilization of Betamethasone Esters in Cream and Gel Formulations
Various materials have been used to stabilize corticosteroids in semisolid preparations against photodegradation (Section 1.8). The photostabilization technology used for the photostabilization of pharmaceutical dosage forms has been dealt in detail by Piechocki and Thoma [132]. Some work was also carried out on the photostabilization of betamethasone esters in cream and gel formulations. In order to observe the effect of excipients as stabilizers on the photodegradation of betamethasone esters in cream and gel formulations, photolysis of these esters was carried out in the presence of 0.1% each of titanium dioxide (Light scatterer), vanillin (Spectral stabilizer) and butyl hydroxytoluene (Spectral stabilizer/ Free radical scavenger/ weak singlet oxygen quencher). The concentrations of the esters determined during the reaction in the presence of stabilizers at various time intervals are given in Table 35-38. The first-order rate constants (Table 39) were determined from the slopes of the log concentration versus time plots (Figure 66-77). 109
12
10
8
6
4
2
0 0 0.3 0.6 0.9 1.2 1.5 1.8 Ionic strength (µ)
Figure 64. Plot of kobs vs ionic strength (µ) of the photodegradation of betamethasone valerate at pH 7.5.
8
7
6
5
4
3
2
1
0 0 0.3 0.6 0.9 1.2 1.5 1.8 Ionic strength (µ)
Figure 65. Plot of kobs vs ionic strength (µ) of the photodegradation of betamethasone dipropionate at pH 7.5.
110
The data provide a better indication of the loss of these drugs in the presence and absence of these stabilizers. It appears that titanium dioxide is the most effective stabilizer used in this study followed by vanillin and butyl hydroxytoluene. In the case of cream formulations of betamethasone valerate and betamethasone dipropionate protected with titanium dioxide, the loss is decreased to the extent of about 12.92% and 10.39%, respectively as compared to 21.4% and 18.09% in the control. Similarly in gel preparations the loss of the two compounds is decreased to the extent of about 11.67% and 7.96%, respectively as compared to 17.64% and 12.38% in the control. Thus it is evident that titanium dioxide acts as an effective stabilizer in the capacity of a photoprotector for controlling the photodegradation of betamethasone valerate and betamethasone dipropionate. It appears to play its role as a light scattering agent in the photodegradation of these compounds and thus protect them from photodegradation. The loss of the esters in vanillin protected cream and gel formulations is decreased to the extent of 15.50% and 12.90%, 13.76% and 9.20%, respectively while with butyl hydroxytoluene protected cream and gel formulations the decrease is upto the extent of 16.96% and 14.18%, 14.41% and 9.84%, respectively. The vanillin and butyl hydroxytoluene are effective as protectors in the formation of spectral overlay (Figure 78-79) for these compounds and in this capacity provide photoprotection to the drugs.
111
Table 35. Assay of betamethasone valerate (M x 10 5) on photodegradation in creams.
Cream containing Cream containing Cream containing Time (min) Titanium dioxide Vanillin Butyl hydroxytoluene
0 9.98 10.06 9.96
9.65 9.67 9.55 30
9.34 9.26 8.12 60
8.98 8.88 8.68 90
120 8.69 8.50 8.27
Table 36. Assay of betamethasone valerate (M x 10 5) on photodegradation in gels.
Gel containing Gel containing Gel containing Time (min) Titanium dioxide Vanillin Butyl hydroxytoluene
0 10.02 9.95 9.99
30 9.72 9.60 9.63
60 9.41 9.27 9.29
90 9.13 8.93 8.92
120 8.85 8.58 8.55
112
Table 37. Assay of betamethasone dipropionate (M x 10 5) on photodegradation in creams.
Cream containing Cream containing Cream containing Time (min) Titanium dioxide Vanillin Butyl hydroxytoluene
0 10.10 9.96 10.08
30 9.83 9.62 9.72
60 9.57 9.30 9.37
90 9.32 8.98 9.03
120 9.05 8.67 8.65
Table 38. Assay of betamethasone dipropionate (M x 10 5) on photodegradation in gels.
Gel containing Gel containing Gel containing Time (min) Titanium dioxide Vanillin Butyl hydroxytoluene
0 9.92 10.00 9.95
30 9.72 9.78 9.70
60 9.53 9.54 9.46
90 9.34 9.31 9.21
8.97 120 9.13 9.08
113
1.02
1
0.98
0.96
0.94
0.92
0.9 0 30 60 90 120 150 Time (min)
Figure 66. First-order plot for the photodegradation of betamethasone-17-valerate in cream (stabilized with 0.1% titanium dioxide).
