Examining the Uptake of Central Nervous System Drugs and Candidates Across the Blood-Brain Barrier S
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Supplemental material to this article can be found at: http://jpet.aspetjournals.org/content/suppl/2016/05/18/jpet.116.232447.DC1 1521-0103/358/2/294–305$25.00 http://dx.doi.org/10.1124/jpet.116.232447 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS J Pharmacol Exp Ther 358:294–305, August 2016 Copyright ª 2016 by The American Society for Pharmacology and Experimental Therapeutics Examining the Uptake of Central Nervous System Drugs and Candidates across the Blood-Brain Barrier s Scott G. Summerfield, Yanyan Zhang, and Houfu Liu GlaxoSmithKline R&D, Platform Technology and Science, Park Road, Ware (S.G.S); and GlaxoSmithKline R&D, Platform Technology and Science, Zhangjiang Hi-Tech Park, Pudong, Shanghai, China (Y.Z., H.L.) Received February 7, 2016; accepted May 17, 2016 ABSTRACT Downloaded from Assessing the equilibration of the unbound drug concentrations of P-gp, highlighting the composite nature of this parameter with across the blood-brain barrier (Kp,uu) has progressively replaced respect to facilitated BBB uptake, efflux, and passive diffusion. the partition coefficient based on the ratio of the total concen- Several molecules with high Kp,uu values share common struc- tration in brain tissue to blood (Kp). Here, in vivo brain distribution tural elements, whereas uptake across the BBB appears more studies were performed on a set of central nervous system prevalent in the CNS-targeted drug set than the chemical (CNS)–targeted compounds in both rats and P-glycoprotein templates being generated within the current lead optimization (P-gp) genetic knockout mice. Several CNS drugs are charac- paradigm. Challenges for identifying high Kp,uu compounds are jpet.aspetjournals.org terized by Kp,uu values greater than unity, inferring facilitated discussed in the context of acute versus steady-state data and uptake across the rodent blood-brain barrier (BBB). Examples cross-species differences. Evidently, there is a need for better are shown in which Kp,uu also increases above unity on knockout predictive models of human brain Kp,uu. Introduction (e.g., atenolol; Summerfield et al., 2007) or carrier-mediated efflux (Loryan et al., 2014). The most widely recognized BBB at ASPET Journals on September 27, 2021 Drug distribution across the blood-brain barrier (BBB) efflux transporter for reducing brain Kp,uu is P-glycoprotein forms a central element in linking pharmacokinetics to the (P-gp), which exerts its effects on many chemical templates pharmacological action of xenobiotics within the central (Raub, 2006). Much of the founding in vivo work characteriz- nervous system (CNS) (Reichel, 2015). Unbound drug that ing P-gp efflux relied on K measurements; however, this crosses the BBB into brain extracellular fluid (ECF) is avail- p parameter in itself does not indicate whether a drug molecule able to distribute to the biophase or bind nonspecifically is the subject of active transport; indeed, additional measure- within the lipid-rich components of brain tissue (Jeffrey and ments are required, such as determining K in an in vivo Summerfield, 2007). Recent examples linking unbound drug p transporter knockout model (chemical or genetic) or deter- brain concentrations with potency, target engagement or mining the P-gp efflux ratios in an in vitro system. effect (Large et al., 2009; Watson et al., 2009; Liu and Chen, AK value approaching unity suggests that passive 2015) have helped to transition the thinking of CNS drug p,uu diffusion is an important distribution property of the drug discovery scientists away from total concentration measure- ments and more toward unbound drug concentrations. Simi- molecule, or that multiple carrier-mediated processes are larly, interpreting brain distribution has shifted away from balanced (e.g., influx and efflux). Although Kp,uu alone cannot the brain-to-blood partition ratio based on total concentra- distinguish these two scenarios, concentration dependency in this value or the permeability-surface product might suggest tions (Kp) toward the unbound ratio (Kp,uu) comprising brain ECF and unbound blood concentrations (Liu and Chen, 2015). transporter involvement (Cisternino et al., 2013). Finally, a Kp,uu value in excess of unity would suggest facilitated trans- Kp,uu is a steady-state distribution term denoting the unbound concentration gradient across the BBB (Gupta fer of unbound drug across a cellular barrier and the estab- lishment of a concentration gradient. An example of this is et al., 2006; Hammarlund-Udenaes et al., 2008). If Kp,uu is substantially lower than unity, then drug passage across the gabapentin, where the uptake into brain intracellular fluid BBB is restricted by some factor, such as poor permeability (Fridén et al., 2007) supported the involvement of LAT1 carrier-mediated transport (Su et al., 1995). Fewer literature examples are available demonstrating dx.doi.org/10.1124/jpet.116.232447. uptake into the CNS relative to drug efflux; presumably, this s This article has supplemental