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. were taken and stored at ca. 280°C prior to HPLC/MS/MS analysis. Compounds used as retrodialysers for microdialysis were purchased as Samples were extracted by a method based on protein precipitation follows: fluperlapine (Biomol International L.P., Plymouth Meeting, using acetonitrile (250 ml containing a suitable internal standard). 13 15 PA) and [ C2, N]acetaminophen (Cambridge Isotope Laboratories, Brain concentrations were corrected for residual blood volume using 2 m Andover, MA). [ H6]Bupropion was synthesized at GlaxoSmithKline. 15 l/g as the vascular space (Brown et al., 1986). All other chemicals were of analytical grade, and all solvents were of Preparation for Steady-State Intravenous In Vivo Brain high-performance liquid chromatography (HPLC) grade. Microdialysis Studies. Adult male SD rats (body weight 274–376 g; Animals. Adult male Sprague-Dawley (SD) rats (body weight Charles River UK Ltd.) were used. Prior to surgery, rats received 274–376 g; Charles River UK Ltd., Tranent Edinburgh, Scotland) antibiotic (ampicillin or Oxytetracycline) and analgesic (carprofen). were used. Male FVB mdr1a/b(2/2) knockout mice and genetically Under isoflurane anesthesia, rats were cannulated via a femoral vein matched mdr1a/b(1/1) wild-type expressing P-gp were purchased into the vena cava (for drug administration) and jugular vein (for blood from Taconic Farms, Inc. (Germantown, NY). Mouse unbound brain sampling) (Griffiths et al., 1996). Following venous cannulation, and blood fraction determinations by means of equilibrium dialysis animals were placed in a stereotaxic frame (David Kopf Instruments, were conducted in male CD-1 mice (Charles River Laboratories, Tujunga, CA), and a 20-mm midline incision was made to expose the Margate, UK); this mouse strain was selected based on availability skull surface. The periosteum was infiltrated with local anesthetic together with the fact that internal and external work has showed (0.15 ml of mepivacaine hydrochloride) and reflected to expose the little impact in nonspecific brain binding across species (Summerfield bregma. Measurements were taken from the bregma using appropri- et al., 2008) and within strain (Kalvass and Maurer, 2002). All animal ate anterior and lateral stereotaxic coordinates (anterior, 13.2 mm; studies were ethically reviewed and carried out in accordance with lateral, 11.2 mm), and a hole was drilled for the guide probe. Four Animals (Scientific Procedures) Act 1986 and the GSK Policy on the further holes were drilled in the skull surface to enable fixing screws to Care, Welfare and Treatment of Animals. be inserted. The guide probe was lowered (ventral, –2.0 mm; all Preparation for Mouse Steady-State Intravenous Brain coordinates according to Pellegrino et al., 1979) and cemented into Distribution Studies. Each mouse (at least 6 weeks old at the time position, and the was skin sutured around the headcap as previously of surgery) was cannulated via the jugular vein (for drug administra- described (Criado et al., 2003). Jugular and femoral cannulae were tion) while under isoflurane anesthesia, with the cannula exteriorized exteriorized at the back of the neck. Animals were placed in jackets at the back of the neck. Animals were placed in a jacket and tether and collars, housed individually, and allowed to recover for 72 hours counterbalance system and allowed to recover for at least 3 days prior prior to dosing. to dosing. Three male control mdr1a/b (1/1) and three male mdr1a/b In vivo Studies to Determine the Concentration of Test (2/2) knockout mice were dosed with each compound. Compounds in Blood, Brain, and Cerebrospinal Fluid Follow- The dose solution for each test compound was prepared on the day ing Intravenous Administration to Steady State. Microdialysis of dosing using an appropriate formulation for intravenous admin- probes (CMA11, 2-mm probe length; CMA Microdialysis, Kista, istration. The infusion rate and infusion time required to reach Sweden) perfused with artificial cerebrospinal fluid (aCSF; 145 mM 296 Summerfield et al.

NaCl, 2.7 mM KCl, 1.2 mM CaCl2.2H20, 1.0 mM MgCl2.6H20, 2.0 mM loss of retrodialyser and recovery of test compound following the Na2HPO4, pH 7.4) at a flow rate of 1.5 ml/min were inserted through in vivo phase: the previously implanted guide cannula into the cortex. Rats were – placed in Perspex bottom Raturn bowls (BASi, West Lafayette, IN), Cin Cout Lossin vitro 5 (2) were freely moving, and had access to food and water ad libitum Cin throughout the experiment. Probes were dialyzed with aCSF for at least 1 hour prior to dosing to allow tissue equilibration (Chaurasia where Cout is the concentration in the dialysate and Cin is the et al., 2007). concentration in the perfusate, and Dose solutions of test compounds and reference solutions for C retrodialysers were prepared on the day of dose administration and Recovery 5 out (3) in vitro C filtered before administration. Aliquots of dose solutions and reference in solutions were collected prefilter, postfilter, and postdose and stored where Cout is the concentration in the dialysate and Cin is the at approximately 280°C prior to analysis by HPLC-MS/MS. Dose concentration in the bathing solution. Recovery values used to solutions of test compounds were prepared in an appropriate formu- estimate Cubrain,ECF were selected based on the most appropriate lation for intravenous administration. Test compounds were admin- method for each test compound. istered as constant rate intravenous infusions over 6 or 8 hours via the Bioanalysis of Blood, CSF, ECF, and Brain Homogenate femoral vein cannula. The infusion rate and infusion time were Samples. Aliquots of blood, brain, and CSF (50 ml) and microdialyste determined for each test compound from pharmacokinetic data (22.5 ml) were extracted by means of protein precipitation (250 ml) generated previously “in house” (data not shown) to achieve targeted containing a suitable internal standard. The performance of internal Downloaded from – m steady-state blood concentrations (Css, range 0.1 1 g/ml) designed to standard chosen was considered to be acceptable by the analyzers in optimize quantitative measurement of test compounds. The actual all cases. Calibration curve ranges and analytical conditions are dose rates used (mg/kg/h) and the subsequent blood clearances detailed in Supplemental Table 2. Quality control samples (n 5 3per (ml/min/kg) were as follows: acetaminophen, 2887 6 12 mg/kg/h and concentration) accompanied the calibration curves at the assay limit of 45 6 2 ml/min/kg; clozapine, 1474 6 19 mg/kg/h and 115 6 quantification (LLQ; 4 LLQ), the geometric mean of the calibration 13 ml/min/kg; and bupropion, 1826 6 106 mg/kg/h and 127 6 range, and the upper limit of quantification. All samples were analyzed 23 ml/min/kg. Reference solutions were prepared at a concentration by means of LC-MS/MS on a PE-Sciex API-4000 tandem quadrupole jpet.aspetjournals.org of 250 ng/ml retrodialyser in aCSF containing 0.1% (v/v) dimethylsulf- mass spectrometer (Applied Biosystems, Ontario, Canada) using a oxide. Reference solutions were administered via the probe prior to Turbo V Ionspray operated at a source temperature of 700°C (80 psi of and throughout the i.v. infusion period. nitrogen). Samples (3–10 ml) were injected using a CTC Analytics HTS Serial blood samples were collected via the jugular cannula at Pal autosampler (Presearch, Hitchin, UK). regular intervals throughout the infusion to characterize the blood Equilibrium Dialysis Experiments. A 96-well equilibrium di- concentration-time profile and confirm the steady-state blood concen- alysis apparatus was used to determine the free fraction in the blood trations had been achieved (Cblood). Blood samples (ca. 90 ml) were and brain for each drug (HT Dialysis LLC, Gales Ferry, CT). at ASPET Journals on September 27, 2021 collected into tubes containing K2EDTA as anticoagulant, a 50-ml Membranes (3-kDA cutoff) were conditioned in deionized water for aliquot diluted with an equal volume of purified water and stored at 40 minutes, followed by conditioning in 80:20 deionized water:ethanol approximately 280°C prior to analysis by HPLC-MS/MS. For the for 20 minutes, and then rinsed in deionized water before use. Rat determination of brain ECF concentrations (Cubrain,ECF) and probe brains were obtained fresh on the day of the experiment and were recovery, serial aCSF samples were collected at regular intervals homogenized with PBS to a final composition of 1:2 brain:PBS by throughout the infusion period into a refrigerated fraction collector means of ultrasonication (Tomtec Autogiser; Receptor Technologies, maintained at 4°C. Adderbury, Oxon, UK) in an ice bath. Diluted brain homogenate Following the end of the infusion, rats were exsanguinated under was spiked with the test compound (1000 ng/g), and 100-ml aliquots anesthesia (Euthatal [Merial Animal Health, Woking, UK] adminis- (n 5 6 replicate determinations) were loaded into the 96-well tered via the jugular vein cannula), CSF samples were collected from equilibrium dialysis plate. Dialysis versus PBS (100 ml) was carried the cisterna magna for determination of CSF concentration (CCSF), and out for 5 hours in a temperature-controlled incubator at 37°C (Stuart the brains were removed. A 50-ml aliquot of exsanguinated blood Scientific, Watford, UK) using an orbital microplate shaker at 125 rpm samples was diluted with an equal volume of deionized water and (Stuart Scientific). At the end of the incubation period, aliquots of stored at approximately 280°C prior to analysis by HPLC-MS/MS for brain homogenate or PBS were transferred to Matrix ScreenMate determination of drug blood concentration. Another aliquot of exsan- tubes (Matrix Technologies, Hudson, NH), and the composition in each guinated blood samples was diluted with an equal volume of phosphate- tube was balanced with control fluid such that the volume of PBS to buffered saline (PBS) for determination of the unbound drug fraction brain was the same. Sample extraction was performed by the addition m (fublood) by equilibrium dialysis and stored at 4°C. Brains were rinsed in of 200 l of acetonitrile containing an internal standard. Samples were chilled saline, and superficial blood vessels were removed, weighed, and allowed to mix for 15 minutes and then centrifuged at 2465 g in homogenized with an equal volume of PBS. Brain homogenate was 96-well blocks for 20 minutes (Eppendorf 5810R; VWR International, stored as either weighed aliquots (50 ml) at 280°C for determination of Poole, Dorset, UK). The unbound fraction was determined as the ratio of the peak area in buffer to that in brain, with correction for dilution total brain concentration (Cbrain) by HPLC-MS/MS, or as bulk at 4°C for factor according to following equation (Kalvass and Maurer, 2002): determination of the unbound drug fraction (fubrain) by equilibrium dialysis. Total brain concentrations were corrected for residual blood ð1=DÞ volume using a value of 15 ml/g as the vascular space (Brown et al., fu 5    (4) 1986). 1 – 1 1 fu 1 D Probe Calibration. Probes were calibrated by retrodialysis apparent in vivo with probe recoveries being calculated as follows: where D 5 dilution factor in brain homogenate, and fuapparent is the – measured free fraction of diluted brain tissue. 5 Cin Cout Lossin vivo (1) MDR1-MDCKII Cell Passive Membrane Permeability and Cin P-gp Transport Measurements. Cell culture media, bovine serum where Cout is the concentration in the dialysate, Cin is the concentra- albumin, medium supplements (L-glutamine, 20 mM sodium bicar- tion in the perfusate, and percentage of loss is assumed to equal bonate, nonessential amino acids, sodium pyruvate, colchicine), and percentage of recovery. Similarly, probes were calibrated in vitro by Hanks’ balanced salt solution was purchased from Invitrogen Uptake of CNS Drugs and Candidates across the BBB 297