1.02
1
0.98
0.96
0.94
0.92
0.9 0 30 60 90 120 150 Time (min)
Figure 67. First-order plot for the photodegradation of betamethasone-17-valerate in cream (stabilized with 0.1% vanillin). 114
1.01 0.99 0.97 0.95 0.93 0.91 0.89
0.87 0.85 0 30 60 90 120 150 Time (min)
Figure 68. First-order plot for the photodegradation of betamethasone-17-valerate in cream (stabilized with 0.1% butyl hydroxytoluene).
1.01
1
0.99
0.98
0.97
0.96
0.95
0.94 0 30 60 90 120 150 Time (min)
Figure 69. First-order plot for the photodegradation of betamethasone-17-valerate in gel (stabilized with 0.1% titanium dioxide).
115
1.02
1
0.98
0.96
0.94
0.92
0.9 0 30 60 90 120 150 Time (min)
Figure 70. First-order plot for the photodegradation of betamethasone-17-valerate in gel (stabilized with 0.1% vanillin).
1.02
1
0.98
0.96
0.94
0.92
0.9 0 30 60 90 120 150 Time (min)
Figure 71. First-order plot for the photodegradation of betamethasone-17-valerate in gel (stabilized with 0.1% butyl hydroxytoluene).
116
1.01
1
0.99
0.98
0.97
0.96
0.95 0 30 60 90 120 150 Time (min)
Figure 72. First-order plot for the photodegradation of betamethasone dipropionate in cream (stabilized with 0.1% titanium dioxide).
1.02
1
0.98
0.96
0.94
0.92
0.9 0 30 60 90 120 150 Time (min)
Figure 73. First-order plot for the photodegradation of betamethasone dipropionate in cream (stabilized with 0.1% vanillin).
117
1.02
1
0.98
0.96
0.94
0.92
0.9 0 30 60 90 120 150 Time (min)
Figure 74. First-order plot for the photodegradation of betamethasone dipropionate in cream (stabilized with 0.1% butyl hydroxytoluene).
1
0.99
0.98
0.97
0.96
0.95
0.94 0 30 60 90 120 150 Time (min)
Figure 75. First-order plot for the photodegradation of betamethasone dipropionate in gel (stabilized with 0.1% titanium dioxide).
118
1.01
1
0.99
0.98
0.97
0.96
0.95
0.94 0 30 60 90 120 150 Time (min)
Figure 76. First-order plot for the photodegradation of betamethasone dipropionate in gel (stabilized with 0.1% vanillin).
1.01
1
0.99
0.98
0.97
0.96
0.95
0.94 0 30 60 90 120 150 Time (min)
Figure 77. First-order plot for the photodegradation of betamethasone dipropionate in gel (stabilized with 0.1% butyl hydroxytoluene).
119
Table 39. Apparent first-order rate constants ( kobs ) for the photodegradation of betamethasone-17-valerate and betamethasone dipropionate in cream and gel formulations containing 0.1% each of titanium dioxide, vanillin and butyl hydroxytoluene*
Betamethasone-17-valerte Betamethasone dipropionate
3 -1 3 -1 Formulation kobs x10 , min Corr. kobs x10 , min Corr. Coefficient Coefficient
Cream containing 1.153 0.999 0.909 0.999 TiO 2 Cream containing 1.393 0.999 1.155 0.999 vanillin Cream containing 1.548 0.999 1.274 0.999 BHT Gel containing 1.019 0.999 0.692 0.998 TiO 2 Gel containing 1.235 0.999 0.806 0.999 vanillin Gel containing 1.297 0.999 0.865 0.999 BHT
* Rates of loss of betamethasone valerate and betamethasone dipropionate in cream and gel formulations in the absence of the photoprotectors (Table 34) are 2.007 x 10 -3 -1.663 x10 -3 min -1 and 1.617 x10 -3 -1.101 x10 -3 min -1, respectively.
120
1 . 0 0 0
A b 0 . 5 0 0 s Betamethasone valerate .
Vanillin
0 . 0 0 0 2 0 0 . 0 2 5 0 . 0 300.0 350.0 400.0 Wavelength (nm.)
Figure 78. UV spectrum of vanillin showing spectral overlay with betamethasone esters.
1 . 0 0 0
A b 0 . 5 0 0 s Betamethasone valerate .
BHT
0 . 0 0 0 2 0 0 . 0 2 5 0 . 0 300.0 350.0 400.0 Wavelength (nm.)
