Summit 062008

2709 Moffett Ct Plano, TX 75093

Tel: 214-405-6479 Fax: 469-366-5998

TEST DATA REPORT For

SUMMIT PLC

07/05/08 02/06/08 12/06/08 Summit PLC Senthil / Serene / Nasreen

Assay SOP ID:

SBW/BIO/007-14 Date Reported:

Sample ID: Requisition No. Date Sample was Received:

SUM 1-5 BIO/SUM/001

Client Name: Test Done By:

05/02/08

Test Results Verified By: Dr.Yashin Sreenivasan

22/04/08 29/04/08 24/05/08

Test Results Filed By:

Date(s) Assay was Run:

Rupam B.

"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 STUDY DIRECTOR

STUDY CODE: SPLI_03-2007

DR. YASHIN SREENIVASAN HEAD – CELL BIOLOGY

Page 1 of 16

2709 Moffett Ct Plano, TX 75093

Tel: 214-405-6479 Fax: 469-366-5998

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: [email protected] 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

STUDY CODE: SPLI_03-2007

Page 2 of 16

2709 Moffett Ct Plano, TX 75093

Tel: 214-405-6479 Fax: 469-366-5998

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.

STUDY CODE: SPLI_03-2007

Page 3 of 16

2709 Moffett Ct Plano, TX 75093

Tel: 214-405-6479 Fax: 469-366-5998

•

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 STUDY CODE: SPLI_03-2007

Page 4 of 16

2709 Moffett Ct Plano, TX 75093

Tel: 214-405-6479 Fax: 469-366-5998

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.

STUDY CODE: SPLI_03-2007

Page 5 of 16

2709 Moffett Ct Plano, TX 75093

Tel: 214-405-6479 Fax: 469-366-5998

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 cells were cultured alone (control) and with different concentrations (10 ( -6M, 10-7M, 10-8M and 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

STUDY CODE: SPLI_03-2007

Page 6 of 16

2709 Moffett Ct Plano, TX 75093

Tel: 214-405-6479 Fax: 469-366-5998

Effect of ATRA and Spironolactone on Colony Per Well Oil Red accumulation studies were initiated on 10 day old sebocyte primary cultures, whereby cells were cultured alone (control) and with different concentrations (10 ( -6M, 10-7M, 10-8M and -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

STUDY CODE: SPLI_03-2007

Page 7 of 16

2709 Moffett Ct Plano, TX 75093

Tel: 214-405-6479 Fax: 469-366-5998

Effect of ATRA and Spironolactone on Nile Red Staining Nile Red studies were initiated on 10 day old sebocyte primary cultures, whereby cells were cultured alone (control) and with different concentrations (10 ( -6M, 10-7M, 10-8M and 10-9M) of 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

STUDY CODE: SPLI_03-2007

Page 8 of 16

2709 Moffett Ct Plano, TX 75093

Tel: 214-405-6479 Fax: 469-366-5998

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 cultured alone (control) and with different concentrations (10 ( -6M, 10-7M, 10-8M and 10-9M) of 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

STUDY CODE: SPLI_03-2007

Page 9 of 16

2709 Moffett Ct Plano, TX 75093

Tel: 214-405-6479 Fax: 469-366-5998

Fig 7

Fig 8

STUDY CODE: SPLI_03-2007

Page 10 of 16

2709 Moffett Ct Plano, TX 75093

Tel: 214-405-6479 Fax: 469-366-5998

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

Compound STUDY CODE: SPLI_03-2007

Control

Page 11 of 16

C (

2709 Moffett Ct Plano, TX 75093

Tel: 214-405-6479 Fax: 469-366-5998

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

Clonidine hydrochloride

Carbamoyl chloride

Oxybutinin

Control

Conc. (μM) 0 0.03 0.1 0.3 1 3 10 30 100 0.03 0.1 0.3 1 3 10 30 100 0.03 0.1 0.3 1 3 10 30 100

Rep 1 0.1811 0.1708 0.1750 0.1669 0.1659 0.1583 0.1554 0.1546 0.1775 0.1903 0.179 0.1634 0.1688 0.1802 0.1550 0.1545 0.1499 0.203 0.1868 0.1846 0.1419 0.1593 0.1597 0.1595 0.1655

Rep 2 0.1860 0.1948 0.1930 0.1869 0.1879 0.1842 0.1779 0.1717 0.147 0.1762 0.169 0.175 0.169 0.1475 0.1730 0.1646 0.1719 0.1846 0.159 0.163 0.2008 0.1833 0.1777 0.1584 0.1585

Mean 0.1836 0.1828 0.1840 0.1769 0.1769 0.1713 0.1667 0.1632 0.1623 0.1833 0.1740 0.1692 0.1689 0.1639 0.1640 0.1596 0.1609 0.1938 0.1729 0.1738 0.1714 0.1713 0.1687 0.1590 0.1620

Oil Red O Assay SD % CV 0.0035 2 0.0170 9 0.0127 7 0.0141 8 0.0156 9 0.0183 11 0.0159 10 0.0121 7 0.0216 13 0.0100 5 0.0071 4 0.0082 5 0.0001 0 0.0231 14 0.0127 8 0.0071 4 0.0156 10 0.0130 7 0.0197 11 0.0153 9 0.0416 24 0.0170 10 0.0127 8 0.0008 0 0.0049 3

Mean-blank % control 0.1159 100 99 0.1151 0.1163 100 0.1092 94 94 0.1092 0.1036 89 0.0990 85 82 0.0955 82 0.0946 0.1156 100 92 0.1063 0.1015 88 0.1012 87 83 0.0962 0.0963 83 0.0919 79 0.0932 80 109 0.1261 0.1052 91 0.1061 92 89 0.1037 0.1036 89 87 0.1010 0.0913 79 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.

STUDY CODE: SPLI_03-2007

Page 12 of 16

2709 Moffett Ct Plano, TX 75093

Tel: 214-405-6479 Fax: 469-366-5998

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

Compound

C (

Control

Table 4: Nile Red O Assay with 8 concentrations of compound treatment

Compound Oxybutinin Control

STUDY CODE: SPLI_03-2007

in

Carbamoyl chloride

Page 13 of 16

C (

2709 Moffett Ct Plano, TX 75093

Tel: 214-405-6479 Fax: 469-366-5998

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. STUDY CODE: SPLI_03-2007

Page 14 of 16

2709 Moffett Ct Plano, TX 75093

Tel: 214-405-6479 Fax: 469-366-5998

Table 5: HPTLC Estimation of Lipids with Compound Treatment

Lane Inform

Untreated (c Carbomyl chlori Clonidine hydrochl Spiranolacton Retinoic acid Oxybutinine( STUDY CODE: SPLI_03-2007

Page 15 of 16

2709 Moffett Ct Plano, TX 75093

Tel: 214-405-6479 Fax: 469-366-5998

LITERATURE CITED: 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 1.

albumin levels restrict the kinetics of 13-cis retinoic acid uptake and intracellular isomerization to all-trans retinoic acid and inhibit its antiproliferative effect on SZ95 sebocytes. J Invest Dermatol. 2002 Jul;119(1):182-5. 4. Zouboulis CC. Exploration of retinoid activity and the role of

5.

6.

7.

8.

inflammation in acne: issues affecting future directions for acne therapy. J Eur Acad Dermatol Venereol. 2001;15 Suppl 3:63-7. 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. 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. 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. 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.

STUDY CODE: SPLI_03-2007

Page 16 of 16