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Metabolism

Absorption Systems uses both in vitro and in vivo metabolism models to characterize the metabolic stability and metabolic fate of our customers' compounds.

We routinely run assays for our customers that determine:

the metabolic stability of a compound in microsomes and hepatocytes,
the cytochrome P450 (CYP) reaction phenotype of a compound,
induction of CYPs in hepatocytes,
inhibition of UGTs,
the identities and relative amounts of metabolites,
CYP inhibition (IC₅₀ and K_i) of a compound (drug-drug interactions),
the pharmacokinetics of a compound in mice, rats, mini-pigs, dogs, and non-human primates.

Assay	Question	Test System
Metabolic stability	How fast and how extensively is the compound metabolized? If not metabolized, how is it eliminated?	Liver microsomes (human and preclinical species) Other subcellular fractions (liver and/or other organs)
CYP reaction phenotyping	Which enzymes metabolize the compound? Are they induced or inhibited by other drugs that are likely to be co-administered? How many enzymes? An important question in terms of clinical drug-drug interaction potential	Human liver microsomes +/- specific inhibitors Supersomes® expressing individual CYPs
CYP induction	Does the compound accelerate its own metabolism or that of other drugs? The consequence could be therapeutic failure	Fresh or cryopreserved hepatocytes (human and/or preclinical species) Enzymes: CYPs 1A2, 2B6, 2C9, 2C19, 3A4 Endpoints: enzyme activity and mRNA (by qPCR)
CYP inhibition	Does the compound interfere with the metabolism of other drugs? The consequence could be toxicity due to elevated levels of the other drugs	Screening, IC50, Ki, reversible vs. time-dependent inhibition Individual or pooled substrates, quantitation by LC/MS
Metabolite ID	Which preclinical species most closely matches human in terms of metabolite profile? Any unique or disproportionately human metabolites? If so, they might need to be evaluated in separate toxicology studies	Liver microsomes or S9 (human and preclinical species) used to generate metabolites in vitro Bioanalysis and structural elucidation of metabolites using LTQ Orbitrap XL

A description of our various metabolism related assays can be found in the Assay Data Sheets listed in the Related Literature section below. Click on the ones you want to download.

Note:

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Metabolism is a major route of drug elimination. In vitro assays are frequently used to study the metabolic fate of NCEs, to rank-order compounds in terms of metabolic stability, and to predict metabolic pathways in vivo. Using appropriate model systems, both phases of metabolism can be studied in vitro: Phase I, in which the parent compound is converted by hydrolysis or oxidation into a more polar derivative, and Phase II, in which the compound and/or its Phase I metabolites are conjugated to a polar group such as glucuronic acid to form a charged, more water-soluble derivative that is subsequently excreted in the urine or bile.

Absorption Systems uses several different test systems to evaluate metabolism in vitro. Microsomes are fragments of the endoplasmic reticulum membrane, containing many Phase I enzymes. In the presence of appropriate cofactors, microsomal enzymes will catalyze the conversion of an NCE to one or more metabolites. These reactions can yield a variety of information about potential metabolism of the NCE in vivo. These include prediction of the rate of clearance of the NCE by liver or other tissues, identification of metabolites and species differences in metabolite structures, and production of metabolites for chemical characterization or pharmacological activity assays. Microsomal assays can be performed in a high-throughput mode, where several dozen to several hundred compounds are simply screened for “metabolic stability” by following the disappearance of the parent compound. Alternatively, they can be used for detailed dissection of the enzymatic pathways responsible for an NCE’s metabolism by carrying out the reactions in the presence and absence of chemical inhibitors or antibodies that selectively delete one or more enzymes from the metabolic scheme.

S9 subcellular fractions contain a full complement of both Phase I and Phase II enzymes. They are the preferred test system for evaluating metabolism by various Phase II enzymatic pathways, or in cases where the metabolic pathway is unknown. Metabolism by Phase II enzymes is evaluated by measuring the rate of disappearance of test compounds from S9 fractions supplemented with appropriate cofactors. One-at-a-time addition of cofactors to the assay can identify which classes of transferases are involved. Specific antibodies to many of these transferases are available and can be used to identify the specific isoform that mediates the reaction. Like microsomal assays, S9 assays can provide information about clearance, metabolite structure and species differences. They can also be used in high-throughput screening, but the protocols are more complicated because of the number of cofactors that have to be evaluated.

