In vitro and in vivo characterization of a novel CCR3 antagonist, YM-344031

BBRC Biochemical and Biophysical Research Communications 339 (2006) 1217–1223 www.elsevier.com/locate/ybbrc

In vitro and in vivo characterization of a novel CCR3 antagonist, YM-344031 Keiko Suzuki a,*, Tatsuaki Morokata a, Koichiro Morihira b, Ippei Sato b, Satoko Takizawa c, Masayuki Kaneko c, Koichiro Takahashi a, Yasuaki Shimizu a

a

Inflammation Research, Pharmacology Laboratories, Institute for Drug Discovery Research, Yamanouchi Pharmaceutical Co., Ltd., Tsukuba, Japan Chemistry Laboratory II, Chemistry Laboratories, Institute for Drug Discovery Research, Yamanouchi Pharmaceutical Co., Ltd., Tsukuba, Japan c Pharmaceutical Research Laboratories, Toray Industries, Inc., Kamakura, Japan

b

Received 17 November 2005 Available online 5 December 2005

Abstract Eosinophils play a prominent proinflammatory role in a broad range of diseases, including atopic dermatitis and asthma. Eotaxin-1 and its receptor CCR3 are implicated in the recruitment of eosinophils from blood into inflammatory tissues, therefore inhibition of Eotaxin-1/CCR3 interaction may have therapeutic potential for allergic inflammation with eosinophil infiltration. YM-344031, a novel and selective small molecule CCR3 antagonist, potently inhibited ligand binding (IC50 = 3.0 nM), ligand-induced Ca2+ flux (IC50 = 5.4 nM), and the chemotaxis of human CCR3-expressing cells (IC50 = 19.9 nM). YM-344031 (1–10 mg/kg) orally administered to cynomolgus monkeys significantly inhibited Eotaxin-1-induced eosinophil shape change in whole blood. Additionally, orally administered YM-344031 (100 mg/kg) prevented both immediate- and late-phase allergic skin reactions in a mouse allergy model. YM-344031 therefore has potential as a novel and orally available compound for the treatment of allergic inflammation, such as atopic dermatitis and asthma.
2005 Elsevier Inc. All rights reserved. Keywords: CCR3; Eotaxin-1; Eosinophil; Small molecule antagonist

Chemokines are a family of small, secreted proteins that control migration of leukocytes, such as monocytes, lymphocytes, eosinophils, and basophils. Eotaxin-1 is an inducible, secreted chemokine that promotes selective recruitment of eosinophils from the blood into inflammatory tissues [1–5]. CCR3, the receptor for Eotaxin-1, is highly expressed on eosinophils [5–7] and is responsible for mediating the biological effects of other eosinophil chemokines, such as Eotaxin-2, Eotaxin-3, MCP-3, MCP-4, and RANTES [8–10]. Expression of CCR3 has also been observed in basophils, mast cells, and Th2 lymphocytes [11–13]. This *

Corresponding author. Present address: Department of Immunology, Pharmacology Research Laboratories, Institute for Drug Discovery Research, Astellas Pharma Inc., 1-6, Kashima 2-Chome, Yodogawa-ku, Osaka 532-8514, Japan. Fax: +81 6 6304 5367. E-mail address: [email protected] (K. Suzuki). 0006-291X/$ - see front matter
2005 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2005.11.141

could explain the coordinated recruitment of these cell types to sites of allergic inflammation. Several clinical studies have suggested a pivotal role for CCR3 ligands/CCR3 in the pathogenesis of atopic dermatitis, asthma, and allergic rhinitis, all characterized by eosinophilic inflammation. An increase in mRNA and protein expression for Eotaxin-1 and CCR3 has been observed in skin lesions from atopic dermatitis [14]. Serum Eotaxin-1 level was significantly correlated with disease activity of atopic dermatitis [15]. Also, serum level of Eotaxin-3 was significantly higher in patients with atopic dermatitis, correlating well with the clinical severity [16]. Enhanced expression of Eotaxin-1 and CCR3, together with an accumulation of eosinophils, was also observed in asthmatics [17,18]. Moreover, preclinical studies using mice have corroborated a critical role for CCR3 and its ligands in allergic inflammation, in particular in the infiltration of

