Identification and Characterization of a Potent ...

THE JOURNAL

OF

BIOLOGICAL CHEMISTRY

Vol. 275, No. 25, Issue of June 23, pp. 19000 –19008, 2000 Printed in U.S.A.

Identification and Characterization of a Potent, Selective, and Orally Active Antagonist of the CC Chemokine Receptor-1* Received for publication, February 11, 2000 Published, JBC Papers in Press, March 29, 2000, DOI 10.1074/jbc.M001222200

Meina Liang‡§, Cornell Mallari¶, Mary Rosser储, Howard P. Ng‡**, Karen May‡, Sean Monahan‡§§, John G. Bauman‡, Imadul Islam‡, Ameen Ghannam‡, Brad Buckman‡, Ken Shaw‡, Guo-Ping Wei‡, Wei Xu‡, Zuchun Zhao‡, Elena Ho¶, Jun Shen¶, Huynh Oanh¶, Babu Subramanyam¶, Ron Vergona¶, Dennis Taub¶¶, Laura Dunning‡, Susan Harvey储, R. Michael Snider‡, Joseph Hesselgesser储储, Michael M. Morrissey‡, H. Daniel Perez储储, and Richard Horuk储储 From the Departments of Discovery ‡Research, 储Biological Research, ¶Pharmacology, and 储储Immunology, Berlex Biosciences, Richmond, Calfornia 94804 and ¶¶Laboratory of Immunology, NIA, National Institutes of Health, Baltimore, Maryland 21224

It is clear that the inappropriate interaction of immune cells, such as T lymphocytes and monocytes, can lead to extensive inflammation and tissue destruction, which is a hallmark of several autoimmune diseases such as rheumatoid arthritis and multiple sclerosis. Immune cells are sent on their destructive journey by chemoattractant molecules known as chemokines,

* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. § To whom correspondence should be addressed: Meina Liang, Berlex Biosciences, Dept. of Molecular Pharmacology, 15049 San Pablo Ave., Richmond, CA 94804. Tel.: 510-669-4091; Fax: 510-262-7844; E-mail: [email protected]. **Current address: Coulter Pharmaceutical, South San Francisco, CA 94080. §§ Current address: Mirus Corp., Madison, WI 53719.

which interact with and signal through specific cell surface chemokine receptors. Chemokine receptors belong to the GPCR1 superfamily and have been viewed as attractive therapeutic targets by the pharmaceutical industry mainly because of their central role in regulating leukocyte trafficking. The premise that drugs that can inhibit the directed migration and activation of immune cells could be useful therapeutically has prompted the search for specific and highly potent chemokine receptor antagonists. Autoimmune diseases like multiple sclerosis and rheumatoid arthritis are characterized by interactions between invading T lymphocytes and tissue macrophages that result in extensive inflammation, tissue damage, and chronic disease pathologies. Numerous studies have demonstrated CCR1 expression in these cell types, and a variety of evidence provides strong in vivo concept validation for a role of this receptor in animal models of these diseases. For example, Karpus et al. (1, 2) were able to show in a mouse EAE model of multiple sclerosis that antibodies to MIP-1␣ prevented the development of both initial and relapsing paralytic disease as well as infiltration of mononuclear cells into the central nervous system. Treatment with MIP-1␣ antibody was also able to ameliorate the severity of ongoing disease. These results led the authors to conclude that MIP-1␣ plays an important role in this T-cell-mediated disease. Furthermore, in a recent study, Rottman et al. (3) were able to demonstrate in a mouse EAE model of multiple sclerosis that CCR1 knockout mice had a significantly reduced incidence of disease compared with wild type mice. The spinal cords of the wild type mice showed non-suppurative myelitis, whereas those from the CCR1 knockouts were minimally inflamed. Taken together these data strongly support the idea that CCR1 plays a role in the pathogenesis of EAE and furthermore suggest a role in the pathophysiology of the human disease, multiple sclerosis. From the data discussed above it is apparent that CCR1 antagonists could potentially be very important therapeutically in multiple sclerosis and in other autoimmune and in1 The abbreviations used are: GPCR, G-protein-coupled receptors; CCR1, CC chemokine receptor-1; MIP-1␣, macrophage inflammatory protein-1␣; MCP-3, monocyte chemotactic protein-3; MCP-1, monocyte chemotactic protein-1; EAE, experimental allergic encephalomyelitis; HEK293, human embryonic kidney cells; FLIPR, fluorometric imaging plate reader; FACScan, fluorescence-activated cell scanner; HPLC-MS, high pressure liquid chromatography-mass spectrometry; BX 471, R-N[5-chloro-2-[2-[4-[(4-fluorophenyl)methyl]-2-methyl-1-piperazinyl]-2oxoethoxy]phenyl]urea hydrochloric acid salt.

19000

This paper is available on line at http://www.jbc.org

Downloaded from http://www.jbc.org/ by guest on May 2, 2018

The CC chemokine receptor-1 (CCR1) is a prime therapeutic target for treating autoimmune diseases. Through high capacity screening followed by chemical optimization, we identified a novel non-peptide CCR1 antagonist, R-N-[5-chloro-2-[2-[4-[(4-fluorophenyl)methyl]-2-methyl-1-piperazinyl]-2-oxoethoxy]phenyl]urea hydrochloric acid salt (BX 471). Competition binding studies revealed that BX 471 was able to displace the CCR1 ligands macrophage inflammatory protein-1␣ (MIP-1␣), RANTES, and monocyte chemotactic protein-3 (MCP-3) with high affinity (Ki ranged from 1 nM to 5.5 nM). BX 471 was a potent functional antagonist based on its ability to inhibit a number of CCR1-mediated effects including Ca2ⴙ mobilization, increase in extracellular acidification rate, CD11b expression, and leukocyte migration. BX 471 demonstrated a greater than 10,000-fold selectivity for CCR1 compared with 28 G-protein-coupled receptors. Pharmacokinetic studies demonstrated that BX 471 was orally active with a bioavailability of 60% in dogs. Furthermore, BX 471 effectively reduces disease in a rat experimental allergic encephalomyelitis model of multiple sclerosis. This study is the first to demonstrate that a non-peptide chemokine receptor antagonist is efficacious in an animal model of an autoimmune disease. In summary, we have identified a potent, selective, and orally available CCR1 antagonist that may be useful in the treatment of chronic inflammatory diseases.

