stability of betamethasone esters in some topical …

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 (40oC) 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 tR 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

C=O

OH

CH3

O

CH3

O

CORTISONE

CH2OH

C=O

OH

CH3

HO

CH3

O

HYDROCORTISONE

CH2OH

C=O

OH

CH3

O

CH3

O

PREDNISONE

CH2OH

C=O

OH

CH3

HO

CH3

O

PREDNISOLONE

CH2OH

C=O

OH

CH3

HO

CH3

O

FLUDROCORTISONE

F

CH2OH

C=O

OH

CH3

HO

CH3

O

METHYLPREDNISOLONE

CH2OH

C=O

OH

CH3

HO

CH3

O

DEXAMETHASONE

CH2OH

C=O

OH

CH3

HO

CH3

O

TRIAMCINOLONE

F

CH3

F

CH3OH

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%

Diflucortolone valerate 0.3%

Fluocinolone valerate 0.2%

Halcinonide 0.1%

Ulobetasol propionate 0.05%

Amcinonide 0.1%

Beclomethasone dipropionate 0.025%

Betamethasone benzoate 0.025%

Betamethasone dipropionate 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%

Betamethasone valerate 0.025%

Fludroxycortide 0.0125%

Flumetasone pivalate 0.002%

Prednicarbate 0.25%

Fluocinolone acetonide 0.0025%

Hydrocortisone 0.5% & 1%

Hydrocortisone acetate 1%

Methylprednisolone acetate 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

characteristics

Betamethasone Valerate Betamethasone

Dipropionate

Molecular formula

C27H37FO6 C28H37FO7

Molecular weight

476.6 504.6

Appearance White or creamy-white

crystalline powder

Almost white crystalline

powder

Solubility

Practically insoluble in water,

soluble in alcohol, freely

soluble in acetone and in

dichloromethane

Practically insoluble in

water, sparingly soluble in

alcohol, freely soluble in

acetone and in

dichloromethane

Melting point

About 190 0C with

decomposition

About 170 to 179 0C with

decomposition

Optical rotation

[�]250 = + 65.7

0

Dioxane

[�]270

= + 89.40

Methanol

[�]250 = + 77

0

Dioxane

UV max (nm) 238 (�1.57 x 104)

239 (�1.592 x 104)

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 C28H37FO7 and it has the following chemical structure [12].

O

H3C

F H

H

HO

H H3C

H3C

O

O

O

O

CH3

O

H

CH3

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 C27H37FO6 and it has the following chemical structure [12].

O

H3C

F H

H

HO

H H3C

H3C

O

O

O

OH

H

CH3

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.

C-17 Steroidal ester C-21 Steroidal ester

Hydrolysis Hydrolysis

Steroid base

Ester group migration

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).

C

OH

OH2C

OH

(1)

C

OH

OC

OH

(2)

H CH

OH

(3)

OHOHC

O

(4)

+

CHO

CH2 OH

GLYCOLALDEHYDE

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

C

OH

OH2C

OH

(1)

COOH

H

(5)

O2

HO-

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).

C

OH

OH2C

OH

(1)

O2

HO-

C

OH

OCHC

(6)

H2O

CH

OH

OHOOC

(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

CO

OHO

O

hν

CH2OR

CO

OHO

O

H

1a , R=H

1b , R=COCH3

2a , R=H

2b , R=COCH3

14

The general scheme for the lumi rearrangements is given as below.

O

hν

O

neutral

O

H

CH3

αααα − attack

H2O

O

HO

H3O

H2O

ββββ − attack

CH3

OH

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.

COCH2OR

OH

O

hν

COCH2OR

OH

O

R1

X

R3

R2

SolidR1

R3

XR2

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

O

hν

X

F

HO

H

H

OCH2OH

O

O

X

F

HO

H

H

OCH2OH

O

O

O

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).

O

HO

OCH2OH

HO

hν

O

HO

HO

O

HO

H2O

HO

HO HO

O

HO

OH

HO

OH

O

O

PREDNISOLONE

Scheme 1

O

hν

HO

OCH2OH

HO

H2O O

HO

OCH2OH

HO

OH

Scheme 2

PREDNISOLONE

17

O

HO

OCH2OH

HO

hν

PREDNISOLONE

Scheme 3a

O

O

O

OHO

H2O

O

O

O

OCH2OH

HO

Scheme 3b

O CH2OH

OH

CH3

HO

HO

CH3

OCH2OH

CH3

hν

H2O

OH

H

H

F

HO

O

DEXAMETHASONE

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

OH

O

hν, N2

X

Solid

X

O

O

H

OH H, COCH3

X R

HYDROCORTISONE/ ACETATE C

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.

