Analysis of the pharmacokinetic/pharmacodynamic relationship of a small molecule CXCR3 antagonist, NBI-74330, using a murine CXCR3 internalization assay

Article abstract of DOI:10.1038/sj.bjp.0707519

Background and purpose: Pharmacokinetic/pharmacodynamic (PK/PD) models are necessary to relate the degree of drug exposure in vivo to target blockade and pharmacological efficacy. This manuscript describes a murine agonist-induced CXCR3 receptor internalization assay and demonstrates its utility for PK/PD analyses. Experimental approach: Activated murine DO11.10 cells were incubated with agonist in the presence or absence of a CXCR3 antagonist and changes in surface CXCR3 expression were detected by flow cytometry. For PK/PD analysis, mice were dosed with a small molecule CXCR3 antagonist, NBI-74330, (100 mg kg^-1) orally or subcutaneously and plasma samples taken at specified timepoints for the CXCR3 internalization assay. Key results: Surface CXCR3 expression was specifically decreased in response to CXCL9, CXCL10 and CXCL11. CXCL11 was the most potent CXCR3 agonist in buffer (pA₅₀=9. 23±0.26) and the pA₅₀ for CXCL11 was unaltered in murine plasma (pA₅₀=9.17±0.15). The affinity of a small molecule CXCR3 antagonist, NBI-74330, was obtained in the absence or presence of plasma (buffer pA₂ value: 7.84±0.14; plasma pK_B value 6.36±0.01). Administration of NBI-74330 to mice resulted in the formation of an N-oxide metabolite, also an antagonist of CXCR3. Both antagonists were detectable up to 7 h post oral dose and 24 h post subcutaneous dose. Measurement of CXCR3 internalization demonstrated significant antagonism of this response ex vivo, 24 h following subcutaneous administration of NBI-74330. Conclusions and implications: The CXCR3 receptor internalization assay provides a robust method for determining agonist potency orders, antagonist affinity estimates and PK/PD analyses, which discriminate between dosing regimens for the CXCR3 antagonist NBI-74330.

Full text of DOI:10.1038/sj.bjp.0707519

British Journal of Pharmacology (2007) 152, 1260–1271
2007 Nature Publishing Group All rights reserved 0007–1188/07 30.00
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RESEARCH PAPER
Analysis of the pharmacokinetic/pharmacodynamic
relationship of a small molecule CXCR3 antagonist,
NBI-74330, using a murine CXCR3 internalization
assay
LA Jopling1, GF Watt1, S Fisher2, H Birch3, S Coggon3 and MI Christie1
1Department of Pharmacology, UCB Inflammation Discovery, Granta Park, Great Abington, Cambridgeshire, UK; 2Department of
Molecular and Cellular Systems, UCB Inflammation Discovery, Granta Park, Great Abington, Cambridgeshire, UK and 3Department
of Drug Metabolism and Pharmacokinetics, UCB Inflammation Discovery, Granta Park, Great Abington, Cambridgeshire, UK
Background and purpose: Pharmacokinetic/pharmacodynamic (PK/PD) models are necessary to relate the degree of drug
exposure in vivo to target blockade and pharmacological efficacy. This manuscript describes a murine agonist-induced CXCR3
receptor internalization assay and demonstrates its utility for PK/PD analyses.
Experimental approach: Activated murine DO11.10 cells were incubated with agonist in the presence or absence of a CXCR3
antagonist and changes in surface CXCR3 expression were detected by flow cytometry. For PK/PD analysis, mice were dosed
with a small molecule CXCR3 antagonist, NBI-74330, (100 mg kgꢀ1) orally or subcutaneously and plasma samples taken at
specified timepoints for the CXCR3 internalization assay.
Key results: Surface CXCR3 expression was specifically decreased in response to CXCL9, CXCL10 and CXCL11. CXCL11 was
the most potent CXCR3 agonist in buffer (pA50 ¼ 9.23 0.26) and the pA50 for CXCL11 was unaltered in murine plasma
(pA50 ¼ 9.17 0.15). The affinity of a small molecule CXCR3 antagonist, NBI-74330, was obtained in the absence or presence
of plasma (buffer pA2 value: 7.84 0.14; plasma pKB value 6.36 0.01). Administration of NBI-74330 to mice resulted in the
formation of an N-oxide metabolite, also an antagonist of CXCR3. Both antagonists were detectable up to 7 h post oral dose
and 24 h post subcutaneous dose. Measurement of CXCR3 internalization demonstrated significant antagonism of this
response ex vivo, 24 h following subcutaneous administration of NBI-74330.
Conclusions and implications: The CXCR3 receptor internalization assay provides a robust method for determining agonist
potency orders, antagonist affinity estimates and PK/PD analyses, which discriminate between dosing regimens for the CXCR3
antagonist NBI-74330.
British Journal of Pharmacology (2007) 152, 1260–1271; doi:10.1038/sj.bjp.0707519; published online 5 November 2007
Keywords: CXCR3; CXCL9; CXCL10; CXCL11; receptor internalization; NBI-74330; pharmacokinetic/pharmacodynamic
Abbreviations: E/[A], agonist concentration effect curve; PD, pharmacodynamic; PK, pharmacokinetic
Introduction
The drug discovery process requires appropriate pharmaco-
kinetic/pharmacodynamic (PK/PD) models to understand
the relationship between drug exposure and pharmacologi-
cal effect. PK/PD models help facilitate the transition of
molecules from research to development and onto the
market by minimizing drug attrition due to poor PK
parameters (Walker, 2004). PK/PD models provide vital
information such as the mode of action of a molecular
target, dose range and regimen, therapeutic window of a
drug and may also enable biomarker identification (Colburn,
2000). Tolerance of blood or plasma within an appropriate
assay system assists with the development of PK/PD model-
ling and may provide information on the properties required
of a potential drug candidate (Walker, 2004).
Approximately 27% of current Food and Drug Adminis-
tration-approved drugs target the G-protein-coupled recep-
tor (GPCR) family (Overington et al., 2006) to which
chemokine receptors belong. The chemokine receptor sub-
family provides a number of attractive drug targets. Chemo-
kines and chemokine receptors have been implicated in a
variety of inflammatory conditions and a role for CXCR3 has
Correspondence: Dr LA Jopling, Department of Pharmacology, UCB
Inflammation Discovery, Granta Park, Great Abington, Cambridgeshire
CB21 6GS, UK.