Figure 79. UV spectrum of butyl hydroxytoluene showing spectral overlay with betamethasone esters.
CHAPTER FIVE
IN VITRO PHOTOTOXICITY TESTING
122
5.1 Introduction
Screening for phototoxicity in vitro is necessary before introducing drugs into clinical therapy. It is not only important for prevention of any untoward drug reaction in humans but is also helpful in investigating new drugs of any pharmacological group with minor phototoxic properties. Corticosteroids have been shown to cause phototoxicity in animals and aquatic organisms [120, 89]. In vitro experiments also reveal the potential phototoxicity of these drugs [116, 133]. Therefore, the common in vitro phototoxicity screening tests i.e. photohemolysis assay, lipids photoperoxidation and protein photodamage, were also performed on betamethasone esters to assess any possible phototoxic effects of these drugs.
5.2 Photohemolysis
The photohemolytic activity of betamethasone esters was evaluated by irradiating mouse RBC (10 6 cells/ ml) in phosphate buffer saline (0.01M Phosphate buffer, 0.135M NaCl, pH 7.4) containing betamethasone esters (50µM). The hemolysis induced by the betamethasone esters and their photoproducts is shown in Figure 80- 83. Hemolysis was not induced by the compounds in the dark or the light alone. Betamethasone valerate showed greater photohemolytic activity (97%) than the betamethasone dipropionate (74%) in 60 minutes. BHA (free radical scavenger) strongly inhibited photohemolysis caused by both compounds (34% and 47% in
betamethasone valerate and betamethasone dipropionate, respectively). NaN 3 (singlet oxygen quencher) also inhibited the process to some extent (7% and 21%, respectively). Photoproducts of both compounds were able to induce hemolysis in the dark. Betamethasone valerate photoproducts caused complete hemolysis in the dark in 30 minutes. On the other hand, 37% hemolysis was produced by betamethasone dipropionate photoproducts. The hemolytic activity of the photoproducts was increased by further irradiation (complete hemolysis in 20 min and 70% hemolysis in 30 min in betamethasone valerate and betamethasone dipropionate photoproducts, respectively). Hence photoproducts of both compounds showed more toxicity than the parent compounds. The exact mechanism of 123
photohemolysis caused by the betamethasone esters could not be confirmed,
however, strong inhibition by BHA and minor role of NaN 3 probably support the involvement of free radical intermediates in the process. The generation of free radical intermediates has already been reported in these compounds [84].
5.3 Lipid Photoperoxidation
The membrane lipids are one of the main targets in membrane photodamage, therefore, lipid photoperoxidation was investigated using linoleic acid as the unsaturated lipid model. Both compounds showed significant photoperoxidation of linoleic acid (Figure84-85) as evidenced by an increase in the absorption of linoleic acid solution at 233nm due to the formation of conjugated dienic hydroperoxides as a function of irradiation dose.
5.4 Protein Photodamage
Membrane proteins are another target in membrane photodamage, therefore, the drug induced photodamage was evaluated on membrane proteins by measuring the photosensitizing cross-linking in erythrocyte ghosts. Densitometric scanning of the polyacrylamide gel electrophoresis of the erythrocyte ghosts irradiated in the presence of both compounds for increasing time intervals or pre-irradiated compounds and then mixed with the ghosts, did not show any cross-linking of proteins. This observation is in agreement with a previous study on triamcinolone acetonide [109]. It is concluded that betamethasone esters have phototoxic potential under UV irradiation as evidenced by the phothemolysis and lipid photoperoxidation tests. The observed photohemolysis is due to the peroxidation of the lipids in the cell membranes. Furthermore, the phototoxicity mechanism for betamethasone esters most probably involves the reaction of free radical species with cellular components. No information is available on these aspects, therefore, further investigations are required to explore the phototoxicity of these drugs on other biological molecules.
124
100 90 80 70 60 50 40 30 20 10 0 0 10 20 30 40 50 60 Time (min)
Figure 80. Effects of additives on the photohemolysis of RBC induced by betamethasone -17-valerate (1). ( ) RBC (hv) ( ) RBC+1(dark) ( ) RBC+1(hv) ( ) RBC+BHA+1(hv)
( ) RBC + NaN3 + 1 (hv).
125
100 90 80 70 60 50 40 30 20 10 0 0 5 10 15 20 25 30 Time (min)
Figure 81. Hemolysis induced by betamethasone-17-valerate photoproducts in the dark ( ) and further UV irradiation ( ).
