Professional Documents
Culture Documents
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
iv
An important aspect of this work has been the evaluation of in vitro phototoxicity of
betamethasone
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
vii
CONTENTS
Abstract
iii
Acknowledgements
CHAPTER
Page
1.
1.1
Introduction
1.2
Physicochemical Characteristics
1.3
Chemical Structure
1.4
Synthesis
1.5
Stability
1.5.1
Chemical Stability
1.5.1.1
Hydrolysis
1.5.1.2
Oxidation
11
1.5.1.3
Photolysis
13
1.5.2
Physical Stability
19
1.6
20
20
1.6.2
21
1.7
22
1.8
Phototoxicity
22
EXPERIMENTAL WORK
25
27
2.1
28
2.2
Methods
29
2.2.1
29
viii
2.2.2
29
2.2.3
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
34
35
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
37
2.2.7.1
Radiation chamber
37
2.2.7.2
Radiation source
37
2.2.7.3
Method
37
2.2.8
38
38
2.2.8.2
Sample preparation
39
2.2.8.3
Chromatographic procedure
39
2.2.9
Photohemolysis
39
2.2.10
40
2.2.11
Protein Photodamage
40
2.2.11.1
40
2.2.11.2
41
2.2.11.3
42
ix
RESULTS AND DISCUSSION
3.
43
44
3.1
Introduction
45
3.2
45
of Betamethasone Esters
3.3
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
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
82
4.
92
4.1
Introduction
93
4.2
93
Betamethasone Esters
4.3
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
108
4.6
108
x
and Gel Formulations
5.
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.
CH2OH
CH3
CH2OH
C=O
CH3
C=O
HO
OH
CH3
OH
CH3
O
CORTISONE
HYDROCORTISONE
CH2OH
CH3
C=O
CH3
OH
CH3
CH2OH
CH2OH
C=O
CH3
C=O
HO
HO
OH
OH
CH3
CH3
F
O
O
PREDNISONE
CH2OH
CH3
HO
FLUDROCORTISONE
PREDNISOLONE
C=O
CH3
OH
C=O
CH3
HO
HO
CH3
CH3
OH
F
O
C=O
OH
OH
CH3
CH3
CH2OH
CH2OH
CH3
METHYLPREDNISOLONE
DEXAMETHASONE
TRIAMCINOLONE
Very potent
Potent
Amcinonide 0.1%
Halcinonide 0.1%
Moderately potent
Mildly potent
Fludroxycortide 0.0125%
Hydrocortisone acetate 1%
Prednicarbate 0.25%
6
1.2 Physicochemical Characteristics
The physicochemical characteristics of selected betamethasone derivatives [12-14]
are shown in Table 2.
Physicochemical
characteristics
Betamethasone Valerate
Betamethasone
Dipropionate
Molecular formula
C27H37FO6
C28H37FO7
Molecular weight
476.6
504.6
Appearance
White or creamy-white
crystalline powder
Solubility
Melting point
Optical rotation
[ ]250 = + 65.70
Dioxane
[ ]270 = + 89.40
Methanol
[ ]250 = + 770
Dioxane
UV max (nm)
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
O
H 3C
O
O
HO
CH3
H3C
O
CH3
F
O
BETAMETHASONE DIPROPIONATE
Betamethasone Valerate
Chemically betamethasone valerate is 9-floro-11 , 17, 21-trihydoxy-16 -methyl
pregna-1, 4-diene-3, 20-dione-17-valerate. The empirical formula of the compound
is C27H37FO6 and it has the following chemical structure [12].
O
H3C
O
H 3C
O
OH
HO
H
H3C
CH3
F
O
BETAMETHASONE-17-VALERATE
8
1.4 Synthesis
Betamethasone Dipropionate
Betamethasone dipropionate is synthesized by reacting betamethasone alcohol with
ethyl orthopropionate and toluene-p-sulphonic acid to yield betamethasone 17, 21ethylorthopropionate [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-psulphonic 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.
Hydrolysis
Hydrolysis
Steroid
base
Figure 2.
10
beclomethasone dipropionate is hydrolyzed rapidly to beclomethasone-17monopropionate initially with subsequent slow hydrolysis to beclomethasone. In
addition
steroidal
esters
hydrocortisone
butyrate
[55],
hydrocortisone
in
various
ointment
betamethasone-17-valerate
to
bases
and
reported
betamethasone-21-valerate
the
and
degradation
of
betamethasone
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
OH
OH
H 2C
O
C
OH
CH
OH
(1)
OHC
OH
OH
(2)
(3)
O
CHO
+
CH2
OH
GLYCOLALDEHYDE
(4)
In alkaline medium, under the effect of oxygen, the dihydroxyacetone group at C-17
breaks oxidatively in a form of glycol cleavage to a hydroxyaldehyde which, by
intramolecular rearrangement, changes to the carboxylic acid anion (5).
12
OH
H 2C
O
C
COOH
OH
H
O2
HO-
(1)
(5)
Oxidative attack at C-21 can result in glyoxal derivative (6) which rearranges in
acid medium and with the addition of water produces the hydroxy acid (7).
OH
H 2C
CHC
O
C
C
OH
HO
O
CH
OH
O2
(1)
HOOC
OH
H 2O
(6)
(7)
13
oxidation. Heating at 75C for 6 months in the presence of air, displayed no change
in colour or in the thin layer chromatogram.
