Muscle to Brain Partitioning as Measure of Transporter-Mediated Efflux at the Rat Blood–Brain Barrier and Its Implementation into Compound Optimization in Drug Discovery

Abstract

Movement of xenobiotic substances across the blood–brain barrier (BBB) is tightly regulated by various transporter proteins, especially the efflux transporters P-glycoprotein (P-gp/MDR1) and breast cancer resistance protein (BCRP). Avoiding drug efflux at the BBB is a unique challenge for the development of new central nervous system (CNS) drugs. Drug efflux at the BBB is described by the partition coefficient of unbound drug between brain and plasma (Kp,uu,brain) which is typically obtained from in vivo and often additionally in vitro measurements. Here, we describe a new method for the rapid estimation of the in vivo drug efflux at the BBB of rats: the measurement of the partition coefficient of a drug between brain and skeletal muscle (Kp,brain/muscle). Assuming a closely similar distribution of drugs into the brain and muscle and that the efflux transporters are only expressed in the brain, Kp,brain/muscle, similar to Kp,uu,brain, reflects the efflux at the BBB. The new method requires a single in vivo experiment. For 64 compounds from different research programs, we show the comparability to other approaches used to obtain Kp,uu,brain. P-gp- and BCRP-overexpressing cell systems are valuable in vitro tools for prescreening. Drug efflux at the BBB can be most accurately predicted based on a simple algorithm incorporating data from both in vitro assays. In conclusion, the combined use of our new in vivo method and the in vitro tools allows an efficient screening method in drug discovery with respect to efflux at the BBB.

Complete inhibition of ABCB1 and ABCG2 at the blood–brain barrier by co-infusion of erlotinib and tariquidar to improve brain delivery of the model ABCB1/ABCG2 substrate [11C]erlotinib

“…The muscle is not protected by ABCB1 and ABCG2 and muscle surrounding the skull has been proposed as a reference region to investigate ABC transporter activity at the BBB, thereby taking any change in peripheral kinetics and/or binding to plasma proteins into account. 34 In our previous study conducted in baboons, infusion of high-dose elacridar increased AUCR brain/muscle of [ 11 C]erlotinib to approximately unity suggesting complete inhibition of efflux transporter activity at the BBB. 18 In the present study, AUCR brain/muscle measured in the two macaques treated with the erlotinib/tariquidar combination was 0.5 and 1, which corresponded to a 2.3-and 4.0-fold increase over baseline, respectively.…”

“…Animals were injected i.v. 34 Moreover, kinetic modeling was performed using Logan plot analysis with the corresponding metabolite-corrected arterial plasma input function to estimate V T , brain . 32 Compartmental models (1-or 2tissue compartment) did not provide accurate estimates of outcome parameters to describe the brain kinetics of [ 11 C]erlotinib.…”

A Bidirectional Permeability Assay for beyond Rule of 5 Compounds

“…Binding of research compounds to plasma protein and tissue homogenates was determined using the equilibrium dialysis method as described previously [18]. Briefly, plasma, brain homogenate, and muscle homogenate were spiked with 1-10 µM of test compound and dialyzed in equilibrium dialysis cells (RED-device, Thermo Scientific/Pierce or Dianorm Equilibrium Device, Harvard Apparatus Holliston, MA, USA) against 100 mM potassium phosphate buffer pH 7.4 for 2-6 h at 37 • C. Fraction unbound in plasma (f u,plasma ) was calculated as: f u,plasma = C bu f f er /C plasma (5) where C plasma and C buffer are the plasma and buffer concentration, respectively. Unbound tissue concentration (f u,tissue ) was calculated as:…”

“…4 Estimated with data from [22]. 5 K p,br and K p,br/mu determined with an oral dose of 10 or 100 mg/kg, as described in Section 2.2.…”

A mechanistic framework for a priori pharmacokinetic predictions of orally inhaled drugs

“…Inhalation device-specific parameters see S1 Appendix, Section 4 #1 in-house data: fraction unbound was determined with an in vitro binding assay as described in [50], permeability was determined based on an in vitro permeability assay with Calu cells, with assay conditions as described for MDCK II cells in [50]. The in vitro assay setup for determining Blood:Plasma ratio and drug solubility in surfactant-containing media is described in S1 Appendix, Section 5.…”