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, transport of Drugs interacting with this protein may be useful two ABC domains. substrates across the membrane takes molecules for the reversal of cancer drug resistance or Order sigma.com/order Technical service sigma.com/techinfo sigma.com/lifescience 5

increasing the absorption or brain entry of various Control for ATPase and vesicular transport assays. ATP as an energy source. ATP hydrolysis yields pharmacological agents. inorganic phosphate (P), which can be detected Distributed for SOLVO Biotechnology, Inc. i Detection of ATPase activity of the MDR1 protein by a simple colorimetric reaction. The amount of M9819-500UL 500 μL P liberated is proportional to the activity of the is a measure of transporter activity. The assay is i transporter. performed using purified membrane vesicles MRP2 human from Sf9 (Spodoptera frugiperda) cells, expressing MRP2 (ABCC2) is an organic anion transporter high levels of MDR1 protein. The ABC transporters The vesicular transport assay determines the found in liver, kidney, and gut epithelium apical pump substrates out of the cell by using hydrolysis interaction of compounds with the MRP2 membranes. The transport of glucuronate of ATP as an energy source. ATP hydrolysis yields transporter. The interaction is detected by changes conjugates plays a role in the detoxification of in the initial rate of 3H-β-estradiol 17-(β-D- inorganic phosphate (Pi), which can be detected endogenous and xenobiotic substances, and may by a simple colorimetric reaction. The amount of glucuronide) transport by MRP2 into membrane cause multidrug resistance (MDR) in tumor cells. vesicles purified from Sf9 cells expressing the Pi liberated is proportional to the activity of the The rat Mrp2 transporter shows 72.3% sequence transporter. transporters. Membrane preparations from identity and 85.6% sequence similarity with infected cells always contain some closed human MRP2. Both transporters are expressed  membrane preparation, for ATPase, recombinant, membrane vesicles that have an inside-out on the canalicular membrane of the liver and are expressed in Sf9 cells orientation (5-10% of total lipid). In the case of known to be responsible for the transport of some Supplied as isolated Sf9 cell membranes these inside-out vesicles, transport of substrates organic molecules and their conjugates to the bile. containing human MDR1 (Pgp) suspended in across the membrane takes molecules from the TMEP solution. surrounding buffer and transports them into the  membrane preparation, for ATPase, recombinant, expressed in Sf9 cells Distributed for SOLVO Biotechnology, Inc. vesicles. Supplied as isolated Sf9 cell membranes MRP2 (ABCC2) is an organic anion transporter M9194-500UL 500 μL containing rat Mpr2 suspended in TMEP solution. found in the liver, kidney, and gut epithelium Mdr1b from rat apical membranes. The transport of glucuronate Distributed for SOLVO Biotechnology, Inc. conjugates plays a role in the detoxification of M9694-500UL 500 μL The MDR1 protein is involved in cancer drug endogenous and xenobiotic substances, and may resistance and in the transport of hydrophobic cause multidrug resistance (MDR) in tumor cells. drugs and xenobiotics in the bowel, kidney, liver, MXR human and the blood-brain barrier. In rodents, there are The quantity of transported molecules can be MXR membrane vesicles are purified from two MDR1 genes, mdr1a and mdr1b, while in determined by methods such as HPLC, LC/MS/ recombinant baculovirus transduced Sf9 cells human, there is a single MDR1 gene. Based on MS separation and detection, and also by labeling or selected, MXR over-expressing mammalian 3 function and tissue distribution in rodents, the with fluorescent or radioactive ( H-β-estradiol cells. Membrane preparations from transporter equivalent of the human MDR1 gene product 17-(β-D-glucuronide) tags. MRP2 mediates the expressing cells always contain some closed (PgP) is the product of the rodent mdr1b transport of β-estradiol 17-(β-D-glucuronide) membrane vesicles that are inside-out orientation (E 17βG) very efficiently. Compounds that interact gene. There have been no reported significant 2 (5–10% of total lipid). In the case of these inside- differences in function, substrate specificity, or with the transporter modulate the initial rate of out vesicles, the transport of substrates across the E 17βG transport measured without any other substrate affinity between these two proteins. 2 membrane takes molecules from the buffer in compounds added. If a substance is a transported Detection of the ATPase activity of the Mdr1b which the membrane is suspended and transports substrate of the transporter, it might compete them into the vesicles. The rate of this transport is protein is a measure of transporter activity. The with E 17βG, thus reducing the rate of E 17βG 2 2 temperature and ATP dependent. assay is performed using purified membrane transport. If a compound is an inhibitor of the vesicles from Sf9 (Spodoptera frugiperda) cells, The quantity of transported molecules can be transporter, it will block the transport of E217βG expressing high levels of Mdr1b protein. The ABC into the membrane vesicles. Some compounds determined by methods such as HPLC, LC/MS/ transporters pump substrates out of the cell by can be co-transported with E 17βG increasing the MS separation and detection, and also by labeling 2 3 using hydrolysis of ATP as an energy source. ATP rate of E 17βG transport compared to the control with fluorescent or radioactive ( H labeled MTX) hydrolysis yields inorganic phosphate (P), which 2 tags. i level. can be detected by a simple colorimetric reaction. Methotrexate (MTX) is a transported substrate of The amount of P liberated is proportional to the  membrane preparation, for Vesicular Transport, i the MXR transporter with low affinity and high recombinant, expressed in Sf9 cells activity of the transporter. capacity. The vesicular transport assay provides Supplied as isolated Sf9 cell membranes  membrane preparation, for ATPase, recombinant, information on any interaction between the MXR containing human MRP2 suspended in TMEP expressed in Sf9 cells transporter and the test compound that would solution. Supplied as isolated Sf9 cell membranes affect the transport of the reporter substrate 3 containing rat Mdr1b suspended in TMEP Distributed for SOLVO Biotechnology, Inc. ( H-Methotrexate ) into the membrane vesicles. solution. M9069-500UL 500 μL If a test compound is an activator or inhibitor of the MXR transporter, it competes with MTX, thus Distributed for SOLVO Biotechnology, Inc. Mrp2 from rat reducing the rate of MXR mediated MTX transport. M9319-500UL 500 μL Distributed for SOLVO Biotechnology, Inc. Detection of ATPase activity of the Mrp2 protein MDR1, MRP, and BSEP Control is a measure of transporter activity. The assay is  membrane preparation, wild type variant, for performed using purified membrane vesicles from Vesicular Transport  membrane preparation, from Sf9 cells Sf9 (Spodoptera frugiperda) cells, expressing high Supplied as isolated mammalian cell membranes Supplied as isolated Sf9 cell membranes levels of Mrp2 protein. ABC transporters pump containing human MXR (wild type variant) containing a defective MRP1 gene suspended in substrates out of the cell by using hydrolysis of suspended in TMEP solution. TMEP solution. 6

The MXR transporter can be produced in sufficient ▼ MXR Control MXR Control quantity by selected, MXR over-expressing Control for ATPase and vesicular transport assays. mammalian cell lines.  membrane preparation, from Sf9 cells Distributed for SOLVO Biotechnology, Inc. Supplied as isolated Sf9 cell membranes M9569-500UL 500 μL containing a defective MXR gene suspended in  membrane preparation, wild type variant, for MXR Control TMEP solution. Vesicular Transport, recombinant, expressed in Sf9  membrane preparation, mammalian M9944-500UL 500 μL cells Supplied as isolated mammalian cell membranes Supplied as isolated Sf9 cell membranes (not selected for transport expression) suspended MXR Control ▲ containing wild type human MXR suspended in in TMEP solution. TMEP solution. C3992-500UL 500 μL The MXR transporter can be expressed in Sf9 insect cells using the baculoviral expression system, yielding high protein levels (up to 5% of total membrane protein) in the cell membrane of infected cells. M9444-500UL 500 μL

Selected Antibodies to Transport Proteins

Species Prestige Product Name Host Clone No. Form Gene Symbol Reactivity Application Antibody Cat. No. Anti-ABCB10 (N-term) rabbit - IgG fraction of ABCB10, human human ELISA (i) - SAB1300312-100UG antiserum Anti-ABCB6 (C-term) rabbit - IgG fraction of ABCB6, human human ELISA (i) - SAB1300078-100UG antiserum IHC WB Anti-ABCB7 (C-term) rabbit - IgG fraction of ABCB7, human human ELISA (i) - SAB1300313-100UG antiserum IHC WB Anti-ABCC4 rabbit - affinity isolated ABCC4, human human IHC (p)  HPA002476-100UL antibody PA Anti-ABCC9 rabbit - affinity isolated ABCC9, human human IHC (p)  HPA007279-100UL antibody PA Anti-AF130358.1 rabbit - affinity isolated AF130358.1, human human IHC (p)  HPA027071 antibody PA Anti-CDH17 rabbit - affinity isolated CDH17, human human IHC (p)  HPA023616-100UL antibody PA Anti-CDH17 rabbit - affinity isolated CDH17, human human IHC (p)  HPA023614-100UL antibody PA Anti-CFTR rabbit - affinity isolated CFTR, human human IHC (p)  HPA021939-100UL antibody PA Anti-Dopamine Transporter rabbit - affinity isolated SLC6A3, human human WB - D6944-25UL (N-terminal) antibody Slc6a3, rat mouse D6944-200UL Slc6a3, mouse (predicted) rat Anti-GABA Transporter GAT-3 rabbit - affinity isolated Slc6a11, rat rat IHC - G8407-.1ML antibody Anti-LST-3TM12 (ab1) rabbit - affinity isolated LST-3TM12, human human WB - SAB2101401-50UG antibody Anti-LST-3TM12 (ab2) rabbit - affinity isolated LST-3TM12, human human WB - SAB2101402-50UG antibody Anti-MRP8/ABCC11 goat - affinity isolated ABCC11, human bovine ELISA (i) - SAB2500647-100UG antibody canine WB human Anti-OSTB rabbit - affinity isolated OSTbeta, human human IHC (p)  HPA008533-100UL antibody PA Anti-PMAT(Slc29a4) (N-term) rabbit - IgG fraction of PMAT, human human ELISA (i) - SAB1300519-100UG antiserum IHC WB Order sigma.com/order Technical service sigma.com/techinfo sigma.com/lifescience 7

