Ointment

ISSN: 2320 3471(Online)
Pasupathi A et.al. Indian J ournal of Research in Pharmacy and Biotechnology

Volume 1 Issue 1 www.ijrpb.com Page 95
FORMULATION, DEVELOPMENT, EVALUATION OF CALCITRIOL AND CLOBETASOL
PROPIONATE OINTMENT
Pasupathi A*, Palanisamy P, B Jaykar, R Margret Chandira, B S Venkateswarlu
Vinayaka Missions College of Pharmacy, vinayaka missions university, Salem, India
*Corresponding author: Email: palanisamy2907@gmail.com
ABSTRACT
Psoriasis vulgaris is a common skin disorder characterized by focal formation of inflamed, raised
plaques that constantly shed scales derived from excessive growth of skin epithelial cells. Prevalence of
Psoriasis is 2-3 % in general population. Currently, Topical Corticosteroids remain a pivotal treatment
due to their effective anti-inflammatory properties; however potential adverse effects associated with
chronic application limit long-term continuous therapy. Vitamin D analogues provide another mechanism
of action reducing lesions through effects on keratinocytes and on cytokine environment. A topical
combination of corticosteroid & Vitamin D derivative appears to provide a balanced approach to
psoriasis treatment. When Calcitriol is used in combination with topical steroids, some improvement in
psoriasis treatment was observed. The main side effect of Calcitriol is skin irritation. Topical steroids
Clobetasol used in conjunction with Calcitriol may lessen skin irritation. Combination reduces hazards
associated with long term use of topical Corticosteroids (atrophy and rebound) as well as irritation
associated with Calcipotriol. Various formulations of Calcitriol (0.0003%) and Clobetasol Propionate
(0.05%) were taken for optimization in relation to ointment base, consistency, ointment stability, stability
with antioxidant, stability with different preservatives, and effect of temperature of Calcitriol phase
addition to bulk white petrolatum base of ointment. Observations of all formulations for physical
characterization and drug release profile had shown that, all comply with the specifications of official
pharmacopeias and or standard reference. It was observed that, formulation batch- F12 selected as
optimized formulation of Ointment, as it fulfills all requirements of Topical ointment.
KEY WORDS: Psoriasis vulgaris, Calcitriol (0.0003%) and Clobetasol Propionate (0.05%).
1. INTRODUCTION
Psoriasis vulgaris (Dr. Peter Pugliese, 2001) is a common skin disorder characterized by focal formation of
inflamed, raised plaques that constantly shed scales derived from excessive growth of skin epithelial cells.
Prevalence of Psoriasis is 2-3%in general population. The disease is defined by a series of linked cellular changes
in the skin, Hyperplasia of epidermal keratinocytes, vascular hyperplasia and ectasia and infiltration of T
lymphocytes, neutrophils, and other types of leucocyte in affected skin. Psoriasis (Dr. Robert E. Connolly, 2009) is
T-cell-mediated autoimmune disorder. The process begins with an environmental factor, perhaps a viral antigen,
which induces T cells to produce cytokines. The cytokines stimulate keratinocyte proliferation and production of
antigenic adhesion molecules in the dermal blood vessels. These adhesion molecules further stimulate T cells to
produce cytokines, thus perpetuating the response.
1.1. Rationale for combination of Calcitriol and Clobetasol propionate: Currently, topical corticosteroids
remain a pivotal treatment due to their effective anti-inflammatory properties; however, potential adverse effects
associated with chronic application limit long therapy. Vitamin D analogues provide another mechanism of action,
reducing lesions through effects on both keratinocytes and on cytokine environment. A topical combination of
corticosteroid and vitamin D derivative appears to provide a balanced approach to psoriasis treatment. When
Calcitriol is used in combination with topical steroids, psoriasis improves more than with either agent alone. The
main side effect of Calcitriol is skin irritation. Topical steroids Clobetasol propionate used in conjunction with
Calcitriol may lessen skin irritation. Combination reduces the hazards associated with the long-term use of topical
corticosteroids. Aim of the present work was to formulate efficacious and safe and stable semisolid dosage form
with combination therapy for a super-potent topical Steroid and a topical Vitamin. In order to
Reduce dosing frequency. To provide synergistic effect.
To provide Ease of application. To increase bioavailability of drug.
1.2. Objective of work: The objectives of the work were to formulate topical preparation of Calcitriol and
Clobetasol propionate combination ointment. The combination in single pharmaceutical composition of Calcitriol
with Clobetasol propionate is not without certain problems. The reason for this is that the Calcitriol is unstable in
aqueous media and more particularly at acidic pH values, where as Clobetasol propionate is unstable in basic
environment. The critical role of vehicle is to enhance therapeutic efficacy and patient compliance. So Attempts
have been made to develop Calcitriol and Clobetasol propionate combination ointment.
2. MATERIALS AND METHODS
Clobetasol Propionate (Micronized) was procured from Sumit Lab and Calcitriol was procured from
Biocon (India), White petrolatum (W.S.P.) was gifted by Unicorn / Wax oils (India), Liquid paraffin was gifted by
Wax oils (India), Cetosteryl alcohol, Sorbiton sesquioleate was gifted by Croda (India), Shea Butter was gifted by
Dow Corning (India), PEG-4000 was gifted by Clariant (India), White bees Wax was gifted by Manali Petro
(India), Propylene glycol was gifted by Wax oils LTD(India), Chlorocresol, Vitamin E Acetate was gifted by
Merck Ltd(India), Caprilic capric tri-glyceride (Fractionated coconut oil) was gifted by Subhash chemicals
(Bangalore), Citric Acid, Monohydrate, Sodium Citrate, Dihydrate was gifted by RFCL LTD (India), Butylated
Hydroxytoluene (BHT) was gifted by Loba chem.(India).
3. FORMULATION OF BATCHES
Table No. 1 Formulation of trial batches from F1 to F6
Ingredient (%w/w) Batches (Quantities in % w/w)
F1 F2 F3 F4 F5 F6
White Petrolatum 64.909 -- -- 64.90 84.90 --
Bees Wax 5.000 -- 1.25 5.00 -- 1.25
Shea Butter 5.000 -- -- 5.00 -- --
Butylated Hydroxytoluene 0.04 0.04 -- -- -- --
Propylene Glycol 10 -- 47.45 10.00 10.00 47.45
Clobetasol propionate 0.05020 0.05020 0.05020 0.05020 0.05070 0.05020
Caprylic capric Triglyceride 15.00 -- -- 15.00 -- --
Calcitriol 0.000307 0.000307 0.000307 0.000307 0.000307 0.000307
Vitamin E Acetate -- -- -- -- 0.05 0.05
Liquid Paraffin -- -- -- -- -- --
Sorbiton Sesquioleate -- -- -- -- -- --
Gelucire 44/14 -- -- -- 0.05 -- --
PEG-400 -- 64.910 -- -- -- --
PEG-4000 -- 35.00 -- -- -- --
Cetostearyl Alcohol -- -- 8.40 -- -- 8.40
Glyceryl Monostearate -- -- 11.00 -- -- 11.00
Stearoyl Macrogol -- -- 1.50 -- -- 1.50
Chlorocresol -- -- 0.08 -- -- 0.08
Citric Acid, monohydrate -- -- 0.05 -- -- 0.05
Sodium Citrate, dehydrate -- -- 0.05 -- -- 0.05
Purified Water -- -- 30.18 -- -- 30.18

