1521-009X/44/9/1536–1542$25.00 http://dx.doi.org/10.1124/dmd.116.070359DRUG METABOLISM AND DISPOSITION Drug Metab Dispos 44:1536–1542, September 2016Copyright ª 2016 by The American Society for Pharmacology and Experimental Therapeutics

Occupancy of Nociceptin/Orphanin FQ Peptide Receptors by theAntagonist LY2940094 in Rats and Healthy Human Subjects s

Eyas Raddad, Amy Chappell, Jeffery Meyer, Alan Wilson, Charles E. Ruegg, Johannes Tauscher,1

Michael A. Statnick, Vanessa Barth, Xin Zhang, and Steven J. Verfaille

Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, Indiana (E.R., A.C., C.E.R., J.T., M.A.S., V.B., X.Z., S.J.V.); ResearchImaging Centre, Campbell Family Mental Health Research Institute, Centre for Addiction and Mental Health, Department of

Psychiatry, University of Toronto, Toronto, Ontario, Canada (J.M., A.W.)

Received March 7, 2016; accepted June 6, 2016

ABSTRACT

Therapeutic benefits from nociceptin opioid peptide receptor (NOP)antagonism were proposed for obesity, eating disorders, anddepression. LY2940094 ([2-[4-[(2-chloro-4,4-difluoro-spiro[5H-thieno[2,3-c]pyran-7,4ʹ-piperidine]-1ʹ-yl)methyl]-3-methyl-pyrazol-1-yl]-3-pyridyl]methanol) is a novel, orally bioavailable, potent, and selectiveNOP antagonist. We studied NOP receptor occupancy (RO) after singleoral LY2940094 doses in rat hypothalamus and human brain by use ofliquid chromatography with tandem mass spectrometry (LC-MS/MS)(LSN2810397) and positron emission tomography (PET) ([11C]NOP-1A)tracers, respectively. A bolusplus constant infusion tracer protocolwithPETwas employed in humans at 2.5 and 26.5 hours after administrationof the LY2940094 dose. The RO was calculated from the change inregional distributional volume (VT) corrected for nondisplaceablevolume using Lasson plots. The RO followed a simple Emax relationship

to plasma LY2940094 concentration, reaching near complete occu-pancy in both species. For rat hypothalamus, the plasma concentrationat half-maximum RO (EC50) was 5.8 ng/ml. In humans, LY2940094 waswell tolerated and safe over the 4–40 mg dose range, and it peaked inplasma at 2 to 6 hours after a 1- to 2-hour lag, with approximate dose-proportional exposure. After 4–40 mg doses, NOP RO was similaracross the prefrontal cortex, occipital cortex, putamen, and thalamus,with EC50 of 2.94 to 3.46 ng/ml, less than 2-fold lower than in rats. Over4–40 mg doses, LY2940094 mean plasma levels at peak and 24 hourswere 7.93–102 and 1.17–14.1 ng/ml, corresponding to the cross-regionaverageNOPROof 73%–97%and28%–82%, respectively. The rat EC50

translates well to humans. LY2940094 readily penetrates the humanbrain, and a once-daily oral dose of 40 mg achieves sustainably high(>80%) NOP RO levels suitable for testing clinical efficacy.

Introduction

Nociceptin/orphanin FQ (nociceptin) is a 17-amino acid neuropeptidethat acts as an endogenous ligand for the opioid-like one receptor(ORL1), also known as NOP (nociceptin opioid peptide receptor). NOPis a class A G-protein–coupled receptor (Meunier et al., 1995;Reinscheid et al., 1995), is encoded by the gene opiate receptor-like1 (OPRL1), and is widely expressed in the central nervous system and

specifically in regions associated with mood disorders and obesity.Symptoms of many neuropsychiatric disorders, such as pain, anxiety,depression, anorexia, obesity, and drug abuse, are believed to be poten-tially linked to the NOP receptor (Lambert, 2008; Murphy, 2010), whichsuggests NOP antagonism as a potential therapeutic strategy (Gavioli andCalo, 2006; Witkin et al., 2014).Interest in the target has spurred the discovery of multiple tracers.

Hostetler et al. (2013) reported the discovery of [18F]MK-0911, a potent,selective, and brain-penetrant positron emission tomography (PET)tracer, which was shown to have NOP binding in monkeys and humans,and was displaceable by the potent NOP antagonist MK-5757. We haverecently discovered both labeled and unlabeled NOP receptor tracerswith subnanomolar binding affinities with no intrinsic activity (i.e.,antagonists), high selectivity, central nervous system penetration, andlow nonspecific binding (Pedregal et al., 2012). LSN2810397 [Com-pound (S)-27 in Pedregal et al., 2012; NOP Ki = 0.114 nM] is anunlabeled tracer based on liquid chromatography with tandem massspectrometry (LC-MS/MS), and [11C]NOP-1A (Kimura et al., 2011;Lohith et al., 2012, 2014; Pike et al., 2011) is a labeled tracer (NOP Ki =0.15 nM) that lacks brain-penetrant radiometabolites and is suitable for

This work was supported by Eli Lilly and Company. E.R., S.J.V., C.E.R., M.A.S.,V.B., and X.Z. are current employees of, and have financial holdings in, Eli Lilly andCompany. A.C. and J.T. were employees and had financial holdings in Eli Lilly andCompany during the conduct of the studies. J.M. and A.W. received operatinggrant funds for this study from Eli Lilly and for other studies from GlaxoSmithKline,Bristol Myers Squibb, Lundbeck, Janssen, and SK Life Sciences in the past5 years. J.M. has consulted to these companies, with the exception of Janssen,as well as Takeda, Sepracor, Trius, Mylan, and Teva. LY2940094 and LSN2810397were sourced from Eli Lilly and Company.

