Benzodiazepine binding site occupancy by the novel GABA A receptor subtypeselective

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1 JPET Fast This Forward. article has not Published been copyedited on and September formatted. The 24, final 2009 version as may DOI: /jpet differ from this version. JPET # of 31 Benzodiazepine binding site occupancy by the novel GABA A receptor subtypeselective drug 7-(1,1-dimethylethyl)-6-(2-ethyl-2H-1,2,4-triazol-3-ylmethoxy)-3-(2- fluorophenyl)-1,2,4-triazolo[4,3-b]pyridazine (TPA023) in rats, primates and man John R. Atack, Dean F. Wong, Tim D. Fryer, Christine Ryan, Sandra Sanabria, Yun Zhou, Robert Dannals, Wai-si Eng, Raymond E. Gibson, H. Donald Burns, Jose Vega, Laura Vessy, Paul Scott- Stevens, John S. Beech, Baron, Jean-Claude, Bindi Sohal, Michael L. Schrag, Franklin I. Aigbirhio, Ruth M. McKernan and Richard J. Hargreaves Merck Sharp & Dohme Research Laboratories, Neuroscience Research Centre, Harlow, Essex. UK (J.R.A., P.S.-S., B.S., R.M.M.) Division of Nuclear Medicine, Johns Hopkins Medical Institutions, Baltimore, MD (D.F.W., Y.Z., R.D.) Wolfson Brain Imaging Center, University of Cambridge, Cambridge, UK (T.D.F., J.S.B., J.C.B., F.I.A.) Imaging Research, Merck Research Laboratories, West Point, PA (C.R., S.S., W.E., R.E.G., H.D.B, R.J.H.) Clinical Pharmacology, Merck Research Labs, North Wales, PA (J.V., L.V.) Drug Metabolism Department, Merck Research Laboratories, West Point, PA (M.L.S.) Copyright 2009 by the American Society for Pharmacology and Experimental Therapeutics.

2 JPET # of 31 Running title: Occupancy of benzodiazepine binding sites by TPA023 Address correspondence, proofs and reprint requests to Dr. John R. Atack at his present address: Neuroscience, Johnson & Johnson Pharmaceutical Research and Development, Turnhoutseweg 30, B Beerse, Belgium Tel: +32 (0) ; Fax: +32 (0) JAtack1@its.jnj.com Number of text pages: 31 Number of tables: 1 Number of figures: 6 Number of references: 40 Words in abstract: 203 Words in Introduction: 649 Words in Discussion: 1812 Abbreviations: TPA023, 7-(1,1-Dimethylethyl)-6-(2-ethyl-2H-1,2,4-triazol-3-ylmethoxy)-3-(2-fluorophenyl)-1,2,4- triazolo[4,3-b]pyridazine; L , 7-(1,1-dimethylethyl)-6-(2-methyl-2H-1,2,4-triazol-3-ylmethoxy)- 3-(2,5-difluorophenyl)-1,2,4-triazolo[4,3-b]pyridazine; PET, positron emission tomography; SPECT, single photon emission computed tomography Recommended Section Assignment: Neuropharmacology

3 JPET # of 31 Abstract The GABA A receptor α2/α3 subtype-selective compound 7-(1,1-Dimethylethyl)-6-(2-ethyl-2H- 1,2,4-triazol-3-ylmethoxy)-3-(2-fluorophenyl)-1,2,4-triazolo[4,3-b]pyridazine (TPA023; also known as MK-0777) is a triazolopyridazine that has similar, subnanomolar affinity for the benzodiazepine binding site of α1-, α2-, α3- and α5-containing GABA A receptors and has partial agonist efficacy at the α2 and α3 but not the α1 or α5 subtypes. The purpose of the present study was to define the relationship between plasma TPA023 concentrations and benzodiazepine binding site occupancy across species measured using various methods. Thus, occupancy was measured using either in vivo [ 3 H]flumazenil binding or [ 11 C]flumazenil small-animal positron emission tomography (micropet) in rats, [ 123 I]iomazenil γ-scintigraphy in rhesus monkey, and [ 11 C]flumazenil PET in baboons and man. For each study, plasma-occupancy curves were derived and the plasma concentration of TPA023 required to produce 50% occupancy (EC 50 ) calculated. The EC 50 values for rat, rhesus monkey and baboon were all similar and ranged from ng/ml although in man, the EC 50 was slightly lower at 9 ng/ml. In man, a single 2 mg dose of TPA023 produced in the region of 50-60% occupancy in the absence of overt sedative-like effects. Considering that non-selective full agonists that are associated with sedation at occupancies of less than 30%, these data emphasize the relatively non-sedating nature of TPA023.

