Methods for estimation of brain active site concentration of drugs and other chemicals
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1
Uppsala University, Division of Pharmacokinetics and Drug Therapy, Department of Pharmaceutical Biosciences, Sweden
Brain active site concentrations are defined as the unbound, pharmacologically active concentrations at the site of action. The site of action may be located extracellularly in the brain interstitial fluid (ISF) or at some intracellular target within the parenchymal cells. Unbound blood concentrations are not representative of the brain ISF concentrations when the drug is exposed to active efflux or influx transport at the blood-brain barrier (BBB). The brain ISF concentrations may neither be representative of the intracellular concentrations depending on type of transport to the intracellular target. Methods intended for estimation of brain active site concentrations have to directly measure or estimate the unbound concentration. Recent development has made it possible to measure and/or estimate brain ISF concentrations, and also to estimate average intracellular concentrations.
When discussing methods, it is important to distinguish between methods for estimation of rate of transport across the BBB and methods for estimation of the extent of equilibration between brain and blood [1]. Examples of "rate methods" are in situ brain perfusion, brain efflux index and cell culture models that measure the permeability surface area product or uptake clearance into the brain. Only the "extent methods" can be used to estimate active site concentrations. The extent of equilibration can be determined for total drug (Kp) or unbound drug (Kp,uu). Kp,uu is by definition the ratio of unbound concentrations in brain ISF and blood.
Total drug ratios can be determined with i.e. logBB methods and with PET. A simple method with which to determine Kp is to administer an i.v. infusion to steady state [1, 2]. A suggestion is to administer an infusion for 4 hours to rats and thereafter take brain and blood samples (Fig. 1). In order to obtain Kp,uu, the infusion should be combined with plasma protein binding determination and brain slice measurement. This method increases the possibility of obtaining good estimations of unbound target concentrations already in the drug discovery setting. Unbound ratios can be measured directly with microdialysis for drugs that do not stick to tubings and probes. The unspecific binding in brain can be determined with Vu,brain or fu,brain measurements, where Vu,brain is the unbound volume of distribution in brain (Fig. 1), and fu,brain is the unbound fraction in brain. Vu,brain is equal to 1/fu,brain under certain circumstances.
CSF has for long been valued as a substitute for unbound brain concentrations but with a lack of comparison. This has mainly been due to the lack of methods to measure brain ISF concentrations. The unbound concentration ratio between CSF and blood (Kp,uu,CSF) was recently evaluated as a substitute for Kp,uu [3]. There was a rather good correlation (Fig. 2 A). However, the CSF ratio over-predicted the brain Kp,uu ratio at low values, and underpredicted the ratio at high values. At low values, this can be explained as caused by the presence of P-glycoprotein (P-gp) at the BBB but a different function of P-gp at the bloodcerebrospinal fluid barrier (BCSFB). At high values, it is speculated that active uptake transporters are present in the BBB but not in the BCSFB. Human and rat data had the same rank-order, but human Kp,uu,CSF values were on average 3-fold higher than those in rats (Fig. 2 B).
Intracellular exposure can be addressed with a combination of the brain slice and the brain homogenate methods [4]. Development is ongoing to further sub-divide estimations into cellular components.
In conclusion, methods for estimation of brain active site concentrations need to measure or estimate the unbound moiety. There is a rather limited availability of methods, but recent development has made it possible to increase the knowledge about the relationship between brain active site concentrations and unbound concentrations in blood.
References
1. Hammarlundâ€Udenaes, M., U. Bredberg, and M. Friden, Methodologies to assess brain drug delivery in lead optimization. Curr Top Med Chem, 2009. 9(2): p. 148â€62.
2. Friden, M., H. Ljungqvist, B. Middleton, U. Bredberg, and M. Hammarlundâ€Udenaes, Improved measurement of drug exposure in the brain using drugâ€specific correction for residual blood. J Cereb Blood Flow Metab, 2009.
3. Friden, M., S. Winiwarter, G. Jerndal, O. Bengtsson, H. Wan, U. Bredberg, M. Hammarlundâ€Udenaes, and M. Antonsson, Structureâ€brain exposure relationships in rat and human using a novel data set of unbound drug concentrations in brain interstitial and cerebrospinal fluids. J Med Chem, 2009. 52(20): p. 6233â€43.
4. Fridén, M., A. Gupta, M. Antonsson, U. Bredberg, and M. Hammarlundâ€Udenaes, In vitro methods for estimating unbound drug concentrations in the brain interstitial and intracellular fluids. Drug Metab Dispos, 2007. 35(9): p. 1711â€9.
Conference:
Pharmacology and Toxicology of the Blood-Brain Barrier: State of the Art, Needs for Future Research and Expected Benefits for the EU, Brussels, Belgium, 11 Feb - 12 Feb, 2010.
Presentation Type:
Oral Presentation
Topic:
Presentations
Citation:
Hammarlund-Udenaes
M
(2010). Methods for estimation of brain active site concentration of drugs and other chemicals.
Front. Pharmacol.
Conference Abstract:
Pharmacology and Toxicology of the Blood-Brain Barrier: State of the Art, Needs for Future Research and Expected Benefits for the EU.
doi: 10.3389/conf.fphar.2010.02.00014
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Received:
01 Mar 2010;
Published Online:
01 Mar 2010.
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Correspondence:
Margareta Hammarlund-Udenaes, Uppsala University, Division of Pharmacokinetics and Drug Therapy, Department of Pharmaceutical Biosciences, Uppsala, Sweden, mhu@farmbio.uu.se