E-mail: louise.jopling@ucb-group.com
Received 20 July 2007; revised 17 September 2007; accepted 21 September
2007; published online 5 November 2007

Antagonism of CXCR3 receptor internalization
LA Jopling et al
1261
been suggested in disorders such as rheumatoid arthritis,
multiple sclerosis and transplant rejection based on receptor
and/or agonist expression profiles within clinical samples
or from murine models of disease (Sorensen et al., 1999;
Hancock et al., 2000; Eriksson et al., 2003; Pease and
Williams, 2006).
downregulation) and gain (re-expression and new synthesis)
of receptor expression (Koenig and Edwardson, 1997). These
processes may occur constitutively but, in the presence of
agonist, the dynamic equilibrium of these events changes
and receptors are rapidly endocytosed. CXCL11-induced
CXCR3 receptor internalization has been reported to occur
within a 30 min incubation period (Sauty et al., 2001).
Furthermore, each CXCR3 agonist appears to utilize different
intra- and extracellular domains for specific signalling and
functional responses. Colvin et al. (2004) demonstrated that
the carboxyl terminus of human CXCR3 was required for
CXCL9- and CXCL10-induced CXCR3 internalization by
mutagenesis studies, while the third intracellular loop of
CXCR3 was required for CXCL11-induced CXCR3 interna-
lization. Previous data from the same group demonstrated
that CXCR3 internalization events were not affected by
pretreatment with Pertussis toxin and were therefore inde-
pendent of G-protein coupling (Sauty et al., 2001). Insensi-
tivity to Pertussis toxin for receptor internalization events
has been reported for other Gai-coupled chemokine receptors
such as CXCR4 (Forster et al., 1998). Studies examining the
extracellular components of CXCR3 required for signalling
events suggest a multistep model of CXCR3 activation,
similar to that reported for CCR2 receptors (Monteclaro and
Charo, 1997; Xanthou et al., 2003; Colvin et al., 2004). The
first step of receptor activation by all CXCR3 agonists
involves high-affinity agonist binding to sulphated tyrosine
residues within the amino terminus and while the extra-
cellular domains required for chemotaxis have been identi-
fied, the corresponding extracellular residues involved in
agonist-induced CXCR3 receptor internalization have not
been fully elucidated (Colvin et al., 2004).
The human CXCR3 receptor was originally identified and
cloned in the mid-1990s (Marchese et al., 1995; Loetscher
et al., 1996) and while CXCR3 mRNA has been detected in
monocytes, neutrophils and mast cells, the T cell is the
predominant cell type that expresses this receptor (Loetscher
et al., 1996). Chemokine receptor expression profiles on
polarized human T cells demonstrated CXCR3 to be strongly
associated with the development of CD4þ Th1 cells (Bone-
cchi et al., 1998; Sallusto et al., 1998). Cellular activation
through CXCR3 occurs in response to binding of the
agonists, CXCL9, CXCL10 or CXCL11 (Rabin et al., 1999;
Tensen et al., 1999), which are produced in response to IFN-g,
implicating the CXCR3-receptor activation axis in Th1-
dominated diseases.
Human CXCR3 receptor function has been extensively
studied using a range of assay systems such as radioligand
binding, intracellular calcium flux, in vitro migration or
receptor internalization assays. CXCL11 and CXCL10 are the
most widely studied CXCR3 ligands and the data suggest
that CXCL11 is the most efficacious agonist for the human
receptor, generally demonstrating full agonism with an A50
range of 0.1–30 nM (Sauty et al., 1999; Proost et al., 2001;
Gonsiorek et al., 2003; Heise et al., 2005). CXCL9 and
CXCL10 have been shown to behave as full or partial
agonists depending upon the assay system tested with A50
ranges of 10–100 and 30–300 nM, respectively (Clark-Lewis
et al., 2003; Gonsiorek et al., 2003; Heise et al., 2005). What is
intriguing about CXCR3, in contrast to many other chemo-
kine receptors, is that approximately 40% of freshly isolated
human peripheral blood T cells express the receptor but
these cells do not possess functional responsiveness to
CXCR3 agonists (Loetscher et al., 1998). It has been
demonstrated that activation of peripheral blood T cells
in vitro induces responsiveness to CXCR3 agonists, through
increasing receptor density (Loetscher et al., 1998). In
addition, Xie et al. (2003) showed that murine T cells
activated under Th2-polarizing conditions expressed high
surface levels of CXCR3 and were able to migrate in vitro to
CXCR3 agonists. However, following adoptive transfer to
naive mice, these Th2 cells were not able to migrate to sites
of inflammation, in contrast to Th1-polarized cells (Xie et al.,
2003). These authors claim that antigen encounter but not
cytokine milieu plays a dominant role in the regulation of
CXCR3 expression levels. These data suggest that surface
expression of CXCR3 does not necessarily predict in vitro
and/or in vivo functional responsiveness of cells.
NBI-74330, a small molecule CXCR3 antagonist, has been
studied using human CXCR3 GTPgS, calcium flux and
cellular migration assays. The data demonstrate that NBI-
74330 inhibited these human CXCR3-dependent processes
with an IC50 range of 7–18 nM (Heise et al., 2005). A second
molecule, TAK-779, while specific for human CCR5 only,
demonstrated dual antagonism of murine CCR5 and CXCR3
in vitro (Gao et al., 2003). TAK-779 was also shown to
significantly inhibit T-cell trafficking in a murine collagen-
induced arthritis model, which may, in part, be due to
antagonism of the murine CXCR3 receptor (Gao et al., 2003).