4 JPET # of 31 Introduction The GABA A receptor is a pentameric assembly of subunits derived from a family of genes of which there are 19 members (α1-6, β1-3, γ1-3, δ, ε, θ, π, ρ1-3), with the predominant composition of native receptors being α, β and γ subunits with a 2:2:1 stoichiometry and an αβαβγ arrangement as viewed from the synapse (Sieghart and Sperk, 2002). Clinically used benzodiazepines, such as diazepam and lorazepam, exert their actions on GABA A receptors containing β and γ2 subunits along with either an α1, α2, α3 or α5 subunit (Sieghart, 1995, 2006), the collective population of which comprises around 75% of total brain GABA A receptors (McKernan and Whiting, 1996; Siegehart and Sperk, 2002). The different GABA A receptors with which benzodiazepines interact are associated with specific aspect of the pharmacology of these drugs. For example, the α1 subtype is associated with sedation (Rudolph et al., 1999; McKernan et al., 2000) whereas the α2 and/or α3 subtypes mediate the anxiolytic effects (Löw et al., 2000; Dias et al., 2005). Accordingly, it has been hypothesized that compounds which selectively modulate the α2 and/or α3 subtypes without effects on α1-containing receptors should prove to be non-sedating anxiolytics (Atack, 2005). This α2/α3 selectivity may be achieved either by compounds having higher affinity at the α2 and α3 compared to α1 and α5 subtypes or, alternatively, by binding to all subtypes but only having modulatory effects (i.e., agonism) at the α2 and α3 subtypes (Atack, 2005). This latter approach has been used to identify a series of triazolopyridazines (Carling et al., 2005) of which the prototypic compound is L (McKernan et al., 2000). Although L is a useful pharmacological tool compound, pharmacokinetic issues precluded its further development. However, the structurally-related compound 7-(1,1-dimethylethyl)-6- (2-ethyl-2H-1,2,4-triazol-3-ylmethoxy)-3-(2-fluorophenyl)-1,2,4-triazolo[4,3-b]pyridazine (TPA023, also known at MK-0777; Lewis et al., 2008) has more favourable pharmacokinetic properties and the in

5 JPET # of 31 vitro and in vivo properties of this compound have been described (Atack et al., 2006b). Thus, TPA023 has equivalent affinity ( nm) for the α1, α2, α3 and α5 subtypes of the GABA A receptor but is an antagonist at the α1 and α5 and a partial agonist at the α2 and α3 subtypes (Atack et al., 2006b). In preclinical studies, the in vitro properties of TPA023 translate into a compound that is a non-sedating anxiolytic in rodent and primate species (Atack et al., 2006b). Moreover, this compound has essentially no abuse liability in baboons trained to a cocaine baseline (Ator, 2005). As part of these preclinical studies, behavioural outcomes were defined in terms of receptor occupancy. For example, TPA023 was anxiolytic in a variety of rodent anxiolytic assays at between 70-88% occupancy but produced no overt sedation and/or myorelaxation at >95% occupancy (Atack et al., 2006b). Moreover, TPA023 has no significant abuse potential even at doses that produced essentially complete receptor occupancy (Ator, 2005). Based upon these data TPA023 was selected as a clinical candidate and has been shown to demonstrate a tendency to increased performance in cognition in a small population of schizophrenia subjects (Lewis et al., 2008). In the course of identifying subtype-selective GABA A modulators, compounds were prioritized for subsequent behavioural analyses based upon their potencies in a rat [ 3 H]flumazenil in vivo binding assay with potency being defined in terms of the dose required to produce 50% occupancy (Occ 50 ) as well as the corresponding plasma drug concentration (EC 50 ). Since the plasma EC50 was a key selection criterion, it was considered important to evaluate to what extent this parameter translates across species. Accordingly, the purpose of the present study was to measure the plasma EC 50 for TPA023 in a number of preclinical species (rat, rhesus monkey, baboon) using in the rat either [ 3 H]flumazenil in vivo binding or [ 11 C]flumazenil small-animal positron emission tomography (micropet), in rhesus monkey [ 123 I]iomazenil γ-scintigraphy or baboon [ 11 C]flumazenil positron emission tomography (PET) and to

6 JPET # of 31 compare these plasma-occupancy relationships to that observed in man measured using [ 11 C]flumazenil PET. Methods Drugs. TPA023 was synthesized as described elsewhere (Carling et al., 2005). Flumazenil was obtained from Sigma-Aldrich (St. Louis, MO) and bretazenil was synthesized in-house (Merck). [ 3 H]Flumazenil ([ 3 H]Ro ; Ci/mmol) was purchased from PerkinElmer LAS (Boston, MA) and [ 11 C]flumazenil was synthesized as described in more detail below. Rat [ 3 H]flumazenil in vivo binding studies. Experiments measuring the inhibition of the in vivo binding of [ 3 H]flumazenil by TPA023 (i.e., TPA023 occupancy) were carried out as described previously (Atack et al., 2006b). In brief, rats were dosed orally with either vehicle (0.5% methyl cellulose) or 1, 3 or 10 mg/kg TPA023 and then 3 min prior to killing at 0.75, 1.5, 3, 6 or 18 h after dosing, animals received a tail vein injection of [ 3 H]flumazenil (1ml/kg of radioligand diluted 1:150 in isotonic saline). Three min later, animals were killed by stunning and decapitation, the brain was rapidly removed, homogenised and aliquots of homogenate were filtered and washed over Whatman GF/B glass fibre filters. Filters were then placed in vials, scintillant cocktail added and radioactivity counted using a Beckman LS6500 scintillation counter. In order to determine the level of non-specific binding, a separate group of animals were given 5 mg/kg bretazenil (i.p. in 100% PEG 300 with a treatment time of 30 min), injected with [ 3 H]flumazenil, killed and the brain processed as described above. The occupancy of TPA023 was defined as the extent by which the specific binding in TPA023-treated animals was reduced relative to the vehicle-treated group.

