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Research article
A selective and potent CXCR3 antagonist SCH 546738 attenuates the development of autoimmune diseases and delays graft rejection
Chung-Her Jenh*, Mary Ann Cox, Long Cui, Eva-Pia Reich, Lee Sullivan, Shu-Cheng Chen, David Kinsley, Shiguang Qian, Seong Heon Kim, Stuart Rosenblum, Joseph Kozlowski, Jay S Fine, Paul J Zavodny and Daniel Lundell
Corresponding author:
Department of Respiratory and Immunology, Merck Research Laboratories, 2015 Galloping Hill Road, Kenilworth, NJ 07033, USA
Department of Medicinal Chemistry, Merck Research Laboratories, 2015 Galloping Hill Road, Kenilworth, NJ 07033, USA
University of Pittsburgh, Starzl Transplantation Institute, Pittsburgh, PA, USA
Current address: Shiguang Qian, Department of Immunology and General Surgery, Cleveland Clinic, Cleveland, OH, USA; Jay S. Fine, Boehringer Ingelheim Pharmaceuticals, Inc., Ridgefield, CT, USA
For all author emails, please .
BMC Immunology 2012, 13:2&
doi:10.72-13-2
The electronic version of this article is the complete one and can be found online at:
Received:23 September 2011
Accepted:10 January 2012
Published:10 January 2012
& 2012 Jenh et al.; licensee BioMed Central Ltd.
This is an open access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
The CXCR3 receptor and its three interferon-inducible ligands (CXCL9, CXCL10 and CXCL11)
have been implicated as playing a central role in directing a Th1 inflammatory response.
Recent studies strongly support that the CXCR3 receptor is a very attractive therapeutic
target for treating autoimmune diseases, such as rheumatoid arthritis, multiple sclerosis
and psoriasis, and to prevent transplant rejection. We describe here the in vitro
and in vivo pharmacological characterizations of a novel and potent small molecule
CXCR3 antagonist, SCH 546738.
In this study, we evaluated in vitro pharmacological properties of SCH 546738 by radioligand
receptor binding and human activated T cell chemotaxis assays. In vivo efficacy of
SCH 546738 was determined by mouse collagen-induced arthritis, rat and mouse experimental
autoimmune encephalomyelitis, and rat cardiac transplantation models. We show that
SCH 546738 binds to human CXCR3 with a high affinity of 0.4 nM. In addition, SCH 546738
displaces radiolabeled CXCL10 and CXCL11 from human CXCR3 with IC50 ranging from 0.8 to 2.2 nM in a non-competitive manner. SCH 546738 potently and specifically
inhibits CXCR3-mediated chemotaxis in human activated T cells with IC90 about 10 nM. SCH 546738 attenuates the disease development in mouse collagen-induced
arthritis model. SCH 546738 also significantly reduces disease severity in rat and
mouse experimental autoimmune encephalomyelitis models. Furthermore, SCH 546738 alone
achieves dose-dependent prolongation of rat cardiac allograft survival. Most significantly,
SCH 546738 in combination with CsA supports permanent engraftment.
Conclusions
SCH 546738 is a novel, potent and non-competitive small molecule CXCR3 antagonist.
It is efficacious in multiple preclinical disease models. These results demonstrate
that therapy with CXCR3 antagonists may serve as a new strategy for treatment of autoimmune
diseases, including rheumatoid arthritis and multiple sclerosis, and to prevent transplant
rejection.
Background
Leukocyte infiltration into inflammatory sites is critical for the initiation and
progression of a variety of inflammatory disorders and is controlled via the activation
and signaling of specific cell-surface chemoattractant receptors by their cognate
protein ligands, termed chemokines. Chemokines, which are produced by a number of
cell types at sites of inflammation, mediate the firm adhesion of leukocytes to activated
endothelial cells, their subsequent transmigration and extravasation into the inflamed
tissue, and possibly several cellular signaling pathways involved in cell activation
and differentiation [-].
CXCR3 is a seven-transmembrane G-protein coupled chemokine receptor which has been
demonstrated to play an important role in a variety of inflammatory and immunological
responses. CXCR3 receptor is predominantly expressed on activated T helper 1 (Th1)
cells. Its ligands, CXCL10 (IP-10), CXCL9 (MIG) and CXCL11 (I-TAC) are expressed by
endothelial cells, epithelial cells and infiltrating leukocytes following stimulation
by interferon (IFN)-γ or Type I IFNs and their expression is synergistically enhanced
by the key pro-inflammatory mediator tumor necrosis factor (TNF)-α [-].