1Current affiliation: Takeda, Deerfield, Illinois.dx.doi.org/10.1124/dmd.116.070359.s This article has supplemental material available at dmd.aspetjournals.org.

ABBREVIATIONS: AE, adverse event; CAMH, Centre for Addiction and Mental Health; Cp, tracer concentration in plasma; Ct, average tracerconcentration in the brain; EC50, plasma concentration at half-maximum receptor occupancy; Emax, maximum occupancy; HPLC, high-performanceliquid chromatography; LC-MS/MS, liquid chromatography with tandem mass spectrometry; LY2940094, [2-[4-[(2-chloro-4,4-difluoro-spiro[5H-thieno[2,3-c]pyran-7,4ʹ-piperidine]-1ʹ-yl)methyl]-3-methyl-pyrazol-1-yl]-3-pyridyl]methanol; MRI, magnetic resonance imaging; NAD-299, (3R)-3-[di(cyclobutyl)amino]-8-fluoro-3,4-dihydro-2H-chromene-5-carboxamide; NOP, nociceptin opioid peptide receptor; PET, positron emissiontomography; RO, receptor occupancy; Ro-64-6198, [(1S,3aS)-2,3,3a,4,5,6-hexahydro-1H-phenalen-1-yl]-1-phenyl-1,3,8-triazaspiro[4.5]decan-4-one; ROI, regions of interest; SB-612111, (5S,7S)-7-[[4-(2,6-dichlorophenyl)piperidin-1-yl]methyl]-1-methyl-6,7,8,9-tetrahydro-5H-benzo[7]annulen-5-ol; TEAE, treatment-emergent adverse event; VT, PET tracer distribution volume.

1536

http://dmd.aspetjournals.org/content/suppl/2016/06/27/dmd.116.070359.DC1Supplemental material to this article can be found at:

at ASPE

T Journals on A

pril 19, 2022dm

d.aspetjournals.orgD

ownloaded from

PET studies. The two tracers are similar structurally, differing only in thepresence or absence of one N-substituted methyl. In humans, [11C]NOP-1A is safe, with favorable kinetics and reproducibility (Lohith et al.,2012, 2014), and exhibits a distribution volume (VT) consistent withknown NOP density distribution (Berthele et al., 2003).Potent and selective NOP antagonists based on the dihydrospiro

(piperidine-4,7ʹ-thieno[2,3-c]pyran) chemical scaffold have beenrecently discovered (Toledo et al., 2014). Among these, ([2-[4-[(2-chloro-4,4-difluoro-spiro[5H-thieno[2,3-c]pyran-7,4ʹ-piperidine]-1ʹ-yl)methyl]-3-methyl-pyrazol-1-yl]-3-pyridyl]methanol), referred to asLY2940094 (Statnick et al., 2016) (Compound 36 in Toledo et al., 2014;NOP Ki = 0.105 nM), was advanced to clinical development for thetreatment of depression and alcohol dependence.We assessed the brain NOP receptor occupancy (RO) after single oral

administration of a range of doses of LY2940094 to healthy volunteersusing [11C]NOP-1A as a PET ligand, and examined the relationshipbetween the NOP RO levels of LY2940094 and its plasma concentra-tions to support dose selection for future efficacy trials. Furthermore, weassessed the translatability of the concentration-RO relationship ofLY2940094 between that in rats (using LSN2810397 as the LC-MS/MS–based tracer) and humans (using [11C]NOP-1A and PET imaging).

Materials and Methods

Preclinical Studies of LY2940094 NOP RO in Rats. The NOP RO ofLY2940094 (Eli Lilly and Company, Indianapolis, IN) was evaluated in thehypothalamus in both dose–response and time-course experiments. MaleSprague-Dawley rats (230–300 g; three to four per dose group or time point)were housed on a 12-hour light/dark cycle (testing during light phase) andreceived free access to normal rat chow and water. Animals received eithervehicle, the reference NOP antagonist (5S,7S)-7-[[4-(2,6-dichlorophenyl)-piperidin-1-yl]methyl]-1-methyl-6,7,8,9-tetrahydro-5H-benzo[7]annulen-5-ol (SB-612111, 30 mg/kg, oral by gavage; Eli Lilly and Company) as a positive control(Spagnolo et al., 2007), or LY2940094 (0.01–10 mg/kg for the dose–responseexperiment; 1 mg/kg for the time-course experiment). For the dose–responseexperiment, NOP RO was assessed at 6 hours after dose administration; for thetime-course experiment, NOPROwas assessed at 0.083, 0.25, 1, 2, 4, 8, and 24 hoursafter dose administration. All experiments were conducted in compliance with theGuide for the Care and Use of Laboratory Animals under protocols approved by alocal animal care and use committee.

For every RO assessment, 3 mg/kg of the tracer LSN2810397 (Eli Lilly andCompany) was administered intravenously 40 minutes before sacrificing theanimals. Tracer concentrations in the harvested hypothalamus, a region with highNOP expression (Neal et al., 1999; Florin et al., 2000), were assessed aspreviously described elsewhere (Pedregal et al., 2012; Toledo et al., 2014). Thebrain tissue (time-course study) and plasma (both studies) LY2940094 concen-tration were determined by use of a specific and validated LC-MS/MS methodthat uses a 1200 HPLC apparatus (high-performance liquid chromatography;Agilent Technologies, Palo Alto, CA) linked to an API 4000 mass spectrometer(Applied Biosystems, Foster City, CA). A C18 column (2.1 mm � 50 mm; partno. 971700-907; Agilent Technologies) was used for the HPLC.

