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Drug Metabolism Biofiles Volume 6, Number 1 Drug Metabolism Drug Transport Phase I Drug Metabolism Drug Conjugate Analysis Biofilesonline Biofilescontents Your gateway to Biochemicals and Reagents for Life Science Research Introduction 3 Biofiles Online allows you to: Drug Transport: Proteins, • Easily navigate the content of Antibodies, and Modulators 4 the current Biofiles issue • Access any issue of Biofiles Phase I Drug Metabolism: Enzymes, • Subscribe for email notifications Substrates, and Antibodies 12 of future eBiofiles issues Register today for upcoming issues and Drug Conjugate Analysis: eBiofiles announcements at Enzymes, Substrates and Inhibitors, sigma.com/biofiles and Derivitization Reagents 22 Highlights from this issue: Cover: Although phase I drug Drug Metabolism metabolism occurs in most tissues, Today’s drug designers are increasingly focused on optimizing drug the primary and first pass site of design to increase bioavailability and circulating time as well as decreasing the incidence of adverse drug metabolism occurs during hepatic reactions. This issue of Biofiles features circulation. selected products from Sigma-Aldrich’s drug metabolism platform. Product areas highlighted include our portfolio of reagents for drug transport, phase I metabolism, and drug conjugate analysis. Coming Next Issue: Prebiotics and Probiotics The next issue of Biofiles will explore the functionality of prebiotics and probiotics. Prebiotics are non-digestible substrates found in foods such Volume 6, Number 2 as soybeans and chicory, while probiotics are live Biofiles microorganisms available as dietary supplements. In addition to their role in maintaining digestive health, prebiotics and probiotics have been evaluated as treatments for inflammatory bowel disorders, chemotherapy-induced diarrhea, and Nutrition Research cancer. Sigma Life Science offers microbial media, enzymes, cell culture reagents, and labeled istopes to support this research. Technical content: Robert Gates, M. S. M. Order sigma.com/order Technical service sigma.com/techinfo sigma.com/lifescience 3 Introduction Robert Gates Market Segment Manager [email protected] In the past two decades, designers of new responders do not effectively metabolize drugs have employed the increasing body codeine and exhibit little analgesic effect of knowledge of how drugs are absorbed, following codeine administration. Ultra-rapid distributed, metabolized, and excreted- metabolizers, who may constitute 4% of the the ADME concept-to optimize drug population, may develop opioid intoxication efficacy. Small molecule organic drugs are following codeine administration. A challenge considered xenobiotics-compounds foreign for drug designers will be to integrate the to our native metabolome. Most require increasing body of information provided enzymatic modification for detoxification by the field of pharmacogenetics to create and elimination. This enzymatic modification safer drugs with lower risks of adverse drug (biotransformation) occurs by two types reaction. of reactions, phase I and phase II, which This issue of Biofiles is devoted to our diverse differ regarding the type of modification portfolio of reagents, enzymes, and labware introduced. Biotransformation is also utilized designed for the analysis of drug delivery, in the metabolism of many prodrugs to absorption, and metabolism. their active form, for example enalapril to enalaprilate. Understanding and Specific product focus for this issue is drug manipulating these enzyme/protein- transport, phase I enzymes, and drug- based metabolic mechanisms is an area of congugate analysis. increasing focus for improved drug targeting • Our portfolio of drug transport-related to specific organs, tissues, and cell types, products includes transporter protein/ as well as enabling creative strategies for membrane preparations as well as selected improving bioavailability and circulating time. antibodies and modulators to transport An added complexity for drug designers proteins. is the effect of human polymorphisms of • Our phase I metabolism products include metabolic enzymes on drug metabolism. enzymes such as cytochrome P450s Several metabolic enzymes have known and related electron transport proteins, common polymorphisms that result in other oxidases, and a select list of related either altered enzymatic activity or protein antibodies. stability. A classic example of differing Drug conjugate analysis focuses mainly pharmacogenetics and drug efficacy, • on sample preparation, hydrolysis, is codeine. Codeine is a prodrug that is derivitization, and subsequent analysis of modified by cytochrome P450 (CYP2D6) glucuronide and sulfate conjugates. to morphine. The CYP2D6 locus is highly polymorphic and approximately 6–10% of the