Pharmacological, pharmacokinetic, and primate analgesic efficacy profile of the novel bradykinin B1 receptor antagonist ELN441958

Article abstract of DOI:10.1124/jpet.107.120352

The bradykinin B₁ receptor plays a critical role in chronic pain and inflammation, although efforts to demonstrate efficacy of receptor antagonists have been hampered by species-dependent potency differences, metabolic instability, and low oral

Full text of DOI:10.1124/jpet.107.120352

0022-3565/07/3222-619–630$20.00
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright © 2007 by The American Society for Pharmacology and Experimental Therapeutics
JPET 322:619–630, 2007
Vol. 322, No. 2
120352/3226329
Printed in U.S.A.
Pharmacological, Pharmacokinetic, and Primate Analgesic
Efficacy Profile of the Novel Bradykinin B1 Receptor Antagonist
ELN441958
Jon E. Hawkinson, Balazs G. Szoke, Albert W. Garofalo, Dennis S. Hom,
Hongbing Zhang, Mark Dreyer, Juri Y. Fukuda, Linda Chen, Bhushan Samant,
Stellanie Simmonds,1 Karla P. Zeitz, Angie Wadsworth, Anna Liao, Raymond A. Chavez,2
Wes Zmolek, Lany Ruslim, Michael P. Bova, Ryan Holcomb,3 Eduardo R. Butelman,
Mei-Chuan Ko, and Annika B. Malmberg4
Elan Pharmaceuticals, South San Francisco, California (J.E.H., B.G.S., A.W.G., D.S.H., H.Z., M.D., J.Y.F., L.C., B.S., S.S.,
K.P.Z., A.W., A.L., R.A.C., W.Z., L.R., M.P.B., R.H., A.B.M.); Laboratory on the Biology of Addictive Diseases, The Rockefeller
University, New York, New York (E.R.B.); and Department of Pharmacology, University of Michigan Medical School, Ann Arbor,
Michigan (M.-C.K.)
Received January 23, 2007; accepted April 27, 2007
ABSTRACT
The bradykinin B1 receptor plays a critical role in chronic pain cated that ELN441958 is selective for primate over rodent B1
and inflammation, although efforts to demonstrate efficacy of receptors with a rank order potency (KB, nanomolar) of human
receptor antagonists have been hampered by species-depen- (0.12 Ϯ 0.02) ϳ rhesus monkey (0.24 Ϯ 0.01) Ͼ rat (1.5 Ϯ 0.4) Ͼ
dent potency differences, metabolic instability, and low oral mouse (14 Ϯ 4). ELN441958 had good permeability and met-
exposure of current agents. The pharmacology, pharmaco- abolic stability in vitro consistent with high oral exposure and
kinetics, and analgesic efficacy of the novel benzamide B1 moderate plasma half-lives in rats and rhesus monkeys. Be-
receptor antagonist 7-chloro-2-[3-(9-pyridin-4-yl-3,9-diaza- cause ELN441958 is up to 120-fold more potent at primate than
spiro[5.5]undecanecarbonyl)phenyl]-2,3-dihydro-isoindol-1- at rodent B1 receptors, it was evaluated in a primate pain
one (ELN441958) is described. ELN441958 competitively model. ELN441958 dose-dependently reduced carrageenan-
inhibited the binding of the B1 agonist ligand [3H]desArg10
-
induced thermal hyperalgesia in a rhesus monkey tail-with-
kallidin ([3H]DAKD) to IMR-90 human fibroblast membranes drawal model, with an ED50 ϳ3 mg/kg s.c. Naltrexone had no
with high affinity (Ki ϭ 0.26 Ϯ 0.02 nM). ELN441958 potently effect on the antihyperalgesia produced by ELN441958, in-
antagonized DAKD (but not bradykinin)-induced calcium dicating a lack of involvement of opioid receptors. ELN441958
mobilization in IMR-90 cells, indicating that it is highly selective is a novel small molecule bradykinin B1 receptor antagonist
for B1 over B2 receptors. Antagonism of agonist-induced cal- exhibiting high oral bioavailability and potent systemic efficacy
cium responses at B1 receptors from different species indi- in rhesus monkey inflammatory pain.
This work was presented in abstract form: Hawkinson JE (2006) The novel
bradykinin B1 receptor antagonist ELN441958 is anti-hyperalgesic in two
viable therapeutic strategy to reduce pain and inflammation.
primate pain models, in the Society for Neuroscience Abstract; 2006 Oct 14–18;
Antagonism of bradykinin receptors may prove to be a
Atlanta, GA. Society for Neuroscience, Washington, DC. This work was re-
viewed by the Animal Care and Use Committee of The Rockefeller University,
the University of Michigan, and Elan Pharmaceuticals.
1 Current affiliation: Rinat Laboratories, Pfizer, Inc., South San Francisco,
California.
Initial interest focused on the constitutively expressed brady-
kinin B2 receptor, which is selectively activated with high
affinity by bradykinin (BK) and kallidin (KD). Recently,
there has been considerable interest in the inducible B1 re-
ceptor, which is selectively activated by desArg9-bradykinin
(DABK) and desArg10-kallidin (DAKD), the N-terminal argi-
nine-cleaved metabolites of BK and KD (Leeb-Lundberg et
al., 2005). The B1 receptor is not normally expressed in most
2 Current affiliation: Avigen, Inc., Alameda, California.
3 Current affiliation: Myriad Genetics, Salt Lake City, Utah.
4 Current affiliation: Amgen, Inc., Cambridge, Massachusetts.
Article, publication date, and citation information can be found at
http://jpet.aspetjournals.org.
doi:10.1124/jpet.107.120352.
ABBREVIATIONS: BK, bradykinin; KD, kallidin; DABK, desArg9-bradykinin; DALBK, desArg9-Leu8-bradykinin; DAKD, desArg10-kallidin; DALKD,
desArg10-Leu9-kallidin; CFA, complete Freund’s adjuvant; P-gp, P-glycoprotein; CNS, central nervous system; ELN441958, 7-chloro-2-[3-(9-
pyridin-4-yl-3,9-diazaspiro[5.5]undecanecarbonyl)phenyl]-2,3-dihydro-isoindol-1-one; MEM, minimum essential medium; FBS, fetal bovine se-
rum; IL, interleukin; HBSS, Hanks’ balanced salt solution; BSA, bovine serum albumin; DMSO, dimethyl sulfoxide; FLIPR, fluorometric imaging
plate reader; LC/MS/MS, liquid chromatography-tandem mass spectrometry; MDCK, Madin-Darby canine kidney; MOPS, 3-(N-morpholino)-
propanesulfonic acid; PK, pharmacokinetic; AUC, area under the concentration-time curve; DRG, dorsal root ganglion; desArg10HOE140, des-
Arg9-D-Arg[Hyp3,Thi5,D-Tic7,Oic8]BK; SSR240612, (2R)-2-[((3R-3-(1,3-benzodioxol-5-yl)-3-[[(6-methoxy-2-naphthyl)sulfonyl]amino]propanoyl)-
amino]-3-(4-[[2R,6S)-2,6-dimethylpiperidinyl]methyl]phenyl)-N-isopropyl-N-methylpropanamide hydrochloride]; LF22-0542, N-[[4-(4,5-
dihydro-1H-imidazol-2-yl)phenyl]methyl-2-[2-[[(4-methoxy-2,6-dimethylphenyl)sulfonyl]methylamino]ethoxy]-N-methylacetamide fumarate.
619

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Hawkinson et al.
tissues, but it is dramatically up-regulated following inflam- duced late-phase licking in the mouse formalin model, inhib-
mation and tissue injury, and it does not readily desensitize. ited thermal hyperalgesia induced by ultraviolet irradiation
The low sequence homology between B1 and B2 receptors in rats and also reduced thermal hyperalgesia in the rat
(36%) is consistent with the high degree of selectivity dis- chronic constriction nerve injury nerve injury model (Gougat
played by many agonists and antagonists (Leeb-Lundberg et et al., 2004). LF22-0542 reduced responding in the late phase
al., 2005).
of the formalin model and in the acetic acid-induced writhing
B1 receptors from different species fall into two pharmaco- model in mice, and it also reduced formalin-induced flinch-
logical classes (Regoli et al., 2001). The rodent-like class ing, carrageenan-induced mechanical hyperalgesia, and
includes rat, mouse, hamster, and dog B1 receptors, whereas CFA-induced thermal hyperalgesia in rats (Porreca et al.,
the primate-like class includes the human, rhesus, pig, and 2006). LF22-0542 reduced thermal, but not mechanical, hy-
rabbit receptors. The rodent B1 receptors are potently acti- peralgesia in the rat spinal nerve ligation model (Porreca et
vated by both DABK and DAKD, whereas DAKD has consid- al., 2006).