The murine CXCR3 receptor has not been extensively
studied and characterized, often due to limitations of
reagents at the time of publication. However, the generation
of CXCR3- or CXCL10-deficient mice has implicated CXCR3
in driving models of viral inflammation of the CNS,
transplant rejection and pulmonary fibrosis (Hancock et al.,
2000, 2001; Christensen et al., 2004; Jiang et al., 2004; Hsieh
et al., 2006). For example, CXCR3-deficient mice survived
intracerebral lymphocytic choriomeningitis virus infection
with susceptibility restored following reconstitution with
wild-type CD8þ T cells (Christensen et al., 2004). More
recently, CXCL10-deficient mice have identified the key
requirement of this agonist for immune surveillance of the
CNS following lymphocytic choriomeningitis virus infection
in CXCL10-deficient mice have been identified (Christensen
et al., 2006). In contrast, intracerebral infection of CXCR3-
or CXCL10-deficient mice with dengue virus showed
This study sets out to describe and validate an agonist-
induced CXCR3 receptor internalization assay. Such assays
have previously been used in a qualitative manner to
elucidate the processes involved in chemokine receptor-
trafficking events. Generally, total surface expression of
chemokine receptors, in common with other members of
the G-protein coupled receptor superfamily, is the net
outcome of processes governing both loss (endocytosis and
British Journal of Pharmacology (2007) 152 1260–1271

Antagonism of CXCR3 receptor internalization
1262
LA Jopling et al
significantly higher mortality rates than wild-type mice,
suggesting a protective role for CXCR3 and CXCL10 in such
viral infections (Hsieh et al., 2006). CXCR3-deficient mice
have also demonstrated profound resistance to acute cardiac
rejection, with graft survival permanently maintained in
these mice following treatment with a subtherapeutic dose
of cyclosporin A (Hancock et al., 2000). Furthermore, the use
of CXCL10-deficient recipient mice showed that donor-
derived CXCL10 was responsible for allograft rejection
(Hancock et al., 2001). Many of the gene-deletion studies
expand upon data generated with anti-ligand antibodies, for
example Hildebrandt et al. (2004) demonstrated that neu-
tralization of CXCL9 or CXCL10 in a model of experimental
idiopathic pneumonia syndrome, significantly inhibited
donor cell recruitment to the lungs comparable to that
demonstrated using CXCR3-deficient mice. Few studies have
completely blocked the CXCR3 receptor in wild-type mice,
although neutralization of murine CXCR3 using a poly-
clonal antibody was carried out in a sheep red blood cell
delayed-type hypersensitivity model. While 70% inhibition
was observed in the anti-CXCR3 antibody treatment group it
was not fully determined whether the antibody had cell-
depletion properties that may have contributed to the
apparent efficacy (Xie et al., 2003).
Cell culture
Spleens from DO11.10 mice were collected and single-cell
splenocyte suspensions were generated following disaggrega-
tion through a 40-mm mesh (BD Biosciences). Cells were
washed twice in DO11.10 medium (RPMI þ 10% fetal bovine
serum þ penicillin (100 U mlꢀ1)–streptomycin (0.1 mg lꢀ1) þ
L-glutamine (2 mM) þ 50 mM 2-mercaptoethanol þ 12.5 mM
HEPES) (Sigma-Aldrich, Dorset, UK), counted and resus-
pended at 1 Â 106 cells mlꢀ1 in DO11.10 medium containing
antigen (ovalbumin323ꢀ339
, ) (University of
200 ng mlꢀ1
Southampton, Southampton, UK). Three days later, cells
were split twofold and resuspended in fresh DO11.10
medium reconstituted with recombinant murine interleu-
kin-2 (rmuIL-2, 10 ng mlꢀ1) (Peprotech, London, UK). Cells
were cultured for a further 5 days prior to use in the receptor
internalization assay.
Murine CXCR3 internalization assay
Activated DO11.10 cells, as described above, were pelleted
and washed in fresh DO11.10 medium. Cells were pooled,
resuspended in medium, overlaid onto sterile Nycoprep
˚
(1.077 A) (Axis Shield, Huntingdon, UK) and centrifuged at
550 g for 20 min at room temperature. Viability of cells
present at the interface was determined by Trypan blue
exclusion. Cells were removed from the interface and
washed twice in assay buffer (0.25% BSA in phosphate-
buffered saline). Cells (5 Â 105) were incubated in a total
assay volume of 100 ml, which included the agonist and
antagonist concentrations as required, at 371 C for the
specified time. A 60 min incubation period was used in all
cases, with the exception of time-course studies. For detec-
tion of surface CXCR3 expression levels, cells were washed
and stained with rhodamine–phycoerythrin-conjugated
There are caveats to many of the approaches undertaken to
validate CXCR3 in inflammatory disease conditions. Eluci-
dation of the feasibility of pharmacological manipulation of
CXCR3 in chronic inflammatory diseases awaits therapeutic
blockade of the receptor using small molecule antagonists.
Here, we describe the characterization of an agonist-induced
CXCR3 receptor internalization assay in activated primary
murine cells, which has been used to determine agonist
potency and antagonist affinity measurements and most
importantly for PK/PD analyses enabling discrimination
between dosing regimens for preclinical validation of
CXCR3 as a drug target.
anti-murine CXCR3 or isotype control antibodies at
a
concentration of 2.5 mg mlꢀ1 for 30 min on ice (R&D Systems,
Abingdon, UK; Invitrogen, Paisley, UK). Unbound antibody
was then removed by washing and the cells were fixed using
CellFix according to manufacturer’s instructions (BD Bios-
ciences), prior to data acquisition using an EPICS-XL flow
cytometer (Beckman Coulter, High Wycombe, UK). Specific
CXCR3 staining was determined by subtracting the isotype
control staining profile for each experimental condition.
For plasma assays, 5 Â 105 cells were pelleted and resus-
pended in 90 ml of sex-matched EDTA-treated mouse plasma.
For in vitro plasma assays, the plasma source was naive
pooled Balb/c plasma. For ex vivo PD studies, plasma was
obtained from mouse blood at appropriate time intervals
following in vivo dosing of CXCR3 antagonist and at each
time point the plasma was pooled. Assays were then carried
out in a total volume of 100 ml, at 371 C for 60 min. Surface
CXCR3 was detected as described previously.
Methods
Animal use
Adult mice (6–8 weeks of age) were used in accordance with
the Animals (Scientific Procedures) Act 1986. Balb/c mice
were purchased from Charles River Laboratories (Margate,
UK) and DO11.10 mice were obtained from the John
Radcliffe Hospital (Oxford, UK). Animals were kept under a
light/dark cycle of 12/12 h and had access to food and water
ad libitum.
Mouse blood was collected into Microvette 600 EDTA
tubes (Sarstedt, Leicester, UK) via cardiac puncture under
terminal gaseous anaesthesia (isoflurane; NVS, Stoke-on-
Trent, UK). For PK/PD experiments, male Balb/c mice were
dosed with 100 ml of dosing solution either via oral gavage
(25 mm; 20 G cannula) (Harvard Apparatus, Edenbridge, UK)
or s.c. using a 25 G needle (BD Biosciences, Oxford, UK). In
each case, the vehicle used for dosing was 0.1% sodium
docusate/99.9% methylcellulose (0.5%; 400 cP). EDTA-trea-
ted naive, Balb/c, sex-matched, plasma was purchased from
B&K (Hull, UK).
[
35S]GTPgS-binding assay
Membranes from murine CXCR3-transfected Chinese Ham-
ster ovary cells were prepared as follows: cell pellets were
resuspended at 1 Â 107cells mlꢀ1 and homogenized using a
glass 10 ml manual homogenizer in ice-cold buffer A (15 mM
Tris-HCl, 2 mM MgCl, 0.3 mM EDTA, 1 mM EGTA, pH 7.5).