Species Prestige Product Name Host Clone No. Form Gene Symbol Reactivity Application Antibody Cat. No. Anti-SLC10A2 goat - affinity isolated Slc10a2*, mouse mouse ELISA (i) - SAB2500947-100UG antibody rat WB Anti-SLC10A5 rabbit - affinity isolated SLC10A5, human human IHC (p)  HPA025966-100UL antibody PA WB Anti-SLC10A5 rabbit - IgG fraction of SLC10A5, human human IHC - AV43773-100UG antiserum WB Anti-SLC10A7 rabbit - affinity isolated SLC10A7, human human WB - SAB2102163-50UG antibody Anti-SLC15A1 rabbit - affinity isolated SLC15A1, human human IHC (p)  HPA002827-100UL antibody PA Anti-SLC16A1 rabbit - affinity isolated SLC16A1, human human IF (i)  HPA003324-100UL antibody IHC (p) PA WB Anti-SLC16A3 rabbit - affinity isolated SLC16A3, human human IHC (p)  HPA021451-100UL antibody PA WB Anti-SLC16A7/MCT2 goat - affinity isolated SLC16A7, human chimpanzee ELISA (i) - SAB2500948-100UG antibody human WB Anti-SLC18A2 rabbit - affinity isolated SLC18A2, human human IHC (p)  HPA016856-100UL antibody PA Anti-SLC1A1 rabbit - affinity isolated SLC1A1, human human IHC (p)  HPA020086-100UL antibody PA Anti-SLC1A2 rabbit - affinity isolated SLC1A2, human human IHC (p)  HPA009172-100UL antibody PA Anti-Slc22A17 rabbit - affinity isolated SLC22A17, human human ELISA (i) - SAB3500306-100UG antibody mouse IF (i) rat WB Anti-SLC22A17 rabbit - affinity isolated SLC22A17, human human IHC (p)  HPA002728-100UL antibody PA Anti-SLC22A2 rabbit - IgG fraction of SLC22A2, human human WB - AV43847-100UG antiserum Monoclonal Anti-SLC22A2 mouse 2D2 purified SLC22A2, human human ELISA (i) - WH0006582M1-100UG immunoglobulin WB Anti-SLC22A3 rabbit - affinity isolated SLC22A3, human human IHC (p)  HPA029750-100UL antibody PA Anti-SLC22A7 rabbit - affinity isolated SLC22A7, human human WB - SAB2102178-50UG antibody Anti-SLC22A8 rabbit - affinity isolated SLC22A8, human human WB - SAB2102179-50UG antibody Anti-SLC25A26 rabbit - affinity isolated SLC25A26, human human IHC (p)  HPA026887-100UL antibody PA Monoclonal Anti-SLC27A4 mouse 1F4-1B10 purified SLC27A4, human human ELISA (i) - WH0010999M1-100UG immunoglobulin WB Anti-SLC29A1 rabbit - affinity isolated SLC29A1, human human IHC (p)  HPA012384-100UL antibody PA WB Anti-SLC29A2 rabbit - affinity isolated SLC29A2, human human IHC (p)  HPA018168-100UL antibody PA Anti-SLC36A4 rabbit - affinity isolated SLC36A4, human human IHC (p)  HPA017887-100UL antibody PA Anti-SLC44A2 rabbit - affinity isolated SLC44A2, human human IHC (p)  HPA003228-100UL antibody PA Anti-SLC6A1 rabbit - affinity isolated SLC6A1, human human WB - SAB2102222-50UG antibody Anti-SLC6A2 rabbit - affinity isolated SLC6A2, human human WB - SAB2102224-50UG antibody 8

Species Prestige Product Name Host Clone No. Form Gene Symbol Reactivity Application Antibody Cat. No. Monoclonal Anti-SLC6A4 mouse 2A9 purified SLC6A4, human human ELISA (i) - WH0006532M6-100UG immunoglobulin Monoclonal Anti-SLC6A5 mouse 3B3 purified SLC6A5, human human ELISA (i) - WH0009152M1-100UG immunoglobulin WB Anti-SLC6A6 rabbit - affinity isolated SLC6A6, human human IHC (p)  HPA015028-100UL antibody PA Anti-SLC6A7 rabbit - affinity isolated SLC6A7, human human IHC (p)  HPA028907-100UL antibody PA Anti-SLC7A3 rabbit - affinity isolated SLC7A3, human human IHC (p)  HPA003629-100UL antibody PA Anti-SLCO3A1 rabbit - affinity isolated SLCO3A1, human human WB - SAB2102229-50UG antibody Anti-SLCO5A1 rabbit - affinity isolated SLCO5A1, human human IHC (p)  HPA025062-100UL antibody PA Anti-TAP1 rabbit - affinity isolated TAP1, human human WB - SAB2102370-50UG antibody Anti-TETRAN rabbit - affinity isolated TETRAN, human human IHC - AV45054-50UG antibody WB Anti-Vesicular Acetylcholine rabbit - affinity isolated Slc18a3, rat human IF (i) - V5387-.2ML Transporter (VAChT) antibody SLC18A3, human rat IHC (p) WB Anti-Vesicular Monoamine guinea - whole antiserum Slc18a2, rat rat IHC (f) - V6637 Transporter (VMAT-2) pig

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Sigma and CompoZr are registered trademarks of Sigma-Aldrich Biotechnology L.P. Order sigma.com/order Technical service sigma.com/techinfo sigma.com/lifescience 9

Mission esiRNA  esiRNA targeting mouse Pgp (esiRNA1) MISSION® esiRNA Solution in low-TE (Tris-HCl (10 mM final), EDTA (1  esiRNA targeting human PGP (esiRNA1) ▼ MISSION® esiRNA mM final), pH to 8.0. Solution in low-TE (10 mM Tris-HCl, pH 8.0, 1 mM concentration...... 200 ng/μL RNA MISSION® esiRNA EDTA) EMU017111-20UG 20 μg concentration...... 200 ng/μL RNA MISSION esiRNA are endo-ribonuclease prepared EMU017111-50UG 50 μg siRNA. MISSION esiRNA are a heterogeneous EHU010411-20UG 20 μg mixture of siRNAs that all target the same mRNA EHU010411-50UG 50 μg sequence. These multiple silencing triggers lead to highly specific and effective gene silencing. MISSION® esiRNA ▲

Selected Transporter Modulators

Name Structure Description Cat. No. 1-Benzoyl-5-methoxy-2-methylindole- O Putative inhibitor of multidrug resistance-associated protein 1 B1183-5MG 3-acetic acid OH (MRP1). H3CO CH3 N

O

Berberine chloride form O Fluorescent stain for heparin in mast cells An alkaloid with weak B3251-5G + Cl– O N antibiotic properties. Substrate for MDR efflux pumps. Antimicrobial B3251-10G activities of berberine is potentiated by the MDR inhibitor OCH3 B3251-25G 5´-methoxyhydnocarpin (5´-MHC). Berberine upregulates the

OCH3 expression of Pgp in hepatoma cells. Treatment with berberine potentially results in the reduced accumulation of chemotherapeutic drugs.

Berberine hemisulfate salt O Fluorescent stain for heparin in mast cells An alkaloid with weak B3412-10G O O antibiotic properties. Substrate for MDR efflux pumps. Antimicrobial S -O O- activities of berberine is potentiated by the MDR inhibitor O 5´-methoxyhydnocarpin (5´-MHC). Berberine upregulates the N+ H3CO expression of Pgp in hepatoma cells. Treatment with berberine OCH3 2 potentially results in the reduced accumulation of chemotherapeutic drugs. 1-(4-Chlorobenzyl)-5-methoxy-2- O Putative inhibitor of multidrug resistance-associated protein 1 C1610-5MG methylindole-3-acetic acid OH (MRP1). H3CO CH3 N

Cl

Chloroquine diphosphate salt CH3 DNA intercalator. Also used to increase transfection efficiency. C6628-25G CH3 NCH Standard anti-malarial drug. Substrate for MRP in multidrug resistant C6628-50G HN 3 cell line and inhibits photoaffinity labeling of MRP by quinoline- C6628-100G • 2H PO 3 4 based photoactive drug IAAQ (N-[4-[1-hydroxy-2-(dibutylamino) C6628-250G Cl N ethyl]quinolin-8-yl]-4-azidosalicylamide).

1-Deoxyforskolin from Coleus forskohlii CH P-GPefflux inhibitor D1290-1MG O 3 CH2 CH 3 O

H OH CH3 O CH3 H3C CH OH 3 O 9-Deoxyforskolin from Coleus forskohlii - P-GPefflux inhibitor D4665-1MG 1-(4-Fluorobenzyl)-5-methoxy-2- O Putative inhibitor of multidrug resistance-associated protein 1 F2927-5MG methylindole-3-acetic acid OH (MRP1). H3CO CH3 N

F 10

Name Structure Description Cat. No. Indomethacin O Cyclooxygenase (COX) inhibitor that is relatively selective for COX-1. I7378-5G

H3CO OH I7378-10G CH3 I7378-25G N I7378-100G O Cl

Ko143 O O Ko143 has been used as a positive control inhibitor on functions K2144-1MG CH H NH 3 of BCRP in MCF7 and BCRP over-expressing MCF7/MX100 cell lines O CH3 K2144-5MG N CH3 using a BCRP prototypical substrate mitoxantrone. O CH3 N H3CO H CH3

OOH Mefenamic acid CH An NSAID. Circumvents MRP-mediated multidrug resistance. M4267-50G 3 H H3C N Specifically and significantly potentiates the cytotoxicity of M4267-500G anthracyclines as well as teniposide, VP-16 and vincristine.

PGP-4008 O Selectively inhibits P-glycoprotein. P6490-2MG

HN

N N

Probenecid O Useful as an inhibitor of the organic anion transporter, MRP. P8761-25G H C OH P8761-100G 3 O N H3C S O Reversin 121 Peptide chemosensitizer, inhibitor of P-glycoprotein. R1276-3MG

O

O O O H t-Bu N t-Bu O N O H H O N O

O Reversin 205 Peptide chemosensitizer, inhibitor of P-glycoprotein. R1401-10MG O O

O O O O CH H 3 N H3C O N OCH3 H O CH O H CH 3 N 3 N O CH3 H O CH3

Staurosporine from Streptomyces sp. CH3 Potent inhibitor of phospholipid/calcium-dependent protein kinase. S4400-.1MG HN Inhibits the upregulation of VEGF expression in tumor cells. S4400-.5MG H3C O Partially reverses MDR, sensitizing cells with MDR phenotype to O S4400-1MG cytotoxic agents. Inhibits Pgp phosphorylation. CH3 N N

O N H

Staurosporine from Streptomyces sp. CH3 Potent inhibitor of phospholipid/calcium-dependent protein kinase. S5921-.1MG HN Inhibits the upregulation of VEGF expression in tumor cells. S5921-.5MG H3C O Partially reverses MDR, sensitizing cells with MDR phenotype to O S5921-1MG cytotoxic agents. Inhibits Pgp phosphorylation. CH3 N N

O N H Staurosporine solution from - Potent inhibitor of phospholipid/calcium-dependent protein kinase. S6942-200UL Streptomyces sp. Inhibits the upregulation of VEGF expression in tumor cells. Potent cell-permeable inhibitor of protein kinase C. Induces apoptosis in Jurkat cells. Order sigma.com/order Technical service sigma.com/techinfo sigma.com/lifescience 11

Name Structure Description Cat. No. Sulindac O Treatment of human colorectal cancer cell lines induces MRP1 S8139-5G OH and MRP3 but not other members of the MRP family. Reported to S8139-25G F significantly increase the cytotoxicity of the anthracyclines as well CH3 as teniposide, VP-16 and vincristine. Nonsteroidal anti-inflammatory; preferential inhibitor of COX-1.