erably higher affinity for the primate receptors than does
In the present study, we describe the in vitro pharmaco-
DABK. Antagonist pharmacology also differs between these logical profile, metabolic stability, P-glycoprotein (P-gp) lia-
two classes. For example, the prototypical B1-receptor antag- bility, CNS penetration, in vivo pharmacokinetics, and pri-
onist desArg9-Leu8-bradykinin (DALBK) is a neutral antag- mate analgesic efficacy of the novel B1 receptor antagonist
onist of primate receptors, but it is a partial agonist at rodent ELN441958 (Fig. 1).
receptors (Meini et al., 1996; MacNeil et al., 1997). In addi-
tion, several classes of small molecule B1-receptor antago-
nists exhibit marked potency differences between primate
Materials and Methods
Synthesis of ELN441958. Addition of tert-butyl 3,9-diaza-
spiro[5.5]undecane-3-carboxylate to 4-chloropyridine hydrochloride
followed by deprotection in neat trifluoroacetic acid affords 3-(4-
pyridinyl)-3,9-diazaspiro[5.5]undecane trifluoroacetate. Addition of
methyl 3-aminobenzoate to methyl 2-(bromomethyl)-6-chlorobenzo-
ate followed by ester hydrolysis gives 3-(7-chloro-1-oxo-1,3-dihy-
droisoindol-2-yl)benzoic acid, which is then coupled with 3-
(4-pyridinyl)-3,9-diazaspiro[5.5]undecane trifluoroacetate to afford
7-chloro-2-[3-(9-pyridin-4-yl-3,9-diazaspiro[5.5]undecanecarbonyl)
phenyl]-2,3-dihydro-isoindol-1-one (ELN441958).
Recombinant Cell Lines. The complete open reading frame
encoding the bradykinin B1 receptor (BKB1R) is contained within a
single exon in both the mouse and rat genomes. Accordingly, the
cDNAs for each BKB1R were amplified from mouse and rat genomic
DNA (Clontech, Mountain View, CA) using primers specific for the 5Ј
and 3Ј ends of the respective coding regions (GenBank, mouse:
U47281; rat: U66107). Polymerase chain reaction amplification was
achieved under standard conditions, using a combination of Taq and
Pfu DNA polymerases for improved fidelity. Amplification products
were subcloned into the TOPO TA cloning vector pCR 2.1 TOPO
(Invitrogen, Carlsbad, CA) and sequenced. The correct BKB1R cDNA
was subcloned out of the TOPO TA vector and introduced into a
pEAK 8 plasmid (Edge Biosystems, Gaithersburg, MD) that was
previously modified by the addition of novel sites in the polylinker
region by an overlapping oligonucleotide strategy. This plasmid is an
episomal vector that drives transgene expression with the eF1␣
promoter, and stable clones can be selected with puromycin. The
mouse and rat BKB1R pEAK plasmids were stably transfected into
PEAK-R and PEAK-S cells (Edge Biosystems), modified human em-
bryonic kidney 293 cells designed specifically to preserve episomal
vector expression. Stable populations of cells were maintained with
puromycin. Stable transfectants of either PEAK-R or PEAK-S cells
were selected based on their responsiveness in the cell-based assay
(see below).
and rodent receptors (Kuduk et al., 2004; Ransom et al.,
2004).
It has been proposed that the B2 receptor is involved in
acute inflammation, whereas the B1 receptor is the primary
kinin receptor mediating chronic inflammation (Perkins et
al., 1993). In peripheral tissues, activation of newly formed
B1 receptors promotes the generation of inflammatory medi-
ators, including prostanoids, nitric oxide, and cytokines, re-
sulting in plasma extravasation, leukocyte trafficking,
edema, and pain (McLean et al., 2000; Eisenbarth et al.,
2004; Lawson et al., 2005; Leeb-Lundberg et al., 2005). B1
receptors are constitutively expressed on primary afferent
pain fibers, and they are up-regulated following nerve injury
(Levy and Zochodne, 2000). Activation of neuronal B1 recep-
tors may contribute to neuropeptide release and subsequent
neurogenic inflammation in peripheral tissues (McLean et
al., 2000) as well as central sensitization at the level of the
spinal cord (Pesquero et al., 2000). B1 agonists injected der-
mally produce pain in humans, which is exacerbated by prior
ultraviolet irradiation of the injection area (Eisenbarth et al.,
2004).
B1 receptor knockout mice have a pain phenotype impli-
cating a role for this receptor in inflammatory and neuro-
pathic pain. These B1 receptor-deficient mice exhibit reduced
responding in several experimental pain models relative to
wild-type mice, including acute tests (hot-plate and tail-
flick), following formalin, capsaicin, or complete Freund’s
adjuvant (CFA) injection into the paw, and in the partial
sciatic nerve ligation model of neuropathic pain (Pesquero et
al., 2000; Ferreira at el., 2001; Porreca et al., 2006).
Peptide antagonists including DALBK and desArg10HOE140
provided early pharmacological evidence in rodents implicating
the role of the B1 receptor in pain. Peptide antagonists are
effective in early and late-phase licking following formalin in-
jection into the paw (Ferreira et al., 2002), CFA-induced ther-
mal and mechanical hyperalgesia (Fox et al., 2003), and carra-
geenan-induced mechanical hyperalgesia (Poole et al., 1999).
Peptide antagonists also reduce thermal and mechanical hyper-
algesia in models of neuropathic pain, e.g., in the rat chronic
constriction nerve injury model (Levy and Zochodne, 2000).
More recently, small molecule B1 receptor antagonists
have shown efficacy in rodent pain models. SSR240612 re-
Cell Culture. IMR-90 human lung fibroblast cells (CCL-186;
American Type Culture Collection, Manassas, VA) and DBS-FRhL-2
rhesus lung fibroblast cells (CL-160; American Type Culture Collec-
tion) were grown in Eagle’s minimum essential medium (MEM)
supplemented with 10% fetal bovine serum (FBS) as recommended
O
Cl
O
N
N
N
N
Fig. 1. Structure of ELN441958.

Bradykinin B1 Receptor Antagonist ELN441958
621
by American Type Culture Collection. Peak-S or -R cells expressing 3.6 ␮M Fluo-4/acetoxymethyl ester for 75 min at 37°C in 1% FBS-
recombinant rat or mouse B1 receptors, respectively, were cultured containing HEPES/HBSS without bicarbonate or phenol red, pH 7.5.
in Dulbecco’s modified Eagle’s medium supplemented with 10% FBS,
400 ␮g/ml hygromycin B, and 1.5 ␮g/ml puromycin.
Loaded cells were washed by centrifugation, resuspended in assay
buffer (0.1% BSA in HEPES/HBSS solution), and plated into 96-well
black/clear plates at 100,000 cells/well. Plates were centrifuged
(200g; 10 min) to move cells to the bottom of wells, and then they
were assayed in the FLIPR as described above, with DABK as
agonist at 3 ϫ EC50 concentration (4–10 nM). Peak height of agonist-
induced fluorescence as a function of antagonist concentration was
fitted to the sigmoidal function (Prism; GraphPad Software Inc.) to
determine IC50 values. KB values were calculated according to the
Cheng-Prusoff equation using the EC50 of the agonist used for each
species of receptor (Table 2).
Solubility. ELN441958 was dissolved in DMSO as a 20 mM stock
solution, diluted 200-fold in HBSS, pH 7.4, shaken overnight at room
temperature, and centrifuged at 14,000g for 60 min. The concentra-
tion of ELN441958 in the supernatant was determined using high-
performance liquid chromatography-UV (aqueous acetonitrile con-
taining 0.1% formic acid mobile phase) by comparing its peak area to
a standard curve.
Metabolic Stability. ELN441958 (1 ␮M) was incubated at 37°C
with rat or human liver microsomes (0.5 mg/ml; BD Biosciences,
Bedford, MA) in the presence of 1 mM NADPH for 60 min. Aliquots
were taken at 0, 5, 12, 20, 30, 45, and 60 min, precipitated with
organic solvent containing internal standard, and analyzed by LC/
MS/MS using a C18 column (2.1 ϫ 50 mm, 5 ␮m; Sepax GPC18;
Sepax Technologies, Newark, DE; flow 900 ␮l/min) with aqueous
acetonitrile containing 0.1% formic acid as mobile phase. The
amount of parent compound remaining at each time point was esti-
mated by the chromatographic peak area ratios and then plotted
using a log-linear fit to determine the first-order rate constant.
Midazolam, a high-clearance positive control, was incubated in par-
allel in each experiment.