1.5.1.3 Photolysis
Photolytic degradation of glucocorticoids has been studied extensively and cited by
different workers both in solutions [74, 75] and in the solid state [76, 77].The
photochemical behavior of glucocorticoids was preliminarily investigated by Barton
and Taylor [78, 79] who focused attention on prednisone acetate. It was found
sensitive to light and converted into a range of novel molecules depending upon the
reaction conditions. Hamlin et al. [80] studied the photolysis of alcoholic solutions
of hydrocortisone, prednisolone and methylprednisolone under ordinary fluorescent
light. They observed that the degradation follows first-order kinetics and the rate of
degradation of prednisolone and methylprednisolone was alike, whereas
hydrocortisone degraded at about 1/7 the rate of the other two steroids. A more
systematic work on prednisone and its 21-acetate was performed by Williams et al.
[81]. They reported that irradiation of prednisone (1a) or prednisone acetate (1b) in
dry dioxane with 254 nm light produced lumiprednisone (2a) and (2b), respectively,
in 65% yield.
CH2OR
CH2OR
CO
CO
O
OH
H
O
h
O
1a , R=H
1b , R=COCH3
2a , R=H
2b , R=COCH3
OH
14
O
h
neutral
O
CH3
OH
CH3
H3O
H2O
H2O
attack
attack
O
HO
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
COCH2OR
OH
X
OH
X
R2
O
R1
h
Solid
R1
O
R3
R3
R2
15
Compound
R1
R2
R3
OH
Pridnisolone / Acetate
H, Ac F
H
OH
Dexamethasone / Acetate
-CH3
-CH3
OH
Betamethasone
C
H
OH
Diflorasone
CH3
CH2OH
CH2OH
O
HO
HO
H
O
F
O
X
16
Degradation in the ring A has also been observed in hydrocortisone in polyethylene
glycol ointment base. [88]. The primary photoproducts may undergo further
transformation with cleavage of the three-membered ring, resulting in
rearomatisation or cleavage of ring A or in the expansion of ring B according to
conditions as shown in prednisolone and dexamethasone [89] (Scheme 1,2,3a,3b).
CH2OH
O
HO
HO
HO
h
O
O
PREDNISOLONE
HO
HO
HO
O
HO
H2O
HO
O
HO
HO
O
OH
OH
Scheme 1
O
HO
CH2OH
O
HO
HO
h
H 2O
O
OH
PREDNISOLONE
Scheme 2
CH2OH
HO
17
CH2OH
HO
HO
HO
O
O
H 2O
h
O
PREDNISOLONE
O
Scheme 3a
CH2OH
CH2OH
OH
HO
HO
HO
CH2OH
CH3
OH
CH3
CH3
HO
h
H2O
O
DEXAMETHASONE
Scheme 3b
18
COCH2R
OH
X
h, N2
Solid
O
X
R
OH H, COCH3
HYDROCORTISONE/ ACETATE
CORTISONE/ ACETATE
CO
H
H, COCH3
COCH2OH
COCH2OH
O
OH
HO
H
HO
CH3
Cl
CH3
CH3
Cl
F
O
F
OH
HO
Cl
h
Solid
F
F
O
F
HALOMETHASONE
Cl
CHOH
CH3
CO
OH
HO
HOCH2CO
HO
HO
H
Cl
F
O
CH3
19
COCH2OCOCH2CH3
COCH2OR
HO
OCO2CH2CH
HO
OR1
h
Solid
O
PREDNICARBATE
R=COCH2CH3,R1=H
R=H,R1=COOCH2CH3
20
1.6
Substance
Betamethasone
valerate
Pure drug/
Dosage
form
Adsorbent
Solvent
System
Rf Values of the
parent compound and
its thermal degrades
Reference
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]
Water:
methanol
ether:dichloromethane
(1.2:8:15:77,
v/v)
[98,99]
Chloroform :
ethylacetate
(1:1, v/v)
Bet-17-valerate = 0.246
Bet- 21-valerate = 0.513
Bet =0.12
Present
work
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
//
Pure drug
//
Pure drug
Betamethasone
dipropionate
Pure drug
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.
Detector
1.0
UV (254nm)
Bet-17-valerate=7min
Bet-21-valerate=9min
1.5
UV (239nm)
[100]
1.5
Diode-array
(239nm)
Bet-17-valerate=5.7min
Bet-21-valerate=6.8min
[101]
Water:
acetonitrile
(45: 55, v/v)
1.5
VariableWavelength
/diode-array
UV detectors
(239nm)
Bet-17-valerate=5.6min
Bet-21-valerate=6.5min
[102]
Substance
Pure drug/
Dosage form
Column
Mobile phase
Betvalerate
Pure drug
//
Ointment
Water:
acetonitrile
(60: 40, v/v)
Water:
acetonitrile
(45:55, v/v)
//
Cream/lotion
Pre-column (RP18) =
3cm x 4.6mm i.d.
packed with
lichrosorb. Analytical
column = 25cm x
4.6mm i.d. packed
with 10 ODS
Pre-column (RP18) =
3cm x 4.6mm i.d.
packed with
lichrosorb. Analytical
column = 25cm x
4.6mm i.d. packed
with 10 ODS
Water:
acetonitrile
(45: 55, v/v)
Reference
[98,99]
//
Isopropyl
Myristate
//
Pure drug/
Cream/ gel
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
Water:
acetonitrile
(3:1, v/v)
0.5
UV (254nm)
Bet-monopropionate=5min
Bet-dipropionate=7min
[59]
//
Pure drug/
Cream/ gel
Water:
acetonitrile
(40:60, v/v)
UV (254nm)
Bet-17-propionate=3.72min
Bet-21-propionate=4.34min
Bet-dipropionate=8.20min
Bet=2.34min
Present
work
Bet = Betamethasone
1.2
22
quinosol,
titanium
dioxide,
colors/pigments,
flavonoids,
23
200
visible
400
500
vanillin
600
(nm)
yellow colourants
red colourants
blue colourants
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
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.