The homogenate was centrifuged at 40 000 g for 25 min at
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Antagonism of CXCR3 receptor internalization
LA Jopling et al
1263
41 C. The supernatant was carefully removed and the pellet
resuspended in buffer and centrifuged as outlined
deionized water before use. Plasma was mixed with test
compound (10 mM) and 50 ml aliquots (n ¼ 6 replicate deter-
minations) were loaded onto the 96-well equilibrium dialysis
block. Dialysis vs phosphate buffer (100 mM, pH 7.4, 50 ml)
was carried out for 4 h at 371 C with constant agitation, after
which time, aliquots of plasma and buffer were transferred to
a 96-deep well plate. The composition in each well was
balanced with control fluid, such that the volume of buffer
and plasma was the same in each sample. Sample extraction
was performed by the addition of 120 ml of acetonitrile
containing internal standard. Samples were mixed, centri-
fuged at 1750 g at 41 C for 10 min and 40 ml of supernatant
was transferred into another 96-well plate followed by
addition of 160 ml of 5% acetonitrile (in water). Analysis of
test compound was performed using HPLC-MS/MS as
described and relative responses of test compound in the
buffer and plasma half-wells were used to determine the
respective drug-free fraction.
A
previously. The supernatant was then removed and the final
pellet resuspended at 1 Â 108 cells mlꢀ1 in ice-cold buffer B
(7.5 mM Tris-HCl, 12.5 mM MgCl, 0.3 mM EDTA, 1 mM EGTA,
250 mM sucrose, pH 7.5). The protein concentration was
determined using BCA Protein Assay Kit according to
manufacturer’s instructions (Perbio Science, Cramlington,
UK). The membrane preparation was divided into aliquots,
frozen and stored at ꢀ801 C until use.
The GTPgS-binding assay was carried out in a 96-well
Optiplate (Perkin Elmer, Beaconsfield, UK) in a final assay
volume of 200 ml, using 1 mg per well of cell membrane
resuspended in binding buffer (20 mM HEPES, 100 mM NaCl,
10 mM MgCl, 1 mM EDTA (pH 7.4), 5 mM GDP, 25 mg mlꢀ1
saponin, 5 mg mlꢀ1 wheat germ agglutinin–polyvinyl to-
luene scintillation proximity assay beads, 0.1% BSA) and
preincubated with agonist (in the absence and presence of
antagonist or antibody) at room temperature for 60 min. At
this time, 0.3 nM [35S]GTPgS (Amersham, Little Chalfont,
UK) was added and incubated for a further 2 h at room
temperature before centrifugation for 10 min at 380 g. Bound
radioactivity was measured using a TopCount NXT micro-
plate scintillation and luminescence counter (Perkin Elmer).
Data analysis and statistics
Agonist concentration effect (E/[A]) curves were fitted to a
three-parameter logistic equation, using GraphPad Prism
(version 4). Data (mean s.e.) from individual experiments
are shown superimposed with a single curve generated using
the mean of the individual parameter estimates (nX3). The
control E/[A] curve parameters, midpoint location ([A]50),
midpoint slope (nH) and maximal asymptote (a), were not
significantly different between individual experiments and
the mean values for each control group depicted, lie within
95% confidence limits of the population (CXCL11 buffer
pA50 ¼ 9.38 0.31, n ¼ 32; plasma pA50 ¼ 9.07 0.21, n ¼ 35;
data expressed as mean s.d.) calculated from a number of
experiments conducted over a 14-month time period by
independent operators.
Bioanalysis of NBI-74330 and metabolite-1
Blood and plasma levels of NBI-74330 and metabolite-1 were
measured using HPLC-MS/MS. Samples (20 ml) were dis-
pensed into a 96-deep well plate (Porvair, King’s Lynn, UK)
and measured against calibration standards. Samples and
calibration standards were precipitated with internal stan-
dard in acetonitrile (120 ml), mixed and then centrifuged at
1750 g at 41 C for 10 min. Blood and plasma sample extracts
were then analysed using a Micromass Mass Spectrometer
(Waters Ltd, Elstree, UK). Extracts (20 ml) were injected using
a CTC analytics HTS Pal Autosampler (Presearch, Hitchin,
UK) onto a Phenomenex Luna C18 50 mm  2.1 mm, 5 mm
column (Phenomenex, Macclesfield, UK) operated at 401 C
and at an eluent flow rate of 1.0 ml minꢀ1 with a split ratio of
5:1 into the mass spectrometer. Analytes were eluted using a
high-pressure linear gradient programme by means of an
HP1100 binary HPLC system (Agilent Technologies, Stock-
port, UK) using water containing 0.1% formic acid (solvent
A) and acetonitrile containing 0.1% formic acid (solvent B).
A 5 min gradient was used for analysis of in vivo blood and
plasma extracts, increasing from 5 to 95% solvent B at 4 min
before returning to the starting conditions at 4.1 min, which
were maintained until 5 min.
Specific experiments were conducted for Schild analysis
and E/[A] curves were constructed in the presence and
absence of increasing antagonist concentrations. A compe-
titive antagonist will shift E/[A] curves to the right with no
significant change in E/[A] curve midpoint slope or max-
imum response, as judged by using one-way ANOVA with
Bonferroni post-test. If no significant changes were obtained,
individual dose ratios (DRs) were calculated from the degree
of rightward shift of the control E/[A] curve in the presence
of antagonist and fitted to the following equation:
logðDR ꢀ 1Þ ¼ Schild slope Á log½antagonistꢁ ꢀ pKB
If the unconstrained Schild slope (b) was not significantly
different from unity, the data were re-fitted constraining the
slope to unity and pKB was determined. If the criteria of
competitive antagonism were not fully satisfied, an apparent
pA2 value was estimated from the lowest antagonist
concentration that produced a significant rightward shift
of the E/[A] curve.
A 3 min gradient was used for analysis of plasma protein-
binding extracts, which increased from 5 to 95% solvent B at
2 min before returning to the starting conditions at 2.1 min,
which were maintained until 3 min.
Plasma protein-binding properties of NBI-74330
Plasma protein binding of NBI-74330 was measured by
equilibrium dialysis using a 96-well Teflon block (HT Dialysis
LLC, Gales Ferry, CT, USA). Membranes (3-kDa cutoff) were
conditioned in deionized water for 60 min, followed by
80:20 deionized water/ethanol for 20 min, and then rinsed in
Drugs
NBI-74330 was synthesized as described elsewhere (Medina
and Johnson, 2002). Metabolite-1 was prepared by reaction
of NBI-74330 (1.20 g) with metachloroperbenzoic acid
(0.48 g) (Schneller and Luo, 1980) and the product was
British Journal of Pharmacology (2007) 152 1260–1271

Antagonism of CXCR3 receptor internalization
1264
LA Jopling et al
purified by preparative HPLC to give the compound as a
colourless solid (0.85 g).