3.1. Evaluation of formulation:
3.1.1.Measurements of pH: (Amra Osmancevic and Kerstin Landin-Wilhelmsen, 2010) (By Extraction
methodology): 2.5gm Ointment sample was taken in 100 ml dry beaker, then 50 ml water was added to it. Beaker
was heated on water bath maintained at about 60C to 70C for 10 minutes, cooled to room temperature, and then
centrifuged at 3000 rpm for 10 minutes. The pH of supernant water extract was measured by using Labindia 200
+

pH meter. The pH measurements were done by using a digital type pH meter by dipping the glass electrode into the
ointment formulation.
3.1.2. Determination of viscosity: ( Zaira Kharaeva, 2009) The measurement of viscosity of the formulated
Ointment was done by using Brookfield Viscometer (CAP 2000+

Viscometer). Spindle No. 1 was used for the
determination of viscosity of ointment. Spindle was rotated at 80 rpm for 30 second for each measurement. The
results are shown in Table No.5.
3.1.2. Determination of extrudability:

(Christina MP, 1994) (Smith, Jan G, 2010) Extrudability test is the
measure of the force required to extrude the material from a collapsible tube when certain amount of force has been
applied on it in the form of weight. In the present study the quantity in percentage of cream extruded from the tube
on application of certain load was determined. More the quantity extruded, better was the extrudability of ointment.
3.1.3. Determination of spreadability: (Christina MP, 1994) (Smith, Jan G, 2010) One of the criteria for a cream,
ointment or gel is that it should possess good spreadability. Spreadability is a term expressed to denote the extent
of area to which the cream readily spreads on application to skin or affected part. The therapeutic efficacy of a
formulation also depends on its spreading value. Hence determination of spreadability is very important in
evaluating ointment characteristics. Special apparatus was designed to study the spreadability of ointment
formulations. The spreadability is expressed in terms of time in seconds taken by two slides to slip off from
ointment, placed in between the slides under the direction of certain load. Lesser the time taken for separation of
twoslides, better the spreadability of ointment.
3.1.4. Microscopic evaluation: Microscopic evaluation of all formulated batches was done by using Compound
microscope with the help of MOTIC software. Evaluation of the sample was carried initially and after each
stability condition.
Procedure:
Clean and dry glass slide and cover slip were taken
Very small quantity of sample was taken on glass slide
Sample was covered by placing cover slip on it. Precaution was taken that there was no entrapment of air
Cover slip was slightly pressed to spread the sample uniformly to prepare the thin film
It was observed under microscope with different power of lenses like 10x, 40x and 100x.