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eosinophils. Gonzalo et al. [19] demonstrated that mouse Eotaxin mRNA levels parallel the kinetics of eosinophil accumulation in lung during experimentally induced allergic inflammation. Targeted disruption of Eotaxin-1 significantly weakened antigen-induced tissue eosinophilia in mouse allergic models, such as experimental allergic airway diseases and onchocercal keratitis [20]. Also, a specific antibody against Eotaxin-1 reduced lung eosinophilia and bronchial hyperreactivity in mice after antigen exposure [21,22]. Recruitment of eosinophils to lung parenchyma and bronchoalveolar lavage fluid was severely impaired in CCR3-deficient mice, that fail to develop airway hyperresponsiveness following antigen inhalation [23]. At a gross level CCR3-deficient mice were normal, with normal T and B cell phenotypes and normal hematological parameters [23]. As the role of CCR3 appears to be largely restricted to the various aspects of eosinophil function and not related to other components of the immune system, blockade of CCR3 ligands and CCR3 interaction has potential as an effective and attractive approach for the treatment of inflammatory diseases caused by eosinophils. Here, a novel and small molecule selective CCR3 antagonist, YM-344031, is evaluated. YM-344031 potently inhibited the binding of Eotaxin-1 and RANTES to human CCR3-expressing cells, plus inhibited ligand-induced Ca2+ flux and chemotaxis. Furthermore, orally administered YM-344031 demonstrated an in vivo pharmacological effect in a mouse allergy model. These results indicate that YM-344031 has therapeutic potential for allergic inflammation such as atopic dermatitis and asthma. Materials and methods Reagents. YM-344031 (Fig. 1) was synthesized by Yamanouchi Pharmaceutical (Tsukuba, Japan). This compound was dissolved in dimethyl sulfoxide (DMSO) before use. All chemokines were purchased from Pepro Tech (Rocky Hill, NJ, USA). Cell culture reagents were obtained from Gibco (Rockville, MD, USA), and general laboratory reagents were purchased from Sigma (St. Louis, MO, USA). Cells and cell culture. All chemokine receptor expressing cells were generated as described previously [24]. Briefly, cDNA for human CCR1, 2, 3, 4, and CCR5 was generated using RT-PCR from human spleen mRNA (Becton–Dickinson, Franklin Lakes, NJ, USA). Amplified cDNA fragments were subcloned into pEF-BOS [25] and stably expressed in murine pre-B cell lymphoma B300-19 cells [26]. Each clonal line was screened for ligand-induced Ca2+ mobilization to identify the best responding cell line and maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, 100 lg/ml streptomycin, 50 lg/ ml b-mercaptoethanol, and 1 mg/ml G418. cDNA for murine and monkey CCR3 was obtained from mRNA of murine spleen (Becton–Dickinson) and monkey peripheral blood leuko-

Fig. 1. Chemical structure of YM-344031, N-{(3R)-1-[(6-fluoro-2-naphthyl)methyl]pyrrolidin-3-yl}-2-{1-[(3-methyl-1-oxidopyridin-2-yl)carbonyl]piperidin-4-ylidene}acetamide.