H C 3 S O

H C Tolmetin sodium salt dihydrate 3 O An NSAID. Circumvents MRP-mediated multidrug resistance. T6779-1G • 2H2O Significantly increases the cytotoxicity of the anthracyclines as well as T6779-5G N ONa teniposide, VP-16 and vincristine. O CH3 R(+)-Verapamil monohydrochloride — Inhibitor of P-glycoprotein; less active enantiomer of (±)-verapamil. V106-5MG hydrate V106-25MG

Zomepirac sodium salt Cl H3C An NSAID. Circumvents MRP-mediated multidrug resistance. Z2625- O Significantly increases the cytotoxicity of the anthracyclines as well as 250MG N ONa teniposide, VP-16 and vincristine. Z2625-1G O CH 3 Z2625-5G

Derivatization Reagents Brochure Listing over 400 Derivatization Reagents

• Products sorted by GC, HPLC, Chiral and TLC techniques • Reagents also listed by “Application” • Vials, syringes and other useful items for derivatization reactions • Up-to-date application information and references

To order a copy, go to, call 800-359-3041 (US and Canada only) or 814-359-3041, email [email protected] 12

Phase I Drug Metabolism

Phase I Drug Metabolism

Phase I biotransformation reactions introduce into more water soluble compounds that or expose functional groups on the drug can be excreted. Typically, oxidation is with the goal of increasing the polarity the most common phase I reaction. The of the compound. Although Phase I drug hepatic cytochrome P450 system is the most metabolism occurs in most tissues, the important of the phase I oxidation systems primary and first pass site of metabolism (Figure 1). occurs during hepatic circulation. Additional metabolism occurs in gastrointestinal Cytochrome P450 Enzymes epithelial, renal, skin, and lung tissues. Within and Reagents Figure 1. Our hepatic microsomal cytochrome P450 cells, most phase I enzymes are located in the enzymes are membrane-bound oxygenases containing The cytochrome P450s are members of a a thiolate heme near the active site. Electron donation endoplasmic reticulum and thus are enriched superfamily of monoxygenases that catalyze for the oxidatve mechanism is supplied from a coupled in microsomal preparations. NAD(P)H reductase and cytochrome b5 system. Until the oxidative metabolism of xenobiotics, recently, the difficulty of isolating and reconstituting Phase I reactions are broadly grouped into the initial step in the biotransformation and native enzymes from human tissue has hampered their use for studies with well-defined in vitro drug metabolism three categories, oxidation, reduction, and elimination of a wide variety of drugs and systems. For this reason, multienzyme microsomal prepa- hydrolysis. As most small molecule drugs environmental pollutants. P450s are located rations are often utilized for the elucidation of the oxidate primarily in the endoplasmic reticulum of fate of drugs and other xenobiotics. Our recombinant are lipophilic in nature, drug metabolism microsomal preparations have the advantage of a single converts these hydrophobic compounds liver tissue. cytochrome P450 isozyme activity.

Formation of HFC in picomoles 700 Formation of CHC in picomoles Formation of CHC in picomoles 120.0 60.0 e e e 600 100.0 50.0 500 80.0 40.0 400 60.0 30.0 300 40.0 20.0 200

20.0 10.0 pmol of metabolit 10 0 pmol of metabolit pmol of metabolit

0.0 0.0 0 0510 15 20 25 30 35 0510 15 20 25 30 35 0510 15 20 25 30 35 Time in minutes Time in minutes Time in minutes CYP 1A2 with Reductase CYP 3A4 with Reductase & b5 CYP 2D6 with Reductase

Formation of CHC in picomoles Formation of CHC in picomoles Formation of CHC in picomoles 18.0 160.0 30.0 e e e 16.0 140.0 25.0 14.0 120.0 12.0 20.0 100.0 10.0 80.0 15.0 8.0 60.0 6.0 10.0

4.0 40.0 5.0 pmol of metabolit 2.0 pmol of metabolit 20.0 pmol of metabolit

0.0 0.0 0.0 0510 15 20 25 30 35 0510 15 20 25 30 35 0510 15 20 25 30 35 Time in minutes Time in minutes Time in minutes CYP 2C9 with Reductase CYP 2C19 with Reductase CYP 2e1 with Reductase

Figure 2. Specific activity determined by a fluorescent product generated from an enzyme-specific non-fluorescent substrate after incubation with cytochrome P450. The fluorescent emission vs. time yields activity per mg protein. Order sigma.com/order Technical service sigma.com/techinfo sigma.com/lifescience 13

Cytochrome P450 Enzymes  2C9 Isozyme, recombinant, expressed in Reductase Activity: One unit will reduce 1 Saccharomyces cerevisiae, Coexpressed with nanomole of cytochrome c per minute in the Cerosomes: P450 Microsomes human NADPH reductase presence of NADPH at pH 7.8 at 25 °C. Expressed in Yeast activity: 2.0–3.0 units/pmol enzyme CYP450 content: 35.0–50.0 picomoles per mg Use of a eukaryotic, yeast host allows secondary activity: 1300–1650 units/mg protein protein efficient expression of the active isozyme P450 Activity: One unit will oxidize 1.0 picomole of C3499-500PMO 500 with more accurate post-translational 7-benzoxymethyloxy-3-cyanocoumarin per minute C3499-1000PMO 1000 at pH 7.4 at 37 °C. modification. Cerosomes are prepared  3A4 Isozyme, recombinant, expressed in Reductase Activity: One unit will reduce 1 Saccharomyces cerevisiae, coexpressed with from Saccharomyces cervisiae expressing nanomole of cytochrome c per minute in the human NADPD reductase the human cytochrome P450 isozyme and presence of NADPH at pH 7.8 at 25 °C. activity: 1.5–2.0 units/pmol enzyme human NADPH reductase and cytochrome CYP450 content: 50.0-65.0 picomoles per mg secondary activity: 1300–1700 units/mg protein protein b5 in some preparations. P450 Activity: One unit will oxidize 1.0 picomole C3874-500PMO 500 of 7-benzoxymethyloxy-3-cyanocoumarin per The eukaryotic system provides an C3874-1000PMO 1000 minute at pH 7.4 at 37 °C. isozyme system with the following  2C19 Isozyme, recombinant, expressed in Reductase Activity: One unit will reduce 1 advantages: Saccharomyces cerevisiae, coexpressed with nanomole of cytochrome c per minute in the human NADPH reductase. presence of NADPH at pH 7.8 at 25 °C. • Recombinant isozyme mimics the active, activity: 4.0–6.0 units/pmol enzyme CYP450 content: 40.0-60.0 picomoles per mg native human enzyme secondary activity: 1300–1650 units/mg protein protein Higher specific activity and better signal- P450 Activity: One unit will oxidize 1.0 picomole C3374-500PMO 500 • of 7-ethyloxymethyloxy-3-cyanocoumarin per C3374-1000PMO 1000 to-noise ratio (Figure 2) minute at pH 7.4 at 37 °C.  2C8 Isozyme, recombinant, expressed in • Consistent and reproducible results Reductase Activity: One unit will reduce 1 Saccharomyces cerevisiae, coexpressed with nanomole of cytochrome c per minute in the human NADPH reductase and cytochrome b5 The Cerosomes cytochrome P450 isozymes presence of NADPH at pH 7.8 at 25 °C. activity: 0.050-0.100 units/pmol enzyme are supplied in a solution containing 50 mM CYP450 content: 50-65 picomoles per mg protein secondary activity: 1700–2300 units/mg protein potassium phosphate, pH 7.4, 5% glycerol, 1 C3749-500PMO 500 P450 Activity: One unit will oxidize 1.0 picomole of mM EDTA, and 1 mM PMSF. C3749-1000PMO 1000 7-Benzoxymethoxy-3-cyanocoumarin per minute at pH 7.4 at 37 °C.  2D6 Isozyme, recombinant, expressed in Cyto­chrome P450 Micro­some Saccharomyces cerevisiae, coexpressed with Reductase Activity: One unit will reduce 1 Pre­para­tion human human NADPH reductase, activity: 0.9–1.2 units/ nanomole of cytochrome c per minute in the pmol enzyme presence of NADPH at pH 7.8 at 25 °C. Cerosomes Supplied as a solution containing secondary activity: 1250–1600 units/mg protein CYP450 content: 55.0–75.0 picomoles per mg 50 mM potassium phosphate, pH 7.4, 5% glycerol, P450 Activity: One unit will oxidize 1.0 picomole protein 1 mM EDTA and 1 mM PMSF of 7-ethyloxymethyloxy-3-cyanocoumarin per Cytochrome b5 content: 200-300 picomoles per  1A2 Isozyme, recombinant, expressed in minute at pH 7.4 at 37 °C. mg protein Saccharomyces cerevisiae, coexpressed with human Reductase Activity: One unit will reduce 1 C6999-500PMO NADPH reductase nanomole of cytochrome c per minute in the C6999-1000PMO activity: 10.0-13.0 units/pmol enzyme presence of NADPH at pH 7.8 at 25 °C. Negative Controls secondary activity: 1100–1400 units/mg protein CYP450 content: 50-60 picomoles per mg protein P450 Activity: One unit will oxidize 1.0 picomole of C3624-500PMO 500 Cyto­chrome P450 Micro­some 7-ethyloxymethyloxy-3-cyanocoumarin per minute C3624-1000PMO 1000 Pre­para­tion human at pH 7.4 at 37 °C.  2E1 Isozyme, recombinant, expressed in Cerosomes Supplied as a solution containing 50 Reductase Activity: One unit will reduce 1 Saccharomyces cerevisiae, coexpressed with mM potassium phosphate, pH 7.4, 5% glycerol, 1 nanomole of cytochrome c per minute in the human NADPH reductase mM EDTA and 1 mM PMSF activity: 3.0–6.0 units/pmol enzyme presence of NADPH at pH 7.8 at 25 °C.  Neagtive Control without NADPH reductase, CYP450 content: 60-80 picomoles per mg protein secondary activity: 1600–1900 units/mg protein Produced in Saccharomyces cerevisiae One unit will oxidize 1.0 picomole of C4499-500UL 500 μL C3999-500PMO 500 7-Ethyloxymethoxy-3-cyanocoumarin per minute C3999-1000PMO 1000 at pH 7.4 at 37 °C.  Negative control with human NADPH reductase, recombinant, expressed in Saccharomyces cerevisiae C4374-500UL 500 μL 14

