Data Source: Sanford-Burnham Center for Chemical Genomics (SBCCG)
Source Affiliation: Sanford-Burnham Medical Research Institute (SBMRI, Lake Nona, FL)
Network: NIH Molecular Libraries Probe Production Network (MLPCN)
Grant Proposal Number:
Assay Provider: Dr. Layton Smith, Sanford-Burnham Medical Research Institute

The binding of test compounds to plasma proteins is an important factor affecting drug efficacy, metabolism and pharmacokinetic properties. In many cases, drug efficacy is determined by the concentration of free drug (unbound), rather than the total concentration in plasma. If the drug is highly bound to plasma proteins, the amount of drug available to reach the target is reduced. Subsequently, the efficacy of that compound may be significantly reduced. Therefore, precise information on the free drug fraction is essential for drug development and may be helpful in correlating with in vivo efficacy.

Rapid equilibrium dialysis is an accurate and reliable method for determining the degree to which a compound binds to plasma proteins. Plasma spiked with test compound is added to the left chamber of a commercial plate based RED (rapid equilibrium dialysis) device. Blank, isotonic sodium phosphate buffer is added to the outer chamber of the RED device and the plate is incubated at 37 oC. Aliquots of the buffer and the plasma are taken at pre-determined time points and the concentration of free and bound test compound is determined by LC/MS/MS analysis.

References:
1) Waters, N.J., et al. (2008). Validation of a rapid equilibrium dialysis approach for the measurement of plasma protein binding. J Pharm Sci 97(10): 4586-95.

2) Brouwer, E.J., et al. (2000). Measurement of fraction unbound paclitaxel in human plasma. Drug Metab Disposition 28(10): 1141-5.

3) Brunton, L., et al. Goodman and Gilman's Pharmacological Basis of Therapeutics. McGraw Hill Publishing: New York, 2005.

4) Clausen, J. and Bickel, M. (1993). Prediction of drug distribution in distribution dialysis and in vivo from binding to tissue and blood. J Pharm Sci 82: 345-9.

5) Sonnichsen, D. and Relling, M. (1994). Clinical pharmacokinetics of paclitaxel. Clin Pharmacokinet 27: 256-69.

6) Steele, W., et al. (1983). The protein binding of vinblastrine in the serum of normal subjects and patients with Hodgkin's disease. Eur J Clin Pharmacol 24: 683-7.

Assay Materials:

1) Teflon Base Plate and cartridges from Thermo Scientific (Pierce)
2) Test compound(s)
Atenolol - Low plasma protein binding
Propranolol - High plasma protein binding
3) Solvent ACN, MeOH, DMSO
4) Isotonic 1X PBS pH 7.4
5) Human plasma
6) NaH2PO4
7) Na2HPO4
8) 37 oC incubator shaker (~300rpm)
9) Tabletop centrifuge with a plate holder-capable rotor
10) 96-deep well plates, 2mL (polystyrene)
11) Multi-channel pipette and reagent reservoirs
12) 96-well autosampler for LC/MS or HPLC

Assay Protocol:
Prepare the base plate.

1) Rinse the Teflon Base Plate wells with 20% ethanol for 10 minutes.
2) Remove ethanol and rinse twice with ultrapure water.
3) Allow plate to dry.
4) Place RED inserts (open end up) into the wells of the base plate

Sample Preparation

1) Prepare samples by mixing test compounds (DMSO stocks) with plasma at 10 uM and 1uM final concentrations (final DMSO concentration 0.1%).

2) Place 300 uL of sample into the sample chamber (red ring)

3) Add 500 ul of dialysis buffer (1X PBS pH 7.4) to the buffer chamber.

4) Cover the unit with sealing tape and incubate at 37 oC on an orbital shaker at 300 RPM for 4.0hr.

5) Remove 100 uL from both the buffer and the plasma chambers and place in separate 96-deep well plate.

6) Add 100 ul of plasma to the buffer samples, and 100 ul of 1X PBS pH 7.4 to the collected plasma samples.

7) Add 400 uL of ACN containing internal standards to precipitate protein and release compound.

8) Vortex and Centrifuge for 10 minutes at 13,000g.

9) Perform quantitative measurements by LC/MS/MS.

Calculation:

% Free = (Concentration buffer chamber/Concentration plasma chamber) x 100%

% Bound = 100% - % Free

In this assay compounds with a % bound >= 90% at 1 uM are considered as active.

Scoring for single-concentration screening:

Activity scoring rules developed at Sanford-Burnham Center for Chemical Genomics employs a 3-tiered system:

1) The first tier (0-40 range) is reserved for primary or single-concentration screening data and is not applicable to this assay.

2) The second tier (41-80 range) is reserved for dose-response confirmation data of the primary hits that are cherry picked from the HTS mother plates and is not applicable to this assay.

3) The third tier (81-100 range) is reserved for dry-powder compounds that represent purchased and resynthesized positives and their analogues.

Compounds that are inactive in this assay receive a score of 81.
Compounds that are active in this assay receive a score of 90.