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
Tris-HCl
(pH
6.8),
2%
SDS,
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.
Stock
Solution
2.5%
5%
7.5%
10%
12.5%
15%
17%
20%
1.25
2.5
3.75
5.0
6.25
7.5
8.75
10.0
2.0
2.0
2.0
2.0
2.0
20.
2.0
2.5
1.0
1.5
1.5
1.5
1.5
1.5
1.5
1.5
0.5
0.75
0.75
0.75
0.75
0.75
0.75
0.75
TEMED
0.005
0.005
0.005
0.005
0.005
0.005
0.005
0.005
Water
5.5
8.5
7.75
6.5
5.25
4.0
2.75
1.5
34
2.2.5 Thermal/Photodegradation of Betamethasone Valerate and Betamethasone
Dipropionate in Aqueous and Organic Media
2x10-4M solutions of the compounds were prepared in phosphate buffer
(pH 7.5) and organic solvents e.g. methanol and acetonitrile. Zero time samples
were withdrawn immediately while the remainder solutions were divided into
100 ml aliquots in 100 ml plastic capped glass bottles. The number of samples was
so that a separate sample could be used for each analysis. The samples bottles were
wrapped with aluminum foil for light protection and then placed in an oven at
40 oC. In the case of photodegradation the samples where stirred and flushed with
purified oxygen gas for 30 minutes and then irradiated in the radiation chamber
under controlled temperature (251 oC). The solutions were removed after regular
time intervals and then subjected to HPLC analysis of the parent compounds and
their major thermal or photodegrades as described in section 2.2.8. In the case of
thermal degradation the samples were brought to room temperature before HPLC
analysis. The effect of pH on the thermal stability of betamethasone esters was
studied with citrophosphate buffers of different pH. 2x10-3M solutions (500 ml) of
the esters were prepared by dissolving the exactly weighed quantities of the
compounds in acetonitrile (100 ml) and then mixed with buffers (400 ml) of
different pH. The pH of the final solutions was adjusted with 20% orthophosphoric
acid or 1N sodium hydroxide to 2.5, 3.5, 4.5, 5.5, 6.5 and 7.5, respectively. The
ionic strength () was kept constant at 0.15M. Zero time samples were taken
immediately. The remainder of the solution was divided into aliquots of 50 ml each
in clean 100 ml volumetric flasks. All the flasks were wrapped with aluminum foil
and then kept in an oven at 40 oC. The samples were removed at regular time
intervals and the reaction was immediately terminated by adding 20%
orthophosphoric acid or 1N sodium hydroxide solution to adjust pH of the samples
to approximately 4.0. The volume of the samples was made upto 100 ml with
acetonitrile after bringing the temperature of the samples to room temperature.
Analysis of the samples was performed by a validated high performance liquid
chromatographic assay method as described in section 2.2.8. A similar method was
35
used to study the effect of ionic strength and buffer concentration on the thermal/
photodegradation of betamethasone esters.
2.2.6.1.1 Formulae
Cream
Gel
Material
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
q.s
Material
Betamethasone ester
0.1
Carbomer (940)
0.7
0.5
Propylene glycol
20
Di-isopropenolamine
0.5
Isopropyl alcohol
2.0
Ethyl paraben
0.2
Deionized water
75.9
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 0.45 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 300400 nm was used in all photolytic studies. The technical data of the bulb is as under:
Construction wattage =300
Construction voltage =230
Dimensions (h x w x l) = 203mm x134mm x131mm
Base (standard designation) = E27
Illumination (at sample location) = approx 16,500 lux
2.2.7.3 Method
a.
38
b.
39
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
50M). The suspension was then irradiated with ultraviolet light (300-400 nm)
under gentle shaking in a controlled temperature (251 oC) chamber for increasing
time intervals. Samples containing scavengers like butyl hydroxyanisole (50M)
and sodium azide (50M) were also irradiated similarly. Hemolysis was determined
by measuring the decreasing optical density of the samples at 650 nm [126].
Control samples were (1) untreated RBC (2) RBC in the presence of untreated
compounds and kept in the dark (3) RBC in the presence of pre-irradiated
compounds and kept in the dark and (4) RBC irradiated without compounds.
2.2.10 Photoperoxidation of Linoleic Acid
Linoleic acid (1x10-3M) in phosphate buffer saline (0.01M phosphate buffer,
0.135M NaCl, pH 7.4) containing 0.05% Tween 20 as emulsifying agent was
irradiated with UV light (300-400 nm) in the presence of the compounds (1x10-5M)
for increasing time intervals. Peroxidaton of linoleic acid was determined by
measuring the increasing absorbance at 233 nm corresponding to the conjugated
dienic hydroperoxides formed during irradiation [127]. The process was repeated
with pre-irradiated compounds also.