(Carlsbad, CA). Transwells (12-well, 1.13-cm2 area, 0.4-mm pores) polar surface area was calculated by the method of P. Ertl, and only N were purchased from Costar (Cambridge, MA). Krebs-Ringer bicar- and O are considered as polar atoms (Ertl et al., 2000). Hydrogen bond bonate buffer was obtained from Sigma-Aldrich (St. Louis, MO), and acceptor and hydrogen bond donor were calculated with any N, O, or S drugs were supplied by GlaxoSmithKline (Stevenage, UK). atoms with a lone pair, excluding basic amines and anilines, etc. Multidrug resistance Madin-Darby canine kidney (MDR1-MDCK) type II cells were obtained from the National Institutes of Health (Bethesda, MD). Cells were maintained in minimum essential Eagle’s Results medium containing 2 mM L-glutamine, 20 mM sodium bicarbonate, 0.1 mM nonessential amino acids, 1 mM sodium pyruvate, and 10% Table 1 shows rat and mouse brain Kp,uu values and in vitro bovine serum albumin supplemented with 0.2 mM colchicine to P-gp efflux ratios (ERs) for a series of CNS-targeted molecules. maintain P-gp expression. For permeability experiments, cells with Kp,uu covers a 320-fold range from 0.022 (ergotamine) to 7.1 passage numbers 24–33 were seeded at a density of 60,000 cells/cm2 on (perphenazine). Atenolol is included as a marker of the rat type I collagen-coated polycarbonate membranes in 12-well trans- extravascular brain volume (Kp,uu 5 0.012; Summerfield well plates. The experiments were performed on the eighth day after et al., 2007). Individual Kp and unbound fraction values are seeding. Prior to the permeation assay, the transepithelial electrical supplied in Supplemental Table 3. resistance (TEER) was measured on each cell monolayer using an In Fig. 1, K is plotted versus increasing in situ BBB Endohm Voltameter (World Precision Instruments, Sarasota, FL). p,uu permeability (data reported in Summerfield et al., 2008) and The MDR1-MDCK cell systems used in transport experiments had a V 2 shows no strong correlation between rate of drug passage

high TEER value ranging from 1800 to 2000 cm , which differs from Downloaded from lower TEER MDR1-MDCK models often used in CNS screens (Braun across the BBB and the extent of distribution at steady state et al., 2000). The permeability assay buffer was Hanks’ balanced salt (Hammarlund-Udenaes et al., 2008; Summerfield and Dong, solution containing 10 mM HEPES and 15 mM glucose at pH 7.4. The 2013). Several compounds that are characterized have low cells were dosed on the apical side and basolateral side, and incubated Kp,uu values (,0.5) and are identified as substrates for at 37°C with 5% CO2 and 90% relative humidity under shaking human P-gp in vitro (ER . 2): sumatriptan (Kp,uu 5 0.034, conditions (150 rpm) to reduce the influence of the unstirred water ER 5 2.9), primidone (Kp,uu 5 0.38, ER 5 3.6), amoxapine layer. The test compounds were prepared to a final concentration of (K 5 0.35, ER 5 4.1), rizatriptan (K 5 0.043, ER 5 jpet.aspetjournals.org m p,uu p,uu 3 M. In the P-gp inhibition assays, the cells were incubated with 8.4), metoclopramide (K 5 0.40, ER 5 13), risperidone 2 mM GF 120918A (9,10-dihydro-5-methoxy-9-oxo-N-[4-[2-(1,2,3,4- p,uu (K 5 0.20, ER 5 21), thiothixene (K 5 0.13, ER 5 48), tetrahydro-6,7-dimethoxy-2-isoquinolinyl) ethyl]phenyl]-4-acridine- p,uu p,uu 5 5 5 carboxamide), a known P-gp inhibitor. Drug permeation was tested ergotamine (Kp,uu 0.022, ER 50), and mesoridazine (Kp,uu in two directions, apical-to-basolateral (A-to-B) and basolateral-to- 0.10, ER 5 87). Gabapentin is also associated with a relatively apical (B-to-A), in triplicate. Sampling was done 60 minutes after low rat brain Kp,uu value of 0.44, although it is subject to dosing. The apparent permeability coefficient (Papp)wascalculated facilitated transport (Su et al., 1995). as follows: Several of the CNS-targeted molecules possess tricyclic at ASPET Journals on September 27, 2021   groups (Fig. 2) and were also characterized by rat brain Kp,uu 5 dCr× Vr values greater than 2; this includes the phenothiazine Papp × (5) dt A C0 moiety of chlorpromazine, perphenazine, thioridazine, and . where dCr/dt is the slope of the cumulative concentration in the trifluorperazine. Related tricyclic ring systems lead to Kp,uu receiver compartment versus time in mM/s, Vr is the volume of 2 for clozapine, olanzapine, doxepin, imipramine, loxepin, transport assay buffer in the receiver compartment, A is the area of mianserin, and mirtazapine. For the tricyclic CNS drugs the cell monolayer, and C0 is the drug concentration in the dosing where Kp,uu is close to or less than unity, BBB efflux appears solution. The recovery of most compounds exceeded 80%. The bi- to occur. From steady-state CNS penetration studies using directional efflux ratio was determined as follows: wild-type and genetic P-gp knockout mice, maprotiline is identified as a moderate P-gp substrate (Table 1). Indeed, the P ð Þ 5 app B-A Efflux ratio (6) mouse brain Kp,uu of maprotiline increases from 1.42 to P ð Þ app A-B 3.06 between the wild-type and mdr1a/b(2/2) knockout mice, P-gp efflux was confirmed by determining the bidirectional efflux ratio which suggests the presence of competing efflux and uptake in the presence of elacridar (2 mM) and observing an efflux ratio of processes. Similarly, amitriptyline has been reported as a unity. P-gp substrate based on in vivo knockout studies (Uhr et al., Data Analysis. Total blood concentrations were measured in 2000, 2007; Abaut et al., 2009). Sun et al. (2006) reported serial blood samples. Total Cblood values were determined using the multiple transporters involved in the passage of carbamaze- mean measured blood concentrations during the final hours of the i.v. pine across cultured rat brain microvascular endothelial cells. infusion once steady state had been achieved. Total Cbrain values were For fluphenazine, the rat brain K is 1.58 but increases to measured in terminal brain homogenate samples, and C was p,uu CSF over 6 in both mdr1a/b(1/1) and mdr1a/b(2/2) mice (Table 1). measured in terminal CSF samples. Cbrain was used with Cblood to determine the CNS penetration of test compounds. Amoxapine and thiothixene are identified as P-gp substrates in vitro (Table 1) but are not entirely excluded from the rat The unbound brain-to-blood concentration, Kp,uu, was determined as follows, where Cu represents the unbound concentration in the brain since their respective Kp,uu values are 0.35 and 0.13. The relevant compartment: brain Kp,uu for tacrine was determined as 1.61 in rats (Table 1), increasing to over 2 in mice (Table 1). Striatal Cubrain;ECF Kp;uu 5 (7) extracellular fluid concentrations of tacrine were shown to Cublood decrease with increasing dose (Sung et al., 2005), and trans- Molecular and Physicochemical Descriptors. Acidic and ba- porter studies in human embryonic kidney 293 cells suggested sic ionization constants (pKa) were calculated from ChemAxon version the involvement of multiple cationic transporter systems in 5.4.1.1 (ChemAxon Kft, Budapest, Hungary). cLogP and cLogD7.4 rats. Quetiapine and midazolam are the only tricyclic exam- were calculated using the ChemAxon .NET 5.11.4.30. Topological ples in this compound set where the rat brain Kp,uu values are 298 Summerfield et al.