O

HO

O

COCH2OH

OH

CH3H

F

F

Cl

O

HO

CH3H

F

F

Cl

O

O

HO

O

COCH2OH

OH

CH3H

F

F

Cl

hν

Solid

HALOMETHASONE

F

F

O

Cl

OH

HOCH2COHO

CH3

O

HOHO

CH3H

F

Cl

CO

CHOH

19

O

HO

COCH2OCOCH2CH3

OCO2CH2CH

hν

Solid

PREDNICARBATE

O

HO

COCH2OR

OR1

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.

Substance

Pure drug/

Dosage

form

Adsorbent Solvent

System

Rf Values of the

parent compound and

its thermal degrades

Reference

Betamethasone

valerate

Semisolid

ointment

bases

Silica gel 60

Chloroform

ethylacetate

(1:1, v/v)

Bet-17-valerate = 0.219


Bet = 0.129

[64]

// Pure drug

Silica gel with

fluorescent

indicator

having an

optimal

intensity of

254nm

Water:

methanol

ether:dichloro-

methane

(1.2:8:15:77,

v/v)

Bet-17-valerate = ---

Bet- 21-valerate = --- [98,99]

// Pure drug

Silica gel with

fluorescent

indicator

having an

optimal

intensity of

254nm.

Chloroform :

ethylacetate

(1:1, v/v)


Bet- 21-valerate = 0.513

Bet =0.12

Present

work

Betamethasone

dipropionate Pure drug

Silica gel with

fluorescent

indicator

having an

optimal

intensity of

254nm.

Chloroform :

ethylacetate

(1:1, v/v)

Bet-17-propionate =

0.18

Bet-21-propionate = 0.4

Bet- dipropionate =

0.48

Bet = 0.12

Present

work

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.

Substance Pure drug/

Dosage form Column Mobile phase

Flow

rate

(ml/min)

Detector

Retention time of the

parent compound and its

thermal degrades

Reference

Bet-

valerate Pure drug

Stainless Steel Column

250mm x 4.6mm i.d.

packed with ODS

Water:

acetonitrile

(60: 40, v/v)

1.0 UV (254nm) Bet-17-valerate=7min

Bet-21-valerate=9min [98,99]

// Ointment 25cm x 4.6mm i.d.

packed with 10 µ ODS

Water:

acetonitrile

(45:55, v/v)

1.5 UV (239nm) Bet-17-valerate= ---

Bet-21-valerate= --- [100]

// Cream/lotion

Pre-column (RP18) =

3cm x 4.6mm i.d.

packed with

lichrosorb. Analytical

column = 25cm x

4.6mm i.d. packed

with 10µ ODS

Water:

acetonitrile

(45: 55, v/v)

1.5 Diode-array

(239nm)

Bet-17-valerate=5.7min

Bet-21-valerate=6.8min [101]

// Isopropyl

Myristate

Pre-column (RP18) =

3cm x 4.6mm i.d.

packed with

lichrosorb. Analytical

column = 25cm x

4.6mm i.d. packed

with 10µ ODS

Water:

acetonitrile

(45: 55, v/v)

1.5

Variable-

Wavelength

/diode-array

UV detectors

(239nm)

Bet-17-valerate=5.6min

Bet-21-valerate=6.5min [102]

// Pure drug/

Cream/ gel

250mm x 4.6mm i.d.

(µBondapak, C18)

packed with 5µ ODS

Water:

acetonitrile

(40:60, v/v)

1.2 UV (254nm)

Bet-17-valerate =5.67min

Bet-21-valerat =7.26min

Bet=2.48min

Present

work

Bet

dipropionate Pure Drug

Stainless Steel column

(Permaphase) 1m x

2mm i.d. packed with

ODS

Water:

acetonitrile

(3:1, v/v)

0.5 UV (254nm) Bet-monopropionate=5min

Bet-dipropionate=7min [59]

// Pure drug/

Cream/ gel

250mm x 4.6mm i.d.

(µBondapak, C18)

packed with 5µ ODS

Water:

acetonitrile

(40:60, v/v)

1.2 UV (254nm)

Bet-17-propionate=3.72min

Bet-21-propionate=4.34min

Bet-dipropionate=8.20min

Bet=2.34min

Present

work

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

600500400300200

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 O7 in 0.01N H2SO4

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 250C. 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

2x10-4

M solutions of the compounds were prepared in phosphate buffer

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. 2x10-3

M 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

(pH 7.5) and organic solvents e.g. methanol and acetonitrile. Zero time samples

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

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.

