Abstract
Estimating the unbound fraction of drugs in brain has become essential for the evaluation and interpretation of the pharmacokinetics and pharmacodynamics of new central nervous system drug candidates. Dialysis-based methods are considered to be accurate for estimating the fraction unbound in brain; however, these techniques are hampered by a low throughput. In this study, we present a novel, matrix-free, high-throughput method for estimating the unbound fraction, based on a sample pooling approach combining the TRANSIL brain absorption assay with liquid chromatography-mass spectrometry. The base measurement of the TRANSIL approach is the affinity to brain membranes, and this method is used directly to predict the free fraction in brain. The method was evaluated by comparing the free fraction of drugs in brain [fu,brain (%)] obtained using the TRANSIL brain absorption assay and equilibrium dialysis methods for a test set of 65 drugs (27 marketed and 38 proprietary drugs). A good correlation (r2 > 0.93) of fu,brain (%) between the TRANSIL brain absorption assay and equilibrium dialysis was observed. Moreover, we compared the lipid composition of rat and porcine brain and analyzed the influence of the brain albumin content on brain tissue binding measurement. The comparison of the lipid composition indicated only minor differences between rat and porcine brain, and albumin appears to have a low impact on brain tissue binding measurements. The TRANSIL brain absorption assay with sample pooling methodology not only significantly reduces the biological matrix required but also increases the throughput, compared with the conventional dialysis methods.
Footnotes
Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
doi:10.1124/dmd.110.036095.
-
ABBREVIATIONS:
- CNS
- central nervous system
- Kp
- brain/plasma ratio
- Cbrain
- brain total concentration
- Cu,brain
- brain unbound concentration
- fu,brain
- free fraction of drugs in brain
- GSK
- GlaxoSmithKline
- HPLC
- high-performance liquid chromatography
- PBS
- phosphate-buffered saline
- CSF
- cerebrospinal fluid
- LC-MS/MS
- liquid chromatography-tandem mass spectrometry
- DMSO
- dimethyl sulfoxide
- UPLC/MS/MS
- ultraperformance liquid chromatography-tandem mass spectrometry
- RSA
- rat serum albumin
- HPTLC
- high-performance thin-layer chromatography
- MALDI-TOF-MS
- matrix-assisted laser desorption and ionization time-of-flight mass spectrometry
- SM
- sphingomyelin
- PC
- phosphatidylcholine
- PS
- phosphatidylserine
- PA
- phosphatidic acid
- TLC
- thin-layer chromatography
- PI
- phosphatidylinositol
- HBA
- number of H-bond acceptors
- HBD
- number of H-bond donors
- PSA
- polar surface area
- cLogP
- predicted log (octanol/water) partition coefficient
- fu
- free fraction of drugs
- CV
- coefficient of variation.
- Received August 25, 2010.
- Accepted November 11, 2010.
- Copyright © 2011 by The American Society for Pharmacology and Experimental Therapeutics
DMD articles become freely available 12 months after publication, and remain freely available for 5 years.Non-open access articles that fall outside this five year window are available only to institutional subscribers and current ASPET members, or through the article purchase feature at the bottom of the page.
|