Transporters

Transporter Assays

Transporters are membrane-bound proteins that enable movement of substances, from nutrients to xenobiotics, in and out of cells. Drug transporter interactions are an important part of the pharmacokinetic properties of drug products. Understanding which transporters drive influx/efflux of your drug can help predict in vivo safety and performance, optimise development, and speed up regulatory approval.

Drug transporters are grouped into two classes:

ATP binding cassette (ABC)

Transporters that are relevant for intestinal absorption, urinary excretion and blood-brain barrier transfer. Examples include P-glycoprotein (P-gp) and BCRP.

Solute Carrier (SLC)

Transporters that mainly impact renal and hepatic uptake of drugs, as well as urinary excretion. Examples include OATP1B1 and OCT1

Pharmaron has extensive experience evaluating the potential interactions of compounds with drug transporter proteins. We have years of experience with in vitro transporter substrate and inhibitor potential assay development. Our expertise spans co-medications, product profiles, development plans, clearance routes, chemical structures, and physicochemical properties.

Our suite of in vitro assays enables robust and comprehensive characterization of drug interactions with both ABC and SLC transporters. The cell lines and inhibitors we use align with all FDA recommendations for these assays.

Other Express Services

ADME Profiling

Metabolism Assays

Metabolite Identification Assays

Binding/Partitioning Assays

Permeability Assays

Physicochemical Assays

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Project Deliverables:

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Available Express Transporter Assays

Inhibition

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

Required from Sponsor

Either a minimum of 100 µL of test article at 10 mM in DMSO or 2 mg of powder
Molecular weight of the test article and its salt form
MSDS or handling and storage information, e.g., light sensitive, store at -20°C, etc.

Deliverables

Cell batch QC results
The percent recovery of digoxin from the assay wells containing Caco-2 monolayers
The apparent permeability (Papp) of digoxin in both directions, in the presence and absence of inhibitor
The efflux ratio [(Papp B to A)/(Papp A to B)] of digoxin in the presence and absence of inhibitor
P-gp substrate classification:
- Positive: Digoxin efflux ratio ≥ 2.0 in the absence of inhibitor and corrected efflux ratio reduced by ≥ 50% in the presence of inhibitor
- Negative: Digoxin efflux ratio ≥ 2.0 in the absence of inhibitor and corrected efflux ratio reduced by <50% in the presence of inhibitor

Substrate

Test article and digoxin each at 10 µM in HBSSg with maximum DMSO concentration ≤ 0.8%

Assay System

Confluent monolayers of Caco-2 (clone C2BBe1) cells, 21-28 days old

Assay Conditions

Bidirectional permeability of Digoxin in Caco-2 cells in the presence and absence of inhibitor
Transport buffer: HBSSg, pH 7.4 ± 0.2
Apical and basolateral side at pH 7.4
Dose two cell monolayers for each direction (n=2), in the presence and absence of inhibitor
- Dose apical side for (A→B) assessment
- Dose basolateral side for (B→A) assessment
Sample from both apical and basolateral sides at 120 minutes
Determine the concentrations of digoxin using a generic LC-MS/MS method with a minimum 6-point calibration curve

Assay QC

The quality of the monolayer batch is verified using control compounds before the monolayers are released for use
The quality of each monolayer used in the assay is verified by calculating the Papp for the control article, lucifer yellow, dosed post-experimentally

Notes

The results from this assay are sent to the sponsor in the ExpressPlus report format, which may include graphical representations of data and comparison with historical data for reference articles.
The solubility of the test article in Hanks’ buffer containing ≤ 0.8% DMSO must be greater than the test concentration. If the solubility of the test article is unavailable, Absorption Systems can conduct a solubility assessment at an additional charge.
Assay conditions with the inhibitor include:
- A 30 minute pre-incubation with test article
Assay conditions without the inhibitor includes
- A 30 minute pre-incubation with buffer
Digoxin can be run in the presence and absence of Valspodar as an optional positive control
- Valspodar dosed at 1 µM

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This MDR 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 MDR1-MDCK cell monolayers.

Required from Customer

Either a minimum of 300 µL of test compound at 10 mM in DMSO, or 5 mg of powder
Exact molecular mass of test compound and its salt form
Solubility and chemical stability of the test compound in Hanks’ buffer
MSDS or handling and storage information, e.g., light sensitive, store at -20°C, etc.

Deliverables

Apparent permeability (P_app) of digoxin in both directions, in the presence and absence of test compound
Efflux ratio [(P_app B to A)/(P_app A to B)] of digoxin in the presence and absence of test compound
Classification of test compound as a likely inhibitor or non-inhibitor of P-gp at the concentration tested

Substrate

Test compound and digoxin each at 10 µM in modified Hanks’ buffer (HBSSg) with final DMSO concentration < 0.8%

Assay System

Confluent monolayers of MDR1-MDCK cells, 7-14 days old, in Transwell^® dual-chamber plates, with apical and basolateral pH 7.4

Assay Conditions

Dose duplicate monolayers (N=2) on apical side for A to B digoxin permeability
Dose duplicate monolayers (N=2) on basolateral side for B to A digoxin permeability
Repeat above steps on monolayers pretreated and co-dosed with test compound on both apical and basolateral sides
Sample both apical and basolateral sides at 120 minutes
Determine the concentration of digoxin using a generic LC-MS/MS method with a minimum 4-point calibration curve

Assay QC

Verify the quality of the cell monolayer batch using control compounds before the monolayers are released for use
Verify the quality of each monolayer used in an assay by a pre-experiment TEER measurement and by calculating the P_app for the co-dosed control compound, Lucifer yellow

Notes

The results from this assay are provided to the customer in the ExpressPlus report format, which may include graphical representations of data and comparison with historical data for reference compounds.
The solubility of the test compound in Hanks’ buffer containing < 0.8% DMSO must be greater than the test concentration. If the solubility of the test compound is unavailable, Absorption Systems can conduct a solubility assessment at an additional charge.

Options

The customer can request that a positive control be performed in parallel (additional fees apply)

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This BCRP transporter assay is used to screen for inhibition of BCRP by a test compound, by measuring its effect on the bidirectional permeability of a BCRP substrate through BCRP-MDCK cell monolayers

Required from Customer

Either a minimum of 300 µL of test compound at 10 mM in DMSO, or 5 mg of powder
Exact molecular mass of test compound and its salt form
Solubility and chemical stability of the test compound in Hanks’ buffer
MSDS or handling and storage information,
e.g., light sensitive, store at -20°C, etc.

Deliverables

Apparent permeability (P_app) of cladribine in both directions, in the presence and absence of test compound
Efflux ratio [(P_app B to A)/(P_app A to B)] of cladribine in the presence and absence of test compound
Classification of test compound as a likely inhibitor or non-inhibitor of BCRP at the concentration tested

Substrate

Test compound and cladribine each at 10 µM in modified Hanks’ buffer (HBSSg) with final DMSO concentration < 0.8%

Assay System

Confluent monolayers of BCRP-MDCK cells, 7-14 days old, in Transwell^® dual-chamber plates, with apical and basolateral pH 7.4

Assay Conditions

Dose duplicate monolayers (N=2) on apical side for A to B cladribine permeability
Dose duplicate monolayers (N=2) on basolateral side for B to A cladribine permeability
Repeat above steps on monolayers pretreated and co-dosed with test compound on both apical and basolateral sides
Sample both apical and basolateral sides at 120 minutes
Determine the concentration of cladribine using a generic LC-MS/MS method with a minimum 4-point calibration curve

Assay QC

Verify the quality of the cell monolayer batch using control compounds before the monolayers are released for use
Verify the quality of each monolayer used in an assay by a pre-experiment TEER measurement and by calculating the P_app for the co-dosed control compound, Lucifer yellow

Notes

The results from this assay are provided to the customer in the ExpressPlus report format, which may include graphical representations of data and comparison with historical data for reference compounds.
The solubility of the test compound in Hanks’ buffer containing < 0.8% DMSO must be greater than the test concentration. If the solubility of the test compound is unavailable, Absorption Systems can conduct a solubility assessment at an additional charge.

Options

The customer can request that a positive control be performed in parallel (additional fees apply)

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This OAT1 transporter assay is used to screen for inhibition of OAT1 by a test compound in OAT1-transfected HEK293 cells at a single concentration

Required from Customer

Either a minimum of 300 µL of test compound at 10 mM in DMSO, or 5 mg of powder
Exact molecular mass of test compound and its salt form
Relevant solubility of the test compound
MSDS or handling and storage information, e.g., light sensitive, store at -20°C, etc.

Deliverables

Normalized uptake of para-aminohippurate (PAH) in the presence and absence of test compound in OAT1-transfected and vector control cells
Percent inhibition by the test compound
>50% inhibition indicates that the test compound is a significant inhibitor of OAT1

Substrate

OAT1 probe substrate PAH at 10 μM
Test compound at 10 μM in modified Hanks’ buffer with less than 0.8% DMSO

Assay System

OAT1-transfected HEK293 cells and vector control cells cultured in 24-well plates

Assay Conditions

Measure uptake of PAH in OAT1-transfected and vector control cells with and without test compound
Single concentration of test compound
Two cell lines: OAT1-transfected and vector control cell lines
Four treatments as follows:
- OAT1-transfected cells with PAH +/-test compound
- Vector control cells with PAH +/- test compound
Perform assay in duplicate (N=2 per treatment)
Wells are equilibrated with blank buffer containing 1% BSA prior to initiating the assay
Buffer is aspirated and replaced with test compound dosing solution
Incubate cells for 5 min at 37°C
Terminate the incubation and lyse the cells
Analyze cell lysate using LC-MS/MS to determine PAH concentration
Determine protein concentration in cell lysate and normalize PAH concentration to protein content

Assay QC

Uptake rate of probe substrate in OAT1-transfected vs. vector control cells

Notes

The results from this assay are provided to the customer in the ExpressPlus report format, which may include graphical representations of data and comparison with historical data for reference compounds.
The solubility of the test compound in Hanks’ buffer containing < 0.8% DMSO must be greater than the test concentration. If the solubility of the test compound is unavailable, Absorption Systems can conduct a solubility assessment at an additional charge.
Percent inhibition is calculated as 100 x [1-(IR_OAT1-IR_VC)_TC / (IR_OAT1 – IR_VC)₀], where IR_OAT1 is the influx rate of PAH in OAT1-transfected cells, IR_VCis the influx rate in vector control cells, “TC” means in the presence of test compound, and “0” means in the absence of test compound.

Options

The customer can request that a positive control be performed in parallel (additional fees apply)

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This OAT3 transporter assay is used to screen for inhibition of OAT3 by a test compound in OAT3-transfected HEK293 cells at a single concentration

Required from Customer

Either a minimum of 300 µL of test compound at 10 mM in DMSO, or 5 mg of powder
Exact molecular mass of test compound and its salt form
Relevant solubility of the test compound
MSDS or handling and storage information, e.g., light sensitive, store at -20°C, etc.