CCR1 Chemokine Receptor Antagonist

EXPERIMENTAL PROCEDURES

Materials—Unlabeled chemokines were from Peprotech (Rocky Hill, NJ). 125I-Labeled chemokines were obtained from NEN Life Science Products. CCR1 Expression Vectors—CCR1 cDNA was obtained as described (5) and inserted into a mammalian expression vector containing the SV40 replication origin, the human cytomegaloviral enhancer with the puromycin-N-acetyl-transferase gene (puromycin resistance) and hygromycin B gene (hygromycin resistance) similar to that described previously (6). Cell Lines—The human monocytic cell line THP-1, Jurkat cells, and the HEK293 cell line were obtained from the American Type Culture Collection and were cultured as described previously (4). For binding assays, cells were harvested and washed once with phosphate-buffered salt solution. Cell viability was assessed by trypan blue exclusion, and cell number was determined by counting the cells in a hemocytometer. CCR1 Expressing Cells—The transfection and selection of HEK293 cells stably expressing human CCR1 was as described previously (4). Chemokine Binding Studies—Binding assays were performed by filtration as described previously (4). Radiolabeled chemokines at a final concentration of approximately 0.1– 0.2 nM were used as ligand. HEK293 cells expressing human CCR1 at 8,000 or 300,000 cells per assay point were used as the receptor source. Nonspecific binding was determined in the presence of 100 nM unlabeled chemokine. The binding data were curve-fitted with the computer program IGOR (Wavemetrics) to determine the affinity and number of sites. Cytosolic Ca2⫹ Measurements—HEK293 cells expressing human CCR1 were plated on poly-D-lysine-coated black wall 96-well plates (Becton Dickinson, Franklin Lakes, NJ) at 80,000 cells/well and were cultured overnight. Cells were then loaded with 4 ␮M Fluo-3 (Molecular Probes, Eugene, OR), a calcium-sensitive fluorescence dye, for 60 min at 37 °C in Hanks’ balanced salts solution (Life Technologies, Inc.) containing 20 mM Hepes, 3.2 mM calcium chloride, 1% fetal bovine serum, 2.5 mM probenecid, and 0.04% pluronic acid. The excess dye was removed by gently washing cells 4 times with assay buffer (Hanks’ balanced salts solution containing 20 mM Hepes, 2.5 mM probenecid, and 0.1% bovine serum albumin) using a Denley washer (Labsystems, Franklin, MA). Changes in intracellular free Ca2⫹ concentration were measured with a FLIPR (Molecular Devices, Sunnyvale, CA) immediately after the addition of agonist at 37 °C. To examine the antagonistic activity of BX 471, the cells were pretreated with the compound for 15 min before the addition of agonist. The intracellular Ca2⫹ concentration in nM was calculated based on the equation Ca2⫹ ⫽ KD (F ⫺ Fmin)/(Fmax ⫺ F) (7). KD is the dissociation constant of the complex of Fluo-3 and Ca2⫹ (390 nM for Fluo-3). F is the measured fluorescence intensity. Fmax is the maximal fluorescence intensity determined in the presence of

0.1% triton X-100. Fmin is the minimum fluorescence intensity determined in the presence of 0.1% Triton X-100 plus 5 mM EGTA. Measurement of Extracellular Acidification in HEK Cells—Extracellular acidification was measured in a microphysiometer as described previously (4). Chemotaxis—Cell migration was examined using a 48-well microchemotaxis assay as described previously (8). CD11b Expression on Peripheral Blood Mononuclear Cells —CD11b expressed on peripheral blood mononuclear cells in a whole blood assay was measured as described (9). Briefly, human whole blood was collected by venipuncture into 2.5-ml Vacutainer tubes containing EDTA. The blood was kept at room temperature and used immediately after phlebotomy. The whole blood samples (200 ␮l) were pretreated with or without 1 ␮M BX 471 at 37 °C for 15 min followed by treatment with or without 100 nM MIP-1␣ for an additional 15 min. The reaction was terminated by the addition of 1 ml of cold phosphate-buffered salt solution wash. The tubes were centrifuged (200 ⫻ g for 7 min at 4 °C), and the supernatant was removed by aspiration. The cell pellet was resuspended in cold phosphate-buffered salt solution, 10 ␮l of 1 mg/ml heat-aggregated IgG was added, and the tubes were incubated for 10 min at 4 °C. Antibodies CD11b FITC (5 ␮l) and CD14 PE (20 ␮l) were added to each assay tube and incubated for 20 min at 4 °C. Finally, 1 ml of ice-cold phosphate-buffered salt solution was added, and the cells were pelleted as above and analyzed by FACScan (Becton Dickinson, San Jose, CA). Determination of Pharmacokinetic Parameters in Dogs—Fasted male beagle dogs (n ⫽ 3 per treatment group) were given BX 471 either by oral gavage or by intravenous injection via the cephalic vein at a dose of 4 mg/kg. The compound was dissolved in a vehicle of 40% aqueous cyclodextrin. Serial blood samples were collected utilizing an in-dwelling catheter in the jugular vein at the indicated time points up to 6 h post-dosing. EDTA was used as an anticoagulant. The samples were centrifuged (1000 ⫻ g for 10 min at 4 °C), and plasma was stored frozen until analyzed for drug levels by HPLC-MS (electrospray mode operated under a positive ion mode). Plasma samples were thawed and denatured by the addition of four parts of ice-cold methanol containing a fixed amount of an internal standard to one part of plasma. The resulting protein precipitate was removed by centrifugation at 5000 ⫻ g, and the supernatants were analyzed directly. Concurrently plasma calibration standards of BX 471 were prepared over the range of quantification, processed, and analyzed under identical conditions. A FISONS, VG Platform single quadrupole instrument was used in these analyses with an electrospray inlet operated at 3.57 kV. Chromatographic separation was accomplished using a YMC AQ octadecyl silane reversed phase column (4.6 ⫻ 250 mm) following a short isocratic elution method (35% methanol, 65% water containing 0.1% trifluoroacetic acid). The total column flow (1 ml/min) was split post-column to infuse 50 ␮l/min into the mass spectrometer. The chromatograms were collected over a total run time of 7.5 min/sample following a 50-␮l injection on the column. The ions were collected in a single ion positive ionization mode. A calibration curve for quantification was generated by plotting ion current ratios between the internal standard peak and the analyte in the plasma standards over the quantification range. Calculations of percent oral availability was deduced from the area under curve measurements. Pharmacokinetic parameters were calculated using WinNonLin version 3.0. WST-1 Staining—THP-1 and HEK293 cells expressing human CCR1 were incubated with various concentrations of BX 471 for 24 and 72 h followed by the addition of 10 ␮l of WST-1 (Roche Molecular Biochemicals) into 100 ␮l of culture medium. After 1 h of incubation with the dye at 37 °C, the optical density was measured by the SpectraMax plate reader at 440 nm. EAE Study in Lewis Rats—Male Lewis rats were immunized subcutaneously into both hind footpads with 50 ␮l of a guinea pig spinal cord homogenate. The guinea pig spinal cord homogenate was prepared by homogenizing guinea pig (male Hartley) whole spinal cords and adjusting the concentration to 1 g/ml with 0.9% physiological saline. This homogenate was then diluted (1:1) with complete Freund’s adjuvant containing 1 mg/ml Mycobacterium tuberculosis. One day after immunization the animals were injected subcutaneously 3 times/day with increasing doses of the CCR1 antagonist BX 471 (5, 20, and 50 mg/kg) dissolved in 40% cyclodextrin insaline solution or with the vehicle as a control. There were 10 animals per treatment group. Rats were weighed, and clinical symptoms were evaluated on a daily basis throughout the study and scored as follows: 0, no symptoms; 1, complete tail paralysis; 2, paraparesis, abnormal gait; 3, paralysis of one hind limb; 4, paralysis of both hind limbs; 5, moribund or dead. Clinical score