Illumination (at sample location) = approx 16,500 lux

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 106 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 (1x10-3

M) 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 (1x10-5

M)

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

BSA

- 0.1 0.2 0.4 0.6 0.8 - - - -

Membrane

protein

- - - - - - 0.3 0.3 0.6 0.6

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

900C 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

Betamethasone dipropionate

Acetonitrile, methanol,

phosphate buffer ( pH 7.5),

cream, gel

Acetonitrile, methanol,

phosphate buffer ( pH 7.5),

cream, gel

Betamethsone-21- valerate

Betamethasone alcohol

Betamethasone-17-propionate



47

5.0

2.5

0

0.0 2.5 5.0 7.5 10.0 12.5min


Betamethasone-17-valerate


Beclomethasonedipropionate

[mV]

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

H3C

HOH3CH

H

F H

OH

O

H

CH3

O

O

H3C

BETAMETHASONE-21-VALERATEO

H3C

HOH3CH

H

F H

O

H

CH3

OH

BETAMETHASONE-17-VALERATE

O

O

H3C

O

H3C

HOH3CH

H

F H

BETAMETHASONE ALCOHOL

O

OH

OHH

CH3


Hydrolysis

Figure 5. Degradation pathway for the thermal transformation of betamethasone-17-

valerate into betamethasone-21-valerate and betamethasone alcohol.

49

20

10

0

0.0 2.5 5.0 7.5 10.0 12.5min




Betamethasonedipropionate

Beclomethasonedipropionate

[mV]

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

HOH3CH

H

F H

O

O

H

CH3

O

BETAMETHASONE DIPROPIONATE

O

H3C

HOH3CH

H

F H

O

H

CH3

BETAMETHASONE-21-PROPIONATE

OH

O

H3C

HOH3CH

H

F H

BETAMETHASONE-17-PROPIONATE

O

OH

OHH

CH3

O

H3C

O

CH3

O

O

CH3

O

H3C

HOH3CH

H

F H

O

OH

CH3

BETAMETHASONE ALCOHOL

OH

H


Hydrolysis

DeacylationDeacylation

O

H3C

O

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 tR 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 dipropionate

Betamethasone-21-propionte


0.0192

0.0165

0.0625

0.0135

0.0241

0.0218

0.994

0.995

0.994

0.998

0.999

0.994

54

Table 9. Recoveries of betamethasone valerate from spiked samples.

Dosage form

µg added µg found % Recovery

Cream

250.3

249.9

250.2

251.5

247.8

249.05

250.65

254.2

247.23

250.03

99.50

100.30

101.59

98.30

100.89

Mean: 100.116

RSD: 1.27%

Gel

249.8

250.9

248.5

251.3

255.1

250.78

249.15

251.3

247.92

255.35

100.39

99.30

101.12

98.65

100.09

Mean: 99.91

RSD: 0.96%

Table 10. Recoveries of betamethasone dipropionate from spiked samples.

Dosage form

µg added

µg found

% Recovery

Cream

253.7

254.1

250.8

250.2

246.5

259.0

257.3

252.52

246.2

244.51

102.08

101.26

100.67

98.40

99.18

Mean: 100.32

RSD: 1.5%

Gel

250.7

251.9

245.2

248.2

253.1

247.85

259.45

249.49

249.19

248.62

98.86

103.00

101.75

100.40

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

Time (Hour)

Betamethasone

-17-valerate

(M x 105)

Betamethasone-21-

valerate

(M x 105)

Betamethasone

alcohol

(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


acetonitrile (40 0C).


Time (Hour)

Betamethasone

-17-valerate

(M x 105)

Betamethasone-21-

valerate

(M x 105)

Betamethasone

alcohol

(M x 105)

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


phosphate buffer (pH 7.5) at 40 0C.


Time (Hour)

Betamethasone

-17-valerate

(M x 105)

Betamethasone-21-

valerate

(M x 105)

Betamethasone

alcohol

(M x 105)

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


cream (40 0C).


Time (Hour)

Betamethasone

-17-valerate

(M x 105)

Betamethasone-21-

valerate

(M x 105)

Betamethasone

alcohol

(M x 105)

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).


Time (Hour)

Betamethasone

-17-valerate

(M x 105)

Betamethasone-21-

valerate

(M x 105)

Betamethasone

alcohol

(M x 105)

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).