TABLE 1

Brain Kp,uu values for rat and mouse [mdr1a/b(+/+) and mdr1a/b(2/2)] quoted as mean 6 standard deviation

Three rats and mice (n = 3) were used for each drug. Kp values derived from oral dosing studies.

Kp,uu Kp,uu Kp,uu

In Vitro P-gp 2 2 CNS Drug Efflux Ratio Rat mdr1a/b(+/+) Mouse mdr1a/b( / ) Mouse Mean S.D. Mean S.D. Mean S.D. Atenolol — 0.012 0.003 ——— — Amantadine 0.8 3.01 0.85 ——— — Amitriptyline 1.9 1.33 0.40 ——— — Amoxapine 4.1 0.35 0.17 0.23 0.06 0.55 0.10 Atomoxetine 3.4 2.13 0.85 2.35 0.17 4.10 0.35 Bupropion 1.4 4.97 0.91 6.14 2.54 5.41 0.52 Carbamazepine 0.8 0.76 0.20 ——— — Chlorpromazine 0.8 3.41 0.81 2.79 0.42 3.19 0.45 Citalopram 20.1 1.30 0.33 1.47 0.17 4.58 0.48 Clozapine 1.3 3.83 0.80 2.51 0.14 2.84 0.32 Diazepam 1.0 0.96 0.16 ——— —

Donepezil 2.3 1.77 0.23 1.19 0.09 2.64 0.29 Downloaded from Doxepin 1.9 3.11 0.59 ——— — Ergotamine 49.6 0.02 0.01 ,0.01 — 0.30 0.12 Ethosuximide 0.8 1.50 0.75 ——— — Fluoxetine 1.2 5.23 1.87 ——— — Fluphenazine 2.3 1.58 0.57 6.12 1.31 7.77 1.25 Gabapentin 0.8 0.44 0.11 ——— — Haloperidol 1.3 1.77 0.71 1.54 0.21 1.89 0.49 a — ——— —

Imipramine 3.52 0.32 jpet.aspetjournals.org Isocarboxazid 1.2 0.18 0.04 ——— — Lamotrigine 1.6 4.86 0.72 1.00 0.11 1.09 0.08 Loxapine 1.2 2.95 0.89 ——— — Maprotiline 4.0 0.98 0.47 1.42 0.44 3.06 0.45 Meprobamate 3.3 0.77 0.10 0.61 0.10 0.80 0.20 Memantinea — 6.21 1.56 ——— — Mesoridazine 87.1 0.10 0.03 0.19 0.02 3.30 0.73 Metoclopramide 13.2 0.40 0.08 0.90 0.15 6.23 0.83 Mianserina — 3.22 0.43 ——— — at ASPET Journals on September 27, 2021 Midazolam 2.0 1.08 0.10 ——— — Mirtazapine 1.3 3.85 0.50 ——— — Olanzapine 4.9 2.13 0.60 6.86 1.52 9.53 1.38 Pemoline 1.2 0.55 0.15 ——— — Pergolide 1.8 3.19 0.59 ——— — Perphenazine 4.7 7.12 2.17 5.99 1.53 6.17 1.58 Phenelzine 1.4 1.48 0.34 ——— — Phenytoin 2.8 0.66 0.11 3.05 0.42 2.79 0.37 Primidone 3.6 0.38 0.10 0.68 0.10 1.25 0.22 Quetiapine 1.5 1.26 0.14 1.15 0.20 1.27 0.34 Risperidone 20.8 0.21 0.04 0.18 0.07 2.29 0.42 Rizatriptan 8.4 0.04 0.01 0.40 0.27 0.53 0.10 Selegiline 0.8 1.08 0.17 ——— — Sertraline 2.6 2.67 0.51 3.87 1.03 4.22 1.52 Sumatriptan 2.9 0.04 0.01 0.02 0.01 0.08 0.02 Tacrine 1.1 1.61 0.56 2.25 0.83 2.68 0.97 Temazepam 1.6 0.80 0.12 ——— — Thioridazine 33.7 3.50 1.67 ——— — Thiothixene 47.7 0.13 0.04 0.34 0.06 3.67 0.47 Tiagabine 25.8 0.62 0.21 0.77 0.14 1.17 0.20 Trazodone 1.1 1.54 0.28 ——— — Trifluoperazine 7.3 3.35 1.10 2.18 0.47 3.35 0.70 Venlafaxine 4.7 1.91 0.30 3.49 0.71 5.43 0.98 Zaleplon 5.6 0.59 0.10 0.77 0.13 1.38 0.23 Ziprasidone 1.7 0.75 0.22 4.10 0.91 7.17 1.85 Digoxin ———0.12 0.03 3.41 0.42

—, not determined. a Kp values derived from oral dosing studies. close to unity but show no propensity for efflux by P-gp in vitro a common tricyclodecanamine moiety, and a concentration or in vivo (Table 1). Currently, no further information is dependency on the BBB permeability of memantine has been reported in the literature to suggest whether quetiapine is reported that arises from facilitated transport via a cationic subject to efflux via another transport system. proton antiporter (Mehta et al., 2013). Eight additional compounds were characterized by Kp,uu The rat brain Kp,uu of lamotrigine was determined as 4.86 in values greater than 2 (Fig. 2): amantadine, atomoxetine, this study, which accords with it being a substrate for OCT1 in bupropion, fluoxetine, lamotrigine, memantine, pergolide, human brain endothelial cells (Dickens et al., 2012). It is and sertraline. Of these, amantadine and memantine share noteworthy that the mouse brain Kp,uu for lamotrigine is much Uptake of CNS Drugs and Candidates across the BBB 299 Downloaded from jpet.aspetjournals.org

Fig. 1. Bar graph showing steady-state Kp,uu values for CNS drugs, ordered along the x-axis in terms of increasing in situ BBB permeability (reference data reported in Summerfield et al., 2008). closer to unity (Table 1), but there is no indication of efflux by active transport (Wright, 1972). Mulac et al. (2012) also

P-gp substrate (Table 1). The unbound blood fractions in both reported ergotamine as a breast cancer resistance protein at ASPET Journals on September 27, 2021 rat and mouse are comparable for lamotrigine (0.28 and 0.29, substrate based on in vitro observations, and hence, bidirec- respectively), as are the unbound brain fractions (0.27 and tional transport may be occurring. Last, sertraline was 0.20, respectively). Taken together, these data may support a measured with a rat brain Kp,uu of 2.67 and a mouse brain species difference in the BBB transport of lamotrigine be- Kp,uu around 4. In contrast, Doran et al. (2005) reported the tween rat and mouse. mouse brain Kp,uu of sertraline as 1.5 following subcutaneous The rat brain Kp,uu of fluoxetine was determined as 5.23 in administration, rather than i.v. infusion to steady-state used this study. In contrast, results from Doran et al. (2005) in this study. suggested that the mouse brain Kp,uu was close to unity in Mdr1a/b Mouse Model. Brain penetration data collected both mdr1a/b(1/1) or mdr1a/b(2/2) mice. Fluoxetine is sub- in mice highlight several CNS-targeted molecules where the ject to an extremely high degree of nonspecific brain tissue brain Kp,uu increases markedly above unity following P-gp binding, and fubrain was measured as 0.00094 in mice (Doran knockout (Table 1); these drugs are citalopram (Kp,uu 5 4.58), et al., 2005) but was around 4-fold higher in this study at risperidone (Kp,uu 5 2.29), ziprasidone (Kp,uu 5 7.17), digoxin 0.0040. Since greater variability is likely to be seen at the (Kp,uu 5 3.41), mesoridazine (Kp,uu 5 2.79), thiothixene (Kp,uu 5 lowest extremity of the fubrain assay, the high Kp,uu could be an 3.67), metoclopramide (Kp,uu 5 6.23), and venlafaxine (Kp,uu 5 experimental artifact in this study, although comparable data 7.59). Mesoridazine and thiothixene both possess the pheno- in a single species are not available for fluoxetine. It is thiazine moiety but are also strong P-gp substrates. For noteworthy that the structural analog atomoxetine was citalopram, Bundgaard et al. (2012) reported an increase in characterized by a rat brain Kp,uu of 2.13 and increased from the mouse brain Kp,uu to 4.8 in P-gp–deficient mice. Although 2.35 to 4.10 in the P-gp knockout mouse model (Table 1). the primary focus of this work was on a P-gp species difference, the Conversely, in vivo microdialysis determined the Kp,uu of putative involvement of an uptake mechanism has been recog- atomoxetine as 0.7 (Kielbasa et al., 2009). The unbound nized (Rochat et al., 1999). blood/plasma fractions are comparable between these studies, For risperidone, the mouse brain Kp increased from 0.78 to although Kp differs almost 2-fold between this study (Kp 5 19) 8.02 in the study reported by Doran et al. (2005), which and the following microdialysis (Kp 5 11). equates a change in Kp,uu from 0.26 to 2.63, respectively, when Pergolide is an ergot derivative characterized by a rat brain correcting for the fuplasma and fubrain data cited in the same Kp,uu value of 3.19 and was not identified as a P-gp substrate, study. In contrast, Bundgaard et al. (2012) reported Kp,uu in agreement with another reported study (Vautier et al., values around unity in P-gp knockout rats and mice following 2008). Although few reports are available on the transport of infusion to steady state. A structural analog, ziprasidone, was ergotamine itself in vivo, an ergot-containing analog, lysergic characterized by a mouse brain Kp,uu value of 7.17 in our acid, was seen to accumulate in frog choroid plexus cells by an present study, which may point to a potential mechanism for Na/K ATPase–dependent process with the characteristics of facilitated uptake for both structurally related drugs. Digoxin 300 Summerfield et al. Downloaded from