P450 Microsomes Expressed in Microsomes containing recombinant human C5110-1VL 1 vial Insect Cells CYP2C9 and recombinant rabbit NADPH-P450 reductase.  4F3B isozyme microsomes, with P450 Reductase and cytochrome b , recombinant, expressed in Cyto­chrome P450 human 5 Solution in 100 mM potassium phosphate buffer, 1 baculovirus infected insect cells (BTI-TN-5B1-4) mM EDTA, 1 mM DTT, 20% (v/v) glycerol, pH 7.4.  1A1 Isozyme Microsomes, with P450 Reductase, The CYP4F3B isoform catalyzes the recombinant, expressed in baculovirus infected One unit will hydroxylate 1 nanomole of diclofenac 20-hydroxylation of leukotriene B . insect cells (BTI-TN-5B1-4) per minute at pH 7.4 at 37 °C. 4 Solution in 100 mM potassium phosphate buffer, The CYP1A1 isoform catalyzes 7-deethylation of C5107-1VL 1 vial pH 7.4. ethoxyresorufin. C5235-1VL 1 vial Solution in 100 mM potassium phosphate buffer,  2E1 isozyme microsomes, with P450 Reductase and cytochrome b , recombinant, expressed in pH 7.4. 5 baculovirus infected insect cells (BTI-TN-5B1-4) Microsomes containing human CYP1A1 and 1.0 nmole P450 2E1 per 0.5 ml vial Cytochrome Reductase and recombinant human NADPH-P450 reductase. buffered aqueous solution (100 mM potassium Cytochrome b C3735-1VL 1 vial phosphate, pH 7.4) 5 The oxidative mechanism of the cytochrome  1A2 Isozyme Microsomes, with P450 Reductase Cytochrome c Reductase, b , P450 content and 5 P450 system requires an electron transport and cytochrome b5, recombinant, expressed in p-Nitrophenol Hydroxylase activity reported on a baculovirus infected insect cells (BTI-TN-5B1-4) lot-specific basis. system of proteins to support catalysis. Microsomes containing recombinant human C5740-1VL 1 vial Cytochrome P450 (NADP) reductase is CYP1A2, recombinant rabbit NADPH-P450 coexpressed with all of our recombinant  3A4 isozyme microsomes, with P450 reductase reductase, and cytochrome b5. and cytochrome b , recombinant, expressed in cytochrome P450 preparations. Cytochrome Solution in 100 mM potassium phosphate buffer 5 , baculovirus infected insect cells (BTI-TN-5B1-4) b is also coexpressed with some pH 7.4. 5 Microsomes containing recombinant human preparations. C5614-1VL 1 vial CYP3A4 and recombinant rabbit NADPH-P450 Cytochrome P450 reductase catalyzes the  1B1 Isozyme Microsomes, with P450 Reductase, reductase recombinant, expressed in baculovirus infected Solution in 100 mM potassium phosphate buffer, reduction of hemethiolate-dependent insect cells (BTI-TN-5B1-4) pH 7.4. monooxygenases such as EC 1.14.14.1 Solution in 100 mM potassium phosphate buffer, One unit will convert 1 nanomole of testosterone (unspecified xenobiotic monooxygenases2) pH 7.4. to 6β-hydroxytestosterone per minute at pH 7.4 and is part of the microsomal hydroxylating Microsomes containing human CYP1B1 and at 37 °C. system. This reductase is a flavoprotein recombinant human NADPH-P450 reductase. C4982-1VL 1 vial containing FMN and FAD. It also reduces Tested for the ability to catalyze the 7-deethylation  4A11 isozyme microsomes, with P450 Reductase, cytochrome b and cytochrome c. of ethoxyresorufin. 5 recombinant, expressed in baculovirus infected C3860-1VL 1 vial insect cells (BTI-TN-5B1-4) Cytochrome b5 is a 15 kDa membrane- bound flavoprotein capable on enhancing  2A6 isozyme microsomes, with P450 Reductase The CYP4A11 isoform catalyzes the ω-hydroxylation of lauric acid. the redox potential of the P450 oxidative and cytochrome b5, recombinant, expressed in baculovirus infected insect cells (BTI-TN-5B1-4) Solution in 100 mM Tris-HCl buffer, pH 7.5. system. Catalyzes 7-hydroxylation of coumarin. Microsomes containing human CYP4A11 and Solution in 100 mM Tris-HCl buffer, pH 7.5. recombinant human NADPH-P450 reductase. Cyto­chrome c Reductase from porcine heart C4735-1VL 1 vial C4610-1VL 1 vial NADH: (acceptor) oxidoreductase; NADH dehydro­  2C9*2 isozyme microsomes, with P450 Reductase,  4F2 isozyme microsomes, with P450 Reductase genase

recombinant, expressed in baculovirus infected and cytochrome b5, recombinant, expressed in [9027‑14‑9] baculovirus infected insect cells (BTI-TN-5B1-4) insect cells (BTI-TN-5B1-4)  Type I, lyophilized powder, activity: ≥1.0 units/ The CYP2C9*2 isoform catalyzes the The CYP4F2 isoform catalyzes the 20-hydroxylation mg protein of leukotriene B . 4-hydroxylation of diclofenac. 4 Crude, lyophilized powder containing potassium Solution in 100 mM Tris-HCl buffer, pH 7.5. Solution in 100 mM potassium phosphate buffer, phosphate, pH approx. 7.0 Microsomes containing human CYP2C9*2 and pH 7.4. One unit will reduce 1.0 μmole of oxidized recombinant human NADPH-P450 reductase. C4985-1VL 1 vial cytochrome c per min at pH 8.5 at 25 °C.

C4235  4F3A isozyme microsomes, with P450 Reductase C3381-25MG 25 mg and cytochrome b , recombinant, expressed in C3381-100MG 100 mg  2C9 isozyme microsomes, with P450 Reductase, 5 baculovirus infected insect cells (BTI-TN-5B1-4) C3381-500MG 500 mg recombinant, expressed in baculovirus infected Sf9 cells The CYP4F3A isoform catalyzes the Cytochrome c Reductase (NADPH) Positive 20-hydroxylation of leukotriene B4. Solution in 100 mM potassium phosphate buffer, Control from rabbit liver pH 7.4. Solution C9363 Order sigma.com/order Technical service sigma.com/techinfo sigma.com/lifescience 15

▼ Cytochrome P450 Reductase Supplied in a solution containing 100 mM The enzyme catalyses electron transfer to potassium phosphate, pH 7.7, 0.1 mM EDTA, 0.2 cytochrome P450. This system plays a major role in Cyto­chrome P450 Reductase human mM DTT, and 20% (v/v) glycerol. detoxification of drugs and xenobiotics, activation of carcinogens, and metabolism of endogenous NADPH: Ferrihemoprotein­ oxidoreductase One unit will cause the reduction of 1.0 μmole of substrates such as steroids. NADPH-P450 reductase is a membrane-bound cytochrome c by NADPH per minute at pH 7.4 at flavoprotein that transfers electrons from NADPH 37 °C. Solution in 30 mM potassium phosphate buffer, pH 7.7, and 0.1 mM EDTA containing 50%(v/v) glycerol. to the various isoforms of cytochrome P450. The C8113-200UG 200 μg ratio of reductase: P450 that is typically used mol wt ~80 kDa ranges from 0.5–5:1. The enzyme contains one Cyto­chrome P450 Reductase from rabbit Package size based on protein content. mole each of FAD and FMN per mole of protein. liver induced with phenobarbital One unit will catalyze the reduction of 1.0 μmole Calculated mol wt 76.5 kDa NADPH:Ferrihemo­protein oxidoreductase [9039‑06‑9] of cytochrome c by NADPH per min at pH 7.7 at  recombinant, expressed in baculovirus infected  ~90% (SDS-PAGE), activity: 25-75 units/mg protein 30 °C. insect cells (Bradford), buffered aqueous glycerol solution C4839-50UG 50 μg

Cytochrome P450 Reductase ▲

Selected Cytochrome P450 Substrates Name Structure Description Cat. No. Debrisoquine sulfate Its metabolite may induce D1306-100MG

N NH2 1 • /2 H2SO4 Parkinsonism. NH

3-Oxo-1-cyclopentanecarboxylic acid O OH Substrate used in a study of 550485-1G biohydroxylation with mutants of 550485-5G cytochrome P450 BM-3. O

Resorufin benzyl ether N - B1532-5MG B1532-10MG O O O

Resorufin ethyl ether N - E3763-1MG E3763-5MG CH3CH2O O O

Resorufin methyl ether N Fluorimetric substrate for M1544-1MG cytochrome P450 linked enzymes. M1544-5MG CH3O O O

Resorufin pentyl ether (CH2)4CH3 Fluorimetric substrate for P0928-1MG O O O cytochrome P450 linked enzymes, P0928-5MG CYP2B and CYP2B4. P0928-10MG N 16

Other Phase I-Related Enzymes Flavin-Containing Monooxygenase-1, phosphorous-containing moiety associated Microsomes human with their respective oxides. These enzymes are Other Oxidases classified as Phase I or oxidative drug metabolism CH3 H3C CH3 enzymes. FMO5 is the only FMO isoform expressed N O N CH Allene Oxide Synthase from FMO 3 O in both adult and fetal human liver. Parthenium argentatum 2 H NADPHNADP H2O FMO-5 is the only FMO isoform expressed in both Potent anti-oxidant enzyme to remove lipid adult and fetal human liver. hydroperoxides in biological samples. Solution in 100 mM potassium phosphate buffer, Flavin Containing Monooxygenase is a broad spectrum  recombinant, expressed in Escherichia coli, activity: pH 7.4. monooxygenase. Substrates include primary, second- 25,000–40,000 units/mg protein ary and tertiary amines, hydrazines, phosphines, boron, Supplied by Gentest Corp. Supplied as a solution in phosphate buffered sale sulfide, selenide, and iodide containing compounds. pH 7.2 F5178-1VL 1 vial  FMO-1 One unit will cause a change in A nm of 1.0 per 234 recombinant, expressed in baculovirus infected ▼ Laccase minute at pH 7.0 at 25 °C in a 13S-HPOD13(S)- insect cells, buffered aqueous solution hydroperoxy-(9Z,11E)-octadecanoic Acid) The FMOs are responsible for catalyzing the reduction assay oxygenation of a variety of xenobiotic and drug OH O A6481-1VL 1 vial compounds including environmental pollutants and carcinogens which contain a nucleophilic Laccase O2 4 4 2H2O (+)-Cam­phor Mono­oxy­genase from nitrogen, sulfur, selenium, or phosphorus- Pseudomonas putida containing moiety associated with their respective oxides. These enzymes are classified as Phase I or [9030‑82‑4] OH OH oxidative drug metabolism enzymes.  activity: ~0.3 U/g Solution in 100 mM potassium phosphate buffer, Laccases are multicopper oxidase complexes with a broad 1 U corresponds to the amount of enzyme which pH 7.4. specificity for one-electron oxidation of phenolic related catalyzes the conversion of 1 μmol (+)-camphor compounds, resulting in the reduction of O to water. (Fluka No. 21300) per minute at pH 7.1 and 30°C Supplied by Gentest Corp. 2 [80498‑15‑3] Stereoselective lactone formation in vitro by F4928-1VL 1 vial coupled enzyme systems1,2 Laccase from Rhus vernicifera Flavin-Containing Monooxygenase-3, Lit cited: 1. G. Grogan et al., Biotechnol. Lett. 14, 1125 (1992); 2. J. Benzene­ ­diol:oxygen oxidoreductase Chem. Soc. Perkin Trans. I 2539 (1994). Microsomes human  21332  FMO-3 crude acetone powder, activity: ≥50 units/mg recombinant, expressed in baculovirus infected solid Cyclo­oxy­genase 1 from sheep insect cells, buffered aqueous solution One unit will produce a ΔA530 of 0.001 per min at pH 6.5 at 30 °C in a 3 mL reaction volume using Prostaglandin­ endoperoxide synthase; Prosta­glandin H The FMOs are responsible for catalyzing the syringaldazine as substrate. synthase 1; COX-1; Constitutive cyclo­oxy­genase oxygenation of a variety of xenobiotic and drug COX-1 catalyzes the conversion of arachidonic compounds including environmental pollutants L2157-10KU 10000 units and carcinogens which contain a nucleophilic acid to prostaglandin H2 (the first step in the biosynthesis of prostaglandins, thromboxanes, and nitrogen, sulfur, selenium, or phosphorus- Laccase from Trametes versicolor containing moiety associated with their respective prostacyclins). It is involved in the homeostatic former nomenclature: Coriolus versicolor role of eicosanoids and constitutively expressed in oxides. These enzymes are classified as Phase I or oxidative drug metabolism enzymes.  powder, activity: >20 units/mg almost all animal tissues. Has an apparent KM of 8.3 μM for arachidonic acid. FMO-3 is the major human hepatic form. One unit corresponds to the amount of enzyme which converts 1 μmole of catechol per minute at dimer subunit mol wt 70 kDa Solution in 100 mM potassium phosphate buffer, pH 4.5 and 25 °C pH 7.4  aqueous solution, ≥95% (SDS-PAGE), activity: 53739-100MG-F 100 mg ≥40,000 units/mg protein Supplied by Gentest Corp. 53739-1G-F 1 g Solution in 80 mM Tris-HCl, pH 8, with 0.1% F5053-1VL 1 vial TWEEN® 20 and 300 μM diethyldithiocarbamate.  powder, activity: ≥0.5 units/mg One unit consumes one nanomole of oxygen Flavin-Containing Monooxygenase-5, One unit corresponds to the amount of enzyme per minute at 37 °C in 0.1 M Tris-HCl buffer, pH 8, Microsomes human which converts 1 μmole of catechol per minute at containing 100 μM arachidonate, 5 mM EDTA, 2 pH 6.0 and 25 °C  FMO-5 mM phenol, and 1 μM hematin. recombinant, expressed in baculovirus infected 38429-1G 1 g C0733-5000UN 5000 units insect cells, buffered aqueous solution (2.5 mg in 38429-10G 10 g 100 mM Potassium Phosphate Buffer) FMOs are responsible for catalyzing the Laccase ▲ oxygenation of a variety of xenobiotic and drug compounds including environmental pollutants and carcinogens, which demonstrate a nucleophilic nitrogen, sulfur, selenium, and Order sigma.com/order Technical service sigma.com/techinfo sigma.com/lifescience 17

Laccase, Coriolus versicolor, CLEA Tyrosinase from mushroom Alcohol Dehydro­genase from Parvibaculum lavamentivorans  Laccase, Coriolus versicolor, cross-linked enzyme O O O O aggregate HC OH HC OH HC OH HC OH  recombinant, expressed in Escherichia coli, activity: ≥0.3 units/mg H3N CH H3N CH H3N CH H3N CH activity: ≥40.0 units/mg 1 U corresponds to the amount of enzyme which CH2 CH2 Tyrosinase CH2 CH2 One unit corresponds to the amount of enzyme O H2O oxidizes 1 μmol ABTS per minute at pH 4.5 and 2 which reduces 1 μmole ethyl acetate per minute 25°C at pH 7.0 and 30 °C.