Permeability. ELN441958 was evaluated for its permeability
across a monolayer of Madin-Darby canine kidney (MDCK) II cells
(American Type Culture Collection). Cells were grown for 4 days in
Dulbecco’s modified Eagle’s medium with GlutaMAX containing 10%
heat-inactivated FBS and 40 units/ml penicillin/streptomycin at
37°C and 5% CO2, and they were plated into 12-mm-diameter 12-
well Transwell plates (Corning Life Sciences). ELN441958 (5 ␮M)
was added in HEPES/HBSS with 0.1% glucose, pH 7.4, and plates
were incubated for 2 h at 37°C with shaking. Donor and receiver
wells were then sampled and assayed by LC/MS/MS to determine
compound levels. From these data, the forward flux (apical to baso-
lateral) was calculated. The extent of transport by P-gp was deter-
mined in the same manner, except that MDCK cells expressing
human multidrug resistance (MDR)-1 (kind gift from Dr. Piet Borst,
The Netherlands Cancer Institute, Amsterdam, The Netherlands)
were used, and the forward flux was calculated in the absence or
presence of the P-gp inhibitor cyclosporine A at 10 ␮M (Sigma-
Aldrich, St. Louis, MO). The known P-gp substrate indinavir was
used a positive control. Integrity of the monolayer was determined
using 100 ␮M lucifer yellow (Sigma-Aldrich) with Ͻ2% leakage ob-
served in all experiments.
Radioligand Binding. Confluent IMR-90 cells were treated with
0.35 ng/ml interleukin (IL)-1␤ in 10% FBS/MEM for 4 h to up-
regulate B1 receptors. Cells were then rinsed with phosphate-buff-
ered saline, collected by scraping in ice-cold TES buffer (25 mM
NaTES, 1 mM phenantroline, 2 ␮M captopril, and 140 ␮g/ml baci-
tracin, pH 7.4), and centrifuged at 3200g for 5 min. Pellets were
homogenized in TES buffer on ice using a polytron-type homogenizer
(VirTis, Gardiner, NY). The membranes were collected and washed
by centrifugation twice at 50,000g for 20 min at 4°C. Pellets were
resuspended in buffer at 0.5 mg/ml protein and stored at Ϫ80°C.
Protein concentration was measured using the bicinchoninic acid
method in the presence of 2% SDS (Pierce Chemical, Rockford, IL).
Displacement binding experiments were carried out in 96-deep well
plates in binding buffer [20 mM HEPES in Hanks’ balanced salt
solution (HBSS) without bicarbonate or phenol red, 0.1% BSA, 1 mM
phenantroline, 2 ␮M captopril, and 140 ␮g/ml bacitracin, pH 7.4] at
room temperature. Approximately 40 ␮g of membranes/well were
incubated with ϳ0.5 nM [3H]DAKD (PerkinElmer Life and Analyt-
ical Sciences, Wellesley, MA) in the absence or presence of different
concentrations of test compounds in a final volume of 300 ␮l. The
actual [3H]DAKD concentration used in each experiment was deter-
mined by sampling the working solution of [3H]DAKD in binding
buffer and counting on a scintillation counter (LS5000; Beckman
Coulter, Fullerton, CA). Test compounds were diluted in seven half-
log steps at 100ϫ final concentration in DMSO, and then they were
assayed in duplicate wells (final 1% DMSO). Nonspecific binding was
determined in the presence of 1 ␮M DAKD. After a 1-h incubation,
binding was terminated by rapid filtration through GF/B filter plates
(Whatman, Florham Park, NJ) presoaked in 0.2% polyethyleneimine
using a Packard Filtermate (PerkinElmer Life and Analytical Sci-
ences). Filters were rinsed four times with ice-cold wash buffer (50
mM Tris-HCl, pH 7.4), and bound radioactivity was counted after the
addition of
a scintillant in a MicroBeta scintillation counter
(PerkinElmer Life and Analytical Sciences). Following correction for
nonspecific binding, IC50 values were calculated using the four-
parameter sigmoidal concentration-response function, and KD and
Bmax values were calculated using the hyperbolic function (Prism;
GraphPad Software Inc., San Diego, CA). Binding Ki values were
calculated according to the Cheng-Prusoff equation using the KD of
[3H]DAKD (0.31 nM) determined in saturation binding experiments.
Calcium Mobilization (FLIPR). Human (IMR-90) and rhesus
(DBS-FRhL-2) lung fibroblast cells expressing native B1 receptors
were harvested by trypsinization and seeded into black wall/clear
bottom 96-well plates (Costar 3904; Corning Life Sciences, Acton,
MA) at approximately 13,000 cells/well. The following day, cells were
treated with 0.35 ng/ml human IL-1␤ in 10% FBS/MEM for 2 h to
up-regulate B1 receptors. Induced cells were loaded with fluorescent
calcium indicator by incubation with 2.3 ␮M Fluo-4/acetoxymethyl
ester (Invitrogen) at 37°C for 1.5 h in the presence of an anion
transport inhibitor (2.5 mM probenecid in 1% FBS/MEM). Extracel-
lular dye was removed by washing with assay buffer (2.5 mM pro-
benecid and 0.1% BSA in 20 mM HEPES/HBSS without bicarbonate
or phenol red, pH 7.5), and cell plates were kept in the dark until
used. Test compounds were assayed at seven concentrations in trip-
licate. Addition of test compounds to the cell plate and incubation for
ATPase. Membranes prepared from Sf9 insect cells expressing
human MDR1 using the baculovirus expression system (Autographa
californica) were purchased from BD Biosciences. The ATPase reac-
tion was carried out in a 35-␮l reaction volume in a 384-well micro-
titer plate containing 1% DMSO, 0.1 to 100 ␮M ELN441958, and
0.67 mg/ml Sf9 membranes in a reaction buffer consisting of 50 mM
KCl, 50 mM MOPS-Tris, pH 7.0, with 5 mM sodium azide, 2 mM
dithiothreitol, 0.1 mM EGTA, and 1 mM ouabain. The reaction was
started with the addition of MgATP (5 mM final), incubated for 50
min at 37°C, and then quenched with SDS (2.2% final). The colori-
metric reaction was developed for 40 min at 37°C following addition
5 min at 35°C, followed by the addition of 2 to 8 nM final (3 ϫ EC50
)
B1 agonist DAKD (Bachem California, Torrance, CA), were carried
out in the fluorometric imaging plate reader (FLIPR; Molecular
Devices, Sunnyvale, CA) while continuously monitoring calcium-
dependent fluorescence. B2 responses were measured in IMR-90 cells
in an identical manner except that the IL-1␤ treatment step was
omitted and BK (0.7 nM final; 3 ϫ EC50) replaced DAKD as agonist.
Cells expressing recombinant rat or mouse B1 receptors were de- of the detection reagent consisting of 60 mM ammonium molybdate
tached using PBS without calcium or magnesium and loaded with and 15 mM zinc acetate, and then the absorbance was determined at

622
Hawkinson et al.
800 nm using a Safire2 microplate reader (Tecan, Durham, NC).
Indinavir was used as a positive control.
100
80
60
40
20
0
desArg10HOE140
ELN441958 Formulation. To achieve maximal exposure for
pharmacokinetic and efficacy studies, ELN441958 was formulated
as a clear solution. For rodent studies, ELN441958 was dissolved in
2% Tween 80 in saline and adjusted to pH 4.5 to 7 with HCl. For
rhesus PK studies, ELN441958 was formulated in 10% polyethylene
glycol 660 hydroxystearate (Solutol; BASF, Ludwigshafen, Ger-
many) in either saline (i.v.) or water (p.o.), adjusted to pH 4.5 to 7
with HCl. For s.c. administration in rhesus monkeys in both PK and
efficacy studies, it was formulated as a clear solution in 20% Captisol
(sulfobutylether-␤-cyclodextrin; CyDex, Lenexa, KS) in water ad-
justed to pH 4.5 to 7 with formic acid.
DALKD
ELN441958
-11
-10
-9
-8
-7
-6
log [Inhibitor] M
Pharmacokinetic Analysis. Aliquots of plasma (100 ␮l) from
rhesus monkeys or Sprague-Dawley rats were added to extraction
plates containing an internal standard in 400 ␮l of methanol/aceto-
nitrile (50:50) and mixed. After centrifugation, the supernatant was
transferred to a 96-deep well plate and evaporated to dryness. The
residue was reconstituted in 20% methanol/0.1% formic acid mobile
phase, and it was analyzed by LC/MS/MS using a C18 column and an
API-3000 triple quadrupole mass spectrometer (Applied Biosystems,
Foster City, CA). Plasma concentrations were determined from a
calibration curve prepared by extracting known amounts of
ELN441958 from control plasma. The sensitivity limit was approx-
imately 0.25 ng/ml or ng/g ELN441958 per sample.
Fig. 2. ELN441958 is a subnanomolar inhibitor of [3H]DAKD binding to
the native human bradykinin B1 receptor. Inhibition of radioligand bind-
ing to membranes prepared from IMR-90 human lung fibroblasts. See
Table 1 for Ki values.
TABLE 1
Affinity of ELN441958 and peptidic antagonists for the native human
bradykinin B1 receptor
Inhibition of radioligand binding to membranes prepared from IMR-90 human lung
fibroblasts. See Fig. 2 for displacement curves. Data are means Ϯ S.E.M. of at least
three experiments.