Time

(Hour)

Betamethasone

dipropionate

(M x 105)

Betamethasone

-17-propionate

(M x 105)

Betamethasone

-21-propionate

(M x 105)

Betamethasone

alcohol

(M x 105)

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


acetonitrile (40 0C).


Time

(Hour)

Betamethasone

dipropionate

(M x 105)

Betamethasone-

17-propionate

(M x 105)

Betamethasone-

21-propionate

(M x 105)

Betamethasone

alcohol

(M x 105)

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


phosphate buffer (pH 7.5) at 40 0C.


Time

(Hour)

Betamethasone

dipropionate

(M x 105)

Betamethasone-

17-propionate

(M x 105)

Betamethasone-

21-propionate

(M x 105)

Betamethasone

alcohol

(M x 105)

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


cream (40 0C).


Time (Hour)

Betamethasone

dipropionate

(M x 105)

Betamethasone-17-

propionate

(M x 105)

Betamethasone-21-

propionate

(M x 105)

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).


Time (Hour)

Betamethasone

dipropionate

(M x 105)

Betamethasone-17-

propionate

(M x 105)

Betamethasone-21-

propionate

(M x 105)

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-

order rate constants, kobs, for the degradation reactions at 40 oC 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

Figure 8. First-order plot for the degradation of betamethasone valerate

in methanol (40 oC).

0

0.2

0.4

0.6

0.8

1

1.2

0 24 48 72 96 120 144 168

Time (Hour)

0

0.2

0.4

0.6

0.8

1

1.2

0 24 48 72 96 120 144 168

Time (Hour)

63


in acetonitrile (40 oC).


in phosphate buffer (pH 7.5) at 40 oC.


in cream (40 oC).

0.97

0.975

0.98

0.985

0.99

0.995

1

1.005

1.01

0 24 48 72 96 120 144 168

Time (Hour)

0

0.2

0.4

0.6

0.8

1

1.2

0 24 48 72 96 120 144 168

Time (Hour)

64


in gel (40 oC).

Figure 13. First-order plot for the degradation of betamethasone dipropionate

in methanol (40 oC).

0.86

0.88

0.9

0.92

0.94

0.96

0.98

1

1.02

0 24 48 72 96 120 144 168

Time (Hour)

0.97

0.975

0.98

0.985

0.99

0.995

1

1.005

0 24 48 72 96 120 144 168

Time (Hour)

65


in acetonitrile (40 oC).


in phosphate buffer (pH 7.5) at 40 oC.

0.955

0.96

0.965

0.97

0.975

0.98

0.985

0.99

0.995

1

0 24 48 72 96 120 144 168

Time (Hour)

0.9

0.92

0.94

0.96

0.98

1

1.02

0 24 48 72 96 120 144 168

Time (Hour)

66


in cream (40 oC).


in gel (40 oC).

0.994

0.996

0.998

1

1.002

1.004

1.006

1.008

1.01

1.012

0 24 48 72 96 120 144 168

Time (Hour)

0.975

0.98

0.985

0.99

0.995

1

1.005

0 24 48 72 96 120 144 168

Time (Hour)

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

Medium Dielectric Constant kobs x103, hr

-1 Corr. kobs x10

3, hr

-1 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

0

1

2

3

4

5

6

7

8

9

10

0 10 20 30 40 50 60 70 80 90

.

.

( ) 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.

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

0 10 20 30 40 50 60 70 80 90

.

.


Figure 19. Dependence of the rate constant of thermal degradation of betamethasone

dipropionate on the solvent dielectric constant.

Dielectric constant

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 k1 [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

Figure 20. pH-rate profile for the degradation of betamethasone dipropionate (40 oC).

Figure 21. pH-rate profile for the degradation of betamethasone-17-valerate (40 oC).

-1

-1.5

-2

-2.5

-3

-3.5

-4

pH

2 3 4 5 6 7 8

-1.1

-1.6

-2.1

-2.6

-3.1

-3.6

-4.1

-4.6

pH

2 3 4 5 6 7 8

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).

pH Betamethasone-21-

valerate

Betamethasone

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).

pH Betamethasone-17-

propionate

Betamethasone-21-

propionate

Betamethasone

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-1

s-1

and 1.305x10-6

M-1

s-1

for betamethasone valerate and

betamethasone dipropionate degradation, respectively. The plots show that buffer

causes inhibition of the reaction. This is evident from the values of k0 {(5.5x10-3

hr-1

(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 (Mx105) at various buffer

(Phosphate) concentration (0.05-0.2M) at 40 oC.