(66 2%, n ¼ 6; Figure 1a) compared to freshly isolated
splenocytes (30 1%, n ¼ 4; data not shown). Surface CXCR3
expression was significantly (ANOVA; Po0.01 (n ¼ 6)) re-
duced following 60 min incubation at 371 C with a single
concentration (300 nM) of each of the CXCR3 agonists
(CXCL9, CXCL10 and CXCL11), but was not significantly
affected by incubation with 300 nM CXCL12, a CXCR4
receptor agonist (Figure 1b). The time course and concentra-
tion dependence of agonist-induced loss of surface CXCR3
expression showed that for each CXCR3 agonist, a 60 min
Results
In vitro characterization of an agonist-induced murine CXCR3
internalization assay
Murine DO11.10 splenocytes activated in vitro for 8 days
displayed higher levels of surface CXCR3 expression
Figure 1 The selectivity and specificity of a murine CXCR3 internalization assay. (a) CXCR3 surface expression on DO11.10 cells activated in
vitro with antigen (ovalbumin323ꢀ339) was measured by flow cytometry using anti-CXCR3 antibody (black line) compared to isotype control
staining (dashed line). (b) A single concentration (300 nM) of CXCL9, CXCL10, CXCL11 and CXCL12 was incubated with activated DO11.10
cells for 60 min at 371 C and the percentage decrease in surface CXCR3 expression was measured compared to cells incubated with buffer
alone (***Po0.001; n ¼ 3–14). (c–e) CXCL9, CXCL10 and CXCL11 concentrations (0.3, 3, 30 and 300 nM) were incubated with activated
DO11.10 cells for 30, 60 or 120 min and CXCR3 expression was compared to cells incubated with buffer alone (100% CXCR3 expression) at
each time point (n ¼ 3). (f) Membranes generated from muCXCR3-transfected Chinese Hamster ovary cells were incubated with 0.1 nM
CXCL11 anti-CXCR3 antibody or isotype control antibody (rat IgG2a) for 60 min and then incubated with 0.3 nM [35S]GTPgS for 2 h followed
by measurement of GTP binding (n ¼ 3). The negative control, 0(ꢀ), was carried out in the absence of agonist while all other samples were
incubated with 0.1 nM CXCL11.
British Journal of Pharmacology (2007) 152 1260–1271

Antagonism of CXCR3 receptor internalization
LA Jopling et al
1265
incubation time was sufficient to achieve steady-state
responses (Figures 1c–e).
Classification of murine CXCR3 using the CXCL11-induced
receptor internalization assay
E/[A] curves were constructed using a 60 min incubation
The specificity of the agonist-induced loss of surface
CXCR3 expression was explored by determining whether
the loss of measured surface CXCR3 expression was due to a
competitive interaction between the anti-CXCR3 antibody
and CXCL11. This was investigated, using a murine CXCR3
GTPgS assay to determine the effect of a range of anti-CXCR3
antibody concentrations (1–30 mg mlꢀ1) or isotype control
antibody on CXCL11 binding to cell membranes expressing
murine CXCR3 (Figure 1f). Individual antibody concentra-
tions were investigated in the presence and absence of a
single CXCL11 concentration, 0.1 nM, equivalent to an A50
in this assay (data not shown). No significant inhibition of
CXCL11 binding by the anti-CXCR3 antibody was observed
compared to the isotype control antibody. This indicated
that the antibody concentration (2.5 mg mlꢀ1) used to detect
surface CXCR3 expression in the receptor internalization
assay was not competing with CXCL11 for the same binding
site.
period. CXCL11 exhibited the greatest potency (pA50
¼
9.23 0.26, n ¼ 12), while CXCL10 (pA50 ¼ 7.74 0.13,
n ¼ 14) and CXCL9 (pA50 ¼ 7.51 0.38, n ¼ 6) were 30- to
50-fold less potent. The maximal responses in the E/[A] curve
for each agonist were equivalent (Figure 2a).
In a separate set of experiments, a small molecule CXCR3
receptor antagonist, NBI-74330 (Heise et al., 2005), was
used to further characterize the CXCL11-induced loss of
surface CXCR3 expression. CXCL11 E/[A] curves were
generated in the presence and absence of increasing
concentrations of NBI-74330 (Figure 2b). NBI-74330
(30–300 nM) produced concentration-dependent, parallel
rightward shifts of the CXCL11 E/[A] curve with no
significant change in the E/[A] curve maximal response
(Figure 2b). However, at the highest concentration (1 mM), a
significant reduction of the CXCL11 E/[A] curve maximal
response was obtained (ANOVA; Po0.05), which was
Figure 2 Murine CXCR3 receptor classification using receptor internalization and GTPgS assays. (a) Increasing agonist concentrations (0.1–
300 nM) were incubated with activated DO11.10 cells for 60 min at 371 C and surface CXCR3 expression was detected by flow cytometry. Data
are expressed as percent decrease in CXCR3 expression (mean s.e.) to generate E/[A] curves for CXCL11, CXCL10 and CXCL9 (n ¼ 6–14). (b)
Increasing concentrations of NBI-74330 (30 nM–1 mM) were included in the CXCR3 internalization assay and CXCL11 E/[A] curves were
generated following 60 min incubation at 371 C. Data are expressed as percent decrease in CXCR3 expression (mean s.e.) in the presence and
absence of 30, 100, 300 nM or 1 mM NBI-74330 (n ¼ 6) (*Po0.05). (c) Schild plot analysis comparing the degree of rightward shift of the
CXCL11 E/[A] curve (log (rꢀ1)) in the presence of NBI-74330 concentrations to the CXCL11 E/[A] curve generated in the absence of
antagonist. Individual data points are plotted, the unconstrained Schild slope is b ¼ 1.25 0.14. An apparent pA2 value of 7.84 0.14 was
estimated from the 100 nM NBI-74330 E/[A] curve (n ¼ 6). (d) Increasing concentrations of NBI-74330 (100 nM–1 mM) were included within the
murine CXCR3 GTPgS assay using the agonist CXCL11. Data are expressed as mean c.p.m. s.e. in the presence and absence of 100, 300 nM
or 1 mM NBI-74330 (n ¼ 3) (***Po0.001). An apparent pA2 value of 8.35 0.04 was estimated from the 100 nM NBI-74330 E/[A] curve.