Deliverables

Normalized uptake of furosemide in the presence and absence of test compound in OAT3-transfected and vector control cells
Percent inhibition by the test compound
>50% inhibition indicates that the test compound is a significant inhibitor of OAT3

Substrate

OAT3 probe substrate furosemide at 5 μM
Test compound at 10 μM in modified Hanks’ buffer with less than 0.8% DMSO

Assay System

OAT3-transfected HEK293 cells and vector control cells cultured in 24-well plates

Assay Conditions

Measure uptake of furosemide in OAT3-transfected and vector control cells with and without test compound
Single concentration of test compound
Two cell lines: OAT3-transfected and vector control cell lines
Four treatments as follows:
- OAT3-transfected cells with furosemide +/-test compound
- Vector control cells with furosemide +/- test compound
Perform assay in duplicate (N=2 per treatment)
Wells are equilibrated with blank buffer containing 1% BSA prior to initiating the assay
Buffer is aspirated and replaced with test compound dosing solution
Incubate cells for 5 min at 37°C
Terminate the incubation and lyse the cells
Analyze cell lysate using LC-MS/MS to determine furosemide concentration
Determine protein concentration in cell lysate and normalize furosemide concentration to protein content

Assay QC

Uptake rate of probe substrate in OAT3-transfected vs. vector control cells

Notes

The results from this assay are provided to the customer in the ExpressPlus report format, which may include graphical representations of data and comparison with historical data for reference compounds.
The solubility of the test compound in Hanks’ buffer containing < 0.8% DMSO must be greater than the test concentration. If the solubility of the test compound is unavailable, Absorption Systems can conduct a solubility assessment at an additional charge.
Percent inhibition is calculated as 100 x [1-(IR_OAT3-IR_VC)_TC / (IR_OAT3 – IR_VC)₀], where IR_OAT3 is the influx rate of furosemide in OAT3-transfected cells, IR_VCis the influx rate in vector control cells, “TC” means in the presence of test compound, and “0” means in the absence of test compound.

Options

The customer can request that a positive control be performed in parallel (additional fees apply)

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This assay is used to screen for inhibition of OCT1 by a test compound in OCT1-transfected HEK293 cells at a single concentration.

Required from Customer

Either a minimum of 300 µL of test compound at 10 mM in DMSO, or 5 mg of powder
Exact molecular mass of test compound and its salt form
Relevant solubility of the test compound
MSDS or handling and storage information, e.g., light sensitive, store at -20°C, etc.

Deliverables

Normalized uptake of MPP+ in the presence and absence of test compound in OCT1-transfected and vector control cells
Percent inhibition by the test compound
>50% inhibition indicates that the test compound is a significant inhibitor of OCT1

Substrate

OCT1 probe substrate 1-methyl-4-Phenylpyridinium (MPP+) at 5 μM
Test compound at 10 μM in modified Hanks’ buffer with less than 0.8% DMSO

Assay System

OCT1-transfected HEK293 cells and vector control cells cultured in 24-well plates

Assay Conditions

Measure uptake of MPP+ in OCT1-transfected and vector control cells with and without test compound
Single concentration of test compound
Two cell lines: OCT1-transfected and vector control cell lines
Four treatments as follows:
- OCT1-transfected cells with MPP+ +/-test compound
- Vector control cells with MPP+ +/- test compound
Perform assay in duplicate (N=2 per treatment)
Wells are equilibrated with blank buffer containing 1% BSA prior to initiating the assay
Buffer is aspirated and replaced with test compound dosing solution
Incubate cells for 2 min at 37°C
Terminate the incubation and lyse the cells
Analyze cell lysate using LC-MS/MS to determine MPP+ concentration
Determine protein concentration in cell lysate and normalize MPP+ concentration to protein content

Assay QC

Uptake rate of probe substrate in OCT1-transfected vs. vector control cells

Notes

The results from this assay are provided to the customer in the ExpressPlus report format, which may include graphical representations of data and comparison with historical data for reference compounds.
The solubility of the test compound in Hanks’ buffer containing < 0.8% DMSO must be greater than the test concentration. If the solubility of the test compound is unavailable, Absorption Systems can conduct a solubility assessment at an additional charge.
Percent inhibition is calculated as 100 x [1-(IR_OCT1-IR_VC)_TC / (IR_OCT1 – IR_VC)₀], where IR_OCT1 is the influx rate of MPP+ in OCT1-transfected cells, IR_VCis the influx rate in vector control cells, “TC” means in the presence of test compound, and “0” means in the absence of test compound.

Options

The customer can request that a positive control be performed in parallel (additional fees apply)

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This assay is used to screen for inhibition of OCT2 by a test compound in OCT2-transfected HEK293 cells at a single concentration.

Required from Customer

Either a minimum of 300 µL of test compound at 10 mM in DMSO, or 5 mg of powder
Exact molecular mass of test compound and its salt form
Relevant solubility of the test compound
MSDS or handling and storage information, e.g., light sensitive, store at -20°C, etc.

Deliverables

Normalized uptake of MPP+ in the presence and absence of test compound in OCT2-transfected and vector control cells
Percent inhibition by the test compound
>50% inhibition indicates that the test compound is a significant inhibitor of OCT2

Substrate

OCT2 probe substrate 1-methyl-4-Phenylpyridinium (MPP+) at 5 μM
Test compound at 10 μM in modified Hanks’ buffer with less than 0.8% DMSO

Assay System

OCT2-transfected HEK293 cells and vector control cells cultured in 24-well plates

Assay Conditions

Measure uptake of MPP+ in OCT2-transfected and vector control cells with and without test compound
Single concentration of test compound
Two cell lines: OCT2-transfected and vector control cell lines
Four treatments as follows:
- OCT2-transfected cells with MPP+ +/-test compound
- Vector control cells with MPP+ +/- test compound
Perform assay in duplicate (N=2 per treatment)
Wells are equilibrated with blank buffer containing 1% BSA prior to initiating the assay
Buffer is aspirated and replaced with test compound dosing solution
Incubate cells for 2 min at 37°C
Terminate the incubation and lyse the cells
Analyze cell lysate using LC-MS/MS to determine MPP+ concentration
Determine protein concentration in cell lysate and normalize MPP+ concentration to protein content

Assay QC

Uptake rate of probe substrate in OCT2-transfected vs. vector control cells

Notes

The results from this assay are provided to the customer in the ExpressPlus report format, which may include graphical representations of data and comparison with historical data for reference compounds.
The solubility of the test compound in Hanks’ buffer containing < 0.8% DMSO must be greater than the test concentration. If the solubility of the test compound is unavailable, Absorption Systems can conduct a solubility assessment at an additional charge.
Percent inhibition is calculated as 100 x [1-(IR_OCT1-IR_VC)_TC / (IR_OCT1 – IR_VC)₀], where IR_OCT1 is the influx rate of MPP+ in OCT1-transfected cells, IR_VCis the influx rate in vector control cells, “TC” means in the presence of test compound, and “0” means in the absence of test compound.

Options

The customer can request that a positive control be performed in parallel (additional fees apply)

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This assay is used to screen for inhibition of OATP1B1 by a test compound in OATP1B1-transfected HEK293 cells at a single concentration

Required from Customer

Either a minimum of 300 µL of test compound at 10 mM in DMSO, or 5 mg of powder
Exact molecular mass of test compound and its salt form
Relevant solubility of the test compound
MSDS or handling and storage information, e.g., light sensitive, store at -20°C, etc.

Deliverables

Normalized uptake of atorvastatin in the presence and absence of test compound in OATP1B1-transfected and vector control cells
Percent inhibition by the test compound
>50% inhibition indicates that the test compound is a significant inhibitor of OATP1B1

Substrate

OATP1B1 probe substrate Atorvastatin at 0.15 μM
Test compound at 10 μM in modified Hanks’ buffer with less than 0.8% DMSO

Assay System

OATP1B1-transfected HEK293 cells and vector control cells cultured in 24-well plates

Assay Conditions

Measure uptake of atorvastatin in OATP1B1-transfected and vector control cells with and without test compound
Single concentration of test compound
Two cell lines: OATP1B1-transfected and vector control cell lines
Four treatments as follows:
- OATP1B1-transfected cells with atorvastatin +/-test compound
- Vector control cells with atorvastatin +/- test compound
Perform assay in duplicate (N=2 per treatment)
Wells are equilibrated with blank buffer containing 1% BSA prior to initiating the assay
Buffer is aspirated and replaced with test compound dosing solution
Incubate cells for 5 min at 37°C
Terminate the incubation and lyse the cells
Analyze cell lysate using LC-MS/MS to determine atorvastatin concentration
Determine protein concentration in cell lysate and normalize atorvastatin concentration to protein content

Assay QC

Uptake rate of probe substrate in OATP1B1-transfected vs. vector control cells

Notes

The results from this assay are provided to the customer in the ExpressPlus report format, which may include graphical representations of data and comparison with historical data for reference compounds.
The solubility of the test compound in Hanks’ buffer containing < 0.8% DMSO must be greater than the test concentration. If the solubility of the test compound is unavailable, Absorption Systems can conduct a solubility assessment at an additional charge.
Percent inhibition is calculated as 100 x [1-(IR_1B1-IR_VC)_TC / (IR_1B1 – IR_VC)₀], where IR_1B1 is the influx rate of atorvastatin in OATP1B1-transfected cells, IR_VCis the influx rate in vector control cells, “TC” means in the presence of test compound, and “0” means in the absence of test compound.

Options

The customer can request that a positive control be performed in parallel (additional fees apply)

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This OATP1B3 transporter assay is used to screen for inhibition of OATP1B3 by a test compound in OATP1B3-transfected HEK293 cells at a single concentration.

Required from Customer

Either a minimum of 300 µL of test compound at 10 mM in DMSO, or 5 mg of powder
Exact molecular mass of test compound and its salt form
Relevant solubility of the test compound
MSDS or handling and storage information, e.g., light sensitive, store at -20°C, etc.

Deliverables

Normalized uptake of atorvastatin in the presence and absence of test compound in OATP1B3-transfected and vector control cells
Percent inhibition by the test compound
>50% inhibition indicates that the test compound is a significant inhibitor of OATP1B3

Substrate

OATP1B3 probe substrate Atorvastatin at 0.15 μM
Test compound at 10 μM in modified Hanks’ buffer with less than 0.8% DMSO

Assay System

OATP1B3-transfected HEK293 cells and vector control cells cultured in 24-well plates

Assay Conditions

Measure uptake of atorvastatin in OATP1B3-transfected and vector control cells with and without test compound
Single concentration of test compound
Two cell lines: OATP1B3-transfected and vector control cell lines
Four treatments as follows:
- OATP1B3-transfected cells with atorvastatin +/-test compound
- Vector control cells with atorvastatin +/- test compound
Perform assay in duplicate (N=2 per treatment)
Wells are equilibrated with blank buffer containing 1% BSA prior to initiating the assay
Buffer is aspirated and replaced with test compound dosing solution
Incubate cells for 5 min at 37°C
Terminate the incubation and lyse the cells
Analyze cell lysate using LC-MS/MS to determine atorvastatin concentration
Determine protein concentration in cell lysate and normalize atorvastatin concentration to protein content

Assay QC

Uptake rate of probe substrate in OATP1B3-transfected vs. vector control cells

Notes

The results from this assay are provided to the customer in the ExpressPlus report format, which may include graphical representations of data and comparison with historical data for reference compounds.
The solubility of the test compound in Hanks’ buffer containing < 0.8% DMSO must be greater than the test concentration. If the solubility of the test compound is unavailable, Absorption Systems can conduct a solubility assessment at an additional charge.
Percent inhibition is calculated as 100 x [1-(IR_1B3-IR_VC)_TC / (IR_1B3 – IR_VC)₀], where IR_1B3 is the influx rate of atorvastatin in OATP1B3-transfected cells, IR_VC is the influx rate in vector control cells, “TC” means in the presence of test compound, and “0” means in the absence of test compound.