Fig. 2. Structures for CNS drugs con- taining tricyclic moieties. Tricyclic CNS- targeted compounds where rat brain Kp,uu $ 2 (A), tricyclic CNS-targeted compounds where rat brain Kp,uu , 2(B), and other CNS-targeted compounds where rat brain Kp,uu $ 2 (C). *Data available to suggest BBB efflux and/or multiple jpet.aspetjournals.org carrier-mediated transport processes. at ASPET Journals on September 27, 2021

overdose is known to induce CNS symptoms (Kelly and Smith, subsequent mechanistic studies suggested carrier-mediated 1992), and despite P-gp playing a key role in maintaining low transport by means of a proton-coupled organic cation anti- brain concentrations of digoxin, high affinity for a specific porter (Kitamura et al., 2014). organic anion transporter may help to increase Kp,uu above In Vivo Microdialysis. To cross-reference the in vitro unity in the mdr1a/b(2/2) model (Noé et al., 1997; Taub et al., determinations of Cubrain (e.g., Cbrain fubrain,homogenate), two 2011). For metoclopramide, the rat brain Kp,uu (0.40) in this high brain Kp,uu drugs were selected for rat brain micro- study agrees with another report (0.37) (Doran et al., 2012), dialysis (clozapine and bupropion). Acetaminophen was also but no other comparative work in P-gp–deficient species is included as a control because Kp,uu was measured as being available, nor could any literature reports be found that close to unity. Clozapine and bupropion were also selected, as suggest the involvement of carrier-mediated transport for they differ greatly in terms of their unbound blood fractions metoclopramide. Work in transfected human embryonic kid- (0.066 vs. 0.391) and unbound brain fractions (0.011 vs.0.171). ney 293 cells has shown metoclopramide is a substrate for The blood and brain ECF unbound concentration-time profiles OATP1A2 along with several triptans, although this trans- for bupropion are displayed in Fig. 3. The corresponding porter does not have rodent homologs (Cheng et al., 2012). For concentration-time profiles for acetaminophen and clozapine venlafaxine, the Kp,uu was determined to be 1.91 in the rat were reported previously (Summerfield and Dong, 2013). brain, 4.87 in mdr1a/b(1/1) mice, and 7.59 in mdr1a/b(2/2) For acetaminophen, the unbound concentrations were mice. Recently, its structural isomer tramadol was character- comparable between blood (4.97 6 1.00 mM), brain ECF (3.52 6 ized by a brain Kp,uu of 2.3 following microdialysis, and 0.52 mM), and cisternal CSF (4.95 60.27 mM) (Table 2). Following Uptake of CNS Drugs and Candidates across the BBB 301 Downloaded from

Fig. 3. Unbound drug concentrations of bupropion in blood and brain extracellular fluid following in vivo microdialysis in male Sprague-Dawley rats. jpet.aspetjournals.org Unbound blood concentrations (d) are reported as the mean 6 standard deviation (n = 4); concentrations at individual brain extracellular fluid are reported for rat #1 (X), rat #2 (s), rat #3 (¤) and rat #4 (◻).

microdialysis, the rat brain Kp,uu of acetaminophen was de- For clozapine, the rat brain Kp,uu was determined as 2.2 6 termined as 0.73 6 0.16. Equilibrium dialysis of ex vivo blood and 0.2 following microdialysis, consistent with the data presented brain homogenates from the rats infused with acetaminophen in Table 1. The initial brain ECF measurements for clozapine realized a mean unbound blood fraction of 0.741 6 0.128 were below the quantification limit of the analytical method, at ASPET Journals on September 27, 2021 and a mean unbound brain fraction of 0.724 6 0.050. These but concentrations were measurable at steady state (albeit values were used to correct the total concentrations in blood with more variability than bupropion and acetaminophen). and brain to unbound concentrations, which yields an The unbound concentration of clozapine was highest in ex vivo rat Kp,uu of 0.613 6 0.136. cisternal CSF (0.156 6 0.005 mM), followed by brain ECF In the case of bupropion, the rat brain Kp,uu was determined (0.077 6 0.008 mM), and then lowest in blood (0.036 6 0.002 as 3.3 6 1.7 following microdialysis (range 2.35–5.77). The mM). Equilibrium dialysis of ex vivo blood and brain homog- particular rat associated with the highest Kp,uu value was also enates from the rats infused with clozapine realized a mean characterized by an approximately 2-fold larger brain Kp unbound blood fraction of 0.059 6 0.012 and a mean unbound (18 vs. 10) and 2-fold higher brain ECF concentrations (2.64 brain fraction of 0.0109 6 0.0015. Using the total concentra- vs. 1.28 mM). The unbound concentrations of bupropion were tions in brain and correcting for the ex vivo brain binding gave higher in brain ECF (1.62 6 0.69 mM) relative to both the blood a comparable ex vivo rat brain Kp,uu of 3.18 6 0.50. (0.504 6 0.066 mM) and cisternal CSF (0.250 6 0. 053 mM). Kp,uu Assessments of CNS Drug Candidates from Equilibrium dialysis of ex vivo blood and brain homogenates Lead Optimization. Rat brain Kp,uu data were available for from the rats infused with bupropion realized a mean unbound 771 proprietary compounds synthesized against CNS targets, blood fraction of 0.537 6 0.056 and a mean unbound brain of which Kp,uu was $2 for 41 compounds (5.5%) and $3 for fraction of 0.149 6 0.008. Using the total concentrations in 19 compounds. Of these, CNS penetration measurements brain and correcting for the ex vivo brain binding gave a were taken at steady state for four compounds, whereas the comparable ex vivo rat brain Kp,uu of 3.08 6 0.71. remainder were from acute studies. Table 3 outlines the

TABLE 2 Summary of pharmacokinetic and brain distribution values for acetaminophen, bupropion, and clozapine in rats

Acetaminophen Bupropion Clozapine fu(blood) 0.741 6 0.128 0.537 6 0.056 0.0590 6 0.0120 fu(brain) 0.724 6 0.050 0.149 6 0.008 0.0109 6 0.0015 Cublood (mM) 4.97 6 1.00 0.504 6 0.066 0.036 6 0.002 Cubrain ECF (mM) 3.52 6 0.52 1.62 6 0.69 0.077 6 0.008 Cisternal CSF (mM) 4.95 6 0.27 0.250 6 0.053 0.156 6 0.005 Kp,uu ECF 0.73 6 0.16 3.30 6 1.70 2.16 6 0.23 Cubrain estimated (mM) 3.13 6 0.21 1.64 6 0.08 0.124 6 0.018 Ex vivo Kp,uu 0.613 6 0.136 3.10 6 0.70 3.18 6 0.50 302 Summerfield et al.

TABLE 3

Proprietary CNS drug candidates with Kp,uu $ 2 in rat and their physicochemical properties

Rat Kp,uu MW cLogP cLogD7.4 tPSA HBA HBD pKa (Most Acidic) pKa (Most Basic) Max 11.6 484 7.5 7.5 85.8 5 2 14 9.9 Min 2.01 221 1 0.3 3.2 0 0 5.9 0 Median 2.91 403 3.7 2.8 47.5 2 1 14 7.6

HBA, hydrogen bond acceptor; HBD, hydrogen bond donor; MW, Molecular Weight; tPSA, topological polar surface area.