The importance of CXCR3 in leukocyte recruitment was first demonstrated in the CXCR3
knockout mouse, where the rejection of a cardiac allograft was significantly delayed
compared to matched wild type animals, and where treatment of the CXCR3-deficient
host with the immunosuppressive agent cyclosporine resulted in permanent allograft
engraftment []. Transplant rejection is caused by infiltration, activation and expansion of host
leukocytes in the grafted organ resulting in destruction of the donor tissue. The
marked upregulation of CXCR3 ligand expression and the predominant expression of CXCR3
on infiltrating T cells during allograft rejection in human and in animal models indicate
a critical role for CXCR3-dependent T cell recruitment in transplant rejection [-]. Similarly, the upregulation of CXCR3 ligands and the increased number of CXCR3+ lymphocytes documented in chronic inflammatory diseases such as rheumatoid arthritis
(RA) [-], multiple sclerosis (MS) [,] and psoriasis [] indicates the potential importance of CXCR3-mediated leukocyte recruitment in the
pathology of these conditions, and suggests the potential utility of the selective
CXCR3 antagonist in the treatment and amelioration of these disorders.
To date, many different classes of small molecule CXCR3 antagonists have been discovered
[-], and it was reported that CXCR3 antagonism reduced inflammation and cartilage damage
in mouse and rat models of collagen-induced arthritis (CIA), attenuated atherosclerotic
plaque formation, prolonged allograft survival, and inhibited lung metastasis [,,,-]. In this report, we described the in vitro and in vivo pharmacological characterizations
of a novel and potent CXCR3 antagonist SCH 546738 (compound 8a) []. So far, SCH 546738 is reported to have the highest affinity of 0.4 nM to human CXCR3
receptor. SCH 546738 inhibits CXCL10 and CXCL11 binding and human activated T cell
chemotaxis with nanomolar potency. In vivo, SCH 546738 shows significant efficacy
in mouse CIA and rat experimental autoimmune encephalomyelitis (EAE) model. More importantly,
we show that combination of IFN-β therapy and CXCR3 inhibition has an additive effect
on delaying disease onset and attenuating disease severity in the mouse EAE model.
Furthermore, SCH 546738 delays graft rejection and in combination with cyclosporine,
permits permanent engraftment in the rat cardiac allograft transplant model. These
results demonstrate that SCH 546738 may offer a tool to evaluate the full therapeutic
potential of CXCR3 antagonism in chronic inflammatory disease and preventing allograft
rejection.
All chemokines were obtained from R & D Systems (Minneapolis, MN). 125I-hCXCL10 was obtained from PerkinElmer Life Science (Waltham, MA) and 125I-hCXCL11 from GE Healthcare Life Sciences (Piscataway, NJ). 35S radiolabeled SCH 535390 (a sulfonamide analog of the CXCR3 compound series) was
made in the lab.
Synthesis of SCH 546738
Synthesis of SCH 546738 was accomplished by the method outlined in Figure . The 2-chlorine of commercially available pyrazine 1 was regioselectively displaced with (S)(+)-2-ethylpiperazine in the presence of Pd-catalyst to afford compound 2. Subsequent reductive amination of compound 2 with N-Boc-piperidin-4-one in the presence of Ti(OiPr)4 followed by removal of Boc gave compound 3. The tricyclic compound 3 was reacted with 4-chlorobenzyl chloride in the presence
of excess base to provide methyl ester 4, which was converted to SCH 546738 by heating with ammonia.
Synthesis of SCH 546738. Synthesis of SCH 546738 was accomplished by the method outlined with details in
Methods section.
CXCR3 expressing cells and membrane preparations
The cDNAs encoding human, mouse and rat CXCR3 were generated based on the published
sequences: human (NM_001504) [], mouse (NM_009910) [], rat (NM_053415) []. The cDNAs for monkey and dog CXCR3 were cloned in the lab. All CXCR3 cDNAs were
cloned into the mammalian expression vector pME18Sneo, a derivative of the SRα expression
vector as described previously []. IL-3-dependent mouse pro-B cells Ba/F3 were transfected to express CXCR3 of different
species and cell membranes were prepared as described previously [].
Radioligand binding assays
A scintillation proximity assay was used for radioligand competition binding assays
as described previously [] with some modifications. For each assay point, 1 ug of membrane was preincubated
for 1 hr with 300 ug wheat germ agglutinin (WGA) coated SPA beads (GE Healthcare Life
Sciences) in the binding buffer (50 mM HEPES, 1 mM CaCl2, 5 mM MgCl2, 125 mM NaCl, 0.002% NaN3, 1.0% BSA) at room temperature. The beads were spun down, resuspended in the binding
buffer and transferred to a 96-well Isoplate (Wallac, Gaithersburg, MD). The indicated
concentrations of 125I-hCXCL10, 125I-hCXCL11 or 35S-SCH 535390 with a series of titrations of SCH 546738 were added to start the reaction.
After indicated reaction times at room temperature, the amount of radioactivity bound
to the SPA beads was determined with a Wallac 1450 Microbeta counter (Wallac).