population either lacks or exhibits little CYP2D6 enzymatic activity. These poor 4 Drug Transport Drug Transport Protein-based drug transporters are found in research. Understanding the specific from the surrounding buffer and transports them into the vesicles. most tissues including liver, kidney, intestine, mechanisms of tumor cell transporters The bile salt export pump (BSEP/ABCB11) and brain. Because of their complexity and is becoming an essential aspect of chemotherapuetic drug design. belongs to the family of ATP-binding-cassette genetic heterogeneity, these proteins are (ABC) transporters and has also been called the often produced as recombinant membrane ABC-type transporters are located in the sister of P-glycoprotein (sister Pgp). Most ABC preparations expressed in Sf9 cells. These plasma membrane Figure 1 and control the transporters transport substrates across the cell membrane using ATP as an energy source. transporters are particularly important in translocation of many classes of molecules. BSEP is the major bile salt transporter in the cancer treatment and multi-drug resistance Some allow the specific passage of inorganic liver canalicular membrane and is inhibited by ions, while others facilitate ATP-dependent a number of drugs or drug metabolites. This is translocation of organic compounds potentially a significant mechanism for drug- induced cholestasis. Dysfunction of individual including short peptides, lipids, bile acids, bile salt transporters such as BSEP, due to genetic glutathione, and glucuronide conjugates. mutation, suppression of gene expression, disturbed signaling, or steric inhibition, is an Typically, ABC transporters are composed important cause of cholestatic liver disease. of multiple transmembrane domains (TMD) The quantity of transported molecules can be and one or more ATP-binding domains determined by methods such as HPLC, LC/MS/ (ABC). MS separation and detection, and also by labeling with fluorescent or radioactive (3H-taurocholic Membrane preparations containing ABC acid) tags. BSEP mediates the transport of transporters show a baseline ATPase taurocholic acid (TC) very efficiently. Compounds activity that varies for different transporters. that interact with the transporter modulate the initial rate of TC transport measured without Transported substrates increase this baseline any other compounds added. If a substance is a ATPase activity, while inhibitors or slowly transported substrate of the transporter, it might transported compounds inhibit the baseline compete with TC, thus reducing the rate of TC Canaliculus ATPase activity and/or the ATPase activity transport. If a compound is an inhibitor of the 5 6 7 8 9 10 11 12 transporter, it will block the transport of TC into 3 4 1 2 measured in the presence of a stimulating the membrane vesicles. Some compounds can be agent. Both activation and inhibition studies co-transported with TC, increasing the rate of TC Cytosol can be performed. transport compared to the control level. A B A B NH2 membrane preparation, for Vesicular Transport, ABC ABC COOH recombinant, expressed in Sf9 cells BSEP Transport Proteins Supplied as isolated Sf9 cell membranes Figure 1.The Bile salt export pump (BSEP), encoded by BSEP human containing human BSEP suspended in 50 mM ABCB11, is a member of the Multi Drug Resistance (MDR)/ HEPES-Tris, 100 mM KNO3, and 50 mM sucrose, TAP subfamily of ATP-binding cassette (ABC) transporters. Bile salt export pump; ABCB11 pH 7.4. Members of the ABC transporter superfamily are defined The vesicular transport assay determines the Distributed for SOLVO Biotechnology, Inc. by the sequence and organization of their ATP-binding interaction of compounds with the BSEP cassette (ABC) domains. The ABC domains contain several transporter. The interaction is detected by changes B2436-500UL 500 μL conserved sequences, including a Walker A motif, Walker B in the initial rate of 3H-taurocholic acid transport motif, and the ABC signature motif. by BSEP into membrane vesicles purified from MDR1 human BSEP is a canalicular-specific exporter and is the major Sf9 cells expressing the transporters. Membrane Pgp; ABCB1 The MDR1 protein is involved in human bile acid transport protein. Mutations in this preparations from infected cells always contain cancer drug resistance and in the transport gene are associated with a severe human disease, type some closed membrane vesicles that have an of hydrophobic drugs and xenobiotics in the 2 Progressive Familial Intrahepatic Cholestasis (PFIC2). inside-out orientation (5–10% of total lipid). In Structurally, BSEP contains two transmembrane domains, bowel, kidney, liver, and the blood-brain barrier. each consisting of six membrane-spanning domains, and the case of these inside-out vesicles,
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