Because no true null region devoid of the NOP receptor was discerned in ratbrain, the ratio method for target occupancy calculations could not be employed.To define 100% occupancy, or full blockade of specific binding, the hypothalamictracer levels (ng/g) of a positive control group were used. Previous experimentsdetermined that an oral dose of 30 mg/kg of SB-612111 fully blocked the tracerLSN2810397 (Pedregal et al., 2012). The vehicle group ng/g tracer levels in thehypothalamus represented 0% occupancy. The hypothalamic tracer levels ofcohorts of animals receiving LY2940094 were linearly interpolated based on theslope of the line between the tracer levels of the vehicle (0%) and positive control(100%) groups to obtain occupancy values based on the ng/g of tracer measured.

Clinical Study Design. The single-dose PET study used an open-label,nonrandomized, and adaptive study design and was conducted on sequentialcohorts of healthy participants, enrolling a total of 14 healthy men, of whom12 completed all the study procedures (3 per dose level). The participants weremen between 18 and 45 years of age (inclusive) in good health. Five of the

participants (41.7%) were white, 6 (50.0%) were black or African American, and1 (8.3%) was Asian. A more complete list of entry criteria can be found atclinicaltrials.gov, identifier: NCT01404091.

Participant recruitment and monitoring was conducted at Kendle Early Stage(Toronto, Canada), and human neuroimaging was conducted at the Centre forAddiction andMental Health (CAMH)Research ImagingCentre, Toronto, Canada.The PET scans were performed at baseline and approximately 2.5 and 26.5 hoursafter dose administration over two visits. The participants consented to the protocolbefore their inclusion, the study was approved by the institutional review boards ofKendle Early Stage and CAMH, and it was performed in accordance with theethical standards laid down in the 1964 Declaration of Helsinki.

There were four cohorts of subjects. The first cohort of three subjects received40 mg of LY2940094, and the subsequent cohorts received dose levels that wereselected based on accumulated RO data in the study, with the objective of exploringa wide dynamic range of RO. Cohort 2 and 3 subjects (n = 3 each) received 10 and4 mg, respectively, and cohort 4 subjects received 20 mg (n = 2) and 40 mg (n = 1).

Safety was monitored regularly, including adverse events (AEs), vital signs,electrocardiograms, physical examinations, clinical laboratory tests, the ColumbiaSuicide Severity Rating Scale, and other routine laboratory tests.

Radiotracer [11C]NOP-1A. [11C]NOP-1Awas synthesized and formulated inthe radiochemistry laboratory of the CAMH PET Centre (Toronto, Canada) by11C-methylation of an N-desmethyl precursor (Eli Lilly and Company), aspreviously described in detail (Pike et al., 2011), and it was administered within60 minutes after the completion of radiosynthesis through an indwelling catheterin the antecubital vein, during which the radiotracer is known to be stable.

[11C]NOP-1Awas administered as an intravenous bolus plus constant infusionover the 2-hour scan.We followed publishedmethods to determine themagnitudeof bolus fraction (i.e., Kbol) to be equivalent to 70 minutes of constant infusion(Carson et al., 1993). Previous assessments have demonstrated that approximatesteady-state conditions could be consistently achieved (R. Innis, personal commu-nication, 2011) using this regimen. The average total injected dose of radioactivityfor the PET radiotracer was less than 20 mCi (740 MBq) of [11C]NOP-1A, whichalong with the transmission scan corresponded to less than 3.382 mSv per scan andless than 10.15 mSv for the study.

Magnetic Resonance Imaging and PET Image Acquisition Procedures.The emission PET images were acquired on a whole body PET camera system,Siemens-Biograph HiRez XVI (Siemens Molecular Imaging, Knoxville, TN).Magnetic resonance imaging (MRI) scans were obtained before the PET scanningprocedures to coregister the PET and MRI images for analysis of the PET data toidentify the volumes of interest using an automated method verified by visualinspection (Rusjan et al., 2006). Additional details regarding the MRI and PETimage acquisition can be found in Supplemental Fig. 3.

One arterial blood sample was collected during the PET scanning after a steadystate had been reached (mean 6 S.D. = 59 6 11 minutes) after the radiotracerinjection. The parent fraction was measured by HPLC to determine the metabolitecorrected plasma input function, as previously described elsewhere (Lohith et al.,2012). Sample chromatogram can be found in Supplemental Fig. 4.

Distribution Volume and RO Determination. PET tracer distributionvolume (VT) was estimated in multiple brain regions: putamen, caudate, occipitalcortex, prefrontal cortex, thalamus, and cerebellum. VT, representing the sum ofspecific and nonspecific binding (Ichise et al., 2001; Innis et al., 2007),was determinedas the ratio of the average tracer concentration in the brain (Ct) during steady statedivided by the tracer concentration in plasma (Cp). For the four regions of interest(ROI), namely, the prefrontal and occipital cortices, putamen, and thalamus, the NOPRO by LY2940094 (OROI

drug) was calculated based on the following equation:

OdrugROI ¼

�Vbase

T;ROI 2VdrugT;ROI

�.�Vbase

T;ROI-VND

�:

where VT,ROIbase, VT,ROI

drug, and VND are the total distribution volume before(baseline) and after LY290094 administration and nondisplaceable volume,respectively.

The nondisplaceable volume (VND) was estimated as the intercept of the Lassenplots (Lassen et al., 1995; Cunningham et al., 2010), where the difference betweenthe baseline and blocked VT values is plotted against baseline VT for differentregions of the brain.

Plasma Concentrations and Pharmacokinetics of LY2940094. Venousblood samples were collected in containers with an appropriate anticoagulantbefore and after each scan as well as serially over a 24-hour period. LY2940094

Occupancy of the NOP Antagonist LY2940094 in Rats and Humans 1537

at ASPE

T Journals on A

pril 19, 2022dm

d.aspetjournals.orgD

ownloaded from

plasma concentrations were measured by a specific and validated LC-MS/MSmethod. The prevailing LY2940094 concentration during the scan was calculatedas the average of the concentration before and after each scan. LY2940094pharmacokinetic parameters were estimated using standard noncompartmentalanalysis methods.