Human activated T cell chemotaxis assays
The preparation of human activated T cells was performed as described previously []. Human peripheral blood lymphocytes were prepared by Ficoll-Hypaque centrifugation,
depleted of monocytes, and stimulated for 2 days with 1 ug/ml PHA (Murex Diagnostics,
Dartford, U.K.) and 100 U/ml IL-2 (Sigma, St. Louis, MO) in RPMI 1640 supplemented
with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 100 ug/ml streptomycin, 100 U/ml
penecillin, 1% non-essential amino acids and 2 mM HEPES. Following stimulation, peripheral
blood lymphocytes were cultured in above media containing 5% conditioned media (Sigma)
for up to 15 days.
Human activated T cell chemotaxis assays were performed using 96-well ChemoTx(R) microplates (Neuro Probe, Inc., Gaithersburg, MD) with a 3 um filter as per manufacturers'
instructions. Activated T cells were washed with RPMI medium twice, and then resuspended
in the medium containing 20% FBS. 1.25 × 105 cells/reaction were mixed with indicated concentrations of the compound and placed
on the filter. The compound and chemokines were mixed and placed in the bottom well
of the ChemoTx system. After 2.5 hours incubation at 37°C/5% CO2, the cells were scraped off and the plate system was centrifuged for 5 minutes at
1000 RPM. The filter screen was then removed and the ChemoTx plate was inverted into
a 96 well plate (Microlite + #7571 from Thermo Labsystems) with a funnel plate. The
plate system was centrifuged for 5 minutes at 1000 RPM. The volume in the wells was
brought to 100 μl with assay buffer and the plates were rested for approximately 15
minutes at room temperature. The number of migrated cells was measured using the Cell
Titer Glo Luminescent Assay from Promega (Madison, WI) as per vendor's instructions.
Chemotaxis is expressed as a chemotactic index, which is a ratio versus the one without
chemokines (spontaneous migration).
Mouse collagen-induced arthritis
Murine collagen-induced arthritis (CIA) was established as previously described []. Briefly, 12-week-old male B10.RIII mice (Jackson Laboratories, Bar Harbor, ME) were
immunized intradermally at five sites with bovine type II collagen (Elastin Products,
Owensville, MO) emulsified with an equal volume of complete Freund's adjuvant (CFA).
CFA was comprised of a mixture of incomplete Freund's adjuvant (Difco, Detroit, MI)
and heat-killed, freeze-dried Mycobacteria tuberculosis (Ministry of Agriculture, Fisheries & Food, Surrey, England). Each mouse received
300 ug/ml bovine type II collagen and 0.5 mg/ml complete Freund's adjuvant. Mice were
boosted intraperitoneally with 100 ug of bovine type II collagen on day 20. Disease
progression was monitored by a standardized visual scoring system with a scale from
0 to 12 reflecting the degree of swelling/redness of each paw (maximal score 3 per
paw) and the number of paws (maximal 4) involved per individual animal.
Histopathological analysis
After euthanasia, front and hind paws of the animals were dissected and fixed by immersion
in 10% phosphate-buffered formalin before decalcification. Following decalcification
with Cal-Rite (Richard Allen Scientific, Kalamazoo, MI), formalin fixed tissues were
processed and sectioned at 5um. Paraffin sections were stained with Hematoxylin and
Eosin (H&E). The criteria of histopathological analysis was carried out as described
[]. The changes in joint structures, including cartilage destruction, bone erosion/remodelling
and pannus formation were scored as follows: 0 = Normal, 1 = Minimal, 2 = Mild, 3
= Moderate, 4 = Marked, 5 = Severe. A grade of 5.5 was also added to address full-thickness
cartilage breach. Cellular infiltrates and inflammation in animal joints were scored
as the following: 0 = Normal, 1 = Minimal, 2 = Mild, 3 = Moderate, 4 = Marked.
Mouse experimental autoimmune encephalomyelitis
Female C57BL/6 mice were purchased from Jackson Laboratory (Bar Harbor, ME). For immunization,
150 μg MOG35-55 peptide prepared by Princeton Biomolecules (Langhorne, PA, USA) and
300 μg killed Mycobacterium tuberculosis (Difco, Detroit, MI) were mixed in CFA (Sigma-Aldrich,
St Louis, MO, USA) and injected s.c. in two 50-μl injections over the flanks on day
1. Also, 200 ng of pertussis toxin (Sigma-Aldrich, St Louis, MO) was injected i.v.
on days 0 and 2. The compound was administered orally twice daily. Dosing with the
compound started at day 0, 24 h prior to MOG35-55 immunization (day 1). Mice were
monitored daily and assessed for clinical signs of disease in a blinded fashion according
to the following criteria: 0, 1, 2, limp tail
an 3, 4, hind limb plu
and 5, moribund or dead. Cumulative clinical scores were calculated by adding daily
scores from the day of immunization until the end of the experiment. Mean clinical
scores at separate days and mean maximal scores were calculated by adding the scores
of individual mice and dividing with the number of mice in each group, including mice
not developing signs of EAE. All animals were used in accordance with protocols and
guidelines established by institute's Animal Care and Use Committee.