7 JPET # of 31 Rat [ 11 C]flumazenil micropet studies. Rat micropet studies were performed as described in more detail elsewhere (Atack et al., 2007). Briefly, cannulated rats (right femoral artery for blood sampling, right femoral vein for compound administration and left femoral vein for [ 11 C]flumazenil administration) were anaesthetised with isoflurane, placed in a micropet P4 scanner (Concorde Microsystems Inc., Knoxville, TN) and maintained under isoflurane anaesthesia for the duration of the experiment. [ 11 C]Flumazenil (specific activity ~6 Ci/µmol at the time of injection) was synthesized as described previously (Atack et al., 2007) and MBq were injected over a 40 sec period to initiate each study. Ten and 26 min after [ 11 C]flumazenil administration, rats were dosed with either vehicle (aqueous solutions in 2% DMSO/10% hydroxypropyl-β-cyclodextrin; n=2) or low- and high-doses of TPA023 (0.05 and 0.2 mg/kg i.v., respectively; n=3). Forty-six min after the study started, rats received an i.v. injection of 0.25 mg/kg flumazenil in order to determine non-specific binding. The time-activity curve after flumazenil injection was extrapolated backwards in order to estimate the level of nonspecific binding throughout the duration of the study. Having corrected for the estimated levels of nonspecific binding, the % occupancy of TPA023 was calculated as the extent by which the TPA023 timeactivity curves (n=3) were reduced during the min (low dose) and min (high-dose) periods relative to vehicle-treated animals (n=2), Rhesus monkey iomazenil [ 123 I]iomazenil γ-scintigraphy. The occupancy of rhesus monkey brain GABA A receptors by TPA023 was carried out as described in more detail elsewhere (Atack et al., 2009a). In brief, four separate studies were performed in the same rhesus monkey. For each study, the monkey was anesthetized with ketamine (10 mg/kg i.m.) followed by propofol (2-5 mg/kg i.v. bolus followed by a constant infusion of 0.5 mg/kg/min i.v.). The monkey was intubated and then ventilated with oxygen at ~50 ml per breath at a rate of respirations per min. Body temperature was

8 JPET # of 31 maintained with circulating water heating pads, and temperature, and heart rate monitored for the duration of the study. The monkey was positioned on the camera head (LEAP collimator, Siemens Orbiter) for lateral brain imaging. [ 123 I]Iomazenil, which was synthesised by oxidative radioidination of the trimethyl-tin precursor essentially as previously described (McBride, et al., 1991; Atack et al., 2009a), was injected as a bolus (~0.4 mci) followed by infusion ( mci/h) in order to reach steady-state. After 2 h, TPA023 was administered for an additional 3-h period using an EtOH:PEG400:water (1:3:6 v/v) vehicle and a bolus plus infusion paradigm (0.04 mg/kg over 5 min then 0.01 mg/kg/h; 0.25 mg/kg over 5 min then mg/kg/h, 0.2 mg/kg over 5 min then 0.05 mg/kg/h and 0.1 mg/kg over 5 min then mg/kg/h). Non-specific binding was defined by the infusion of 1 mg/kg flumazenil (using the same vehicle as TPA023) at the end of the mg/kg/h scan. Occupancy was defined as the % inhibition of steady-state [ 123 I]iomazenil uptake having subtracted non-specific binding. Baboon [ 11 C]flumazenil PET. The baboon [ 11 C]flumazenil PET studies were conducted as part of a series of studies to evaluate the abuse potential of TPA023 (Ator et al., 2009). During the course of these PET scans, blood samples were collected, centrifuged, and plasma removed and stored prior to subsequent bioanalysis of drug concentrations. Human PET Imaging Studies Synthesis of [ 11 C]Flumazenil. [ 11 C]Flumazenil was synthesized in the Laboratory of Nuclear Chemistry at Johns Hopkins Medical Institutions by N-alkylation of Ro , the desmethyl precursor of flumazenil (ABX GmbH Dresden). Briefly, [ 11 C]Carbon dioxide was produced by the

9 JPET # of N(p,α) 11 C reaction using a nitrogen gas target containing oxygen. Irradiation with 17 MeV protons, produced by the General Electric PETtrace cyclotron at the Johns Hopkins PET Center, gave 11 C in the form of [ 11 C]carbon dioxide. After bombardment, the [ 11 C]carbon dioxide was swept at a flow rate of 100 ml/min under nitrogen gas pressure to a hot cell and collected on activated molecular sieves in a PETtrace MeI MicroLab then immediately reduced in the presence of a nickel catalyst and hydrogen gas to [ 11 C]methane. The [ 11 C]methane was converted to [ 11 C]methyl iodide by recirculation across heated molecular iodine collected on a Porapak Q column. After complete collection, the column was heated to release the [ 11 C]methyl iodide under a stream of helium which was then trapped in a cooled (-78 C ) solution of the precursor (Ro ) dissolved in dimethylformamide (DMF) whereupon aqueous tetrabutylammmonium hydroxide (0.4M) was added and the reaction mixture heated for 2 min. After dilution with HPLC buffer, the solution was injected onto a semipreparative HPLC, the appropriate fraction was collected and transferred remotely to a rotary evaporator to remove the solvent. The residue was redissolved in sterile normal saline (7 ml) and the solution passed through a sterile 0.22 μm filter into a sterile evacuated vial. After the addition of 3 ml of sterile 8.4% sodium bicarbonate, a sample was taken for determination of identity, radiochemical and chemical purity, ph and assessment of specific radioactivity. Radiochemical purity and specific activity for each batch was >90% and >1,000 Ci/mmol, respectively. Imaging Studies. This was a double-blind, single oral dose, randomized, placebo controlled study in 6 healthy subjects (5 male, 1 female, average age = 34 years). The ethics committee of Johns Hopkins University approved the protocol and each volunteer gave written informed consent. For each participant studies were conducted in one, ~10-h day after fasting (except for water) from midnight of the preceding day. Subjects were cannulated under local anaesthesia in a radial artery for blood