LY2940094 Plasma Concentration–NOP RO Relationship. The relation-ship between the prevailing LY2940094 concentrations in plasma during the PETscan and the OROI

drug values was characterized using a mixed-effect Emax model,where Emax was fixed to the theoretical value of 100%. The model estimated EC50

values and additive intersubject and within-subject variances using SAS version9.2 (SAS Institute, Cary, NC).

Results

LY2940094 NOP RO in Rat Brain. LY2940094 potently, dose-dependently, and fully occupy the NOP receptor (Fig. 1A). In the time-course study, LY2940094 plasma and brain concentrations were generallyparallel (Fig. 1B). Following its peak, RO followed an approximate lineardecline (Fig. 1C), maintaining .50% RO for at least 12 hours (byinterpolation). A linear decline in RO is typical when drug levels declineexponentially, while the relationship between concentration and ROfollows an Emax model. The Emax fit to the NOP RO vs. plasmaLY2940094 levels (Fig. 1D) resulted in an EC506 SE estimate of 5.7561.25 ng/mL.ROwas practically 100%between 5minutes and 2 hours postdose, when LY2940094 was at its highest levels in plasma and braintissue, approximately 99–206 ng/mL and 84–123 ng/g, respectively.LY2940094 Human Plasma Pharmacokinetics. LY2940094

plasma levels started to rise after a 1- to 2-hour lag time that was quite

variable across subjects (Fig. 2). Peak levels were achieved between 2and 6 hours, followed by a biphasic decline: A rapid phase up toapproximately 12 hours, followed by a slow terminal phase.The first postdose PET scan, which was a priori set at 2.5 hours to

coincide with the peak drug levels, appears to have occurred too early inmany subjects. The mismatch was most significant for the subjectstaking the 4- and 40-mg doses.The pharmacokinetic parameter estimates for LY2940094 are sum-

marized in Table 1. Cmax and area under the plasma concentration–timecurve from 0 to 28.5 hours after dose administration (AUC0–28.5) valuesappeared to increase approximately linearly as the LY2940094 doseincreased, although the sample size was too small to test this hypothesisformally.Radiosynthesis and Measurement in Plasma of [11C]NOP-1A.

Mean tracer specific activity of [11C]NOP-1A at the end of the synthesiswas 5720 mCi/mmol (S.D. = 1600 mCi/mmol), and the mean massinjected was 1.57 mg (S.D. = 0.66 mg) with a radiochemical purity of98.2% (S.D. = 1.0%). Parent [11C]NOP-1A typically formed 64% of thetotal radioactivity in arterial blood samples.[11C]NOP-1A Time-Activity Curves and LY2940094 NOP RO.

Sample PET images (Fig. 3) obtained from the same subject demon-strated the overall brain uptake of [11C]NOP-1A and its blockade byLY2940094.Time-activity curves, presented as the ratio of radioactivity to the

parent-related radioactivity in plasma, are shown in Supplemental Fig. 1.At baseline, the brain tracer uptake ratio levels in various regions ofinterest rapidly increased upon the regimen initiation to a peak level

Fig. 1. NOP RO in rat hypothalamus after single oral dosing of LY2940094. (A) NOP RO 6 hours after dose administration as a function of dose, with Emax model fit (dottedline). (B) Plasma and brain LY2940094 concentrations over time after 1 mg/kg dose. (C) Time course of NOP RO and brain concentration after 1 mg/kg dose. (D) NOP ROas a function of time-matched LY2940094 plasma concentration, with Emax model fit (dotted line).

1538 Raddad et al.

at ASPE

T Journals on A

pril 19, 2022dm

d.aspetjournals.orgD

ownloaded from

typically after 10 to 20 minutes (due to the bolus) and subsequentlyslightly decreased to steady-state levels. The steady-state level washighest in receptor-richer regions like the thalamus, occipital cortex,prefrontal cortex, and putamen, followed by the caudate and hippocam-pus; that in the cerebellar cortex was the lowest. Postdose scans revealeda reduction in radiotracer uptake (lower steady-state levels) that wasmore profound after higher LY2940094 doses and in peak time scans,which was consistent with the higher NOP blockade at higher plasmaconcentrations.Supplemental Figure 2 shows several typical Lassen plots obtained.

Linear regression fits were good, with R2 ranging between 0.83 and0.99, except for one scan after the 4-mg dose where the relationship wasquite flat, indicating very low NOP occupancy.Table 2 summarizes the mean NOPRO values by ROI, dose, and time

point. LY2940094 exhibited dose-dependent target engagement of theNOP receptors after single oral doses (4–40 mg), exceeding 80% onaverage in the top dose levels. The NOP RO seemed to decreasemoderately from the first to the second scan. The RO was similar acrossthe different brain ROIs.The Emax model fitted the NOP RO-plasma concentration data well

(Fig. 4). Table 3 summarizes the derived parameters from model fits.Safety. LY2940094 was safe and well tolerated after single admin-

istrations of doses ranging from 4–40 mg. There were no deaths, serious

AEs, or severe treatment-emergent adverse events (TEAEs). The overallincidence of TEAEs was 25%. TEAEs were mild in severity, with norelationship to dose, and were considered either unrelated or unlikely tobe related to LY2940094. The most frequent TEAE was vessel puncturesite pain, reported by two participants.