Hepatocytes, available from humans as well as various animal species, offer an additional, more physiological, test system for evaluating hepatic clearance of NCEs. Fresh hepatocytes, plated or in suspension, are considered the “gold standard” for in vitro metabolic assays because they express virtually all of the important enzymes and transporters implicated in drug metabolism, at physiological concentrations, along with all necessary cofactors. However, the supply of fresh hepatocytes, particularly from humans, is limited and their availability is unpredictable, which limits the usefulness of this test system. Cryopreserved hepatocytes are an alternative test system, are commercially available from inventory at any time, and retain metabolic activity and viability for years. Individual lots can be pre-characterized for expression of metabolic functions of interest and stored in large quantities for repeated use. An additional desirable feature of cryopreserved human hepatocytes is that cells from several different donors can be pooled for assay, thereby eliminating the variable of differences in enzyme activity between donors. In vitro to in vivo correlation studies have demonstrated a good correlation between metabolic clearance predicted from cryopreserved hepatocyte assays and the values observed in in vivo studies.

A typical NCE follows a pathway like this during characterization of its metabolism:

Stability in human and animal liver microsomes or S9 fraction
Identification of metabolites present in human and/or animal microsomes or S9 fraction
Confirmation of metabolite formation using cryopreserved or fresh hepatocytes
Dissection of the metabolic pathway (enzyme phenotyping assays)

Click here to link to the 2006 FDA draft guidance on drug interaction studies: http://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/UCM072101.pdf

Click here to link to the 2008 FDA guidance on safety testing of drug metabolites: http://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/ucm079266.pdf

Please contact Absorption Systems if you would like our help in determining the metabolic profile of your compounds.

Related Literature

TITLE

EA151

Stability in Plasma Assay Data Sheet

This assay is used to determine the stability of test compound in blood plasma for various species, including humans.
Click Here to Download

EA153

Express Plus Stability in Plasma or Whole Blood

This assay is used to determine the stability of a test compound in whole blood or plasma from mouse, rat, rabbit, dog, monkey, or human.
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EA222

Express P-gp Inhibitor Assessment Across Cell Monolayers

This assay is used to screen for inhibition of P-gp by a test compound, by measuring its effect on the bidirectional permeability of a P-gp substrate through Caco-2 cell monolayers.
Click Here to Download

EA224

Express PepT1 Substrate Assessment in Caco-2 Cell Monolayers

This assay is used to identify active uptake of a test compound by PepT1, using the Caco-2 cell monolayer system.
Click Here to Download

EA232

BCRP Interaction Assessment Level 2

This assay uses CPT-B1 (BCRP-knockdown), CPT-P1 (P-gp-knockdown), and Caco 2 cell monolayers to determine if a test compound is a substrate and/or inhibitor of BCRP.
Click Here to Download

EA233

BCRP Interaction Assessment Level 3

This assay uses CPT-B1 (BCRP-knockdown), CPT-P1 (P-gp-knockdown), and Caco-2 cell monolayers to determine if a test compound is a substrate and/or inhibitor of BCRP.
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EA401

Express Plus Metabolic Stability (human liver microsomes) Assay Data Sheet

This assay is used to determine the percent remaining and intrinsic clearance of a test compound incubated with pooled human liver microsomes in the presence of NADPH.
Click Here to Download

EA402

Express Plus Metabolic Stability (rat liver microsomes) Assay Data Sheet

This assay is used to determine the percent remaining and intrinsic clearance of a test compound incubated with pooled rat liver microsomes in the presence of NADPH.
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EA403

Metabolic Stability in Liver Microsomes Assay Data Sheet

This assay is used to determine the percent remaining and half-life of a test compound incubated with liver microsomes in the presence and absence of NADPH.
Click Here to Download

EA404

Metabolic Stability in the Presence of S9 Fractions Assay Data Sheet

This assay is used to determine the percent remaining and half-life of a test compound incubated with S9 fraction in the presence and absence of cofactors.
Click Here to Download

EA405

Metabolic Stability in the Presence of Hepatocytes Assay Data Sheet

This assay is used to determine the percent remaining and half-life of a test compound incubated with either cryopreserved or fresh hepatocytes.
Click Here to Download

EA406

IC50 Determination Using Fluorogenic Substrates Assay Data Sheet

This assay uses fluorogenic substrates to determine the potency with which a test compound inhibits recombinant human cytochrome P450 (CYP) enzymes.
Click Here to Download

EA407

CYP Inhibition Screen in Human Liver Microsomes Using LC-MS-MS Assay Data Sheet

This assay is used to screen for inhibition of cytochrome P450 (CYP) enzymes by a test compound.
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EA408

CYP 450 Phenotyping Using Supersomes Assay Data Sheet

Cytochrome P450 reaction phenotyping using CYP-specific Supersomes™
Click Here to Download