10 JPET # of 31 sampling and also had a venous cannula inserted for [ 11 C]flumazenil injection and three scans were performed commencing 1 h prior to or 1.5 and 6 h after receiving a single oral dose of either placebo (n=1), TPA023 (2 mg; n=3) or Lorazepam (2 mg; n=2). Scans were performed on a GE Advance PET scanner and were initiated by the injection of [ 11 C]flumazenil (~15 mci) with 22 scans being acquired over a one hour period (4 x 0.25 min, 4 x 0.5 min, 3 x 1 min, 2 x 2 min, 5 x 4 min and 6 x 5 min). The measurement of the arterial plasma concentration of [ 11 C]flumazenil and its labeled metabolites during the scan allowed a metabolite-corrected arterial input function for the tracer to be generated (data on file at Johns Hopkins University PET Center). Venous plasma samples were collected at 0.5, 1.5, 2, 2.5, 3, 4, 5, 6.5, 7, and 7.5 h post dosing for determination of plasma levels of TPA023 using LC-MS/MS. Image Analysis. Generation of Regions of Interest (ROI) Prior to the PET scans, standard non-contrast magnetic resonance images (MRI) were obtained for all subjects and these were aligned with [ 11 C]-flumazenil PET images from the first scan to allow the manual delineation of regions of interest (ROI) on the occipital cortex, frontal cortex, cerebellum and pons. Images from the second and third PET scans were aligned with the first study in order to obtain the same ROIs in all studies and from these aligned images time-activity curves (TACs) were generated. Modeling and Occupancy Calculation The [ 11 C]flumazenil TACs were used together with the metabolite corrected plasma curve obtained from blood sampling during the corresponding scan to calculate the in vivo ligand binding of [ 11 C]flumazenil to the benzodiazepine site of GABA A receptors using a one-tissue linear compartmental model (Koeppe et al., 1991). This permitted an estimation of the rate constant for forward capillary exchange (K 1 ), the volume of distribution of flumazenil (V D ) and the blood volume. V D is proportional

11 JPET # of 31 to the ratio of the number of benzodiazepine binding sites and the equilibrium dissociation constant, B max /K d (and can be estimated independent of any flow changes, which will be reflected in changes of K 1 ). VD K k B + K = 1 max 1 + λ Equation 1 2 d Where B max is the density of benzodiazepine binding sites available for binding, K d is the equilibrium dissociation rate constant and λ corresponds to the non-receptor-bound volume of distribution. The λ value for [ 11 C]flumazenil may be considered as constant in all gray matter regions and also in different subjects. Comparison of the V D values obtained from the three [ 11 C]flumazenil scans were used to estimate receptor occupancy at the studied times and percentage of receptor occupancy at T=2 and 6.5 h after drug administration where calculated from equation 1 using λ=0.8 in 2 as: Occupancy( T) DV(baseline) DV(T) = x100% Equation 2 DV(baseline) λ Results Rat [ 3 H]flumazenil in vivo binding and plasma pharmacokinetics of TPA023 in rat. The occupancy by TPA023 of GABA A receptor benzodiazepine binding sites in rat brain was clearly time- and dosedependent (Figure 1). Hence, at all three doses, maximum occupancy was observed 0.75 h after dosing with respective occupancies at 1, 3 and 30 mg/kg being 74 ± 3%, 89 ± 1% and 97 ± 0% and the extrapolated Occ 50 being 0.29 mg/kg. Thereafter, occupancy at 1 and 3 mg/kg doses dropped rapidly, being 34 ± 5% and 54 ± 2% 3 h post-dosing. On the other hand, occupancy at 10 mg/kg was more

12 JPET # of 31 sustained such that at 6 h post dose, 72 ± 10% occupancy remained but this decreased to only 12 ± 7% after 18 h. Measurement of plasma TPA023 concentrations showed that there was rapid absorption of compound following oral dosing, with C max being achieved at 0.75 h at each dose. Moreover, peak plasma concentrations were roughly dose-proportional in that doses of 1, 3 and 10 mg/kg resulted in C max values of 48 ± 9, 131 ± 15 and 430 ± 41 ng/ml, respectively. Rat [ 11 C]flumazenil micropet. Figure 2 shows the results of the rat micropet experiments. Following administration of low and high doses of TPA023 (0.05 and 0.2 mg/kg i.v., respectively), plasma drug concentrations (~15 and 65 ng/ml) were relatively constant following an initial distribution phase (Figure 2A). The time-activity curves for a representative vehicle- and TPA023-treated animal show that there is displacement of [ 11 C]flumazenil binding after injections of the low and high doses of TPA023 (0.05 and 0.2 mg/kg i.v., respectively; Fig. 2B). The occupancies under the low dose and high dose conditions were 27 ± 1% and 92 ± 3% (Fig. 2C), giving an estimated Occ 50 of 0.07 mg/kg i.v. Rhesus monkey [ 123 I] γ-scintigraphy Figure 3 shows the displacement of [ 123 I]iomazenil binding in the rhesus monkey brain produced by infusions of 0.01, and mg/kg/h TPA023. These doses produced respective occupancies of 20, 72 and 97%. Human PET. Figure 4 shows the images of [ 11 C]flumazenil binding in all subjects and illustrates the widespread distribution of benzodiazepine binding sites. The time-activity curves for single representative subjects given either placebo, lorazepam (2 mg; lorazepam subject # 1) or TPA023 (2

