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-21-valerate = 0.454
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-17-valerate = 0.246
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
Betamethasone-21-propionate
Betamethasone alcohol
47
5.0
2.5
0
0.0 2.5 5.0 7.5 10.0 12.5min
Betamethasone alcohol
Betamethasone-17-valerate
Betamethasone-21-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
Ester group migration
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
Betamethasone alcohol
Betamethasone-17-propionate
Betamethasone-21-propionate
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
Ester group migration
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-17-valerate
Betamethasone-21-valerate
Betamethasone alcohol
Betamethasone dipropionate
Betamethasone-21-propionte
Betamethasone-17-propionate
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
Table 12. Assay of betamethasone-17-valerate and degradation products formed in
acetonitrile (40 0C).
Degradation products
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
Table 13. Assay of betamethasone-17-valerate and degradation products formed in
phosphate buffer (pH 7.5) at 40 0C.
Degradation products
Time (Hour)
Betamethasone
-17-valerate
(M x 105)
Betamethasone-21-
valerate
(M x 105)
Betamethasone
alcohol
(M x 105)
0 10.14 0.00 0.00
24 9.20 0.79 0.12
48 8.29 1.58 0.87
72 7.31 2.19 1.45
96 6.45 2.73 2.30
120 5.48 2.90 3.65
144 4.60 3.38 4.50
Table 14. Assay of betamethasone-17-valerate and degradation products formed in
cream (40 0C).
Degradation products
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).
Degradation products
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).
Degradation products
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
Table 17. Assay of betamethasone dipropionate and degradation products formed in
acetonitrile (40 0C).
Degradation products
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
Table 18. Assay of betamethasone dipropionate and degradation products formed in
phosphate buffer (pH 7.5) at 40 0C.
Degradation products
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
Table 19. Assay of betamethasone dipropionate and degradation products formed in
cream (40 0C).
Degradation products
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).
Degradation products
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
Figure 9. First-order plot for the degradation of betamethasone valerate
in acetonitrile (40 oC).
Figure 10. First-order plot for the degradation of betamethasone valerate
in phosphate buffer (pH 7.5) at 40 oC.
Figure 11. First-order plot for the degradation of betamethasone valerate
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
Figure 12. First-order plot for the degradation of betamethasone valerate
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
Figure 14. First-order plot for the degradation of betamethasone dipropionate
in acetonitrile (40 oC).
Figure 15. First-order plot for the degradation of betamethasone dipropionate
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
Figure 16. First-order plot for the degradation of betamethasone dipropionate
in cream (40 oC).
Figure 17. First-order plot for the degradation of betamethasone dipropionate
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
.
.
( ) Methanol ( ) Acetonitrile ( ) Water (Phosphate buffer (pH 7.5)
Figure 19. Dependence of the rate constant of thermal degradation of betamethasone
dipropionate on the solvent dielectric constant.
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).
Figure 23. First-order plot of the thermal degradation of betamethasone valerate (40 oC)
at pH 7.5 (0.1M 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
Figure 24. First-order plot of the thermal degradation of betamethasone valerate (40 oC)
at pH 7.5 (0.15M phosphate buffer).
Figure 25. First-order plot of the thermal degradation of betamethasone valerate (40 oC)
at pH 7.5 (0.2M 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)
78
Figure 26. First-order plot of the thermal degradation of betamethasone dipropionate
(40 oC) at pH 7.5 (0.05M phosphate buffer).
Figure 27. First-order plot of the thermal degradation of betamethasone dipropionate
(40 oC) at pH 7.5 (0.1M 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
Figure 28. First-order plot of the thermal degradation of betamethasone dipropionate
(40 oC) at pH 7.5 (0.15M phosphate buffer).
Figure 29. First-order plot of the thermal degradation of betamethasone dipropionate
(40 oC) at pH 7.5 (0.2M phosphate buffer).
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
Phosphate concentration (M)
81
Table 26. Apparent first-order rate constants (kobs) for the thermal degradation of
betamethasone-17-valerate and betamethasone dipropionate at various
phosphate concentrations (40 oC).
Betamethasone-17-valerte Betamethasone dipropionate
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
Figure 32. First-order plot of the thermal degradation of betamethasone valerate (40 oC)
at pH 7.5 (µ = 0.3M).