Rat experimental autoimmune encephalomyelitis
Male Lewis rats challenged by injection of 50 ul (30 mg) of a guinea pig spinal cord
homogenate in CFA into one footpad. The animals were treated starting at day 0 and
oral dosing continued throughout the 3-week disease course, with varying amounts of
SCH 546738 in 0.4% methylcellulose (MC) p.o. Animals were scored for disease severity:
0, 1, 2, 3, complete hind limb
4, complete hind limb paralysis, forelimb w 5, death.
Statistical analysis
For CIA and EAE models, unpaired t-tests were performed using GraphPad InStat version
5.0.1 for Windows 98, GraphPad Software, San Diego California USA ( ). Statistical significance was evaluated by comparing the vehicle-treated group with
the experimental group using unpaired t-test. Differences were considered significant
when p values were &0.05.
Cardiac transplantation in rats
Cardiac graft of ACI (RT1a) rats was heterotopically transplanted into the abdominal
cavity of Lewis (RT1l) recipients employing a microvascular surgical technique as described []. The grafts were monitored daily by abdominal palpation, and the complete cessation
of heart contraction was defined as graft rejection. SCH 546738 or 0.4% methylcellulose
(vehicle) was orally administered at the indicated dose (0.2 ml) twice a day, starting
on the day before transplantation until the day of graft rejection. To test whether
SCH 546738 enhanced the effect of conventional immunosuppressive reagent, the recipients
were received treatment with subtherapeutic dose of CsA for one week combined with
treatment with SCH 546738. Graft survival was analyzed using the log-rank test. The
parametric data were analyzed by Student t test (2-tailed) using GraphPad InStat version
5.0.1 for Windows 98, GraphPad Software, San Diego California USA ( ). p & 0.05 was considered statistically significant.
To identify CXCR3 antagonists, we have generated a mouse Pro-B cell line Ba/F3 stably
expressing a high level of human CXCR3 receptor. The membranes were prepared for establishing
a sensitive binding assay using [125I]hCXCL10 based on the scintillation proximity assay []. From high throughput screening of small molecule compound libraries, several lead
compounds were discovered []. Through the optimization of the lead compound, we have found SCH 546738 (compound
8a) [] to be a selective and potent CXCR3 antagonist with a good PK for in vivo studies.
Its structure is shown in Figure .
Affinity of SCH 546738 for CXCR3 receptor
The affinity of SCH 546738 binding to human CXCR3 receptor was determined by competition
binding analysis using 35S radiolabeled SCH 535390 (a sulfonamide analog of the CXCR3 compound series with
a Kd of 0.6 nM) as a competitive tracer. In multiple experiments, the affinity constant
(Ki) of SCH 546738 binding to human CXCR3 receptor was determined to be 0.4 nM (data
not shown).
Inhibition of CXCL10 and CXCL11 binding to CXCR3 receptor
Competition of human CXCL10 and CXCL11 binding to human CXCR3 by SCH 546738 was determined
at various concentrations of [125I]hCXCL10 and [125I]hCXCL11 around the Kd (50-100 pM) for the receptor. The IC50 of SCH 546738 is constant (~1 or 2 nM) and independent of the input concentrations
of either [125I]hCXCL10 (25-500 pM) or [125I]hCXCL11 (12.5-250 pM) (Figure ), respectively. These results indicate that SCH 546738 is a non-competitive antagonist
of both CXCL10 and CXCL11 binding to CXCR3, suggesting that SCH 546738 binds to CXCR3
receptor at an allosteric site and change its conformation which prevents the binding
of both CXCL10 and CXCL11.
Effect of concentrations of [125I]hCXCL10 and [125I]hCXCL11 on the IC50 of SCH 546738 binding to human CXCR3 receptor. The ability of SCH 546738 to compete the binding of [125I]hCXCL10 and [125I]hCXCL11 to human CXCR3 receptor was determined using various concentrations of [125I]hCXCL10 and [125I]hCXCL11 as described in Methods. After 3 hr reaction, specific counts relative to
input counts (B/B0) in the presence of increasing concentrations of SCH 546738 are plotted. The IC50 for each concentration of [125I]hCXCL10 and [125I]hCXCL11 are shown.
It is important to investigate species specificity of SCH 546738 to design in vivo
preclinical studies. As shown in Table , SCH 546738 has strong cross-species activities with IC50 of 1.3 nM, 6.4 nM, 5.9 nM and 4.2 nM in inhibiting the binding of [125I]hCXCL10 to CXCR3 of monkey, dog, mouse and rat origin, respectively.