Antagonist
Ki
Hill Slope
Mouse CNS Penetration. To assess the contribution of P-gp to
CNS penetration, the brain and plasma levels of ELN441958 in
female FVB wild-type mice were compared with MDR1 a/b (Ϫ/Ϫ)
knockout mice (Taconic Farms, Germantown, NY) at 5 min following
a single 2.5 mg/kg i.v. bolus injection. To assess the time course of
CNS penetration, brain, spinal cord, and plasma levels were mea-
sured in male FVB wild-type mice at 0.5, 1, 2, and 4 h following s.c.
administration of 10 mg/kg ELN441958. LC/MS/MS quantitation
nM
ELN441958
DALKD
0.26 Ϯ 0.02
0.49 Ϯ 0.06
15 Ϯ 3
1.1 Ϯ 0.1
0.98 Ϯ 0.06
0.88 Ϯ 0.07
desArg10HOE140
whereas desArg10HOE140 had more than 50-fold lower af-
finity. Saturation binding studies were performed to deter-
and tissue sample preparation were as described above, except that mine the type of inhibition (Fig. 3). KD and Bmax values
ELN441958 levels in brain and spinal cord were quantitated follow-
ing homogenization of the preweighed tissue in ethyl acetate, and
calibration curves were prepared by extracting known amounts of
ELN441958 from ethyl acetate homogenates of control tissue. Mouse
tissues were perfused, and the reported brain concentration was
adjusted for the concentration of ELN441958 in plasma. The total
area under the concentration-time curve from time 0 to 4 h (AUC0-4
(mean Ϯ S.E.M.; n ϭ 3) were 0.31 Ϯ 0.03 nM and 1.8 Ϯ 0.5
pmol/mg, respectively, in the control condition. In the pres-
ence of 0.5 nM ELN441958, KD and Bmax values were 1.3 Ϯ
0.1 nM and 1.7 Ϯ 0.4 pmol/mg, respectively, consistent with
competitive inhibition.
ELN441958 is a potent, neutral antagonist of B1 receptor
activation based on the inhibition of agonist-induced in-
creases in intracellular calcium in native and recombinant
cells. The potencies for stimulation of calcium mobilization
h
) was estimated from plasma, brain, and spinal cord concentrations
using standard methods. These values were then used to calculate
brain/plasma and spinal cord/plasma ratios.
Carrageenan-Induced Hyperalgesia. Adult male and female by the appropriate agonist for B1 receptors from the different
rhesus monkeys (Macaca mulatta) (weight range 8–11 kg; n ϭ 3)
were trained in the tail-withdrawal procedure described by Ko and
Lee (2002). A 20-s maximal tail-withdrawal latency (baseline cut-off)
was used with a normally non-noxious thermal stimulus of 46°C. To
produce inflammation-associated hyperalgesia, carrageenan (2 mg/
tail in 0.1 ml of saline s.c.) was injected into the terminal 1 to 4 cm
of the tail, and tail-withdrawal latencies were measured every 30
min for 3.5 h. ELN441958 (1, 3, or 10 mg/kg) or naproxen (10 mg/kg)
was injected s.c. on the back 30 min before carrageenan injection. In
the opioid-receptor antagonist study, 1 mg/kg naltrexone s.c. was
species examined, i.e., DAKD at the human and rhesus mon-
key B1 receptors and DABK at rat and mouse B1 receptors,
are shown in Table 2. In IMR-90 cells expressing the native
human B1 receptor, ELN441958 produced a concentration-
dependent antagonism of the DAKD-induced calcium mobi-
lization with a KB of 0.12 Ϯ 0.02 nM (Figs. 4A and 5A; Table
3). ELN441958 itself does not produce an agonist response at
concentrations completely blocking the agonist-induced re-
sponse at all four species of receptor tested, indicating that it
administered on the back just before ELN441958. Blood samples for is a neutral antagonist in this assay (data for the rat receptor
compound analysis were drawn 4 h after carrageenan injection (4.5
h after the ELN441958 administration).
is shown in Fig. 4B). In contrast, the peptide antagonist
DALKD exhibits clear agonism at rodent B1 receptors (Fig.
4B) as reported previously (Meini et al., 1996; MacNeil et al.,
1997). A very small agonist response was generally observed
following application of desArg10HOE140 at high concentra-
Results
Potency and Selectivity of ELN441958 at Bradykinin tions. The rank order potency for ELN441958 antagonism of
B1 Receptors. ELN441958 inhibited the binding of the ag- B1 receptors from different species was human ϳ rhesus Ͼ
onist radioligand [3H]DAKD to the human B1 receptor rat Ͼ mouse; it was 120-fold less potent at the mouse relative
present in IMR-90 lung fibroblast cell membranes with high to human receptor (Fig. 5; Table 3). In contrast, the peptide
affinity (Ki ϭ 0.26 Ϯ 0.02 nM) (Fig. 2; Table 1). The peptide antagonists showed much less species difference in antago-
antagonist DALKD had a similar subnanomolar affinity, nist potency. The rank order potency for DALKD antagonism

Bradykinin B1 Receptor Antagonist ELN441958
623
TABLE 3
Potency of ELN441958 and peptidic antagonists for inhibition of
agonist-induced calcium mobilization at rodent and primate bradykinin
B1 receptors
A
3.0
Total
Inhibition of DAKD (human and rhesus) or DABK (rat and mouse)-stimulated
calcium mobilization in cells expressing native (human and rhesus) or recombinant
(rat and mouse) B1 receptors. See Fig. 5 for concentration-response curves. Data are
means Ϯ S.E.M. of at least three experiments
2.5
2.0
1.5
1.0
0.5
0.0
Specific
Control
+ 0.5 nM ELN441958
Antagonist
Species
KB
Hill Slope
nM
ELN441958
Human
Rhesus
Rat
Mouse
Human
Rhesus
Rat
0.12 Ϯ 0.02
0.24 Ϯ 0.01
1.5 Ϯ 0.4
14 Ϯ 4
0.28 Ϯ 0.02
0.17 Ϯ 0.01
1.9 Ϯ 0.2
1.1 Ϯ 0.1
22 Ϯ 3
1.12 Ϯ 0.04
0.67 Ϯ 0.07
1.4 Ϯ 0.2
1.6 Ϯ 0.2
1.7 Ϯ 0.5
1.5 Ϯ 0.3
1.1 Ϯ 0.1
1.4 Ϯ 0.2
0.77 Ϯ 0.06
0.85 Ϯ 0.10
1.4 Ϯ 0.2
2.0 Ϯ 0.2
Nonspecific
0
5
10
15
20
DALKD
[3H]DAKD concentration (nM)
Mouse
Human
Rhesus
Rat
desArg10HOE140
B
5.3 Ϯ 1.1
11 Ϯ 2
5.4 Ϯ 1.0
1.0
Mouse
0.8
0.6
0.4
0.2
0.0
Control
Human B1 antagonism
A
25000
Control
0.03
0.1
+ 0.5 nM ELN441958
20000
15000
0.3
0.0
0.5
1.0
1.5
2.0
2.5
1
3
[3H]DAKD bound (pmol/mg)
10
30
10000
0.0
Fig. 3. ELN441958 competitively displaces the selective B1-receptor pep-
tide agonist ligand [3H]DAKD from the native human B1 receptor. A,
representative [3H]DAKD saturation experiment in IMR-90 cell mem-
branes. Specific binding in the absence (control) and presence of 0.5 nM
ELN441958 were calculated by subtraction of the linear regression of the
nonspecific binding (determined in the presence of 1 ␮M DAKD) from the
total binding. Total binding shown is from the control condition. Data
points represent averages of duplicates. B, Scatchard transformation of
the specific binding data from Fig. 3A. In this experiment, KD and Bmax
values were 0.36 nM and 2.6 pmol/mg, respectively, in the control condi-
tion and 1.6 nM and 2.4 pmol/mg, respectively, in the presence of
ELN441958.