OH OH O O 75449-1ML 1 mL H Monoamine Oxidase OH OH OH O R N ' ' H O H O RCH RNH H O 75449-5ML 5 mL C R 2 2 3 2 2 Tyrosinase in a copper-containing monophenol oxygenase H2 with activity for both catechols and cresol. Alcohol Dehydro­genase from 38837-100MG 100 mg Mono­phenol, dihydroxy­phenyl­ala­nine:oxygen Saccharomyces cerevisiae Monoamine oxidases are a group of enzymes that oxidoreductase; Cate­chol Oxi­dase; Poly­phenol Oxi­ Alcohol:NAD+ oxidoreductase; Alcohol Dehydro­ catalyze the oxidative deamination of primary dase; Mono­phenol Mono­oxy­genase [9002‑10‑2] genase from yeast monoamines. Isoelectric point (pI): 4.7-5 [9031‑72‑5] pH optimum is 6-7 A 141 kDa tetramer containing 4 equal subunits. Mono­amine Oxi­dase A human The active site of each subunit contains a zinc Molecular weight: 128 kDa by sedimentation atom. Each active site also contains 2 reactive MAO-A velocity diffusion; 133 kDa by light-scattering sulfhydryl groups and a histidine residue. MAOs are proteins of the mitochondrial measurements, and 119.5 kDa by electrophoresis. Isoelectric point: 5.4-5.8 membrane. These enzymes are responsible for Tyrosinase is a copper-containing oxidase, which catalyzing oxidative deamination of endo- and has activity for both catechols and cresol. It is Optimal pH: 8.6-9.0 xenobiotic amines. Substrate specificity differs for responsible for browning reactions. The enzyme Substrates: Yeast ADH is most active with ethanol each isozyme. is reported to have two binding sites for aromatic and its activity decreases as the size of the alcohol  recombinant, expressed in baculovirus infected substrates and a different binding site for oxygen- increases or decreases. Branched chain alcohols BTI insect cells copper. The copper is probably Cu(I), with and secondary alcohols also have very low activity. inactivation involving oxidation to Cu(II) ion. -3 ~2.5 mg per vial KM (ethanol) = 2.1 × 10 M  lyophilized powder, activity: ≥1000 unit/mg solid -1 M7316-1VL 1 vial KM (methanol = 1.3 × 10 M Converts tyrosine to L-DOPA or tyramine to K (isopropanol) = 1.4 × 10-1 M Mono­amine Oxi­dase B human dopamine M Inhibitors: Compounds that react with free One unit = ΔA of 0.001 per min at pH 6.5 at 25 °C MAO-B 280 sulfhydryls, including N-alkylmaleimides and in 3 mL reaction mix containing L-tyrosine. MAOs are proteins of the mitochondrial iodoacetamide. membrane. These enzymes are responsible for T3824-25KU 25000 units Zinc chelator inhibitors, including catalyzing oxidative deamination of endo- and T3824-50KU 50000 units 1,10-phenanthroline, xenobiotic amines. Substrate specificity differs for T3824-250KU 250000 units 8-hydroxyquinoline, 2,2′-dipyridyl, and thiourea. each isozyme. T3824-500KU 500000 units T3824-1MU 1000000 units Substrate analogue inhibitors, including β-NAD  recombinant, expressed in baculovirus infected analogs, purine and pyrimidine derivatives, BTI insect cells Dehydrogenases chloroethanol, and fluoroethanol. ~2.5 mg per vial Extinction Coefficient: E1% = 14.6 (water, 280 nm) M7441-1VL 1 vial ▼ Alcohol Dehydrogenase  lyophilized powder (contains buffer salts), activity: ≥300 units/mg protein Mono­amine Oxi­dase Insect Cell Control Alcohol Dehydro­genase equine Solids containing ≤ 2% citrate buffer salts MAO negative control [9031‑72‑5] 1 U corresponds to the amount of enzyme which suitable for conventional determination of β-NAD, This product is suitable as a negative control for reduces 1 µ mol benzaldehyde per minute at pH β-NADH, ethanol and acetaldehyde MAO-A and MAO-B. 7.0 and 30°C. One unit will convert 1.0 μmole of ethanol to  recombinant, expressed in wild type baculovirus  recombinant, expressed in Escherichia coli, activity: acetaldehyde per min at pH 8.8 at 25 °C. transfected BTI insect cells ≥0.5 units/mg Contains bound β-NAD and β-NADH and is not ~2.5 mg per vial 55689-100MG 100 mg suitable for the recycling microassay of β-NAD and M7566-1VL 1 vial 55689-500MG 500 mg β-NADH. If you require ADH for this purpose, see Cat. No. A3263.  recombinant, expressed in Escherichia coli, activity: A7011-7.5KU 7500 units ≥10.0 U/mL A7011-15KU 15000 units 79854-1ML 1 mL A7011-30KU 30000 units 79854-5ML 5 mL A7011-75KU 75000 units A7011-150KU 150000 units A7011-300KU 300000 units 18

 activity: ≥300 units/mg protein  lyophilized powder, activity: 30–90 units/mg (S)-Aroma­tic Alcohol Dehydrogenase, + Solids containing <2% citrate buffer salts protein NADP dependent from Purified Thermoanaerobium sp. suitable for recycling micro-assay of β-NAD and Alcohol:NADP+ oxidoreductase β-NADH Contains sodium citrate and dithioerythritol. [9028‑12‑0] mol. wt. ~141,000 (four subunits) A9287-100UN 100 units  lyophilized powder, activity: 1–5 units/mg solid One unit will convert 1.0 μmole of ethanol to A thermostable enzyme with broad specificity for acetaldehyde per min at pH 8.8 at 25 °C. Alcohol Dehydro­genase, recombinant from E. coli alcohols and ketones. Highly enantioselective for A3263-7.5KU 7500 units many (S)-aromatic alcohols. Chiral selectivity Vs/Vr Alcohol:NADP+ oxidoreductase A3263-15KU 15000 units ≥40 using (S)- and (R)-sec-phenethyl alcohol. [9028‑12‑0] A3263-30KU 30000 units Lyophilized powder containing raffinose,  activity: ≥500 U/mL A3263-75KU 75000 units dithiothreitol, and potassium phosphate buffer A3263-150KU 150000 units 1 U corresponds to the amount of enzyme which salt. reduces 1 μmol acetone per minute at pH 7.0 and For Gel Filtration Chromatography 30 °C (NADPH as cofactor) One unit will oxidize 1.0 μmole of 2-propanol to Gel filtration molecular weight marker acetone per min at pH 9.0 at 50 °C. 49641-1ML 1 mL A9685-2UN 2 units mol. wt. ~150,000 49641-5ML 5 mL A9685-10UN 10 units A8656-1VL 1 vial Alde­hyde Dehydro­genase from Hydroxy­steroid Dehydro­genase from Alcohol Dehydrogenase ▲ baker's yeast Pseudomonas testosteroni ATCC 11996 [9028‑88‑0] Alcohol dehydro­genase–Agar­ose from [9001‑56‑3]  lyophilized powder, activity: ≥1 units/mg baker's yeast (S. cerevisiae) α-Enzyme oxidizes only the 3α-hydroxysteroids of 1 U corresponds to the amount of enzyme which the C19, C21, and C24 series. β-Enzyme oxidizes the Alcohol:NAD+ oxidoreductase; ADH oxidizes 1 μmol acetaldehyde (Fluka No. 00070) 3β-hydroxysteroids of the C , and C series, the to acetic acid per minute at pH 8.0 and 25 °C. 19 21  lyophilized powder 17β-hydroxysteroids of the C18, C19, and C21 series, One unit will convert 1.0 μmole of ethanol to 82884-25MG-F 25 mg and certain 16β-hydroxysteroids. acetaldehyde per min at pH 8.8 at 25 °C. 82884-100MG-F 100 mg Mixture of α- and β-hydroxysteroid dehydrogenases A2529-25UN 25 units  solution, ~1 unit/mL contains 50 mM K-phosphate, 50% glycerol  α-enzyme activity: 0.05–0.10 unit/mg solid (using Alcohol Dehydro­genase, Deinococcus androsterone as substrate) 1 U corresponds to the amount of enzyme which radiodurans, recombinant from E. coli oxidizes 1 μmol acetaldehyde (Fluka No. 00070) α- and β-Enzymes can be extracted by grinding ADH to acetic acid per minute at pH 8.0 and 25°C and/or sonication in water or buffer. [9031‑72‑5] Crude dried cells grown on a medium containing 88307  activity: ≥10000 units/mL testosterone One unit corresponds to the amount of Alde­hyde Dehydro­genase, potas­sium- One unit will oxidize 1.0 μmole of substrate per enzyme which reduces 1 μmole ethyl-2-oxo-4- activated from baker's yeast (S. cerevisiae) min in the presence of β-NAD at pH 8.9 at 25°C. phenylbutyrate per minute at pH 7.0 and 37 °C Alde­hyde:NAD[P]+ oxidoreductase Protein determined by biuret. 16892-1ML 1 mL [9028‑88‑0] H7127-100MG 100 mg 16892-5ML 5 mL  lyophilized powder, activity: ≥2.0 units/mg protein β-Hydroxysteroid dehydrogenase from Alcohol Dehydro­genase, NADP+ Pseudomonas testosteroni dependent from Thermoanaerobium brockii Contains trehalose, potassium phosphate and citrate buffer salts, dithiothreitol, and traces of +  β-Hydroxy­steroid: NAD(P)+ oxidoreductase Alcohol:NADP oxidoreductase; TBADH β-NAD and propionic acid. [9028‑12‑0] lyophilized powder, activity: 20–50 units/mg One unit will oxidize 1.0 μmole of acetaldehyde An extremely thermostable enzyme with broad protein to acetic acid per min at 25 °C at pH 8.0 in the substrate specificity for alcohols, ketones and One unit will oxidize 1.0 μmole of testosterone per presence of β-NAD+, potassium and thiols. + acetaldehyde. min at pH 8.9 at 25°C, in the presence of β-NAD . One unit will oxidize 1.0 μmole of 2-propanol to A6338-25UN 25 units H5133 acetone per min at pH 7.8 at 40 °C in the presence A6338-100UN 100 units of NADP+. A6338-250UN 250 units 3α-Hydroxy­steroid Dehydro­genase from Pseudomonas testosteroni  lyophilized powder, activity: 5-15 units/mg  lyophilized powder + protein Contains potassium phosphate salts 3α-Hydroxy­steroid:NAD(P) oxidoreductase [9028‑56‑2] Contains phosphate buffer salts and activity: ≥15 units/mg protein dithioerythritol  activity: ≥15 units/mg protein A9770-25UN 25 units Lyophilized powder containing potassium A8435-100UN 100 units A9770-100UN 100 units phosphate buffer salt and EDTA A8435-250UN 250 units A9770-250UN 250 units Order sigma.com/order Technical service sigma.com/techinfo sigma.com/lifescience 19