126
100 90 80 70 60 50 40 30 20 10 0 0 10 20 30 40 50 60 Time (min)
Figure 82. Effects of additives on the photohemolysis of RBC induced by betamethasone Dipropionate (2).
( ) RBC (hv) ( ) RBC+2(dark) ( ) RBC+2(hv) ( ) RBC+BHA+2(hv)
( ) RBC + NaN3 +2 (hv).
127
100 90 80 70 60 50 40 30 20 10 0 0 5 10 15 20 25 30 35 40 Time (min)
Figure 83. Hemolysis induced by betamethasone dipropionate photoproducts in the dark ( ) and further UV irradiation ( ).
128
1 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 0 10 20 30 40 50 60 Time (min)
Figure 84. Photoperoxidation of linoleic acid (10 -3M) sensitized by betamethasone -17-Valerate (1). ( ) Linoleic acid+1 (dark) ( ) Linoleic acid (hv) ( ) Linoleic acid+1 (hv).
129
0.5
0.4
0.3
0.2
0.1
0 0 10 20 30 40 50 60 Time (min)
Figure 85. Photoperoxidation of linoleic acid (10 -3M) sensitized by betamethasone dipropionate (2). ( ) Linoleic acid+2 (dark) ( ) Linoleic acid (hv) ( ) Linoleic acid+2 (hv).
CONCLUSIONS
131
CONCLUSIONS
Betamethasone esters are extensively used in various topical formulations for a variety of dermatological disorders. These compounds are sensitive to heat and light, and may undergo a change in potency in these formulations under adverse storage conditions. The present work involves the study of the following aspects of betamethasone esters.
1. Identification of Degradation Products of Betamethasone Esters
An HPLC method has been used for the characterization of the thermal and photodegradation products of betamethasone esters in aqueous and organic solvents and in cream and gel formulations. The thermal degradation products of betamethasone valerate and betamethasone dipropionate are betamethasone -21- valerate and betamethasone alcohol and betamethasone-17-propionate, betamethasone -21-propionate and betamethasone alcohol , respectively indicating a difference in the mode of degradation of these compounds. The photodegradation of these esters leads to the formation of two products each of betamethasone valerate
and betamethasone dipropionate. These products are unknown and their t R values have been reported. The identification of thermal degradation products was based
on the comparison of the t R values with those of the reference standards.
2. Assay of Betamethasone Esters and Their Degradation Products
The USP HPLC method, validated under the present experimental conditions, has been applied to the assay of betamethsone esters and their thermal and photodegradation products. The RSD of the method has been found to be within 2%. The product distribution at 10% degradation of both esters has been reported indicating a difference in the nature of thermal and photodegradation products. This may involve a change in the mode of these degradation reactions
132
3. Kinetics of Degradation Reactions
The thermal and photodegradation of betamethasone esters follows first-order
kinetics. The values of apparent first-order rate constants (kobs ) of thermal and photochemical reactions in different media (methanol, acetonitrile, phosphate buffer and cream and gel formulations) are in the range of 0.239-9.07x10 -3 hr -1 and 1.101-11.303x10 -3 min -1, respectively. The pH-rate profiles of these esters may be represented by V-shaped curves indicating acid-base catalyzed reactions.
4. Solvent Effect
The plots of (kobs ) versus solvent dielectric constants are linear for both esters and indicate a decrease in the rate of thermal and photodegradation as a function of solvent polarity. This suggests the involvement of a non-polar intermediate in the degradation reactions.
5. Buffer and Ionic Strength Effects
A study of the effect of concentration and ionic strength of phosphate buffer indicates that the rate of reaction is inhibited by the buffer species. This could be explained on the basis of the interaction of buffer with the excited species causing deactivation and hence a decrease in the rate of reaction.
6. Photostabilization of Betamethasone Esters in Cream and Gel Formulations
The use of vanillin and butyl hydroxytoluene as agents causing spectral overlay of betamethasone esters and titanium dioxide acting as a light scattering agent are effective in the photostabilization of these esters. Titanium dioxide is more effective as a photostabilizer compared with the other agents (vanillin and butyl hydroxytoluene).
133
7. Phototoxicity
The evaluation of the phototoxicity of betamethasone esters using the in vitro phototoxicity tests such as photohemolysis, lipid peroxidation and protein photodamage indicates that these esters are phototoxic and cause hemolysis of mouse red blood cells. Photoproducts of these esters have been found to be toxic in the dark also. The phototoxicity mechanism for betamethasone esters could not be confirmed, however, it may involve the reaction of free radical species with cellular components. Appropriate precautions should be taken in the use of dermatological preparations containing these compounds.
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