cytes, respectively. Amplified cDNA fragments for murine and monkey CCR3 were subcloned into pCR3.1 and pEF-BOS, and then stably expressed in CHO cells and B300-19 cells, respectively. Transfectants that expressed murine or monkey CCR3 were obtained and maintained in the same manner as human CCR3-expressing cells. Radioligand binding assays. [125I]-Eotaxin-1 and [125I]-RANTES were purchased from Amersham Pharmacia Biotech (Buckinghamshire, UK). Radioligand binding assays were performed using a scintillation proximity assay (SPA). Wheat germ agglutinin-coated SPA beads (1 mg; Amersham Pharmacia Biotech) in 100 ll of binding assay buffer [25 mM Hepes (pH 7.4), 1 mM CaCl2, 5 mM MgCl2, 0.5% bovine serum albumin (BSA), and 0.05% NaN3] were prepared in a 96-well microtiter plate. YM-344031 was dissolved in 100% DMSO solution and 1 ll of that solution was added to each well, then cells (2 · 105 cells, 50 ll) were also transferred to wells. Reactions were started by adding 50 ll of 100 pM [125I]-Eotaxin-1 or 50 pM [125I]-RANTES at room temperature. One hour later, radioactivity was counted using a Packard TopCount NXT plate reader (Packard, Meriden, CT, USA). Calcium mobilization experiments. Human CCR3-expressing B300-19 cells were loaded with 5 lM fura-2 acetoxymethyl ester in RPMI 1640 media containing 1% FBS for 30 min at 37 C in the dark. After washing twice, cells were diluted to 2 · 106 cells/ml with 20 mM Hepes buffer containing 0.1% BSA, 130 mM NaCl, 5.4 mM KCl, 1 mM MgCl2, 2.5 mM CaCl2, and 5.5 mM glucose. The cell suspension (490 ll) was then transferred to cuvettes. YM-344031 was dissolved in 100% DMSO solution, then 1 ll of the solution was added to a cuvette 1 min prior to the addition of 10 ll Eotaxin-1 (final concentration 3 nM) with constant agitation. Changes in fluorescence were monitored at 25 C using a spectrophotometer CAF-110 (Nihon Bunkoh, Tokyo, Japan) at excitation wavelengths of 340 nm and 380 nm, and an emission wavelength of 510 nm. Calculation of Ca2+ concentration was performed using Kd for Ca2+ binding at 224 nm. To assess the inhibitory effect of YM-344031 on other CCR3 ligands, Eotaxin-2 (3 nM), Eotaxin-3 (10 nM), MCP-4 (1 nM), and RANTES (10 nM) were used. To evaluate selectivity, human CCR1, CCR2, CCR4, and CCR5-expressing cells were loaded with fura-2 and stimulated with RANTES (6 nM), MCP-1 (6 nM), MDC (6 nM), and RANTES (6 nM), respectively. To assess species cross reactivity, murine or monkey CCR3-expressing cells loaded with fura-2 were stimulated with murine Eotaxin-1 (10 nM) or human Eotaxin-1 (10 nM). Chemotaxis assays. Measurement of chemotaxis using human CCR3expressing B300-19 cells was performed using a modified Boyden chamber procedure as described previously [27]. Briefly, lower chambers were filled with 33 ll of each chemokine, Eotaxin-1 (3 nM), Eotaxin-2 (10 nM), Eotaxin-3 (100 nM), MCP-4 (10 nM) or RANTES (100 nM), separated from the upper chamber by a polycarbonate filter with 5 lm pores (Neuro Probe, Gaithersburg, MD, USA). The upper chamber was filled with 200 ll of cell suspension (5 · 105 cells) in chemotaxis assay buffer (RPMI 1640 supplemented with 1 mg/ml BSA), containing 0.2% DMSO or YM-344031. Cell migration proceeded for 3 h at 37 C in a 5% CO2 incubator, after which the chamber was disassembled. The number of cells that migrated to the lower chamber was quantified by measuring ATP content using an ATPlite ATP detection system (Perkin Elmer, Wallesley, MA, USA). Eotaxin-1-induced shape change of eosinophils in monkey whole blood. Male cynomolgus monkeys, weighing approximately 5 kilograms, were orally administered vehicle (distilled water) or YM-344031 (1, 3, and 10 mg/kg). One hour later, a 2 ml sample of blood was taken and the reactivity of eosinophils to human Eotaxin-1 was assessed using a gated autofluorescence forward scatter (GAFS) assay, as previously described for human blood [28]. Briefly, 90 ll of fresh monkey blood was mixed with various concentrations of human Eotaxin-1 (10 ll) in 1.2 ml polypropylene tubes, incubated at 37 C for 10 min in a shaking water bath, and then transferred to ice. To preserve any change in shape for the eosinophils, cells were fixed in 250 ll of ice-cold fixative solution containing 2.5% CellFIX (Becton–Dickinson) and then left on ice for 1 min. Ice-cold lysis buffer (2 ml; 168 mM NH4Cl, 10 mM KHCO3) was added and then the mixture was left on ice for another 10 min to achieve red cell lysis. Samples were sorted by a FACSCalibur flow cytometer (Becton–Dickinson) and then data from 500 eosinophils, selected by high autofluorescence, were