Effect of SCH 546738 on CXCR3 from various species
Functional inhibition of CXCR3-mediated chemotaxis
The functional activity of SCH 546738 was investigated by CXCR3-mediated chemotaxis
assays using human activated T cells. SCH 546738 at fixed concentrations of 1, 10
or 100 nM was evaluated for its ability to inhibit human activated T cell chemotaxis
induced by various concentrations of the three CXCR3 ligands CXCL9, CXCL10 and CXCL11
and the CCR7 ligand CCL19 (MIP-3β). SCH 546738 at 10 nM inhibited T cell chemotaxis
induced by all three CXCR3 ligands about 90% (Figure ). In contrast, SCH 546738 did not affect T cell chemotaxis induced by the CCR7 ligand
CCL19. Furthermore, SCH 546738 inhibited T cell chemotaxis induced by the three CXCR3
ligand among all tested ligand concentrations in an insurmountable manner, suggesting
that SCH 546738 is a non-competitive antagonist, as has been characterized in the
competition binding analyses (Figure ). It is critical to have a nonocompetitive antagonist which will inhibit binding
of multiple endogenous ligands and inhibit its function (or activation) at any possible
high local concentration of the ligand in the disease stage.
Effect of SCH 546738 on human Activated T cell chemotaxis induced by CXCL10, CXCL9,
CXCL11 and CCL19. Human peripheral blood lymphocytes were prepared by Ficoll-Hypaque centrifugation,
depleted of monocytes, and stimulated by PHA/IL-2. After 7-9 days of stimulation,
activated T cell chemotaxis assay was carried out as described in Methods. The medium
containing 20% fetal bovine serum was used for all dilutions of chemokines and compounds.
Various concentrations of each indicated chemokine were added to the bottom wells
in the presence of fixed concentrations of SCH , 10, 100 nM) (added both
on the filter with cells and in the bottom wells). Chemotaxis of each sample is expressed
relative to spontaneous response (without chemokines) as chemotactic index.
Biochemical selectivity and pharmacokinetic properties
SCH 546738 was tested at concentrations of 1-10 μM against a panel of 49 GPCR binding
assays. Most of the assays were not affected by SCH 546738 (Table ). These results indicate that SCH 546738 is a highly selective antagonist of CXCR3.
In addition, SCH 546738 has a favourable pharmacokinetic profile in rodents. Figure
shows the plasma concentrations of SCH 546738 in Lewis rat and C57BL/6 mouse over
24 hr post-dose. The AUC (0-24 hr) is 7.7 μM.hr in Lewis rat @ 10 mg/kg (mpk) and
is 12.6 μM.hr in C57BL/6 mouse @ 30 mpk. Therefore, SCH 546738 is suitable for in
vivo preclinical studies.
GPCR counterscreens of SCH 546738
Plasma concentration versus time profiles of SCH 546738 in Lewis rat and C57BL/6 mouse. SCH 546738 in 0.4% methylcellulose was administered orally at 10 mg/kg (mpk) in
Lewis rats or 30 mpk in C57BL/6 mice. The plasma concentration of SCH 546738 in the
blood was calculated as the mean of 3 animals (n = 3) at indicated time points post-dose.
Their AUC from 0 to 24 hr is also calculated and indicated.
Administration of SCH 546738 attenuates disease in mouse collagen-induced arthritis
and protects joint structure
Collagen-induced arthritis (CIA) was induced in male B10.RIII mice by immunization
with bovine collagen type II (BC II) which resulted in the development of poly-arthritis
in the paw. Sixteen days later which was 4 days prior to receiving a BC II boost (day
-4); mice were randomized into treatment groups with approximately 10% of the animals
in each group having developed at least one swollen paw. Oral twice daily treatment
with SCH 546738 was initiated at this time (day -4) and continued through day 9, with
a BC II antigen boost on day 0. Figure
shows that SCH 546738 attenuated disease development in a dose-dependent fashion,
with significant reduction of the disease score evident at 40 mpk on days 4, 7 and
9, while it protected significantly on days 7 and 9 at 10 mpk. SCH 546738 administration
at 3 mpk had no statistically significant effect on disease severity.
SCH 546738 attenuates disease in mouse collagen-induced arthritis. SCH 546738 in 0.4% methylcellulose was administered orally twice daily at 3, 10
and 40 mg/kg (mpk). Dosing was initiated 16 days (day -4) postimmunization and continued
through day 9. The disease score was significantly decreased in SCH 546738-treated
animals as compared with vehicle-treated animals (* p & 0.05, two-tailed t test) at
day 4, 7 and 9.
Paws collected on day 9 from the vehicle and 40 mpk SCH 546738 groups of two independent
experiments were analyzed by histopathology. Statistical analysis of the combined
histopathology scores demonstrated that in animals treated with 40 mpk SCH 546738,
both leukocyte infiltration into the joint and the structural damage to the bone and
cartilage was significantly attenuated (Figure ). This data demonstrates that therapeutic treatment with a CXCR3 antagonist significantly
impairs the development of disease in an animal model of rheumatoid arthritis, and
supports the clinical development of SCH 546738 in this disease.
SCH 546738 in mCIA: Histopathological analysis of paws on day 9. Paws collected at day 9 from the vehicle (0.4% methylcellulose) and 40 mpk SCH 546738
groups of two independent experiments were analyzed by histopathology. (A) compares
the histopathological scores of both groups of tissues. Both leukocyte infiltration
into the joint and the structural damage to the bone and cartilage was significantly
attenuated in SCH 546738-treated animals (* p & 0.05; ** p & 0.005, two-tailed t test).