One unit will oxidize 1.0 μmole of androsterone Ester­ase from porcine liver Epoxide Hydrol­ase, Agro­bac­ter­ium per min at pH 8.9 at 25 °C in the presence of  PLE radio­bac­ter, recombinant from E. coli + β-NAD . ammonium sulfate suspension, activity: [9048‑63‑9] ≥150 units/mg protein (biuret) H1506-50UN 50 units  activity: ≥50 U/mL Suspension in 3.2 M (NH ) SO , pH 8 4 2 4 1 U corresponds to the amount of enzyme which ▼ 7α-Hydroxysteroid dehydrogenase E2884-200UN 200 units turns over 1 μmol p-nitrostyreneoxide per minute E2884-1KU 1000 units at pH 7.2 and 25°C 7α-Hydroxysteroid dehydrogenase from E2884-5KU 5000 units 73118-100UL-F 100 μL Escherichia coli E2884-20KU 20000 units  activity: 5-20 units/mg protein (biuret) Epoxide Hydrol­ase, Aspergillus niger Esterase ▲ one unit will oxidize 1.0 μmole of sp., recombinant from Aspergillus niger chenodeoxycholic acid per min in the presence Hydrolases [9048‑63‑9] of β-NAD+ at pH 8.9 at 25 °C. No activity with  powder, activity: ≥1.5 units/mg deoxycholic acid, androsterone or testosterone. ▼ Epoxide Hydrolase 1 U corresponds to the amount of enzyme that H9506 O HO hydrolizes 1 μmol p-nitrostyrene oxide per minute Epoxide Hydrolase at pH 7.2 and 25°C H2O 7α-Hydroxysteroid dehydrogenase from R R OH Pseudomonas sp. licensed from CNRS 71832-25MG-F 25 mg  lyophilized powder, activity: 3-5 units/mg protein Epoxide hydrolases catalyze the hydration of reactive epoxides to their corresponding dihydrodiol products. 71832-100MG-F 100 mg One unit will oxidize 1.0 μmole of chenodeoxycholic acid per min in the presence [9048‑63‑9] Microsomal Epoxide Hydrolase of β-NAD+ at pH 8.9 at 25 °C. No activity with deoxycholic acid, androsterone or testosterone. Epoxide Hydrol­ase  mEH ; Epoxide Hydratase; Xenobiotic Epoxide Hydrol­ase  Hydratase, epoxide H2140-1UN 1 unit from human, recombinant, expressed in human activity: ≥0.13 units/mg H2140-10UN 10 units lymphoblast cell line recombinant, expressed in Escherichia coli Epoxide hydrolase functions to render epoxides ▲ 7α-Hydroxysteroid dehydrogenase 1 U corresponds to the amount of enzyme which less chemically reactive. Two forms of human hydrolizes 1 μmol p-nitrostyrene per minute at pH hydrolase are involved in this process, microsomal Esterases 7.2 and 25°C epoxide hydrolase (mEH) and soluble epoxide 50727-10MG 10 mg hydrolase. mEH is an enzyme of the endoplasmic ▼ Esterase 50727-50MG 50 mg reticulum. Although typically expressed in the Carboxyl­ ester­ase; Carboxy­lic-ester hydrol­ase liver, it is also present at significant levels in other [9016‑18‑6] Epoxide Hydrol­ase from tissues such as the adrenal gland. The primary One unit will hydrolyze 1.0 μmole of ethyl butyrate Rhodococcus rhodochrous characteristics of mEH substrates include marked to butyric acid and ethanol per min at pH 8.0 at lipophilicity and a lack of any trans-substitutions 25 °C.  lyophilized powder, activity: ≥0.5 U/g on the oxirane ring. 1 U corresponds to the amount of enzymes which buffered aqueous solution (100 mM potassium Ester­ase from rabbit liver hydrolizes 1 μmol (S)-NEPC [(2S,3S)-trans-3-phenyl- phosphate, pH 7.4)  lyophilized powder, activity: ≥75 units/mg protein 2-oxiranylmethyl-4-nitrophenyl carbonate, Prod. E7404-3MG 3 mg Lyophilized powder containing Tris buffer salts No. 04088] per minute at pH 8.0 and 25 °C Asymmetric hydrolysis of epoxides to optically E0887-500UN 500 units active diols1,2 E0887-1KU 1000 units Lit cited: 1. Hechtberger, P., Tetrahedron Asymmetry 4, 1161 (1993); 2. Mischitz, M., et al., Tetrahedron Asymmetry 7, 2041 (1996). 45299-250MG 250 mg

Epoxide Hydrolase ▲ 20

Selected Antibodies to Phase I Enzymes Clone Species Prestige Product Name Host No. Form Gene Symbol Reactivity Application Antibody Cat. No. Monoclonal Anti-AKAP9 mouse 1A6 purified immunoglobulin AKAP9, human human ELISA (i) - WH0010142M1-50UG WB Anti-AKAP9/AKAP450/ goat - affinity isolated antibody AKAP9, human human ELISA (i) - SAB2500052-100UG CG-NAP mouse IHC WB Anti-Aromatase rabbit - affinity isolated antibody CYP19A1, human human IF (i) - A7981-25UL WB A7981-200UL Anti-Aromatase/CYP19A1 goat - affinity isolated antibody CYP19A1, human human ELISA (i) - SAB2500110-100UG WB Anti-CYP11A1 rabbit - affinity isolated antibody CYP11A1, human human IHC (p)  HPA016436-100UL PA Anti-CYP17A1 goat - affinity isolated antibody CYP17A1, human canine ELISA (i) - SAB2500285-100UG cat WB human Anti-CYP1A1 (AB2) rabbit - affinity isolated antibody CYP1A1, human human WB - AV41404-50UG Anti-CYP1A1 (AB3) rabbit - affinity isolated antibody CYP1A1, human human WB - AV41405-50UG Anti-CYP1B1 rabbit - affinity isolated antibody CYP1B1, human human WB - AV51761-50UG Monoclonal Anti-CYP1B1 mouse 2F8 purified immunoglobulin CYP1B1, human human ELISA (i) - WH0001545M3-100UG Anti-CYP24A1 rabbit - affinity isolated antibody CYP24A1, human human IHC (p)  HPA022261-100UL PA Monoclonal Anti-CYP24A1 mouse 1F8 purified immunoglobulin CYP24A1, human human ELISA (i) - WH0001591M7-100UG WB Anti-CYP26A1 (481-495) rabbit - IgG fraction of antiserum CYP26A1, human human WB - C6498-200UL Anti-CYP26B1 rabbit - affinity isolated antibody CYP26B1, human human IHC (p)  HPA012567-100UL PA WB Anti-CYP2C19 rabbit - affinity isolated antibody CYP2C19, human human IHC (p)  HPA015066-100UL PA WB Anti-CYP2C8 rabbit - affinity isolated antibody CYP2C8, human human IHC (p)  HPA013547-100UL PA WB Anti-CYP2D6 rabbit - IgG fraction of antiserum CYP2D6, human human IHC - AV41675-100UG WB Anti-CYP2E1 rabbit - affinity isolated antibody CYP2E1, human human IHC (p)  HPA009128-100UL PA WB Anti-CYP2E1 rabbit - affinity isolated antibody CYP2E1, human human WB - AV41424-50UG Monoclonal Anti-CYP2J2 mouse 2D10 purified immunoglobulin CYP2J2, human human ELISA (i) - WH0001573M1-100UG WB Anti-CYP3A4 mouse - IgG fraction of antiserum CYP3A4, human human ELISA (c) - SAB1400064-50UG IHC (p) WB Anti-CYP3A4 rabbit - IgG fraction of antiserum CYP3A4, human human ELISA (c) - SAB1400065-100UG WB Anti-CYP4F2 rabbit - affinity isolated antibody CYP4F2, human human IHC (p)  HPA014048-100UL PA Anti-CYP51A1 (495-509) rabbit - IgG fraction of antiserum CYP51A1, human human WB - C8873-200UL Anti-CYP7A1 rabbit - affinity isolated antibody CYP7A1, human human IHC (p)  HPA010970-100UL PA WB Anti-CYP7B1 rabbit - affinity isolated antibody CYP7B1, human human IHC (p)  HPA017761-100UL PA WB Monoclonal Anti-Cytochrome mouse 3B8C1 purified immunoglobulin CYP1A2, human human IHC - C5118-25UL P450 1A2 Cyp1a2, rat rat WB C5118-200UL Order sigma.com/order Technical service sigma.com/techinfo sigma.com/lifescience 21

Clone Species Prestige Product Name Host No. Form Gene Symbol Reactivity Application Antibody Cat. No. Monoclonal Anti-Cytochrome mouse K1 purified immunoglobulin Cyp2c6, rat human ELISA (i) - C0746-25UL P450 2C6 (CYP2C6) rat WB C0746-200UL Monoclonal Anti-Cytochrome mouse F18 P3 purified immunoglobulin CYP3A5, human human ELISA (i) - C4743-25UL P450 3A5 B6 IHC C4743-200UL WB Anti-DBH rabbit - affinity isolated antibody DBH, human human IHC (p)  HPA005960-100UL PA Anti-FMO1 rabbit - affinity isolated antibody FMO1, human human IHC (p)  HPA023680-100UL PA Anti-FMO3 rabbit - affinity isolated antibody FMO3, human human IHC (p)  HPA013750-100UL PA WB Anti-KMO rabbit - affinity isolated antibody KMO, human human IHC (p)  HPA031115-100UL PA Anti-PAM rabbit - affinity isolated antibody PAM, human human WB - SAB2101711-50UG Anti-POR rabbit - affinity isolated antibody POR, human human IHC (p)  HPA010136-100UL PA WB Anti-TPH1 rabbit - affinity isolated antibody TPH1, human human IHC (p)  HPA022483-100UL PA WB Anti-YWHAZ rabbit - IgG fraction of antiserum YWHAZ, human human WB - AV48046-100UG