Table No. 2. Formulation of trial batches from F7 to F12
FIngredient (%w/w) Batches (Quantities in % w/w)
F7 F8 F9 F10 F11 F12
White Petrolactum 89.41 84.90 89.90 89.399 84.45 89.399
Bees Wax -- -- -- -- --
Shea Butter -- -- -- -- -- --
Butylated Hydroxytoluene -- -- -- -- -- 0.05
Propylene Glycol 5.000 10.00 5.00 5.00 10.00 5.00
Clobetasol propionate 0.05020 0.05070 0.05020 0.05070 0.05070 0.05070
Caprylic capric Triglyceride 5.000 -- -- -- -- --
Calcitriol 0.000307 0.000307 0.000307 0.000307 0.000307 0.000307
Vitamin E Acetate 0.05 0.05 0.05 0.05 0.05 --
Liquid Paraffin -- 5.00 5.00 5.00 5.00 5.00
Sorbiton Sesquioleate 0.50 -- -- -- 0.50 0.50
Gelucire 44/14 -- -- -- 0.50 -- --
3.1.5. Microbiological enumeration test: 10 gm of product was transferred aseptically to make 100ml of buffered
phosphate buffer solution pH 7.2 or fluid soyabean casein digest medium containing 4% polysorbate 20 (solution-
A)
3.1.6. Pour-plate method: 1ml of prepared sample was taken and transferred in to sterile Petri dishes. 15 to 20ml
of liquefied soyabean-casein digest agar was poured in to sterile petri dishes, suitable for cultivation of bacteria and
liquefied sabouraud dextrose agar suitable for cultivation of fungi at not more than 45C then covered them. The
sample was mixed with media by tintling or rotating the dishes and contents were allowed to solidify at room
temperature. The plates of soyabean-casein digest agar were incubated at 30 to 35C for 3 to 5 days and the plates
of sabouraud dextrose agar at 20 to 25C for 5 to 7 days. Plates were selected corresponding to given dilution and
showing the highest number of colonies less than 250 for total aerobic microbial count and 50 for total yeast and
mould count (TYMC). The arithmetic mean per culture medium of counts was taken and the number of colony
forming units (cfu) per gram of sample was calculated.
3.1.7. Interpretation of the results: The total aerobic microbial count (TAMC) was considered equal to the
number of cfu found using soyabean-casein digest agar. If colonies of fungi were detected on this medium, they
were counted as part of TAMC. The total yeast and mould count (TYMC) was considered equal to the number of
cfu found using sabouraud dextrose agar. If colonies of bacteria were detected on this medium, they were counted
as a part of TYMC. When the TYMC was expected to exceed the acceptance criteria due to bacterial growth,
sabouraud dextrose agar containing antibiotics needed to be use.
3.2. Test for specified micro-organisms
3.2.1. Sample preparation and pre-incubation: A sample was prepared using a 1 in 10 dilution of quantity
corresponding not less than 1 gm of product to be examined or 10ml of solution A was transferred aseptically to
make 100ml soyabean-casein digest broth. If the product was known to have antimicrobial activity, an inactivating
agent such as polysorbate 80, soyalecithin needed to be adding. It was incubated at 30 to 35C for 18 to 24 hours.
After 24 hours incubation, the test for Pseudomonas aeruginosa and Staphylococcus aureus were carry out.
3.2.2. Test for Pseudomonas aeruginosa
Selection and subculture: After 24 hours of incubation of soyabean-casein digest broth, a loopful of enriched
culture was streaked on a plate of centrimide agar. The plates were incubated at 30-35C for 18 to 72 hours.
Interpretation: The presence of Pseudomonas aeruginosa was indicated by growth of colonies. This was
confirmed by identification tests. The product comply the test if the colonies were not present.
Identification tests: Any colony showing greenish colour was subcultured on the surface of pseudomonas agar
medium for detection of pyocyanin and pseudomonas agar medium for detection of fluroresin, plates were covered
and inverted and incubated at 35-37C for not less than three days. Observation and result: The streaked surface
was examined under UV light to determine whether colonies are having characteristics given below.
Morphological characteristics: Addition of fluroresin to pseudomonas agar medium shows generally colourless
to yellowish colonies and yellowish fluorescence in UV light. Morphological characteristics pseudomonas agar
medium for detection of pyocyanin shows generally greenish colonies and bluish fluorescence pseudomonas agar
medium in UV light.

Table No. 2 Morphological characteristics
Medium Characteristic colonial
Morphology
fluorescence in UV light
Pseudomonas Agar medium for
detection of Pyocyanin
Generally colourless to
yellowish colonies
Yellowish
Pseudomonas Agar medium for
detection of Pyocyanin
Generally greenish
colonies
Blue
Any suspect colonial growth was confirmed by oxidase test.
Oxidase test: if growth of suspect colonies occurs, smear the suspected colony on oxidase disc. If there is no
development of purple colour within 30 seconds, sample meets the requirement for the test of absence of
pseudomonas aeruginosa.
3.2.3. Test for Staphylococcus aureus
Selection and subculture: After 24 hours of incubation of Soyabean-Casein Digest Broth, a loopful of enriched
culture was streaked on a plate of mannitol salt agar. The plates were incubated at 30-35C for 18 to 72 hours.
Interpretation:The presence of staphylococcus aureus was indicated by growth of yellow or white colonies
surrounded by yellow zone. This was confirmed by identification tests. The product comply the test if the colonies
were not present. Identification tests: In case the colonies characteristics matches with the description as mentioned
above, then presence of positive cocci was confirmed by gram staining.
Coagulase Test (Staphylococcus aureus): With the aid of an inoculating loop, transfer representative suspect
colonies from the agar surfaces of the mannitol salt agar medium to individual tubes, each containing 0.5ml of
mammalian, preferably rabbit or horse plasma with or without suitable additives. Incubate in water bath at 37C,
examining the tubes at 3 hours and subsequently at suitable intervals up to 24 hours. Test positive and negative
sample meets the requirements of the test for absence of Staphylococcus aureus.
Table No. 3 Culture Conditions for Inoculum Preparation
Organism Suitable Medium Incubation
Temperature
Inoculum
Incubation Time
Microbial Recovery
Incubation Time
Pseudomonas aeruginosa
(ATCC No. 9027)
SoybeanCasein
Digest Agar
32.5 2.5
0
C 18 to 24 hours 3 to 5 days
Staphylococcus aureus
(ATCC No. 6538)
SoybeanCasein
Digest Agar
32.5 2.5
0
C 18 to 24 hours 3 to 5 days
Interpretation criteria:
Table No. 4 Acceptance criteria for microbiology interpreted as follows.
Limit Maximum
acceptable count
10
1
CFU 20
10
2
CFU 200
10
3
CFU 2000
3.2.4. Stability Study:Stability study was performed as per ICH guideline. The purpose of stability testing is to
provide evidence on how the quality of a drug substance or drug product varies with time under the influence of a
variety of environmental factors such as temperature, humidity and light. Therefore, stability studies provide data
to justify the storage condition and shelf-life of the drug product. For drug substance, such studies establish the
retest date in addition to the storage condition of raw material.
4. RESULT AND DISCUSSION
4.1. Related Substances calculation:

Figure No. 1: Chromatogram for RS of mixed Standard

Table No. 5 Peak results for RS of mixed standard

Chromatogram for RS of sample (Optimized batch F12):
Figure No. 2 Chromatogram for RS of Sample for Clobetasol propionate (Optimized batch F12)
Table No. 6 Peak results for RS of sample (optimized batch F12)

Single maximum impurity- 0.12
Total impurities- 0.2867
RS study of sample (optimized batch, F12) after accelerated stability study at the condition 40
0
C/75 %RH showed
that related substances formed due to degradation of active ingredients were below the limits prescribed.
4.2. Evaluation of Ointment: The evaluated parameters were pH, viscosity, spreadability, extrudability and assay.

Table No.7 Initial evaluation of formulation batches

Note: * = Mean S.D., CP=Clobetasol propionate, C=Calcitriol

Name RT Area % Area USP
Tailing
USP Plate
Count
Clobetasol propionate 12.85 25468 10.7 1.13 6746
Calcitriol 17.4 255216 90.3 1.093 12416
Peak No. Name RT Area % Area % Impurity
1 Unknown-1 4.7 6052 0.41 0.0297
2 Unknown-2 5.3 7241 0.49 0.031
3 Unknown-3 13.9 20389 13.93 0.100
4 Clobetasol propionate 12.9 253981 83.20 --
5 Unknown-4 5.7 28706 1.96 0.126
Batch
N0.
pH *

Viscosity*
(Poise)
% Drug content*
(Clobetasol Propionate)
%Drug content*
(Calcitriol)
F1 6.15 0.01 - - -
F2 6.46 0.03 1.29 0.22 - -
F3 6.43 0.04 3.57 0.26 - -
F4 6.21 0.12 5.05 0.12 - -
F5 6.63 0.15 3.58 0.25 98.28 0.17 98.37 1.08
F6 6.75 0.15 4.10 0.14 97.26 0.13 98.46 1.15
F7 6.10 0.15 3.56 0.17 98.41 0.08 97.89 1.23
F8 5.70 0.15 3.55 0.27 98.29 0.05 99.19 0.94
F9 5.25 0.26 3.50 0.37 98.17 0.35 99.42 0.29
F10 5.45 0.26 3.55 0.52 99.40 0.23 98.94 0.54
F11 5.10 0.26 3.49 0.45 99.27 0.25 99.94 0.19
F12 5.32 0.26 3.61 0.57 99.83 0.23 99.62 0.86
4.2.1.In-vitro Drug release profile of Calcitriol:
Table No.8 Diffusion Profile for Calcitriol (Trial Batch F7, F8, F9)
Figure No. 3 Diffusion Profile for Calcitriol (Trial Batch
F7, F8, F9)
Time (min) Galderma (innovator) F7 F8 F9
0 0 0 0 0
30 4.3 2.6 3.1 3.4
60 12.4 7.4 8.7 9.9
120 37.2 19.9 25.3 28.2
180 53.9 32.7 44.5 46.7
240 70.9 45.3 59.4 60.7
300 86.3 55.6 69.8 75.9
Diffusion Drug Release Profile of Calcitriol
0 100 200 300 400
0
10
20
30
40
50
60
70
80
90
100
Innovator
F7
F8
F9
Time
%

D
r
u
g

R
e
l
e
a
s
e

Table No.9 Diffusion Profile for Calcitriol (Trial Batch
F10, F11, F12)
Figure No. 4 Diffusion Profile for Calcitriol
(Trial Batch F10, F11, F12)
Time (min) Galderma
(Innovator)
F10 F11 F12
0 0 0 0 0
30 4.3 3.5 3.9 4.1
60 12.4 9.9 11.1 12.6
120 37.2 28.7 32.4 37.1
180 53.9 47.9 50.2 54.3
240 70.9 62.7 66.6 70.5
300 86.3 77.9 84.8 87.3
Diffusion drug release profile of Calcitriol
0 100 200 300 400
0
10
20
30
40
50
60
70
80
90
100
INNOVATOR
F10
F11
F12
Time(Min)
%
D
r
u
g

R
e
l
e
a
s
e

Table No.10 Diffusion Profile for Calcitriol (Trial Batch F12) Figure No. 5 Diffusion Profile for Calcitriol
(Trial Batch F12 & Innovator)
Time (min) Galderma (Innovator) F12
0 0 0
30 4.3 4.1
60 12.4 12.6
120 37.2 37.1
180 53.9 54.3
240 70.9 70.5
300 86.3 87.3
Diffusion drug release profile of Calcitriol
0 100 200 300 400
0
10
20
30
40
50
60
70
80
90
100
INNOVATOR
F12
Time(Min)
%
D
r
u
g