2.2.11 Protein Photodamage
For determination of protein photocross linking white membranes (ghosts) were
irradiated with the untreated and pre-irradiated compounds and then subjected to
polyacrylamide gel electrophoresis as described as under.
2.2.11.1 Preparation of white membranes (ghosts)
White membranes (ghosts) were prepared by gradual osmotic lysis method [128].
Whole blood collected from untreated albino mouse using heparin as anticoagulant,
was centrifuged at 2500rpm for 15minutes for the separation of RBCs. The RBCs
were washed three times with saline (0.9%NaCl) at 4 oC and subsequently lysed
1:40 with 50mM phosphate buffer (pH 8.3) at 4 oC. The membranes (ghosts) were
41
washed with the same lysis buffer at least five times at 25000g at 4 oC until a
colorless solution of ghosts was obtained .The ghosts were aliquoted and stored at
-70 oC . Ghosts were resuspended in phosphate buffer saline before use.
2.2.11.2 Determination of membranes protein contents
Membrane protein contents were determined by Bradford protein assay method
using bovine serum albumin (BSA) as a standard [129]. Water and proteins were
added into ten colorimetric tubes (10x100mm) according to the top three rows of
table 6. Tube1 was used as a blank while tube 2 to tube 6 were for construction of a
standard calibration curve. Tubes 7 to tube 10 were duplicates of two different
concentrations of the ghosts 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.
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
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.
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,
46
Compound
Betamethasone valerate
Medium
Degradation Products
Acetonitrile, methanol,
Betamethsone-21- valerate
Betamethasone alcohol
cream, gel
Betamethasone dipropionate
Acetonitrile, methanol,
Betamethasone-17-propionate
Betamethasone-21-propionate
cream, gel
Betamethasone alcohol
47
[mV]
Betamethasone alcohol
5.0
Betamethasone
-17-valerate
Betamethasone
-21-valerate
Beclomethasone
dipropionate
2.5
0
0.0
Figure 4.
2.5
5.0
7.5
10.0
12.5
min
48
O
O
H3C
H
HO
H3C
H3C
OH
O
O
H
H3C
H3C
CH3
CH3
OH
H3C
HO
O
BETAMETHASONE-21-VALERATE
BETAMETHASONE-17-VALERATE
H
y
d
r
o
l
y
s
i
s
O
H 3C
HO
OH
OH
H3C
H
CH3
O
BETAMETHASONE ALCOHOL
Figure 5. Degradation pathway for the thermal transformation of betamethasone-17valerate into betamethasone-21-valerate and betamethasone alcohol.
49
[mV]
Betamethasone-17-propionate
20
10
Betamethasone
alcohol
Betamethasone-21-propionate
Betamethasone
dipropionate
Beclomethasone
dipropionate
0
0.0
2.5
5.0
7.5
10.0
12.5
min
50
O
H3C
O
H3 C
H
HO
O
CH3
H
H3C
O
CH3
Deacylation
BETAMETHASONE DIPROPIONATE
Deacylation
O
O
H3C
HO
OH
H3C
H3 C
OH
OH
H3C
HO
H
H3C
CH3
CH3
CH3
F
O
BETAMETHASONE-17-PROPIONATE
BETAMETHASONE-21-PROPIONATE
H
y
d
r
o
l
y
s
i
s
HO
OH
H3C
OH
H3C
CH3
F
O
BETAMETHASONE ALCOHOL
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
0.0192
0.994
Betamethasone-21-valerate
0.0165
0.995
Betamethasone alcohol
0.0625
0.994
Betamethasone dipropionate
0.0135
0.998
Betamethasone-21-propionte
0.0241
0.999
Betamethasone-17-propionate
0.0218
0.994
54
Dosage form
Cream
Gel
g added
g found
% Recovery
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%
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%
Dosage form
Cream
Gel
g added
g found
% Recovery
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%
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
Degradation products
Time (Hour)
Betamethasone
-17-valerate
(M x 105)
10.04
Betamethasone-21valerate
(M x 105)
0.00
Betamethasone
alcohol
(M x 105)
0.00
24
48
72
96
120
144
8.69
1.06
0.34
7.40
6.00
2.00
2.43
1.16
2.19
4.75
3.65
2.90
3.35
3.04
4.10
2.72
3.73
4.97
Degradation products
Time (Hour)
Betamethasone
-17-valerate
(M x 105)
9.97
Betamethasone-21valerate
(M x 105)
0.00
24
48
72
96
120
144
8.80
7.62
6.51
5.40
4.23
3.24
0.91
1.84
2.35
2.73
2.92
3.18
Betamethasone
alcohol
(M x 105)
0.00
0.00
0.32
1.12
2.06
3.23
3.97
56
Degradation products
Time (Hour)
Betamethasone
-17-valerate
(M x 105)
10.14
Betamethasone-21valerate