physicochemical properties of these high Kp,uu compounds. Donepezil poses an interesting example of potential cross- Generally, the number of hydrogen bond donors and acceptors species differences in Kp,uu. Being a modest P-substrate in the was low (median values of 1 and 2, respectively), and many mouse, the donepezil Kp,uu increases from 1.19 and 2.64 were characterized by reasonably acidic pKa values. Of these (Table 1), whereas Kim et al. (2010) noted concentration- CNS discovery candidates, a wide range of structures were dependent transport of donepezil and proposed this to be noted that were mostly distinct from the CNS compound set, mediated by organic cation transporters, such as the choline presumably because of the novel CNS targets being prose- transport system. Recently, we reported the minipig Kp of cuted in lead optimization. Four of these proprietary mole- donepezil as 14.9, identified as compound “L” following a 1-hour Downloaded from cules were characterized by fused ring systems, but in general, i.v. infusion at a dose of 1 mg/kg (Summerfield and Dong, 2013). the chemotypes were not comparable to those detailed in the The unbound fractions in plasma and brain were 0.260 6 0.027 CNS compound set. and 0.162 6 0.017, realizing a minipig brain Kp,uu of 7.2. In In comparison, the CNS-targeted compound set was char- humans, the brain Positron Emission Tomography (PET) bio- 11 acterized by 19 out of 53 (36%) with Kp,uu $ 2, and 15 (28%) distribution of C-donepezil was approximately 20 in healthy jpet.aspetjournals.org with Kp,uu $ 3. Analysis of physicochemical properties elderly subjects (Okamura et al., 2008), which translates to a associated with rat Kp,uu values revealed that drugs with Kp,uu estimate of 10, after correcting for plasma protein bind- Kp,uu . 2 had reduced topological polar surface area as ing (Nagy et al., 2004) and assuming the brain tissue binding compared with drugs with Kp,uu , 0.5 or 0.5 # Kp,uu # 2 is comparable across species (Summerfield et al., 2008). (Supplemental Fig. 1; Supplemental Table 1). However, the Given the interplay between passive transfer across the compound set is not large enough to form a definitive conclu- BBB and the potential for competing efflux, experimental sion. A more detailed analysis in relationships between com- conditions are likely to play an important part in whether a pound physicochemical properties and rat Kp,uu values in a drug candidate is identified as a substrate for drug uptake. at ASPET Journals on September 27, 2021 larger data set was published elsewhere (Zhang et al., 2016). Absolute Kp,uu values may be dependent on the circulating Selection of the compound set was originally conceived to unbound concentration bathing the BBB (e.g., the dose represent the span of physiochemical properties of molecules administered) and whether any of these transport processes targeting the CNS, e.g., rather than maximizing the span of are subject to saturation. At the limit, brain Kp,uu would targets themselves. The compound set comprises agonists, approach unity if the transport processes are saturated and antagonists, inhibitors, and reuptake inhibitors of various passive diffusion becomes the dominant process of drug important CNS receptors, such as dopamine (e.g., clozapine passage across the BBB. Interestingly, a review by the and perphenazine), serotonin (e.g., risperidone and citalopram), International Transporter Consortium noted that, other than gamma aminobutyric acid (e.g., midazolam and meprobamate), carrier-mediated transfer via P-gp, decreases or increases in noradrenaline (e.g., amoxapine and venlafaxine), and mono- brain Kp were generally less than a few-fold in many trans- amine oxidase (e.g., selegiline and isocarboxazid). Many of these porter knockout models (Kalvass et al., 2013; Tweedie et al., CNS drugs were also approved in an era when pharmacology 2013). screening played a more prominent role in progressing early It is important to recognize not only that Kp,uu is a steady- leads, whereas today’s paradigm relies far more heavily on state parameter, but also that it is most often generated batteries of in vitro Drug Metabolism and Pharmacokinetics indirectly, e.g., by correcting the total brain concentration for (DMPK) screens to triage drug candidates. Evidently, during the unbound brain fraction. Hence, it is imperative that that period, the concept of Kp,uu had not been conceived, and steady-state conditions are achieved to accurately reflect the hence serendipity played a large part in selecting drug brain Kp,uu, which is exemplified in Fig. 4. The BBB initiative candidates subject to uptake across the BBB. that led to many of the publications reported by GSK performed a large battery of in vivo and in vitro tests on this Discussion set of CNS-targeted molecules, including both acute (oral) and steady-state (i.v.) brain distribution studies. Acute studies The introduction of the Kp,uu parameter has provided new were included because many of the early estimates of brain insights into the processes that occur during drug passage across distribution during lead optimization were derived from short- the BBB. This work focused on molecules in which there is term pharmacology models, whereas steady-state brain dis- evidence to support facilitated passage across the BBB—namely, tribution studies were supplemented for promising molecules. where Kp,uu is markedly greater than unity. However, the Comparison of the acute and steady-state brain Kp data present study also highlights examples in which there are clearly highlighted a strong correlation between the two data sets; at least two transport processes acting in opposing directions, however, the slope of correlation was approximately 0.5 such that the overall Kp,uu is closer to unity. Examples of this are rather than unity, the steady-state brain Kp values generally citalopram, metoclopramide, and thiothixene. being higher than the acute Kp values. Figure 4 shows this Uptake of CNS Drugs and Candidates across the BBB 303 Downloaded from

Fig. 4. Scatter plot showing rat brain Kp,uu values derived from brain penetration studies in male Sprague-Dawley rats; the x-axis denotes steady-state jpet.aspetjournals.org data, and the y-axis shows acute data following oral dosing. The dashed line represents the line of unity, and the solid line is a best fit to the data by linear regression. AUC, area under the curve.

correlation in terms of Kp,uu, where the difference is most would still lead to an efficacious effect in vivo, albeit at lower marked for the higher Kp,uu compounds. Similarly, the mouse doses. The traditional Kp measurement would have provided data set originally reported by Doran et al. (2005) has been a no indication of uptake, and discovery pharmacokinetic/ valuable resource for understanding brain distribution in pharmacodynamic models are rarely refined enough to high- at ASPET Journals on September 27, 2021 terms of unbound concentrations, although only one com- light efficacy at an unexpectedly low dose. Indeed, prior to the pound was described by a brain Kp,uu greater than 2 in the inception of Kp,uu, carrier-mediated transport may have only mdr1a/b(1/1) mouse (e.g., methylphenidate; Liu et al., 2008). been speculated when the extent of brain penetration In the corresponding mdr1a/b(2/2) strain, eight compounds are appeared at odds with the physicochemical properties (e.g., characterized by Kp,uu $ 2, e.g., cyclobenzaprine, fluvoxamine, gabapentin and lamotrigine). In the case of lipophilic mole- hydrocodone, methylphenidate, metoclopramide, nortriptyline, cules, which readily traverse the BBB, a difference of a few- propoxyphene, and risperidone. These Kp,uu values were de- fold in brain penetration arising from facilitated uptake termined from the area under the curve measurements follow- mechanism would not be discernible without the determina- ing subcutaneous dosing over 5 hours (concentration sampling tion of Kp,uu. from four time points: 0.5, 1, 2.5, or 5 hours). The acute studies Predicting difference in Kp,uu across species remains a shown in Fig. 4 were performed following oral dosing with blood significant challenge, especially when only rodent data or and brain samples collected up to 8 hours. acute brain distribution studies are performed. So how does Many CNS drugs possessing tricyclic functions, such as the industry obtain better estimates for the human Kp,uu? chlorpromazine and olanzapine, appear to undergo facilitated Take an example such as donepezil, where literature data transfer across the rodent BBB. An uptake mechanism for suggest Kp,uu is far higher in humans than would be antici- clozapine across HL-60 cells has been proposed previously pated from the rodent data. The early estimates of the human (Henning et al., 2002), but further work is required to dose are highly sensitive to the value of brain Kp,uu; a 3-fold characterize whether there is a common carrier-mediated change in Kp,uu would attenuate the corresponding human system or whether this structural element would be useful in dose prediction 3-fold (assuming unbound brain concentra- other CNS-targeted drug candidates. Since the pH values of tions are driving efficacy). In vitro BBB models for human blood and brain ECF are comparable, these increases in brain Kp,uu have been reported based on stem cells (Cecchelli et al., Kp,uu for tricyclic molecules are unlikely to be the result of ion 2014), and the promise is immense for helping progress the trapping, and in general, their acidities and basicities vary next generation of CNS drugs. Although the number of widely (see Supplemental Data File 1; Supplemental Table S2). reported in vitro and in vivo human brain Kp,uu compounds Not all of the examples highlighted in this study (where is currently small, it is noteworthy that both bupropion and Kp,uu is significantly greater than unity) can be supported lamotrigine showed human brain Kp,uu estimates close to by literature data, but perhaps this is unsurprising. Drug unity. However, the estimates for human Kp,uu were made efflux, particularly P-gp, has blighted drug discovery because using CSF concentrations, whereas this may not be a reliable the inherent reduction in brain penetration leads to a corre- surrogate for a variety of reasons (de Lange, 2013). A question sponding reduction in efficacy. However this is not the case for also remains concerning the junction tightness in human cell- uptake across the BBB; higher unbound brain concentrations based systems. Low TEER values could lead to increased 304 Summerfield et al. directional flux of drug across the in vitro BBB system; in Fridén M, Gupta A, Antonsson M, Bredberg U, and Hammarlund-Udenaes M (2007) In vitro methods for estimating unbound drug concentrations in the brain in- particular, Kp,uu values tend to be only a few-fold greater than terstitial and intracellular fluids. Drug Metab Dispos 35:1711–1719. unity. Griffiths R, Lewis A, and Jeffrey P (1996) Models of drug absorption in situ and in conscious animals. Pharm Biotechnol 8:67–84. In summary, the introduction of Kp,uu has enabled a deeper Gupta A, Chatelain P, Massingham R, Jonsson EN, and Hammarlund-Udenaes M insight into the factors affecting the unbound concentration (2006) Brain distribution of cetirizine enantiomers: comparison of three different tissue-to-plasma partition coefficients: K(p), K(p,u), and K(p,uu). Drug Metab gradient across the BBB. In vivo microdialysis studies on Dispos 34:318–323. clozapine and bupropion confirmed the corresponding in vitro Hammarlund-Udenaes M, Fridén M, Syvänen S, and Gupta A (2008) On the rate and K estimates (Table 2). Clearly, examples such as citalo- extent of drug delivery to the brain. Pharm Res 25:1737–1750. p,uu Henning U, Löffler S, Krieger K, and Klimke A (2002) Uptake of clozapine into HL-60 pram and maprotiline demonstrate that a measured brain promyelocytic leukaemia cells. Pharmacopsychiatry 35:90–95. Jeffrey P and Summerfield SG (2007) Challenges for blood-brain barrier (BBB) Kp,uu around unity need not necessarily be the result of a screening. Xenobiotica 37:1135–1151. purely passive transfer process (Table 1). Differences in brain Kalvass JC and Maurer TS (2002) Influence of nonspecific brain and plasma binding distribution across species, concentration dependency of brain on CNS exposure: implications for rational drug discovery. Biopharm Drug Dispos 23:327–338. distribution (in vitro or in vivo), or evidence of uptake from Kalvass JC, Polli JW, Bourdet DL, Feng B, Huang SM, Liu X, Smith QR, Zhang other pharmacokinetic information may highlight the poten- LK, and Zamek-Gliszczynski MJ; International Transporter Consortium (2013) Why clinical modulation of efflux transport at the human blood-brain tial for uptake across the BBB. barrier is unlikely: the ITC evidence-based position. Clin Pharmacol Ther 94: 80–94. Kelly RA and Smith TW (1992) Recognition and management of digitalis toxicity. Am Acknowledgments – –