R
e
l
e
a
s
e

4.2.2. In-vitro Drug release profile of Clobetasol propionate:

Table No.11 Diffusion Profile for Clobetasol propionate
Figure No. 6 Diffusion Profile for Clobetasol
propionate (Trial Batch F7, F8, F9)
Time
(min)
Galderma
(Innovator) F7 F8 F9
0 0 0 0 0
30 9.1 7.0 6.4 7.3
60 18.4 12.8 11.3 13.4
120 42.4 37.1 30.7 38.7
180 60.2 53.2 47.5 55.8
240 76.6 69.4 63.7 70.1
300 91.1 81.4 78.2 83.8
Diffusion Drug Release Profile of Clobetasol propionate
0 100 200 300 400
0
10
20
30
40
50
60
70
80
90
100
Innovator
F7
F8
F9
Time
%

D
r
u
g

R
e
l
e
a
s
e

In-vitro Drug release profile of Clobetasol propionate:
Table No.12 Diffusion Profile for Clobetasol propionate
Figure No. 7 Diffusion Profile for Clobetasol
propionate (Trial Batch F10, F11, F12)
Time
(min)
Galderma
(Innovator)
F10 F11 F12
0 0 0 0 0
30 9.1 7.4 8.5 9.4
60 18.4 13.5 15.6 18.9
120 42.4 38.4 39.8 42.7
180 60.2 55.9 58.2 61.1
240 76.6 70.3 73.5 77.0
300 91.1 83.5 87.4 92.1
0 100 200 300 400
0
10
20
30
40
50
60
70
80
90
100
110
120
Innovator
F10
F11
F12
Time(Min)
%

D
r
u
g

R
e
l
e
a
s
e

In-vitro Drug release profile of Clobetasol propionate:

Table No.13 Diffusion Profile for Clobetasol
propionate (Trial Batch F12)
Figure No. 8 Diffusion Profile for Calcitriol (Trial
Batch F12 & Innovator)
Time(min) Galderma(Innovator) F12
0 0 0
30 9.1 9.4
60 18.4 18.9
120 42.4 42.7
180 60.2 61.1
240 76.6 77.0
300 91.1 92.1
0 100 200 300 400
0
10
20
30
40
50
60
70
80
90
100
Innovator
F12
Time(min)
%

D
r
u
g

R
e
l
e
a
s
e

4.3. Optimization of Ointment base for consistency: The base selection was carried out in F3, F4 and F5. First
batch was formulated as per the Galderma ointment and Calcitriol was incorporated in Caprylic capric triglyceride,
consistency suddenly found to be dropped and drug phase was not dispersed uniformly. In next batch F2,
micronized form of both the drugs was used and Calcitriol phase was incorporated in PEG base after emulsification
at 70
0
C. Micronized form of drug gave smooth texture to ointment but consistency again found to be dropped.
After that to improve the consistency, in trial batch F3 Dermovat Cream base with 8.4% w/w Cetostearyl alcohol
was incorporated in water phase. Viscosity was found good but after the stability testing (e.g. 25C/60%RH,
30C/65%RH, 40C/75%RH) assay results indicated that concentration of both drugs were decreasing day by day.
F1 & F3 has better consistency than PEG base. In next trial F4 white petrolatum base Gelucire 44/14 as emulsifier
was used, but consistency was increased too high due to Shea butter and bees wax. After evaluation it was found
that consistency was needed to reduce slightly and spreadability needed to improve as it showed tackiness. Results
showed that F5 exhibited good spreadability property as compared to previous batches.
4.4. Batches with the antioxidant: In the Innovator formula, BHT was used as antioxidant. The antioxidant was
used to find out the more stable formulation as both the drugs are prone to oxidative degradation. The continued
degradation of drugs results in a total loss of its therapeutic activity. Previous batch F2 was prepared with single
antioxidant i.e., BHT. And next trial batch F5 was taken with antioxidant Vitamin E Acetate were evaluated for the
assay initially and after the stability testing (e.g. 25C/60%RH, 30C/65%RH, 40C/75%RH). The antioxidant
vitamin E was added to WSP base, PEG base, & Cream base formulation batches. (F2, F5, F6). But according to
microbiological enumeration test, it was observed that batch F5 with white petrolatum and Vitamin E Acetate
having better stability than PEG base or Cream base. Finally Vitamin E Acetate was selected as antioxidant for
next formulation trial batches.

