Using intestinal subcellular fractions as a test system is highly recommended due to the importance of intestinal enzymes in the first-pass metabolism of many orally ingested xenobiotics, including cytochrome P450 (CYP), UDP-glucuronosyltransferase (UGT), and esterase enzymes. Consequences of intestinal biotransformation can be very significant, including toxification by bioactivation and detoxification by aiding in excretion.
Human Intestinal Microsomes, S9, and Cytosol for Preclinical Drug Development
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We offer pooled intestinal subcellular fractions demonstrating high enzyme activity levels. Options include microsomes, S9, and cytosol, from matching large donor pools to minimize variability.
Intestinal microsomes are supplied in 250 mM sucrose buffer. S9 and cytosolic fractions are supplied in a homogenization buffer with or without Phenylmethylsulfonyl flouride (PMSF)– find out more about elution and sample preparation on page 80 of our Product Tech Guide.
Highest Activity Levels on the Market
Our designated products team is trained to carefully extract intestinal subcellular fractions only from mature enterocytes of the villus tips, ensuring the highest possible CYP and UGT enzyme activity levels. In addition to human tissue, we also offer intestinal fractions from cynomolgus monkey, beagle dog, IGS Sprague-Dawley rat, and CD-1 mouse. Each lot comes with documented characterization and can maintain superior enzymatic activity levels through multiple freeze-thaw cycles.
Why are some lots PMSF-free?
Most ester-type drugs are hydrolyzed by esterases bound in the intestinal mucosa1, so esterase activity in intestinal test systems can be useful in understanding a drug candidate’s extrahepatic metabolic profile. Phenylmethylsulfonyl flouride (PMSF), a serine protease inhibitor, is included in buffers used during tissue processing and subcellular fraction because it protects CYP and other important drug-metabolizing enzymes from being broken down by the high levels of naturally occurring proteases found in the intestinal compartment. The compromise to protecting those metabolic activities is that the PMSF also inhibits esterases.
To remedy this, we offer PMSF-free fractions from human, monkey, canine, rat, and mouse. The PMSF-free fractions have optimal esterase activity, however it comes at the detriment of other metabolic activities due to increased protease liability and it is recommended to use PMSF-free fractions for use in assays that are specifically investigating esterase interactions. For all assays focused on other enzymes (e.g. CYP, UGT, SULT) we recommend our standard intestinal subcellular fractions prepared with PMSF.
Read more about elution methods and activity characterization in a recent blog post by Senior Manager of Technical Support, Dr. Chris Bohl: Considerations when studying esterase activities in the intestine.
Make sure your microsomes and S9 fractions are properly activated
For best results from assays using microsomes and S9 fractions, we recommend using NADPH regeneration to support high metabolism levels by ensuring that the NADPH levels are not a limiting factor in your incubations. Our preferred system, RapidStartTM , uses an enzymatic reaction that converts NADP to NADPH, which is then used as a cofactor by various metabolic enzymes and oxidized back to NADP, where the cycle continues ensuring stable NADPH levels throughout the incubation. RapidStartTM NADPH regeneration is easily activated by simply adding water. Our easy-to-use formula is the most convenient and flexible NADPH regenerating system you’ll find, allowing users to easily tailor the system’s capability by simply varying the amount of water added. RapidStartTM is perfect for long- and short-term incubations and supports the function of recombinant enzymes and subcellular fractions, allowing you to achieve the most accurate and reliable data during your in vitro assays.
Don’t see what you need? Other preparations can be made available!
If you require subcellular test systems not available through our standard product offerings, Custom Subcellular Preparations can be easily ordered to meet the specific needs of your study.
Our designated Custom Products team regularly prepares and isolates cells and/or subcellular fractions from more than 45 different species and has capabilities to meet unique needs that are unavailable through most vendors– non-standard rodent strains, farm animals, insects, precarious tissue isolations such as adrenal gland or jejunum. Our specialists can carry out over 40 characterization assays, providing the end user with a good foundation for their DMPK assays. See our Custom Products page for examples of past custom preparations or get in touch with a specialist to find out how we can meet your needs.
References
- “CFR – Code of Federal Regulations Title 21.” Accessdata.fda.gov, Apr. 2019, www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfcfr/CFRSearch.cfm?FR=361.1
- Solon, Eric G. “Quantitative Whole Body Autoradiography (QWBA) and Imaging Mass Spectrometry (IMS): Can IMS Replace QWBA to Support Regulated Drug Discovery and Development.” Bioanalysis Zone, Bioanalysis Zone, 21 Mar. 2018, www.bioanalysis-zone.com/2018/03/09/quantitative-whole-body-autoradiography-qwba-imaging-mass-spectrometry-ims-can-ims-replace-qwba-support-regulated-drug-discovery-development/.
Access more details about extrahepatic subcellular fraction preparations
For more information about how our product specialists ensure quality inHuman intestine subcellular preparations, as well as enzyme activity characterization assays, refer to pages 79-85 in our Product Tech Guide
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