EA409

Induction of CYP1A2, 2B6, and 3A4 in Human Hepatocytes Assay Data Sheet

This assay is used to assess the potential of a test compound to induce CYP1A2, CYP2B6, and CYP3A4 in fresh or cryopreserved human hepatocytes.
Click Here to Download

EA410

Ki Determination in Human Liver Microsomes Using LC-MS-MS Assay Data Sheet

This assay is used to determine the inhibition constant (Ki ) of a test compound and the mechanism of inhibition of CYPs in human liver microsomes (HLM). The IC50 of the test compound for a given CYP must be known or determined with EA407, CYP Inhibition, prior to Ki determination.
Click Here to Download

EA411

Metabolic Stability in Human Intestinal Microsomes Assay Data Sheet

This assay is used to determine the percent remaining and half-life of a test compound incubated with human intestinal microsomes in the presence and absence of NADPH.
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EA413

CYP Inhibition IC50 Using LC-MS-MS in Human Liver Microsomes

This assay is used to determine the potency with which a test compound inhibits cytochrome P450 (CYP) enzymes.
Click Here to Download

EA414

Mechanism-based CYP Inhibition Using LC-MS-MS in Human Liver Microsomes

This assay is used to determine whether a test compound is a mechanism-based inhibitor of cytochrome P450 (CYP) enzymes.
Click Here to Download

EA415

Express Plus Metabolic Stability (mouse liver microsomes) Assay Data Sheet

This assay is used to determine the percent remaining and intrinsic clearance of a test compound incubated with pooled mouse liver microsomes in the presence of NADPH.
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EA416

Express Metabolic Stability (primate liver microsomes) Assay Data Sheet

This assay is used to determine the percent remaining and intrinsic clearance of a test compound incubated with pooled primate liver microsomes in the presence of NADPH.
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EA417

Express Plus Metabolic Stability (dog liver microsomes) Assay Data Sheet

This assay is used to determine the percent remaining and intrinsic clearance of a test compound incubated with pooled dog liver microsomes in the presence of NADPH.
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EA418

Express Plus IC50 Determination Using Fluorogenic Substrates

This assay uses fluorogenic substrates to determine the potency with which a test compound inhibits recombinant human cytochrome P450 (CYP) enzymes.
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EA419

Biomimetic Oxidation

This assay is used to generate, isolate, and characterize multi-mg quantities of targeted metabolite(s) of a test compound using biomimetic oxidations.
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EA421

UGT Inhibition Using Recombinant Enzymes and LC/MS/MS

This assay is used to assess the potential of a test compound to inhibit individual human UDP-glucuronosyltransferase (UGT) isoforms.
Click Here to Download

EA424

Express Plus Metabolic Stability in Liver S9 Fractions

This assay is used to determine the percent remaining and intrinsic clearance of a test compound incubated with liver S9 fraction (mouse, rat, dog, monkey, or human) in the presence of cofactors.
Click Here to Download

EA425

Express Plus Metabolic Stability in Hepatocytes

This assay is used to determine the percent remaining and intrinsic clearance of a test compound incubated with cryopreserved hepatocytes (mouse, rat, dog, monkey, or human).
Click Here to Download

EA426

Express Plus CYP Inhibition Using LC-MS-MS in Human Liver Microsomes

This assay is used to screen for reversible inhibition of individual cytochrome P450 (CYP) enzymes by a single concentration of a test compound.
Click Here to Download

EA427

Express Plus CYP IC50 Using LC-MS-MS in Human Liver Microsomes

This assay is used to determine the IC50 for reversible inhibition of individual cytochrome P450 (CYP) enzymes by a test compound.
Click Here to Download

EA430

Express Plus Time Dependent Inhibition With & without NADPH

This assay is used to screen for time-dependent inhibition of individual cytochrome P450 (CYP) enzymes by a test compound after pre-incubation with and without NADPH.
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EA431

Time Dependent Inhibition Using IC50 Shift Approach

This assay is used to identify time-dependent inhibition of individual cytochrome P450 (CYP) enzymes by a test compound using the IC50 shift approach.
Click Here to Download

EA432

Express Plus UGT Inhibition

This assay is used to assess the potential of a test compound to inhibit individual human UDP-glucuronosyltransferase (UGT) isoforms.
Click Here to Download

EA433

Express Plus Detection of Glucuronides

This assay is used to detect glucuronidation of a test compound after incubation with human liver microsomes in the presence of UDPGA.
Click Here to Download

EA434

Express Plus Glutathione Trapping

This assay is used to detect a glutathione conjugate of a test compound, due to formation of a reactive metabolite, after incubation with human liver microsomes in the presence of NADPH and GSH.
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EA438