flammatory diseases in which target cells expressing the receptor play a role. For this reason we have established a program to develop highly potent and specific CCR1 antagonists. Recently we described a functional CCR1 antagonist belonging to the 4-hydroxypiperidine class (4). This molecule was shown to have a Ki of 40 nM for CCR1 and a 250-fold selectivity against other GPCR tested. In this report, we describe the discovery and pharmacological characterization of a novel, potent, and selective functional CCR1 antagonist. The compound, BX 471, is able to displace the CCR1 ligands MIP-1␣, RANTES, and MCP-3 with high affinity (Ki ranged from 1 nM to 5.5 nM) and is a potent functional antagonist based on a number of in vitro biological assays including the inhibition of Ca2⫹ mobilization, extracellular acidification rate, CD11b expression, and leukocyte migration. BX 471 demonstrated a greater than 10,000-fold selectivity for CCR1 versus other GPCR in both receptor binding assays and functional assays. Pharmacokinetic studies revealed that BX 471 had an oral bioavailability of 60% in dogs. Finally, even though BX 471 binds to rat CCR1 with a 100-fold reduced affinity (Ki ⫽ 121 ⫾ 60 nM), it is still able to effectively reduce disease in a rat EAE model of multiple sclerosis. The in vivo demonstration of efficacy for a CCR1-specific receptor antagonist could represent a major advance in the treatment of multiple sclerosis, where peripheral leukocyte recruitment and activation are critical to disease pathology.

19001

19002


FIG. 1. Structure of BX 471.

data were analyzed using an analysis of variance and Fisher’s least significant difference. RESULTS

FIG. 2. Radiolabeled human RANTES binding to human CCR1 was displaced by unlabeled RANTES, MIP-1␣ and BX 471. HEK293 cells expressing human CCR1 were incubated for 30 min at room temperature with 125I-RANTES in the presence of increasing concentrations of unlabeled RANTES, MIP-1␣ or BX 471. The binding reactions were terminated by filtration of cells through a GFB glass fiber filter soaked in 3% polyethyleneimine. The data shown are specific binding as a percentage of total binding ⫾ S.D. Specific binding was ⬃10% of total 125I-RANTES added. Total binding was approximately 2500 cpm, and nonspecific binding was approximately 800 cpm. The results shown in each case were from two separate studies. The inset shows a Scatchard plot for BX 471 displacement of 125I-RANTES binding to CCR1. TABLE I The displacement of radiolabeled MIP-1␣, RANTES, and MCP-3 binding to CCR1-transfected HEK293 cells by BX 471 HEK293 cells expressing human CCR1 were incubated with radiolabeled ligands MIP-1␣, RANTES, and MCP-3 in the presence of increasing concentrations of BX 471. The binding assay was terminated by filtration as described under “Experimental Procedures.” The Ki values were calculated by fitting the data with the computer program IGOR (wavemetrics) and presented as the mean of two experiments ⫾ S.E. Competing ligands

Ki ⫾ S.E. nM

MIP-1␣

1.0 ⫾ 0.03

RANTES

2.8 ⫾ 0.8

MCP-3

5.5 ⫾ 2.2

BX 471 and then stimulated with 100 nM MIP-1␣. The CCR1 antagonist inhibited CD11b up-regulation by MIP-1␣ by 100% and 65%, respectively (Fig. 6, B and C). Chemokines were originally defined and classified as potent leukocyte chemoattractants mediating their effects through GPCR like CCR1 (11). Thus, characterization of a putative CCR1 antagonist would be incomplete without demonstrable inhibition of leukocyte migration elicited by a chemokine stimulus. Fig. 7 shows that BX 471 is able to inhibit the directed migration of both human lymphocytes and monocytes in response to the CCR1 ligands MIP-1␣ and RANTES but has no effect on the CCR5 ligand MIP-1␤, the CCR2 ligand MCP-1, or the CXCR4 ligand stromal-derived factor 1␣. Thus, BX 471 is a potent inhibitor of leukocyte migration and is specific for the CCR1 receptor since it is unable to affect the directed migration of cells in response to various chemokine ligands for CCR5, CCR2, CXCR1, and CXCR4. It thus shows functional selectivity as well as functional antagonism. Schild Analysis of BX 471—The mechanism of the antagonistic effect of BX 471 was examined on CCR1-transfected cells by Schild analysis (12). The concentration-response curves for