K. Suzuki et al. / Biochemical and Biophysical Research Communications 339 (2006) 1217–1223

Potency, selectivity, and species cross reactivity In both the [125I]-Eotaxin-1 and [125I]-RANTES radioligand binding assays, binding to human CCR3-expressing B300-19 cells was inhibited by YM-344031 (Fig. 1) in a concentration-dependent manner, with IC50 values of 3.0 and 16.3 nM, respectively (Fig. 2A, Table 1). YM-344031 inhibition of the ligand-induced transient rise in intracellular Ca2+ concentration ([Ca2+]i) in cells expressing human CCR3 was examined. Response to CCR3 ligands, such as Eotaxin-1, Eotaxin-2, Eotaxin-3, MCP-4, and RANTES, was inhibited by YM-344031 with IC50 values of 5.4, 4.1, 3.5, 2.0, and 2.3 nM, respectively (Fig. 2B, Table 1). Potency of YM-344031 for inhibition of chemotaxis mediated by CCR3 was investigated further. Cells expressing human CCR3 readily caused migration toward all five CCR3 ligands. Chemotaxis induced by Eotaxin-1, Eotaxin-2, Eotaxin-3, MCP-4, and RANTES was strongly inhibited by YM-344031 with IC50 values of 19.9, 9.2, 17.4, 15.8, and 17.4 nM, respectively (Fig. 2C, Table 1). Taken together, these results suggest that YM-344031 is a potent and functional antagonist of CCR3. To determine the specificity of YM-344031 against CCR3, its ability to inhibit other chemokine receptor responses was assessed. At concentrations up to 10 lM, YM-344031 failed to significantly inhibit binding of [125I]RANTES to cells expressing CCR1 or CCR5 (data not shown). Similarly, up to 10 lM, there was no effect on Ca2+ mobilization via CCR1, CCR2, CCR4 or CCR5, which were induced by corresponding chemokines (Table 1). Therefore, YM-344031 is a minimum of 1000-fold more selective for CCR3 than other chemokine receptors tested. To examine species specificity of YM-344031, cells expressing monkey and murine CCR3 were produced. B300-19 cells expressing monkey and murine CCR3 dem-

% of Control Binding

80

60

40

20

0 -11

-10

-9

-8

-7

-6

-5

YM-344031 (log M) B

80

2+

Inf lux

100

% of Control Ca

Results

100

60

40

20

0 -11

-10

-9

-8

-7

-6

-5

-6

-5

YM-344031 (log M) C 120 % of Control Chemotaxis

acquired from each sample. The mean forward scatter for eosinophils in each sample was detected and a dose–response curve for human Eotaxin-1 generated. Measurement of skin reaction. Male Balb/c mice (6 weeks; n = 10; Charles River, Hamamatsu, Japan) were immunized by intraperitoneal injection with 1 lg ovalbumin (OVA) and 1 mg aluminum hydroxide gel (alum). At 14 days after immunization, both immunized and non-immunized mice were challenged by intradermal injection of 10 ll saline in the presence or absence of 10 lg OVA, to each ear. YM-344031 (10 and 100 mg/kg) was orally administered 1 h before, plus 8 and 16 h after antigen challenge. Prednisolone (10 mg/kg) or ketotifen (10 mg/kg) was given once at 1 h before OVA challenge. Ear thickness was measured using a dial thickness gauge (Mitsutoyo, Kanagawa, Japan) 1 and 24 h after challenge. The average increase in ear thickness at each point was estimated and results were expressed as ear swelling. Statistical analysis. Results were expressed as means ± standard error (SE). Either StudentÕs t test or DunnetÕs multiple range test was employed for evaluation of data. A value of p < 0.001, p < 0.01 or p < 0.05 was considered to be statically significant. Animal handling. All experiments were performed in accordance with regulations of the Animal Ethical Committee of Yamanouchi Pharmaceutical.