(M x 105)
0.00
24
48
72
96
120
144
9.20
8.29
7.31
6.45
5.48
4.60
0.79
1.58
2.19
2.73
2.90
3.38
Betamethasone
alcohol
(M x 105)
0.00
0.12
0.87
1.45
2.30
3.65
4.50
Degradation products
Time (Hour)
Betamethasone
-17-valerate
(M x 105)
10.26
Betamethasone-21valerate
(M x 105)
0.00
24
48
72
96
120
144
10.08
9.91
9.75
9.60
9.43
9.29
0.21
0.20
0.24
0.33
0.31
0.32
Betamethasone
alcohol
(M x 105)
0.00
0.00
0.06
0.15
0.20
0.30
0.59
57
Degradation products
Time (Hour)
Betamethasone
-17-valerate
(M x 105)
10.02
Betamethasone-21valerate
(M x 105)
0.00
24
48
72
96
120
144
9.92
9.83
9.75
9.63
9.56
9.45
0.13
0.21
0.24
0.20
0.29
0.31
Betamethasone
alcohol
(M x 105)
0.00
0.00
0.00
0.11
0.18
0.25
0.38
Degradation products
Time
(Hour)
Betamethasone
dipropionate
(M x 105)
0
24
48
72
96
120
144
9.98
Betamethasone
-17-propionate
(M x 105)
0.00
Betamethasone
-21-propionate
(M x 105)
0.00
Betamethasone
alcohol
(M x 105)
0.00
9.55
9.19
8.78
8.42
8.00
7.63
0.00
0.00
0.15
0.29
0.38
0. 43
0.18
0.43
0.60
0.77
0.94
1.13
0.00
0.00
0.00
0.00
0.13
0.28
58
Degradation products
Time
(Hour)
Betamethasone
dipropionate
(M x 105)
10.01
Betamethasone17-propionate
(M x 105)
0.00
24
48
72
96
120
144
9.69
9.36
9.00
8.70
8.41
8.10
0.00
0.00
0.00
0.09
0.16
0.21
Betamethasone- Betamethasone
21-propionate
alcohol
(M x 105)
(M x 105)
0.00
0.00
0.11
0.20
0.39
0.45
0.52
0.63
0.00
0.00
0.00
0.00
0.00
0.04
Degradation products
Time
(Hour)
Betamethasone
dipropionate
(M x 105)
9.93
Betamethasone17-propionate
(M x 105)
0.00
24
48
72
96
120
144
9.78
9.65
9.49
9.37
9.20
9.09
0.00
0.14
0.23
0.25
0.28
0.28
Betamethasone- Betamethasone
21-propionate
alcohol
5
(M x 10 )
(M x 105)
0.00
0.00
0.15
0.24
0.40
0.51
0.67
0.69
0.00
0.10
0.18
0.34
0.52
0.74
59
Degradation products
Time (Hour)
Betamethasone
dipropionate
(M x 105)
10.04
Betamethasone-17propionate
(M x 105)
0.00
24
48
72
96
120
144
9.98
9.88
9.79
9.71
9.65
9.58
0.00
0.00
0.00
0.00
0.00
0.03
Betamethasone-21propionate
(M x 105)
0.00
0.04
0.04
0.09
0.13
0.19
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)
10.26
Betamethasone-17propionate
(M x 105)
0.00
24
48
72
96
120
144
10.19
10.14
10.07
10.00
9.96
9.91
0.00
0.00
0.00
0.00
0.12
0.08
Betamethasone-21propionate
(M x 105)
0.00
0.00
0.05
0.13
0.17
0.21
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 firstorder 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-17propionate. In these reactions betamethasone-21-propionate is the major reaction
61
product which is further degraded to betamethasone alcohol and betamethasone-17propionate is the minor degradation product which is converted to betamethasone21-propionate during the reaction. Therefore, the overall degradation of
betamethasone dipropionate could be considered to follow first-order kinetics. This
has been observed in the treatment of the analytical data for betamethasone
dipropionate and on the basis the values of apparent first-order rate constants for the
overall degradation have been reported.
62
1.2
1
0.8
0.6
0.4
0.2
0
0
24
48
72
96
120
144
168
Time (Hour)
1.2
1
0.8
0.6
0.4
0.2
0
0
24
48
72
96
Time (Hour)
120
144
168
63
Figure 9. First-order plot for the degradation of betamethasone valerate
in acetonitrile (40 oC).
1.2
1
0.8
0.6
0.4
0.2
0
0
24
48
72
96
120
144
168
Time (Hour)
1.01
1.005
1
0.995
0.99
0.985
0.98
0.975
0.97
0
24
48
72
96
120
144
168
Time (Hour)
64
1.005
1
0.995
0.99
0.985
0.98
0.975
0.97
0
24
48
72
96
120
144
168
Time (Hour)
1.02
1
0.98
0.96
0.94
0.92
0.9
0.88
0.86
0
24
48
72
96
120
144
168
Time (Hour)
65
1.02
1
0.98
0.96
0.94
0.92
0.9
0
24
48
72
96
120
144
168
Time (Hour)
1
0.995
0.99
0.985
0.98
0.975
0.97
0.965
0.96
0.955
0
24
48
72
96
120
144
168
Time (Hour)
66
1.005
1
0.995
0.99
0.985
0.98
0.975
0
24
48
72
96
120
144
168
Time (Hour)
1.012
1.01
1.008
1.006
1.004
1.002
1
0.998
0.996
0.994
0
24
48
72
96
120
144
168
Time (Hour)
67
Table 21.
Betamethasone-17-valerte
Betamethasone dipropionate
Corr.
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
3.6 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.
69
10
9
8
7
6
5
4
3
2
1
0
0
10
20
30
40
50
60
70
80
90
.