Figure 33. First-order plot of the thermal degradation of betamethasone valerate (40 oC)
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
Figure 34. First-order plot of the thermal degradation of betamethasone valerate (40 oC)
at pH 7.5 (µ = 0.9M).
Figure 35. First-order plot of the thermal degradation of betamethasone valerate (40 oC)
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
Figure 36. First-order plot of the thermal degradation of betamethasone valerate (40 oC)
at pH 7.5 (µ = 1.5M).
Figure 37. First-order plot of the thermal degradation of betamethasone dipropionate
(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
Figure 38. First-order plot of the thermal degradation of betamethasone dipropionate
(40 oC) at pH 7.5 (µ = 0.6M).
Figure 39. First-order plot of the thermal degradation of betamethasone dipropionate
(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
Figure 40. First-order plot of the thermal degradation of betamethasone dipropionate
(40 oC) at pH 7.5 (µ = 1.2M).
Figure 41. First-order plot of the thermal degradation of betamethasone dipropionate
(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
Table 29. Apparent first-order rate constants (kobs) for the thermal degradation of
betamethasone-17-valerate and betamethasone dipropionate at various ionic
strength ( 40 oC ).
Betamethasone-17-valerte Betamethasone dipropionate
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
Betamethasone-17-valerate
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
W a v e l e n g t h ( n m )
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
W a v e l e n g t h ( n m )
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.
W a v e l e n g t h ( n m )
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
W a v e l e n g t h ( n m )
( 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
W a v e l e n g t h ( n m )
( 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.
Figure 51. First-order plot for the photodegradation of betamethasone valerate in
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
Figure 52. First-order plot for the photodegradation of betamethasone valerate in
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
Figure 56. First-order plot for the photodegradation of betamethasone dipropionate in
acetonitrile.
Figure 57. First-order plot for the photodegradation of betamethasone dipropionate in
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
Figure 58. First-order plot for the photodegradation of betamethasone dipropionate in
cream.
Figure 59. First-order plot for the photodegradation of betamethasone dipropionate in
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.
Betamethasone-17-valerte 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.
( ) Methanol ( ) Acetonitrile ( ) Water (Phosphate buffer (pH 7.5)
Figure 61. Dependence of the rate constant of photodegradation of betamethasone
dipropionate on the solvent dielectric constant.
( ) Methanol ( ) Acetonitrile ( ) Water (Phosphate buffer (pH 7.5)
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
Phosphate concentration (M)
0
2
4
6
8
10
12
14
16
0 0.05 0.1 0.15 0.2
Phosphate concentration (M)
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
Butyl hydroxytoluene
0
10.02 9.95 9.99
30
9.72
9.60
9.63
60
9.41
9.27
9.29
90
9.13 8.93 8.92
120 8.85 8.58
8.55
112
Table 37. Assay of betamethasone dipropionate (M x 105) on photodegradation in
creams.
Time (min) Cream containing
Titanium dioxide
Cream containing
Vanillin
Cream containing
Butyl hydroxytoluene
0
10.10 9.96 10.08
30
9.83
9.62
9.72
60
9.57
9.30
9.37
90
9.32
8.98
9.03
120 9.05 8.67
8.65
Table 38. Assay of betamethasone dipropionate (M x 105) on photodegradation in gels.
Time (min) Gel containing
Titanium dioxide
Gel containing
Vanillin
Gel containing
Butyl hydroxytoluene
0
9.92
10.00
9.95
30
9.72
9.78
9.70
60
9.53
9.54
9.46
90
9.34
9.31
9.21
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).
Figure 67. First-order plot for the photodegradation of betamethasone-17-valerate
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
Figure 68. First-order plot for the photodegradation of betamethasone-17-valerate
in cream (stabilized with 0.1% butyl hydroxytoluene).
Figure 69. First-order plot for the photodegradation of betamethasone-17-valerate
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
Figure 70. First-order plot for the photodegradation of betamethasone-17-valerate
in gel (stabilized with 0.1% vanillin).
Figure 71. First-order plot for the photodegradation of betamethasone-17-valerate
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).
Figure 73. First-order plot for the photodegradation of betamethasone dipropionate
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
Figure 74. First-order plot for the photodegradation of betamethasone dipropionate
in cream (stabilized with 0.1% butyl hydroxytoluene).