Options

The customer can request that a positive control be performed in parallel (additional fees apply)

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This assay is used to determine the IC₅₀ of a test compound for inhibition of P-gp in MDR1-MDCK cells

Required from Customer

Either a minimum of 300 µL of test compound at 20 mM in DMSO, or 10 mg of powder
Exact molecular mass of test compound and its salt form
Solubility and chemical stability of the test compound in Hanks’ buffer
MSDS or handling and storage information, e.g., light sensitive, store at -20°C, etc.

Deliverables

Apparent permeability (P_app) of digoxin in the basolateral-to-apical direction, in the presence and absence of test compound
IC₅₀ of the test compound

Substrate

P-gp probe substrate digoxin at 10 µM
Test compound at six concentrations (serial diluations from 100 µM) in modified Hanks’ buffer with less than 0.8% DMSO

Assay System

Confluent monolayers of MDR1-MDCK cells, 7-14 days old, in Transwell^® dual-chamber plates, with apical and basolateral pH 7.4

Assay Conditions

Monolayers pretreated and co-dosed with test compound on both apical and basolateral sides
Six concentrations of test compound
- Single incubation (n=1) per concentration
Negative control: vehicle only
- Duplicate incubations (n=2)
Positive control: single concentration of known inhibitor Valspodar
- Duplicate incubations (n=2)
Dose digoxin on basolateral side only for B to A permeability
Sample both apical and basolateral sides at 120 minutes
Determine the concentration of digoxin using a generic LC-MS/MS method with a minimum 4-point calibration curve

Assay QC

Verify the quality of the cell monolayer batch using control compounds before the monolayers are released for use
Verify the quality of each monolayer used in an assay by a pre-experiment TEER measurement and by calculating the P_app for the co-dosed control compound, Lucifer yellow

Notes

The results from this assay are provided to the customer in the ExpressPlus report format, which may include graphical representations of data and comparison with historical data for reference compounds.
The solubility of the test compound in Hanks’ buffer containing < 0.8% DMSO must be greater than the test concentration. If the solubility of the test compound is unavailable, Absorption Systems can conduct a solubility assessment at an additional charge.

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This P-gp transporter assay is used to determine the IC₅₀ of a test compound for inhibition of P-gp in Caco-2 cells.

Required from Customer

Either a minimum of 300 µL of test compound at 20 mM in DMSO, or 10 mg of powder
Exact molecular mass of test compound and its salt form
Solubility and chemical stability of the test compound in Hanks’ buffer
MSDS or handling and storage information, e.g., light sensitive, store at -20°C, etc.

Deliverables

Apparent permeability (P_app) of digoxin in the basolateral-to-apical direction, in the presence and absence of test compound
IC₅₀ of the test compound

Substrate

P-gp probe substrate digoxin at 10 µM
Test compound at six concentrations (serial diluations from 100 µM) in modified Hanks’ buffer with less than 0.8% DMSO

Assay System

Confluent monolayers of Caco-2 cells, 21-28 days old, in Transwell^® dual-chamber plates, with apical and basolateral pH 7.4

Assay Conditions

Monolayers pretreated and co-dosed with test compound on both apical and basolateral sides
Six concentrations of test compound
- Single incubation (n=1) per concentration
Negative control: vehicle only
- Duplicate incubations (n=2)
Positive control: single concentration of known inhibitor Valspodar
- Duplicate incubations (n=2)
Dose digoxin on basolateral side only for B to A permeability
Sample both apical and basolateral sides at 120 minutes
Determine the concentration of digoxin using a generic LC-MS/MS method with a minimum 4-point calibration curve

Assay QC

Verify the quality of the cell monolayer batch using control compounds before the monolayers are released for use
Verify the quality of each monolayer used in an assay by a pre-experiment TEER measurement and by calculating the P_app for the co-dosed control compound, Lucifer yellow

Notes

The results from this assay are provided to the customer in the ExpressPlus report format, which may include graphical representations of data and comparison with historical data for reference compounds.
The solubility of the test compound in Hanks’ buffer containing < 0.8% DMSO must be greater than the test concentration. If the solubility of the test compound is unavailable, Absorption Systems can conduct a solubility assessment at an additional charge.

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This assay is used to determine the IC₅₀ of a test compound for inhibition of BCRP in BCRP-MDCK cells.

Required from Customer

Either a minimum of 300 µL of test compound at 20 mM in DMSO, or 10 mg of powder
Exact molecular mass of test compound and its salt form
Solubility and chemical stability of the test compound in Hanks’ buffer
MSDS or handling and storage information, e.g., light sensitive, store at -20°C, etc.

Deliverables

Apparent permeability (P_app) of cladribine in the basolateral-to-apical direction, in the presence and absence of test compound
IC₅₀ of the test compound

Substrate

BCRP probe substrate cladribine at 10 µM
Test compound at six concentrations (serial dilutions from 100µM) in modified Hanks’ buffer with less than 0.8% DMSO

Assay System

Confluent monolayers of BCRP-MDCK cells, 7-14 days old, in Transwell^® dual-chamber plates, with apical and basolateral pH 7.4

Assay Conditions

Monolayers pretreated and co-dosed with test compound on both apical and basolateral sides
Six concentrations of test compound
- Single incubation (n=1) per concentration
Negative control: vehicle only
- Duplicate incubations (n=2)
Positive control: single concentration of known inhibitor Ko143
- Duplicate incubations (n=2)
Dose cladribine on basolateral side only for B to A permeability
Sample both apical and basolateral sides at 120 minutes
Determine the concentration of cladribine using a generic LC-MS/MS method with a minimum 4-point calibration curve

Assay QC

Verify the quality of the cell monolayer batch using control compounds before the monolayers are released for use
Verify the quality of each monolayer used in an assay by a pre-experiment TEER measurement and by calculating the P_app for the co-dosed control compound, Lucifer yellow

Notes

The results from this assay are provided to the customer in the ExpressPlus report format, which may include graphical representations of data and comparison with historical data for reference compounds.
The solubility of the test compound in Hanks’ buffer containing < 0.8% DMSO must be greater than the test concentration. If the solubility of the test compound is unavailable, Absorption Systems can conduct a solubility assessment at an additional charge.

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This assay is used to determine the IC₅₀ of a test compound for inhibition of OAT1 in OAT1-transfected HEK293 cells

Required from Customer

Either a minimum of 300 µL of test compound at 20 mM in DMSO, or 10 mg of powder
Exact molecular mass of test compound and its salt form
Relevant solubility of the test compound
MSDS or handling and storage information, e.g., light sensitive, store at -20°C, etc.

Deliverables

Normalized uptake of para-aminohippurate (PAH) in the presence and absence of test compound in OAT1-transfected and vector control cells
IC₅₀ of the test compound

Substrate

OAT1 probe substrate PAH at 10 μM
Test compound at six concentrations (serial diluations from 100 µM) in modified Hanks’ buffer with less than 0.8% DMSO

Assay System

OAT1-transfected HEK293 cells and vector control cells cultured in 24-well plates

Assay Conditions

Measure uptake of PAH in OAT1-transfected and vector control cells with and without test compound
Six concentrations of test compound
- Single incubation (n=1) per concentration
Negative control: vehicle only
- Duplicate incubations (n=2)
Positive control: single concentration of known inhibitor probenecid
- Duplicate incubations (n=2)
Two cell lines: OAT1-transfected and vector control cell lines
Treatments performed in each cell line:
- Wells are equilibrated with blank buffer containing 1% BSA prior to initiating the assay
- Buffer is aspirated and replaced with dosing solution
- Incubate cells for 5 min at 37°C
- Terminate the incubation and lyse the cells
- Analyze cell lysate using LC-MS/MS to determine PAH concentration
- Determine protein concentration in cell lysate and normalize PAH concentration to protein content

Assay QC

Uptake rate of probe substrate in OAT1-transfected vs. vector control cells
Inhibition of probe substrate uptake by a known inhibitor of OAT1

Notes

The results from this assay are provided to the customer in the ExpressPlus report format, which may include graphical representations of data and comparison with historical data for reference compounds.
The solubility of the test compound in Hanks’ buffer containing < 0.8% DMSO must be greater than the test concentration. If the solubility of the test compound is unavailable, Absorption Systems can conduct a solubility assessment at an additional charge.

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This assay is used to determine the IC₅₀ of a test compound for inhibition of OAT3 in OAT3-transfected HEK293 cells.

Required from Customer

Either a minimum of 300 µL of test compound at 20 mM in DMSO, or 10 mg of powder
Exact molecular mass of test compound and its salt form
Relevant solubility of the test compound
MSDS or handling and storage information, e.g., light sensitive, store at -20°C, etc.

Deliverables

Normalized uptake of furosemide in the presence and absence of test compound in OAT3-transfected and vector control cells
IC₅₀ of the test compound

Substrate

OAT3 probe substrate PAH at 10 μM
Test compound at six concentrations (serial diluations from 100 µM) in modified Hanks’ buffer with less than 0.8% DMSO

Assay System

OAT3-transfected HEK293 cells and vector control cells cultured in 24-well plates

Assay Conditions

Measure uptake of PAH in OAT3-transfected and vector control cells with and without test compound
Six concentrations of test compound
- Single incubation (n=1) per concentration
Negative control: vehicle only
- Duplicate incubations (n=2)
Positive control: single concentration of known inhibitor probenecid
- Duplicate incubations (n=2)
Two cell lines: OAT3-transfected and vector control cell lines
Treatments performed in each cell line:
- Wells are equilibrated with blank buffer containing 1% BSA prior to initiating the assay
- Buffer is aspirated and replaced with dosing solution
- Incubate cells for 5 min at 37°C
- Terminate the incubation and lyse the cells
- Analyze cell lysate using LC-MS/MS to determine PAH concentration
- Determine protein concentration in cell lysate and normalize PAH concentration to protein content

Assay QC

Uptake rate of probe substrate in OAT3-transfected vs. vector control cells
Inhibition of probe substrate uptake by a known inhibitor of OAT3

Notes

The results from this assay are provided to the customer in the ExpressPlus report format, which may include graphical representations of data and comparison with historical data for reference compounds.
The solubility of the test compound in Hanks’ buffer containing < 0.8% DMSO must be greater than the test concentration. If the solubility of the test compound is unavailable, Absorption Systems can conduct a solubility assessment at an additional charge.

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This assay is used to determine the IC₅₀ of a test compound for inhibition of OCT1 in OCT1-transfected HEK293 cells.

Required from Customer

Either a minimum of 300 µL of test compound at 20 mM in DMSO, or 10 mg of powder
Exact molecular mass of test compound and its salt form
Relevant solubility of the test compound
MSDS or handling and storage information, e.g., light sensitive, store at -20°C, etc.

Deliverables

Normalized uptake of MPP+ in the presence and absence of test compound in OCT2-transfected and vector control cells
IC₅₀ of the test compound

Substrate

OCT2 probe substrate 1-methyl-4-Phenylpyridinium (MPP+) at 5 μM
Test compound at six concentrations (serial diluations from 100 µM) in modified Hanks’ buffer with less than 0.8% DMSO

Assay System

OCT2-transfected HEK293 cells and vector control cells cultured in 24-well plates

Assay Conditions

Measure uptake of MPP+ in OCT1-transfected and vector control cells with and without test compound
Six concentrations of test compound
- Single incubation (n=1) per concentration
Negative control: vehicle only
- Duplicate incubations (n=2)
Positive control: single concentration of known inhibitor repaglinide
- Duplicate incubations (n=2)
Two cell lines: OCT1-transfected and vector control cell lines
Treatments performed in each cell line:
- Wells are equilibrated with blank buffer containing 1% BSA prior to initiating the assay
- Buffer is aspirated and replaced with dosing solution
- Incubate cells for 2 min at 37°C
- Terminate the incubation and lyse the cells
- Analyze cell lysate using LC-MS/MS to determine MPP+ concentration
- Determine protein concentration in cell lysate and normalize MPP+ concentration to protein content

Assay QC

Uptake rate of probe substrate in OCT2-transfected vs. vector control cells
Inhibition of probe substrate uptake by a known inhibitor of OCT2

Notes

The results from this assay are provided to the customer in the ExpressPlus report format, which may include graphical representations of data and comparison with historical data for reference compounds.
The solubility of the test compound in Hanks’ buffer containing < 0.8% DMSO must be greater than the test concentration. If the solubility of the test compound is unavailable, Absorption Systems can conduct a solubility assessment at an additional charge.