Example images of paw tissue sections collected from both groups of animals are shown
in (B) (top, bottom, tarsal area). Massive cellular infiltrates and
bone/cartilage erosions were evident in both tarsal and phalanges areas of the vehicle
treated mouse paw (left panels). In contrast, cellular infiltrates were mainly observed
in the phalanges region (right top), and rarely in the tarsal region (right bottom)
of SCH546738-treated animals.
Administration of SCH 546738 reduces disease in experimental autoimmune encephalomyelitis
Experimental autoimmune encephalomyelitis (EAE) is an animal model for human MS and
development of disease is dependent on T cell infiltration into the CNS. In the murine
model of EAE, SCH 546738 was tested in combination with interferon-β (IFN-β), a current
first-line therapeutic for the amelioration of relapsing-remitting MS. C57BL/6 mice
were primed by intravenous injection of pertussis toxin on day 0 and day 2. EAE was
induced on day 1 by subcutaneous injection of the myelin peptide MOG 35-55 emulsified
in CFA in the back of primed mice. Disease progression was monitored by a scoring
system as described in Methods. IFN-β administered at 1700 ng by daily intramuscular
injection significantly delayed disease onset and attenuated disease severity at peak
of disease compared to vehicle treated animals (Figure ). Similarly, SCH 546738 at 30 mpk orally twice daily delayed disease onset and attenuated
disease severity on days 17 and 19 (Figure ). Combination treatment with SCH 546738 and IFN-β had a significant additive effect
in delaying disease onset and attenuating disease severity compared to treatment with
either SCH 546738 or IFN-β alone (Figure ) suggesting that a CXCR3 antagonist may offer substantial 'add-on' efficacy onto
existing IFN-β therapy and further delay the occurrence of relapses in MS patients.
In addition, EAE was induced in Lewis rats by subcutaneous injections of guinea pig
spinal cord emulsified in CFA into one hind paw. SCH 546738 reduced the severity of
the disease in a dose-dependent manner as well (data not shown).
Combination of IFN-β therapy and CXCR3 inhibition has an additive effect on delaying
disease onset and attenuating disease severity in the mouse EAE model. IFN-β was administered at 1700 ng by daily intramuscular injection and SCH 546738
was orally twice daily at 30 mpk. The mouse EAE was conducted as described in Methods.
Treatment with either IFN-β or SCH 546738 alone or the combination significantly delayed
disease onset and attenuated disease severity (p & 0.05, two-tailed t test) at day
16, 17 and 19.
Inhibition of CXCR3 delays graft rejection and in combination with cyclosporine, permits
permanent engraftment
Published data demonstrated that in the CXCR3 knockout mouse rejection of cardiac
allografts was significantly delayed []. Based on this observation SCH 546738 was tested at various doses by twice daily
oral administration in a rat cardiac allograft model starting at the day of transplantation.
SCH 546738 significantly increased the mean survival time of the graft at 1 mpk (MST
= 11 days) when compared to the vehicle control (MST = 6 days), and further delayed
graft rejection at a dose of 5 mpk (MST = 14 days) (Figure ). Cyclosporine is the current gold standard in organ transplant therapies in human.
A cyclosporine dose response was conducted earlier in a rat cardiac allograft model
and 2.5 mpk of cyclosporine is a low and suboptimal dose (data not shown). Figure
shows that cyclosporine significantly delayed graft rejection in the rat model at
a daily suboptimal dose of 2.5 mpk and permitted the permanent engraftment of approximately
40% of the grafts (&100 days graft survival). In combination with 2.5 mpk cyclosporine
a suboptimal dose of 5 mpk SCH 546738 twice daily increased the rate of permanent
engraftment to 100% (Figure ). These data indicate that the selective inhibition of CXCR3 would have a beneficial
effect on allograft survival and may offer the possibility of reducing the dose of
cyclosporine used in patients, thereby limiting the potential for serious side effects.
SCH 546738 delays graft rejection and in combination with cyclosporine, permits permanent
engraftment in the rat cardiac allograft transplant model. SCH 546738 was administered orally twice daily at 1, 5 and 15 mpk. Cyclosporine
was administered daily at 2.5 mpk. In the combination study, 5 mpk SCH 546738 and
2.5 mpk cyclosporine were administered. SCH 546738 significantly increased the mean
survival time of the graft at 1 mpk (MST = 11 p & 0.05), 5 mpk (MST = 14
p & 0.05) and 15 mpk (MST = 14.9 p & 0.05) when compared with the vehicle control
(MST = 6 days). Graft survival was analyzed using the log-rank test.