BX 471 Displaced Ligand Binding to CCR1—Chemical optimization of compounds identified from high capacity screening of our compound libraries, using a CCR1 binding assay as described (4), yielded the lead compound, designated BX 471 (Fig. 1). In competition binding experiments with HEK293 cells expressing human CCR1, BX 471 was able to displace 125IRANTES binding in a concentration-dependent manner with a Ki of 2.8 nM, which is similar to the KD for RANTES of 1 nM (Fig. 2, Table I). In addition to RANTES, other ligands that bind to CCR1 with high affinity include MIP-1␣ and MCP-3. Competition binding studies showed that BX 471 could displace both 125I-MIP-1␣ and 125I-MCP-3 binding from human CCR1 with Ki values of 1 and 5.5 nM, respectively (Table I). These data demonstrated that BX 471 was a potent inhibitor of CCR1. Functional Antagonism for CCR1—To demonstrate that BX 471 is a functional antagonist for human CCR1, we measured the ability of the compound to inhibit agonist-induced Ca2⫹ mobilization in CCR1-expressing cells. As shown in Fig. 3, MIP-1␣ at 30 nM induced a rapid and transient increase in intracellular Ca2⫹ (Fig. 3, inset). BX 471 inhibited the Ca2⫹ transients induced by submaximal concentrations of CCR1 ligands, 30 nM MIP-1␣, 300 nM RANTES, and 100 nM MCP-3 in a concentration-dependent manner with IC50 values of 5, 2, and 6 nM, respectively, demonstrating functional antagonism for CCR1 (Fig. 3). When given alone, the compound did not induce Ca2⫹ transients, indicating that the compound has no intrinsic agonistic activity (data not shown). The reversibility of receptor inhibition by an antagonist is a desirable property for a therapeutic agent. The Ca2⫹ mobilization assay was used to test the reversibility of the BX 471 effect. HEK293 cells expressing human CCR1 were treated with 300 nM BX 471 for 30 min at 37 °C, and under these conditions CCR1 was completely inhibited. The compound was then removed from cells by 8 washes with media and assay buffer. The Ca2⫹ transients in response to 30 nM MIP-1␣ was then measured. As shown in Fig. 4, MIP-1␣ at 30 nM increased intracellular Ca2⫹ concentration, and the response was completely inhibited by 300 nM BX 471. After removal of BX 471, the MIP-1␣-induced Ca2⫹ transients recovered, indicating the reversibility of the CCR1 inhibition by BX 471. To further examine the functional antagonism of BX 471, we measured its ability to inhibit the extracellular acidification rate in response to MIP-1␣ in THP-1 cells using the microphysiometer (10). As shown in Fig. 5, MIP-1␣ induced a rapid increase in the extracellular acidification rate, reaching a maximum after about 2 min and returning close to base-line levels within 8 min. The response elicited by MIP-1␣ was inhibited by BX 471 in a concentration-dependent manner with an IC50 of approximately 1 nM (Fig. 5). As an additional measurement of the functional antagonism of BX 471 for CCR1, the expression of the integrin CD11b on monocytes was measured in a whole blood assay using FACScan analysis (Fig. 6). MIP-1␣ dose responsively induced the expression of CD11b on monocytes with an EC50 of 110 nM (Fig. 6A). Blood from two separate donors was incubated with 1 ␮M


FIG. 4. Reversibility of BX 471 inhibition on CCR1-mediated increase in Ca2ⴙ transients on HEK293 cells expressing human CCR1. Fluo-3-loaded cells were pretreated with or without 300 nM BX 471 for 30 min during loading. After 30 min of pretreatment, BX 471 was removed by rinsing cells 4 times with media at 37 °C followed by rinsing 4 times with assay buffer. Some of the untreated cells were incubated with 300 nM BX 471 for 15 min. Then all the cells were stimulated with or without 30 nM MIP-1␣. The changes in fluorescence representing the changes in Ca2⫹ concentration were measured by FLIPR at 37 °C. Data shown were average fluorescence counts (percent of the counts in the absence of BX 471) ⫾ S.D.

Ca2⫹ transients induced by MIP-1␣ in the presence of increasing concentrations of BX 471 were determined (Fig. 8). BX 471 shifted the concentration-response curves to the right. However, the CCR1 inhibition by BX 471 could be overcome by increasing concentrations of MIP-1␣, suggesting surmountable antagonism (Fig. 8A). After transformation of the data, a linear Schild plot was generated with a slope of 1.29, which is not significantly different from unity (Fig. 8B). The pA2 value of 8.45 was obtained from the intercept on the x axis. The deduced Ki of 3.5 nM from the Schild plot is consistent with the Ki predicted from the receptor binding assay (Fig. 2 and Table I). Selectivity of BX 471 for CCR1—Since CCR1 belongs to a large family of GPCR that numbers well over 450 members (13), it is important to determine the specificity of the CCR1 antagonist to establish its therapeutic utility. The selectivity of BX 471 was thus tested for inhibition of radioligand binding against a panel of 28 GPCR, including related chemokine receptors. Although BX 471 had a Ki of inhibition for CCR1

FIG. 5. The CCR1 antagonist, BX 471, inhibits the ability of MIP-1␣ to increase the extracellular acidification rate in THP-1 cells. Cells were pretreated with increasing concentrations of BX 471 for 30 min and then stimulated with 1 nM MIP-1␣. The increase in acidification rate was monitored using a microphysiometer. Data have been normalized as percentage biological response. Data shown are representative of at least three separate studies.