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This OCT transporter assay is used to determine the IC₅₀ of a test compound for inhibition of OCT2 in OCT2-transfected HEK293 cells.

Required from Customer

Either a minimum of 300 µL of test compound at 20 mM in DMSO, or 10 mg of powder
Exact molecular mass of test compound and its salt form
Relevant solubility of the test compound
MSDS or handling and storage information, e.g., light sensitive, store at -20°C, etc.

Deliverables

Normalized uptake of MPP+ in the presence and absence of test compound in OCT2-transfected and vector control cells
IC₅₀ of the test compound

Substrate

OCT2 probe substrate 1-methyl-4-Phenylpyridinium (MPP+) at 5 μM
Test compound at six concentrations (serial diluations from 100 µM) in modified Hanks’ buffer with less than 0.8% DMSO

Assay System

OCT2-transfected HEK293 cells and vector control cells cultured in 24-well plates

Assay Conditions

Measure uptake of MPP+ in OCT1-transfected and vector control cells with and without test compound
Six concentrations of test compound
- Single incubation (n=1) per concentration
Negative control: vehicle only
- Duplicate incubations (n=2)
Positive control: single concentration of known inhibitor repaglinide
- Duplicate incubations (n=2)
Two cell lines: OCT1-transfected and vector control cell lines
Treatments performed in each cell line:
- Wells are equilibrated with blank buffer containing 1% BSA prior to initiating the assay
- Buffer is aspirated and replaced with dosing solution
- Incubate cells for 2 min at 37°C
- Terminate the incubation and lyse the cells
- Analyze cell lysate using LC-MS/MS to determine MPP+ concentration
- Determine protein concentration in cell lysate and normalize MPP+ concentration to protein content

Assay QC

Uptake rate of probe substrate in OCT2-transfected vs. vector control cells
Inhibition of probe substrate uptake by a known inhibitor of OCT2

Notes

The results from this assay are provided to the customer in the ExpressPlus report format, which may include graphical representations of data and comparison with historical data for reference compounds.
The solubility of the test compound in Hanks’ buffer containing < 0.8% DMSO must be greater than the test concentration. If the solubility of the test compound is unavailable, Absorption Systems can conduct a solubility assessment at an additional charge.

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This assay is used to determine the IC₅₀ of a test compound for inhibition of OATP1B1 in OATP1B1-transfected HEK293 cells.

Required from Customer

Either a minimum of 300 µL of test compound at 20 mM in DMSO, or 10 mg of powder
Exact molecular mass of test compound and its salt form
Relevant solubility of the test compound
MSDS or handling and storage information, e.g., light sensitive, store at -20°C, etc.

Deliverables

Normalized uptake of atorvastatin in the presence and absence of test compound in OATP1B1-transfected and vector control cells
IC₅₀ of the test compound

Substrate

OATP1B1 probe substrate atorvastatin at 0.15 μM
Test compound at six concentrations (serial diluations from 100 µM) in modified Hanks’ buffer with less than 0.8% DMSO

Assay System

OATP1B1-transfected HEK293 cells and vector control cells cultured in 24-well plates

Assay Conditions

Measure uptake of atorvastatin in OATP1B1-transfected and vector control cells with and without test compound
Six concentrations of test compound
- Single incubation (n=1) per concentration
Negative control: vehicle only
- Duplicate incubations (n=2)
Positive control: single concentration of known inhibitor rifamycin SV
- Duplicate incubations (n=2)
Two cell lines: OATP1B1-transfected and vector control cell lines
Treatments performed in each cell line:
- Wells are equilibrated with blank buffer containing 1% BSA prior to initiating the assay
- Buffer is aspirated and replaced with dosing solution
- Incubate cells for 5 min at 37°C
- Terminate the incubation and lyse the cells
- Analyze cell lysate using LC-MS/MS to determine atorvastatin concentration
- Determine protein concentration in cell lysate and normalize atorvastatin concentration to protein content

Assay QC

Uptake rate of probe substrate in OATP1B1-transfected vs. vector control cells
Inhibition of probe substrate uptake by a known inhibitor of OATP1B1

Notes

The results from this assay are provided to the customer in the ExpressPlus report format, which may include graphical representations of data and comparison with historical data for reference compounds.
The solubility of the test compound in Hanks’ buffer containing < 0.8% DMSO must be greater than the test concentration. If the solubility of the test compound is unavailable, Absorption Systems can conduct a solubility assessment at an additional charge.

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This assay is used to determine the IC₅₀ of a test compound for inhibition of OATP1B3 in OATP1B3-transfected HEK293 cells.

Required from Customer

Either a minimum of 300 µL of test compound at 20 mM in DMSO, or 10 mg of powder
Exact molecular mass of test compound and its salt form
Relevant solubility of the test compound
MSDS or handling and storage information, e.g., light sensitive, store at -20°C, etc.

Deliverables

Normalized uptake of atorvastatin in the presence and absence of test compound in OATP1B3-transfected and vector control cells
IC₅₀ of the test compound

Substrate

OATP1B3 probe substrate atorvastatin at 0.15 μM
Test compound at six concentrations (serial diluations from 100 µM) in modified Hanks’ buffer with less than 0.8% DMSO

Assay System

OATP1B3-transfected HEK293 cells and vector control cells cultured in 24-well plates

Assay Conditions

Measure uptake of atorvastatin in OATP1B3-transfected and vector control cells with and without test compound
Six concentrations of test compound
- Single incubation (n=1) per concentration
Negative control: vehicle only
- Duplicate incubations (n=2)
Positive control: single concentration of known inhibitor rifamycin SV
- Duplicate incubations (n=2)
Two cell lines: OATP1B3-transfected and vector control cell lines
Treatments performed in each cell line:
- Wells are equilibrated with blank buffer containing 1% BSA prior to initiating the assay
- Buffer is aspirated and replaced with dosing solution
- Incubate cells for 5 min at 37°C
- Terminate the incubation and lyse the cells
- Analyze cell lysate using LC-MS/MS to determine atorvastatin concentration
- Determine protein concentration in cell lysate and normalize atorvastatin concentration to protein content

Assay QC

Uptake rate of probe substrate in OATP1B3-transfected vs. vector control cells
Inhibition of probe substrate uptake by a known inhibitor of OATP1B3

Notes

The results from this assay are provided to the customer in the ExpressPlus report format, which may include graphical representations of data and comparison with historical data for reference compounds.
The solubility of the test compound in Hanks’ buffer containing < 0.8% DMSO must be greater than the test concentration. If the solubility of the test compound is unavailable, Absorption Systems can conduct a solubility assessment at an additional charge.

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This BSEP transporter assay is used to screen for inhibition of BSEP by a test compound in hBSEP-expressing vesicles at a single concentration

Required from Customer

Either a minimum of 300 µL of test compound at 10 mM in DMSO, or 5 mg of powder
Exact molecular mass of test compound and its salt form
Relevant solubility of the test compound
MSDS or handling and storage information, e.g., light sensitive, store at -20°C, etc.

Deliverables

Normalized uptake of ³H-taurocholic acid in the presence and absence of test compound in hBSEP-expressing vesicles
Percent inhibition by the test compound
>50% inhibition indicates that the test compound is a significant inhibitor of BSEP

Substrate

BSEP probe substrate ³H-taurocholic acid at 1 μM
Test compound at 10 μM in Hepes Influx Buffer

Assay System

hBSEP-expressing vesicles in the presence of ATP vs. AMP

Assay Conditions

Measure uptake of ³H-taurocholic acid in hBSEP-expressing vesicles with and without test compound, in the presence of ATP vs. AMP
Single concentration of test compound
Four treatments as follows:
- hBSEP-expressing vesicles cells with ³H-taurocholic acid +/-test compound, in the presence of ATP and AMP (separately)
Perform assay in duplicate (N=2 per treatment)
Incubate cells for 30 min at 37°C
Terminate the incubation
Measure radioactivity of processed samples to determine 3H-taurocholic acid concentration

Assay QC

Uptake rate of probe substrate in hBSEP-expressing vesicles in the presence of ATP vs. AMP

Notes

The results from this assay are provided to the customer in the ExpressPlus report format, which may include graphical representations of data and comparison with historical data for reference compounds.
The solubility of the test compound in Hepes Influx Buffer must be greater than the test concentration. If the solubility of the test compound is unavailable, Absorption Systems can conduct a solubility assessment at an additional charge.
Percent inhibition is calculated as 100 x [1-(IR_ATP-IR_AMP)_TC / (IR_ATP – IR_AMP)₀], where IR_ATP is the influx rate of ³H-taurocholic acid in hBSEP-expressing vesicles in the presence of ATP, IR_AMP is the influx rate of ³H-taurocholic acid in hBSEP-expressing vesicles in the presence of AMP, “TC” means in the presence of test compound, and “0” means in the absence of test compound.

Options

The customer can request: that a positive control be performed in parallel (additional fees apply)

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This assay is used to determine the IC₅₀ of a test compound for inhibition of BSEP in hBSEP-expressing vesicles.

Required from Customer

Either a minimum of 300 µL of test compound at 10 mM in DMSO, or 10 mg of powder
Exact molecular mass of test compound and its salt form
Solubility of test compound in Hanks’ buffer
MSDS or handling and storage information, e.g., light sensitive, store at -20°C, etc.

Deliverables

Normalized uptake of ³H-taurocholic acid in the presence and absence of test compound in hBSEP-expressing vesicles
IC₅₀ of the test compound

Substrate

BSEP probe substrate ³H-taurocholic acid at 1 μM
Test compound at six concentrations (serial diluations from 100 µM) in Hepes Influx Buffer

Assay System

hBSEP-expressing vesicles in the presence of ATP vs. AMP

Assay Conditions

Measure uptake of ³H-taurocholic acid in hBSEP-expressing vesicles with and without test compound, in the presence of ATP vs. AMP
Six concentrations of test compound
- Single incubation (n=1) per concentration
Negative control: vehicle only
- Duplicate incubations (n=2)
Positive control: single concentration of known inhibitor CsA
- Duplicate incubations (n=2)
Treatments performed in in the presence of ATP and AMP (separately):
- Incubate cells for 30 min at 37°C
- Terminate the incubation
- Measure radioactivity of processed samples to determine ³H-taurocholic acid concentration

Assay QC

Uptake rate of probe substrate in hBSEP-expressing vesicles in the presence of ATP vs. AMP
Inhibition of probe substrate uptake by a known inhibitor of BSEP

Notes

The results from this assay are provided to the customer in the ExpressPlus report format, which may include graphical representations of data and comparison with historical data for reference compounds.
The solubility of the test compound in Hepes Influx Buffer must be greater than the test concentration. If the solubility of the test compound is unavailable, Absorption Systems can conduct a solubility assessment at an additional charge.

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This assay is used to screen for inhibition of MATE1 by a test compound in MATE1-transfected HEK293 cells at a single concentration.