F2 F5 F6
Figure No. 9: Microscopic evaluation of F2, F5 and F6

4.5. Batches with different Solubilizer: To evaluate the effect of Solubilizer on stability of formulation, batches
with the different Solubilizer concentrations were evaluated for assay to find out the effect of Solubilizer on %
drug content after stability study. Initial batches showed that variation in Solubilizer effects on assay and variation
of drug content at different points of the SS container as such top, middle and bottom. In trial batch F7 Fractionated
coconut oil (5%) was used as Solubilizer for Calcitriol with propylene glycol (5%) for Clobetasol propionate as
Solubilizer. In trial batch F8 Liquid paraffin (5%) used as Solubilizer for Calcitriol with propylene glycol (10%)
for Clobetasol propionate as Solubilizer. In trial batch F9 Liquid paraffin (5%) was used as Solubilizer for
Calcitriol with propylene glycol (5%) for Clobetasol propionate as Solubilizer. It provides uniformity of both drugs
dispersion in ointment base. The trial batches were evaluated initially and after the stability period of 3 month at
different stability conditions.
4.6. Selection of Emulsifier for stabilization of Ointment (F10): As in trial batch F7, F8 & F9 oily appearance
was observed on the top surface of the ointment, due to separation of Solubilizer to the some extent, after keeping
over night. This was due to separation of propylene glycol or liquid paraffin from white petrolatum base. So it was
needed to add an Emulsifying agent. The two emulsifiers were selected as Gelucire 44/14 & Sorbiton sesquioleate.
HLB of Sorbiton sesquioleate = 03.7 HLB of Gelucire 44/14= 14
As the formulation to be prepared was consist of a combination of more than one oleaginous material such
as water-insoluble hydrophobic oils and fats. Most of the early ointment bases used to be exclusively oleaginous in
nature. All the previous batches were formulated without the surfactant. Except trial batch F4. Consistency was
found good but separation of propylene glycol or liquid paraffin from white petrolatum base. To overcome the non
uniformity of Solubilizer with white petrolatum base, trial batch F10 was not found good. But Trial batch F11
given better uniformity and homogenousity of Solubilizer with white petrolatum base. Fraction of surfactant was
added in both the drug phases (i.e., Clobetasol propionate and Calcitriol).
4.7. Batches with different Temp. of addition of Calcitriol phase to the bulk white petrolatum base:In, Trial
batch F11 & F12 the Calcitriol in liquid paraffin phase was added to the bulk White petrolatum base at 70
o
C to
72
o
C & 35
o
C to 37
o
C. F12 batch was observed more consistent than F11. Hence effect of Temp. On formulation
was optimized. Depending on the antimicrobial stability, assay and In-vitro Drug release profile, final optimized
batch F12 was selected.
4.8. Stability study of different batches: Stability study was carried out according to the ICH guidelines at
25
0
C/60% RH, 30
0
C/75% RH, 40
0
C/75% RH for three months. The formulation was packed in aluminum
lacquered tube and kept in stability chamber. All the parameters were evaluated after stability study.
Table No. 14 Stability results after 1 month
Parameters Stability result at 25
0
C/60 %RH
F7 F8 F9 F10 F11 F12
pH* 7.110.04 7.720.05 5.160.10 5.220.25 5.620.12 5.620.05
Viscosity* (Poise) 3.700.11 3.620.08 3.640.05 3.570.04 3.620.04 3.610.18
Spreadability.* (gm cm/sec) 4.110.16 4.200.08 4.120.07 4.200.23 4.270.11 4. 500.08
Extrudability* (%) 760.94 800.54 820.16 810.26 160.33 870.16
%Assay (CP)* 99.461.25 99.470.72 99.621.01 98.210.72 99.740.22 98.780.62
%Assay(C)* 98.831.05 99.530.89 99.630.96 98.100.49 99.000.31 99.200.43
Stability result at 30
0
C/75 %RH
pH* 7.210.04 7.760.12 5.180.02 5.190.25 5.330.17 5.600.23
Viscosity* (Poise) 3.660.15 3.600.27 3.590.04 3.580.71 3.620.26 3.610.08
Spreadability.* (gm cm/sec) 4.160.14 4.240.26 4.180.08 4.180.21 4.200.18 4.210.26
Extrudability* (%) 780.87 801.01 900.84 810.42 840.06 860.05
%Assay (CP)* 98.981.43 99.450.14 99.541.03 99.140.04 98.700.17 99.710.42
%Assay(C)* 98.781.21 99.530.04 99.620.78 99.050.26 99.000.42 99.660.19
0
C/75 %RH
pH* 7.920.28 8.300.18 5.160.15 5.190.25 5.310.54 5.580.52
Viscosity* (Poise) 3.600.12 3.550.34 3.620.54 3.580.06 3.540.06 3.610.87
Spreadability.* (gm cm/sec) 4.250.11 4.180.12 4.120.65 4.180.21 4.160.27 4.210.06
Extrudability* (%) 800.87 811.12 910.33 810.87 840.06 860.05
%Assay (CP)* 97.341.12 99.191.53 99.541.34 99.140.06 98.640.42 99.700.27
%Assay(C)* 97.111.01 99.231.19 99.520.23 99.050.26 99.040.42 99.580.06
Note: * = Mean Standard deviation, CP=Clobetasol propionate, C=Calcitriol
Once the ointment base was optimized, trials with different antioxidants, with different Solubilizer and
temperature of addition of Calcitriol phase to bulk phase (F7-F12) were formulated and kept for the stability study.
Results of the stability studies are shown in Tables 14, 15 and 16. Assay of F3 and F4 was found to be
dropped at the accelerated stability conditions of 40
0
C/75 %RH. This drop in assay may be due to ineffective
protection against oxidative degradation. Use of antioxidants in F5 and F6 gave better results. Also the dispersion
of both drugs in ointment base was not uniform and homogenous so in F7, F8 and F9 different Solubilizer with
different concentration were used. 5% propylene glycol and 5% liquid paraffin for Clobetasol propionate and
Calcitriol found better results. In F10 to overcome the non uniformity of solubilizer with white petrolatum base
was optimized by using Emulsifying agent Gelucire 44/14 and Sorbiton sesquioleate. Result of Sorbiton
sesquioleate was found better. In trial F11, F12 Temp. of addition of Calcitriol phase to the bulk white petrolatum
base was optimized. Also % drug content was affected to minor extent was considerable. Final optimized
formulation was selected on the basis of Microbiological enumeration testing of F9-F12.
4.9. Related Substances: Determination of related substances of Clobetasol propionate (active ingredient) was
carried out for the batches kept for stability study at different conditions (i.e., F7-F12). Determination of related
substances of Calcitriol (active ingredient) was not carried out due to conc. was too much low 0.0003% w/w. All
the stability batches showed the total impurities for Clobetasol propionate below the prescribed limit
4.10. Microbiological Enumeration Test: The preservative efficacy test was designed to perform the quantitative
and qualitative estimation of specific viable microorganisms present in formulation by using various cultures of
bacteria and fungi (Staphylococcus aureus, Pseudomonas aeruginosa) for 28 days. Cfu/gm of sample was counted
at the interval of 7 days. Results are tabulated in Table No. 17.
Table No. 15 Stability results after 2 months
0
C/60 %RH
F7 F8 F9 F10 F11 F12
pH* 7.250.54 8.130.18 5.160.09 5.220.19 5.280.19 5.580.12
Viscosity* (Poise) 3.650.02 3.510.96 3.640.04 3.570.22 3.600.18 3.620.04
Spreadability.*
(gm cm/sec)
4.410.17 4.180.08 4.120.41 4.200.33 4.250.08 4.501.21
Extrudability* (%) 760.27 800.04 820.12 810.21 820.49 860.32
%Assay (CP)* 99.350.65 99.310.72 99.640.15 98.210.22 98.70.62 99.700.07
%Assay(C)* 98.640.21 99.370.21 99.630.31 98.100.16 99.000.89 99.200.31
0
C/75 %RH
pH* 7.210.81 7.760.12 5.180.02 5.190.78 5.330.23 5.560.12
Viscosity* (Poise) 3.660.11 3.600.07 3.590.04 3.58 0.11 3.620.53 3.610.05
Spreadability.*
(gm cm/sec)
4.160.31 4.240.13 4.180.18 4.180.25 4.200.12 4.210.21
Extrudability* (%) 780.35 801.01 900.71 810.42 840.06 860.08
%Assay (CP)* 98.981.02 99.011.03 99.541.03 99.140.24 98.500.06 99.600.37
%Assay(C)* 98.780.22 99.140.41 99.621.05 99.050.41 99.000.23 99.150.12
0
C/75 %RH
pH* 7.920.28 8.300.31 5.200.15 5.270.25 5.310.54 5.550.02
Viscosity* (Poise) 3.600.12 3.550.34 3.610.54 3.340.06 3.540.06 3.610.15
Spreadability.*
(gm cm/sec)
4.250.11 4.180.12 4.020.42 4.110.21 4.160.27 4.210.12
Extrudability* (%) 800.12 801.09 910.33 810.87 840.23 860.65
%Assay (CP)* 97.040.06 98.860.42 99.451.04 99.140.06 98.640.17 99.430.27
%Assay(C)* 97.050.01 98.940.13 99.020.21 99.050.46 99.040.65 99.100.02