13 JPET # of 31 mg; TPA023 subject # 1) and receiving [ 11 C]flumazenil injections prior to, or 1.5 or 6 h after dosing (Scans 1, 2 and 3, respectively) are shown in Figure 5. The time-activity curves for the subject given placebo were very reproducible. Furthermore, subjects given lorazepam had time activity curves and 6-7 h after 2 mg lorazepam treatment (Scans 2 and 3, respectively) very similar to baseline scan prior to lorazepam treatment (Scan 1), suggesting that lorazepam produced levels of occupancy that were below the reliable level of detection using PET (<10%). In contrast, 2 mg TPA023 produced a marked alteration in the time-activity curve, indicative of a reduction of [ 11 C]flumazenil binding due to the occupancy of benzodiazepine binding sites by TPA023. When averaged across brain regions for the three subjects, the mean occupancies 2 and 6.5 h after oral administration of TPA023 were 58 ± 6% and 46 ± 7%, respectively, with the estimated Occ 50 (t = 2 h) being 1.5 mg total dose (or ~0.02 mg/kg). Plasma-occupancy relationships. For each species, the occupancy data obtained was plotted as a function of plasma TPA023 concentrations and these data are presented in Figure 6 and the plasma TPA023 concentrations required to produce 50% occupancy (the EC 50 ) values, and their associated 95% confidence intervals, are summarized in Table 1. The data used for the rat [ 3 H]flumazenil in vivo binding analysis was derived from the occupancy time course experiment (Figure 1). This permitted the plasma-occupancy relationship to be measured at various time after dosing and but there was no tendency for the plasma-occupancy relationship to vary as a function of time with EC 50 values 0.75, 1.5, 3 and 6 h post-dose being 27, 17, 25 and 23 ng/ml, respectively. Accordingly, all the data from this experiment was pooled for the analysis shown in Figure 6A. These data clearly show that in preclinical species and despite the method of radioactivity measurement (ex vivo in brain homogenates or in vivo using PET or SPECT) or the choice of radioligand ([ 3 H]flumazenil, [ 11 C]flumazenil or [ 123 I]iomazenil)

14 JPET # of 31 the EC 50 values are relatively comparable across species, ranging from 19 mg/ml in baboon to 30 ng/ml in rhesus monkey, with rat being, depending on the methodology, ng/ml (Table 1). Discussion In rats, the plasma EC 50 was essentially the same at different times between 0.75 and 6 h postdose and ranged from ng/ml. These data suggest that TPA023 did not have a slow off-rate (i.e., did not have prolonged occupancy), in which case the EC 50 value would decrease as a function of time. In this regard, TPA023, with an affinity of nm, is comparable to flumazenil itself, which, with a comparable affinity ( nm; Atack et al., 2005) clearly has a rapid rate of dissociation in vivo as evidenced by the fact that not only is flumazenil cleared rapidly from the rat brain with a half-life of 16 min (Lister et al., 1984) but also the displacement of the in vivo binding of [ 11 C]flumazenil binding occurs rapidly not only in rats (Figure 2) but also baboons and man (Figures 4 and 5). In addition, the GABA A α5 subtype-selective triazolophthalazine α5ia, which is structurally-related to TPA023, also showed no tendency for a slow off-rate since the plasma EC 50 was, like TPA023, time-independent (Atack et al., 2009a). The similarity of the EC 50 values obtained in rat using either [ 3 H]flumazenil in vivo binding or [ 11 C]flumazenil micropet (respective EC 50 s = 25 and 21 ng/ml) suggests that despite the marked methodological differences between these techniques, they produce similar results, consistent with analogous data obtained for lorazepam (Atack et al., 2007). Furthermore, the comparable EC 50 values for rhesus monkey and baboon (30 and 19 ng/ml) obtained by [ 123 I]iomazenil γ-scintigraphy and [ 11 C]flumazenil PET, respectively, indicate that these differing radioligands and imaging modalities

