Measurement of Unbound Drug Exposure in Brain: Modeling of pH Partitioning Explains Diverging Results between the Brain Slice and Brain Homogenate Methods

Abstract

ABSTRACT:Currently used methodology for determining unbound drug exposure in brain combines measurement of the total drug concentration in the whole brain in vivo with estimation of brain tissue binding from one of two available in vitro methods: equilibrium dialysis of brain homogenate and the brain slice uptake method. This study of 56 compounds compares the fraction of unbound drug in brain (f u,brain ), determined using the brain homogenate method, with the unbound volume of distribution in brain (V u,brain ), determined using the brain slice method. Discrepancies were frequent and were primarily related to drug pH partitioning, attributable to the preservation of cellular structures in the slice that are absent in the homogenate. A mathematical model for pH partitioning into acidic intracellular compartments was derived to predict the slice V u,brain from measurements of f u,brain and drug pK a .This model allowed prediction of V u,brain from f u,brain within a 2.2-fold error range for 95% of the drugs compared with a 4.5-fold error range using the brain homogenate f u,brain method alone. The greatest discrepancies between the methods occurred with compounds that are actively transported into brain cells, including gabapentin, metformin, and prototypic organic cation transporter substrates. It was concluded that intrabrain drug distribution is governed by several diverse mechanisms in addition to nonspecific binding and that the slice method is therefore more reliable than the homogenate method. As an alternative, predictions of V u,brain can be made from homogenate f u,brain using the pH partition model presented, although this model does not take into consideration possible active brain cell uptake.

Development of a Rat Plasma and Brain Extracellular Fluid Pharmacokinetic Model for Bupropion and Hydroxybupropion Based on Microdialysis Sampling, and Application to Predict Human Brain Concentrations

“…Taking the average of these two values, and assuming that brain ECF is 0.1 log unit more acidic than plasma (pH 7.3 versus pH 7.4; Fridén et al, 2011), results in a predicted diffusional equilibrium preference on the brain side of 1.01, which supports that the CL in /CL out ratios of approximately 2 are due to an uptake transporter for bupropion and hydroxybupropion at the BBB. The net uptake asymmetry observed is similar to oxycodone BBB transport in rats, (Boström et al, 2006), which showed a net 3-fold asymmetry favoring rat brain ECF relative to unbound plasma concentrations.…”

“…Transfer characteristics between the plasma and brain were modeled using either a single intercompartmental clearance (Q) or separate parameters for the uptake and efflux apparent distributional clearances (CL in and CL out , respectively). The unbound volume of distribution of bupropion in the brain (V b,B ) was tested with versus without fixing this parameter to a literature-reported value of 16 ml/g brain (Fridén et al, 2011). For hydroxybupropion, a computational approach was used to estimate its volume of distribution in the brain (V b,HB ) (Spreafico and Jacobson, 2013).…”

“…The experimentally obtained pK a of bupropion has been estimated at 7.9 (Gondaliya and Pundarikakshudu, 2003) and 8.6 (Fridén et al, 2011). Taking the average of these two values, and assuming that brain ECF is 0.1 log unit more acidic than plasma (pH 7.3 versus pH 7.4; Fridén et al, 2011), results in a predicted diffusional equilibrium preference on the brain side of 1.01, which supports that the CL in /CL out ratios of approximately 2 are due to an uptake transporter for bupropion and hydroxybupropion at the BBB.…”

Species Independence in Brain Tissue Binding Using Brain Homogenates

“…Brain slices are considered more physiologically relevant than brain homogenates because the cellular structures (cell membrane, influx and efflux transporters, and intracellular fluids) are preserved in brain slices, whereas these are disrupted in brain homogenates. Nevertheless, data generated using the brain homogenate method have good correlation with brain slice data, especially when cytosolic pH partition was corrected for basic compounds Lin, 2008;Fridén et al, 2011). This finding suggests that nonspecific binding to lipophilic components in the brain is the dominant mechanism for brain tissue binding and that the presence of intact structural elements plays a less significant role in determining brain binding.…”

Use of the Cassette-Dosing Approach to Assess Brain Penetration in Drug Discovery

“…The K p values calculated from the instrument response were almost identical to the ones from the measured concentration, supporting the use of this simplified bioanalytical method in drug discovery. A similar analytical approach has been used in the determination of unbound fractions in brain homogenate or slices where the absolute concentrations are not critical but the concentration ratios are important (Fridén et al, 2011). Similar Brain Penetration after Discrete or Cassette Dosing in P-gp, Bcrp, and P-gp/Bcrp Knockout Mice.…”