Discussion

We report here the results of our preclinical and clinical studiesdesigned to demonstrate the NOP RO by the novel potent and selectiveNOP receptor antagonist LY2940094, which is currently under clinicaldevelopment for the treatment of depression. Our studies relied on a pairof structurally similar NOP receptor tracers, LSN2810397 and [11C]NOP-1A, both containing spiropiperidine, a common structural motif for NOPantagonists (Toledo et al., 2014) that is also shared by LY2940094. Thestudies examine the ability of rat NOP RO potency to predict human NOPRO potency. Additionally, these studies shed light on key attributes ofLY2940094, including its systemic availability and pharmacokinetics,brain penetration, and the level of NOPRO in various regions of interest inthe brain, providing a basis for dose selection for future development intarget indications.Preclinical to Clinical Translation of NOP RO. In translational

drug development, it is desirable to use the RO results of a candidatedrug in preclinical species for predicting its human behavior (Melhem,2013). Cross-species potency differences could arise from differences inplasma protein binding, transport into the brain, and the degree ofreceptor homology. Good cross-species concordance has been found inmultiple applications (Burns et al., 2007; Hostetler et al., 2013; Melhem,2013). In others, significant differences existed but were explained bydifferences in protein binding. For example, the serotonin 1A (5-HT1A)receptor antagonist (3R)-3-[di(cyclobutyl)amino]-8-fluoro-3,4-dihydro-2H-chromene-5-carboxamide (NAD-299) was shown to possess about10-fold higher in vivo potency in monkeys than in humans, which couldbe accounted for by a 10-fold higher plasma free fraction (Andrée et al.,2003). In contrast, it was found that three structurally related inverseagonists for the GABAA receptors had EC50 values in human that are athird to a fifth of those in rats and rhesus monkeys, which could not be

Fig. 2. Individual LY2940094 plasma concentrations overtime by dose in human subjects after single oral adminis-tration. Vertical lines are drawn to represent the timing ofthe two post-dose scans.

TABLE 1

Mean (%CV) plasma noncompartmental LY2940094 pharmacokinetic parametersby dose after single oral administration to human subjects

Dose n Cmax Tmaxa AUC0–28.5

mg ng/ml h h*ng/ml

4 3 7.93 (44.2) 6.00 (13.1) 63.4 (27.5)10 3 22.4 (24.7) 4.48 (0.8) 176 (24.0)20 2 38.5 NC 2.32 NC 316 NC40 4 102 (26.2) 6.00 (12.7) 811 (16.3)

AUC0–28.5 = area under the plasma concentration–time curve from 0–28.5 hours after doseadministration; NC, not calculated; Tmax = time of maximum concentration.

aMedian.

Occupancy of the NOP Antagonist LY2940094 in Rats and Humans 1539

at ASPE

T Journals on A

pril 19, 2022dm

d.aspetjournals.orgD

ownloaded from

accounted for by differences in protein binding or binding affinity(Atack et al., 2010; Eng et al., 2010).In our studies, the LY2940094 NOP RO EC50 values in rats and humans

were approximately 2-fold apart; 5.75 and 2.64–3.46 ng/ml, respectively.We assessed the binding affinity of LY2940094 in the membrane ofChinese hamster ovary cells expressing recombinant human NOP receptorand membranes isolated from whole rat brain with Ki values of 0.11 and0.71 nM, respectively (Statnick et al., 2016). The free fraction in plasmawasalso assessed in both species: 0.89%and 0.56%, respectively. Correcting therat NOP RO EC50 value by both factors gives a human-equivalent EC50 of0.56 ng/ml, overpredicting the human RO potency by 5-fold.A larger dataset for different receptor types and ligands would be needed

before generalized translatability conclusions can be made. However, it isclear that a few-fold error in either direction should be expected. In thislight, rats were reasonably predictive of human potency of NOP RO andcan thus support using preclinical RO data to help design early clinicalstudies. Contributing factors to translation error include relatively imprecisein vitro methods as well as differences in the methods used to assess RO.Brain Penetration and RO Kinetics. Multiple pieces of evidence

suggest that LY2940094 equilibrates across the blood-brain barrierquickly, likely through passive diffusion. Data generated in ourlaboratories demonstrated that LY2940094 exhibited fast, passive

permeability with high partitioning in Madin-Darby canine kidney cells,andwas not a P-glycoprotein substrate (unpublished). Consistentwith theseresults, plasma and brain concentrations were generally parallel in rats (Fig.1B). Furthermore, during the model building of human data, we confirmedthat the EC50 values separately estimated after the first and second postdosescans were quite similar, suggesting a direct relationship between NOPRO and plasma concentrations with no hysteresis or lag, a phenomenonconsistent with rapid equilibration between brain and plasma.LY2940094 Pharmacokinetics, NOP RO, and Dose Selection.

Overall, our PET study in healthy volunteers demonstrated brainpenetrance and specific target engagement of NOP receptors after singleoral doses (4–40 mg) of LY2940094. The NOP RO increased with doseand concentration, and was generally higher at the first postdose PETscan (2.5 hours) compared with the second (26.5 hours). The NOP ROlevels appeared similar at the 20- and 40-mg doses at approximately 80%.The RO remained high 26.5 hours after the 40-mg dose (above 70%).On a plasma LY2940094 concentration basis, LY2940094 potently

occupied the NOP receptors, with EC50 values consistent across ROIs,ranging between 2.64 and 3.46 ng/ml. Unlike the relationship with dose,the LY2940094 concentration–RO relationship clearly suggests thathigher drug levels would produce an NOP RO approaching 100%,consistent with rat data. The discrepancy can be explained by

Fig. 3. Sample image of [11C]NOP-1A uptake in the samehuman subject at baseline and at 2.5 hours and 26.5 hoursafter oral dosing of LY2940094 (40 mg), respectively.

TABLE 2

Regional NOP receptor occupancy values (%) by time point and LY2940094 dose after single oral administration tohuman subjects

Model: Occupancy (%) = Emax * Concentration/(EC50 + Concentration); additive random subject.

