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An Effective Screening Approach to Assess the Impact of α-1 Acid Glycoprotein Binding on the Fraction Unbound of a Drug
Karin D. Brown*, Gary P. Hingorani and Ronald B. Franklin
Abstract # 25022
Array BioPharma Inc., Boulder, CO
Available at www.arraybiopharma.com
METHODS
ABSTRACT
Binding to plasma proteins can be an important determinant of drug action and pharmacokinetics.
Human Serum Albumin (HSA) and α-1 Acid Glycoprotein (AAG) are the most abundant plasma
proteins with average physiological concentrations of approximately 40 g/L and 0.6-1.2 g/L,
respectively. AAG plasma levels can vary due to physiological, pathological and genetic factors. The
fluctuations in AAG plasma levels can have a direct effect on concentrations of unbound drug, and
consequently alter its pharmacokinetics and pharmacodynamics [1].
Rapid Equilibrium Dialysis (RED): Human Serum Albumin (HSA) with
Increasing Concentrations of α-1 Acid Glycoprotein (AAG)
• A stock concentration of 40 g/L HSA was made using PBS
buffer, pH 7.4.
• Concentrations of AAG (0, 0.6, 1.2, 2.0 and 3.0 g/L) were
dissolved into 40 g/L HSA.
• HSA/AAG concentrations were spiked with test article at a 2
µM (0.1% DMSO final) concentration.
• 300 µL of plasma/500 µL of buffer were added to appropriate
chamber (n=3).
• Plate was sealed and agitated on a plate shaker at 300 rpm
within a humidified CO2 (5%) incubator for 6 hours.
• Samples were extracted with 800 µL acetonitrile with an
internal standard, labetalol (0.2 µM final).
• The plates were spun in a centrifuge for 10 minutes at 3000 x
g.
• The supernatant was transferred into a shallow 96-well plate
and diluted 1:1 with water.
• Plasma/buffer levels were quantified via LC-MS/MS
(API4000) and protein binding was calculated.
Two techniques were utilized to determine the binding affinity (Kd) and free fraction for test
compounds. The TRANSIL® binding methodology was used to establish the Kd values for AAG and
HSA in order to ascertain which compounds have a greater affinity for AAG. To understand the effect
of AAG binding on the fraction unbound (Fu) of a drug, protein binding was assessed by Rapid
Equilibrium Dialysis (RED) using physiological concentrations of HSA (40 g/L) in the presence of
increasing concentrations of AAG
(0-3 g/L).
Literature compounds with a greater than 10-fold preference for AAG demonstrated a greater than
three-fold decrease in the free fraction with increasing AAG levels: ritonavir, imatinib, nelfinavir,
docetaxel, chlorpromazine, imipramine, erythromycin, and propranolol. Test compounds that had a
moderate preference (5-10 fold) for AAG showed two-fold or lower decreases in free fraction with
increasing AAG concentrations: paclitaxel, saquinavir and ketoconazole. While those compounds that
displayed similar affinity for AAG and HSA did not exhibit a decrease in the free fraction with an
increase in AAG concentrations: sulfasalazine, phenytoin, warfarin, and carbamazepine. These
techniques were used to assess the extent of AAG binding on the free fraction for several in-house
compounds.
INTRODUCTION
• Binding of drugs to plasma proteins can play an important role in drug distribution.
• AAG is the second most abundant plasma protein and can have a pronounced effect on the fraction
unbound, particularly for basic drugs.
Figure 3: Equilibrium Dialysis Results for Marketed Drugs which Demonstrate a Greater
Than 3-Fold Decrease in Free Fraction using HSA with Increasing Concentrations of AAG
(n=4).
Table 2. TRANSIL® Binding Results with AAG and HSA for Array BioPharma
Inc. Compounds.
A
B
a
pKa values estimated using ACD/pKa DB software
* Not determined
Table 3: Rapid Equilibrium Dialysis: Free Fraction of Drugs using HSA with
Increasing Concentrations of AAG for Marketed Drugs.
Figure 4: Equilibrium Dialysis Results for Array BioPharma Inc. Compounds (n=4).
Analytical Quantitation
• LC-MS/MS (API 4000), ESI+ ionization mode
• Column: Kinetex 2.6 micron C18 100Å, 30 x 3.0 mm column (Phenomenex, Torrance,
CA)
• Linear gradient conditions using mobile phases A (water containing 1% IPA and 0.1%
formic acid by volume) and B (acetonitrile containing 0.1% formic acid).
Figure 1: Strategy for Assessing Impact of AAG Binding.
• Determining the affinity for AAG and HSA is important for understanding the potential effects of
increasing concentrations of AAG in vivo.
*Standard Deviation
Table 4: Rapid Equilibrium Dialysis: Free Fraction of Drugs using HSA with
Increasing Concentrations of AAG for Array BioPharma Inc. Compounds.
SUMMARY & CONCLUSIONS
• Understanding of the free fraction and concentration of the drug in relation to increasing levels of
AAG in vitro may help in understanding PK/PD and efficacy in the test patient population.
•
• Here, two techniques were combined to determine the Kd values for AAG and HSA using the
TRANSIL® binding methodology and to assess the potential effect on free fraction with increasing
concentrations of AAG through the use of rapid equilibrium dialysis (RED).
• The TRANSIL® binding method provides a simple assessment for the affinity of a compound for
AAG and HSA and to evaluate the preference for AAG versus HSA.
METHODS
TRANSIL®
Table 1: TRANSIL® Binding Results with AAG and HSA for Marketed Drugs.
Methodology [2]
Binding
(www.admecell.com)
•
•
•
•
•
• By evaluating compounds that demonstrate a substantial preference for AAG, using RED with
physiological concentrations of HSA in the presence of increasing AAG concentrations, allows for
understanding the effect of AAG binding on the free fraction.
*Standard Deviation
Figure 2: Equilibrium Dialysis Results for Marketed Drugs which Demonstrate
Little or No Decrease in Free Fraction using HSA with Increasing Concentrations of
AAG (n=4).
Each well was spiked with test article at a 2
µM (1% DMSO final) concentration in PBS,
pH 7.4
Wells were mixed according to the protocol
and placed in a humidified CO2 (5%)
incubator at 37°C for 2 minutes.
The plates were spun in a centrifuge for 10
minutes at 750 x g.
Supernant was diluted 2:1 with acetonitrile
spiked with labetalol (0.4 µM final
concentration), as the internal standard.
Samples were quantified via LC-MS/MS
(API4000) and Kd values were calculated.
• Drugs being developed for disease states in which there are elevated AAG concentrations may be
tested using these new techniques to determine their affinity to AAG and the potential effect to the
unbound fraction of drug, and hence pharmacokinetics and efficacy.
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a-pKa
values estimated using ACD/pKa DB software
measured by LC-MS/MS
b-values
The Kd and Fu values for marketed drugs determined from the TRANSIL® method and by RED,
correlated well with published values.
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