ranging from 1 to 5.5 nM (Table I), it had less than 50% inhibitory activity for all receptors tested at a concentration of 10 ␮M (Table II). These data indicated that BX 471 had a greater than 10,000-fold selectivity for CCR1 compared with all other GPCR tested. Effect of BX 471 on General Toxicity—To demonstrate that CCR1 antagonism by BX 471 was not due to the cellular toxicity of the compound, THP-1- or CCR1-transfected HEK293 cells were treated with BX 471 at concentrations up to 10 ␮M for 24 h, and cellular toxicity was monitored by measuring WST-1 staining. No significant toxicity was observed (data not shown). The toxicity for BX 471 was further examined in vivo by a battery of serum diagnostic tests including hepatic and renal function tests and blood electrolytes on rabbits that had been dosed with BX 471 at 20 mg/kg/day for 30 days. The test results all fell within the normal range (data not shown). The results suggest that the inhibition of BX 471 on CCR1 activation was not due to cellular toxicity and that chronic treatment with the drug had no adverse effects on the normal physiology of the animals. Pharmacokinetics of BX 471 in Dogs—The oral bioavailability of BX 471 was examined in conscious dogs. BX 471 was administered to fasted male beagle dogs at 4 mg/kg in a vehicle of 40% cyclodextrin in saline by bolus intravenous injection via the cephalic vein or by oral gavage. The plasma samples were prepared, and compound concentrations in the plasma were determined by HPLC-MS. As shown in Fig. 9, BX 471 reached peak plasma levels approximately 2 h after oral dosing and maintained measurable concentrations for up to 6 h. BX 471 exhibits a volume of distribution (0.5 l/kg) close to the volume of body water (0.6 l/kg), suggesting that the compound is confined primarily to the aqueous volume (Table III). Low clearance, 2 ml/min/kg (which represents less than 10% of the total liver blood flow) in the dog resulted in a moderate terminal half-life of 3 h (Fig. 9 and Table III). For dogs that were orally dosed, the half-life for BX 471 was approximately 3 h. Calculations of percent oral availability using area under curve measurements obtained from analysis using TOPFIT software indicated that BX 471 is an orally absorbed drug in fasted dogs with an oral bioavailability of approximately 60% (Fig. 9 and Table III). Efficacy of BX 471 in a Rat EAE Model of Multiple Sclerosis—Before determining the efficacy of BX 471 in a rat EAE model of multiple sclerosis, we tested its ability to inhibit MIP-1␣ binding to rat CCR1 receptors. Scatchard analysis of


FIG. 3. BX 471 inhibited the ability of CCR1 agonists MIP-1␣, RANTES, and MCP-3 to increase Ca2ⴙ transients in HEK293 cells expressing human CCR1. Fluo-3-loaded cells were pretreated with increasing concentrations of BX 471 for 15 min and then stimulated with CCR1 agonists MIP-1␣ (30 nM), RANTES (300 nM), and MCP-3 (100 nM). The changes in fluorescence representing the changes in Ca2⫹ concentration were measured by FLIPR at 37 °C. Data shown were average fluorescence counts (percent of the counts in the absence of BX 471) ⫾ S.D. The inset graph shows the time course of the MIP-1␣-induced increase of intracellular Ca2⫹ concentration. The Ca2⫹ concentration was shown as nM, calculated as described under “Experimental Procedures.”

19003

19004


FIG. 6. BX 471 inhibits the MIP-1␣-induced up-regulation of CD11b in human blood. A, MIP-1␣ dose-response curve. Human whole blood was collected and pretreated with increasing concentrations of MIP-1␣ at 37 °C for 15 min. The reaction was terminated, and the expression of CD11b was determined by FACScan analysis using FITC-labeled antibody against CD11b. B and C, inhibition of MIP-1␣-induced CD11b up-regulation by BX 471. Human whole blood from two separate donors was collected and pretreated with or without 1 ␮M BX 471. The blood was then stimulated with or without 100 nM MIP-1␣. Data are expressed as fluorescence intensity.

DISCUSSION

FIG. 7. The CCR1 antagonist, BX 471, inhibits chemotaxis of human lymphocytes and monocytes induced by MIP-1␣ and RANTES. A, lymphocytes were examined for their ability to migrate in response to various CC and CXC chemokines (100 nM) in the presence or absence of the CCR1 antagonist BX 471 (100 nM) as described under “Experimental Procedures.” B, monocytes were examined for their ability to migrate in response to various CC and CXC chemokines (100 nM) in the presence or absence of the CCR1 antagonist BX 471 (100 nM) as described under “Experimental Procedures.” The results shown are the mean of three experiments ⫾ S.E. HPF, high power field.

competition binding studies with BX 471 demonstrated that the compound was able to inhibit chemokine binding to rat CCR1 with a Ki of 121 ⫾ 60 nM (data not shown), which was approximately 100 times less effective for rat CCR1 than for human CCR1. In addition, BX 471 does not inhibit chemokine