Figure 75. First-order plot for the photodegradation of betamethasone dipropionate
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
Figure 76. First-order plot for the photodegradation of betamethasone dipropionate
in gel (stabilized with 0.1% vanillin).
Figure 77. First-order plot for the photodegradation of betamethasone dipropionate
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*
Betamethasone-17-valerte Betamethasone dipropionate
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
Cream containing 1.393 0.999 1.155 0.999
vanillin
Cream containing 1.548 0.999 1.274 0.999
BHT
Gel containing 1.019 0.999 0.692 0.998
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
Betamethasone valerate
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
Betamethasone valerate
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.
REFERENCES
135
REFERENCES
1. Grollman, A., Grollman, E.F. (1970). Pharmacology and Therapeutics, 7th
ed.,
Lea & Febiger, Philadelphia, Chap. 35.
2. Hench, P.S., Kendall, E.C., Slocumb, C.H., Polley, H.F. (1949). Proc. Staff Meet.
Mayo Clin. 24, 181-197.
3. Katzung, B.G. (1995). Basic and Clinical Pharmacology, 6th
ed., Appleton and
Lange, East Norwalk, pp. 590-607.
4. Fausi, A.S. (1976). Ann. Ind. Med. 84, 304-315.
5. Bernard, P., Parker, K.L. (2001). Adrenocorticotropic hormone, adrenocortical
steroids and their synthetic analogs, inhibitors of the synthesis and actions of
adrenocortical hormones, in: Hardman, J.G., Limpard, L.E., Eds., Goodman &
Gilman’s Pharmacological Basis of Therapeutics, 10th
ed., Mc Graw Hill Medical
Publishing Division, New York., pp. 1671-1672.
6. Schimmer, B.P., George, S.R. (2007). Principles of Medical Pharmacology, 7th
ed., Elsevier Saunders, Canada, Chap. 48.
7. Micheal, E.L., Rokea, A.E. (1997). Mayo. Clin. Proc. 72, 1141-1144.
8. Gilchrest, B.A., Goldwyn, R.M. (1981). Plast. Reconstr. Surg. 67, 435-439.
9. Katz, H.I. (1995). Dermatol. Clin. 13, 805-815.
10. Fusaro, R.M. (1988). Drug Intell. Clin. Pharm. 22, 412-418.
11. Sweetman, S.C., Ed. (2007). Martindale The Complete Drug Reference, 35th
ed.,
Pharmaceutical press, London., pp. 1347-1350, 1369-1370.
12. United States Pharmacopeia 32 (2009). United States Pharmacopeial Convention,
Rockville, MD, pp. 1660-1661, 1665-1666.
13. Budavari, S., Ed. (1989). The Merck Index. An Encyclopedia of chemicals,
Drugs, and Biologicals, 11th
ed. Merck & Co., Rahway. N.J., p. 1202.
14. Moffat, A.C., Osselton, M.D., Widdop, B. (2004). Clarke’s Analysis of Drugs and
Poisons, 3rd
ed., Pharmaceutical Press, London., p. 694.
15. Elks, J., May, P.J., Weir, N.G. (1967). US Patent, 3, 312, 591.
16. Elks, J., May, P.J., Weir, N.G. (1967). US Patent, 3, 312, 590.
136
17. Lachman, L., DeLuca, P., Akers, M.J. (1986). Kinetic Priciples and Drug
Stability, in: Lachman, L., Lieberman, H.A., Kanig, J.L., Eds., The Theory and
Practice of Industrial Pharmacy, 3rd
ed., Lea & Febiger Philadelphia, Chap. 26.
18. Kreienbaum, M.A. (1986). Am. J. Hosp. Pharm. 43, 1747-1750.
19. Kizu, J., Ichihara, W., Tomonaga, E., Abe, J., Watanabe, T., Inone, T., Hori, S.
(2004). Yakagaku Zasshi. 124, 93-97.
20. Boreen, A.L., Arnold, W.A., McNeil, K. (2003). Aquat. Sci. 65, 320-341.
21. Gupta, P., Garg, S. (2002). Pharm. Technol. 26, 144-162. On-line.
22. Olin, B.R., Ed. (1992). Topical Corticosteroid Formulations, Drug Facts and
Comparisons, St. Louis, Missouri, pp. 2241-2242.