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100 80 60 40 20 0 -11

-10

-9

-8

-7

YM-344031 (log M) Fig. 2. CCR3 antagonistic activity of YM-344031 in vitro. (A) Displacement of 100 pM [125I]-Eotaxin and 50 pM [125I]-RANTES from human CCR3-expressing B300-19 cells with increasing concentrations of YM344031. (B) Inhibition of Ca2+ mobilization stimulated with various ligands in CCR3-B300-19 cells. (C) Inhibition of ligand-induced chemotaxis of CCR3-B300-19 cells. Data are means ± SE of multiple experiments (n = 3– 4). Chemokines are designated using the following symbols: Eotaxin-1 (d), Eotaxin-2 (m), Eotaxin-3 (j), MCP-4 (), and RANTES (.).

onstrated an increase in [Ca2+]i similar to that produced by human and murine Eotaxin-1, respectively. In this assay system, YM-344031 inhibited [Ca2+]i elevation via monkey

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Table 1 Selectivity of YM-344031 for chemokine receptors

Eotaxin-1 (CCR3) Eotaxin-2 (CCR3) Eotaxin-3 (CCR3) MCP-4 (CCR3) RANTES (CCR3) RANTES (CCR1) MCP-1 (CCR2) MDC (CCR4) RANTES (CCR5)

IC50 (nM) 2+

Binding

[Ca ]i

Chemotaxis

3.0 NT NT NT 16.3 >10,000 NT NT >10,000

5.4 4.1 3.5 2.0 2.3 >10,000 >10,000 >10,000 >10,000

19.9 9.2 17.4 15.8 17.4 NT NT NT NT

% of Eosinophil Shape Change

Ligand (receptor)

NT: not tested. Results are means of three experiments.

130 125 120 115

IC50 (nM) [Ca2+]i

Human Eotaxin-1 (Human CCR3) Human Eotaxin-1 (Monkey CCR3) Murine Eotaxin-1 (Murine CCR3)

5.4 9.9 74

Results are means of three experiments.

and murine CCR3 in a concentration-dependent fashion, with IC50 values of 9.9 and 74 nM, respectively (Table 2).

**

105

*** **

100 0

Table 2 Cross-reactivity of YM-344031 to cynomolgus monkey and murine CCR3 Ligand (receptor)

**

110

10 30 100 Eotaxin-1 (ng/ml)

300

Fig. 3. Inhibition of Eotaxin-1-induced eosinophil shape change in whole blood from cynomolgus monkey. Cynomolgus monkeys were orally administered placebo (s), 1 mg/kg (m), 3 mg/kg (j) or 10 mg/kg () YM344031. One hour after dosage, blood samples were collected and stimulated with human Eotaxin-1. Increase in eosinophil shape change in whole blood was determined by GAFS, as described in Materials and methods. Response to Eotaxin-1 is shown as percentage increase in basal FSC values and displayed as means ± SE (n = 5 separate monkeys). **p < 0.01, ***p < 0.001 compared to placebo group (DunnetÕs multiple range test).

Inhibition of eosinophil shape change in cynomolgus monkeys To assess CCR3 antagonist activity of YM-344031 at the whole-animal level, cynomolgus monkeys were orally administered YM-344031 (1–10 mg/kg). Collected whole blood samples were stimulated with human Eotaxin-1 and then analyzed in the GAFS assay to assess the ratio of shape change in eosinophils. As illustrated in Fig. 3, cynomolgus eosinophils from a placebo group exhibited a shape change response to human Eotaxin-1 (10–300 ng/ ml). However, eosinophils from YM-344031 (1–10 mg/kg) administered groups were significantly protected from 100 ng/ml Eotaxin-1-induced eosinophil shape change. YM-344031 (10 mg/kg) also prevented eosinophil response caused by 300 ng/ml of Eotaxin-1. Protection against allergic skin reaction in mice Leukocyte shape change is an essential process for leukocyte migration from the microcirculation into sites of inflammation. Effect of YM-344031 on antigen-induced cutaneous inflammation in mice was also examined. As potency of antagonistic activity of YM-344031 on murine CCR3 was about eight times weaker than monkey CCR3 (Table 2), YM-344031 was orally administered (10– 100 mg/kg). Abnormal behavior in animals was not observed at any of the tested doses. In this model, a biphasic response occurred 1 and 24 h after OVA challenge in OVA-immunized mice [29]. As shown in Table 3, 100 mg/ kg YM-344031 significantly prevented the development of both immediate-phase responses (IPR) and late-phase responses (LPR). In contrast, 10 mg/kg prednisolone

