In Vitro ADMET Laboratories LLC

Evaluation of Inhibitory Potential of Test Article in Pooled Human Hepatocytes TEST ARTICLES:
In Vitro ADMET Laboratories, LLC Columbia, Maryland SPONSOR:
Protocol No. 1300-092111
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In Vitro ADMET Laboratories LLC SPONSOR

Company Name:
Sponsor's Representative:

In Vitro ADMET Laboratories, LLC 9221 Rumsey Road, Suite 8 Columbia, Maryland 21044 Utkarsh Doshi, Ph.D. Phone: 410 -869-9037 Fax: 410 -869-9034 Email: Protocol No. 1300-092111
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In Vitro ADMET Laboratories LLC OBJECTIVE

The objective of this study is to evaluate inhibitory potential of test article on CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4 enzymes in human hepatocytes. GOOD LABORATORY PRACTICES (GLP)
This study will not be a GLP study. ABBREVIATIONS
Universal Cryopreserved Cell Recovery Medium Dimethylsulfoxide Electrospray Ionization Hepatocyte Incubation Media HPLC/MS/MS - HPLC Coupled with Mass Spectrometry

3.1. In Vitro Test System Studies will be performed in 30-donor pooled suspension lot of human hepatocytes. Pooled human hepatocytes (pool of 30 donors, mixed gender) will be obtained from IVAL, Columbia, MD.

4.1. Identification The following test articles will be tested as follows: Name/Identification 4.2. Special Handling Instructions Protocol No. 1300-092111
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In Vitro ADMET Laboratories LLC Handling instructions will be provided by the Sponsor. If no instructions are provided by the Sponsor, test articles will be handled according to applicable laboratory standard operating procedures. TEST ARTICLE PREPARATION
5.1. Test Article Concentrations The test articles will be dissolved in DMSO and a 10mM stock solution will be prepared. The stock solution will be further diluted with HQM to prepare a working solution of 100 µM. The final concentration of DMSO in the reaction mixture will be 0.1%. Seven concentrations (e.g. 0.1, 0.3, 1, 3, 10, 30 and 100 µM) of the test article will be used. The total volume of the incubation will be 250µL. There will be an initial solubility study to determine if the highest concentration of 100 µM is soluble. 6.0 POSITIVE ARTICLE (REFERENCE INHIBITORS)