Required from Customer

Either a minimum of 300 µL of test compound at 10 mM in DMSO, or 5 mg of powder
Exact molecular mass of test compound and its salt form
Relevant solubility of the test compound
MSDS or handling and storage information, e.g., light sensitive, store at -20°C, etc.

Deliverables

Normalized uptake of Metformin in the presence and absence of test compound in MATE1-transfected and vector control cells
Percent inhibition by the test compound
>50% inhibition indicates that the test compound is a significant inhibitor of MATE1

Substrate

MATE1 probe substrate Metformin at 50 μM
Test compound at 10 μM in modified Hanks’ buffer with less than 0.8% DMSO

Assay System

MATE1-transfected HEK293 cells and vector control cells cultured in 24-well plates

Assay Conditions

Measure uptake of Metformin in MATE1-transfected and vector control cells with and without test compound
Single concentration of test compound
Two cell lines: MATE1-transfected and vector control cell lines
Four treatments as follows:
- MATE1-transfected cells with Metformin +/-test compound
- Vector control cells with Metformin +/- test compound
Perform assay in duplicate (N=2 per treatment)
Wells are equilibrated with blank buffer containing 1% BSA prior to initiating the assay
Buffer is aspirated and replaced with test compound dosing solution
Incubate cells for 5 min at 37°C
Terminate the incubation and lyse the cells
Analyze cell lysate using LC-MS/MS to determine Metformin concentration
Determine protein concentration in cell lysate and normalize Metformin concentration to protein content

Assay QC

Uptake rate of probe substrate in MATE1-transfected vs. vector control cells

Notes

The results from this assay are provided to the customer in the ExpressPlus report format, which may include graphical representations of data and comparison with historical data for reference compounds.
The solubility of the test compound in Hanks’ buffer containing < 0.8% DMSO must be greater than the test concentration. If the solubility of the test compound is unavailable, Absorption Systems can conduct a solubility assessment at an additional charge.
Percent inhibition is calculated as 100 x [1-(IR_1B1-IR_VC)_TC / (IR_1B1 – IR_VC)₀], where IR_1B1 is the influx rate of Metformin in MATE1-transfected cells, IR_VCis the influx rate in vector control cells, “TC” means in the presence of test compound, and “0” means in the absence of test compound.

Options

The customer can request: that a positive control be performed in parallel (additional fees apply)

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This assay is used to screen for inhibition of MATE2K by a test compound in MATE2K-transfected HEK293 cells at a single concentration

Required from Customer

Either a minimum of 300 µL of test compound at 10 mM in DMSO, or 5 mg of powder
Exact molecular mass of test compound and its salt form
Relevant solubility of the test compound
MSDS or handling and storage information, e.g., light sensitive, store at -20°C, etc.

Deliverables

Normalized uptake of Metformin in the presence and absence of test compound in MATE2K-transfected and vector control cells
Percent inhibition by the test compound
>50% inhibition indicates that the test compound is a significant inhibitor of MATE2K

Substrate

MATE2K probe substrate Metformin at 50 μM
Test compound at 10 μM in modified Hanks’ buffer with less than 0.8% DMSO

Assay System

MATE2K-transfected HEK293 cells and vector control cells cultured in 24-well plates

Assay Conditions

Measure uptake of Metformin in MATE2K-transfected and vector control cells with and without test compound
Single concentration of test compound
Two cell lines: MATE2K-transfected and vector control cell lines
Four treatments as follows:
- MATE2K-transfected cells with Metformin +/-test compound
- Vector control cells with Metformin +/- test compound
Perform assay in duplicate (N=2 per treatment)
Wells are equilibrated with blank buffer containing 1% BSA prior to initiating the assay
Buffer is aspirated and replaced with test compound dosing solution
Incubate cells for 5 min at 37°C
Terminate the incubation and lyse the cells
Analyze cell lysate using LC-MS/MS to determine Metformin concentration
Determine protein concentration in cell lysate and normalize Metformin concentration to protein content

Assay QC

Uptake rate of probe substrate in MATE2K-transfected vs. vector control cells

Notes

The results from this assay are provided to the customer in the ExpressPlus report format, which may include graphical representations of data and comparison with historical data for reference compounds.
The solubility of the test compound in Hanks’ buffer containing < 0.8% DMSO must be greater than the test concentration. If the solubility of the test compound is unavailable, Absorption Systems can conduct a solubility assessment at an additional charge.
Percent inhibition is calculated as 100 x [1-(IR_1B1-IR_VC)_TC / (IR_1B1 – IR_VC)₀], where IR_1B1 is the influx rate of Metformin in MATE2K-transfected cells, IR_VCis the influx rate in vector control cells, “TC” means in the presence of test compound, and “0” means in the absence of test compound.

Options

The customer can request: that a positive control be performed in parallel (additional fees apply)

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This assay is used to determine the IC₅₀ of a test compound for inhibition of MATE1 in MATE1-transfected HEK293 cells.

Required from Customer

Either a minimum of 300 µL of test compound at 20 mM in DMSO, or 10 mg of powder
Exact molecular mass of test compound and its salt form
Relevant solubility of the test compound
MSDS or handling and storage information, e.g., light sensitive, store at -20°C, etc.

Deliverables

Normalized uptake of Metformin in the presence and absence of test compound in MATE1-transfected and vector control cells
IC₅₀ of the test compound

Substrate

MATE1 probe substrate Metformin at 50 μM
Test compound at six concentrations (serial diluations from 100 µM) in modified Hanks’ buffer with less than 0.8% DMSO

Assay System

MATE1-transfected HEK293 cells and vector control cells cultured in 24-well plates

Assay Conditions

Measure uptake of Metformin in MATE1-transfected and vector control cells with and without test compound
Six concentrations of test compound
- Single incubation (n=1) per concentration
Negative control: vehicle only
- Duplicate incubations (n=2)
Positive control: single concentration of known inhibitor Cimetidine
- Duplicate incubations (n=2)
Two cell lines: MATE1-transfected and vector control cell lines
Treatments performed in each cell line:
- Wells are equilibrated with blank buffer containing 1% BSA prior to initiating the assay
- Buffer is aspirated and replaced with dosing solution
- Incubate cells for 5 min at 37°C
- Terminate the incubation and lyse the cells
- Analyze cell lysate using LC-MS/MS to determine Metformin concentration
- Determine protein concentration in cell lysate and normalize Metformin concentration to protein content

Assay QC

Uptake rate of probe substrate in MATE1-transfected vs. vector control cells
Inhibition of probe substrate uptake by a known inhibitor of MATE1

Notes

The results from this assay are provided to the customer in the ExpressPlus report format, which may include graphical representations of data and comparison with historical data for reference compounds.
The solubility of the test compound in Hanks’ buffer containing < 0.8% DMSO must be greater than the test concentration. If the solubility of the test compound is unavailable, Absorption Systems can conduct a solubility assessment at an additional charge.

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This assay is used to determine the IC₅₀ of a test compound for inhibition of MATE2K in MATE2K-transfected HEK293 cells.

Required from Customer

Either a minimum of 300 µL of test compound at 20 mM in DMSO, or 10 mg of powder
Exact molecular mass of test compound and its salt form
Relevant solubility of the test compound
MSDS or handling and storage information, e.g., light sensitive, store at -20°C, etc.

Deliverables

Normalized uptake of Metformin in the presence and absence of test compound in MATE2K-transfected and vector control cells
IC₅₀ of the test compound

Substrate

MATE2K probe substrate Metformin at 50 μM
Test compound at six concentrations (serial diluations from 100 µM) in modified Hanks’ buffer with less than 0.8% DMSO

Assay System

MATE2K-transfected HEK293 cells and vector control cells cultured in 24-well plates

Assay Conditions

Measure uptake of Metformin in MATE2K-transfected and vector control cells with and without test compound
Six concentrations of test compound
- Single incubation (n=1) per concentration
Negative control: vehicle only
- Duplicate incubations (n=2)
Positive control: single concentration of known inhibitor Pyrimethamine
- Duplicate incubations (n=2)
Two cell lines: MATE2K-transfected and vector control cell lines
Treatments performed in each cell line:
- Wells are equilibrated with blank buffer containing 1% BSA prior to initiating the assay
- Buffer is aspirated and replaced with dosing solution
- Incubate cells for 5 min at 37°C
- Terminate the incubation and lyse the cells
- Analyze cell lysate using LC-MS/MS to determine Metformin concentration
- Determine protein concentration in cell lysate and normalize Metformin concentration to protein content

Assay QC

Uptake rate of probe substrate in MATE2K-transfected vs. vector control cells
Inhibition of probe substrate uptake by a known inhibitor of MATE2K

Notes

The results from this assay are provided to the customer in the ExpressPlus report format, which may include graphical representations of data and comparison with historical data for reference compounds.
The solubility of the test compound in Hanks’ buffer containing < 0.8% DMSO must be greater than the test concentration. If the solubility of the test compound is unavailable, Absorption Systems can conduct a solubility assessment at an additional charge.

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Objective

To determine the IC₅₀ of the Customer’s test article towards BCRP in Caco-2 cell monolayers

Required from Customer

A minimum of 100 µL of test article at 100 mM in DMSO stock solution or 5 mg powder
Molecular weight of the test article and its salt form
MSDS or handling and storage information

Deliverables

Express Plus containing:

Materials and methods
Results:
- The apparent permeability (P_app) of cladribine in the presence and absence of test article and/or known inhibitor
- The IC₅₀ of the test article towards BCRP
Appendix including results of cell batch certification

Substrate

Cladribine and test article in HBSSg with maximum DMSO concentration ≤ 0.8%

Test System

Twenty-one (21) to twenty-eight (28) days-in-culture Caco-2 cells plated in Transwell^™ dual chamber plates
Each batch of Caco-2 cell monolayers will be certified using internally established criteria

Assay Conditions

Unidirectional permeability assessment of cladribine in cultured Caco-2 cells with and without test article
Transport buffer: HBSSg, pH 7.4 ± 0.2
Treatments:

Treatment	Permeability	Replicates	Test article Concentration	Cladribine Concentration	Donor and Receiver Sampling Time Point
With Test article	B➞A	2	3-fold serial dilutions from 100 µM (6 concentrations)	10 µM	120 min
Without Test article		2	N/A
With Ko143 (0.5 µM)		2	N/A

NOTE: Cells treated with inhibitors will be pre-incubated with inhibitor for 30 minutes prior to assay onset. Cells used for testing with no inhibitors will be pre-incubated with blank Hanks buffer for 30 minutes prior to assay onset.

LC/MS/MS analysis of cladribine with a minimum 6-point standard curve
At least 60% standards must be accurate to within ±20%, except at the Lower Limit of Quantitation (LLOQ), where ±25% will be considered acceptable
Monolayer integrity confirmed via post-experiment Lucifer Yellow permeability assessment

Assay QC

The quality of the monolayer batch is verified using control compounds before the monolayers are released for use
The quality of each monolayer used in the assay is verified by calculating the P_app for the control compound lucifer yellow post-experiment

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Objective

To determine the bidirectional permeability of cladribine across Caco-2 cell monolayers in the absence and presence of test article, in order to assess if the Sponsor’s test article is a possible BCRP inhibitor

Required from Customer

A minimum of 100 µL of test article at 10 mM in DMSO stock solution or 1 mg powder
Molecular weight of the test article and its salt form
MSDS or handling and storage information

Deliverables

Express Plus containing:

Materials and methods
Results:
- The apparent permeability (P_app) of cladribine in both directions
  P_app = (dC_r/dt) x V_r/(A x C_A)
  
  Where,
  
  dC_r/dt is the slope of the cumulative concentration in the receiver compartment versus time in µM s^-1;
  
  V_ris the volume of the receiver compartment in cm³;
  
  A is the area of the insert (1.13 cm²for 12-well Transwell^®);
  
  C_Ais the average of the nominal dosing concentration and the measured 120 minute donor concentration in µM
- The efflux ratio (ER)
  
  ER =
- The BCRP inhibitor potential of a test article classified as either positive or negative. If the corrected efflux ratio of cladribine (C_ERC) is reduced by more than 50% in the presence of the test article (C_ERC/TC), then the test article is classified as a BCRP inhibitor.
  