15 JPET # of 31 produce similar results, although others have suggested that these ligands have different in vivo properties (Hosoi et al., 1999). Studies using [ 11 C]flumazenil PET in man have shown that a dose of clonazepam which produces sleep and ataxia (0.03 mg/kg p.o.) occupies 15-23% of BZ sites (Shinotoh et al., 1989) whereas sedative doses of diazepam (30 mg p.o.) and alprazolam (0.5 mg every 6 h) have respective occupancies of 24% and 16% (Pauli et al., 1991; Fujita et al., 1999). In addition, the usual clinical dose of zolpidem (10 mg) is predicted to correspond to 15% occupancy based on the fact that a 20 mg dose produced 26-29% occupancy (Abadie et al., 1996). Furthermore, midazolam doses of 12.5 and 50 µg/kg i.v. (the latter of which induced sleep) produced occupancies, depending on the brain region, of 3-16% and 17-35% respectively (Malizia et al., 1996). Interestingly, comparable data (7% and 39% occupancy at the low- and high-dose) were obtained using total head counts (Malizia et al., 1996), suggesting that non-tomographic methods, which require lower levels of radioactivity compared to tomographic techniques, may be used to determine levels of occupancy in man (Malizia et al., 1995). In a [ 123 I]iomazenil SPECT study, a sleep-inducing, steady-state infusion of midazolam (6 mg/h) gave cortical occupancy in the region of 20-30% (Videbaek et al., 1993), which is comparable to [ 11 C]flumazenil data (Malizia et al., 1996). Taken together these data clearly demonstrate that for classical benzodiazepines (clonazepam, diazepam, alprazolam and midazolam) as well as the α1- subtype preferring drug zolpidem, occupancies of >15-30% are associated with sedation or sleep. Pagoclone (0.4 mg) gives occupancy of 11-15% in man with a reduced effect on saccadic eye movements relative to lorazepam (Lingford-Hughes et al., 2005) and although it is unclear to what extent this occupancy is related to parent compound or the pharmacologically active metabolite 5 - hydroxy pagoclone (Atack et al., 2006a), these data are consistent with an in vivo partial agonist pharmacological profile (Lingford-Hughes et al., 2005).

16 JPET # of 31 In contrast to the classical benzodiazepines, a 2 mg dose of TPA023 produced occupancy in the region of 50-60% yet showed no overt sedative-like effects suggesting that it is the intrinsic efficacy of this compound rather than a lack of sufficient occupancy that accounts for the lack of sedation seen in man. These data are consistent with the lack of efficacy of TPA023 at α1-containing receptors, the subtype of GABA A receptor responsible for the sedative effects of diazepam (Rudolph et al., 1999; McKernan et al., 2000) and are in agreement with the pharmacodynamic effects of TPA023, which showed that doses of 0.5 and 1.5 mg did not produce the sedation like effects (e.g., increased body sway, alertness measured using a visual analogue scale) observed with lorazepam (de Haas et al., 2007). Similarly, data with the α2/α3-selective compound TPA023B, which is an imidazotriazine follow-up compound to TPA023, showed that levels of occupancy in the region of 50% could be achieved in the absence of overt sedation (van Laere et al., 2008; Atack et al., 2009b). However, in contrast to the relatively high levels of occupancy achieved in man TPA023 and TPA023B in the absence of overt sedation, MRK-409 (also known as MK-0343, de Haas et al., 2008) produced sedation and somnolence at doses of 1.5 and 2 mg with occupancy at the maximum tolerated dose of 1 mg being below the levels of detection ( 10%; Atack et al., 2009c). The sedation produced by MRK-409 was attributed to the weak partial agonist efficacy that this compound had at the α1 subtype and clearly demonstrate the value of the measurement of receptor occupancy in interpreting clinical data (Atack et al., 2009c). Pharmacodynamic studies similar to those described for TPA023 and MRK-409 (de Haas et al., 2007, 2008) have also been reported for SL (de Haas et al., 2009), a compound with full agonist efficacy at the a2 and a3 subtypes and partial agonist efficacy at a1- and a5-containing GABAA receptors (Griebel et al., 2001). The fact that a 2 mg dose of lorazepam produced occupancy that was below the limits of detection (i.e., <10%) in the human PET study is consistent with previous studies showing that

17 JPET # of 31 therapeutically-relevant (anxiolytic) doses of lorazepam produce levels of occupancy of <3% (Sybirska et al., 1993) or, depending on the brain region, 6-9% (1 mg; Lingford-Hughes et al., 2005). The maximum plasma concentrations of lorazepam after a single, 2 mg dose are in the region of 20 ng/ml (Greenblatt et al., 1988; de Haas et al, 2007) and a similar estimated concentration of TPA023, ~20 ng/ml, is achieved following a 2 mg dose of TPA023 (calculated by extrapolating from plasma pharmacokinetics of single doses of 0.5 mg and 1.5 mg TPA023 (respective C max values = ~5 and ~15 ng/ml, respectively; de Haas et al, 2007). However, despite 2 mg doses of TPA023 and lorazepam achieving comparable plasma concentrations (20 ng/ml), TPA023 gave much higher levels of occupancy in man compared to lorazepam (50-60% vs. <10%). This is consistent with the lorazepam plasma EC 50 in rats ( ng/ml; lorazepam micropet data) being greater than for TPA023 (21-25 ng/ml). Since the binding of TPA023 to human plasma protein is comparable (~90%; unpublished observations and Greenblatt, 1981), the greater potency of TPA023 relative to lorazepam is presumably related to the higher affinity of TPA023 for the benzodiazepine binding site ( nm; Atack et al., 2006b) compared to lorazepam (2-6 nm; unpublished observations). The high affinity of TPA023 also presumably accounts for why effects of TPA023 on saccadic eye movement peak velocity were seen at doses of 0.5 and 1.5 mg (de Haas et al., 2007) that were an order of magnitude less than the minimally effective dose of 25 mg required for the lower-affinity, α2/α3-preferring compound SL (de Haas et al., 2009). Hence, the affinity of TPA023 for the different subtypes of GABA A receptors ranges from nm whereas SL has affinity at the α1, α2 and α3 subtypes ranging from nm, with even lower affinity, 215 nm, at the α5 subtype (Griebel et al., 2001). The C max values for the 0.5 and 1.5 mg doses of TPA023 were 5 and 13 ng/ml (de Haas et al., 2007) whereas respective values for the 2.5, 7.5 and 25 mg doses of SL were 37, 126 and 375 ng/ml (de Haas et al., 2009). Accordingly, the plasma drug concentrations required for

