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TEST DATA REPORT

For

SUMMIT PLC

07/05/08
Assay SOP ID: SBW/BIO/007-14 Date Reported: 02/06/08
12/06/08
Sample ID: SUM 1-5 Client Name: Summit PLC
Requisition No. BIO/SUM/001 Test Done By: Senthil / Serene / Nasreen
Date Sample was
05/02/08 Test Results Verified By: Dr.Yashin Sreenivasan
Received:
22/04/08
Date(s) Assay was
29/04/08 Test Results Filed By: Rupam B.
Run:
24/05/08

"This study was conducted according to the procedures described in this report. All data
presented are authentic, accurate and correct to the best of our knowledge."

DR. SWATI BHATTACHARYYA DR. YASHIN SREENIVASAN

STUDY DIRECTOR HEAD – CELL BIOLOGY

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Work Order Number: BIO/SUM/001

Services Being Reported: Individual Tests
Compound Information:
Reference compounds – ATRA, Spironolactone.
Test compounds - Chlonidine hydrochloride, Carbamoyl chloride, Oxybutinin.
Date received – 05/02/08
Alternative Code 1: Sum 1
SBI Internal #: SUM 1/SEB
Lot: Sigma/Seb/001

Sponsor: Summit PLC

91 Milton Park
Abingdon
Oxfordshire
OX14 4RY
UK

Tel: +44 (0) 1235 44 39 39

Fax: +44 (0) 1235 44 39 99

e-mail: info@summitplc.com
Undertaken at: Simbiosys Biowares Inc - India
Simbiosys Biowares India Pvt Ltd
Unit 1 & 2, Innovator Building, ITPL, Bangalore 560066, India

Date of Study: 06/02/08 - 20/04/07

Date Reported: 12/06/08
Study Directors: Yashin Sreenivasan, Ph.D. Simbiosys Biowares India Pvt Ltd.
Distribution: Summit PLC

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STUDY OBJECTIVE
Evaluate the effect of compounds on primary cultures of sebocytes for the following
parameters:

 Cell growth kinetics

 Oil Red staining
 Nile Red staining
 HPTLC study of the lipids
 Reference compounds - ATRA and Spironolactone
 Test compounds - Chlonidine hydrochloride, Carbamoyl chloride, Oxybutinin

METHODS
Methods employed in this study have been adapted from the scientific literature to maximize
reliability and reproducibility. The brief protocols for the methods used are as follows.

1. Nile Red staining

• Remove the medium completely.
• Wash the cells with 200 µl of 1 X PBS.
• Add 100µl of dye (10µg/ml) into all wells and 3 wells as Blank (with out cells) and
mix well.

Blank - 100 µl of 10µg/ml dye in 1X PBS (3 wells).

Background control - just cells + 100µl 1X PBS (in duplicates).
Experimental control – just cells +100µl of 10µg/ml dye in 1X PBS (in
duplicates).

• Incubate for 10minutes.

• Measure the fluorescence: excitation 485 nm and emission: 535 nm.
2. Oil red O staining
• Remove the medium completely.

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• Wash the cells with 100 µl of 1 X DPBS.

• Add 100 µl of 10% formalin in DPBS and incubate at 30 minutes (RT).
• Remove all the formalin.
• Wash the cells with 100 µl of 60% isopropanol.
• Add 50µl freshly diluted Oil red O working solution and incubate for 15 minutes in
room temperature.
• Remove all the Oil red O.
• Wash six times with 200µl of 1X DPBS. (View under microscope) .Make sure that
no Oil red O is sticking to the walls.
• Elute Oil red O by adding 100 µl of 4% triton in 100% isopropanol and incubate
for 15 minutes at 370C.
• Pipette the isopropanol with Oil red O up and down several times to be sure that all
Oil red O is in the solution.
• Transfer 70 µl to a fresh 96 well plate. (Add 70 µl of 4% triton in 100%
isopropanol as a blank)
• Measure the O.D. at 530 nm.