Table 3 Effect of YM-344031 on antigen-induced skin inflammation in mice Immunization Treatment

+ + + + +

mg/kg, po Ear swelling (·10 2mm)

— — Vehicle — YM-344031 10 YM-344031 100 Prednisolone 10 Ketotifen 10

IPR

LPR

2.80 ± 0.75 14.05 ± 0.99## 12.75 ± 0.42 10.05 ± 1.87* 11.65 ± 1.20 10.10 ± 0.33+

2.70 ± 0.80 8.40 ± 0.47## 6.30 ± 0.60* 2.55 ± 0.53** 2.65 ± 1.82$$ 6.30 ± 0.37+

##

p < 0.01 compared to the non-immunized group (StudentÕs t test); p < 0.01; *p < 0.05 compared to vehicle control (DunnetÕs multiple range test); $$p < 0.01, +p < 0.05 compared to vehicle control (StudentÕs t test).

**

strongly inhibited development of the late response; however, the effect on the early response was not significant. Ketotifen (10 mg/kg), an anti-allergy agent, weakened development of the early response as well as YM-344031, but its inhibitory effect on the late response was only partial. Discussion There has been intensive effort over the last 5 years to develop non-peptide, low molecular weight CCR3 antagonists for the treatment of inflammatory conditions, such as atopic dermatitis, asthma, and allergic rhinitis. To date, three groups have reported potent and selective small molecule antagonists of CCR3 [30–32], but they could not be used in in vivo experiments. Therefore, the usefulness of CCR3 antagonists has not been demonstrated at

K. Suzuki et al. / Biochemical and Biophysical Research Communications 339 (2006) 1217–1223

the whole animal level. Here, a novel and orally available CCR3 antagonist, YM-344031, is described. Since YM-344031 displaced radio-iodinated Eotaxin-1 and RANTES on CCR3-expressing cells, plus inhibited [Ca2+]i elevation and chemotaxis induced by various CCR3 ligands with similar potency, it is plausible that YM-344031 binds directly to CCR3, rather than to chemokines. YM-344031 was highly selective for CCR3 and did not inhibit any [Ca2+]i elevation via CCR1, CCR2, CCR4 or CCR5. As CCR1, CCR2, CCR4, and CCR5 are quite divergent to CCR3 at the amino acid sequence level, with only 62%, 47%, 41%, and 58% of amino acid homology [6,33], respectively, it is reasonable to conclude that YM344031 did not interact with other chemokine receptors. Recently, Zhang et al. [34] reported ligand-induced shape change for rhesus macaque eosinophils in whole blood using GAFS assay. This assay is based on the fact that eosinophils, like all leukocytes, undergo rapid cytoskeletal rearrangement upon chemokine receptor activation, resulting in a change in cellular morphology [35]. Here it was demonstrated that cynomolgus monkey eosinophils in whole blood also exhibited a shape change response to human Eotaxin-1 using GAFS assay. When whole blood from monkey administered YM-344031 was used, Eotaxin-1-induced eosinophil shape change was significantly reduced. This indicates that YM-344031 is orally bioavailable and exerts powerful CCR3 antagonistic activity even in whole blood, with its large number of blood cells and serum proteins. This ex vivo assay system is also suitable for estimation of pharmacodynamics of test compounds in human. When allergy patients are challenged with an allergen they have previously been sensitized with, they exhibit an immediate phase response (IPR), such as the appearance of wheals and flares on the skin and bronchoconstriction. Within several hours after IPR, they also often develop a delayed, more sustained local inflammation, known as the late-phase response (LPR). It is interesting to note that YM-344031 inhibited both immediate- and late-phase allergic cutaneous reaction to OVA in mice. The IPR is mainly due to chemical mediators, such as histamine and serotonin, released from mast cells. This was confirmed in a report demonstrating that IPR could not be induced in mast cell-deficient W/Wv mice [29]. Inhibition of IPR by the CCR3 antagonist probably reflects the fact that CCR3 is expressed on mast cells and YM-344031 inhibited the function of mast cells in vivo. However, LPR is characterized by local accumulation of activated inflammatory cells, including eosinophils, monocytes, and T lymphocytes [36]. Of these cells, eosinophils are of particular importance in terms of tissue damage [37]. Although histological examinations were not performed, accumulation of eosinophils and Th2 lymphocytes at the site of OVA challenge could be reduced by this compound. As LPR is important to inflammatory processes in allergic disease, including atopic dermatitis and asthma, YM-344031 has strong therapeutic