Time (Hour) Phosphate

Concentration

(M) 0 24 48 72 96

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 (Mx105) at various buffer

(Phosphate) concentration (0.05-0.2M) at 40 oC.

Time (Hour) Phosphate

Concentration

(M) 0 24 48 72 96

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

Figure 22. First-order plot of the thermal degradation of betamethasone valerate (40 oC)

at pH 7.5 (0.05M phosphate buffer).



0.4

0.5

0.6

0.7

0.8

0.9

1

1.1

0 24 48 72 96

Time (Hour)

0.4

0.5

0.6

0.7

0.8

0.9

1

1.1

0 24 48 72 96

Time (Hour)

77





0.4

0.5

0.6

0.7

0.8

0.9

1

1.1

0 24 48 72 96

Time (Hour)

0.4

0.5

0.6

0.7

0.8

0.9

1

1.1

0 24 48 72 96

Time (Hour)

78

Figure 26. First-order plot of the thermal degradation of betamethasone dipropionate

(40 oC) at pH 7.5 (0.05M phosphate buffer).



0.965

0.97

0.975

0.98

0.985

0.99

0.995

1

1.005

0 24 48 72 96

Time (Hour)

0.955

0.96

0.965

0.97

0.975

0.98

0.985

0.99

0.995

1

1.005

0 24 48 72 96

Time (Hour)

79





0.988

0.99

0.992

0.994

0.996

0.998

1

1.002

0 24 48 72 96

Time (Hour)

0.975

0.98

0.985

0.99

0.995

1

1.005

0 24 48 72 96

Time (Hour)

80

Figure 30. Plot of kobs vs phosphate concentration of thermal degradation of

betamethasone valerate (40 oC) at pH 7.5.

Figure 31. Plot of kobs vs phosphate concentration of thermal degradation of

betamethasone dipropionate (40 oC) at pH 7.5.

0

0.2

0.4

0.6

0.8

1

1.2

1.4

0 0.05 0.1 0.15 0.2 0.25

Phosphate concentration (M)

2

2.5

3

3.5

4

4.5

5

5.5

6

0 0.05 0.1 0.15 0.2 0.25


81


betamethasone-17-valerate and betamethasone dipropionate at various

phosphate concentrations (40 oC).


Phosphate kobs x103, hr

-1 Corr. Coefficient kobs x10

3, hr

-1

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 ZA ZB, 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 (Mx105) at different ionic

strength (0.3-1.5M) at 40 oC.

Time (Hour) Ionic Strength

(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 (Mx105) at different ionic

strength (0.3-1.5M) at 40 oC.

Time (Hour) Ionic Strength

(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


at pH 7.5 (µ = 0.3M).


at pH 7.5 (µ = 0.6M).

0.4

0.5

0.6

0.7

0.8

0.9

1

1.1

0 24 48 72 96

Time (Hour)

0.4

0.5

0.6

0.7

0.8

0.9

1

1.1

0 24 48 72 96

Time (Hour)

85


at pH 7.5 (µ = 0.9M).


at pH 7.5 (µ = 1.2M).

0.4

0.5

0.6

0.7

0.8

0.9

1

1.1

0 24 48 72 96

Time (Hour)

0.4

0.5

0.6

0.7

0.8

0.9

1

1.1

0 24 48 72 96

Time (Hour)

86


at pH 7.5 (µ = 1.5M).


(40 oC) at pH 7.5 (µ = 0.3M).

0.88

0.9

0.92

0.94

0.96

0.98

1

1.02

0 24 48 72 96

Time (Hour)

0.4

0.5

0.6

0.7

0.8

0.9

1

1.1

0 24 48 72 96

Time (Hour)

87


(40 oC) at pH 7.5 (µ = 0.6M).


(40 oC) at pH 7.5 (µ = 0.9M).

0.88

0.9

0.92

0.94

0.96

0.98

1

1.02

0 24 48 72 96

Time (Hour)

0.88

0.9

0.92

0.94

0.96

0.98

1

1.02

0 24 48 72 96

Time (Hour)

88


(40 oC) at pH 7.5 (µ = 1.2M).


(40 oC) at pH 7.5 (µ = 1.5M).

0.88

0.9

0.92

0.94

0.96

0.98

1

1.02

0 24 48 72 96

Time (Hour)

0.88

0.9

0.92

0.94

0.96

0.98

1

1.02

0 24 48 72 96

Time (Hour)

89


betamethasone-17-valerate and betamethasone dipropionate at various ionic

strength ( 40 oC ).