3. Lipid extraction

Lipids were extracted by the Folch-Lees method (Folch et al, 1957). The cells were
homogenized with chloroform/methanol (2/1) to a final volume 20 times the volume of the
cell sample (1g in 20ml of solvent mixture). The whole mixture was agitated for 45
minutes in a vortex machine at room temperature. The homogenate was centrifuged at
2000 rpm for 10 minutes to recover the liquid phase. The solvent was washed with 0.2
volume (4 ml for 20 ml) of water or 0.9% NaCl solution. After vortexing briefly, the
mixture was centrifuged at 2000 rpm for 10 minutes to separate the two phases. The upper
phase was removed by siphoning and the lower chloroform phase containing lipids was
evaporated at 370C. After proper drying the lipids were dissolved in an appropriate volume
of chloroform/methanol (2/1) mixture and stored at -200C.

4. Lipid analysis by thin layer chromatography

Lipid analysis was done by thin layer chromatography according to the protocol followed
by Downie et al, 1998. Chromatography was carried out on 20 × 20 cm TLC pre-coated

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silica gel plates. Separation of neutral and polar lipids was done by developing half-way
in polar solvent using methyl acetate/isopropanol/ chloroform/ methanol/0.25% KCl (aq)
25:25:25:10:9 (vol/vol) followed by three runs in the following neutral solvents: (і)
toluene/ diethyl ether/ethanol/acetic acid 60:40:1:0.23 (vol/vol), (іі) hexane/diethyl ether
96:4 (vol/vol), and (ііі) hexane alone. The separated lipid classes were visualized by
spraying the plates with 3% cupric acetate in 8% orthophosphoric acid followed by heating
in an oven at 1600C for 15 minutes. The lipid classes were quantified densitometrically
using the ImageJ software developed at the National Institute of Health, USA.

VALIDATION:
ATRA and Spironolactone are both well compounds used for the treatment of acne. This
initial study was done to ensure the validity of the assay whereby these 2 compounds will
be run on every plate as control for the rest of the compounds asked by the client. The
activity of the compound(s) was estimated in 4 different doses – 10-6M, 10-7M, 10-8M and
10-9M. Assays were performed under conditions described in the accompanying "
Experimental Results" section of this report.

SCREENING
For screening the assay was done with 8 concentrations in triplicates (0.03, 0.1, 0.3, 1, 3,
10, 30 and 100 µ M) in triplicates. Assays were performed under conditions described in
the accompanying " Experimental Results" section of this report.

RESULTS
A summary of results meeting the significance criteria is presented in the following section.
Complete results are presented under the section labeled "Experimental Results" as mean
percent inhibition in all experiments.

SUMMARY / CONCLUSION
The assay has been validated with ATRA and Spironolactone as reference compounds.
The sebocytes have been treated with test compounds and screened for Oil red, Nile
red and HPTLC. This completes phase 1 & 2 of the project.

Effect of ATRA and Spironolactone on Cell Growth Kinetics of Sebocytes

Cell growth kinetics studies were done with sebocytes isolated from midline chest skin from 3
different volunteers after obtaining all required regulatory approvals. The cells were cultured
alone (control) and with 10-7M of ATRA and Spironolactone for upto 15 days. The results of the
study have been shown in Fig 1.

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Fig 1

Effect of ATRA and Spironolactone on Oil Red Staining

Oil Red accumulation studies were initiated on 10 day old sebocyte primary cultures, whereby
( -6M, 10-7M, 10-8M and
cells were cultured alone (control) and with different concentrations (10
10-9M) of of ATRA and Spironolactone for 48 hours. The 12 day old culture at the end of
compound treatment were stained for Oil Red. The results of the study have been shown in Fig
2.

Fig 2

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Effect of ATRA and Spironolactone on Colony Per Well

Oil Red accumulation studies were initiated on 10 day old sebocyte primary cultures, whereby
( -6M, 10-7M, 10-8M and
cells were cultured alone (control) and with different concentrations (10
-9
10 M) of of ATRA and Spironolactone for 48 hours. The 12 day old culture at the end of
compound treatment were stained for Oil Red and counted as individual colonies per well. The
cells were graded in 3 major classes <6, 6-50 and >50. The results of the study have been shown
in Fig 3.