Region of Interest

Dose

4 10 20 40

mg

Scheduled postdose time

2.5(n = 3)

26.5(n = 3)

2.5(n = 3)

26.5(n = 3)

2.5(n = 2)

26.5(n = 2)

2.5(n = 4)

26.5(n = 3)

h

Prefrontal cortexMean 48.8 38.9 74.4 30.2 89.0 62.4 86.5 76.6S.D. 42.6 17.3 13.0 14.4 12.6 26.3 7.66 8.69

Occipital cortexMean 44.1 39.1 73.3 23.1 82.5 56.7 83.2 74.0S.D. 42.3 15.0 13.4 19.9 18.2 27.8 7.13 5.54

PutamenMean 51.3 43.9 65.1 27.5 80.6 56.9 80.9 71.5S.D. 34.6 13.5 17.5 18.7 13.7 28.5 8.12 7.92

ThalamusMean 49.3 42.7 66.5 29.5 82.5 60.0 83.7 75.5S.D. 40.4 14.2 17.4 13.9 13.0 21.1 7.25 8.49

1540 Raddad et al.

at ASPE

T Journals on A

pril 19, 2022dm

d.aspetjournals.orgD

ownloaded from

LY2940094 plasma levels during the PET scans. LY2940094 absorp-tion lagged by 1 to 2 hours and peaked 2 to 6 hours after oral dosing.Although the intent was for the first postdose scan to concur with thepeak drug levels, the PET scan largely preceded the peak in mostsubjects, oftentimes starting at the beginning of the rising phase of theLY2940094–concentration relationship due to the significant absorp-tion lag time (Fig. 2). Thus, the dose–RO relationship significantlyunderestimates the potency of LY2940094 at peak levels. By chance, thedrug levels during the first postdose scan in the 20- and 40-mg dosegroups were similar and hence the RO was similar (Table 2).Given the apparent quick equilibration of LY2940094 and constant RO

potency over time, NOP RO can be predicted from LY2940094 plasmaconcentration, using the established concentration–RO relationship. Giventhis relationship, we predict that the RO at mean observed peak concen-tration for the doses of 4, 10, 20, and 40 mg averaged over the four testedregions to be 73%, 88%, 93%, and 97%, respectively. Similarly, theplasma LY2940094 concentration 24 hours after dose administration isconsistent with 28%, 49%, 65%, and 82%, respectively.We measured RO after a single dose of LY2940094. However, the

RO after multiple-dose administration is of greater interest for a drugintended for chronic administration. The data in this study alone areinadequate to make such prediction, as a multiple-dose pharmacoki-netic examination would be necessary. However, it is clear from theconcentration data up to 28.5 hours after dose administration that theterminal half-life of LY2940094 is significantly in excess of 24 hours;thus, a significant accumulation of 2-fold or higher is to be expected

upon once-daily dosing of LY2940094. Accounting for 2-fold concen-tration accumulation, RO at trough levels would likely be in the vicinityof 90%. If NOP is similar to other G protein-coupled receptors such asdopamine D2 receptor antagonists as antipsychotic agents, a level ofoccupancy between 50% and 90% is associated with clinically effica-cious doses (Grimwood and Hartig, 2009).In rats, LY2940094 reversed thewell-documented hypothermic effect of

theNOP agonist 8-[(1S,3aS)-2,3,3a,4,5,6-hexahydro-1H-phenalen-1-yl]-1-phenyl-1,3,8-triazaspiro[4.5]decan-4-one (Ro-64-6198) 2 hours after doseadministration at 0.1 and 0.3 mg/kg, doses that are consistent with 50% to80% NOP RO in our study (Toledo et al., 2014). Therefore, a 40-mgonce-daily dose of LY2940094, producing NOP RO in excess of 80%throughout the dosing interval, is likely sufficient to test the therapeutichypotheses of LY2940094, but a lower dose may also produce such a levelof RO. Based on this information, a 40-mg daily dose of LY2940094 wasrecently used in a proof-of-concept study ofmajor depressive disorder (Postet al., 2015),whichwas conducted after the studiesmentioned in this report.In that study, an 8-week treatment with a once-daily 40-mg dose ofLY2940094 provided some evidence of antidepressant effect (Post et al.,2015), further supporting the current hypothesis.

Conclusion

The [11C]NOP-1A tracer has proven useful for the assessment of therelationship between NOP RO, dose, and plasma concentration for thenewly discovered NOP antagonist LY2940094. LY2940094 penetrates

Fig. 4. Relationship between NOP RO and LY2940094 plasma concentration after single oral administration to human subjects in (A) prefrontal cortex, (B) occipital cortex,(C) putamen, and (D) thalamus (data from 12 subjects).

Occupancy of the NOP Antagonist LY2940094 in Rats and Humans 1541

at ASPE

T Journals on A

pril 19, 2022dm

d.aspetjournals.orgD

ownloaded from

the brain with apparent quick equilibration. Rat NOP RO potency wasreasonably predictive of human potency. At oral doses that appeared safein human volunteers (4–40 mg), LY2940094 occupied up to a meanof 87% at 2.5 hours after dose administration, with an expectation ofachieving approximately 97% at peak levels in various regions of thebrain. The plasma concentration potency (EC50) ranged between2.64 and 3.46 ng/ml. At this potency and pharmacokinetics, a 40-mgLY2940094 dose would be expected to maintain NOP RO above 80%throughout the dosing interval upon once-daily oral dosing, a dose that isappropriate to test the therapeutic hypotheses in future studies.

Acknowledgments

The authors thank Dr. Robert Innis (National Institute of Mental Health,Bethesda, MD) for assisting with the transfer of the [11C]NOP-1A tracer toCAMH and his general guidance on the methods, Dr. Francois Vandenhende(ClinBAY, Belgium) for conducting some of the calculations, and Dr. Wai-si Eng(YourEncore, Inc., Indianapolis, IN) for his editorial support of this manuscript.All experiments referred to in this manuscript fully comply with current laws ofthe countries in which they were performed.