Increasing evidence suggests a role for the chemokines MIP-1␣ and RANTES in autoimmune diseases like multiple sclerosis and rheumatoid arthritis (1, 2, 14, 15) and in organ transplant rejection (16 –18). A receptor for these chemokines, CCR1, could therefore be a major therapeutic target. Indeed, recent studies with CCR1 knockout mice in animal models of multiple sclerosis (3) and organ transplant rejection (19) have implicated this receptor in the pathogenesis of these diseases. Based on these data, we established a program to discover potent, selective, small molecule antagonists of CCR1 for the treatment of autoimmune diseases and organ transplant rejection. The approach to identify CCR1 antagonists was to screen our compound library with a high throughput 125I-MIP-1␣ binding assay (4). The compounds identified from screening were chemically optimized, and this led to the discovery of BX 471 (Fig. 1). BX 471 was a potent inhibitor of chemokine binding to CCR1 (Fig. 2 and Table I) and inhibited the activation of CCR1


binding to rat CCR5 (data not shown) and is thus specific for rat CCR1. From pharmacokinetic studies in rats, we determined that the subcutaneous administration of BX 471 three times a day would give blood drug levels of 1 to 5 ␮M (data not shown), which we calculated would be about 10 –50 times the Ki on rat CCR1 receptors and should be sufficient for inhibition of MIP-1␣ binding. Unfortunately in contrast to the pharmacokinetic data obtained in the dog, BX 471 was poorly orally available in the rat (⬍20%, data not shown). Based on these studies, a rat EAE model of multiple sclerosis was set up. Animals were dosed subcutaneously three times a day with vehicle or with BX 471 at 5, 20, and 50 mg/kg. The CCR1 antagonist, BX 471, dose-dependently decreased the severity of the disease (Fig. 10A). At the highest dose, 50 mg/kg, there was a marked reduction in the clinical score that was statistically significant at p ⫽ 0.05 (analyzed by analysis of variance) compared with the vehicle control. However, even at the lower two doses of BX 471, 20 mg/kg and 5 mg/kg, there were still noticeable decreases in the clinical score. This is more readily observed by expressing the data as the average accumulated clinical score per treatment group (Fig. 10B). Statistical analysis of the average accumulated clinical score data by t test revealed that the 50 and 20 mg/kg doses were statistically significant compared with the vehicle control p ⫽ 0.003 and p ⫽ 0.014, respectively.


measured by Ca2⫹ mobilization, extracellular acidification, CD11b expression, and cell migration (Figs. 3, 5, 6, and 7). The direct mechanism by which BX 471 inhibits the activation of human CCR1 has not yet been established. However, based on our studies, we favor the idea that the compound directly binds to CCR1, leading to the blockade of receptor activation. MIP-1␣ was able to bind to both CCR1 and CCR5 with high affinity. However, BX 471 was able to inhibit the binding and activation of CCR1 but not CCR5. Therefore, it is unlikely that BX 471 inhibits CCR1 by direct binding to the CCR1 ligand, MIP-1␣. In addition, BX 471 did not demonstrate toxicity either in vitro or in vivo. Furthermore, BX 471 showed greater than a 10,000-fold selectivity for CCR1 when tested in 28 other GPCR binding assays and in cell migration assays in response to MIP-1␤, MCP-1, interleukin-8, and stromal-derived factor 1␣. These data suggest that CCR1 inhibition by BX 471 is not due to either cellular toxicity or nonspecific interaction with other cell surface receptors. Furthermore, a radiolabeled active analogue of BX 471 was able to bind to CCR1 with

high affinity (data not shown). The antagonism of CCR1 by BX 471 appeared to be reversible and surmountable (Figs. 4 and 8). The data is consistent with competitive antagonism. Mechanistically, competitive antagonism is observed when an antagonist binds to the same site on a receptor as the agonist but is incapable of activating the receptor, therefore preventing receptor binding and subsequent activation of the receptor by its agonist. In addition, similar Schild data could also be obtained if the antagonist were to bind to a separate but interacting site on the receptor from the agonist binding site. Studies with a number of GPCR have revealed that the receptor binding sites for antagonists can be distinct from the agonist binding sites (20 –22). For example, Gether et al. (21) demonstrate that non-conserved residues in transmembrane segments V and VI were essential for the binding of the neurokinin-1 receptor antagonist, CP-96345, to its receptor but were not important for the binding of the natural peptide ligand, substance P. Since the chemical structure of BX 471 does not resemble the natural ligands of CCR1, it is tempting to speculate that BX 471 binds to separate or overlapping sites on CCR1 rather than to those involved in chemokine binding. Although peptide antagonists of chemokine receptors have been described (23, 24), their sub-optimal metabolic stability and oral bioavailability have limited their therapeutic utility. Non-peptide antagonists could potentially overcome these disadvantages. Studies show that BX 471 was 60% orally available when tested in conscious dogs. It appeared to be relatively stable in plasma with a clearance rate of 2 ml/min/kg and maintained a measurable level for at least 6 h (Fig. 9 and Table III). Therefore, if these profiles of BX 471 are confirmed in humans, this compound would provide a better alternative as a therapeutic candidate than the peptide antagonists reported. We have previously identified a small molecule CCR1 antagonist, 2–2-diphenyl-5-(4-chlorophenyl)piperidin-1-yl)valeronitrile (4, 25). In addition, a patent filed by Takeda Chemical Industries, Ltd. reported a CCR1 antagonist, a urea piperidine derivative of the nitrile group, that is structurally similar to our 4-hydroxypiperidine series (26). This compound, however, demonstrated potential cross-reactivity with several biogenic amine neurotransmitter receptors. Furthermore, Banyu Pharmaceutical Co. Ltd. has recently reported small molecule CCR1 antagonists (27). This patent disclosure reports a group of tricyclic amides that inhibited receptor binding with an IC50 of 1.8 nM. These compounds were also reported to inhibit binding to CCR3 with a similar IC50 (1.7 nM), making them less specific than the Berlex compound. The fact that these compounds are quaternary ammonium salts may further limit their therapeutic use due to potential pharmacokinetic problems such as poor oral absorption and rapid elimination. Thus, based on these studies, BX 471 appears to be more highly specific and superior on pharmacological and pharmacokinetic grounds to the other reported CCR1 antagonists. Multiple sclerosis is an autoimmune disease mediated by extensive infiltration of T lymphocytes and monocytes into the central nervous system followed by resident macrophage and microglia activation. This results in an extensive inflammation and subsequent demyelination of the white matter in the central nervous system (28). Although the mechanisms responsible for causing this immunologic damage in the central nervous system are still unknown, regulation of the recruitment of activated immune cells and the extravasation of these cells into the central nervous system has been readily studied. Chemokine-induced recruitment of leukocyte subsets clearly plays roles in chronic inflammatory diseases and has been implicated in the pathogenesis of multiple sclerosis (29 –31). Based on


FIG. 8. Concentration-response curves for the ability of MIP-1␣ to increase Ca2ⴙ transients in HEK293 cells expressing human CCR1 in the presence of increasing concentrations of BX 471. A, Fluo-3-loaded cells were pretreated with increasing concentrations of BX 471 for 15 min followed by increasing concentrations of MIP-1␣. The changes in fluorescence representing the changes in Ca2⫹ concentration were measured by FLIPR at 37 °C. Data shown were average fluorescence counts. B, Schild plot of transformed data from A. The results shown are representative of three separate studies.

