Equilibrium dialysis is used in this assay to determine the percentage of test compound that binds to human, rat, dog, primate or mouse plasma proteins.

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 bound of test compound and QC compound

Substrate

• The test compound at 5 μM in human, rat, dog, primate or mouse blood plasma at pH 7.4 with final DMSO concentration less than 1%

Assay System

• Pooled plasma (≥3 donors)
  • Human: mixed gender
  • Rat, dog, primate or mouse plasma: male only
• Dialysis: Rapid Equilibrium Dialysis (RED) device at 37°C

Assay Conditions

• Use two separate wells (N=2), with one side of each dialysis well containing phosphate-buffered saline (PBS) at pH 7.4 and the other side of the well containing plasma
• Use appropriate anticoagulant (typically sodium heparin)
• Adjust pH of PBS and plasma to 7.4 prior to initiation of experiment
• Sample both the plasma side and the buffer side of each well 4 hours after the system reaches 37°C
• Use LC-MS/MS to determine the peak area response ratio (peak area corresponding to the test compound divided by that of an analytical internal standard) without running a calibration curve

Assay QC

• Warfarin is co-dosed with the test compound in each well

Dynamic Range

• 5 to 99.9% bound

Notes

1. 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.
2. This assay uses frozen / thawed pooled blood plasma.
3. This assay can yield ambiguous results with test compounds that are unstable in plasma. Absorption Systems can determine the plasma stability of a test compound at an additional cost.
4. The following equation is used to calculate the percentage of the test compound bound to plasma proteins based on the peak area response ratio (PARR): % bound =  ([PARR (donor)] – [PARR (receiver)]  x 100)/[PARR (donor)]

Options

1. The customer can request that the experiment be performed using a Teflon chamber microdialysis apparatus with an equilibration time of >22 hours.

This assay uses equilibrium dialysis to determine the unbound fraction of test compound in a rat or mouse brain homogenate, a parameter that helps predict brain penetration in vivo.

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

• The calculated percent unbound of test compound and control compound

Substrate

• Test compound at a single concentration (5 μM) in rat or mouse brain homogenate at pH 7.4 with DMSO concentration less than 1%

Assay System

• 96-well equilibrium dialysis or equivalent apparatus at 37°C
• Rat or mouse brain homogenate (≥ 3 male donors  pooled) in phosphate-buffered saline (PBS) at pH 7.4

Assay Conditions

• Adjust pH of PBS to 7.4 prior to initiation of the experiment
• Perform equilibrium dialysis in duplicate (N=2)
• Homogenize brain tissue in 2 volumes of PBS
• Spike test compound into diluted brain homogenate (final concentration 5μM)
• Dialyze the test compound in brain homogenate against PBS at 37°C
• Sample brain homogenate and buffer in each well at >22 hours after the system reaches 37°C
• Normalize samples to achieve the same analytical matrix
• Use LC-MS/MS to determine the peak area response ratio for the test compound and control compound (relative to the peak area of an analytical internal standard) without running a calibration curve

Assay QC

• Co-dose test compound with a control compound with low fraction unbound

Dynamic Range

• Up to 99.0% bound

Notes

1. 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.
2. This assay uses a frozen/thawed rat brain homogenate.
3. Results from this assay, when combined with results from EA702, ExpressPlus Plasma Protein Binding (Rat Plasma) and EA203, ExpressPlus BBB Penetration Potential, may be used to predict brain permeation in vivo.

This assay is used to determine the blood-to-plasma partition coefficient of a test compound in mice, rats, and humans in vitro

Required from Customer

• Either a minimum of 300 μL of test compound at 10 mM in DMSO or 5 mg of powder
• The exact molecular mass of the test compound and its salt form
• MSDS or handling and storage information, e.g., light-sensitive, store at -20°C, solubility, etc.
• Stability of test compound in whole blood and/or plasma from the test species, if known

Deliverables

• The concentrations of test compound and control compound in whole blood and plasma
• The calculated blood-to-plasma partition coefficient (KRBC/P) for the test compound and control compound
• Hematocrit of the whole blood lot used in the study

Substrate

• Test compound at a single concentration (typically 5 μM) in  heparinized blood and plasma with final DMSO concentration < %

Assay System

• Fresh, whole heparinized blood and plasma from mice, rats ( ≥3 donors pooled; male only) or humans ( ≥3 donors pooled; mixed gender
• LC-MS/MS is used to determine the peak area response ratio (PARR; peak area corresponding to test compound or control divided by that of an analytical internal standard) without running a standard curve

Assay Conditions

• Adjust pH of blood and plasma to 7.4 prior to addition of test compound
• Spike whole blood and reference plasma (from the same lot of whole blood) with test compound, mix, and incubate at 37°C
• Co-incubate control compound with test compound
• Duplicate incubation (N=2 separate incubations) in whole blood and reference plasma
• Sample blood and prepare plasma  (test plasma) by centrifugation at 60 minutes
• Sample reference plasma at 60 minutes

Assay QC

• Results are accepted only when the blood-to-plasma partition coefficient of the reference compound meets internal acceptance criteria

Notes

1. 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.