Ionic strength kobs x103, hr

-1 Corr. Coefficient kobs x10

3, hr

-1 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

Figure 42. Plot of kobs vs ionic strength (µ) of thermal degradation of betamethasone

valerate (40 oC) at pH 7.5.

Figure 43. Plot of kobs vs ionic strength (µ) of thermal degradation of betamethasone

dipropionate (40 oC) at pH 7.5.

0

0.2

0.4

0.6

0.8

1

0 0.3 0.6 0.9 1.2 1.5 1.8

Ionic strength (µ)

0

1

2

3

4

5

6

0 0.3 0.6 0.9 1.2 1.5 1.8

Io n ic s t r e n g th ( µ )

91

Figure 44. Plot of log (kobs/ko) vs of thermal degradation of betamethasone

valerate (40 oC) at pH 7.5.

Figure 45. Plot of log (kobs/ko) vs of thermal degradation of betamethasone

dipropionate (40 oC) at pH 7.5.

-1.2

-1

-0.8

-0.6

-0.4

-0.2

0

0.2

0.4

0 0.2 0.4 0.6 0.8 1 1.2 1.4

µ

µ

µ

-0.4

-0.3

-0.2

-0.1

0

0.1

0.2

0 0.2 0.4 0.6 0.8 1 1.2 1.4

µ

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 tR values of 14.1 and 20.9min, respectively. In the

case of betamethasone dipropionate two major products (C and D) were detected

with tR values of 19.28 and 31.2min, respectively. It appears from the tR 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 tR values and

consequently different chemical structures. In view of the absence of reference

94

100

0

Time (min)

20 30

50

100

150

200

mAU


Photoproduct APhotoproduct B

Figure 46. HPLC chromatogram showing betamethasone-17-valerate and its

photoproducts A and B.

100

0

Time (min)

20 30

50

100

150

200

mAU

Betamethasonedipropionate

Photoproduct CPhotoproduct D

Figure 47. HPLC chromatogram showing betamethasone dipropionate and its

photoproducts C and D.

95

2 0 0

W a v e l e n g t h ( n m )

2 5 0 3 0 0 3 5 0 4 0 00 . 0 0

1 . 0 0

0 . 9 0

0 . 8 0

0 . 7 0

0 . 6 0

0 . 5 0

0 . 4 0

0 . 3 0

0 . 2 0

0 . 1 0


0 . 0 0

4 . 0 0

3 . 6 0

3 . 2 0

2 . 8 0

2 . 4 0

2 . 0 0

1 . 6 0

1 . 2 0

0 . 8 0

0 . 4 0


0 . 0 0

0 . 1 0

0 . 0 8

0 . 0 6

0 . 0 4

0 . 0 2

( 1 ) ( A ) ( B )

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

Figure 48. UV spectra of betamethasone-17-valerate (1) and its photoproducts A and B.


0 . 0

2 . 0

1 . 8

1 . 6

1 . 4

1 . 2

1 . 0

0 . 8

0 . 6

0 . 4

0 . 2

( 2 )

2 0 0 2 5 0 3 0 0 3 5 0 4 0 00 . 0 0

1 . 0 0

0 . 9 0

0 . 8 0

0 . 7 0

0 . 6 0

0 . 5 0

0 . 4 0

0 . 3 0

0 . 2 0

0 . 1 0


( C )

2 0 0 2 5 0 3 0 0 3 5 0 4 0 0

0 . 0

2 . 0

1 . 8

1 . 6

1 . 4

1 . 2

1 . 0

0 . 8

0 . 6

0 . 4

0 . 2


( D )

2 0 0 2 5 0 3 0 0 3 5 0 4 0 0

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

(min)

Acetonitrile

(M x 105)

Methanol

(M x 105)

Phosphate buffer, pH

7.5 (M x 105)

Cream

(M x 105)

Gel

(M x 105)

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

(min)

Acetonitrile

(M x 105)

Methanol

(M x 105)

Phosphate buffer,

pH 7.5 (M x 105)

Cream

(M x 105)

Gel

(M x 105)

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.

Medium

Photoproduct

A

Photoproduct

B

Minor

Photoproducts

Acetonitrile

7.90 1.46 0.64

Methanol

2.80 6.30 0.90

Phosphate

buffer (pH7.5)

2.60 6.30 1.10

Cream

6.60 3.40 --

Gel

5.70 4.30 --

Table 33. Product distribution at 10% photodegradation of betamethasone dipropionate in

different media.