All batches with different preservative combinations showed decrease in Cfu count but F12 containing
Vitamin E Acetate (0.5% w/w) was found to be most effective in inhibiting the microbial growth.
Preservative in F12 was found to be effective as,
The concentration of viable bacteria was not more than 0.1% of initial concentration by the 14
th
day.
The concentration of each test microorganism remained at or below the designated levels during the
remainder of 28 day test period.
On the basis all the evaluation parameters of formulation including assay, RS, viscosity, spreadability, extrudability
and emulsion stability initially and after exposure to accelerated stability conditions and finally on the basis of
antimicrobial preservative efficacy testing, F12 was selected as optimized formula for topical delivery of
Clobetasol propionate and Calcitriol.
4.11. Microscopic evaluation of optimized batch: From microscopy it was observed that the uniform sized
globules were distributed throughout White petrolatum base (Figure 10). Microscopic evaluation after accelerated
stability studies showed that ointment was stable as retaining its integrity and globule size did not increase to
considerable extent.

Table No. 16 Stability results after 3 months
0
C/60 %RH
F7 F8 F9 F10 F11 F12
pH* 7.300.26 8.130.18 5.460.09 5.320.19 5.280.19 5.510.12
Viscosity* (Poise) 3.650.14 3.510.96 3.640.04 3.570.22 3.600.18 3.620.04
Spreadability.* (gm
cm/sec)
4.501.21 4.060.08 4.120.41 4.200.33 4.250.08 4.410.81
Extrudability* (%) 760.27 800.04 820.12 810.21 820.49 860.32
%Assay (CP)* 99.040.65 99.120.72 99.640.15 98.210.22 98.70.62 99.700.07
%Assay(C)* 98.130.21 99.190.21 99.630.31 98.100.16 99.000.89 99.200.31
0
C/75 %RH
pH* 7.210.81 7.760.12 5.380.02 5.280.78 5.330.23 5.500.12
Viscosity* (Poise) 3.660.11 3.600.07 3.590.02 3.58 0.11 3.620.53 3.610.05
Spreadability.* (gm
cm/sec)
4.160.31 4.240.13 4.180.18 4.180.25 4.200.12 4.210.21
Extrudability* (%) 780.32 801.01 900.71 810.42 840.06 860.08
%Assay (CP)* 98.79.17 98.450.23 99.541.03 99.140.24 98.700.06 99.610.37
%Assay(C)* 98.720.05 98.710.37 99.621.05 99.050.41 99.000.23 99.150.12
0
C/75 %RH
pH* 7.920.06 8.300.31 5.200.36 5.270.25 5.390.54 5.510.02
Viscosity* (Poise) 3.600.12 3.550.34 3.610.54 3.340.06 3.540.18 3.610.21
Spreadability.* (gm
cm/sec)
4.250.11 4.180.23 4.020.42 4.110.21 4.360.27 4.220.12
Extrudability* (%) 800.13 801.09 910.15 810.11 840.31 860.65
%Assay (CP)* 97.000.06 98.290.23 99.391.04 99.140.16 98.520.06 99.310.31
%Assay(C)* 96.870.15 98.580.37 99.120.26 99.020.13 99.000.28 99.140.32