23. Velaga, S.P., Bergh, S., Carlfors, J. (2004). Eur. J. Pharm. Sci. 21, 501-509.
24. Gupta, V.D., (1978). J.Pharm. Sci. 67, 299-302.
25. Gupta, V.D., (1979). J.Pharm.Sci. 68, 908-910.
26. Gupta, V.D., (1983). J.Pharm.Sci. 72, 1453-1456.
27. Dekker, D., Beunen, J.H. (1980). Pharm. Weekblad., Sci. Ed., 2, 112-116.
28. Kenley, R.A., Lee, M.O., Sukumar, L., Powel, M.F. (1987). Pharm. Res. 4, 342-
347.
29. Timmins, P., Gray, E.A. (1983). J. Pharm. Pharmacol. 35, 175-177.
30. 6 , 307-319.
31. Mahesh, N.S., Matthias, S., Peter, W.N., William, J.J. (2004). J. Pharm. Sci. 726-
732.
32. Boonsaner, P., Remon, P.J., De Rudder, D. (2006). J. Clin. Ther. 101-106.
33. Guillory, J.K., Poust, R.I. (2003). Chemical Kinetics and Drug Stability, in:
Banker, G.S., Rhodes, C.T., Eds., Modern Pharmaceutics, 4th
ed., Marcel Dekker,
New York, Chap. 6.
34. Ohtani, M., Nakai, T., Ohsawa, K., Kim, S., Matsumoto, M., Etoh, T., Kariya, S.,
Kanou, S., Uchino, K. (2002). Yakugaku Zasshi. 122, 1153-1158.
35. Rabouan-Guyon, M.S., Fauvaud, M.C., Courtois, Y.P., Barthes, M.D. (1997). J.
Clin. Pharm. Ther. 22, 53-59.
Hansen, J., Bundgaard, H. (1980). Int. J. Pharm.
137
36. Connors, K.A., Amidon, G.L., Stella, V.J. (1986). Chemical stability of
Pharmaceuticals, A Hand Book for Pharmacists, 2nd
ed., Wiley, New York, Chap.
3.
37. Carstensen, J.T. (2000). Solution Kinetics, in: Carstensen, J.J., Rhodes, C.T.,
Eds., Drug Stability Principles and Practices, 3rd
ed, Marcel Dekker, New York,
Chap. 2.
38. Florence, A.T., Attwood, D. (1988). Physicochemical Principles of Pharmacy, 2nd
ed., Macmillan, London, Chap. 4.
39. Garrett, E.R. (1967). Kinetics and mechanism in stability of drugs, in: Bean, H.S.,
Beckett, A.H., Carless, J.E., Eds., Advances in Pharmaceutical Sciences,
Academic Press, New York, Chap. 1.
40. Lintner, C.J. (1973). Product stability, in: Cooper, M.S., Ed., Quality Control in
Pharmaceutical Industry, Academic Press, New York, Chap. 4.
41. Martin, A. (1993). Kinetics, in: Physical Pharmacy, 4th
ed., Waverly International,
Baltimore, Chap. 12.
42. Rawlins, E.A., Ed. (1977). Drug Stability, Bentley’s Textbook of Pharmaceutics,
8th
ed., Bailliere Tindall, London, Chap. 10.
43. Vadas, E.B. (2000). Stability of Pharmaceutical products, in: Gennero, A.R., Ed.,
Remington The Science and Practice of Pharmacy, 20th
ed., Lippincott Williams
& Wilkins, Philadelphia, Chap. 52.
44. World Health Organization (2002). WHO guidelines for stability testing of
pharmaceutical products containing well-established drug substances in
conventional dosages forms, WHO, Geneva.
45. ICH Expert working group. Q1A (R) (2000). Stability testing of new drug
substances and products (draft). International Conference on Harmonization of
technical requirements for the registration of pharmaceuticals for human use.
46. Wells, J. (2002). Kinetics and product stability, in: Aulton, M.E., Ed.,
Pharmaceutics The Science of Dosage From Design, 2nd
ed., Churchill
Livingstone, London, Chap. 7.