19005

19006


TABLE II Specificity of BX 471 binding to GPCR BX 471 was tested in receptor binding assays on a number of GPCR. All receptors were expressed in HEK293 cells except for the following: CCR5 in human peripheral blood mononuclear cells; CXCR4 in Jurkat, adrenergic ␤2 in NBR1 cells, bradykinin in HS729 cells, endothelin in Chinese hamster ovary cells, muscarinic M1 and M2 in Sf9 insect cells. Binding assays were carried out in whole cells at 4 °C and at compound concentrations of 1 and 10 ␮M. DARC, Duffy antigen receptor for chemokines; Me, methyl; SDF1␣-stromal-derived factor 1␣; AB-MECA, 4-aminobenzyl-5⬘-N-methylcarboxamidoadenosine; NMS, N-methyl-scopolamine; 8-OH-DPAT, 8-hydroxy-2-(di-n-propyl-amino)-tertralin; MGSA, melanoma growth stimulatory activity. % Inhibition of Binding Receptor

a

125

[ I]AB-MECA [3H]Prazosin [3H]MK912 [3H]MK912 125 I-cyanopindolol [3H]des-Arg10 kallidin 3 [ H]WIN-55,212–2 [3H]Me-N(⫾)L364,718 [3H]SCH23390 [3H]Spiperone [3H]Spiperone [3H]Spiperone [3H]SCH23390 [125I]Endothelin-1 [3H]LTB4 [3H]NMS [3H]NMS [3H]NMS [3H]NMS [3H]NMS [3H]SR-140333 [125I]neuropeptide Y [3H]8-OH-DPAT [3H]NMS [125I]interleukin-8 [125I]SDF-1␣ [125I]MIP-1␤ [125I]MGSA

Tissue

Human Rat Human Human Human Human Human Human Human Human Human Human Human Human Human Human Human Human Human Human Human Human Human Human Human Human Human Human

10 ␮M

1 ␮M

Selectivitya

38 ⫺7 24 22 1 ⫺11 13 16 13 9 17 21 23 8 14 ⫺7 21 ⫺8 15 18 2 19 8 19 ⫺4 5 2 6

⫺6 5 ⫺8 21 2 ⫺9 23 ⫺14 ⫺2 6 21 2 29 ⫺15 ⫺13 ⫺9 12 7 15 11 5 12 4 19 ⫺2 1 3 ⫺3

⬎10,000 ⬎10,000 ⬎10,000 ⬎10,000 ⬎10,000 ⬎10,000 ⬎10,000 ⬎10,000 ⬎10,000 ⬎10,000 ⬎10,000 ⬎10,000 ⬎10,000 ⬎10,000 ⬎10,000 ⬎10,000 ⬎10,000 ⬎10,000 ⬎10,000 ⬎10,000 ⬎10,000 ⬎10,000 ⬎10,000 ⬎10,000 ⬎10,000 ⬎10,000 ⬎10,000 ⬎10,000

Represents Ki BX 471 on test receptor/Ki BX 471 on CCR1. TABLE III Pharmacokinetic parameters for BX 471 in dog Absolute bioavailability (%F) was estimated to be 60%. Pharmacokinetic parameters were determined using WinNonLin version 3.0 software. AUC, area under curve. Pharmacokinetic parameters

Terminal plasma half-life AUC (0–6 h) AUC (0–8 extrapolated) Volume of distribution (Vz) Clearance rate (Clt)

FIG. 9. Time course of plasma concentrations of BX 471 after dosing dogs intravenously or orally. Fasted male beagle dogs (n ⫽ 3 per treatment group) were administered BX 471 by either oral gavage (po) or intravenous injection (iv) via the cephalic vein at a dose of 4 mg/kg. Serial blood samples were collected at the indicated time points up to 6 h post-drug. The samples were analyzed for drug levels by HPLC-MS. Data are shown as plasma concentrations of BX 471 at the indicated time points post-drug. Calculations of percent oral availability using area under curve measurements were obtained from analysis using TOPFIT software.

chemokine antibody neutralization experiments, there is evidence for a role of MIP-1␣ in animal models of multiple sclerosis (1, 2). Although recruitment into the vasculature of the brain is likely to be directed by these chemoattractant proteins, leukocyte activation also takes place and results in the upregulation of adhesion molecules. Neutralization of leukocyte integrins has demonstrated an ability to inhibit or prevent EAE in rodent models (32–35) and has even been proposed as

Intravenous

3h 27 ␮g ⫻ h/ml 39 ␮g ⫻ h/ml 0.5 l/kg 2 ml/min/kg

Oral

3h 16 ␮g ⫻ h/ml 21 ␮g ⫻ h/ml

a possible therapy in the treatment of multiple sclerosis (36). Since we have data showing that BX 471 is able to inhibit the MIP-1␣-induced up-regulation of adhesion molecules like CD11b on primary human leukocytes (Fig. 6), we rationalized that the CCR1 antagonist BX 471 could be effective in an EAE animal model of multiple sclerosis. The CCR1 antagonist, BX 471, at 50 mg/kg gives a 50% reduction of clinical score in the rat EAE model (Fig. 10). The much higher doses of BX 471 that are required to be effective in rat EAE could be due to the fact that the compound has a Ki for inhibition of MIP-1␣ binding for rat CCR1 of 121 nM, compared with a compound Ki of 1–2 nM for human CCR1. Based on these considerations, it is likely that much lower doses of BX 471 (500 ␮g/kg or less) would be required to be therapeutically effective in treating multiple sclerosis in humans. The complex pathology of the development of multiple sclerosis demonstrates the involvement of several factors that are both peripheral and central nervous system-specific. Clearly, inhibition of peripheral leukocyte activation and recruitment can effect the development of central nervous system pathologies. Inhibition of central nervous system-specific glial activa-