Table No.17 Results of Microbiological Enumeration Test
Evaluation time Trial
Batch
Culture used
Staph. aureus
ATCC 6538
Pseudomonas aeruginosa
ATCC 9027
0 hour count Cfu/gm of
sample Cfu x 10
3
F9 213 210
F10 215 208
F11 243 234
F12 209 201
7 days count Cfu/gm of
sample Cfu x 10
3
F9 212 221
F10 189 203
F11 189 196
F12 183 195
sample
F9 132 113
F10 45 39
F11 49 41
F12 39 45
sample
F9 59 54
F10 9 5
F11 7 5
F12 3 2
sample
F9 21 12
F10 0 1
F11 1 0
F12 0 0

Figure No. 10: Microscopy of
optimized batch (F12)

Cfu = Colony forming unit

5. SUMMARY AND CONCLUSSION
A topical combination of corticosteroid & Vitamin D derivative appears to provide a balanced approach to
psoriasis treatment. When Calcitriol is used in combination with topical steroids, Psoriasis improves more than one
agent alone. The main side effect of Calcitriol is skin irritation. Topical steroids Clobetasol used in conjunction
with Calcitriol may lessen skin irritation. Combination reduces hazards associated with long term use of topical
Corticosteroids. Various formulations of Calcitriol (0.0003%) and Clobetasol Propionate (0.05%) were taken for
optimization in relation to ointment base, consistency, ointment stability, stability with antioxidant, stability with
different preservatives, and effect of temperature of Calcitriol phase addition to bulk white petrolatum base of
ointment. Initial batch based on innovator Galderma showed sudden drop in consistency and also the drugs were
not dispersed uniformly in formulation as it showed grittiness. From trial batch F2 micronized form of both the
drugs was used to prevent grittiness. In F2, F3, F4 and F5 selection of base was carried out to improve the
consistency.
Homogeneity and consistency were improved in F5 than F1, F2, F3 and F4 but it showed tackiness as
previous batches. To improve spreadability, Bees wax, Shea butter was not added in F5. Homogeneity of F5 & F6
was satisfactory with uniform globule size and drug dispersion. In trial batch F7 Fractionated coconut oil (5%) was
used as Solubilizer for Calcitriol with propylene glycol (5%) for Clobetasol propionate as Solubilizer. In trial batch
F8 Liquid paraffin (5%) used as Solubilizer for Calcitriol with propylene glycol (10%) for Clobetasol propionate as
Solubilizer. In trial batch F9 Liquid paraffin (5%) was used as Solubilizer for Calcitriol with propylene glycol (5%)
for Clobetasol propionate as Solubilizer. Trial batch F11 & F12 the Calcitriol in liquid paraffin phase was added to
the bulk White petrolatum base at 70
o
C to 72
o
C & 35
o
C to 37
o
C. F12 batch was observed more consistent than
F11. Hence effect of Temperature on formulation was optimized. Depending on the antimicrobial stability, assay
and In-vitro Drug release profile, final optimized batch F12 was selected. Observations of all formulations for
physical characterization, assay & in-vitro drug release had shown that, all comply with the specifications of
official pharmacopeias and or standard reference. It was observed that, formulation batch- F12 selected as
optimized formulation of Ointment, as it fulfills all requirements of Topical ointment identical with innovator
product ( GALDERMA). It was concluded that ointment of Calcitriol & Clobetasol propionate could be formulated
components and processing aspects.
6. ACKNOWLEDGEMENTS
Authors are thankful to Prof.(Dr.) B.Jayakar, Principal, Vinayaka missions college of pharmacy, Salem,
Tamilnadu, India providing all the facilities for this research work.

REFERENCES
Amra Osmancevic and Kerstin Landin-Wilhelmsen, Vitamin D status in psoriasis patients during different
treatments with phototherapy, Journal of Photochemistry and Photobiology, B Biology, 101(2), 2010, 117-121.
Christina MP, Mouse Model of psoriasis, Research in Immunology, 1994, 145(5), 357-362.
Dr. Peter Pugliese, Physiology of the Skin, Workbook and Study Guide, Allured Pub Corp, 1
st
edition, 2001,
35-41.
Dr. Robert E. Connolly, Psoriasis Can Be Cured, 2009, 350-353.
Smith Jan G, United States Application US2010/0249060 A1, Topical formulation of low level Clobetasol
propionate for treating disorders of the skin and mucous membranes, 2010.
The United State Pharmacopoeia 2007, Volume 32(I) The official compendia of standard, USP-30, NF-25,
1787.
Zaira Kharaeva, Elena Gostova, Chiara De Luca, Desanka Raskovic, Liudmila Korkina, Clinical and
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Pasupathi A et.al. Indian J ournal of Research in Pharmacy and Biotechnology

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