  C_ERC = ER-1

Appendix including:
- Analytical method
- Results of batch certification

Substrate

Cladribine and test article each at 10 µM in HBSSg with DMSO concentration ≤ 0.8%

Test System

Twenty-one (21) to twenty-eight (28) days-in-culture Caco-2 cells plated in Transwell^™ dual chamber plates
Each batch of Caco-2 cell monolayers will be certified using internally established criteria

Assay Conditions

Bidirectional permeability assessment of cladribine in cultured Caco-2 cells with and without test article
Transport buffer: HBSSg, pH 7.4 ± 0.2
Test article treatments:

Treatment	Permeability	Replicates	Test article Concentration	Cladribine Concentration	Donor and Receiver Sampling Time Point
With Test article	Bidirectional	2	10 µM	10 µM	120 min
Without Test article	Bidirectional	2	N/A	10 µM	120 min

LC/MS/MS analysis of cladribine in donor and receiver samples only with a minimum 6-point standard curve
- At least 60% standards must be accurate to within ±20%, except at the Lower Limit of Quantitation (LLOQ), where ±25% will be considered acceptable
Monolayer integrity confirmed via post-experiment Lucifer Yellow permeability assessment

Assay QC

The quality of the monolayer batch is verified using control test articles before the monolayers are released for use
The quality of each monolayer used in the assay is verified by calculating the P_app for the control test article lucifer yellow post-experiment

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Objectives

To determine if the Customer’s test article is a potential inhibitor of the multi drug resistance protein 2 (MRP2) transporter using MRP2-expressing membrane vesicles

Required from Customer

Test article either in powder form (1 mg) or stock solution (100 µL of 10 mM DMSO)
Molecular weight of the test article and its salt form
Relevant solubility of test article
The MSDS and handling and storage information, e.g., light sensitive, store at -20ºC, etc.

Deliverables

Express Plus report including:

Materials and methods
Results: Net influx rate of probe substrate in the presence and absence of test article

Assay System

MRP2-expressing membrane vesicles
MRP2 probe substrate: Estradiol 17β-glucuronide
96-well format at 37°C

Assay Conditions

Influx Assessment of Probe Substrate in the Presence of Test article (Single Concentration)

Assay is performed using MRP2-expressing vesicles
Assay buffer: Hepes Influx Buffer (10mM Hepes-Tris, pH 7.4)
Treatment groups:

Treatment	Replicates	Probe Substrate	Concentration of Inhibitor	Incubation Duration
With test article and ATP	2	Estradiol 17β-glucuronide (50 µM)	10 µM	30 min
With test article and AMP	2		10 µM	30 min
Negative Control (solvent only) with ATP	2		N/A	30 min
Negative Control (solvent only) with AMP	2		N/A	30 min

Samples are processed and transferred to a 96-well plate
Radioactivity is measured using a scintillation counter
Determination of percent inhibition using influx rate

Net Influx Rate (NIR) = Uptake activity with ATP – Uptake activity with AMP

Percent Inhibition =

Where,
TC: Test article

Optional Positive Control Performed in Parallel, additional fees apply

Influx Assessment of Probe Substrate in the Presence of a known inhibitor (Single Concentration)
Each treatment run in duplicate (n=2)

Treatment	Replicates	Probe Substrate	Concentration of Inhibitor	Incubation Duration
Positive Control (MK571) with ATP	2	Estradiol 17β-glucuronide (50 µM)	50 mM	30 min
Positive Control (MK571) with AMP	2	Estradiol 17β-glucuronide (50 µM)	50 mM	30 min

Determine net influx rate of probe substrate in the presence and absence of inhibitor. Calculate percent inhibition.
Acceptance criteria:
- Percent inhibition > 50% when co-incubated with a known inhibitor.

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Objectives

To determine if the Customer’s test article is a potential inhibitor of the multi drug resistance protein 2 (MRP2) transporter using MRP2-expressing membrane vesicles

Required from Customer

Test article either in powder form (5 mg) or stock solution (100 µL of 100 mM DMSO)
Molecular weight of the test article and its salt form
Relevant solubility of test article
The MSDS and handling and storage information, e.g., light sensitive, store at -20ºC, etc.

Deliverables

Express Plus report including:
Materials and methods
Results: Determination of IC₅₀for test article

Assay System

MRP2-expressing membrane vesicles
MRP2 probe substrate: Estradiol 17β-glucuronide
96-well format at 37°C

Assay Conditions

Influx assessment of probe substrate in the presence and absence of test article (IC₅₀Determination)
Assay is performed using MRP2 -expressing vesicles
Assay buffer: Hepes Influx Buffer (10mM Hepes-Tris, pH 7.4)

Treatment Groups

Treatment	Replicates	Probe Substrate	Concentration of Inhibitor	Incubation Duration
With test article and ATP	2	Estradiol 17β-glucuronide (50 µM)	6 concentrations	30 min
With test article and AMP	2		6 concentrations	30 min
Negative Control (solvent only) with ATP	2		N/A	30 min
Negative Control (solvent only) with AMP	2		N/A	30 min
Positive Control (MK571) with ATP	2		50 µM	30 min
Positive Control (MK571) with AMP	2		50 µM	30 min

Samples are processed and transferred to a 96-well plate
Radioactivity is measured using a scintillation counter
Percent activity remaining determined for the test and control compound

Net Influx Rate (NIR) = Uptake activity with ATP – Uptake activity with AMP

Percent activity remaining =
- Positive control acceptance criteria: Percent inhibition > 50% when co-incudated with a known inhibitor
Determination of IC₅₀ by nonlinear regression using GraphPad Prism (version 5.0)

Y =

X = logarithm of the nominal concentration of test article

Y = value of the average percent remaining of the transporter at a given concentration

Y_L = the lowest response; this value is set to zero (0)

Y_H = the highest response at the lowest concentration of the inhibitor

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Substrate

This assay is used to determine the interaction of a test article with P-gp using MDR1- MDCK cell monolayers with and without a P-gp inhibitor

Required from Sponsor

Either a minimum of 100 µL of test article at 10 mM in DMSO or 2 mg of powder
Molecular weight of the test article and its salt form
MSDS or handling and storage information e.g., light sensitive, store at -20°C, etc.

Deliverables

Cell batch QC results
The percent recovery of the test article from the assay wells containing MDR1-MDCK monolayers
The apparent permeability (Papp) of the test article in both directions in the presence and absence of inhibitor
The efflux ratio in the presence and absence of inhibitor
The brain penetration potential of a test article classified as either low, moderate, or high using the following criteria
- Low: Papp A→B < 3.0 x 10-6 cm/s
- Low: Papp A→B ≥ 3.0 x 10-6 cm/s, Efflux ≥ 10
- Moderate: Papp A→B ≥ 3.0 x 10-6 cm/s, 10 > Efflux ≥ 3.0
- High: Papp A→B ≥ 3.0 x 10-6 cm/s, Efflux < 3.0
P-gp substrate classification:
- Positive: Efflux ratio ≥2.0 in the absence of inhibitor and corrected efflux ratio reduced by ≥50% in the presence of inhibitor
- Negative: Efflux ratio ≥2.0 in the absence of inhibitor and corrected efflux ratio reduced by <50% in the presence of inhibitor
- Negative: Efflux ratio < 2.0 in both the absence and presence of inhibitor

Substrate

Test article at 5 µM in HBSSg with maximum DMSO concentration ≤ 0.8%

Assay System

Confluent monolayers of MDR1-MDCK cells, 7 to 12 days old

Assay Conditions

Bidirectional permeability of the test article in MDR1-MDCK cells in the presence and absence of inhibitor
Transport buffer: HBSSg, pH 7.4 ± 0.2
Receiver well containing 1% BSA
Apical and basolateral side at pH 7.4
Dose two cell monolayers for each direction(n=2), in the presence and absence of inhibitor
- Dose apical side for (A→B) assessment
- Dose basolateral side for (B→A) assessment
Sample from both apical and basolateral sides at 120 minutes
Determine the concentration of test article using a generic LC-MS/MS method with a minimum 6-point calibration curve

Assay QC

The quality of the monolayer batch is verified using control compounds before the monolayers are released for use
The quality of each monolayer used in the assay is verified by calculating the Papp for the control article, lucifer yellow, dosed post-experimentally

Notes

The results from this assay are sent to the sponsor in the ExpressPlus report format, which may include graphical representations of data and comparison with historical data for reference articles.
This screening assay does not provide definitive information about the P-gp article interactions or the apparent Km.
Assay conditions with the inhibitor include:
A 30 minute pre-incubation with Valspodar
Assay conditions without the inhibitor include:
A 30 minute pre-incubation with buffer
Digoxin can be run as an optional positive control
Dosed at 10 µM
BSA is not included in the receiver chamber

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

Required from Customer

Either a minimum of 300 µL of test compound at 10 mM in DMSO, or 5 mg of powder
Exact molecular mass of test compound and its salt form
MSDS or handling and storage information, e.g., light sensitive, store at -20°C, etc.

Deliverables

Percent recovery of test compound from Transwell^® wells containing Caco-2 cell monolayers
Apparent permeability (P_app) of test compound in the apical-to-basolateral (A to B) direction in the presence and absence of a PepT1 inhibitor
PeptT1 interaction potential of test compound classified as either positive or negative

Substrate

Test compound at 5 µM in modified Hanks’ buffer (HBSSg) with final DMSO concentration <0.8%

Assay System

Confluent monolayers of PepT1-induced Caco-2 cells, 21-28 days old, in Transwell dual-chamber plates

Assay Conditions

Receiver well with 1% BSA in modified Hank’s buffer (HBSSg)
Pre-incubate monolayers with assay buffer (apical pH 5.0, basolateral pH 7.4), with or without GlySar, for 30 minutes prior to initiation of the assay
Dose duplicate monolayers (N=2) with test compound on the apical side only
Dose two other monolayers (N=2) with test compound and a PepT1 inhibitor, GlySar, on the apical side only
Sample both apical and basolateral sides at 120 minutes
Determine the concentration of test compound using a generic LC-MS/MS method with a minimum 4-point calibration curve

Assay QC

Verify the quality of the cell monolayer batch using control compounds before the monolayers are released for use
Verify the quality of each monolayer by pre-experiment TEER and P_app of a co-dosed control compound

Notes

The results from this assay are provided to the customer in the ExpressPlus report format, which may include graphical representations of data and comparison with historical data for reference compounds.

Options

The customer can request: that a positive control (valacyclovir) be run in parallel

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This assay is used to determine the interaction of a test compound with BCRP using BCRP-MDCK cell monolayers with and without a BCRP inhibitor

Required from Customer

Either a minimum of 300 µL of test compound at 10 mM in DMSO, or 5 mg of powder
Exact molecular mass of test compound and its salt form
Solubility of test compound in Hanks’ buffer
MSDS or handling and storage information, e.g., light sensitive, store at -20°C, etc.