Authorship ContributionsParticipated in research design: Chappell, Ruegg, Meyer, Wilson, Tauscher,

Statnick, Barth, Verfaille, Raddad.Conducted experiments: Meyer, Wilson, Statnick, Barth.Contributed new reagents or analytic tools: Meyer, Wilson.Performed data analysis: Raddad, Meyer, Wilson, Barth, Zhang.Wrote or contributed to the writing of the manuscript: Raddad, Chappell,

Meyer, Wilson, Statnick, Barth, Verfaille.

References

Andrée B, Hedman A, Thorberg SO, Nilsson D, Halldin C, and Farde L (2003) Positron emissiontomographic analysis of dose-dependent NAD-299 binding to 5-hydroxytryptamine-1A recep-tors in the human brain. Psychopharmacology (Berl) 167:37–45.

Atack JR, Wong DF, Fryer TD, Ryan C, Sanabria S, Zhou Y, Dannals RF, Eng WS, Gibson RE,Burns HD, et al. (2010) Benzodiazepine binding site occupancy by the novel GABAA receptorsubtype-selective 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 humans. JPharmacol Exp Ther 332:17–25.

Berthele A, Platzer S, Dworzak D, Schadrack J, Mahal B, Büttner A, Assmus HP, Wurster K,Zieglgänsberger W, Conrad B, et al. (2003) [3H]-nociceptin ligand-binding and nociceptinopioid receptor mrna expression in the human brain. Neuroscience 121:629–640.

Burns HD, Van Laere K, Sanabria-Bohórquez S, Hamill TG, Bormans G, Eng WS, Gibson R,Ryan C, Connolly B, Patel S, et al. (2007) [18F]MK-9470, a positron emission tomography(PET) tracer for in vivo human PET brain imaging of the cannabinoid-1 receptor. Proc Natl AcadSci USA 104:9800–9805.

Carson RE, Channing MA, Blasberg RG, Dunn BB, Cohen RM, Rice KC, and Herscovitch P(1993) Comparison of bolus and infusion methods for receptor quantitation: application to[18F]cyclofoxy and positron emission tomography. J Cereb Blood Flow Metab 13:24–42.

Cunningham VJ, Rabiner EA, Slifstein M, Laruelle M, and Gunn RN (2010) Measuring drugoccupancy in the absence of a reference region: the Lassen plot re-visited. J Cereb Blood FlowMetab 30:46–50.

Eng W, Atack JR, Bergstrom M, Sanabria S, Appel L, Dawson GR, Sciberras D, Hargreaves RJ,Langstrom B, and Burns HD (2010) Occupancy of human brain GABA(A) receptors bythe novel a5 subtype-selective benzodiazepine site inverse agonist a5IA as measured using[¹¹C]flumazenil PET imaging. Neuropharmacology 59:635–639.

Florin S, Meunier J, and Costentin J (2000) Autoradiographic localization of [3H]nociceptinbinding sites in the rat brain. Brain Res 880:11–16.

Gavioli EC and Calo G (2006) Antidepressant- and anxiolytic-like effects of nociceptin/orphaninFQ receptor ligands. Naunyn Schmiedebergs Arch Pharmacol 372:319–330.

Grimwood S and Hartig PR (2009) Target site occupancy: emerging generalizations from clinicaland preclinical studies. Pharmacol Ther 122:281–301.

Hostetler ED, Sanabria-Bohórquez S, Eng W, Joshi AD, Patel S, Gibson RE, O’Malley S, Krause SM,Ryan C, Riffel K, et al. (2013) Evaluation of [¹⁸F]MK-0911, a positron emission tomography (PET)tracer for opioid receptor-like 1 (ORL1), in rhesus monkey and human. Neuroimage 68:1–10.

Ichise M, Meyer JH, and Yonekura Y (2001) An introduction to PET and SPECT neuroreceptorquantification models. J Nucl Med 42:755–763.

Innis RB, Cunningham VJ, Delforge J, Fujita M, Gjedde A, Gunn RN, Holden J, Houle S, HuangSC, Ichise M, et al. (2007) Consensus nomenclature for in vivo imaging of reversibly bindingradioligands. J Cereb Blood Flow Metab 27:1533–1539.

Kimura Y, Fujita M, Hong J, Lohith TG, Gladding RL, Zoghbi SS, Tauscher JA, Goebl N, Rash KS,Chen Z, et al. (2011) Brain and whole-body imaging in rhesus monkeys of 11C-NOP-1A, apromising PET radioligand for nociceptin/orphanin FQ peptide receptors. J Nucl Med 52:1638–1645.

Lambert DG (2008) The nociceptin/orphanin FQ receptor: a target with broad therapeutic potential.Nat Rev Drug Discov 7:694–710.

Lassen NA, Bartenstein PA, Lammertsma AA, Prevett MC, Turton DR, Luthra SK, Osman S,Bloomfield PM, Jones T, Patsalos PN, et al. (1995) Benzodiazepine receptor quantificationin vivo in humans using [11C]flumazenil and PET: application of the steady-state principle. JCereb Blood Flow Metab 15:152–165.

Lohith TG, Zoghbi SS, Morse CL, Araneta MD, Barth VN, Goebl NA, Tauscher JT, Pike VW,Innis RB, and Fujita M (2014) Retest imaging of [11C]NOP-1A binding to nociceptin/orphaninFQ peptide (NOP) receptors in the brain of healthy humans. Neuroimage 87:89–95.

Lohith TG, Zoghbi SS, Morse CL, Araneta MF, Barth VN, Goebl NA, Tauscher JT, Pike VW,Innis RB, and Fujita M (2012) Brain and whole-body imaging of nociceptin/orphanin FQ peptidereceptor in humans using the PET ligand 11C-NOP-1A. J Nucl Med 53:385–392.

Melhem M (2013) Translation of central nervous system occupancy from animal models: appli-cation of pharmacokinetic/pharmacodynamic modeling. J Pharmacol Exp Ther 347:2–6.