British Journal of Pharmacology (2007) 152 1260–1271

Antagonism of CXCR3 receptor internalization
1266
LA Jopling et al
accompanied by a significant flattening of the E/[A] curve
midpoint slope (ANOVA; Po0.001). Although the uncon-
strained Schild slope was not significantly different from
unity (b ¼ 1.25 0.14, n ¼ 6) (Figure 2c), the criteria for
competitive antagonism were not fully satisfied (Jenkinson,
1991). Consequently, an apparent pA2 value of 7.84 0.14
(n ¼ 6) was determined from the lowest antagonist concen-
tration (100 nM) that produced a significant rightward shift
of the CXCL11 E/[A] curve.
Compatibility of the murine CXCR3 internalization assay with
plasma
To determine the utility of the murine CXCR3 internaliza-
tion assay for PK/PD modelling, we investigated the inclu-
sion of whole blood or plasma within the assay system. The
magnitude of basal CXCR3 expression was substantially
reduced in mouse whole blood (data not shown), whereas
the basal receptor expression was unchanged by the presence
of mouse plasma. The impact of mouse plasma on agonist
potency and antagonist affinity estimates was determined;
CXCL11 demonstrated similar potency in the presence and
absence of mouse plasma (pA50 values: buffer 9.23 0.26,
n ¼ 12; plasma 9.17 0.15, n ¼ 16; Figure 3a), with no
significant change in the E/[A] curve midpoint slope or
maximal response. Consequently, CXCL11 was employed for
all subsequent experiments. In a separate set of experiments,
plasma CXCL11 E/[A] curves were generated in the presence
and absence of increasing concentrations of NBI-74330. NBI-
74330 (1–10 mM) produced concentration-dependent, paral-
lel rightward shift of the CXCL11 E/[A] curve, with no
significant change in the E/[A] curve maximal response
(Figure 3b). The relationship between the degree of right-
ward shift of the CXCL11 E/[A] curve and the concentration
For comparison, the NBI-74330–CXCL11 interaction was
studied using a murine GTPgS assay. CXCL11 E/[A] curves
were obtained in the presence and absence of increasing
concentrations of NBI-74330 (100 nM–1 mM). NBI-74330
produced concentration-dependent, parallel rightward shift
of the CXCL11 E/[A] curve (Figure 2d). However, at the
highest concentration of 1 mM NBI-74330,
a significant
reduction of the CXCL11 E/[A] curve maximal response
was observed (ANOVA; Po0.001) (Figure 2d). Again,
although the unconstrained Schild slope was not signifi-
cantly different from unity (b ¼ 0.89 0.05, n ¼ 3), the
behaviour was not consistent with the expectations for
competitive antagonism. Therefore, an apparent pA2 value of
8.35 0.04 (n ¼ 3) was obtained.
Figure 3 Tolerance of the murine CXCR3 internalization assay to plasma. (a) CXCL11 E/[A] curves were generated following 60 min
incubation at 371 C of agonist with activated DO11.10 cells in the presence (n ¼ 16) and absence (n ¼ 12) of 90% EDTA-treated, naive mouse
plasma, followed by surface CXCR3 staining by flow cytometry. (b) Increasing concentrations of NBI-74330 (1–10 mM) were included within
the CXCR3 internalization assay and CXCL11 E/[A] curves were generated following 60 min incubation at 371 C. Data are expressed as percent
decrease in CXCR3 expression (mean s.e.) in the presence and absence of 1, 3 or 10 mM NBI-74330 (n ¼ 6). (c) Schild plot analysis comparing
the degree of rightward shift of the CXCL11 E/[A] curve in the plasma receptor internalization assay format, in the presence of NBI-74330
concentrations to the CXCL11 E/[A] curve generated in the absence of antagonist (n ¼ 6). Individual data points are plotted and the
unconstrained Schild slope was b ¼ 1.10 0.12. Constraining the Schild slope to a value of unity generated a pKB value of 6.36 0.01.
British Journal of Pharmacology (2007) 152 1260–1271

Antagonism of CXCR3 receptor internalization
LA Jopling et al
1267
of NBI-74330 was linear and an unconstrained Schild slope
(b ¼ 1.10 0.12, n ¼ 6) not significantly different from unity
was obtained (Figure 3c). Constraining the Schild slope to a
value of unity, generated a pKB value of 6.36 0.01 (n ¼ 6),
demonstrating a B30-fold reduced affinity of NBI-74330 for
murine CXCR3 receptor in the presence of plasma compared
to that using buffer in the receptor internalization assay.
analysis of blood samples by HPLC-MS/MS (Figures 4a and
5a, respectively). NBI-74330 blood exposure was greater
following p.o. compared to s.c. administration, as indicated
by
maximum
concentration
(Cmax
)
(p.o.
7051–
13,010 ng mlꢀ1
;
s.c. 1047–4737 ng mlꢀ1
; data shown as
ranges) and area under the curve (p.o. 21,603–
41,349 ng h mlꢀ1; s.c. 5702–21,600 ng h mlꢀ1; data shown as
ranges). However, dosing of NBI-74330 (Figure 4b) generated
an N-oxide metabolite (metabolite-1; Figure 4c) that was
active against murine CXCR3, and apparent pA2 values
for metabolite-1 were calculated from the effects of
a single antagonist concentration in the CXCR3 receptor
PK and PD profiles of the CXCR3 antagonist, NBI-74330
following administration to mice
The murine PK profile of NBI-74330 (100 mg kgꢀ1) was
determined following p.o. and s.c. administration and
Figure 4 PK/PD parameters of NBI-74330 (100 mg kgꢀ1) following oral administration to mice. (a) Blood concentration of NBI-74330 and
metabolite-1 at time (h) post-oral dose of 100 mg kgꢀ1 of NBI-74330 in 0.1% sodium docusate/99.9% methyl cellulose (n ¼ 3). (b and c)
Chemical structures of NBI-74330 and metabolite-1. (d) Effect of plasma samples taken at 1, 4 and 24 h post-oral dose with 100 mg kgꢀ1 NBI-
74330 on CXCL11-induced CXCR3 internalization response compared to the E/[A] curve generated in naive mouse plasma (n ¼ 3). (e and f)
PK/PD relationship of NBI-74330 and metabolite-1, respectively, following oral administration of NBI-74330 (100 mg kgꢀ1). Data are
presented as plasma concentration of each antagonist at 1, 4 and 24 h post p.o. dose compared to the degree of inhibition of the CXCR3
internalization response induced by 100 nM CXCL11 (n ¼ 3).