0.5
1.0
time (min)
Rat B1 agonism
B
100
DABK
75
50
25
DALKD
TABLE 2
Agonist potency for mobilization of intracellular calcium at rodent and
primate bradykinin B1 receptors
Potencies for stimulation of calcium mobilization measured using the FLIPR using
DAKD as agonist in lung fibroblasts cell lines expressing native B1 receptors from
human (IMR-90) and rhesus monkey (DBS-FRhL-2) or using DABK as agonist in
HEK293 cells expressing recombinant rat and mouse B1 receptors. Data are
means Ϯ S.E.M. of at least four to five experiments.
desArg10HOE140
ELN441958
0
Control
0.0
0.5
1.0
time (min)
Species
Agonista
EC50
Hill Slope
Fig. 4. ELN441958 is a neutral antagonist of B1 agonist-induced calcium
mobilization. Traces represent changes of Fluo-4 fluorescence in cells
expressing B1 receptors measured using FLIPR. A, concentration-depen-
dent antagonism of DAKD-induced calcium release by 0.03 to 30 nM
ELN441958 at the native human receptor present in IMR-90 cells. KB
and Hill slope values for this experiment were 0.17 nM and 0.86, respec-
tively. B, partial agonism of DALKD and desArg10HOE140, but not
ELN441958, at the recombinant rat B1 receptor. Traces shown are from
a single representative experiment. In this experiment, 300 nM DALKD
and 3 ␮M desArg10HOE140 exhibited calcium-related fluorescent peak
responses of 54 and 14%, respectively, relative to the full agonist DABK
at 300 nM. DALKD and desArg10HOE140 agonism ranged from 20 to 55%
(n ϭ 3) and from Ͻ1 to 19% (n ϭ 7), respectively, relative to 300 nM
DABK. Responses to ELN441958 up to 300 nM were indistinguishable
from that of control in all experiments (n ϭ 4).
nM
Human
Rhesus
Rat
DAKD
DAKD
DABK
DABK
0.88 Ϯ 0.14
0.83 Ϯ 0.10
3.0 Ϯ 0.4
1.1 Ϯ 0.1
0.85 Ϯ 0.07
1.0 Ϯ 0.1
1.1 Ϯ 0.1
Mouse
1.7 Ϯ 0.3
a Receptors were activated using the probable physiologic agonist. For reference,
the EC50 values for activation of human and rhesus B1 receptors by DABK were 118
and 34 nM, respectively, and for rat and mouse B1 receptors by DAKD were 2.6 and
2.2 nM, respectively (n ϭ 1–4).
of B1 receptors was rhesus ϳ human Ͼ mouse ϳ rat, with a
7-fold difference between rhesus and rat receptors. The less
potent antagonist desArg10HOE140 showed very little spe-
cies difference, a range of only 4-fold across the four species. lization induced by the selective B2-receptor agonist BK at
ELN441958 is highly selective for B1 over B2 receptors. In concentrations up to 10 ␮M (data not shown). Likewise,
IMR-90 cells, ELN441958 failed to inhibit the calcium mobi- desArg10HOE140 and DALKD were inactive at inhibit-

624
Hawkinson et al.
Rhesus
Human
100
80
60
40
20
0
100
80
desArg10HOE140
desArg10HOE140
60
DALKD
ELN441958
DALKD
40
ELN441958
20
0
-11
-10
-9
-8
-7
-6
-11
-10
-9
-8
-7
-6
log [Antagonist] M
log [Antagonist] M
Mouse
Rat
100
80
60
40
20
0
100
ELN441958
80
60
40
20
0
desArg10HOE140
DALKD
desArg10
HOE140
DALKD
ELN441958
-11
-10
-9
-8
-7
-6
-11
-10
-9
-8
-7
-6
log [Antagonist] M
log [Antagonist] M
Fig. 5. ELN441958 exhibits species differences in potency for antagonism of selective B1 agonist-induced calcium mobilization. Antagonism of DAKD
(human and rhesus) or DABK (rat and mouse)-induced calcium increase in cells expressing either native (human and rhesus) or recombinant (rat and
mouse) B1 receptors indicated by Fluo-4 fluorescence using FLIPR. Means Ϯ S.E.M. of at least three experiments. Calculated KB values are shown
in Table 3.
ing BK-induced responses up to 5 and 30 ␮M, respectively. labile positive control midazolam was cleared much more
In contrast, the selective B2 receptor antagonist HOE140 rapidly. ELN441958 had moderate permeability in MDCK II
potently inhibited the BK-induced response (IC50 ϭ 2.3 Ϯ cells (Table 5). However, the permeability of ELN441958 in
0.4 nM). Regarding other nontarget receptors, 10 ␮M MDCK cells expressing the recombinant human MDR1 gene
ELN441958 produced Ն50% inhibition at 20 of more than coding for P-gp (MDR1-MDCK cells) was substantially lower.
100 enzyme and receptor assays tested in a broad panel Cyclosporine A, an inhibitor of P-gp, increased the perme-
(data not shown). Of these, IC50 values were determined ability in these cells 13-fold. The known P-gp substrate indi-
for five receptors in single experiments. The most potent navir exhibited similar behavior in these assays, but it had
off-target activity identified was at the human ␮-opioid substantially lower intrinsic permeability than ELN441958.
receptor (binding Ki ϭ 0.13 ␮M), suggesting Ͼ500-fold In addition, ELN441958 stimulated P-gp ATPase activity
selectivity for B1 over ␮-opioid receptors. Lower potency 2-fold in membranes expressing the recombinant human
inhibition was measured at the human muscarinic M1, MDR1 gene. Taken together, these data indicate that
␦-opioid, and ␬-opioid receptors (binding Ki values of 0.37, ELN441958 is a substrate for P-gp.
0.69, and 1.5 ␮M). Because in vivo analgesia studies could
be complicated by agonism at opioid receptors, ELN441958
was tested in guanosine 5Ј-O-(3-[35S]thio)triphosphate
binding assays and found to be an agonist at the ␦-opioid
receptor (EC50 ϭ 0.76 ␮M) and also exhibited weak ago-
nism at the ␮-opioid receptor (Ͻ50% stimulation at 10 ␮M)
(data not shown).
P-gp is one of the components of the blood-brain barrier,
TABLE 4
ELN441958 has good in vitro metabolic stability across species
Metabolic clearance determined by decay of parent compound incubated with micro-
somes as measured by LC/MS/MS. Data are means Ϯ S.E.M. (n ϭ 4).
Compound
Rat
Rhesus
Human
Metabolic Stability, Permeability, P-Glycoprotein
Liability, and CNS Penetration. ELN441958 was suffi-
ciently soluble (69 Ϯ 16 ␮M) in aqueous buffer at pH 7.4 for
further characterization of metabolic stability and perme-
ability. ELN441958 demonstrated good metabolic stability
with low in vitro intrinsic clearance in rat, rhesus, and hu-
man microsomes (Table 4). In contrast, the metabolically
ELN441958
t1/2 (min)
50 Ϯ 2
28 Ϯ 1
43 Ϯ 4
33 Ϯ 3
48 Ϯ 3
29 Ϯ 2
Intrinsic clearance
(␮l/mg/min)
Midazolam
t1/2 (min)
Intrinsic clearance
(␮l/mg/min)
1.6 Ϯ 0.4
1200 Ϯ 400
3.3 Ϯ 1.3
650 Ϯ 230
3.7 Ϯ 0.8
430 Ϯ 100

Bradykinin B1 Receptor Antagonist ELN441958
625
TABLE 5
ELN441958 is a substrate for P-glycoprotein
See Materials and Methods for description of the assays. Values are means Ϯ S.E.M. of at least three independent in vitro experiments or single in vivo studies (n ϭ 3–4).
Assay or Model
ELN441958
Indinavir
P-gp ATPase (-fold stimulation over basal)
P-gp ATPase EC50 (nM)
2.1 Ϯ 0.2
N.A.a
7.1 Ϯ 0.9
1.5 Ϯ 0.1
24 Ϯ 4
3.1 Ϯ 0.3
27 Ϯ 7
9 Ϯ 3
MDCK II permeabilityb (nm/s)
54 Ϯ 8
MDR1-MDCK permeabilityc (nm/s)
MDR1-MDCK permeability ϩ cyclosporine A (nm/s)
Cyclosporine A permeability ratio
7.8 Ϯ 2.0
110 Ϯ 50
13 Ϯ 3
Ͻ0.002
1.5 Ϯ 0.3
Brain level in wild-type FVB moused (␮M)
0.042 Ϯ 0.008
0.40 Ϯ 0.03
Brain level in MDR1 a/b (Ϫ/Ϫ) mousee (␮M)
a Stimulation produced by ELN441958 was too low to calculate an EC50 value.
b Percentage of recoveries for ELN441958 and indinavir were 44 Ϯ 5 and 82 Ϯ 3, respectively.
c Percent recoveries for ELN441958 and indinavir were 41 Ϯ 4 and 59 Ϯ 16, respectively.
d Plasma levels for ELN441958 and indinavir were 7.4 Ϯ 0.5 and 3.2 Ϯ 0.1 ␮M, respectively.
e Plasma levels for ELN441958 and indinavir were 20.0 Ϯ 0.3 and 3.8 Ϯ 0.1 ␮M, respectively.
and it is known to limit CNS penetration of certain com-
pounds (Demeule at al., 2002). Because in vitro assays pre-
dicted that the CNS exposure of ELN441958 may be limited
by P-gp, an in vivo study comparing brain levels in wild-type
mice to MDR1 knockout mice was done. Five minutes after
an i.v. dose of 2.5 mg/kg, ELN441958 achieved low micromo-
lar brain levels in MDR1 knockout mice, but it was below the
limit of quantitation in wild-type mice (Table 5), consistent
with P-gp-mediated extrusion from the CNS. To further char-
acterize the CNS penetration by this compound, a brain and
spinal cord time-course study was performed in wild-type
mice. As shown in Fig. 6, the levels of ELN441958 were
considerably lower in brain and spinal cord relative to
plasma at all of the time points measured. Based on the AUC
ratios, ELN441958 exposure in the spinal cord and brain was
1.4 and 6.7%, respectively, of plasma exposure (Table 6).