Metabolite Identification without Structure Elucidation

This assay is used to identify metabolites of a test compound produced by pooled liver microsomes, S9 fraction, or hepatocytes.
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EA439

Metabolite Identification with Structure Elucidation

This assay is used to identify and elucidate structures of metabolites of a test compound after incubation with pooled liver microsomes, S9 fraction, or hepatocytes.
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EA440

Induction CYP Rabbit or Dog Hepatocytes

This assay is used to assess the potential of a test compound to induce CYP3A activity in rabbit or dog hepatocytes.
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EA442

MAO Substrate Assessment Using Human Liver S9 Fraction

This assay uses human liver S9 fraction and a chemical inhibitor to determine if a test compound is metabolized by monoamine oxidase (MAO).
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EA443

CYP Inhibition Screen Using Fluorogenic Substrates

This assay uses fluorogenic substrates to screen for inhibition of recombinant human cytochrome P450 (CYP) enzymes by a single concentration of a test compound.
Click Here to Download

EA444

Express Plus CYP Reaction Phenotyping using Supersomes

This screening assay is used to identify the CYP(s) involved in the metabolism of a test compound, through the use of CYP-specific Supersomes.
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EA445

Express Plus CYP Reaction Phenotyping using Chemical Inhibitors

This screening assay is used to identify the CYP(s) involved in the metabolism of a test compound, through the use of human liver microsomes and CYP-specific chemical inhibitors.
Click Here to Download

EA446

FMO Substrate Assessment Using Supersomes

This assay uses recombinant human enzymes (Supersomes) to determine if a test compound is metabolized by flavin-containing monooxygenase (FMO).
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EA447

MAO Substrate Assessment Using Supersomes

This assay uses recombinant human enzymes (Supersomes) to determine if a test compound is metabolized by monoamine oxidase (MAO).
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EA801

Relative Bioavailability in Rats Assay Data Sheet

This screening assay is used to determine the bioavailability of test compounds relative to a reference compound after oral administration to rats.
Click Here to Download

EA804

Absolute Bioavailability in Mice Assay Data Sheet

This assay is used to determine the absolute bioavailability of a test compound in mice.
Click Here to Download

EA805

Bioavailability in Dogs Assay Data Sheet

This assay is used to determine the bioavailability of a test compound in dogs when the compound is administered by at least two different routes.
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EA807

Express Plus Bioavailability-Exposure in Mice Assay Data Sheet

This screening assay is used to determine the bioavailability / exposure of test compounds after two routes of administration to male mice.
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EA808

Express Plus Bioavailability-Exposure in Rats Assay Data Sheet

This screening assay is used to determine the bioavailability / exposure of test compounds after two routes of administration to male rats.
Click Here to Download

EA810

Express Plus Bioavailability-Exposure in Dogs Assay Data Sheet

This assay is used to determine the bioavailability / exposure of test compounds in male dogs after two routes of administration.
Click Here to Download

EA812

Express Plus Bioavailability-Exposure in Dogs Non-crossover

This assay is used to determine the bioavailability / exposure of a test compound in dogs after two routes of administration (non-crossover).
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EA813

Express Plus ABT Exposure in Rats

This screening assay uses the general CYP inhibitor, 1-aminobenzotriazole (ABT), to determine the role of absorption vs. first-pass metabolism in limiting the systemic exposure of a test compound in rats.
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OS010

Index of Assays and Services

Click Here to Download

OS011

Specialized ADME Assessment

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OS106

Biomimetic Oxidation: Drug Interaction Testing

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OS107

New Metabolism Assays

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OS400b

Overview of Absorption Systems' Rat Biliary Excretion Model

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PP04

Metabolizing_Activities_in_Rabbit_Cornea_and_Conjunctiva (ISSX 2007)

ISSX 2007
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RR1

Induction of CYP 1A and 3A4 Human Hepatocytes

Click Here to Download

RR3

Induction of Cytochrome P450- 2C9- 2C19 and 2B6 Activities in Cryopreserve Human Hepatocytes

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RR9

Identification of Cytochrome P450 Isoforms Responsible for Canadine Metabolism Using Human Supersomes

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Brochures

Type

TITLE

ADDED

Size

ABT Rat Model

A quick and cost-effective way to distinguish between poor absorption and rapid first-pass metabolism for compounds with poor oral bioavailability
Click Here to Download

04/22/10

193 KB

Biomimetic Metabolite Generation and Characterization

A novel way to generate multi-mg quantities of oxidative metabolites
Click Here to Download

05/28/10

928 KB

Metabolite ID

Structural identification and/or quantification of drug metabolites
Click Here to Download

12/03/09

695 KB