Dielectric constant
) Methanol (
) Acetonitrile (
1.2
1
0.8
0.6
0.4
0.2
0
0
10
20
30
40
50
60
70
80
90
.
Dielectric constant
) Methanol (
) Acetonitrile (
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
-1.1
-1.6
-2.1
-2.6
-3.1
-3.6
-4.1
-4.6
2
pH
Figure 20. pH-rate profile for the degradation of betamethasone dipropionate (40 oC).
-1
-1.5
-2
-2.5
-3
-3.5
-4
2
pH
Figure 21. pH-rate profile for the degradation of betamethasone-17-valerate (40 oC).
72
3.6.2 Product Distribution
The product distribution (% ratio) at 10% thermal degradation of betamethasone17-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 (8.339.65%) and betamethasone alcohol (0.17-0.9%). The degradation of betamethasone17-valerate
leads
to
the
formation
of
betamethasone
alcohol
through
73
pH
Betamethasone-21valerate
Betamethasone
alcohol
2.5
8.33
0.17
3.5
9.10
0.90
4.5
5.5
9.55
9.65
0.45
0.35
2.5
Betamethasone-17propionate
-
Betamethasone-21propionate
10.00
Betamethasone
alcohol
-
3.5
4.5
9.20
6.80
0.80
3.20
5.5
0.48
8.68
0.83
6.5
7.5
3.18
5.39
6.69
4.61
0.13
-
pH
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 firstorder rate constants (Table 26) were determined from the slopes of the log
concentration versus time plots (Fig 22-29). The second-order rate constants
determined from the slopes of the plots of kobs versus phosphate concentration are
reported as 3.02x10-6 M-1s-1 and 1.305x10-6 M-1s-1 for betamethasone valerate and
betamethasone dipropionate degradation, respectively. The plots show that buffer
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.
Phosphate
Concentration
(M)
Table 25.
Time (Hour)
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
Phosphate
Concentration
(M)
Time (Hour)
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
1.1
1
0.9
0.8
0.7
0.6
0.5
0.4
0
24
48
72
96
Time (Hour)
Figure 22. First-order plot of the thermal degradation of betamethasone valerate (40 oC)
at pH 7.5 (0.05M phosphate buffer).
1.1
1
0.9
0.8
0.7
0.6
0.5
0.4
0
24
48
72
96
Time (Hour)
Figure 23. First-order plot of the thermal degradation of betamethasone valerate (40 oC)
at pH 7.5 (0.1M phosphate buffer).
77
1.1
1
0.9
0.8
0.7
0.6
0.5
0.4
0
24
48
72
96
Time (Hour)
Figure 24. First-order plot of the thermal degradation of betamethasone valerate (40 oC)
at pH 7.5 (0.15M phosphate buffer).
1.1
1
0.9
0.8
0.7
0.6
0.5
0.4
0
24
48
72
96
Time (Hour)
Figure 25. First-order plot of the thermal degradation of betamethasone valerate (40 oC)
at pH 7.5 (0.2M phosphate buffer).
78
1.005
1
0.995
0.99
0.985
0.98
0.975
0.97
0.965
0.96
0.955
0
24
48
72
96
Time (Hour)
1.005
1
0.995
0.99
0.985
0.98
0.975
0.97
0.965
0
24
48
72
96
Time (Hour)
79
1.005
1
0.995
0.99
0.985
0.98
0.975
0
24
48
72
96
Time (Hour)
1.002
1
0.998
0.996
0.994
0.992
0.99
0.988
0
24
48
72
96
Time (Hour)
80
6
5.5
5
4.5
4
3.5
3
2.5
2
0
0.05
0.1
0.15
0.2
0.25
1.4
1.2
1
0.8
0.6
0.4
0.2
0
0
0.05
0.1
0.15
0.2
0.25
81
Table 26.
Betamethasone-17-valerte
Betamethasone dipropionate
Phosphate
concentration
(M)
Corr. Coefficient
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
Corr. Coefficient
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
83
Table 27.
Table 28.
Ionic Strength
(M)
Time (Hour)
24
48
72
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
96
Ionic Strength
(M)
Time (Hour)
24
48
72
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
96
84
1.1
1
0.9
0.8
0.7
0.6
0.5
0.4
0
24
48
72
96
Time (Hour)
Figure 32. First-order plot of the thermal degradation of betamethasone valerate (40 oC)
at pH 7.5 ( = 0.3M).
1.1
1
0.9
0.8
0.7
0.6
0.5
0.4
0
24
48
72
96
Time (Hour)
Figure 33. First-order plot of the thermal degradation of betamethasone valerate (40 oC)
at pH 7.5 ( = 0.6M).
85
1.1
1
0.9
0.8
0.7
0.6
0.5
0.4
0
24
48
72
96
Time (Hour)
Figure 34. First-order plot of the thermal degradation of betamethasone valerate (40 oC)
at pH 7.5 ( = 0.9M).
1.1
1
0.9
0.8
0.7
0.6
0.5
0.4
0
24
48
Time (Hour)
72
96
Figure 35. First-order plot of the thermal degradation of betamethasone valerate (40 oC)
at pH 7.5 ( = 1.2M).
86
1.1
1
0.9
0.8
0.7
0.6
0.5
0.4
0
24
48
72
96
Time (Hour)
Figure 36. First-order plot of the thermal degradation of betamethasone valerate (40 oC)
at pH 7.5 ( = 1.5M).