18 JPET # of 31 effects on saccadic eye movement peak velocity for TPA023 and SL were 5 and 375 ng/ml, respectively, again emphasizing the relative potency of TPA023. The plasma-occupancy relationship for TPA023 in man is slightly more potent than in preclinical species (EC 50 in man = 9 ng/ml vs ng/ml in preclinical species). Similarly, the structurally-related compound α5ia was also more potent in man compared to preclinical species (respective EC 50 values = 10 ng/ml vs ng/ml; Atack et al., 2009a) as was the imidazotriazine TPA023B (EC 50 values of 19, 10 and 5.8 ng/ml in rat, baboon and human, respectively; Atack et al., 2009b). On the other hand, lorazepam appears to have a similar potency in rats and man, albeit that the low levels of occupancy achieved in man limit the reliability of such a comparison. Nevertheless, the relatively low levels of occupancy of 6-9% produced by 1 mg lorazepam (Lingford-Hughes et al., 2005) occur, assuming dose-proportional plasma drug concentrations in man (Greenblatt et al., 1988; de Haas et al, 2007), at a plasma concentration of around 10 ng/ml and this is comparable to the ~10% occupancy produced at a concentration of 10 ng/ml in rats (Atack et al., 2007). The greater potency of TPA023 in man compared to lorazepam is also reflected in comparison with other benzodiazepine site ligands. For example, in man a plasma concentration of zolpidem ~100 ng/ml produced 26-29% occupancy and a plasma alprazolam concentration of ~20 ng/ml gives 16% occupancy (Fujita et al., 1999). The relative potency of TPA023 in man is further emphasized by the fact that a dose of 2 mg (or ~0.03 mg/kg p.o.) produces 50-60% occupancy whereas in rats an approximately 10-fold higher dose (0.42 mg/kg) was required to give 50% occupancy (Atack et al., 2006b). In summary, the occupancy of brain benzodiazepine binding sites was measured in a number of preclinical species and man using a variety of methods and was related to plasma drug concentrations. Despite the different detection methods (ex vivo or in vivo PET or γ-scintigraphy) and radioligands

19 JPET # of 31 ([ 3 H]flumazenil, [ 11 C]flumazenil or [ 123 I]iomazenil) employed, the plasma TPA023 EC 50 was relatively constant in preclinical species, ranging from ng/ml whereas TPA023 was slightly more potent in man with an EC 50 of 9 ng/ml. This latter EC 50 is much more potent in terms of receptor occupancy than existing benzodiazepine site ligands for which comparable data is available (lorazepam, zolpidem and alprazolam) and suggest that despite TPA023 being an α2/α3-selective GABA A receptor partial agonist, the higher levels of occupancy required for such a compound to have putative clinical efficacy relative to non-selective full agonists should be readily achievable with comparatively small doses. More generally, the present study clearly demonstrates that receptor occupancy is a translational marker that can be applied across species and aids the interpretation not only of preclinical (Atack et al., 2006b; Ator et al., 2009) but also clinical data (Atack et al., 2009b,c) and which in the case of TPA023 clearly emphasize the fact that GABA A α2/α3 subtype-selective compounds have novel pharmacological profiles.

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27 JPET # of 31 Footnotes This work was conducted either while authors were employees of Merck & Co., Inc. or was funded by Merck & Co., Inc. Present addresses Johnson & Johnson Pharmaceutical Research and Development, Beerse, Belgium (J.A.) GSK, Stevenage, UK (P.S.-S.) Novartis, Horsham, Surrey, UK (B.S.) Pfizer Global Research and Development, Sandwich, Kent, UK (R.M.M.)

28 JPET # of 31 Legends for Figures Figure 1. Occupancy of GABA A receptor benzodiazepine sites and corresponding plasma concentrations following oral dosing of TPA023. A. Occupancy of rat brain GABA A receptor benzodiazepine binding sites was measured as the extent by which the in vivo binding of [ 3 H]flumazenil was reduced by dosing of TPA023 (1, 3 or 10 mg/kg p.o. in 0.5% methyl cellulose vehicle). B. Trunk blood was collected from animals used for the occupancy study and plasma concentrations determined. For comparison, the open circles shows the kinetics of TPA023 in plasma of cannulated rats following an i.v. dose (1.2 mg/kg in 50% PEG 300 vehicle, n =6). Values shown are mean ± SEM (n=5-10/group). Figure 2. Rat plasma TPA023 concentrations and cortex radioactivity time-activity curves measured using [ 11 C]flumazenil micropet. A. Mean (± SEM, n=3) plasma TPA023 concentration during the low dose (10-26 min) and high dose (26-52 min) phases of the study. B. Cortex time-activity curves in a representative vehicle- and TPA023-treated rat. Arrows indicate injections of the low dose (0.05 mg/kg i.v.; t=10 min) and high dose (0.2 mg/kg i.v.; t=26 min) of TPA023 as well as a dose of flumazenil (0.25 mg/kg i.v.) sufficient to block all binding sites and therefore define non-specific binding (NSB). Non-specific binding values prior to t=52 min were estimated through backwards exponential extrapolation. C. Mean (± SEM, n=3) TPA023 occupancy derived from the min and min periods of the low- and high-dose periods of the experiment. Figure 3. Displacement by TPA023 of [ 123 I]iomazenil radioactivity from brain rhesus monkey brain as measured using γ-scintigraphy. Two h after the bolus/infusion of [ 123 I]iomazenil, TPA023 was infused