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Drug Conjugate Analysis

Drug Conjugate Analysis

Glucuronidation by the human UDP- prior to analysis by enzyme immunoassay, ▼ β-Glucuronidase glucuronosyltransferase (UGT) family of mass spectrometry, gas chromatography, high β-Glucuronidase from limpets enzymes plays an important role in the performance liquid chromatography, or other (Patella vulgata) metabolic fate of many drugs and other means (Figure 1). Typically, between 1 and 20 β-d-Glucuronide glucuronosohydrol­ ase­ xenobiotics. This biosynthetic reaction also units of glucuronidase is used per μl of plasma, [9001‑45‑0] has a role in the conjugation and excretion urine, or bile for the enzymatic hydrolysis Optimal pH: of endogenous substrates, such as steroids, of glucuronides present in these samples. glucuronidase activity: 4.5 to 5.0 sulfatase activity: ~6.2 bilirubin, and bile acids. UGT activity results The exact amount needed will depend on in the conjugation of glucuronic acid to the specific conditions used and must be Inhibitors: d-glucuronic acid, d-galacturonic acid, d-glucaro-1,4-lactone substrates containing sulfhydryl, hydroxyl, determined empirically. The enzyme from patella vulgata is reported to be aromatic amino, or carboxylic acid moieties. Molluskan Source β−Glucuronidase more effective in hydrolyzing opioid-glucuronides The glucuronides formed are more polar than the Helix pomatia, bovine liver and E. coli (water soluble) than the parent organic ß-Glucuronidase preparations isolated from enzymes mollusks also contain sulfatase activity. For substrate and are generally excreted through  Type L-II, lyophilized powder, activity: the kidney. Similar conjugation reactions occur this reason, the sulfatase activity of these 1,000,000–3,000,000 units/g solid with isoforms of sulfotransferases yielding the preparations is also reported on the certificate Contains sulfatase activity which is inhibited by 0.1 M phosphate. sulfate conjugate. of analysis. One Sigma or modified Fishman unit will liberate The enzyme from patella vulgata is reported to Enzymes for Conjugate Hydrolysis 1.0 μg of phenolphthalein from phenolphthalein be much more effective in hydrolyzing opioid- glucuronide per hr at 37 °C at pH 3.8 (30 min. assay). β-Glucuronidase glucuronides. Whereas the Helix pomatia and G8132-100KU 100000 units Sigma β-Glucuronidases are routinely used E. coli enzymes are reported to be slightly G8132-250KU 250000 units for the enzymatic hydrolysis of glucuronides more effective in hydrolyzing steroid- G8132-500KU 500000 units G8132-1MU 1000000 units from urine, plasma, and other biological fluids glucuronides. G8132-2MU 2000000 units

General β-Glucuronidase Hydrolysis Reaction

COOH COOH O O R β-Glucuronidase O OH OH +H2O OH + ROH OH OH OH OH

β-Glucuronidase Hydrolysis Reaction for Morphine 3 -β-glucuronide

CH3 COOH N CH3 β-Glucuronidase O OH N OH +

OH OH OH COOH O O O OH OH HO O OH OH

Figure 1. β-Glucuronidase is routinely used to hydrolyze xenobiotic glucuronide metabolites to increase volitility and organic solvent solubility prior to GC analysis. Order sigma.com/order Technical service sigma.com/techinfo sigma.com/lifescience 23

β-Glucuronidase from Helix pomatia  Type H-3, aqueous solution, activity: be useful for determining the presence of β-d-Glucuronide glucuronoso­hydrol­ase ≥90,000 units/mL androsterone, 17-hydroxycorticosteroids, [9001‑45‑0] secondary activity: ≤1,000 units/mL sulfatase and estriol in urine. The E. coli enzyme has β-Glucuronidase from Helix pomatia is a mixture of G8885-1ML 1 mL also been shown to be more active against enzymes derived from the Roman snail. G8885-10ML 10 mL estrogen conjugates than other sources of Optimal pH: G8885-25ML 25 mL the enzyme. • Glucuronidase activity: 4.5 to 5.0  Type H-5, lyophilized powder, activity: Optimal pH: 6–7 • Sulfatase activity: ~6.2 ≥400,000 units/g solid secondary activity: ≤40,000 units/g solid sulfatase β-Glucuronidase from Escherichia coli Inhibitors: d-glucuronic acid, d-galacturonic acid, A further purification of G0876 by gel permeation d-glucaro-1, 4-lactone β-D-Glucuronide glucuronoso­hydrol­ase [9001‑45‑0] chromatography. Used for the hydrolysis of glucuronide conjugates Effective in the hydrolysis of steroid glucuronides. in urinary metabolite analysis G1512-100KU 100000 units Used for the hydrolysis of glucuronide conjugates G1512-500KU 500000 units Many β-glucuronidases derived from mollusks also in urinary metabolite analysis contain sulfatase activity.  Type H-3AF, aqueous solution, activity: One Sigma or modified Fishman unit will liberate ≥60,000 units/mL Some preparations have been reported to contain 1.0 μg of phenolphthalein from phenolphthalein acid phosphatase activity. A further processing of Type H-3 to remove glucuronide per hr at 37 °C at the pH 6.8 (30 min agglutinin. assay). One Sigma or modified Fishman unit will liberate 1.0 μg of phenolphthalein from phenolphthalein G0762-100KU 100000 units  Type IX-A, lyophilized powder, activity: 1,000,000- glucuronide per hr at 37 °C at pH 5.0 (30 min 5,000,000 units/g protein (30 min assay) assay). β-Glucuronidase from Helix aspersa Contains buffer salts and stabilizer. (garden snail) Sulfatase Unit Definition: One unit of sulfatase will G7396-25KU 25000 units hydrolyze 1.0 μmole p-nitrocatechol sulfate per hr One Sigma or modified Fishman unit will liberate G7396-125KU 125000 units at pH 5.0 at 37 °C. 1.0 μg of phenolphthalein from phenolphthalein G7396-250KU 250000 units glucuronide per hr at 37°C at the pH 5.0 (30 min  Type HP-2, aqueous solution, activity: G7396-500KU 500000 units assay). ≥100,000 units/mL G7396-1MU 1000000 units secondary activity: ≤7,500 units/mL sulfatase Used for the hydrolysis of glucuronide conjugates G7396-2MU 2000000 units in urinary metabolite analysis Similar to G0876, but produced by Sigma.  Type VII-A, lyophilized powder, activity: 5,000,000-  Type HA-4 20,000,000 units/g protein, pH 6.8 (30 min assay) G7017-1ML 1 mL G7017-2ML 2 mL partially purified powder Contains buffer salts and stabilizer. G7017-5ML 5 mL activity: ≥300,000 units/g solid solid G7646-5KU 5000 units G7017-10ML 10 mL secondary activity: ≤7,500 units/g solid sulfatase G7646-25KU 25000 units G7017-25ML 25 mL G7646-100KU 100000 units One unit of sulfatase will hydrolyze 1.0 μmole G7646-500KU 500000 units  Type H-2, aqueous solution, activity: p-nitrocatechol sulfate per hr at pH 5.0 at 37 °C. ≥85,000 units/mL G4259-1MU 1000000 units  aqueous glycerol solution, activity: 20,000,000- secondary activity: ≤7,500 units/mL sulfatase 60,000,000 units/g protein, pH 6.8 (biuret) G0876-1ML 1 mL β-Glucuronidase ▲ Highly purified solution in 50% glycerol G0876-2ML 2 mL G8162-25KU 25000 units G0876-5ML 5 mL E. coli Source β-GLucuronidase G8162-100KU 100000 units G0876-10ML 10 mL E. coli derived β-Glucuronidase is a ~290 kDa  Type X-A, lyophilized powder, activity: 20,000,000– G0876-25ML 25 mL tetrameric protein with an isoelectric point 60,000,000 units/g protein, pH 6.8 (30 min assay)  Type HP-2S, aqueous solution, activity: of 4.8. Unlike the enzyme preparations Highly purified lyophilized powder containing ≥90,000 units/mL from mollusks that naturally contain buffer salts and stabilizer secondary activity: ≤7,500 units/mL sulfatase β-glucuronidase and sulfatase activities in G7896-25KU 25000 units G7770-2ML 2 mL almost equal amounts, the preparation of G7896-100KU 100000 units G7770-10ML 10 mL G7896-200KU 200000 units β-glucuronidase from E. coli is essentially G7770-25ML 25 mL free of sulfatase activity. The enzyme from  Type VIII-A, aqueous glycerol solution, activity: 5,000,000–20,000,000 units/g protein, pH 6.8  Type H-1, partially purified powder, activity: E. coli has a high rate of hydrolytic activity ≥300,000 units/g solid (biuret, 30 min assay) and it retains this activity during hydrolysis secondary activity: ≥10,000 units/g solid sulfatase G7771-100KU 100000 units better than similar enzymes that are more G0751-100KU 100000 units G0751-500KU 500000 units sensitive to changes in the concentration G0751-1MU 1000000 units of β-glucuronide conjugates. The enzyme G0751-2MU 2000000 units preparation from E. coli has been shown to 24

 activity: ≥10,000,000 units/g protein (30 min  Type B-10, activity: ~10,000 units/mg solid Sulfa­tase from Helix pomatia assay), recombinant, expressed in Escherichia coli One Sigma or modified Fishman unit will liberate Aryl-sulfa­tase; Phenol­sulfa­tase; Aryl-sulfate sulfo­ overproducing strain, lyophilized powder 1.0 μg of phenolphthalein from phenolphthalein hydrol­ase Contains buffer salts and stabilizer. glucuronide per hr at 37 °C at pH 5.0 (30 min [9016‑17‑5] G8295-25KU 25000 units assay). One unit will hydrolyze 1.0 μmole of G8295-500KU 500000 units G0501-100KU 100000 units p-nitrocatechol sulfate per hour at pH 5.0 at 37 °C (30 min assay). G8295-2MU 2000000 units G0501-500KU 500000 units G0501-1MU 1000000 units  Type H-1, lyophilized powder, sulfatase activity:  activity: ≥20,000 units/mg protein, recombinant, ≥10,000 units/g solid expressed in Escherichia coli overproducing strain, Sulfatase lyophilized powder secondary activity: ≥300 unit/mg solid Contains buffer salts and stabilizer. Molluskan sources of sulfatase also contain β-glucuronidase (at pH 5.0) β-glucuronidase activity. Lot-specific S9626-5KU 5000 units G8420-25KU 25000 units S9626-10KU 10000 units G8420-100KU 100000 units activities are reported for both enzymes on the certificate of analysis. S9626-100KU 100000 units Bovine Liver Source β-GLucuronidase  Type H-2, aqueous solution, ≥2,000 units/mL Bovine ß-Glucuronidase is a 290 kD protein ▼ Sulfatase secondary activity: ≥100,000 units/mL with an isoelectric point of 5.1. β-glucuronidase (at pH 5.0) Sulfatase Bovine preparations typically contain small S9751-5ML 5 mL  lyophilized powder, activity: 30–80 units/mg solid amounts of sulfatase activity, usually less S9751-10ML 10 mL S9629 S9751-25ML 25 mL than 0.5%. Optimal pH Sulfa­tase from abalone entrails Sulfa­tase from Patella vulgata (keyhole limpet) β-glucuronidase activity: 4.4 Aryl-sulfa­tase; Phenol­sulfa­tase; Aryl-sulfate sulfo­hydrol­ase Aryl-sulfa­tase; Phenol­sulfa­tase; Aryl-sulfate sulfo­ sulfatase activity: 4.4 [9016‑17‑5] hydrol­ase [9016‑17‑5] β-Glucuronidase from bovine liver  Type VIII, lyophilized powder, activity: 20–40 units/mg solid One unit will hydrolyze 1.0 μmole of β-D-Glucuronide glucuronoso­hydrol­ase p-nitrocatechol sulfate per hr at pH 5.0 at 37 °C [9001‑45‑0] One unit will hydrolyze 1.0 μmole of p-nitrocatechol sulfate per hr at pH 5.0 at 37 °C (30 min assay). Used for the hydrolysis of glucuronide conjugates (30 min assay). in urinary metabolite analysis  Type V, essentially salt-free, lyophilized powder, S9754-500UN 500 units activity: ≥5 units/mg solid  Type B-1, activity: ≥1,000,000 units/g solid S9754-1KU 1000 units S8629-500UN 500 units One Sigma or modified Fishman unit will liberate S9754-5KU 5000 units S8629-1KU 1000 units 1.0 μg of phenolphthalein from phenolphthalein glucuronide per hr at 37 °C at pH 5.0 (30 min Sulfa­tase from Aerobacter aerogenes  Type IV, essentially salt-free, lyophilized powder, assay). activity: ≥10 units/mg solid Aryl-sulfa­tase; Phenol­sulfa­tase; Aryl-sulfate sulfo­ G0251-100KU 100000 units hydrol­ase S8504 G0251-500KU 500000 units [9016‑17‑5] Sulfatase ▲ G0251-1MU 1000000 units  Type VI, buffered aqueous glycerol solution, G0251-5MU 5000000 units activity: 2-5 units/mg protein (biuret), 10–20 units/mL  Type B-3, activity: ≥2,000,000 units/g solid One Sigma or modified Fishman unit will liberate Solution in 50% glycerol containing 0.01 M Tris, 1.0 μg of phenolphthalein from phenolphthalein pH 7.5. glucuronide per hr at 37 °C at pH 5.0 (30 min One unit will hydrolyze 1.0 μmole of p-nitrophenyl assay). sulfate per min at pH 7.1 at 37 °C. G0376-500KU 500000 units S1629-10UN 10 units G0376-1MU 1000000 units S1629-50UN 50 units G0376-5MU 5000000 units Order sigma.com/order Technical service sigma.com/techinfo sigma.com/lifescience 25