British Journal of Pharmacology (2007) 152 1260–1271

Antagonism of CXCR3 receptor internalization
1268
LA Jopling et al
Figure 5 PK/PD parameters of NBI-74330 (100 mg kgꢀ1) following subcutaneous administration to mice. (a) Blood concentration of NBI-
74330 and metabolite-1 at time (h) post-subcutaneous dose of 100 mg kgꢀ1 of NBI-74330 in 0.1% sodium docusate/99.9% methyl cellulose
(n ¼ 3). (b) Effect of plasma samples taken at 2 and 24 h post-subcutaneous dose with 100 mg kgꢀ1 NBI-74330 on CXCL11-induced CXCR3
internalization response compared to the E/[A] curve generated in naive mouse plasma (n ¼ 3).
internalization assay (buffer pA2: 7.42 0.10 (n ¼ 3); plasma
pA2: 7.04 0.16 (n ¼ 3); data not shown). Blood Cmax for
metabolite-1 was higher following p.o. compared to s.c.
receptor internalization assay and using this to generate
PK/PD information to discriminate between dosing regimens.
For many G-protein coupled receptors, receptor internali-
zation assays have been described to help assign newly
identified agonists to their appropriate receptor(s) and to
determine agonist potency orders. The selectivity of the
murine CXCR3 internalization response was demonstrated
using the CXCR3 agonists, CXCL9, CXCL10 and CXCL11,
compared to the CXCR4 agonist CXCL12, and showed that
only the CXCR3 agonists modulated surface expression of
CXCR3 (Figure 1b). The extracellular domains of murine
CXCR3 recognized by the CXCR3 antibody or CXCL11, used
in our assay, have not been fully characterized. However,
further examination of the specificity of the receptor
internalization assay showed that altered surface CXCR3
detection was not due to hindrance of antibody staining by
CXCL11 binding to the receptor (Figure 1f). In agreement
with similar receptor internalization studies, our findings
demonstrate that CXCR3 agonists decrease the proportion of
surface-expressed CXCR3 on activated murine cells in a
concentration- and time-dependent manner (Dorland et al.,
1979; Arai et al., 1997; Sauty et al., 2001). Interestingly,
CXCL10 and CXCL11 reached the maximal internalization
response within 30 min of incubation, while CXCL9 reached
maximal effect by 60 min (Figures 1c–e). Sauty et al. (2001)
previously reported that human CXCL11 induced rapid
internalization of human CXCR3 with maximal effects
within a similar time frame to that observed for murine
CXCR3. CXCL9 has been described as a partial agonist in a
human CXCR3 receptor internalization assay (Sauty et al.,
2001), although a prolonged incubation time may have been
required by CXCL9 in this instance. The delayed response of
CXCL9-induced internalization of murine CXCR3, com-
pared to CXCL10 and CXCL11, may be due to utilization
of different receptor domains. Distinct receptor domains
have been described for CXCL9-, CXCL10- and CXCL11-
induced human CXCR3 internalization events (Colvin et al.,
2004), while those required for murine CXCR3 internaliza-
tion responses have not been identified. The murine CXCR3
internalization assay also showed that CXCL11 was at least
30-fold more potent than either CXCL10 or CXCL9
administration (p.o. 4846–6276 ng mlꢀ1
ng mlꢀ1
while the area under the curve was similar
; s.c. 1005–2842
)
between dosing regimens (p.o. 8149–14,495 ng h mlꢀ1; s.c.
8897–17,704 ng h mlꢀ1) as a consequence of the prolonged
profile of both NBI-74330 and metabolite-1 following s.c.
administration, with significant concentrations of both
antagonists maintained at 24 h post-dose (Figure 5a).
The corresponding PD readout, the degree of antagonism
of the CXCL11 E/[A] curve, was measured using plasma
collected at fixed time points (1, 4 and 24 h) following p.o.
administration of NBI-74330 (100 mg kgꢀ1). Significant
antagonism of the CXCL11 E/[A] curve (430-fold rightward
shift; ANOVA; Po0.01 (n ¼ 3)) was obtained at 1 and 4 h
but not at 24 h post-administration of NBI-74330 p.o.
(Figure 4d). PK/PD analysis for NBI-74330 and metabolite-1
following p.o. administration did not demonstrate hysteresis
but showed a linear relationship, indicating a direct relation-
ship between exposure and effect (Figures 4e and f,
respectively). In addition, following s.c. administration of
NBI-74330, the corresponding PD readout was determined at
2 and 24 h post-dose and significant antagonism of the
CXCL11 E/[A] curve (430-fold rightward shift; ANOVA;
Po0.001) was obtained at both time points (Figure 5b). This
PK/PD analysis suggested that, compared to p.o. dosing, s.c.
administration of NBI-74330 would generate a more pro-
longed CXCR3 receptor antagonism in vivo.
Discussion
Understanding the role and effect of antagonism of CXCR3
in preclinical models of chronic inflammation helps to
determine the pharmacological feasibility of this target in a
drug discovery environment. We therefore set out to develop
a cell assay that would discriminate between small molecule
CXCR3 antagonists within a research-screening cascade. To
our knowledge, this study provides the first example of
applying receptor pharmacology principles to a chemokine
British Journal of Pharmacology (2007) 152 1260–1271

Antagonism of CXCR3 receptor internalization
LA Jopling et al
1269
(Figure 2a), which is in agreement with studies examining
the human CXCR3 agonist potency order (Clark-Lewis et al.,
2003; Heise et al., 2005). The potency of CXCL11 and its
tolerance of plasma in the assay allowed a large experimental
window for antagonism of the CXCR3 receptor internaliza-
tion response using both in vitro and ex vivo assay formats.
The effect of different anticoagulants in mouse plasma did
not significantly affect CXCL11-induced CXCR3 receptor
internalization, while the potency of CXCL10 was decreased
in EDTA-treated plasma and completely inhibited by hepar-
inized plasma (data not shown), presumably due to the
heparin-binding domains possessed by CXCL10 (Campanel-
la et al., 2003). Therefore, the use of CXCL11 for further
studies provided technical flexibility within the assay.
tion (Kaumann and Frenken, 1988), a heterogenous receptor
population or antagonist-induced reduction in the function
of existing receptors (Liu et al., 1992; Robertson et al., 1994),
which account for saturable and non-saturable insurmoun-
table antagonism. Further pharmacological analyses, such as
receptor protection and ‘washout’ studies, could be em-
ployed to determine whether the reduction in the CXCL11
E/[A] curve by NBI-74330 in common with the rightward
shift of the CXCL11 E/[A] curve is due to a reversible,
syntopic mechanism. Nevertheless, the affinity estimates for
NBI-74330 calculated as apparent pA2 values in our murine
CXCR3 receptor internalization and GTPgS assays were
7.84 0.14 and 8.35 0.04, respectively, which are consis-
tent with the values obtained at human CXCR3 receptors
(Heise et al., 2005). In addition, the antagonism of CXCL11-
mediated migration of activated DO11.10 cells was also
investigated (data not shown). Interestingly, CXCL11 was
B30-fold less potent compared to receptor internalization
assay, suggesting that the cell migration response is poorly
coupled. Consequently, this limited the experimental win-
dow available in which the criteria for competitive antagon-
ism was to be fully tested. Not withstanding this, an
apparent pA2 value (7.42 0.05, n ¼ 3) was obtained (data
not shown), which was similar to the values obtained in the
receptor internalization and GTPgS assays.