47. Winfield, A.J. (2004). Storage and stability of medicines, in: Winfield, A.J., Ed.,
Pharmaceutical Practice, 3rd
ed., Churchill Livingstone, London, Chap. 20.
138
48. Mussan, D.G., Bidgood, A.M., Olegniko. (1991). J. Chromatogr. 565, 89-102.
49. Gaysler, A., Kleuser, B., Sippl, W., Lange, K., Korting, H.C., Holtje, H.D. (1999).
Pharm. Res. 16, 1386-1391.
50. Purkysta, A.R., Martin, K.O., Goldberg, A., Monder, C. (1982). J. Ster. Biochem.
17, 51-59.
51. Thoma, K., Kerker, R., Weissbach, C. (1987). Pharm. Ind. 49, 961-963.
52. Teng, X.W., David, C.C., Davies, N.M. (2003). Int. J. Pharm. 259, 129-141.
53. Donald, P., Jager., Kontny., James, M., Nagel., Hubert, J. (1997). US Patent. 5,
676,930.
54. Wurthwein, G., Rohdewald, P. (1990). Biopharm. Drug. Dispos. 11, 381-394.
55. Yip, Y. W., Li Wan Po, A., Irwin, W. J. (1983). J. Pharm. Sci. 72, 776-781.
56. Mauger, J. W., Paruta, A. N., Gerraughty, R.J. (1969). J. Pharm. Sci. 58, 574-578.
57. Johnson, K., Amidon, G.L., Pogany, S. (1985). J. Pharm. Sci. 74, 87-89.
58. Foe, K., Chueng, H.T.A., Tattam, B. N., Brown, K.F., Seale, J.P. (1988). Drug
Metab. Dispos. 26, 132-137.
59. Ferrante, M.G., Rudy, B.C. (1977). Betamethasone dipropionate, in: Florey, K.,
Ed., Analytical profiles of Drug Substances, Academic Press, New York. p. 57.
60. Barnes, A.R., Nash, S., Watkiss, S.B. (2006). J. Clin. Ther. 103-109.�
61. Gardi, R., Vitali, R., Ercoli, A. (1963). Gazz. Chim. Ital. 93, 431-450.
62. British Pharmaceutical Codex (1973). The Pharmaceutical Press, London, p. 55.
63. Bundgaard, H., Hansen, J. (1981). Int. J. Pharm. 7, 197-203.
64. Yip, Y.W., Li Wan Po, A. (1979). J. Pharm. Pharmacol. 31, 400-402.
65. Ryatt, K.S., Feather, J.W., Mehta, A., Dawson, J.B., Cotterill, J.A., Swallow, R.
(1982). Br. J. Dermatol. 107, 71-76.
66. Kubota, K., Ademola, J., Maibach, H.I. (1994). Dermatology, 188, 13-17.
67. Roth, H.J., Eger, K., Troschutz, R. (1979). Pharmaceutical Chemistry. Vol. 2.
Drug Analysis. Ellis Horwood. pp. 466-467.
68. Guttman, D.E., Meister, P.D. (1958). J. Am. Pharm. Assoc. Sci. Ed., 47, 773-778.
69. Oesterling, T.O., Guttman, D.E. (1964). J. Pharm. Sci. 53, 1189-1192.
70. Monder, C. (1968). Endocrinology, 82, 318-326.
71. 6, 307-319. Hansen, J., Bundgaard, H. (1980). Int. J. Pharm.
139
72. Mc Ginity, J.W., Hill, J.A., La via, L.A. (1975). J. Pharm. Sci. 64, 356-357.
73. Mc Ginity, J.W., Patel, T.R., Naqvi, A.H., Hill. J.A. (1976). Drug Dev. Comm. 2,
505.
74. Dulter, H., Ganter, C., Ryf, H., Uttzinger, E.C., Weinberg, K.S., Schffner, K.,
Arigoni, D., Jeger, O. (1962). Helv. Chim. Acta 45, 2346.
75. Chambers, R.J., Marples, B.A. (1972). Chem. Commun. 1122.
76. Cornell, D.G., Evram, E., Filipeseu. N. (1979). Steroids. 33, 485-486.
77. Ogata, M., Noro, Y., Yamada, M., Tahara, T., Nishimura, T. (1998). J. Pharm.
Sci. 87, 91-95.