TABLE 6
ELN441958 has low CNS exposure in the mouse
See Fig. 6 and Materials and Methods for experimental details.
Tissue
Cmax
AUC0–4 h
Plasma Ratio
␮g/ml
␮g ⅐ h/ml
Plasma
Brain
Spinal cord
4.1 Ϯ 0.2
0.32 Ϯ 0.16
0.049 Ϯ 0.02
8.9 Ϯ 0.7
0.60 Ϯ 0.12
0.12 Ϯ 0.02
1.0
0.067
0.014
1
0.1
10 mg/kg, PO
0.01
0.001
Pharmacokinetics. ELN441958 exhibits
a favorable
2.5 mg/kg, IV
pharmacokinetic profile in the rat (Fig. 7; Table 7). After an
i.v. dose of 2.5 mg/kg, ELN441958 had a moderate volume of
distribution (2.7 l/kg, approximately four times total body
water) and a moderate clearance (0.96 l/h/kg, approximately
0
4
8
12
16
20
24
time (h)
24% of hepatic blood flow). The terminal plasma half-life of Fig. 7. ELN441958 exhibits a favorable pharmacokinetic profile in the
rat. Rats were dosed i.v. or orally with ELN441958 in 2% Tween 80/saline
this compound in rats was 1.7 h. When dosed orally, concen-
trations of ELN441958 increased to a maximum of 1.2 ␮g/ml
at 2 h after dosing. The oral availability of ELN441958 in
at 2.5 or 10 mg/kg, respectively (n ϭ 3). See Materials and Methods for
analytical details.
rats was 57%.
compound was 3.9 h in rhesus monkeys. After oral dosing,
ELN441958 had an excellent pharmacokinetic profile in
concentrations of ELN441958 increased to a maximum of 3.6
rhesus monkeys (Fig. 8; Table 7). When dosed i.v. at 1 mg/kg,
␮g/ml at 3.3 h after dosing. The calculated oral bioavailabil-
ELN441958 had a moderate volume of distribution (2.7 l/kg)
ity was greater than 100%. The pharmacokinetics of
and a moderate clearance (0.49 l/h/kg, approximately 32% of
ELN441958 was also examined in rhesus monkeys by the s.c.
hepatic blood flow). The terminal plasma half-life of this
route of administration to support efficacy studies. At an s.c.
dose of 10 mg/kg, ELN441958 achieved peak plasma levels of
5.6 ␮g/ml at 2.2 h postdose. The calculated s.c. bioavailability
was also greater than 100%.
10
Plasma
Carrageenan-Induced Hyperalgesia. Because ELN-
441958 has lower potency at rodent than primate B1 recep-
tors, it was evaluated in a primate pain model. ELN441958
1
Brain
dose-dependently reduced the thermal hyperalgesia elicited
0.1
Spinal cord
by carrageenan injected into the tail (Fig. 9). In this tail-
withdrawal procedure, carrageenan injection (2 mg/tail) pro-
duced a thermal hyperalgesia manifested by a reduced tail-
withdrawal latency from 20 s (baseline) to 1 to 2 s in 46°C
water. A 30-min pretreatment with 10 mg/kg ELN441958 s.c.
completely blocked this hyperalgesia after 1 to 1.5 h postcar-
rageenan injection, and this antihyperalgesic effect was
maintained for at least 3.5 h, consistent with sustained pla-
0.01
0
1
2
3
4
Time (h)
Fig. 6. ELN441958 CNS exposure correlates with plasma levels in the
mouse. ELN441958 was administered at 10 mg/kg s.c. (n ϭ 3). Tissue
levels were determined by LC/MS/MS.

626
Hawkinson et al.
TABLE 7
Baseline
20
Pharmacokinetic parameters for ELN441958 in the rat and rhesus
monkey
10 mg/kg
See Materials and Methods for description of the assays. Values are presented as
means Ϯ S.E.M. from a single study (rat, n ϭ 3; rhesus, n ϭ 3).
15
10
5
Parameter
Rat
Rhesus
i.v. dose (mg/kg)
i.v. t1/2 (h)
i.v. AUC0-t (␮g ⅐ h/ml)
i.v. Vz (l/kg)
i.v. clearance (l/h/kg)
Oral dose (mg/kg)
Oral Cmax (␮g/ml)
Oral Tmax (h)
Oral AUC0-t (␮g ⅐ h/ml)
Oral bioavailability (%)
s.c. dose (mg/kg)
s.c. Cmax (␮g/ml)
s.c. Tmax (h)
2.5
2.0 Ϯ 0.8
2.6 Ϯ 0.3
2.7 Ϯ 0.7
0.96 Ϯ 0.12
10
1.3 Ϯ 0.5
1.7 Ϯ 0.6
6.0 Ϯ 1.4
57
1
3 mg/kg
3.9 Ϯ 0.3
2.1 Ϯ 0.3
2.7 Ϯ 0.4
0.49 Ϯ 0.06
5
1 mg/kg
vehicle control
0
4.0 Ϯ 0.1
3.3 Ϯ 0.7
14 Ϯ 0
0
1
2
3
ϳ100a
time (h)
ELN441958 carrageenan
10
5.6 Ϯ 1.8
2.2 Ϯ 1.0
27 Ϯ 6
Fig. 9. ELN441958 dose-dependently reduced carrageenan-induced hy-
peralgesia in rhesus monkeys. ELN441958 or vehicle was injected s.c. 30
min before local injection of carrageenan into the tail. Tail-withdrawal
latencies in 46°C water were determined at 30-min intervals following
carrageenan injection. Vehicle control data (open squares) partially su-
perimposes on the 1 mg/kg dose (closed diamonds). Each symbol repre-
sents the mean Ϯ S.E.M. for three subjects in a single dosing session.
s.c. AUC0-t, (␮g ⅐ h/ml)
s.c. bioavailability (%)
ϳ100a
a The oral and s.c. bioavailability of ELN441958 exceeded the maximum possible
(100%), and they were calculated to be 138 Ϯ 17 and 137 Ϯ 40%, respectively.
10
between 0.5 and 2.5 h postcarrageenan injection. Finally, one
monkey at 10 mg/kg seemed to be slightly sedated during the
entire test session as evidenced by occasional eye closure,
although this animal was readily aroused for testing tail-
withdrawal latency. The cause of the facial paling and ap-
parent sedation are not known. Although the signs observed
at the 10 mg/kg dose are of some concern for the safety of the
compound, it is unlikely that they complicated the interpre-
tation of the increase in withdrawal latencies, because they
were not observed at the 3 mg/kg dose, which was efficacious
in all subjects. In addition, facial flushing had resolved in all
subjects before the first tail-withdrawal latency measure-
ment.
To determine whether opioid receptors are involved in the
antihyperalgesic effect of ELN441958, the effect of pretreat-
ment with the opioid receptor antagonist naltrexone was
determined. Naltrexone (1 mg/kg s.c.) administered just be-
fore ELN441958 (10 mg/kg s.c.) had no effect on the reduction
of carrageenan-induced hyperalgesia by ELN441958, sug-
gesting that opioid receptors are not involved in this effect
(Fig. 10).
Naproxen was selected as a reference nonsteroidal anti-
inflammatory drug for comparison with ELN441958.
Naproxen (10 mg/kg s.c.) produced a time-dependent reduc-
tion in the carrageenan-induced thermal hyperalgesia in rhe-
sus monkeys (Fig. 11). The reduction in hyperalgesia was
apparent 2 h postcarrageenan injection, and all subjects
reached baseline thresholds by 2.5 to 3 h. Similar antihyper-
algesic effects were observed by 10 mg/kg naproxen and 3
mg/kg ELN441958 (Fig. 9). At the identical dose of 10 mg/kg,
ELN441958 reduced hyperalgesia from 0.5 to 3.5 h after
carrageenan injection, whereas naproxen was only effective
at times Ն2 h postcarrageenan, indicating that ELN441958
is more effective than an equivalent dose of naproxen in
reducing carrageenan-induced hyperalgesia at early time
points.