Discussion
The CXCR3 receptor and its three interferon-inducible ligands (CXCL9, CXCL10 and CXCL11)
have been implicated in several Th1-mediated inflammatory diseases. Recently, the
efficacy of the anti-IP-10 antibody MDX-1100 reported in a phase 2 clinical trial
for RA [] reinforced the crucial role of the CXCL10-CXCR3 axis in this disease, and the therapeutic
potential of small molecule CXCR3 antagonists []. So far, only one of the CXCR3 antagonists, AMG487 (T487), progressed to Phase II
clinical trials but has been halted because of lack of efficacy. Since this may have
been due to variability in drug exposure, it is clear that this failure is not a misrepresentation
of CXCR3 as a drug target. In this regard, SCH 546738 is a small molecule non-competitive
CXCR3 antagonist with much higher affinity than AMG487 and therefore may have better
chance to achieve the in vivo efficacy.
In the mouse CIA model, SCH 546738 is efficacious in reducing disease development
by attenuating leukocyte infiltration into the joint and the structural damage to
the bone and cartilage. It is of interest to note that SCH 546738 demonstrated efficacy
even though dosing was started after the disease process was initiated and when mice
had already started to show signs of paw swelling. It was reported that T487 reduced
inflammation and cartilage damage in mouse and rat models of CIA []. In rat adjuvant arthritis, blockade of CXCR3 by anti-CXCR3 mAb significantly inhibits
T cell infiltration of arthritic joints and reduces the severity of arthritis []. All these data directly demonstrate an important role of CXCR3 in the development
of arthritis and CXCR3 blockade reduces the disease severity in the arthritis. It
is likely that small molecule CXCR3 antagonists may achieve the efficacy of the anti-IP-10
antibody MDX-1100 reported in a phase 2 clinical trial for RA.
The available functional data for the role of CXCR3 and its ligands in EAE are contradictory.
Different investigators have reported conflicting results when using IP-10-/- mice, anti-IP-10 antibody, anti-sense RNA and vaccines [,]. The recent results from CXCR3-/- mice show that CXCR3 is not required for the recruitment of immune cells to the CNS
in MOG-EAE. The work by Liu et al. [] showed exacerbation of EAE disease in CXCR3-/- mice and with neutralizing anti-CXCR3 Abs. It indicates that the exacerbation in the
CXCR3-/- mice correlates with enhanced effector T cell proliferation and reduced peripheral
and CNS expression of IFN-γ, but with no impact on leukocyte migration to CNS. A subsequent
study by Muller et al. [] showed that CXCR3-/- mice had more severe chronic disease with increased demyelination and axonal damage,
although the number of CD4+ and CD8+ T cells infiltrating the CNS were similar in
CXCR3-/- and wild type mice. In contrast to MOG-EAE, CXCR3 appears to promote the lymphocyte
accumulation inside the CNS in some virus-induced demyelinating disease models []. This may point to disease-specific functions of CXCR3 and its ligands, which can
vary depending on the nature of the pathogenic insult. These varied results probably
reflect the complex and perhaps divergent roles for the chemokine system in the pathogenesis
of EAE and virus-induced neuroinflammatory diseases. Recently, a nonspecific small
molecule antagonist of CCR5, CCR2 and CXCR3 (TAK-779) was reported to reduce incidence
and severity of EAE by decreasing migration of inflammatory cells into the CNS []. Our study is the first report that a specific small molecule CXCR3 antagonist SCH
546738 consistently inhibits both mouse and rat EAE clinical disease with no evidence
of exacerbation. Furthermore, combination of IFN-β therapy and CXCR3 inhibition has
an additive effect on delaying disease onset and attenuating disease severity in the
mouse EAE model. At least for small molecule antagonists including SCH 546738, the
beneficial effect of CXCR3 blockade has been observed in EAE. Maybe studies using
CXCR3-/- mice and neutralizing anti-CXCR3 Abs offer some hints as to other possible function
of CXCR3 receptor and its ligands. Beyond leukocyte recruitment, CXCR3 may modulate
T cell IFN-γ production, regulation between Th1 vs. Th17 cells, or control T cells
at the perivascular space in the CNS. It is not unlikely that a small molecule antagonist,
a neutralizing antibody or a genetic deletion can perturb a receptor's activity in
different ways, leading to different conclusion about the protein's biological function.
The role of CXCR3 in leukocyte recruitment was first demonstrated in the CXCR3 knockout
mouse in year 2000, where the rejection of a cardiac allograft was significantly delayed,
and resulted in permanent allograft engraftment with cyclosporine []. In addition, lack of CXCL10 in the graft led to prolonged allograft survival []. However, two recent studies published in 2008 [,] questioned the importance of CXCR3 in allograft rejection and found moderate to little
increase in graft survival using CXCR3-/- mice or small molecule CXCR3 antagonist MRL-957 and anti-CXCR3 antibody targeting
in human CXCR3 knock-in mice. These two studies conclude that CXCR3 is not essential
for leukocyte recruitment in the cardiac allograft rejection. In contrast, Uppaluri
et al. [] demonstrates that a CXCR3 blocking antibody significantly prolonged both cardiac
and islet allograft survival, and induced long-term graft survival greater than 100
days when combined with rapamycin. In 2009, one study shows that TAK-779 attenuates
cardiac allograft vasculopathy in part by reducing CCR5+ and CXCR3+ T lymphocyte subset infiltration into the graft []. The other study by Rosenblum et al. [] shows that small molecule CXCR3 antagonist AMG1237845 prolong
however, it does not inhibit leukocyte recruitment into the graft. The difference
in the contribution of CXCR3 to mouse allograft rejection observed in similar models
in different laboratories can not be explained by current data sets and additional
experiments are required to clarify these conflicting results.