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Sun JJ, Xie L, and Liu XD (2006) Transport of carbamazepine and drug interactions deficiency due to mdr1a P-glycoprotein gene disruption. Neuropsychopharmacology at blood-brain barrier. Acta Pharmacol Sin 27:249–253. 22:380–387. Sung JH, Yu KH, Park JS, Tsuruo T, Kim DD, Shim CK, and Chung SJ (2005) Vautier S, Milane A, Fernandez C, Buyse M, Chacun H, and Farinotti R (2008) Saturable distribution of tacrine into the striatal extracellular fluid of the rat: Interactions between antiparkinsonian drugs and ABCB1/P-glycoprotein at evidence of involvement of multiple organic cation transporters in the transport. the blood-brain barrier in a rat brain endothelial cell model. Neurosci Lett 442: Drug Metab Dispos 33:440–448. 19–23. Taub ME, Mease K, Sane RS, Watson CA, Chen L, Ellens H, Hirakawa B, Reyner EL, Watson J, Wright S, Lucas A, Clarke KL, Viggers J, Cheetham S, Jeffrey P, Porter R, Jani M, and Lee CA (2011) Digoxin is not a substrate for organic anion- and Read KD (2009) Receptor occupancy and brain free fraction. Drug Metab transporting polypeptide transporters OATP1A2, OATP1B1, OATP1B3, and Dispos 37:753–760. OATP2B1 but is a substrate for a sodium-dependent transporter expressed in Wright EM (1972) Active transport of lysergic acid diethylamide. Nature 240:53–54. HEK293 cells. Drug Metab Dispos 39:2093–2102. Zhang Y, Liu H, Summerfield SG, Luscombe CN, and Sahi J (2016) Integrating Tweedie D, Polli JW, Berglund EG, Huang SM, Zhang L, Poirier A, Chu X, and Feng in silico and in vitro approaches to predict drug accessibility to the central nervous B; International Transporter Consortium (2013) Transporter studies in drug de- system. Mol Pharm 13:1540–1550. DOI: 10.1021/acs.molpharmaceut.6b00031. velopment: experience to date and follow-up on decision trees from the In- ternational Transporter Consortium. Clin Pharmacol Ther 94:113–125. Uhr M, Grauer MT, Yassouridis A, and Ebinger M (2007) Blood-brain barrier pen- Address correspondence to: Dr. Scott G. Summerfield, Drug Metabolism etration and pharmacokinetics of amitriptyline and its metabolites in and Pharmacokinetics Department, GlaxoSmithKline, Park Road, Ware, p-glycoprotein (abcb1ab) knock-out mice and controls. J Psychiatr Res 41:179–188. Hertfordshire, United Kingdom, SG12 0DP. E-mail: Scott.G.Summerfield@ Uhr M, Steckler T, Yassouridis A, and Holsboer F (2000) Penetration of amitripty- GSK.com line, but not of fluoxetine, into brain is enhanced in mice with blood-brain barrier Downloaded from jpet.aspetjournals.org at ASPET Journals on September 27, 2021 Examining the Uptake of CNS Drugs and Candidates Across the Blood‐Brain Barrier Scott G Summerfield, Yanyan Zhang, Houfu Liu JPET Table S1

Table S1: Brain and blood unbound fraction and Kp values for rat and mouse (mdr1a/b(+/+) and mdr1a/b(‐/‐)) quoted as mean ± standard deviation. Rat brain Kp mdr1a/b(+/+) mouse brain Kp mdr1a/b(‐/‐) mouse brain Kp Rat fu(blood) Rat fu(brain) Mouse fu(blood) Mouse fu(brain) CNS Drugs (steady‐state) (steady‐state) (steady‐state) Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD Amantadine 0.521 0.123 0.233 0.035 ‐ ‐ ‐ ‐ 6.73 0.287924 ‐ ‐ ‐ ‐ Amitriptyline 0.068 0.012 0.009 0.001 ‐ ‐ ‐ ‐ 10.07 2.160048 ‐ ‐ ‐ ‐ Amoxapine 0.071 0.009 0.010 0.004 0.04 0.01 0.007 0.001 2.51 0.59 1.37 0.28 3.24 0.22 Atomoxetine 0.172 0.036 0.019 0.006 0.12 0.01 0.02 0.00 19.30 2.32 13.68 0.58 23.89 1.45 Buproprion 0.557 0.049 0.171 0.027 0.47 0.03 0.19 0.01 16.20 0.39 15.38 6.23 13.55 0.67 Carbamazepine 0.218 0.055 0.119 0.007 ‐ ‐ ‐ ‐ 1.39 0.10 ‐ ‐ ‐ ‐ Chlorpromazine 0.016 0.002 0.002 0.000 0.020 0.002 0.003 0.000 27.30 0.61 20.40 1.47 23.38 0.95 Citalopram 0.258 0.063 0.049 0.004 0.21 0.01 0.054 0.004 6.83 0.12 5.72 0.33 17.87 0.66 Clozapine 0.066 0.010 0.011 0.001 0.067 0.002 0.012 0.000 22.96 2.56 14.40 0.34 16.33 1.64 Diazepam 0.107 0.006 0.036 0.005 ‐ ‐ ‐ ‐ 2.86 0.23 ‐ ‐ ‐ ‐ Donepezil 0.245 0.009 0.126 0.015 0.253 0.001 0.0861 0.0060 3.44 0.16 3.49 0.08 7.75 0.65 Doxepin 0.166 0.023 0.025 0.003 ‐ ‐ ‐ ‐ 20.65 1.10 ‐ ‐ ‐ ‐ Ergotamine 0.045 0.005 0.013 0.002 0.021 0.001 0.007 0.001 0.08 0.01 <0.01 ND 0.939 0.312 Ethosuximide 0.521 0.094 0.740 0.227 ‐ ‐ ‐ ‐ 1.06 0.37 ‐ ‐ ‐ ‐ Fluoxetine 0.028 0.005 0.004 0.001 ‐ ‐ ‐ ‐ 36.58 6.77 ‐ ‐ ‐ ‐ Fluphenazine 0.014 0.004 0.001 0.000 0.0070 0.0008 0.0010 0.0001 24.63 0.15 43.37 7.37 55.08 5.14 Gabapentin 1.161 0.151 0.748 0.149 ‐ ‐ ‐ ‐ 0.69 0.07 ‐ ‐ ‐ ‐ Haloperidol 0.104 0.039 0.011 0.001 0.075 0.006 0.013 0.001 16.72 2.10 8.73 0.75 10.69 2.50 Imipramine* 0.103 0.002 0.015 0.001 ‐ ‐ ‐ ‐ 23.65 1.64 ‐ ‐ ‐ ‐ Isocarboxazid 0.517 0.056 0.211 0.038 ‐ ‐ ‐ ‐ 0.43 0.05 ‐ ‐ ‐ ‐ Lamotrigine 0.284 0.010 0.273 0.034 0.29 0.01 0.20 0.01 5.06 0.35 1.42 0.12 1.55 0.03 Loxapine 0.053 0.004 0.011 0.003 ‐ ‐ ‐ ‐ 14.23 1.52 ‐ ‐ ‐ ‐ Maprotiline 0.086 0.012 0.006 0.003 0.0384 0.0013 0.0044 0.0006 14.04 1.29 12.41 3.42 26.81 0.17 Meprobamate 0.719 0.012 0.638 0.073 0.69 0.09 0.59 0.10 0.87 0.04 0.71 0.05 0.93 0.12 Memantine* 0.375 0.060 0.116 0.012 20.07 3.34 ‐ ‐ ‐ ‐ Mesorizadine 0.163 0.043 0.016 0.001 0.125 0.010 0.024 0.002 1.04 0.09 0.95 0.06 16.90 3.22 Metaclopramide 0.724 0.081 0.365 0.052 0.43 0.04 0.49 0.04 0.79 0.05 0.79 0.09 5.48 0.25 Mianserin* 0.080 0.001 0.014 0.000 18.41 2.39 ‐ ‐ ‐ ‐ Midazolam 0.043 0.004 0.023 0.000 ‐ ‐ ‐ ‐ 2.02 0.02 ‐ ‐ ‐ ‐ Mirtazapine 0.142 0.011 0.080 0.003 ‐ ‐ ‐ ‐ 6.83 0.66 ‐ ‐ ‐ ‐ Olanzapine 0.135 0.009 0.034 0.009 0.25 0.02 0.082 0.003 8.47 0.56 21.33 4.39 29.62 3.52 Pemoline 0.558 0.034 0.558 0.084 ‐ ‐ ‐ ‐ 0.55 0.12 ‐ ‐ ‐ ‐ Pergolide 0.110 0.011 0.027 0.004 ‐ ‐ ‐ ‐ 12.99 0.69 ‐ ‐ ‐ ‐ Perphenazine 0.014 0.002 0.004 0.000 0.0115 0.0009 0.0012 0.0002 24.91 6.58 59.38 8.38 61.18 8.56 Phenelzine 0.121 0.008 0.076 0.015 ‐ ‐ ‐ ‐ 2.34 0.25 ‐ ‐ ‐ ‐ Phenytoin 0.153 0.019 0.082 0.006 0.016 0.002 0.026 0.002 1.23 0.11 1.86 0.08 1.70 0.04 Primidone 0.757 0.076 0.622 0.137 0.62 0.05 0.75 0.10 0.46 0.05 0.56 0.02 1.03 0.10 Quetiapine 0.080 0.003 0.025 0.002 0.102 0.002 0.033 0.003 4.03 0.25 3.55 0.55 3.94 0.99 Risperidone 0.144 0.018 0.099 0.003 0.33 0.03 0.14 0.01 0.30 0.04 0.44 0.17 5.52 0.75 Rizatriptan 0.630 0.042 0.293 0.088 0.76 0.06 0.61 0.08 0.09 0.01 0.50 0.32 0.66 0.08 Selegiline 0.181 0.023 0.074 0.003 ‐ ‐ ‐ ‐ 2.65 0.23 ‐ ‐ ‐ ‐ Sertraline 0.013 0.002 0.001 0.000 0.0062 0.0006 0.00072 0.00018 38.59 0.46 33.30 0.50 36.53 8.71 Sumatriptan 0.599 0.013 0.724 0.069 0.61 0.04 0.56 0.08 0.03 0.01 0.02 0.01 0.09 0.01 Tacrine 0.457 0.042 0.124 0.040 0.31 0.08 0.13 0.03 5.94 0.51 5.21 0.42 6.22 0.31 Temazepam 0.153 0.017 0.054 0.004 ‐ ‐ ‐ ‐ 2.27 0.16 ‐ ‐ ‐ ‐ Thioridazine 0.004 0.001 0.001 0.000 ‐ ‐ ‐ ‐ 14.01 1.03 ‐ ‐ ‐ ‐ Thiothixene 0.031 0.005 0.003 0.000 0.017 0.001 0.0025 0.0001 1.51 0.24 2.26 0.40 24.63 2.62 Tiagabine 0.093 0.015 0.040 0.012 0.14 0.020 0.06 0.01 1.44 0.09 1.97 0.14 3.00 0.18 Trazodone 0.124 0.010 0.055 0.006 ‐ ‐ 3.47 0.41 ‐ ‐ ‐ ‐ Trifluoperazine 0.004 0.001 0.001 0.000 0.0035 0.0004 0.0006 0.0001 19.13 3.02 12.95 0.74 19.91 0.66 Venlafaxine 0.588 0.048 0.216 0.023 0.77 0.03 0.25 0.04 5.20 0.43 10.60 1.17 16.52 0.93 Zaleplon 0.666 0.064 0.471 0.044 0.51 0.06 0.45 0.06 0.83 0.10 0.86 0.03 1.55 0.04 Ziprasidone 0.232 0.057 0.115 0.014 0.029 0.003 0.037 0.007 1.51 0.13 3.22 0.21 5.63 0.83 Digoxin ‐ ‐ ‐ ‐ 0.064 0.003 0.157 0.014 ‐ ‐ 0.05 0.01 1.4 0.1