material available at jpet.aspetjournals.org. is because drug efflux has a far more negative impact on drug ABBREVIATIONS: aCSF, artificial cerebrospinal fluid; BBB, blood-brain barrier; CNS, central nervous system; ECF, extracellular fluid; ER, efflux ratio; GF 120918A, XXX 9,10-dihydro-5-methoxy-9-oxo-N-[4-[2-(1,2,3,4-tetrahydro-6,7-dimethoxy-2-isoquinolinyl) ethyl]phenyl]-4-acridine-carboxamide; GSK, GlaxoSmithKline; HPLC, high-performance liquid chromatography; Kp, total concentration of gradient across the BBB; Kp,uu, unbound concentration of gradient across the BBB; MS/MS, tandem mass spectrometry; PBS, phosphate-buffered saline; P-gp, P-glycoprotein; pKa, acidic and basic ionization constants; SD, Sprague-Dawley; TEER, transepithelial electrical resistance. 294 Uptake of CNS Drugs and Candidates across the BBB 295 efficacy measurements during lead optimization and early steady-state blood concentrations (taken as at least 3.5 half-lives) drug development and is therefore identified more readily in were determined separately for each compound, and were based on favorable chemical templates. However, K provides a prior studies to establish the intravenous pharmacokinetics. Blood p,uu m powerful tool for probing the prevalence of facilitated uptake samples (25 l) were taken from the tail vein at 0.5-hour intervals in CNS drugs or lead optimization compounds and whether during the last 2 hours of the infusion to confirm steady-state blood concentrations had been achieved. At the end of the infusion period, there are any key structural features or physicochemical the mice were decapitated and the brain removed and homogenized properties that promote the passage of unbound drug across with an equal weight of purified water (ELGA maxima; ELGA Lab- the BBB. In this study, we evaluated Kp,uu across approxi- Water, High Wycombe, UK). Triplicate weighed aliquots (50 ml) of brain mately 50 CNS-targeted compounds. These Kp,uu values were homogenate were stored at ca. 280°C prior to HPLC/tandem mass derived from steady-state intravenous CNS penetration stud- spectrometry (MS/MS) analysis. Samples were extracted by a method ies in rodents [rat, mdr1a/b(2/2) mice, and mdr1a/b(1/1) based on protein precipitation using acetonitrile (250 mlcontaininga mice] to yield Kp, values which were then corrected for suitable internal standard). Brain concentrations were corrected for nonspecific binding to blood and brain. Intracerebral micro- residual blood volume using 15 ml/g of brain tissue as the vascular space (Brown et al., 1986). dialysis was performed on two examples in which Kp,uu was substantially greater than unity (bupropion and clozapine) Preparation for Rat Steady-State Intravenous Brain Distri- bution Studies. Adult male SD rats (body weight 274–376 g; with paracetamol included as a control (e.g., K 1). An p,uu Charles River UK Ltd.) were used. Each rat was cannulated via the assessment of Kp,uu across the lead optimization space within jugular vein (for drug administration) and femoral vein (for blood Downloaded from GlaxoSmithKline (GSK) is also presented as a guide to the sampling) while under isoflurane anesthesia, the cannulae being likelihood of other structural hotspots promoting increased exteriorized at the back of the neck. Animals were placed in a jacket brain distribution of unbound drug. In this case, brain and tether and allowed to recover for at least 2 days prior to dosing. distribution information was collated from both steady-state The dose solution for each test compound was prepared on the day of and acute CNS penetration studies. dosing using an appropriate formulation for intravenous administra- tion. Three rats were dosed for each compound. The infusion rate and infusion time were determined separately for each compound. Blood jpet.aspetjournals.org samples were taken at 0.5-hour intervals during the last 2 hours of the Materials and Methods infusion to confirm steady-state blood concentrations had been Compound Selection. Fifty-three molecules were examined in achieved. At the end of the infusion period, the rats were exsangui- the CNS compound set (Supplemental Table 1 for structures) and were nated and decapitated, and the brain was removed. Blood samples (ca. selected on the basis of their spread in physicochemical properties 80 ml) were collected into tubes containing K2EDTA as anticoagulant (Summerfield et al., 2006). The following compounds were obtained and aliquoted immediately after collection. An aliquot (50 ml) of each from commercial sources: risperidone, chlorpromazine, bupropion, blood sample was diluted with an equal volume of purified water and pergolide mesylate, amitriptyline, diazepam, meprobamate, and tema- stored at ca. 280°C to await analysis. Each brain was homogenized with at ASPET Journals on September 27, 2021 zepam (Sigma Chemical Company, Poole, Dorset, UK); and venlafaxine, an equal weight of purified water (ELGA maxima; ELGA LabWater, citalopram, pemoline, and midazolam (Sequoia Research Products Ltd., High Wycombe, UK). Weighed aliquots (50 ml) of brain homogenate Oxford, UK). All other compounds were available from in-house sources.