Meunier JC, Mollereau C, Toll L, Suaudeau C, Moisand C, Alvinerie P, Butour JL, Guillemot JC,Ferrara P, Monsarrat B, et al. (1995) Isolation and structure of the endogenous agonist of opioidreceptor-like ORL1 receptor. Nature 377:532–535.

Murphy NP (2010) The nociceptin/orphanin FQ system as a target for treating alcoholism. CNSNeurol Disord Drug Targets 9:87–93.

Neal CR, Jr, Mansour A, Reinscheid R, Nothacker HP, Civelli O, Akil H, andWatson SJ, Jr (1999) Opioidreceptor-like (ORL1) receptor distribution in the rat central nervous system: comparison of ORL1receptor mRNA expression with (125)I-[(14)Tyr]-orphanin FQ binding. J Comp Neurol 412:563–605.

Pedregal C, Joshi EM, Toledo MA, Lafuente C, Diaz N, Martinez-Grau MA, Jiménez A, Benito A,Navarro A, Chen Z, et al. (2012) Development of LC-MS/MS-based receptor occupancy tracersand positron emission tomography radioligands for the nociceptin/orphanin FQ (NOP) receptor.J Med Chem 55:4955–4967.

Pike VW, Rash KS, Chen Z, Pedregal C, Statnick MA, Kimura Y, Hong J, Zoghbi SS, Fujita M,Toledo MA, et al. (2011) Synthesis and evaluation of radioligands for imaging brain nociceptin/orphanin FQ peptide (NOP) receptors with positron emission tomography. J Med Chem 54:2687–2700.

Post A, Smart TS, Krikke-Workel J, Dawson GR, Harmer CJ, Browning M, Jackson K, Kakar R,Mohs R, Statnick M, et al. (2016) A selective nociceptin receptor antagonist to treat depression:evidence from preclinical and clinical studies. Neuropsychopharmacology 41:1803–1812.

Reinscheid RK, Nothacker HP, Bourson A, Ardati A, Henningsen RA, Bunzow JR, Grandy DK,Langen H, Monsma FJ, Jr, and Civelli O (1995) Orphanin FQ: a neuropeptide that activates anopioidlike G protein-coupled receptor. Science 270:792–794.

Rusjan P, Mamo D, Ginovart N, Hussey D, Vitcu I, Yasuno F, Tetsuya S, Houle S, and Kapur S(2006) An automated method for the extraction of regional data from PET images. PsychiatryRes 147:79–89.

Spagnolo B, Carrà G, Fantin M, Fischetti C, Hebbes C, McDonald J, Barnes TA, Rizzi A, Trapella C,Fanton G, et al. (2007) Pharmacological characterization of the nociceptin/orphanin FQ receptorantagonist SB-612111 [(-)-cis-1-methyl-7-[[4-(2,6-dichlorophenyl)piperidin-1-yl]methyl]-6,7,8,9-tetrahydro-5H-benzocyclohepten-5-ol]: in vitro studies. J Pharmacol Exp Ther 321:961–967.

Statnick MA, Chen Y, Ansonoff M, Witkin JM, Rorick-Kehn L, Suter TM, Song M, Hu C,Lafuente C, Jiménez A, et al. (2016) A novel nociceptin receptor antagonist LY2940094 inhibitsexcessive feeding behavior in rodents: a possible mechanism for the treatment of binge eatingdisorder. J Pharmacol Exp Ther 356:493–502.

Toledo MA, Pedregal C, Lafuente C, Diaz N, Martinez-Grau MA, Jiménez A, Benito A, TorradoA, Mateos C, Joshi EM, et al. (2014) Discovery of a novel series of orally active nociceptin/orphanin FQ (NOP) receptor antagonists based on a dihydrospiro(piperidine-4,79-thieno[2,3-c]pyran) scaffold. J Med Chem 57:3418–3429.

Witkin JM, Statnick MA, Rorick-Kehn LM, Pintar JE, Ansonoff M, Chen Y, Tucker RC,and Ciccocioppo R (2014) The biology of Nociceptin/Orphanin FQ (N/OFQ) related to obesity,stress, anxiety, mood, and drug dependence. Pharmacol Ther 141:283–299.

Address correspondence to: Dr. Eyas Raddad, Chorus, Lilly Corporate Center,Indianapolis, IN 46285. E-mail: eyas@lilly.com

TABLE 3

Parameter estimates by Emax model for LY2940094 plasma concentration-receptoroccupancy (%) after single oral administration to human subjects

Region of Interest Estimate S.E.95% CI

Lower Upper

Prefrontal cortexEmax 100% (Fixed)EC50 (ng/ml) 2.94 0.512 1.81 4.07Residual S.D. (%) 6.91 1.46 3.70 10.1Intersubject S.D. (%) 12.5 2.98 5.96 19.1

Occipital cortexEmax 100% (fixed)EC50 (ng/ml) 3.46 0.685 1.96 4.97Residual S.D. (%) 7.81 1.66 4.14 11.5Intersubject S.D. (%) 13.9 3.35 6.48 21.2

PutamenEmax 100% (fixed)EC50 (ng/ml) 2.97 0.572 1.71 4.23Residual S.D. (%) 5.77 1.25 3.02 8.52Intersubject S.D. (%) 17.0 3.76 8.76 25.3

ThalamusEmax 100% (fixed)EC50 (ng/ml) 2.64 0.480 1.58 3.70Residual S.D. (%) 5.42 1.18 2.82 8.03Intersubject S.D. (%) 15.42 3.44 7.84 23.0

Emax, maximum % NOP receptor occupancy; CI, confidence interval; EC50, LY2940094concentration that induces 50% receptor occupancy.

1542 Raddad et al.

at ASPE

T Journals on A

pril 19, 2022dm

d.aspetjournals.orgD

ownloaded from