* Oral dosing studies Examining the Uptake of CNS Drugs and Candidates Across the Blood‐Brain Barrier Scott G Summerfield, Yanyan Zhang, Houfu Liu JPET Table S2

Table S2: Chemical structures and their physicochemical properties of CNS drugs. pKa pKa Mol. CNS Drugs Chemical structure cLogP cLogD tPSA HBA HBD (most (most wt. 7.4 acidic) basic)

Amantadine 151 1.47 ‐1.38 26.0 0 1 14 10.71

Amitriptyline 277 4.81 2.48 3.2 0 0 14 9.76

Amoxapine 314 3.08 1.64 36.9 1 1 14 8.83

Atomoxetine 255 3.81 1.46 21.3 0 1 14 9.8

Bupropion 240 3.27 2.39 29.1 1 1 14 8.22

Carbamazepine 236 2.77 2.77 46.3 1 1 14 0

Chlorpromazine 319 4.54 2.74 6.5 0 0 14 9.2

Citalopram 324 3.76 1.41 36.3 2 0 14 9.78

Clozapine 327 3.4 3.12 30.9 0 1 14 7.35

Diazepam 285 3.08 3.08 32.7 2 0 14 2.92

Donepezil 379 4.21 2.96 38.8 1 0 14 8.62 Doxepin 279 3.84 1.51 12.5 0 0 14 9.76

Ergotamine 581 2.43 2.11 115.3 6 2 9.69 7.44

Ethosuximide 141 0.55 0.55 46.2 2 1 10.73 0

Fluoxetine 309 4.17 1.83 21.3 0 1 14 9.8

Fluphenazine 438 3.97 3.09 30.0 1 1 14 8.21

Gabapentin 171 ‐1.51 ‐1.51 63.3 2 1 4.63 9.91

Haloperidol 376 3.66 2.93 40.5 2 1 13.96 8.05

Imipramine 280 4.28 2.48 6.5 0 0 14 9.2

Isocarboxazid 231 1.43 1.43 67.2 3 2 12.02 3.12

Lamotrigine 256 1.93 1.91 90.7 3 2 14 5.87

Loxapine 328 3.46 3.26 28.1 1 0 14 7.18

Maprotiline 277 4.37 1.48 12.0 0 1 14 10.54 Memantine 179 2.07 ‐0.78 26.0 0 1 14 10.7

Meprobamate 218 0.93 0.93 104.6 2 2 14 0

Mesoridazine 387 3.57 2.72 23.6 1 0 14 8.19

Metoclopramide 300 1.4 ‐0.25 67.6 1 2 14 9.04

Mianserin 264 3.83 3.71 6.5 0 0 14 6.92

Midazolam 326 3.33 3.02 30.2 2 0 14 7.42

Mirtazapine 265 3.21 3.13 19.4 0 0 14 6.67

Olanzapine 312 3.39 3.16 30.9 0 1 14 7.24

Pemoline 176 0.8 0.8 64.7 2 1 14 0

Pergolide 313 3.96 2.24 16.1 1 0 14 9.12

Perphenazine 404 3.69 2.82 30.0 1 1 14 8.21

Phenelzine 136 1.2 1.2 38.1 2 2 14 5.55 Phenytoin 252 2.15 1.2 58.2 2 2 6.46 0

Primidone 218 1.12 1.12 58.2 2 2 11.5 0

Quetiapine 384 2.81 2.64 48.3 2 1 14 7.06

Risperidone 410 2.63 1.25 64.2 3 0 14 8.76

Rizatriptan 269 1.77 ‐0.37 49.7 2 1 14 9.56

Selegiline 187 2.85 1.55 3.2 0 0 14 8.67

Sertraline 306 5.15 2.76 12.0 0 1 14 9.85

Sumatriptan 295 0.74 ‐1.24 65.2 2 2 11.24 9.54

Tacrine 198 2.63 1.25 38.9 0 1 14 8.95

Temazepam 301 2.79 2.79 52.9 3 1 10.68 0

Thioridazine 371 5.47 3.93 6.5 0 0 14 8.93

Thiothixene 444 3.36 2.18 43.9 2 0 14 8.56 Tiagabine 376 2.47 2.47 40.5 2 0 4.14 9.26

Trazodone 372 3.13 2.96 45.8 2 0 14 7.09

Trifluoperazine 408 4.66 3.63 9.7 0 0 14 8.39

Venlafaxine 277 2.74 1.22 32.7 1 1 14 8.91

Zaleplon 305 1.53 1.53 74.3 4 0 14 0.3

Ziprasidone 413 4.3 4.13 48.5 2 1 13.18 7.09 Examining the Uptake of CNS Drugs and Candidates Across the Blood‐Brain Barrier Scott G Summerfield, Yanyan Zhang, Houfu Liu JPET Table S3

Q1 Q3 Pos/ Column Column Solvent Solvent Run time Inj. Gradient Elution Analyte MWt Mass Mass Neg Type Size A B (min) Vol Time (min) % Solv.B (uL) 05 195 Amantadine 151.3 152.1 79.2 Pos Hypersil Aquastar 3x30 1mM NH4Ac + 0.1% formic acid Acetonitrile 2.5 1 1.6 95 1.61 5 010 1 100 Amitriptyline 277.4 278.1 91.1 Pos ACE 3, C18 50x3 1mM NH4A c + 0.1% formic acid Acetonitrile 2.5 2.5 1.59 100 1.6 10 020 1.2 80 Amoxapine 313.8 314.2 271 Pos Hypersil Aquastar 3x30 1mM NH4Ac + 0.1% formic acid Acetonitrile 2.5 3 1.6 80 1.6 20 020 1.2 80 Aricept 379.5 380 91 Pos Hypersil Aquastar 3x30 10mM NH4Ac + 0.1% formic acid Acetonitrile 2.5 1 1.6 80 1.6 20 010 1 100 Atomoxetine 255.4 256.1 148.1 Pos ACE 3, C18 50x3 1mM NH4Ac + 0.1% formic acid Acetonitrile 2.6 2.5 1.59 100 1.6 10 05 1.2 90 Bupropion 239.7 240.2 184.1 Pos Hypersil Aquastar 3x30 1mM NH4Ac + 0.1% formic acid Acetonitrile 2.5 1 1.6 90 1.61 5 05 1.2 99 Carbemazepine 236.3 237.3 194.1 Pos Polaris C3 Ether 3x30 1mM NH4Ac + 0.1% formic acid Acetonitrile 2.5 3 1.8 99 1.81 5 020 0.6 80 Chlorpromazine 318.9 319.1 58.2 Pos Hypersil Aquastar 3x30 10mM NH4Ac + 0.1% formic acid Acetonitrile 2.5 ? 180 1.1 20

Citalopram 324.4 325.1 108.8 Pos ACE 3, C18 50x4.6 10mM NH4Ac + 0.1% formic acid MeCN 1.8 2 - -