1
10 mg/kg, SC
0.1
5 mg/kg, PO
0.01
1 mg/kg, IV
0.001
0
4
8
12
16
20
24
time (h)
Fig. 8. ELN441958 exhibits a favorable pharmacokinetic profile in the
rhesus monkey. Male rhesus monkeys were dosed i.v. at 1 mg/kg in 10%
polyethylene glycol 660 hydroxystearate/HCl/saline or orally at 5 mg/kg
in 10% polyethylene glycol 660 hydroxystearate/HCl/water or s.c. at 10
mg/kg in 20% Captisol/water (n ϭ 3). See Materials and Methods for
analytical details. Facial flushing was noted in all animals 0 to 3 min
following i.v. bolus injection, and in one of three animals 16 min after the
s.c. injection.
teau plasma levels up to 4 h following s.c. administration
(Fig. 8). At 3 mg/kg, ELN441958 produced a clear antihyper-
algesic effect only at times greater than 1.5 h postcarrag-
eenan injection and complete block was achieved in all three
subjects by 3.5 h. The large error bars at this dose were the
result of different time intervals required to reach baseline
by each of the three subjects. Because a dose of 1 mg/kg had
no effect on carrageenan-induced hyperalgesia, the minimal
effective dose for ELN441958 was 3 mg/kg s.c. in this model.
Plasma levels of ELN441958 at the 4-h time point were
0.20 Ϯ 0.06, 1.1 Ϯ 0.26, and 5.0 Ϯ 2.0 ␮M at the 1, 3, and 10
mg/kg doses, respectively.
ELN441958 produced signs in certain subjects at doses
reducing carrageenan-induced hyperalgesia. Facial flushing
was observed in two of three monkeys at the 10 mg/kg dose,
occurring within 30 min after compound injection. The ob-
served facial flushing may be due to a release of histamine by
the compound as elevated plasma histamine levels were de-
tected 1 to 5 min following i.v. administration of 1 mg/kg
ELN441958 in rhesus monkeys in the PK study (range 30–
160 ng histamine/ml plasma; n ϭ 3). This possibility was not
explored further in efficacy studies. In addition, facial paling
Discussion
The benzamide ELN441958 is an optimized member of a
was observed in all three subjects at the 10 mg/kg dose new class of nonpeptide bradykinin B1 receptor antagonists.

Bradykinin B1 Receptor Antagonist ELN441958
627
Baseline
20
LF22-0542 (Porreca et al., 2006). ELN441958 is also a sub-
nanomolar antagonist of agonist (DAKD)-induced calcium
mobilization in IMR-90 cells expressing the native human B1
receptor. No agonist-like activity was detected by ELN-
441958 alone in calcium mobilization studies, indicating that
ELN441958 is a neutral antagonist of the B1 receptor. In the
same cells, ELN441958 does not inhibit the activation of the
human bradykinin B2 receptor at concentrations up to 10
␮M, showing that it is highly selective for B1 over B2 recep-
tors. ELN441958 also displays good selectivity for B1 over
other receptors examined in a broad screening panel. It is
Ͼ500-fold selective for the B1 receptor over the human ␮-opi-
oid receptor, the most potent off-target activity identified.
ELN441958 has marked species differences for B1 receptor
antagonist potency compared with the peptidic B1 receptor
antagonists DALKD and desArg10HOE140. Agonist-induced
calcium mobilization experiments indicate that ELN441958
ELN441958
ELN441958 + naltrexone
15
10
5
vehicle control
0
0
1
2
3
time (h)
naltrexone carrageenan
ELN441958
Fig. 10. The opioid-receptor antagonist naltrexone does not affect the
antihyperalgesic effect of ELN441958 in the rhesus carrageenan model.
Naltrexone (1 mg/kg s.c.) was injected just before ELN441958 (10 mg/kg
s.c.). The vehicle control and ELN441958 alone data are the same as in is 120-fold less potent at the mouse B1 receptor relative to the
Fig. 9. Each symbol represents the mean Ϯ S.E.M. for three subjects in a
single dosing session.
human receptor. Similar species differences have been noted
previously for other series of small molecule B1 receptor
antagonists, including the quinoxalines and the diaminopy-
Baseline
ridines (Kuduk et al., 2004; Ransom et al., 2004). These
findings are consistent with the relatively low sequence ho-
mology between B1 receptors from human and mouse (72%;
Hess et al., 1996) or rat (73%; Jones et al., 1999). Because
20
naproxen
15
there is high sequence homology between the mouse and rat
receptor (90%; Jones et al., 1999), it is surprising that
10
ELN441958 is 10-fold more potent at the rat than mouse B1
receptor. Of residues known to be involved in the binding of
peptides or small molecule ligands (Ha et al., 2005), only
Ile97 in the human sequence shows divergence between the
corresponding locations in the rat (Ile107) and mouse
(Val104) sequences. Site-directed mutagenesis studies would
be required to determine whether this conserved substitution
can account for the species differences observed.
MDCK-MDR1 permeability and ATPase assays indicate
that ELN441958 is a substrate for human P-gp. Consistent
with these in vitro results, ELN441958 had low CNS pene-
tration in the mouse. Antagonism of spinal B1 receptors may
be important to achieve full analgesic efficacy of an orally
5
carrageenan alone
0
0
1
2
3
time (h)
naproxen carrageenan
Fig. 11. Naproxen reduces carrageenan-induced hyperalgesia in rhesus
monkeys. Naproxen (10 mg/kg s.c.) was injected 30 min before local
injection of carrageenan into the tail. See Fig. 9 and Materials and
Methods for details. Each symbol represents the mean Ϯ S.E.M. for three
individuals in a single dosing session.
The benzamide core was predicted to confer activity based on delivered antagonist as suggested by the efficacy of intrathe-
molecular modeling studies involving the crystal structure of cally delivered B1 receptor antagonists in pain models (Fox et
a series of potent B1 receptor antagonists, the 3,4-dihydro- al., 2003; Conley et al., 2005). Alternatively, it is possible that
quinoxalinones (Grant et al., 2006). This modeling suggested brain penetration may lead to increased central side effects,
that the quinoxalinone core and its substituents could be because there is a high level of constitutive B1 receptor ex-
effectively replaced by a substituted benzamide, and initial pression in monkey brains (Shughrue et al., 2003), and the B1
compounds synthesized proved to be low micromolar B1 an- receptor is expressed in human brain (Raidoo and Bhoola,
tagonists. Modifications designed to increase structural ri- 1997). Nevertheless, peripherally acting peptidic B1 receptor
gidity resulted in increased activity and also improved oral antagonists given systemically are effective in pain models.
bioavailability, ultimately leading to ELN441958.
For example, desArg10HOE140 is effective against CFA-in-
ELN441958 is a potent, selective, neutral antagonist of the duced mechanical allodynia by either the intrathecal or s.c.
human bradykinin B1 receptor. It has subnanomolar affinity route (Fox et al., 2003). An antagonist that is excluded from
for displacement of the agonist ligand [3H]DAKD. In satura- the spinal cord could still block B1 receptors present on
tion binding studies, ELN441958 reduced the apparent af- peripheral terminals of sensory fibers, and it may also gain
finity of [3H]DAKD more than 4-fold with little change in access to receptors present on cell bodies in the dorsal root
measured receptor density, consistent with reversible, com- ganglion (DRG).
petitive displacement of the agonist radioligand from the
ELN441958 possesses a favorable pharmacokinetic profile
human B1 receptor. Other types of small molecule antago- as predicted by in vitro assays. ELN441958 is well absorbed
nists whose mode of binding has been investigated have also after oral administration in rats and rhesus monkeys consis-
been shown to compete with agonist, including the sulfon- tent with the moderate solubility and permeability in MDCK
amide SSR240612 (Gougat et al., 2004), the quinoxaline com- cells. In particular, ELN441958 is essentially completely ab-
pound A (Ransom et al., 2004), and the N-methylacetamide sorbed and produces high plasma levels after oral adminis-

628
Hawkinson et al.
tration in rhesus monkeys. The bioavailabilities calculated
ELN441958 dose-dependently reduces carrageenan-in-
following oral and s.c. administration in monkeys were duced thermal hyperalgesia in rhesus monkeys, with a min-
greater than 100%, possibly due to lack of dose proportion- imal effective dose of 3 mg/kg s.c. At this dose, ELN441958
ality by one or more route of administration as different dose does not affect the initial hyperalgesia produced by carra-
levels used for each route. ELN441958 has a moderate clear- geenan, but it reduces hyperalgesia beginning 2 h after car-
ance and volume of distribution in both species following i.v. rageenan injection, and it completely blocks the hyperalgesia
administration, consistent with the high metabolic stability by 3.5 h postinjection. The delayed antihyperalgesic effect
in rat, rhesus, and human microsomes. The high oral bio- observed at 3 mg/kg ELN441958 s.c. may be due to a pro-
availability and half-life of ϳ4 h in rhesus monkeys coupled gression of inflammatory mediators producing hyperalgesia
with the good metabolic stability in human microsomes pre- following carrageenan injection. Specifically, generation of
dict a favorable pharmacokinetic profile for ELN441958 in prostanoids may play a dominant role in carrageenan-in-
humans.