Fig 3

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Effect of ATRA and Spironolactone on Nile Red Staining

Nile Red studies were initiated on 10 day old sebocyte primary cultures, whereby cells were
( -6M, 10-7M, 10-8M and 10-9M) of
cultured alone (control) and with different concentrations (10
of ATRA and Spironolactone for 48 hours. The 12 day old culture at the end of compound
treatment were stained for Nile Red. The results of the study have been shown in Fig 4.

Fig 4

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Effect of ATRA and Spironolactone on Lipids as Esimated by HPTLC

HPLTC studies were performed with 12 day old sebocyte primary cultures, whereby cells were
( -6M, 10-7M, 10-8M and 10-9M) of
cultured alone (control) and with different concentrations (10
of ATRA and Spironolactone for 48 hours. The 12 day old culture at the end of compound
treatment were extracted with different solvent for estimation of squalene, wax, cholesterol,
Triglyceride and free fatty acid. The solvent fractions were run on HPTLC and were analyzed
through densitometry scan. The mean area of the peak were estimated for each lipid type for
each individuals with or without treatment. The results of mean percent inhibition have been
shown as Fig 5-9.

Fig 5

Fig 6

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Fig 7

Fig 8

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Fig 9

Screening of compounds for Oil Red Staining on sebocytes

Oil Red accumulation studies were initiated on 7 day old sebocyte primary cultures, whereby
cells were cultured alone (control) and with compounds. The compound treatment was done in
two batches – a) with 3 doses (1, 10 and 100 µ M)) and b) with 8 different concentrations (0.03,
0.1, 0.3, 1, 3, 10, 30 and 100 µ M) of 3 compounds (Chlonidine hydrochloride, Carbamoyl
chloride, Oxybutinin) in triplicates of for 120 hours. The 12 day old culture, at the end of
compound treatment, were stained for Oil Red. The results of the study have been shown in
Table 1 & 2.

Table 1: Oil Red O Assay with 3 concentrations of compound

treatment

C
Compound
(
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Table 2: Oil Red O Assay with 8 concentrations of compound

treatment
Conc. Oil Red O Assay
Compound
(μM) Rep 1 Rep 2 Mean SD % CV Mean-blank % control
Control 0 0.1811 0.1860 0.1836 0.0035 2 0.1159 100
0.03 0.1708 0.1948 0.1828 0.0170 9 0.1151 99
0.1 0.1750 0.1930 0.1840 0.0127 7 0.1163 100
Oxybutinin

0.3 0.1669 0.1869 0.1769 0.0141 8 0.1092 94

1 0.1659 0.1879 0.1769 0.0156 9 0.1092 94
3 0.1583 0.1842 0.1713 0.0183 11 0.1036 89
10 0.1554 0.1779 0.1667 0.0159 10 0.0990 85
30 0.1546 0.1717 0.1632 0.0121 7 0.0955 82
100 0.1775 0.147 0.1623 0.0216 13 0.0946 82
0.03 0.1903 0.1762 0.1833 0.0100 5 0.1156 100
Carbamoyl chloride

0.1 0.179 0.169 0.1740 0.0071 4 0.1063 92

0.3 0.1634 0.175 0.1692 0.0082 5 0.1015 88
1 0.1688 0.169 0.1689 0.0001 0 0.1012 87
3 0.1802 0.1475 0.1639 0.0231 14 0.0962 83
10 0.1550 0.1730 0.1640 0.0127 8 0.0963 83
30 0.1545 0.1646 0.1596 0.0071 4 0.0919 79
100 0.1499 0.1719 0.1609 0.0156 10 0.0932 80
0.03 0.203 0.1846 0.1938 0.0130 7 0.1261 109
0.1 0.1868 0.159 0.1729 0.0197 11 0.1052 91
hydrochloride

0.3 0.1846 0.163 0.1738 0.0153 9 0.1061 92

Clonidine

1 0.1419 0.2008 0.1714 0.0416 24 0.1037 89

3 0.1593 0.1833 0.1713 0.0170 10 0.1036 89
10 0.1597 0.1777 0.1687 0.0127 8 0.1010 87
30 0.1595 0.1584 0.1590 0.0008 0 0.0913 79
100 0.1655 0.1585 0.1620 0.0049 3 0.0943 81

Screening of compounds for Nile Red Staining

Nile Red staining studies were initiated on 7 day old sebocyte primary cultures, whereby cells
were cultured alone (control) and with different concentrations of compound treatment. The
compound treatment was done in two batches – a) with 3 doses (1, 10 and 100 µ M)) and b) with
8 different concentrations (0.03, 0.1, 0.3, 1, 3, 10, 30 and 100 µ M) of 3 compounds
(Chlonidine hydrochloride, Carbamoyl chloride, Oxybutinin) in triplicates of for 120 hours. The 12
day old culture, at the end of compound treatment, were stained for Nile Red. The results of the
study have been shown in Table 3 & 4.