Substrates and Inhibitors Name Structure Description Cat. No.

5-Bromo-4-chloro-3-indolyl β-d- HO OH Chromogenic substrate for β-glucuronidase B8174-5TAB Cl glucuronide sodium salt O OH Br O O- Na+ N O H 5-Bromo-6-chloro-3- H Chromogenic substrate for β-glucuronidase; B4532-10MG OH N indolyl β-d-glucuronide O Cl produces a magenta color in GUS+ bacterial colonies. cyclohexylammonium salt O NH2 HO O Br

HO OH 5-Bromo-6-chloro-3-indolyl sulfate O Chromogenic substrate for β-glucuronidase 16674-25MG - potassium salt OOS K+ 16674-100MG Br O

Cl N H d-Glucaro-δ-lactam potassium salt OH Inhibitor for bovine liver β-glucuronidase. G7166-5MG HO OH

OK O N H O d-Glucuronic acid HO O β-glucuronidase inhibitor G5269-10MG OOH G5269-10G OH G5269-25G OH OH G5269-50G G5269-100G 8-Hydroxyquinoline glucuronide - Chromogenic substrate for sulfatase H1254-50MG

4-Methylumbelliferyl β-d- CH3 Fluorescent substrate for β-glucuronidase M5664 glucuronide hydrate HO O

OO OO OH • xH2O OH OH

4-Methylumbelliferyl sulfate CH3 Fluorescent substrate for sulfatase M7133-500MG potassium salt M7133-1G K+ -O O S O O O O

4-Nitrocatechol sulfate K+ O- O Chromogenic substrate for β-glucuronidase N7251-100MG dipotassium salt O S O- K+ N7251-500MG O N7251-1G

NO2 N7251-5G

4-Nitrophenyl β-d-glucuronide O Chromogenic substrate for β-glucuronidase N1627-25MG HO O N1627-50MG HO O NO2 N1627-100MG

HO OH N1627-250MG N1627-1G N1627-2G Phenolphthalein β-D-glucuronide O Chromogenic substrate for β-glucuronidase P0501-25MG

O P0501-100MG O HO P0501-500MG

HO O O OH HO OH

Phenolphthalein β-d-glucuronide O Chromogenic substrate for β-glucuronidase P0376-25MG sodium salt O P0376-100MG P0376-250MG NaO O O OH P0376-1G O OH HO OH 26

Reagents and Labware for Dimethyl sulf­oxide 2,2,3,3,3-Penta­fluoro-1-pro­panol O DMSO; Methyl sulf­oxide PFPOH [422‑05‑9] CF CF CH OH F F S 3 2 2 Drug Metabolite Analysis [67‑68‑5] (CH ) SO FW 78.13 H C CH OH 3 2 3 3 FW 150.05 F3C N,O-Bis(tri­methyl­silyl)tri­fluoro­acet­amide Supercools easily and remelts slowly at room  97% temperature. Solidified product can be re-liquified with tri­methyl­chloro­silane Generates fluorinated alpha-keto ethers with by warming to room temperature without alkenes1 and has wide applications in the CF BSTFA + TMCS 3 detriment to the product. 2 CH3 CH3 expanding fluorous research area [25561‑30‑2] CF3C[=NSi H3C Si NSO i CH3 CH3 CH3  puriss., for GC, ≥99.7% (GC) Preparation of trifluoromethyl ynamines which, in (CH3)3]OSi(CH3)3 FW 257.40 41638-100ML 100 mL turn, converted aldehydes to α-trifluoromethyl-α,β- The position of the silyl groups has not been unsaturated amides.2 unequivocally established. It has been referred to Hepta­fluoro­butyric acid Lit cited: 1. Manandhar, S.; et al., J. Org. Chem. 67, 6415 (2002); 2. as both N,N and N,O. Ishihara, T.; et al., Tetrahedron 62, 3783 (2006). HFBA; Per­fluoro­butyric acid; Edman FF O Sealed ampules contain 1 ml. Gram sizes are in 257478-5G 5 g Reagent No. 3 F3C OH screw-cap bottles. F F 257478-25G 25 g [375‑22‑4] CF3CF2CF2COOH FW 214.04 T6381-1AMP 1 amp  for ion chromatography, ≥99.5% (GC) T6381-10AMP 10 amp TMAH 52411-5ML-F 5 mL T6381-5G 5 g 52411-25ML-F 25 mL Tri­methyl­phenyl­ammo­nium OH CH3 T6381-25G 25 g N CH hydroxide solution; Phenyl­tri­methyl- 3 CH3 T6381-100G 100 g Iodo­meth­ane ­ammo­nium hydroxide [1899‑02‑1] (CH ) N(OH)C H FW 153.22 N-tert-Butyl­dimethyl­silyl-N-methyl­tri- Methyl iodide ICH3 3 3 6 5 [74‑88‑4] CH I FW 141.94  0.2 M in methanol fluoro­acet­amide with 1% tert-Butyl- 3 dimethyl­chloro­silane  ReagentPlus®, 99.5% 33358-U 10 × 1 mL 33097-U 10 mL 289566-100G 100 g MTBSTFA + 1% TBDMSCl; Silylating­ O CH3 289566-500G 500 g mixture Fluka VI; N-Methyl-N-tert-butyl­- F3C N Si t-Bu dimethyl­silyl­tri­fluoro­acet­amide H3C CH3 N-Methyl-bis(tri­fluoro­acet­amide) [77377‑52‑7] C9H18F3NOSi FW 241.33 MBTFA [685‑27‑8] CF CON(CH )COCF O O MTBSTFA+TBDMSCl, 99:1 3 3 3 FW 223.07 F C NCF  3 3 ≥95% CH3 1 Selective O-silylation of N,O-diacylhydroxylamines. Reagent for introducing trifluoroacetyl group. Preparation of N-tert-butyldimethylsilyl  ~98% (GC), suitable for (derivatization) ethanolamines resulting from hydrolysis of nitrogen mustards.2 M0789-10X1ML 10 × 1 mL Lit cited: 1. Organometallics 23, 2157-2161 (2004); 2. J. Chromatogr. M0789-5ML 5 mL Insert with bottom spring. A. 1122, 242 (2006); 375934-10X1ML 10 × 1 mL Certi­fied glass inserts for 12 x 32 mm, 375934-5ML 5 mL large opening vials (fits crimp seal, snap 375934-25ML 25 mL ring, and 9 mm threaded vials) flat bottom 29441-U 100 ea Order sigma.com/order Technical service sigma.com/techinfo sigma.com/lifescience 27

Coming in Spring to Sigma® Life Science

A New Tool for Comprehensive ADME Analysis

IdMOC or Integrated discrete Multiple • Hepatocytes drugs with desirable ADMET (or ADME/ Organ Co-culture is an in vitro, cell Tox) properties. In vitro toxicity screening • Kidney proximal tubule epithelial cells culture based experimental model for using hepatocytes in conjunction with the study of intercellular communication. • Astrocytes other primary cells such as cardiomyocytes In conventional in vitro systems, each • Endothelial cells (cardiotoxicity model), kidney proximal cell type is studied in isolation ignoring tubule epithelial cells (nephrotoxicity critical interactions between organs or • Airway epithelial cells model), astrocytes (neurotoxicity model), cell types. IdMOC technology is based on • Various tumor cell lines (to model tumor endothelial cells (vascular toxicity model), the concept that multiple organs signal or bearing conditions) and airway epithelial cells (pulmonary communicate via the systemic circulation toxicity model) is invaluable to the drug The IdMOC system has numerous (i.e., blood). design and discovery (early drug discovery applications in drug development, such or rationale drug discovery) process. The IdMOC plate consists of multiple as the evaluation of drug efficacy, ADME, inner wells within a large interconnecting and toxicity. It can simultaneously evaluate References: chamber (Figure 1). Multiple cell types the toxic potential of a drug on cells from (1) Li AP, Bode C, Sakai Y. A novel in vitro system, the are first individually seeded in the inner multiple organs, examine drug stability and integrated discrete multiple organ cell culture (IdMOC) system, for the evaluation of human drug wells and, when required, flooded with an metabolite (hide metabolism) formation, toxicity: comparative cytotoxicity of tamoxifen overlying medium to facilitate well-to-well or estimate drug distribution. By modeling towards normal human cells from five major organs communication. Test material can be added and MCF-7 adenocarcinoma breast cancer cells. multiple-organ interactions, IdMOC can Chem Biol Interact. 2004 Nov 1;150(1):129-36. to the overlying medium and both media examine the pharmacological (hide (2) Li, AP. In vitro evaluation of metabolic drug-drug and cells can be analyzed individually. pharmacology) effects of a drug and its interactions: a descriptive and critical commentary. Current Protocols in Toxicology 2007 33:4.25.1- Plating of hepatocytes with other organ- metabolites on target and off-target organs 4.25.11. specific cells allows evaluation of drug as well as evaluate drug-drug interactions Look for these products metabolism and organotoxicity. (drug interactions keyword) by measuring in Spring 2011! IdMOC technology has been successfully CYP (cytochrome P450 oxidases) induction Z683140 – IdMOC 24-well plates, used in co-culturing the following cell or inhibition in hepatocytes. 6 per pack types and can be applied to both human IdMOC can also be used for routine and and animal models. high-throughput screening (HTS) of Z683159 – IdMOC 96-3well plates, 6 per pack Take advantage of our new product introductory offer of 25% off until Overlying Medium April 30th, 2011!

Check out all of our cell culture products and get a total ADME/Tox Culture solution at Cell A Cell B Cell C sigma.com/cellculture

Figure 1: Schematic representation of an IdMOC assay. Sigma-Aldrich® Worldwide Offices

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