Deliverables

The percent recovery of the test compound from Transwell® wells containing cell monolayers
The apparent permeability (P_app) in both directions with and without inhibitor
The efflux ratio (P_app B to A)/(P_app A to B) with and without inhibitor
BCRP substrate classification:
- Positive when efflux ratio ≥3.0 in the absence of inhibitor and relative efflux ratio ≥2.0
- Negative when efflux ratio ≥3.0 in the absence of inhibitor and relative efflux ratio <2.0, or when efflux ratio <3.0 in the absence and presence of inhibitor
- Requires further study when the effux ratio ≥3.0 in the absence and presence of inhibitor

Substrate

Test compound at 5 µM in HBSSg with maximum DMSO concentration not greater than 1%

Assay System

Confluent monolayers of BCRP-MDCK cells, 7 to 11 days old

Assay Conditions

Receiver well with 1% BSA in modified Hanks buffer (HBSSg)
Apical and basolateral side at pH 7.4 ± 0.2
Assay run in duplicate (N=2)
Single test compound concentration at 5 µM
Dose apical side for A to B assessment and basolateral side for B to A assessment
Sample both donor and receiver sides at 120 minutes
Determine the concentrations of test compound using a generic LC-MS/MS method with a minimum 6-point calibration curve

Assay QC

The quality of the cell monolayer batch is verified using control compounds before the monolayers are used
The quality of each monolayer used in the assay is verified by a TEER measurement and by calculating the P_app for a control compound

Notes

The results from this assay are sent to the customer in the ExpressPlus report format, which may include graphical representations of data and comparison with historical data for reference compounds.
Monolayer integrity will be verified by a pre-experimental TEER.
This screening assay does not provide definitive information about the BCRP compound interactions or the apparent K_m.
Assay conditions with the inhibitor include: a 30 minute pre-incubation with Ko143
Assay conditions without the inhibitor include: a 30 minute pre-incubation with buffer

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This assay is used to determine the interaction of a test compound with BCRP using Caco-2 cell monolayers with and without a BCRP inhibitor

Required from Customer

Either a minimum of 300 µL of test compound at 10 mM in DMSO, or 5 mg of powder
Exact molecular mass of test compound and its salt form
Solubility of test compound in Hanks’ buffer
MSDS or handling and storage information, e.g., light sensitive, store at -20°C, etc.

Deliverables

The percent recovery of the test compound from Transwell® wells containing cell monolayers
The apparent permeability (P_app) in both directions with and without inhibitor
The efflux ratio (P_app B to A)/(P_app A to B) with and without inhibitor
BCRP substrate classification:
- Positive when efflux ratio ≥3.0 in the absence of inhibitor and relative efflux ratio ≥2.0
- Negative when efflux ratio ≥3.0 in the absence of inhibitor and relative efflux ratio <2.0, or when efflux ratio <3.0 in the absence and presence of inhibitor
- Requires further study when the effux ratio ≥3.0 in the absence and presence of inhibitor

Substrate

Test compound at 5 µM in HBSSg with maximum DMSO concentration not greater than 1%

Assay System

Confluent monolayers of Caco-2 cells, 21 to 28 days old

Assay Conditions

Receiver well with 1% BSA in modified Hanks buffer (HBSSg)
Apical and basolateral side at pH 7.4 ± 0.2
Assay run in duplicate (N=2)
Single test compound concentration at 5 µM
Dose apical side for A to B assessment and basolateral side for B to A assessment
Sample both donor and receiver sides at 120 minutes
Determine the concentrations of test compound using a generic LC-MS/MS method with a minimum 6-point calibration curve

Assay QC

The quality of the cell monolayer batch is verified using control compounds before the monolayers are used
The quality of each monolayer used in the assay is verified by a TEER measurement and by calculating the P_app for a control compound

Notes

The results from this assay are sent to the customer in the ExpressPlus report format, which may include graphical representations of data and comparison with historical data for reference compounds.
Monolayer integrity will be verified by a pre-experimental TEER.
This screening assay does not provide definitive information about the BCRP compound interactions or the apparent K_m.
Assay conditions with the inhibitor include: a 30 minute pre-incubation with Ko143
Assay conditions without the inhibitor include: a 30 minute pre-incubation with buffer

This assay is used to determine the interaction of a test compound with P-gp using Caco-2 cell monolayers with and without a P-gp inhibitor.

Required from Customer

Either a minimum of 300 µL of test compound at 10 mM in DMSO, or 5 mg of powder
Exact molecular mass of test compound and its salt form
Solubility of test compound in Hanks’ buffer
MSDS or handling and storage information, e.g., light sensitive, store at -20°C, etc.

Deliverables

The percent recovery of the test compound from Transwell® wells containing cell monolayers
The apparent permeability (P_app) in both directions with and without inhibitor
The efflux ratio (P_app B to A)/(P_app A to B) with and without inhibitor
P-gp substrate classification:
- Positive when efflux ratio ≥3.0 in the absence of inhibitor and relative efflux ratio ≥2.0
- Negative when efflux ratio ≥3.0 in the absence of inhibitor and relative efflux ratio <2.0, or when efflux ratio <3.0 in the absence and presence of inhibitor
- Requires further study when the effux ratio ≥3.0 in the absence and presence of inhibitor

Substrate

Test compound at 5 µM in HBSSg with maximum DMSO concentration not greater than 1%

Assay System

Confluent monolayers of Caco-2 cells, 21 to 28 days old

Assay Conditions

Receiver well with 1% BSA in modified Hanks buffer (HBSSg)
Apical and basolateral side at pH 7.4 ± 0.2
Assay run in duplicate (N=2)
Single test compound concentration at 5 µM
Dose apical side for A to B assessment and basolateral side for B to A assessment
Sample both donor and receiver sides at 120 minutes
Determine the concentrations of test compound using a generic LC-MS/MS method with a minimum 6-point calibration curve

Assay QC

The quality of the cell monolayer batch is verified using control compounds before the monolayers are used
The quality of each monolayer used in the assay is verified by a TEER measurement and by calculating the P_app for a control compound

Notes

The results from this assay are sent to the customer in the ExpressPlus report format, which may include graphical representations of data and comparison with historical data for reference compounds.
Monolayer integrity will be verified by a pre-experimental TEER.
This screening assay does not provide definitive information about the P-gp compound interactions or the apparent K_m.
Assay conditions with the inhibitor include: a 30 minute pre-incubation with Valspodar
Assay conditions without the inhibitor include: a 30 minute pre-incubation with buffer

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This assay uses OAT1-transfected HEK293 cells to assess whether the test compound is a potential substrate of the hepatic uptake transporter OAT1

Required from Customer

Either a minimum of 300 µL of test compound at 10 mM in DMSO, or 5 mg of powder
Exact molecular mass of test compound and its salt form
Solubility of test compound in Hanks’ buffer
MSDS or handling and storage information, e.g., light sensitive, store at -20°C, etc.

Deliverables

Normalized uptake of test compound at each concentration in OAT1-transfected and vector control cells
Determination of substrate classification: positive when the ratio of the influx rates in OAT1-transfected cells vs. vector control cells >2.0

Substrate

Test compound at 0.5 and 5 µM in HBSSg (pH 7.4) with maximum DMSO concentration not greater than 1%

Assay System

OAT1-transfected HEK293 cells and vector control cells cultured in 24-well plates

Assay Conditions

Measure uptake of test article in-transfected and vector control cells
Two concentrations of test article and two sampling time points
Treatments per transporter as follows (repeated for each concentration and incubation duration):
Transfected cells with test article
Vector control cells with test article
Perform assay in duplicate (N=2 per treatment)
Wells are equilibrated with blank buffer containing 1% BSA prior to initiating the assay
Buffer is aspirated and replaced with test article dosing solution
Incubate cells for 2 and 10 min at 37°C
Terminate the incubation and lyse the cells
Analyze cell lysate using LC-MS/MS to determine test article concentration
Determine protein concentration in cell lysate and normalize probe substrate concentration to protein content

Batch QC

The quality of the cell batch is verified before the cells are used (uptake rate of a probe substrate in OAT1-transfected vs. vector control cells)

Options The customer can request:

An in-study positive control for an additional fee

Notes

The results from this assay are sent to the customer in the ExpressPlus report format, which may include graphical representations of data.

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This assay uses OAT3-transfected HEK293 cells to assess whether the test compound is a potential substrate of the hepatic uptake transporter OAT3

Required from Customer

Either a minimum of 300 µL of test compound at 10 mM in DMSO, or 5 mg of powder
Exact molecular mass of test compound and its salt form
Solubility of test compound in Hanks’ buffer
MSDS or handling and storage information, e.g., light sensitive, store at -20°C, etc.

Deliverables

Normalized uptake of test compound at each concentration in OAT3-transfected and vector control cells
Determination of substrate classification: positive when the ratio of the influx rates in OAT3-transfected cells vs. vector control cells >2.0
Substrate
Test compound at 0.5 and 5 µM in HBSSg (pH 7.4) with maximum DMSO concentration not greater than 1%

Assay System

OAT3-transfected HEK293 cells and vector control cells cultured in 24-well plates

Assay Conditions

Measure uptake of test article in-transfected and vector control cells
Two concentrations of test article and two sampling time points
Treatments per transporter as follows (repeated for each concentration and incubation duration):
Transfected cells with test article
Vector control cells with test article
Perform assay in duplicate (N=2 per treatment)
Wells are equilibrated with blank buffer containing 1% BSA prior to initiating the assay
Buffer is aspirated and replaced with test article dosing solution
Incubate cells for 2 and 10 min at 37°C
Terminate the incubation and lyse the cells
Analyze cell lysate using LC-MS/MS to determine test article concentration
Determine protein concentration in cell lysate and normalize probe substrate concentration to protein content

Batch QC

The quality of the cell batch is verified before the cells are used (uptake rate of a probe substrate in OAT3-transfected vs. vector control cells)

Options The customer can request:

An in-study positive control for an additional fee

Notes

The results from this assay are sent to the customer in the ExpressPlus report format, which may include graphical representations of data.

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This assay uses OCT1-transfected HEK293 cells to assess whether the test compound is a potential substrate of the hepatic uptake transporter OCT1.

Required from Customer

Either a minimum of 300 µL of test compound at 10 mM in DMSO, or 5 mg of powder
Exact molecular mass of test compound and its salt form
Solubility of test compound in Hanks’ buffer
MSDS or handling and storage information, e.g., light sensitive, store at -20°C, etc.

Deliverables

Normalized uptake of test compound at each concentration in OCT1-transfected and vector control cells
Determination of substrate classification: positive when the ratio of the influx rates in OCT1-transfected cells vs. vector control cells >2.0

Substrate

Test compound at 0.5 and 5 µM in HBSSg (pH 7.4) with maximum DMSO concentration not greater than 1%

Assay System

OCT1-transfected HEK293 cells and vector control cells cultured in 24-well plates

Assay Conditions

Measure uptake of test article in-transfected and vector control cells
Two concentrations of test article and two sampling time points
Treatments per transporter as follows (repeated for each concentration and incubation duration):
Transfected cells with test article
Vector control cells with test article
Perform assay in duplicate (N=2 per treatment)
Wells are equilibrated with blank buffer containing 1% BSA prior to initiating the assay
Buffer is aspirated and replaced with test article dosing solution
Incubate cells for 2 and 10 min at 37°C
Terminate the incubation and lyse the cells
Analyze cell lysate using LC-MS/MS to determine test article concentration
Determine protein concentration in cell lysate and normalize probe substrate concentration to protein content

Batch QC

The quality of the cell batch is verified before the cells are used (uptake rate of a probe substrate in OCT1-transfected vs. vector control cells)

Options The customer can request:

An in-study positive control for an additional fee

Notes

The results from this assay are sent to the customer in the ExpressPlus report format, which may include graphical representations of data.