29 JPET # of 31 at rates of 0.01, and mg/kg/h (vehicle = EtOH:PEG400:water in a ratio of 1:3:6 v/v). The extent of the displacement of [ 123 I]iomazenil by TPA023 (i.e. the occupancy of benzodiazepine binding sites) was measured and plasma samples removed once relative steady-state conditions had been achieved (i.e., after 3 h of TPA023 infusion). Non-specific binding was estimated by injection of flumazenil (1 mg/kg in EtOH:PEG:water) at t=5 h. Data shown are 3 separate experiments performed using the same monkey. Figure 4. Pseudocoloured images of horizontal sections of the brain of subjects receiving either placebo, lorazepam (2 mg) or TPA023 (2 mg) illustrating the uptake of [ 11 C]flumazenil into the brain. Images were acquired over the duration of each 60 min scan. Figure associated with the 2- and 6.5-h scans represent the average reduction in cumulative radioactivity relative to each subjects baseline scan. Figure 5. Representative time-activity curves for the uptake of [ 11 C]flumazenil into occipital cortex of subjects treated orally with placebo, lorazepam (2 mg) and TPA023 (2 mg). Scans 1, 2 and 3 refer to the periods h before dosing (= baseline), and and 6-7 h post-dosing, respectively. Figure 6. Plasma-occupancy relationship for TPA023 across species. A. Occupancy in rat brain as measured using [ 3 H]flumazenil in vivo binding. Data from each animal used in the time-course study (Figure 1) was plotted as a function of the corresponding plasma TPA023 concentration. These data were fit with a single-site model which gave an EC 50 of 25 ng/ml (Hill slope = 1.17, n=104). B. Rat brain occupancy measured using [ 11 C]flumazenil micropet. EC 50 = 21 ng/ml. C. Occupancy in rhesus monkey brain measured using [ 123 I]iomazenil γ-scintigraphy. Plasma drug concentrations were measured once steady-state levels had been achieved (i.e., 3 h after initiation of TPA023 infusion). EC 50

30 JPET # of 31 = 30 ng/ml. D. Baboon brain occupancy as measured using [ 11 C]flumazenil PET. EC 50 = 19 ng/ml. E. Plasma-occupancy curve for TPA023 in man. Occupancy was measured as the extent to which uptake of [ 11 C]flumazenil PET (calculated from the time-activity curves) was reduced relative to baseline 2 and 6.5 h after a single dose of TPA023 (2 mg). Plasma drug concentrations were the average of those at the start and end of each 60 min acquisition period ( h and 6-7 h post dose). Data shown are from three TPA023-treated subjects, each of which generated two data points (i.e., measurements 2 and 6.5 h post-dose). F. A comparison of plasma-occupancy curves across species.

31 JPET # of 31 Table 1: Summary of plasma concentrations of TPA023 required for 50% occupancy in different preclinical species and man Plasma TPA023 conc., ng/ml Species Assay EC 50 (Hill 95% C.I. a slope) Rat [ 3 H]flumazenil in vivo binding 25 (1.2) Rat [ 11 C]flumazenil micropet 21 (2.1 ) Rhesus monkey [ 123 I]iomazenil γ-scintigraphy 30 (1.5) Baboon [ 11 C]flumazenil PET 19 (1.6) Man [ 11 C]flumazenil PET 9 (1.0 b ) 7-12 a, 95% C.I. = 95% confidence intervals b, Due to the clustering of the human data within a relatively narrow range of occupancies (34-64%), these data were fitted to a single-site model with the Hill slope fixed at All curve-fitting was constrained to minimum and maximum occupancy values of 0 and 100%, respectively.

32 Figure 1 A. B % Occupancy 10 mg/kg 3 mg/kg 1 mg/kg Time, h mg/kg 3 mg/kg 1 mg/kg Plasma TPA023, ng/ml Time, h

33 Figure 2 A. B. C Plasma TPA023, ng/ml Time after [ 11 C]flumazenil, min vehicle/ low-dose mg/kg 0.2 mg/kg % Occupancy vehicle/ high-dose Time, min flu. Cortical radioactivity

34 Figure mg/kg/h mg/kg/h flumazenil mg/kg/h Time, min Brain radioactivity, cpm

35 Figure 4 Placebo Lorazepam, Subject #1 Lorazepam, Subject #2 Baseline 2 h 6.5 h Baseline 2 h 6.5 h ~0% ~-15% 63% 59% TPA023, Subject #1 ~5% ~6% 47% 46% TPA023, Subject #2 ~5% ~-9% TPA023, Subject #3 64% 34%