Chris Bohl, Ph.D. Global Technical Support Manager- Products
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1 Chris Bohl, Ph.D. Global Technical Support Manager- Products
2 Sekisui XenoTech Overview GLP-compliant in vitro ADME-DMPK CRO founded in 1994 at the University of Kansas Medical Center Provide services and products relevant to ADME-DMPK studies In August 2008 XenoTech was acquired by Sekisui, Inc. (part of Sekisui Medical Division, Ltd) In the fall of 2015, Sekisui XenoTech relocated to current facility in Kansas City, KS (pictured right) ~130 employees at US facility Kansas City Global client base, including companies (> 300) involved in the development of pharmaceuticals, cosmetics, food additives Global distributors in Europe, Japan, Korea, India, China and Singapore
3 XenoTech is a provider of key reagents and test systems for in vitro drug metabolism studies Subcellular Fractions Microsomes, S9, Cytosol Human and pre-clinical species Hepatic and extrahepatic (intestine, kidney, lung, etc.) Lysosomes/tritosomes Hepatocytes Human Numerous pre-clinical species (mouse, rat, dog, monkey, etc.) Fresh or cryopreserved Attaching or suspension Individual or pooled Other ADME Products Immortalized hepatocytes (Fa2N-4 cells) Multiple reagents (e.g. NADPH-generating system) Biobank Custom products We use these products in our drug metabolism services. This enables a comprehensive understanding of the test systems that we provide to valued customers
4 CryostaX Hepatocytes
5 What are they?
6 How is CryostaX going to help me and my research?
7 Traditional Method for Preparing Animal Primary Hepatocytes
8 Method for Preparing CryostaX Animal Primary Hepatocytes Larger lot sizes and larger pools
9 Simplified Product Offering CryostaX hepatocytes have 1 standardized Assured Minimal Yield (AMY) post thaw per species. Sprague Dawley Rat = 5.0 x 10^6 per vial CD1 Mouse = 4.0 x 10^6 per vial Cynomolgus Monkey = TBD Rodents have a single format for use in either suspension or attaching cultures. Same extensive characterization as non-cryostax hepatocytes.
10 Simplified Thaw Procedure
11 Same Great Quality Recent vial lot New CryostaX lot
12 Less Cryoinjury in hepatocyte Pools = higher activity Isolate hepatocytes Freeze and store First Freeze Thaw frozen heps Pool hepatocytes Freeze and store pooled hepatocytes Second Freeze
13 Cryoinjury and Effects on Phase I and II Enzymes in Pooled Human Hepatocytes Average of four donors frozen and refrozen individually Percent Control (%) Single freeze (control) Two freezes Three freezes
14 Freezing Effects Effects of Cryopreservation on Phase I and II Enzymes in Pooled Human Hepatocytes Activity (pmol/10^6 cells/min) Single freeze pool (5 donors) Multiple freezes pool (5 donors)
15 Less Cryoinjury to Pooled Human Hepatocytes Isolate hepatocytes Freeze and store CryostaX pellets Only One Freeze Pool one (or more) frozen pellet from each donor directly into vial
16 CryostaX TM Customization at no extra cost Quick turn around (3-4 business days) Examples of custom pools Gender specific (male or female) Specific ethnicity (i.e, Asian, Caucasian, Hispanic) High or low specific enzyme activity (i.e., high CYP3A4) Number of donors in pool (n=2 20) Smaller or larger number of hepatocytes in vial (standard number is >5 million per vial) Serology specific, such as CMV negative donors
17 CryostaX TM Customization Hepatocytes from each donor are fully characterized for CYP activities
18 CryostaX TM Customization Theoretical Activity Data vs. Actual Activity Data Enzymatic Activity (pmol/10 6 cells/min) Theoretical Actual
19 CryostaX TM Pooled Plateable Hepatocytes Activity (pmol/min/10 6 cells) Hepatocyte Lot 1A2 2B6 2C8 2C9 2C19 2D6 3A4 SULT UGT Donor A Donor B Donor C Donor D Donor E Individual Avg Pooled Enzyme activity rates of the pool closely match the average enzyme activity rates of all individuals in the pool, suggesting that each donor is well represented and that one donor does not over-contribute or under-contribute.
20 CryostaX TM Pooled Plateable Hepatocytes Fold change (relative to DMSO control) Activity mrna Activity mrna Activity mrna Activity mrna Activity mrna Activity mrna CYP1A2 CYP2B6 CYP2C8 CYP2C9 CYP2C19 CYP3A4 0.1 µm Rifampin 1 µm Rifampin 5 µm Rifampin 20 µm Rifampin 50 µm Omeprazole 750 µm Phenobarbital CYP1A2, 2B6, 2C8, 2C9, 2C19 and 3A4 enzyme activity and mrna were all inducible by prototypical inducers
21 CryostaX TM Pooled Plateable Hepatocytes Cl int values for 14 compounds with CryostaX pooled plateable hepatocytes CL int (ml/min/kg) ND Low Mid High Able to measure CL int of almost all low turnover drugs over 48 hour time course without media change. The rank order of CL int values was consistent with literature classification as high, medium or low clearance drugs
22 CryostaX TM Pooled Plateable Hepatocytes CL int (ml/min/kg) Good agreement with other currently available test systems for low turn over drug clearance assessment 0 Warfarin Tolbutamide Diazepam Pooled plateable hepatocytes Micro-patterned hepatocytes Hepatocyte Relay Pooled plateable hepatocytes Cost effective and easy to use Micro-patterned hepatocytes Cost prohibitive Hepatocyte relay method Labor intensive
23 CryostaX TM Pooled Hepatocytes CryostaX Pooled Human Hepatocytes Available in suspension or plateable formats. Available as individual donors with custom AMY. The only available human pooled hepatocytes subjected to only ONE cryopreservation process. Easily custom assembled according to customer specified criteria activity, ethnicity, demographics, genotype Effective for evaluating low, mid, and high clearance drugs CryostaX Plateable Hepatocytes: suitable for hepatic uptake studies. can be utilized for CYP induction screening. Simple thaw and utilization procedure
24 Opti Media Improved Attachment Efficiency Improved Confluency and Morphology Extended Expiration dates
25 Customizable, single-freeze pooled hepatocytes (n = 2 to 20) or individual donors Animal hepatocytes -Sprague Dawley Rat CD-1 Mouse Cynomolgus Monkey (soon!) Customizable, PLATEABLE single-freeze pooled hepatocytes (n = 2 to 20) or individual donors Geneknown Human Hepatocytes Pooled genotyped human hepatocytes
26 Featuring publications, posters, webinars and other useful resources. Webinar Topic Request Form: Sekisui XenoTech s Services & Products:
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