78. Barton, D.H.R., Taylor, W.C. (1958). J. Am. Chem. Soc. 80, 244-245.
79. Barton, D.H.R., Taylor, W.C. (1958). J. Am. Chem. Soc. 2500-2510.
80. Hamlin, W.E., Chulski, T., Johnson, R.H., Wagner J.G. (1960) J. Am. Pharm.
Assoc. Sci. Ed. 49, 253-255.
81. Willams, J.R., Moore, R.H., Blount, J.F. (1979). J. Am. Chem Soc. 15, 5019-
5025.
82. Willams, J.R., Moore, R.H., Li, R., Weeks, C.M. (1980). J. Org. Chem. 45, 2324-
2331.
83. Reisch, J., Topaloglu, Y., Henkel, G. (1986). Acta Pharm. Technol. 32, 115-123.
84. Arakawa, Y., Fukaya, C., Yamanouchi, K., Yokoyama, K. (1985). Yakagaku
Zasshi. 105, 1029.
85. Hidaka, T., Huruumi, S., Tamaki, S., Shiraishi, M., Minato, H. (1980).Yakagaku
Zasshi. 100, 72.
86. Thoma, K., Kerker, R., Weissbach, C. (1987). Pharm. Ind. 49, 961-972.
87. Ricci, A., Fasani, E., Mella, M., Albini, A. (2001). J. Org. Chem. 66, 8086-8093.
88. Allen., Gupta, V.D. (1974). J. Pharm. Sci. 63, 107-109.
89. DellaGreca, M., Fiorentino, A., Isidori, M., Lavorgna, A., Previtera, L., Rubino,
A., Temussi, F. (2004). Chemosphere, 54, 629-637.
90. Reisch, J., Iranshani, L., Ekiz-Guecer, N. (1992). Liebigs Ann. Chem. 1199.
91. Reisch, J., Henkel, G., Ekiz-Guecer, N., Nolte, G. (1992). Liebigs Ann. Chem. 63.
92. Takacs, M., Ekiz-Guecer, N., Riesch, J., Gergety-zobin, A. (1991). Pharm. Acta.
Helv. 66, 137.
140
93. Thoma, K., Kerker, R. (1992). Pharm. Ind. 54, 551-554.
94. Kuhnert, M., Randstatter, B.R. (1971). Thermo-Microscopy in the Analysis of
Pharmaceuticals. Pergamon press, oxford, pp. 37-42.
95. Haleblian, J., Koda, R.T., Biles, J.A. (1971). J. Pharm. Sci. 60, 1485.
96. Macek, T.J. (1954). U.S. Patent. 2, 671-750.
97. Ohtani, M., Nakai, T., Ohsawa, K., Kim, S., Matsumoto, M., Etoh, T., Kariya, S.,
Kanou, S., Uchino, K. (2002). Yakugaku Zasshi. 122, 1153-1158.
98. British Pharmacopeia (2009). Her Majesty’s Stationary office, London, pp. 251-
252, 254-255.
99. European Pharmacopeia (2004). Council of Europe, 5th
ed., Strasbourg Cedex,
France, pp.1090-1092, 1094-1095.
100. Yip, Y.W., Li Wan Po, A., Irwin, W.J. (1979). J. Chromatogr. 176, 399-405.
101. Smith, E.W., Haigh, J.M., Kanfer, I. (1985). Int. J. Pharm. 27, 185-192.
102. Smith, E.W., Haigh, J.M. (1989). Pharm. Res. 6, 431-435.
103. Popov, A.P., Priezhev, A.V., Lademann, J., Myllyla, R. (2005). J. Phys. D: Appl.
Phys. 38, 2564-2570.
104. Thoma, K. (1985). Stability of Drugs-Current problems in pharmaceutical
technology, Kongressband, 10th
Conference on pharmaceutical technology,
Shirakabako, Japan. 1-20.
105. Thoma, K., Klimek, R. (1991). Pharm. Ind. 53, 388-396.
106. Thoma, K., Klimek, R. (1991). Int. J. Pharm. 67, 169-175.
107. Edlich, R.F., Winter, K.L., Lim, H.W., Cox, M.J., Becker, D.G., Horovitz, J.H.,
Nichter, L.S., Britt, L.D., Long, W.B. (2004). J. Long-Term Effects Med Inplants.