Further classification of murine CXCR3 with the receptor
internalization assay employed Schild analysis using
a
known human CXCR3 small molecule antagonist NBI-
74330 (Heise et al., 2005). This antagonist had previously
been studied in GTPgS, calcium flux and chemotaxis assays
using cell lines expressing human CXCR3 and generated IC50
values against human CXCL11 of 5.5,
7 and 3.9 nM,
respectively. In addition, Heise et al. (2005) reported that
NBI-74330 demonstrated insurmountable antagonism in
both human CXCR3 calcium flux and GTPgS assays. In both
assays, the profile of NBI-74330 antagonism showed a
concentration-dependent reduction of the maximal re-
sponse without rightward shift of the CXCL11 E/[A] curve
(Heise et al., 2005). However, at the murine CXCR3 receptor,
we observed a different antagonism profile of the CXCL11
response by NBI-74330 in GTPgS and receptor internaliza-
tion assays (Figures 2b and d). Significant rightward shift
(1.88 log units) of the GTPgS E/[A] curve was obtained with
300 nM NBI-74330 without concomitant reduction in the
E/[A] curve maximal response, whereas 1 mM NBI-74330
further rightward shifted the E/[A] curve (2.28 log units)
and significantly reduced the maximal response. This profile
was also obtained in the murine CXCR3 receptor internaliza-
tion assay. Significant rightward shift (1.33 log units) of the
CXCR3 receptor internalization E/[A] curve was obtained
with 300 nM NBI-74330 without concomitant reduction of
the E/[A] curve maximal response, whereas 1 mM NBI-74330
further rightward shifted the E/[A] curve (1.64 log units) and
significantly reduced the maximal response. In both murine
CXCR3 assays, the E/[A] curves generated in the presence of
1 mM NBI-74330 had a pA50 value of 8.15, suggesting that this
may be related to the degree of receptor reserve within the
assays (Figures 2b and d, respectively). Insurmountable
antagonism, which is a concentration-dependent reduction
of the E/[A] curve maximal response, with or without
rightward shift of the CXCL11 E/[A] curve, has been
described extensively within the literature for a wide range
of receptor systems such as angiotensin II receptors (Pendle-
ton et al., 1989), a1-adrenoceptors (Blue et al., 1995) and
5-HT2 receptors (Kaumann and Frenken, 1985). Historically,
insurmountable antagonism was recognized to be due to a
variety of mechanisms, both receptor and non-receptor
mediated (Schild, 1949; Gaddum et al., 1955). A number of
different explanations exist within the literature; irreversible
competitive antagonism (Furchgott R, 1966), pseudo-irrever-
sible antagonism (Paton and Rang, 1965), allosteric modula-
The murine CXCR3 receptor internalization assay toler-
ated plasma and permitted the generation of affinity
estimates using CXCL11 as the agonist (Figure 3a). Subse-
quent Schild analysis of NBI-74330 generated an affinity
estimate (pKB value 6.36 0.01), which was B30-fold lower
than in the absence of plasma. In addition, the antagonist
concentration range employed did not demonstrate
a
reduction in the CXCL11 E/[A] curve maximal response
(Figure 3b). This is consistent with expectations based on the
degree of rightward shift of the CXCL11 E/[A] curves in this
study compared to the corresponding study in buffer. The
maximal degree of rightward shift obtained in the plasma
assay format (1.42 log units) was within the range (o1.64 log
units) outlined previously in the buffer receptor internaliza-
tion assay where there was no concomitant reduction of the
E/[A] curve maximal response. Interestingly mouse plasma
protein binding for NBI-74330 was estimated at 97.3%
(
0.5%) suggesting that in the presence of plasma only 2–
3% of any NBI-74330 concentration is available for receptor
binding. This may explain the 30-fold difference in affinity
estimated for NBI-74330 in the absence and presence of
plasma.
Dosing of NBI-74330 to mice yielded the formation of a
previously unreported active metabolite (metabolite-1). The
exposure profiles of parent (NBI-74330) and metabolite
(metabolite-1) tracked with each other over time and a
linear PK/PD relationship was demonstrated following p.o.
dosing of NBI-74330 (100 mg kgꢀ1
) (Figures 4e and f).
Hysteresis of the PK/PD relationship is often observed with
the formation of an active metabolite leading to a delayed
effect over time (Campbell, 1990). However, because the
exposure profiles for each antagonist are parallel, one
hypothesis for the linear PK/PD relationship is that there is
rapid inter-conversion between NBI-74330 and metabolite-1
(Figures 4a and 5a). Comparison of dosing regimen by
British Journal of Pharmacology (2007) 152 1260–1271

Antagonism of CXCR3 receptor internalization
1270
LA Jopling et al
adapting the CXCR3 internalization assay into an ex vivo
format enabled distinction between p.o. and s.c. adminis-
tration of NBI-74330. Oral dosing of NBI-74330 demon-
strated slightly higher exposure levels of parent compared to
metabolite-1 (threefold), while both antagonist profiles were
equivalent following subcutaneous administration. This may
reflect some saturation of metabolic activity resulting from
the higher concentrations of NBI-74330 obtained following
p.o. administration.
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enabled us to determine that a once daily subcutaneous
administration of the small molecule CXCR3 antagonist
NBI-74330 would provide approximately 82% CXCR3 occu-
pancy. Based on this PK/PD readout, this dosing regimen
may be the most appropriate to establish the pharmacolo-
gical tractability of this target in murine models of chronic
inflammatory disease.
This finding demonstrates the utility of the CXCR3
receptor internalization assay to discriminate between
dosing regimens and enable the evaluation of the role of
CXCR3 in murine models of chronic inflammatory disease.
We believe that these types of studies are essential to
underpin target validation experiments in more complex
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Acknowledgements
We gratefully acknowledge the technical assistance of Paul
Green for conducting PK experiments, Kevin Minton for
conducting the antibody–agonist competition studies and
Bob Watson for synthesis of NBI-74330 and metabolite-1. We
are also extremely grateful to Gayle Chapman for helpful
discussions.
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Conflict of interest
The authors state no conflict of interest.
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