Medium

Photoproduct

C

Photoproduct

D

Minor

Photoproducts

Acetonitrile

4.95 4.20 0.85

Methanol

3.30 5.70 1.00

Phosphate

buffer (pH7.5)

9.40 -- 0.60

Cream

7.30 2.70 --

Gel

8.34 1.66 --

99

Figure 50. First-order plot for the photodegradation of betamethasone valerate in

methanol.


acetonitrile.

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

1.1

0 30 60 90 120 150

Time (min)

0

0.2

0.4

0.6

0.8

1

1.2

0 30 60 90 120 150

Time (min)

100


phosphate buffer (pH 7.5).

Figure 53. First-order plot for the photodegradation of betamethasone valerate in cream.

0.86

0.88

0.9

0.92

0.94

0.96

0.98

1

0 30 60 90 120 150

Time (min)

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

1.1

0 30 60 90 120 150

Time (min)

101

Figure 54. First-order plot for the photodegradation of betamethasone valerate in gel.

Figure 55. First-order plot for the photodegradation of betamethasone dipropionate in

methanol.

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

1.1

0 30 60 90 120 150

Time (min)

0.88

0.9

0.92

0.94

0.96

0.98

1

0 30 60 90 120 150

Time (min)

102


acetonitrile.


phosphate buffer (pH 7.5).

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

1.1

1.2

0 30 60 90 120 150

Time (min)

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

1.1

0 30 60 90 120 150

Time (min)

103


cream.


gel.

0.8

0.85

0.9

0.95

1

1.05

1.1

0 30 60 90 120 150

Time (min)

0.8

0.85

0.9

0.95

1

1.05

1.1

0 30 60 90 120 150

Time (min)

104

Table 34. Apparent first-order rate constants (kobs) for the photodegradation of

betamethasone-17-valerate and betamethasone dipropionate.


Medium Dielectric kobs x103, min

-1 Corr. kobs x10

3, min

-1 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

Figure 60. Dependence of the rate constant of photodegradation of betamethasone

valerate on the solvent dielectric constant.


Figure 61. Dependence of the rate constant of photodegradation of betamethasone

dipropionate on the solvent dielectric constant.


0

1

2

3

4

5

6

7

8

9

0 10 20 30 40 50 60 70 80 90

Dielectric constant

0

2

4

6

8

10

12

0 10 20 30 40 50 60 70 80 90

Dielectric constant

107

Figure 62. Plot of kobs vs phosphate concentration of photodegradation of

betamethasone valerate at pH 7.5.

Figure 63. Plot of kobs vs phosphate concentration of photodegradation of

betamethasone dipropionate at pH 7.5.

0

2

4

6

8

10

12

0 0.05 0.1 0.15 0.2


0

2

4

6

8

10

12

14

16

0 0.05 0.1 0.15 0.2


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

Figure 64. Plot of kobs vs ionic strength (µ) of the photodegradation of

betamethasone valerate at pH 7.5.

Figure 65. Plot of kobs vs ionic strength (µ) of the photodegradation of

betamethasone dipropionate at pH 7.5.

0

1

2

3

4

5

6

7

8

0 0.3 0.6 0.9 1.2 1.5 1.8

Ionic strength (µ)

0

2

4

6

8

10

12

0 0.3 0.6 0.9 1.2 1.5 1.8

Ionic strength (µ)

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 105) on photodegradation in creams.

Time (min)

Cream containing

Titanium dioxide

Cream containing

Vanillin

Cream containing

Butyl hydroxytoluene

0

9.98

10.06

9.96

30

9.65

9.67

9.55

60

9.34

9.26

8.12

90

8.98

8.88

8.68

120

8.69 8.50

8.27

Table 36. Assay of betamethasone valerate (M x 105) on photodegradation in gels.

Time (min)

Gel containing

Titanium dioxide

Gel containing

Vanillin

Gel containing


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 105) on photodegradation in

creams.

Time (min) Cream containing

Titanium dioxide

Cream containing

Vanillin

Cream containing


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 105) on photodegradation in gels.

Time (min) Gel containing

Titanium dioxide

Gel containing

Vanillin

Gel containing


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

120 9.13 9.08 8.97

113

Figure 66. First-order plot for the photodegradation of betamethasone-17-valerate

in cream (stabilized with 0.1% titanium dioxide).


in cream (stabilized with 0.1% vanillin).