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potential. Potency of the antagonistic activity of YM344031 for human CCR3 is about 14 times stronger than murine CCR3 in vitro, therefore a lower dosage of YM344031 is likely to be more effective for human than mice. Recently, Cambridge Antibody Technology has developed bertilimumab (CAT-213), an anti-Eotaxin-1 monoclonal antibody, for potential treatment of allergic disorders, including asthma, conjunctivitis, and allergic rhinitis [38]. In Phase I/IIa clinical studies, bertilimumab was well tolerated, plus reduced allergen-induced infiltration of submucosal eosinophils, and mast cells in patients with seasonal allergic rhinitis. However, a monoclonal antibody directed against IL-5 [39–42] is possibly less efficient than an anti-Eotaxin-1 monoclonal antibody, as not only eosinophils, but also basophils, mast cells and Th2 lymphocytes are thought to participate in the pathogenesis of asthma and other allergic diseases. CCR3 antagonists can suppress not only the effect of Eotaxin-1, but also the effect of other chemokines, such as Eotaxin-2, Eotaxin-3, MCP-3, MCP4, and RANTES, but are still likely to be more potent than bertilimumab. In summary, YM-344031 is a potent, selective, and an orally active CCR3 antagonist. It blocks antigen-induced skin inflammation in both IPR and LPR. The pharmacological profile of YM-344031 indicates its potential utility as an effective oral therapy for atopic dermatitis and asthma. Further biological, pharmacokinetic, and pharmacotoxicity studies are currently being performed. References [1] P.J. Jose, D.A. Grififiths-Johnson, P.D. Collins, D.T. Walsh, R. Moqbel, N.R. Totty, O. Truong, J.J. Hsuan, T.J. Williams, Eotaxin: a potent eosinophil chemoattractant cytokine detected in a guinea-pig model of allergic airways inflammation, J. Exp. Med. 179 (1994) 881– 887. [2] P.J. Jose, I.M. Adcock, D.A. Griffiths-Johnson, N. Berkman, T.N.C. Wells, T.J. Williams, C.A. Power, Eotaxin: cloning of an eosinophil chemoattractant cytokine and increased mRNA expression in allergen-challenged guinea pig lungs, Biochem. Biophys. Res. Commun. 205 (1994) 788–794. [3] P.D. Ponath, S. Qin, D.J. Ringler, I. Clark-Lewis, J. Wang, N. Kassam, H. Smith, X. Shi, J.A. Gonzalo, W. Newman, J.C. Gutierrez-Ramos, C.R. Mackay, Cloning of the human eosinophil chemoattractant, eotaxin. Expression, receptor binding and functional properties suggest a mechanism for the selective recruitment of eosinophils, J. Clin. Invest. 97 (1996) 604–612. [4] E.A. Garcia-Zepeda, M.E. Rothenberg, R.T. Ownbey, J. Celestin, P. Leder, A.D. Luster, Human eotaxin is a specific chemoattractant for eosinophil cells and provides a new mechanism to explain tissue eosinophilia, Nat. Med. 2 (1996) 449–456. [5] M. Kitaura, T. Nakajima, T. Imai, S. Harada, C. Combadiere, H.L. Tiffany, P.M. Murphy, O. Yoshie, Molecular cloning of human eotaxin, an eosinophil-selective CC chemokine, and identification of a specific eosinophil eotaxin receptor, CC chemokine receptor 3, J. Biol. Chem. 271 (1996) 7725–7730. [6] P.D. Ponath, S. Qin, T.W. Post, J. Wang, L. Wu, N.P. Gerard, W. Newman, C. Gerard, C.R. Mackay, Molecular cloning and characterization of a human eotaxin receptor expressed selectively on eosinophils, J. Exp. Med. 183 (1996) 2437–2448. [7] B.L. Daugherty, S.J. Sicilliano, J. DeMartino, L. Malkowitz, A. Sirontino, M.S. Springer, Cloning, expression and characterization of

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