duced hyperalgesia after about 2 h postcarrageenan injec-
Several approaches have been taken to demonstrate effi- tion, but not earlier. This idea is supported by a similar
cacy for B1 antagonists with low potency at rodent B1 recep- time-dependent reduction in hyperalgesia observed with the
tors, precluding their evaluation in standard rodent pain nonselective cyclooxygenase inhibitor naproxen at 10 mg/kg
models. For example, Su et al. (2003) exploited the pharma- s.c., which probably reflects the time course for prostanoid
cological similarity between primate and rabbit B1 receptors production caused by carrageenan. The time-dependent re-
by demonstrating the potent efficacy of a quinoxaline B1 duction in carrageenan hyperalgesia observed at the 3 mg/kg
receptor antagonist in a CFA-induced hyperalgesia in rab- dose of ELN441958 probably represents antagonism of B1
bits. In an elegant study by Fox et al. (2005), the highly receptor-mediated prostanoid production.
human-selective B1 receptor antagonist NVP-SAA164 re-
The early carrageenan-induced hyperalgesia may be unre-
versed CFA-induced hyperalgesia in transgenic mice ex- lated to prostanoid production, and it is thus not sensitive to
pressing only the human B1 receptor. An alternative ap- treatment with 10 mg/kg naproxen s.c. It has been shown that
proach is to evaluate compounds in primate pain models.
during the first hour following carrageenan injection into the
Use of primate pain and inflammation models to evaluate rat paw, edema develops that is mediated by histamine, sero-
potential therapeutic agents is advantageous for several rea- tonin, and bradykinin release (Salvemini et al., 1996). Subse-
sons. In addition to species differences in receptor potency, a quently, neutrophil infiltration and production of NO, tumor
clear advantage of the use of primate models is the more necrosis factor-␣, interferon-␥, cytokines (IL-1 and IL-2), and
direct applicability to humans. There are often major differ- prostaglandins contribute significantly to the edema 1 to 6 h
ences in the pharmacology of neurotransmitter systems in- after carrageenan injection. In particular, NO production has
volved in pain transmission between humans and rodents, been shown to dramatically increase within 1 h, whereas pros-
e.g., cholecystokinin (Ho¨kfelt et al., 2001) and substance P taglandin E2 levels increase slowly, peaking at 3 h after carra-
(Hill, 2000). In addition, scaling from monkey pharmacoki- geenan injection (Salvemini et al., 1996). Thus, it is likely that
netic data is generally the most accurate method to predict the high dose of 10 mg/kg ELN441958 s.c. reduces early carra-
human clearance (Evans et al., 2006). In the bradykinin geenan-induced hyperalgesia by mechanisms other than pro-
system, human and monkey B1 receptors are functionally stanoid production. Because B1 agonists promote generation of
indistinguishable, whereas rodent receptors exhibit a differ- NO (Sangsree et al., 2003), cytokine release (Tsukagoshi et al.,
ent agonist and antagonist pharmacology (Regoli et al., 1999), and neutrophil infiltration (Ehrenfeld et al., 2006), B1
2001). Demonstration of efficacy in primates using experi- antagonists may reduce the hyperalgesia elicited by these
mental analgesic agents with novel mechanisms of action not proinflammatory factors.
validated clinically increases the likelihood of analgesic effi-
cacy in humans.
The potential contribution of receptor induction to the an-
tihyperalgesic effects of ELN441958 in the carrageenan
The rhesus monkey tail-withdrawal procedure is a well model must be considered. The peripheral non-neuronal ef-
characterized method for assessing antinociception in pri- fects of B1 antagonists require receptor induction as receptor
mates. Initially established as an acute pain model, the pro- expression is normally very low or nondetectable. B1 receptor
cedure involves measuring the latency to withdrawal of the induction in peripheral tissues can occur rapidly following
tail immersed in water at an acutely noxious temperature tissue insult. For example, B1 receptor mRNA is dramatically
(e.g., 50°C) (Ko et al., 2004). Trained monkeys respond to increased in the paw within 1 h following paw injection of
standard analgesic agents such as opioids with an increased IL-1␤ (Campos et al., 2002). Carrageenan paw injection in-
latency to tail withdrawal. To evaluate analgesics with a creases B1 mRNA paw expression at 3 h in rats (J. E.
broader range of mechanisms, the procedure has been mod- Hawkinson, unpublished observations). Taken together,
ified by pretreatment with agents that sensitize the tail to these data suggest that carrageenan injection may rapidly
thermal stimuli. For example, topical application of capsaicin (i.e., within about 1 h) induce B1 receptors in peripheral
produces a gradual and relatively prolonged hyperalgesia, tissues. In contrast to non-neural tissues, constitutive ex-
and it closely mimics the experimental use of capsaicin in pression of the B1 receptor in nociceptors has been demon-
humans (Butelman et al., 2003). Alternatively, carrageenan strated (Wotherspoon and Winter, 2000). Paw injection of
injection into the tail of rhesus monkeys produces a thermal CFA results in up-regulation of B1 receptor in the DRG after
hyperalgesia manifested as a reduced tail-withdrawal la- 24 h (Fox et al., 2003). Efficacy of 3 mg/kg ELN441958 in
tency to a normally non-noxious water temperature (Ko and rhesus monkeys observed at times later than 1 h postcarra-
Lee, 2002). The latency to withdrawal in 46°C water is re- geenan injection are consistent with the time course for local
duced to 1 to 2 s from a baseline of 20 s within 1 h after B1 receptor induction, again suggesting a peripheral action
carrageenan injection and is maintained for at least 3.5 h.
at this dose.

Bradykinin B1 Receptor Antagonist ELN441958
629
Rapid effects of 10 mg/kg ELN441958 observed within 1 h tent with low CNS exposure. ELN441958 reduced carrag-
after carrageenan injection may require blockade of consti- eenan-induced hyperalgesia in rhesus monkeys, probably
tutive B1 receptors expressed on pain afferents. Consistent acting primarily at peripheral B1 receptors. Validation of the
with this idea, ELN441958 (3 and 10 mg/kg s.c.) produced a therapeutic utility of bradykinin B1 antagonists in the treat-
marked reduction in topically applied capsaicin-induced al- ment of human pain states will require evaluation in clinical
lodynia in rhesus monkeys within 5 min after removal of trials.
capsaicin. Antiallodynic activity of ELN441958 was observed
in two of the four rhesus monkeys tested in a tail-withdrawal
procedure using both 38 and 42°C stimulation (E. R. Butel-
man, unpublished observations). Because ELN441958 has
poor penetration into the spinal cord, the antihyperalgesic
activity at low doses is more likely mediated by B1 receptors
expressed on nociceptors at their peripheral terminals or at
the cell bodies in the DRG, although high doses may be
sufficient to penetrate the spinal cord and exert a central
antihyperalgesic effect. Intrathecally administered B1 recep-
tor antagonists are effective in acute and inflammatory pain
models (Fox et al., 2003; Conley et al., 2005).
Plasma levels achieved by ELN441958 at efficacious doses
were substantially greater than its in vitro potency. This
difference may be explained by a number of factors related to
the compound and/or the receptor. For example, ELN441958
has restricted access to neuronal B1 receptors expressed in
spinal cord and access to receptors expressed on cell bodies in
the DRG may also be limited. Accessibility to peripheral B1
receptors may also be restricted if the compound is highly
bound to plasma proteins. In addition, time-dependent up-
regulation of B1 receptor expression and the progression of
inflammatory mediators with varying sensitivities to modu-
lation by B1 receptor antagonism following carrageenan in-
jection may also contribute to the relatively high doses of
ELN441958 required for efficacy.
Because ELN441958 has low affinity for ␮- and ␦-opioid
receptors (i.e., it is Ͼ500- and Ͼ2000-fold selective for the
human B1 receptor over ␮- and ␦-opioid receptors, respec-
tively), opioid receptors may play a minimal role in its anti-
hyperalgesic effects. This notion is further supported by the
finding that naltrexone 1 mg/kg s.c. was inactive in blocking
the antihyperalgesic effects of ELN441958. Based on the
binding affinity of naltrexone in monkey brain membranes
and its in vivo antagonist potency in rhesus monkeys (Em-
merson et al., 1994; Ko et al., 1998), this dose of naltrexone is
sufficient to antagonize ␮-, ␬-, or ␦-opioid receptor-mediated
behavioral effects in monkeys. Although in this study we did
not conduct a control experiment to show blockade of opioid
receptor-mediated effects by this dose of naltrexone, several
studies have shown that much smaller doses (i.e., 0.0032–0.1
mg/kg) antagonized a variety of opioid-mediated behavioral
effects in rhesus monkeys (Ko et al., 1998, 2004; Bowen et al.,
2002). For example, naltrexone 0.1 mg/kg produced a 10-fold
rightward shift of the dose-response curve for ␮-opioid ago-
nist-induced antinociception (Ko et al., 1998). Taken to-
gether, it is unlikely that the opioid receptors significantly
contribute to the antihyperalgesic effects of ELN441958 in
monkeys.
Acknowledgments
We thank Dr. Barbara Jagodzinska (Elan Pharmaceuticals) for
the synthesis of indinavir.
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