0.9

0.92

0.94

0.96

0.98

1

1.02

0 30 60 90 120 150

Time (min)

0.9

0.92

0.94

0.96

0.98

1

1.02

0 30 60 90 120 150

Time (min)

114


in cream (stabilized with 0.1% butyl hydroxytoluene).


in gel (stabilized with 0.1% titanium dioxide).

0.94

0.95

0.96

0.97

0.98

0.99

1

1.01

0 30 60 90 120 150

Time (min)

0.85

0.87

0.89

0.91

0.93

0.95

0.97

0.99

1.01

0 30 60 90 120 150

Time (min)

115


in gel (stabilized with 0.1% vanillin).


in gel (stabilized with 0.1% butyl hydroxytoluene).

0.9

0.92

0.94

0.96

0.98

1

1.02

0 30 60 90 120 150

Time (min)

0.9

0.92

0.94

0.96

0.98

1

1.02

0 30 60 90 120 150

Time (min)

116

Figure 72. First-order plot for the photodegradation of betamethasone dipropionate

in cream (stabilized with 0.1% titanium dioxide).


in cream (stabilized with 0.1% vanillin).

0.9

0.92

0.94

0.96

0.98

1

1.02

0 30 60 90 120 150

Time (min)

0.95

0.96

0.97

0.98

0.99

1

1.01

0 30 60 90 120 150

Time (min)

117


in cream (stabilized with 0.1% butyl hydroxytoluene).


in gel (stabilized with 0.1% titanium dioxide).

0.94

0.95

0.96

0.97

0.98

0.99

1

0 30 60 90 120 150

Time (min)

0.9

0.92

0.94

0.96

0.98

1

1.02

0 30 60 90 120 150

Time (min)

118


in gel (stabilized with 0.1% vanillin).


in gel (stabilized with 0.1% butyl hydroxytoluene).

0.94

0.95

0.96

0.97

0.98

0.99

1

1.01

0 30 60 90 120 150

Time (min)

0.94

0.95

0.96

0.97

0.98

0.99

1

1.01

0 30 60 90 120 150

Time (min)

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*


Formulation kobs x103, min

-1 Corr. kobs x10

3, min

-1 Corr.

Coefficient Coefficient

Cream containing 1.153 0.999 0.909 0.999

TiO2


vanillin


BHT

Gel containing 1.019 0.999 0.692 0.998

TiO2

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

Figure 78. UV spectrum of vanillin showing spectral overlay with betamethasone esters.

Figure 79. UV spectrum of butyl hydroxytoluene showing spectral overlay with

betamethasone esters.

2 0 0 . 0

W a v e l e n g t h ( n m . )

2 5 0 . 0 3 0 0 . 0 3 5 0 . 0 4 0 0 . 0

0 . 0 0 0

0 . 5 0 0

1 . 0 0 0

A

b

s

.

BHT


2 0 0 . 0

W a v e l e n g t h ( n m . )

2 5 0 . 0 3 0 0 . 0 3 5 0 . 0 4 0 0 . 0

0 . 0 0 0

0 . 5 0 0

1 . 0 0 0

A

b

s

.

Vanillin


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 (106

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). NaN3

(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 NaN3 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

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).

0

10

20

30

40

50

60

70

80

90

100

0 10 20 30 40 50 60

Time (min)

125

Figure 81. Hemolysis induced by betamethasone-17-valerate photoproducts in the dark

( ) and further UV irradiation ( ).

0

10

20

30

40

50

60

70

80

90

100

0 5 10 15 20 25 30

Time (min)

126

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).

0

10

20

30

40

50

60

70

80

90

100

0 10 20 30 40 50 60

Time (min)

127

Figure 83. Hemolysis induced by betamethasone dipropionate photoproducts in the dark

( ) and further UV irradiation ( ).

0

10

20

30

40

50

60

70

80

90

100

0 5 10 15 20 25 30 35 40

Time (min)

128

Figure 84. Photoperoxidation of linoleic acid (10-3

M) sensitized by betamethasone

-17-Valerate (1).

( ) Linoleic acid+1 (dark) ( ) Linoleic acid (hv) ( ) Linoleic acid+1 (hv).

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

0 10 20 30 40 50 60

Time (min)

129

Figure 85. Photoperoxidation of linoleic acid (10-3

M) sensitized by betamethasone

dipropionate (2).

( ) Linoleic acid+2 (dark) ( ) Linoleic acid (hv) ( ) Linoleic acid+2 (hv).

0

0.1

0.2

0.3

0.4

0.5

0 10 20 30 40 50 60

Time (min)

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 tR values

have been reported. The identification of thermal degradation products was based

on the comparison of the tR 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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135

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