1.02
1
0.98
0.96
0.94
0.92
0.9
0.88
0
24
48
72
96
Time (Hour)
87
1.02
1
0.98
0.96
0.94
0.92
0.9
0.88
0
24
48
Time (Hour)
72
96
1.02
1
0.98
0.96
0.94
0.92
0.9
0.88
0
24
48
72
96
Time (Hour)
88
1.02
1
0.98
0.96
0.94
0.92
0.9
0.88
0
24
48
72
96
Time (Hour)
1.02
1
0.98
0.96
0.94
0.92
0.9
0.88
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
Ionic strength
(M)
Betamethasone dipropionate
0.3
4.414
0.997
0.779
0.999
0.6
3.989
0.998
0.645
0.999
0.9
3.452
0.998
0.525
0.999
1.2
3.070
0.998
0.455
0.999
1.5
2.734
0.999
0.359
0.998
90
6
5
4
3
2
1
0
0
0.3
0.6
0.9
1.2
1.5
1.8
Io n ic s tre n g th ( )
1
0.8
0.6
0.4
0.2
0
0
0.3
0.6
0.9
1.2
1.5
1.8
Ioni c strength ( )
91
0.2
0.1
0
-0.1
-0.2
-0.3
-0.4
0
0.2
0.4
0.6
0.8
1.2
1.4
0.4
0.2
0
-0.2
-0.4
-0.6
-0.8
-1
-1.2
0
0.2
0.4
0.6
0.8
1.2
1.4
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
mAU
200
Betamethasone
-17-valerate
150
100
Photoproduct A
Photoproduct B
50
0
20
10
30
Time (min)
mAU
Betamethasone
dipropionate
200
150
100
Photoproduct C
Photoproduct D
50
0
0
10
20
30
Time (min)
95
1.00
0.10
4 . 00
0.90
3.60
0.80
3.20
0.70
2. 8 0
0.60
2.40
0.50
2.00
0.40
1.60
0.30
1.20
0. 2 0
0.80
0.10
0.40
0.00
0.08
0. 0 6
0.04
0.02
0. 0 0
0.00
300
250
200
350
400
20 0
Wavelength (nm)
(1)
250
300
350
200
400
250
300
3 50
400
Wavelength (nm)
(B)
Wavelength (nm)
(A)
1. 0 0
2.0
2.0
0.90
1.8
1.8
0 . 80
1. 6
1.6
0.70
1.4
1.4
0.60
1.2
1.2
0.50
1.0
1.0
0.40
0.8
0. 8
0.30
0.6
0.6
0.20
0.4
0.4
0.10
0. 2
0.2
0. 0
0.0
0.00
200
250
300
Wavelength (nm)
(2)
350
400
200
250
300
Wa vele ngt h (n m)
(C)
350
4 00
200
25 0
30 0
350
400
Wavelength (nm)
(D)
Figure 49. UV spectra of betamethasone dipropionate (2) and its photoproducts C and D.
96
standards a complete identification of the photodegradation products of
betamethasone esters could not be achieved.
4.3 Assay of Betamethasone Esters and Photodegradation Products
In order to quantify betamethasone valerate, betamethasone dipropionate and their
photodegradation products, all the compounds were assayed by HPLC method
(USP 2009). Since the nature of photodegradation products is not known, it was
assumed that these products give a similar detector response as that of
betamethasone valerate and betamethasone dipropionate. Therefore, the degradation
products were assayed with reference to the peak height of the parent compounds,
respectively. The values of assay data on the photodegradation of betamethasone
valerate and betamethasone dipropionate are given in Table 30-31.
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)
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
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)
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
4.19
3.98
4.78
8.10
8.84
120
98
Table 32. Product distribution at 10% photodegradation of betamethasone-17-valerate
in different media.
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
--
Medium
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
--
Medium
99
1.2
1
0.8
0.6
0.4
0.2
0
0
30
60
90
120
150
Time (min)
1.1
1
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0
30
60
90
120
150
Time (min)
100
1.1
1
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
30
60
90
120
150
Time (min)
1
0.98
0.96
0.94
0.92
0.9
0.88
0.86
0
30
60
90
120
150
Time (min)
Figure 53. First-order plot for the photodegradation of betamethasone valerate in cream.
101
1
0.98
0.96
0.94
0.92
0.9
0.88
0
30
60
90
120
150
Time (min)
Figure 54. First-order plot for the photodegradation of betamethasone valerate in gel.
1.1
1
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
30
60
90
120
150
Time (min)
102
1.1
1
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0
30
60
90
120
150
Time (min)
1.2
1.1
1
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
0
30
60
90
120
150
Time (min)
103
1.1
1.05
1
0.95
0.9
0.85
0.8
0
30
60
90
120
150
Time (min)
1.1
1.05
1
0.95
0.9
0.85
0.8
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
kobs x103, min-1
Betamethasone dipropionate
Corr.
Coefficient
Medium
Dielectric
Constant
(25 0C)
Corr.
Coefficient
Methanol
32.6
11.303
0.999
7.657
0.999
Acetonitrile
40.1
10.651
0.999
7.254
0.999
Phosphate
buffer (pH 7.5)
78.5
9.288
0.999
6.314
0.999
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.