14, 317-340.
108. Thoma, K., Klimek, R. (1991). OS DE 3136282 (German patent Number).
109. Tonnsen, H.H., Karlsen, J. (1988). Int. J.Pharm. 41, 75-81.
110. Thoma, K. (1996). Photodecomposition and stabilization of compounds in dosage
forms, in: Tonnesen, H.H., Ed. Photostability of Drugs and Drug Formulations.
Taylor & Francis, London. pp. 126-133.
141
111. Moan, J. (1996). Benefits and adverse effects from the combination of drugs and
light, in: Tonnesen, H.H., Ed., Photostability of Drugs and Drug Formulations.
Taylor and Francis, London. pp. 177-178.
112. Trush, M.A., Mimnaugh, E.S., Gram, T.E. (1982). Biochem. Pharmacol. 31,
3333-3343.
113. Rahn, R. O., Landry, L. C and Carrier, W. L. (1974). Photochem. Photobiol. 19,
75-78.
114. Epstein, J.H., Wintroub, B.U. (1985). Drugs. 30, 42-57.
115. Harber, L.C., Kochevar, I.E., Shalita, A.R. (1982). Mechanisms of
photosensitization to drugs in humans, in: Regan, J.D., Parrish, J.A., Eds., The
Science of Photomedicine, Plenum Press, New York, pp. 323-347.
116. Albini, A., Fasani. E. (1998). Drugs: Photochemistry and Photostability, The
Royal Society of Chemistry, Cambridge.
117. Albini, A., Fasani. E., Miolo, G., Riicci, A., Caffieri, S. (2003). Photochem.
Photobiol. 78, 425-430.
118. Suzuki, T., Kato, T., Kitagachi, T., Ono, M., Shirawaka, K., Nagata, M.,
Konischi, R. (1996). J. Toxicol. Sci. 21, 475.
119. Keknes, A., Jahn, P., Lange, L. (1993). J. Am. Acad. Dermatol. 28, 475-484.
120. Uchiyama, H. Tanaka, T. Uehara, N. Nakamura, M. Tsuji, M. Sinomiya, M.
Tanaka, H. (1992). J. Toxicol. Sci. 17, 283-312.
121. Johnson, B.E., Wallker, E.M., Hetherington, A. (1986). In vitro models for
cutaneous phototoxicity, in: Marks., Plewig, G. Eds., Skin Models, Springer,
Berlin. pp. 263-281.
122. Bernard, F.X., Barrault, C., Deguercy, A., De Wever, B., Rosdy, M. (2000). Cell
biol. Toxicol. 16, 390-400.
123. Spielmann. (1994). In vitro phototoxicity testing. ECVAM workshop Report 2,
ATLA. 22, 314-348.
124. Bjorn, P.H. (2002). In vitro phototoxicity testing. Development and validation of
new concentration response analyses software and biostatistical analyses related
to the use of various prediction models, ATLA. 30, 415-432.
125. Rand, R.N. (1969). Clin.Chem.15, 839-863.
142
126. Valenzeno, D.P., Trank, J.W. (1985). Photochem. Photobiol. 42, 335-339.
127. Recknagel, R.O., Glenden, E.A. (1984). in: Packer, L., Ed., Oxygen radicals in
biological systems, Methods in Enzymology., Vol. 105, Academic Press, New
York. p. 331.
128. Steck, T.L., Kant, J.A. (1974). Methods Enzymol. 31, 172-180.
129. Bradford, M. (1976). Anal. Biochem. 72, 248-254.
130. Yoshioka, S., Stella, V.J. (2000) Stability of drugs and dosage forms. Kluwer
Academic/ Plenum publishers, New York, Chap. 2.
131. Mollica, J.A., Rehn, C.R., Smith, J.B. (1969). J. Pharm. Sci, 58, 635-636.
132. Thoma, K., Stielgies, H. (2007). Photostabilization of solid and semisolid dosage
forms, in: Piechocki, J.T., Thoma, K., Eds., Pharmaceuticals photostability and
stabilization technology, informa healthcare, New York, Chap. 16.
133. Miolo, G., Caffieri, S., Dalzoppo, D., Riicci, A., Fasani. E., Albini, A. (2005).
Photochem. Photobiol. 2, 291-298.
Top Related