In the rat cardiac allograft transplant model, a small molecule CXCR3 antagonist TLRK-A
was reported to prolong graft survival, but was active only in combination with cyclosporine
[]. However, another small molecule CXCR3 antagonist NIBR2130 did not prolong graft
survival []. In this study, we demonstrate that SCH 546738 delays graft rejection and in combination
with cyclosporine, permits permanent engraftment in the rat cardiac allograft transplant
In summary, our study demonstrates that administration of SCH 546738 attenuates disease
in mouse CIA, rat and mouse EAE, and rat cardiac allograft rejection. Combination
of IFN-β therapy and SCH 546738 has an additive effect in the mouse EAE model. Furthermore,
in combination with cyclosporine, SCH 546738 permits permanent engraftment in the
rat cardiac allograft transplant model.
The findings from our study and others indicate that targeting the CXCR3 receptor
by small molecule antagonists and antibodies can be a promising approach to RA. Since
the results from CXCR3 inhibition in EAE and allograft rejection remains contradictory,
we need to better understand the roles of the chemokine system operating in the pathogenesis
of EAE and allograft rejection that truly reflects the molecular mechanism in human
diseases and enhance the chance of success in human clinical trials.
Conclusions
In the present study, we describe the in vitro and in vivo pharmacological characterizations
of a novel and potent small molecule CXCR3 antagonist, SCH 546738. It binds to human
CXCR3 with an affinity of 0.4 nM, which is the most potent small molecule CXCR3 antagonist
reported so far. Competition binding studies show that SCH 546738 is able to displace
radiolabeled CXCL10 and CXCL11 from human CXCR3 with high affinity (IC50 ranged from 0.8 to 2.2 nM) in a non-competitive manner. In addition, SCH 546738 has
strong cross-species activity with IC50 of 1.3 nM, 5.9 nM, 4.2 nM and 6.4 nM for monkey, mouse, rat and dog CXCR3 receptor,
respectively. SCH 546738 potently and specifically inhibits CXCR3-mediated chemotaxis
in human activated T cells with IC90 about 10 nM. SCH 546738 has a favorable pharmacokinetic profile in rodents. We utilized
multiple preclinical disease models relevant to human rheumatoid arthritis, multiple
sclerosis, transplantation to assess in vivo efficacy of SCH 546738. We demonstrate
that SCH 546738 attenuates the disease development in mouse collagen-induced arthritis
model by decreasing both leukocyte infiltration into the joint and the structural
damage to the bone and cartilage. SCH 546738 also significantly reduces disease severity
in rat experimental autoimmune encephalomyelitis model, and in combination with IFN-β
in mouse experimental autoimmune encephalomyelitis model. Furthermore, SCH 546738
alone achieves dose-dependent prolongation of rat cardiac allograft survival. Most
significantly, SCH 546738 in combination with cyclosporine supports permanent engraftment.
Taken together, the results show that therapy with potent small molecule CXCR3 antagonists
may serve as a new strategy for treatment of autoimmune diseases, including rheumatoid
arthritis and multiple sclerosis, and to prevent transplant rejection.
Abbreviations
IP-10: IFN-γ inducible protein 10; MIG: monokine induced by IFN-γ; I-TAC: interferon-inducible
T cell a RA:
collagen- EAE: experimental autoimmu MOG: myelin
oligoden MIP-3β: macrophage inflammatory protein-3β; GPCR: G
protein-coupled receptor.
Competing interests
All the authors (except SQ) are employees of Merck and the work was funded by Merck.
Authors' contributions
CHJ performed radioligand binding assays, coordinated the study and wrote the manuscript.
MAC performed human activated T cell chemotaxis assay, cloned monkey and dog CXCR3
cDNAs and made membrane preparations. LC and EPR carried out mouse and rat experimental
autoimmune encephalomyelitis models. LS carried out mouse collagen-induced arthritis
model. SCC and DK performed histopathological analysis of samples from mouse collagen-induced
arthritis model. SQ carried out cardiac transplantation in rats. SHK, SR and JK discovered
and synthesized SCH 546738. JSF, PJZ and DL supervised animal models, participated
in the design of the study and supported the preparation of the manuscript. All authors
read and approved the final manuscript.
Acknowledgements
We would like to thank Drs. Inhou Chu, Satwant Narula and Maria Wiekowski for their
support and help.
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