In each experiment, the respective reference compounds will be tested concurrently with test article, and the data will be compared with historical values determined at in Vitro ADMET Laboratories. The experiment will be accepted in accordance with in Vitro ADMET Laboratories Standard Operating Procedures. The following isoform specific inhibitors will be used for enzymatic reactions: CYP1A inhibition Human hepatocytes (phenacetin substrate) CYP2A6 inhibition Human hepatocytes (coumarin substrate) CYP2B6 inhibition Human hepatocytes (bupropion substrate) CYP2C8 inhibition Human hepatocytes (paclitaxel substrate) CYP2C9 inhibition Human hepatocytes (diclofenac substrate) Protocol No. 1300-092111
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In Vitro ADMET Laboratories LLC CYP2C19 inhibition Human hepatocytes (mephenytoin substrate) CYP2D6 inhibition (dextromethorphan Human hepatocytes CYP2E1 inhibition Human hepatocytes CYP3A inhibition Human hepatocytes (midazolam substrate) CYP3A inhibition Human hepatocytes (testosterone substrate) EXPERIMENTAL PROCEDURES
Thaw required vial of pooled human hepatocytes as per the thawing and recovery protocol using 37°C water bath. Empty the contents of vial in a 50 ml tube of UCRM by inversion using one tube of UCRM/vial of hepatocytes, rinse the vials with 1 ml of UCRM and add the contents directly back into the 50 ml tube again by inversion (do not use pipette). Spin the cells at 100 x g for 10 minutes at 4°C. Empty the supernatant in waste container and resuspend the cell pellet in 2 ml of HQM. Perform viable cell count using Tryphan blue dye exclusion method and adjust the concentration to 2x106 cells/ml. Store the cells on ice until use. Prepare 4X concentration of test article/reference inhibitor/vehicle control and substrate and mix them in equal volume to prepare 2X concentration of test article/reference inhibitor/vehicle control and substrate mixture. Keep this mixture in a 370C water bath or 370C incubator until use. Add 125 µL of cell suspension to an uncoated low binding 48-well plate and allow them to acclimatize in a 370 C incubator in a humidified atmosphere of 95% balance air/5% CO2.The reaction will be initiated by adding 125 µL of test article+ substrate, or reference article+ substrate or vehicle + substrate to the wells containing hepatocytes (final concentration of hepatocyte will be 1 x 106 cells/ml. Eight different concentrations (0.03, 0.1, 0.3, 1, 3, 10, 30, and 100 µM) of reference inhibitors and test article will be used for IC50 determination. Incubation will be performed in triplicates in an incubator maintained at 37 0C for different time points (see table underneath). The final incubation volume will be 250µL. After incubation, the reaction will be terminated by adding 250µL of the acetonitrile containing internal standard (0.1µM terfenadine for positive ionization mode and Protocol No. 1300-092111
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In Vitro ADMET Laboratories LLC 0.01µM tolbutamide for negative ionization mode). Samples will be mixed on a plate shaker for 5 minutes and stored at -20C for 15 minutes followed by centrifugation at 3000 rpm for 5 minutes at room temperature. The supernatant (350µL) will be transferred to a clean cluster tube followed by LC/MS/MS analysis. Detected Compound Test article (eight concentrations), Phenacetin (100 µM) (n=3) Test article (eight concentrations), Coumarin (2 µM) (n=3) 7-hydroxycoumarin Test article (eight concentrations), Bupropion (500 µM (n=3) hydroxybupropion Test article (eight concentrations), 6α-hydroxypaclitaxel Paclitaxel (10 µM), (n=3) Test article (eight concentrations), 4-hydroxydiclofenac Diclofenac (10 µM), (n=3) Test article (eight concentrations), 4-hydroxymephenytoin Mephenytoin (25 µM), (n=3) Test article (eight concentrations), Dextromethorphan (5 µM), (n=3) Test article (eight concentrations), 6-hydroxychlorzoxazone Chlorzoxazone (20 µM), (n=3) Test article (eight concentrations), 1'-hydroxymidazolam Midazolam (5 µM), (n=3) Test article (eight concentrations), 6β-hydroxytestosterone Testosterone (50 µM), (n=3) 8.0 SAMPLE ANALYSIS
LC/MS/MS Analysis The metabolites will be detected by HPLC-MS/MS analysis on an API5000 triple quadrupole mass spectrophotometer system (Applied Biosystems). Liquid Protocol No. 1300-092111
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In Vitro ADMET Laboratories LLC chromatography will be performed on an Acquity UPLC. Analytes will be separated on a Kinetec C18,50x 3mm, 2.6 micron column (Phenomenex, CA) using the following mobile phases. Mobile Phase A: 0.1% formic acid in dH2O Mobile Phase B: 0.1% formic acid in CH3CN
Energy (V)

Ratio of peak areas corresponding to the metabolites of each substrate to the internal standard (IS) for all treatment groups (test article, reference article and vehicle control) will be calculated and the percentage (%) inhibition will be calculated using the following equation: Percentage inhibition in the activity: [Area of analyte/Area of IS] vehicle control-[Area of analyte/Area of IS]test article/reference [Area of analyte/Area of IS] vehicle control Protocol No. 1300-092111
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In Vitro ADMET Laboratories LLC IC50 values (concentration causing a half-maximal inhibition of control values) will be determined by non-linear regression analysis of the concentration-response curve using Graphpad Prism.
10.1. Study Report A study report containing the study design, a brief summary of findings and data figures (as needed) will be submitted to the Sponsor using the format provided by the Sponsor. The final study report will be provided as a PDF file.
Li, A. P. (2001). Screening for human ADME/Tox drug properties in drug discovery. Drug Discov Today. 6: 357-366. Li, A. P. (2004). In vitro approaches to evaluate ADMET drug properties. Curr Top Med Chem. 4: 701-706. Li, A. P. (2007). Human-based in vitro experimental systems for the evaluation of human drug safety. Curr Drug Sar 2:193- 199. Protocol No. 1300-092111
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