05 1.2 90 Clozapine 326.8 327.2 270 Pos Hypersil Aquastar 3x30 1mM NH4Ac + 0.1% formic acid Acetonitrile 2.5 1 1.6 90 1.61 5 010 1 100 Diazepam 284.8 285 193.1 Pos ACE 3, C18 50x3 1mM NH4Ac + 0.1% formic acid Acetonitrile 2.5 2.5 1.59 100 1.6 10 010 1.5 90 Doxepin 279.4 280.2 115.2 Pos Hypersil Aquastar 3x30 1mM NH4Ac + 0.1% formic acid Acetonitrile 2.5 2 1.8 90 1.81 10 05 1.2 99 Ergotamine 581.7 582.3 223.3 Pos Polaris C3 Ether 3x30 1mM NH4Ac + 0.1% formic acid Acetonitrile 2.5 3 1.8 99 1.81 5 05 1.5 100 Ethosuximide 141.2 142.1 72.1 Pos SILC-PRIMESEP 200A 5um 150x2.1 0.1% trifluroacetic acid 0.1%Tformic acid 3.8 5 2.5 100 2.6 5 010 1 100 Fluoxetine 309.3 310 148 Pos ACE 3 C13 1mM NH4Ac Acetonitrile 2.5 1.55 100 1.6 10 020 1.2 80 Fluphenazine 437.5 438.3 171.1 Pos Hypersil Aquastar 3x30 1mM NH4Ac + 0.1% formic acid Acetonitrile 2.5 3 1.6 80 1.61 20 010 1 100 Gabapentin 171.3 172.4 154.1 Pos ACE 3, C18 50x3 1mM NH4Ac + 0.1% formic acid Acetonitrile 2.6 2.5 1.59 100 1.6 10 0 1.2 Haloperidol 375.9l 376 165 Pos Hypersil Aquastar 3x30 1mM NH4Ac+0.1%formic acid Acetonitrile 2.5 2 1 1.6 1.61 020 1.2 90 Imipramine 280.4 281 86 Pos Cyano Discovery 50x4.6 10mM NH4Ac + 0.1% formic acid Acetoitrile 21 1.6 90 1.61 20 05 190 Isocarboxazid 231.3 232.2 91.2 Pos Acquity C18 BEH 50x2.1 10mM NH4Ac Native Acetoitrile 2 2 1.5 90 1.51 10 05 1.2 90 Lamotrigine 256.1 256 211.1 Pos Hypersil Aquastar 3x30 1mM NH4Ac + 0.1% formic acid Acetonitrile 2.5 1 1.6 90 1.61 5 010 1.5 90 Loxapine 328.8 328.1 193.2 Pos Hypersil Aquastar 3x30 1mM NH4Ac + 0.1% formic acid Acetonitrile 2.5 2 1.8 90 1.81 10 010 0.8 90 Maprotiline 277.4 278.3 250.1 Pos Acquity C18 BEH 50x2.1 10mM NH4Ac native Acetonitrile 2 1 1.2 90 1.21 10 05 190 Meprobamate 218.3 219.2 158.1 Pos Hypersil Aquastar 3x30 1mM NH4Ac + 0.1% formic acid Acetonitrile 2.5 3 1.6 90 1.6 5 01 1.2 90 Memantine 179.3 180 107.1 Pos Cyano Discovery 50x4.6 10mM NH4Ac + 0.1% formic acid Acetoitrile 2 6 1.6 90 1.61 1 0 1.5 10 Mesoridazine 386.6 387.2 98.2 PosPolaris C3 Ether 30x3 10mM NH4Ac + 0.1% formic acid Acetonitrile 2.5 5 1.8 70 1.81 70 10 05 1.2 99 Metoclopramide 299.8 300.3 227.1 Pos Polaris C3 Ether 3x30 1mM NH4Ac + 0.1% formic acid Acetonitrile 2.5 1 1.8 99 1.81 5 030 1.2 90 Mianserin 264.4 264.9 207.9 Pos Cyano Discovery 50x4.6 10mM NH4Ac + 0.1% formic acid Acetoitrile 2 3 1.6 90 1.61 30 010 1 100 Midazolam 325.8 326.5 128.2 Pos ACE 3 C13 1mM NH4Ac Acetonitrile 2.5 1.55 100 1.6 10 010 1.2 80 Mirtazapine 265.4 266.2 195.1 Pos Hypersil Aquastar 3x30 1mM NH4Ac + 0.1% formic acid Acetonitrile 2.5 1 1.6 80 1.61 10 05 195 Olanzapine 312.4 313.1 256.3 Pos Hypersil Aquastar 3x30 1mM NH4Ac + 0.1% formic acid Acetonitrile 2.5 1 1.6 95 1.61 5 01 1.2 80 Pemoline 176.2 176.9 105.7 Neg Hypersil Aquastar 3x30 1mM NH4Ac + 0.1% formic acid Acetoitrile 2.5 7 1.6 80 1.61 1 020 0.8 80 Pergolide Mesylate 314.5 315.2 208.1 Pos Hypersil Aqustar 3x30 1mM NH4Ac + 0.1% formic acid Acetonitrile 2.5 3 1.2 80 1.21 20 05 190 Perphenazine 404 404.2 171.1 Pos Acquity C18 BEH 50x2.1 10mM NH4Ac Native Acetoitrile 2 2 1.5 90 1.51 10 50% Phenelzine 136.2 137.2 105.2 Pos Cyano discovery 50x4.6 50% Acetonitrile 1.8 2 - - 10mM NH4Ac + 0.1% formic acid

010 1.5 90 Phenytoin 252.3 250.9 101.9 Neg Polaris C3 Ether 3x30 1mM NH4Ac + 0.1% formic acid Acetonitrile 2.5 3 1.8 90 1.81 10 01 1.2 99 Primidone 218.3 219.1 162.1 Pos Hypersil Aquastar 3x30 1mM NH4Ac + 0.1% formic acid Acetonitrile 2.5 1 1.6 99 1.61 1 05 1.2 90 Quetiapine 383.5 384.1 253.1 Pos Hypersil Aquastar 3x30 1mM NH4Ac + 0.1% formic acid Acetonitrile 2.5 1 1.6 90 1.61 5 05 1.2 90 Risperidone 410.5 411.3 190.8 Pos Hypersil Aquastar 3x30 1mM NH4Ac + 0.1% formic acid Acetonitrile 2.5 1 1.6 90 1.61 5 010 1.5 95 Rizatriptan 269.4 270 201.3 Pos Synergi Polar-RP 50x2 10mM NH4Ac native MeCN/MeOH 2.5 5 1.8 95 1.81 10 010 1.2 80 Selegiline 187.3 188.1 119.1 Pos Hypersil Aquastar 3x30 1mM NH4Ac + 0.1% formic acid Acetonitrile 2.5 1 1.6 80 1.61 10 010 1.5 95 Sertraline 306.2 306.1 159 Pos Phenomenex Synergi 50x20 10mM NH4Ac native Acetonitrile 2.5 1 1.8 95 1.81 10 05 1.2 90 Sumatriptan 295 296.2 58 Pos Hypersil Aquastar 3x30 1mM NH4Ac + 0.1% formic acid Acetonitrile 2.5 5 1.6 90 1.61 5 010 1.5 95 Tacrine 198.3 199 144.1 Pos Synergi Polar-RP 50x2 10mM NH4Ac native MeCN/MeOH 2.5 5 1.8 95 1.81 10 010 1 100 Temazopam 300.7 301.4 255.1 Pos ACE 3 C13 1mM NH4Ac Acetonitrile 2.5 1.55 100 1.6 10 010 1.2 80 Thioridazine 370.6 371.2 126.3 Pos Polaris C3 Ether 3x30 1mM NH4Ac + 0.1% formic acid Acetonitrile 2.5 3 1.6 80 1.6 10 05 1.2 80 Thiothixine 443.6 444.3 69.7 Pos Hypersil Aqustar 3x30 1mM NH4Ac + 0.1% formic acid Acetonitrile 2.5 3 1.6 80 1.61 5 05 1.2 99 Tiagabine 375.6 376.2 247.1 Pos Polaris C3 Ether 3x30 1mM NH4Ac + 0.1% formic acid Acetonitrile 2.5 3 1.8 99 1.81 5 05 1.2 99 Trazadone 371.9 372.2 176.3 Pos Polaris C3 Ether 3x30 1mM NH4Ac + 0.1% formic acid Acetonitrile 2.5 1 1.8 99 1.81 5 01 1.2 60 Triflouperazine 407.5 408.3 112.9 Pos Hypersil Aquastar 3x30 1mM NH4Ac + 0.1% formic acid Acetonitrile 2.5 1 1.6 60 1.61 1 05 1.2 80 Venlafaxine 277.4 278.3 57.8 Pos Hypersil Aqustar 3x30 1mM NH4Ac + 0.1% formic acid Acetonitrile 2.5 3 1.6 80 1.61 5 0 110 Zaleplon 305.3 306.2 264.1 Pos ACE 3, C18 50X3 1mM NH4Ac + 0.1% formic acid Acetonitrile 3 5 1.59 100 1.6 100 10 05 1.2 90 Ziprasidone 412.9 413.1 194.1 Pos Hypersil Aquastar 3x30 1mM NH4Ac + 0.1% formic acid Acetonitrile 2.5 1 1.6 90 1.61 5 Examining the Uptake of CNS Drugs and Candidates Across the Blood‐Brain Barrier Scott G Summerfield, Yanyan Zhang, Houfu Liu JPET Figure S1

Figure S1: Physicochemical properties of CNS drugs binned with Kp,uu. Number of CNS drugs in each bin is 11, 23, and 19 for bin Kp,uu < 0.5, bin 0.5 ≤ Kp,uu ≤ 2, bin Kp,uu ≥ 2, respectively.