106
12
10
8
6
4
2
0
0
10
20
30
40
50
60
70
80
90
Dielectric constant
9
8
7
6
5
4
3
2
1
0
0
10
20
30
40
50
60
70
80
90
Dielectric constant
107
16
14
12
10
8
6
4
2
0
0
0.05
0.1
0.15
0.2
12
10
8
6
4
2
0
0
0.05
0.1
0.15
0.2
108
an increase in the buffer concentration in both cases. Therefore, the buffer causes an
inhibition in the rate of reaction. This may be due to deactivation of the excited
species with an increase in buffer concentration. A decrease in the rate of
degradation of such compounds has been observed with an increase in phosphate
buffer [52]. It may be concluded that phosphate buffer has a significant effect on the
photodegradation kinetics of betamethasone ester.
109
12
10
8
6
4
2
0
0
0.3
0.6
0.9
1.2
1.5
1.8
Ionic strength ()
8
7
6
5
4
3
2
1
0
0
0.3
0.6
0.9
1.2
1.5
1.8
Ionic strength ()
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
9.98
10.06
9.96
9.65
9.67
9.55
9.34
9.26
8.12
8.98
8.88
8.68
8.69
8.50
8.27
30
60
90
120
Time (min)
Gel containing
Titanium dioxide
Gel containing
Vanillin
Gel containing
Butyl hydroxytoluene
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
Cream containing
Titanium dioxide
Cream containing
Vanillin
Cream containing
Butyl hydroxytoluene
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
Gel containing
Titanium dioxide
Gel containing
Vanillin
Gel containing
Butyl hydroxytoluene
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
1.02
1
0.98
0.96
0.94
0.92
0.9
0
30
60
90
120
150
Time (min)
1.02
1
0.98
0.96
0.94
0.92
0.9
0
30
60
90
120
150
Time (min)
114
1.01
0.99
0.97
0.95
0.93
0.91
0.89
0.87
0.85
0
30
60
90
120
150
Time (min)
1.01
1
0.99
0.98
0.97
0.96
0.95
0.94
0
30
60
90
120
150
Time (min)
115
1.02
1
0.98
0.96
0.94
0.92
0.9
0
30
60
90
120
150
Time (min)
1.02
1
0.98
0.96
0.94
0.92
0.9
0
30
60
90
120
150
Time (min)
116
1.01
1
0.99
0.98
0.97
0.96
0.95
0
30
60
90
120
150
Time (min)
1.02
1
0.98
0.96
0.94
0.92
0.9
0
30
60
90
120
150
Time (min)
117
1.02
1
0.98
0.96
0.94
0.92
0.9
0
30
60
90
120
150
Time (min)
1
0.99
0.98
0.97
0.96
0.95
0.94
0
30
60
90
120
150
Time (min)
118
1.01
1
0.99
0.98
0.97
0.96
0.95
0.94
0
30
60
90
120
150
Time (min)
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
Formulation
Cream containing
TiO2
Cream containing
vanillin
Cream containing
BHT
Gel containing
TiO2
Gel containing
vanillin
Gel containing
BHT
*
Betamethasone dipropionate
Corr.
Coefficient
Corr.
Coefficient
1.153
0.999
0.909
0.999
1.393
0.999
1.155
0.999
1.548
0.999
1.274
0.999
1.019
0.999
0.692
0.998
1.235
0.999
0.806
0.999
1.297
0.999
0.865
0.999
120
1.000
A
b
0.500
Vanillin
0.000
200.0
250.0
300.0
350.0
400.0
Wavelength (nm.)
Figure 78. UV spectrum of vanillin showing spectral overlay with betamethasone esters.
1.000
A
b
0.500
s
Betam ethasone valerate
BHT
0.000
200.0
250.0
300.0
350.0
400.0
Wavelength (nm.)
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 (50M). The hemolysis
induced by the betamethasone esters and their photoproducts is shown in Figure 8083. 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].
UV
irradiation
as
evidenced
by
the
phothemolysis
and
lipid
124
100
90
80
70
60
50
40
30
20
10
0
0
10
20
30
40
50
60
Time (min)
) RBC (hv)
) RBC+1(dark)
) RBC+1(hv)
) RBC+BHA+1(hv)
125
100
90
80
70
60
50
40
30
20
10
0
0
10
15
20
25
30
Time (min)
126
100
90
80
70
60
50
40
30
20
10
0
0
10
20
30
40
50
60
Time (min)
) RBC (hv)
) RBC+2(dark)
) RBC+2(hv)
) RBC+BHA+2(hv)
127
100
90
80
70
60
50
40
30
20
10
0
0
10
15
20
25
30
35
40
Time (min)
128
1
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
0
10
20
30
40
50
60
Time (min)
129
0.5
0.4
0.3
0.2
0.1
0
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.
and
betamethasone
alcohol
and
betamethasone-17-propionate,
132
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.
133
7. Phototoxicity
The evaluation of the phototoxicity of betamethasone esters using the in vitro
phototoxicity tests such as photohemolysis, lipid peroxidation and protein
photodamage indicates that these esters are phototoxic and cause hemolysis of
mouse red blood cells. Photoproducts of these esters have been found to be toxic in
the dark also. The phototoxicity mechanism for betamethasone esters could not be
confirmed, however, it may involve the reaction of free radical species with cellular
components. Appropriate precautions should be taken in the use of dermatological
preparations containing these compounds.
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135
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