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Table 3: Nile Red O Assay with 3 concentrations of compound

treatment

C
Compound
(
Control
Table 4: Nile Red O Assay with 8 concentrations of compound
treatment

C
Compound
Oxybutinin (
Control

Carbamoyl
chloride
in

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Screening of compounds for Lipid Esimation by HPTLC

HPLTC studies were performed with 7 day old sebocyte primary cultures, whereby cells were
cultured alone (control) with 100 µ M of Chlonidine hydrochloride, Carbamoyl chloride and
Oxybutinin. The 12 day old culture at the end of compound treatment were extracted with
different solvent for estimation of squalene, wax, cholesterol, Triglyceride and phospholipids.
The solvent fractions were run on HPTLC and were analyzed through densitometry scan (Fig
10). The mean area of the peak were estimated for each lipid type for each individuals with or
without treatment. The results of mean percent inhibition have been shown as Table 5.

Fig 10 – HPTLC of lipids isolated from sebocytes with compound

treatment.

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Table 5: HPTLC Estimation of Lipids with Compound Treatment

Lane Inform
Untreated (c
Carbomyl chlori
Clonidine hydrochl
Spiranolacton
Retinoic acid
Oxybutinine(
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LITERATURE CITED:

1. Nelson AM, Gilliland KL, Cong Z, Thiboutot DM. 13-cis Retinoic acid
induces apoptosis and cell cycle arrest in human SEB-1 sebocytes. J Invest
Dermatol. 2006 Oct;126(10):2178-89.
2. Wróbel A, Seltmann H, Fimmel S, Müller-Decker K, Tsukada M, Bogdanoff
B, Mandt N, Blume-Peytavi U, Orfanos CE, Zouboulis CC. Differentiation
and apoptosis in human immortalized sebocytes. J Invest Dermatol. 2003
Feb;120(2):175-81.
3. Tsukada M, Schröder M, Seltmann H, Orfanos CE, Zouboulis CC. High
albumin levels restrict the kinetics of 13-cis retinoic acid uptake and
intracellular isomerization to all-trans retinoic acid and inhibit its anti-
proliferative effect on SZ95 sebocytes. J Invest Dermatol. 2002
Jul;119(1):182-5.
4. Zouboulis CC. Exploration of retinoid activity and the role of
inflammation in acne: issues affecting future directions for acne
therapy. J Eur Acad Dermatol Venereol. 2001;15 Suppl 3:63-7.
5. Ridden J, Ferguson D, Kealey T. Organ maintenance of human sebaceous
glands: in vitro effects of 13-cis retinoic acid and testosterone. J Cell Sci.
1990 Jan;95 ( Pt 1):125-36.
6. Zouboulis CC, Xia L, Akamatsu H, Seltmann H, Fritsch M, Hornemann S,
Rühl R, Chen W, Nau H, Orfanos CE. The human sebocyte culture model
provides new insights into development and management of seborrhoea
and acne. Dermatology. 1998;196(1):21-31.
7. Zouboulis CC, Akamatsu H, Stephanek K, Orfanos CE. Androgens affect the
activity of human sebocytes in culture in a manner dependent on the
localization of the sebaceous glands and their effect is antagonized by
spironolactone. Skin Pharmacol. 1994;7(1-2):33-40.
8. Akamatsu H, Zouboulis CC, Orfanos CE. Spironolactone directly inhibits
proliferation of cultured human facial sebocytes and acts antagonistically
to testosterone and 5 alpha-dihydrotestosterone in vitro. J Invest Dermatol.
1993 May;100(5):660-2.

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