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This assay uses OCT2-transfected HEK293 cells to assess whether the test compound is a potential substrate of the hepatic uptake transporter OCT2.

Required from Customer

Either a minimum of 300 µL of test compound at 10 mM in DMSO, or 5 mg of powder
Exact molecular mass of test compound and its salt form
Solubility of test compound in Hanks’ buffer
MSDS or handling and storage information, e.g., light sensitive, store at -20°C, etc.

Deliverables

Normalized uptake of test compound at each concentration in OCT2-transfected and vector control cells
Determination of substrate classification: positive when the ratio of the influx rates in OCT2-transfected cells vs. vector control cells >2.0

Substrate

Test compound at 0.5 and 5 µM in HBSSg (pH 7.4) with maximum DMSO concentration not greater than 1%

Assay System

OCT2-transfected HEK293 cells and vector control cells cultured in 24-well plates

Assay Conditions

Measure uptake of test article in-transfected and vector control cells
Two concentrations of test article and two sampling time points
Treatments per transporter as follows (repeated for each concentration and incubation duration):
Transfected cells with test article
Vector control cells with test article
Perform assay in duplicate (N=2 per treatment)
Wells are equilibrated with blank buffer containing 1% BSA prior to initiating the assay
Buffer is aspirated and replaced with test article dosing solution
Incubate cells for 2 and 10 min at 37°C
Terminate the incubation and lyse the cells
Analyze cell lysate using LC-MS/MS to determine test article concentration
Determine protein concentration in cell lysate and normalize probe substrate concentration to protein content

Batch QC

The quality of the cell batch is verified before the cells are used (uptake rate of a probe substrate in OCT2-transfected vs. vector control cells)

Options The customer can request:

An in-study positive control for an additional fee

Notes

The results from this assay are sent to the customer in the ExpressPlus report format, which may include graphical representations of data.

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This assay uses OATP1B1-transfected HEK293 cells to assess whether the test compound is a potential substrate of the hepatic uptake transporter OATP1B1

Required from Customer

Either a minimum of 300 µL of test compound at 10 mM in DMSO, or 5 mg of powder
Exact molecular mass of test compound and its salt form
Solubility of test compound in Hanks’ buffer
MSDS or handling and storage information, e.g., light sensitive, store at -20°C, etc

Deliverables

Normalized uptake of test compound at each concentration in OATP1B1-transfected and vector control cells
Determination of substrate classification: positive when the ratio of the influx rates in OATP1B1-transfected cells vs. vector control cells >2.0

Substrate

Test compound at 0.5 and 5 µM in HBSSg (pH 7.4) with maximum DMSO concentration not greater than 1%

Assay System

OATP1B1-transfected HEK293 cells and vector control cells cultured in 24-well plates

Assay Conditions

Measure uptake of test article in-transfected and vector control cells
Two concentrations of test article and two sampling time points
Treatments per transporter as follows (repeated for each concentration and incubation duration):
Transfected cells with test article
Vector control cells with test article
Perform assay in duplicate (N=2 per treatment)
Wells are equilibrated with blank buffer containing 1% BSA prior to initiating the assay
Buffer is aspirated and replaced with test article dosing solution
Incubate cells for 2 and 10 min at 37°C
Terminate the incubation and lyse the cells
Analyze cell lysate using LC-MS/MS to determine test article concentration
Determine protein concentration in cell lysate and normalize probe substrate concentration to protein content

Batch QC

The quality of the cell batch is verified before the cells are used (uptake rate of a probe substrate in OATP1B1-transfected vs. vector control cells)

Options

The customer can request:

An in-study positive control for an additional fee

Notes

The results from this assay are sent to the customer in the ExpressPlus report format, which may include graphical representations of data.

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This assay uses OATP1B3-transfected HEK293 cells to assess whether the test compound is a potential substrate of the hepatic uptake transporter OATP1B3

Required from Customer

Either a minimum of 300 µL of test compound at 10 mM in DMSO, or 5 mg of powder
Exact molecular mass of test compound and its salt form
Solubility of test compound in Hanks’ buffer
MSDS or handling and storage information, e.g., light sensitive, store at -20°C, etc.

Deliverables

Normalized uptake of test compound at each concentration in OATP1B3-transfected and vector control cells
Determination of substrate classification: positive when the ratio of the influx rates in OATP1B3-transfected cells vs. vector control cells >2.0

Substrate

Test compound at 0.5 and 5 µM in HBSSg (pH 7.4) with maximum DMSO concentration not greater than 1%

Assay System

OATP1B3-transfected HEK293 cells and vector control cells cultured in 24-well plates

Assay Conditions

Measure uptake of test article in-transfected and vector control cells
Two concentrations of test article and two sampling time points
Treatments per transporter as follows (repeated for each concentration and incubation duration):
Transfected cells with test article
Vector control cells with test article
Perform assay in duplicate (N=2 per treatment)
Wells are equilibrated with blank buffer containing 1% BSA prior to initiating the assay
Buffer is aspirated and replaced with test article dosing solution
Incubate cells for 2 and 10 min at 37°C
Terminate the incubation and lyse the cells
Analyze cell lysate using LC-MS/MS to determine test article concentration
Determine protein concentration in cell lysate and normalize probe substrate concentration to protein content

Batch QC

The quality of the cell batch is verified before the cells are used (uptake rate of a probe substrate in OATP1B3-transfected vs. vector control cells)

Options | The customer can request:

An in-study positive control for an additional fee

Notes

The results from this assay are sent to the customer in the ExpressPlus report format, which may include graphical representations of data.

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This assay uses MATE1-transfected HEK293 cells to assess whether the test compound is a potential substrate of the hepatic uptake transporter MATE1

Required from Customer

Either a minimum of 300 µL of test compound at 10 mM in DMSO, or 5 mg of powder
Exact molecular mass of test compound and its salt form
Solubility of test compound in Hanks’ buffer
MSDS or handling and storage information, e.g., light sensitive, store at -20°C, etc.

Deliverables

Normalized uptake of test compound at each concentration in MATE1-transfected and vector control cells
Determination of substrate classification: positive when the ratio of the influx rates in MATE1-transfected cells vs. vector control cells >2.0

Substrate

Test compound at 0.5 and 5 µM in HBSSg (pH 8.5) with maximum DMSO concentration not greater than 1%

Assay System

MATE1-transfected HEK293 cells and vector control cells cultured in 24-well plates

Assay Conditions

Measure uptake of test article in-transfected and vector control cells
Two concentrations of test article and two sampling time points
Treatments per transporter as follows (repeated for each concentration and incubation duration):
Transfected cells with test article
Vector control cells with test article
Perform assay in duplicate (N=2 per treatment)
Wells are equilibrated with blank buffer containing 1% BSA prior to initiating the assay
Buffer is aspirated and replaced with test article dosing solution
Incubate cells for 2 and 10 min at 37°C
Terminate the incubation and lyse the cells
Analyze cell lysate using LC-MS/MS to determine test article concentration
Determine protein concentration in cell lysate and normalize probe substrate concentration to protein content

Batch QC

The quality of the cell batch is verified before the cells are used (uptake rate of a probe substrate in MATE1-transfected vs. vector control cells)

Options The customer can request:

An in-study positive control for an additional fee

Notes

The results from this assay are sent to the customer in the ExpressPlus report format, which may include graphical representations of data.

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This assay uses MATE2K-transfected HEK293 cells to assess whether the test compound is a potential substrate of the hepatic uptake transporter MATE2K

Required from Customer

Either a minimum of 300 µL of test compound at 10 mM in DMSO, or 5 mg of powder
Exact molecular mass of test compound and its salt form
Solubility of test compound in Hanks’ buffer
MSDS or handling and storage information, e.g., light-sensitive, store at -20°C, etc

Deliverables

Normalized uptake of test compound at each concentration in MATE2K-transfected and vector control cells
Determination of substrate classification: positive when the ratio of the influx rates in MATE2K-transfected cells vs. vector control cells >2.0

Substrate

Test compound at 0.5 and 5 µM in HBSSg (pH 8.5) with maximum DMSO concentration not greater than 1%

Assay Systems

MATE2K-transfected HEK293 cells and vector control cells cultured in 24-well plates

Assay Condition

Measure uptake of test article in-transfected and vector control cells
Two concentrations of test article and two sampling time points
Treatments per transporter as follows (repeated for each concentration and incubation duration):
Transfected cells with test article
Vector control cells with test article
Perform assay in duplicate (N=2 per treatment)
Wells are equilibrated with blank buffer containing 1% BSA prior to initiating the assay
Buffer is aspirated and replaced with test article dosing solution
Incubate cells for 2 and 10 min at 37°C
Terminate the incubation and lyse the cells
Analyze cell lysate using LC-MS/MS to determine test article concentration
Determine protein concentration in cell lysate and normalize probe substrate concentration to protein content

Batch QC

The quality of the cell batch is verified before the cells are used (uptake rate of a probe substrate in MATE2K-transfected vs. vector control cells)

Options

The customer can request:

An in-study positive control for an additional fee

Notes

The results from this assay are sent to the customer in the ExpressPlus report format, which may include graphical representations of data.

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Treatment	Replicates	Incubation Duration
Test article (10 µM) and ATP	2	30 min
Test article (10 µM) and AMP	2	30 min

Treatment	Replicates	Incubation Duration
Taurocholic acid (5 µM) with ATP	2	30 min
Taurocholic acid (5 µM) with AMP	2	30 min

Treatment	Replicates	Incubation Duration
Test article (10 µM) and ATP	2	30 min
Test article (10 µM) and AMP	2	30 min

Treatment	Replicates	Incubation Duration
Estradiol 17β-glucuronide (50 µM) with ATP	2	30 min
Estradiol 17β-glucuronide (50 µM) with AMP	2	30 min

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Transporters

Transporter Assays

ATP binding cassette (ABC)

Solute Carrier (SLC)

Other Express Services

Project Deliverables:

Available Express Transporter Assays

Caco-2 (Pgp Inhibition Single Concentration)

Required from Sponsor

Deliverables

Substrate

Assay System

Assay Conditions

Assay QC

Notes

MDR1-MDCK Inhibition (Single Concentration)

Required from Customer

Deliverables

Substrate

Assay System

Assay Conditions

Assay QC

Notes

Options

BCRP-MDCK Inhibition (Single Concentration)

Required from Customer

Deliverables

Substrate

Assay System

Assay Conditions

Assay QC

Notes

Options

OAT1 Inhibition (Single Concentration)

Required from Customer

Deliverables

Substrate

Assay System

Assay Conditions

Assay QC

Notes

Options

OAT3 Inhibition (Single Concentration)

Required from Customer

Deliverables

Substrate

Assay System

Assay Conditions

Assay QC

Notes

Options

OCT1 Inhibition (Single Concentration)

Required from Customer

Deliverables

Substrate

Assay System

Assay Conditions

Assay QC

Notes

Options

OCT2 Inhibition (Single Concentration)

Required from Customer

Deliverables

Substrate

Assay System

Assay Conditions

Assay QC

Notes

OATP1B1 Inhibition (single Concentration)

Required from Customer

Deliverables

Substrate

Assay System

Assay Conditions

Assay QC

Notes

Options

OATP1B3 Inhibition (Single Concentration)

Required from Customer

Deliverables