Adenosine A3 Adrenergic ␣1a Adrenergic ␣2a Adrenergic ␣2c Adrenergic ␤1 Bradykinin B1 Cannabinoid Cholecystokinin Dopamine D1 Dopamine D2 Dopamine D3 Dopamine D4.2 Dopamine D5 Endothelin B Leukotriene B4 Muscarinic M1 Muscarinic M2 Muscarinic M3 Muscarinic M4 Muscarinic M5 Neurokinin Neuropeptide Y Serotonin 5-HT1A Serotonin 5-HT16 CXCR2 CXCR4 CCR5 DARC

Ligand


19007

REFERENCES

FIG. 10. The CCR1 antagonist, BX 471, decreases the severity of disease in a rat EAE model of multiple sclerosis. Disease was induced in male Lewis rats as described under “Experimental Procedures.” Animals were either treated with vehicle (40% cyclodextrin in saline) or with solutions of BX 471 (5, 20, and 50 mg/kg) three times a day as described under “Experimental Procedures.” A, average clinical scores in Lewis rats treated with BX 471. B, average cumulative clinical score in Lewis rats treated with BX 471. For details see under “Experimental Procedures.”

tion can also ameliorate the effects of neuronal and oligodendrocyte dysfunction and destruction. CCR1 expression within demyelinating plaques of multiple sclerosis brains on T-lymphocytes, macrophage/microglia, or vascular expression on T lymphocytes implicate this receptor in the pathology of this disease. It is highly likely that a stepwise process of leukocyte accumulation in the central nervous system occurs in multiple sclerosis. Lymphocyte expression of CCR1 in the periphery leads to accumulation of activated cells in blood vessels of multiple sclerosis brains; after transendothelial migration to the perivascular regions, the receptor would likely be downmodulated. Here the expression of other chemokine receptors such as CCR5 and CXCR3, markers of Th1 type T lymphocytes, would be necessary for disease progression. Although a number of chemokines, MIP-1␣, RANTES, MCP-1, interferon inducible protein of 10KD, monokine induced by interferon ␥ (reviewed in Refs. 37–39) and various receptors CCR1, CCR5,

1. Karpus, W. J., Lukacs, N. W., McRae, B. L., Strieter, R. M., Kunkel, S. L., and Miller, S. D. (1995) J. Immunol. 155, 5003–5010 2. Karpus, W. J., and Kennedy, K. J. (1997) J. Leukocyte Biol. 62, 681– 687 3. Rottman, J. B., Silva, R., Slavin, A., Weiner, H. L., Gerard, C. G., and Hancock, W. W. (1999) FASEB J. 13, 666 (abstr.) 4. Hesselgesser, J., Ng, H. P., Liang, M., Zheng, W., May, K., Bauman, J. G., Monahan, S., Islam, I., Wei, G. P., Ghannam, A., Taub, D. D., Rosser, M., Snider, R. M., Morrissey, M. M., Perez, H. D., and Horuk, R. (1998) J. Biol. Chem. 273, 15687–15692 5. Neote, K., DiGregorio, D., Mak, J. Y., Horuk, R., and Schall, T. J. (1993) Cell 72, 415– 425 6. Armatruda, T. T., Gerard, N. P., Gerard, C., and Simon, M. I. (1993) J. Biol. Chem. 268, 10139 –10144 7. Grynkiewicz, G., Poenie, M., and Tsien, R. Y. (1985) J. Biol. Chem. 260, 3440 –3450 8. Taub, D. D., Key, M. L., Clark, D., and Turcovski-Corrales, S. M. (1995) J. Immunol. Methods 184, 187–198 9. Conklyn, M. J., Neote, K., and Showell, H. J. (1996) Cytokine 8, 762–766 10. Parce, J. W., Owicki, J. C., Kercso, K. M., Sigal, G. B., Wada, H. G., Muir, V. C., Bousse, L. J., Ross, K. L., Sikic, B. I., and McConnell, H. M. (1989) Science 246, 243–247 11. Baggiolini, M. (1998) Nature 392, 565–568 12. Arunlakshana, O., and Schild, H. O. (1959) Br. J. Pharmacol. 14, 48 –58 13. Horn, F., Weare, J., Beukers, M. W., Horsch, S., Bairoch, A., Chen, W., Edvardsen, O., Campagne, F., and Vriend, G. (1998) Nucleic Acids Res. 26, 275–279 14. Barnes, D. A., Tse, J., Kaufhold, M., Owen, M., Hesselgesser, J., Strieter, R., Horuk, R., and Perez, D. H. (1998) J. Clin. Invest. 101, 2910 –2919 15. Plater-Zyberk, C., Hoogewerf, A. J., Proudfoot, A. E. I., Power, C. A., and Wells, T. N. C. (1997) Immunol. Lett. 57, 117–120 16. Wiedermann, C. J., Kowald, E. N., Reinish, N., Kaehler, C. M., von Luettichau, I., Pattison, J. M., Huie, P., Sibley, R. K., Nelson, P. J., and Krensky, A. M. (1993) Curr. Biol. 3, 735–739 17. Pattison, J., Nelson, P. J., Huie, P., Von, L. I., Farshid, G., Sibley, R. K., and Krensky, A. M. (1994) Lancet 343, 209 –211 18. Grone, H. J., Weber, C., Weber, K. S., Grone, E. F., Rabelink, T., Klier, C. M., Wells, T. N., Proudfood, A. E., Schlondorff, D., and Nelson, P. J. (1999) FASEB J. 13, 1371–1383 19. Gao, W., Topham, P. S., King, J. A., Smiley, S. T., Csizmadia, V. B. L., Gerard, C. J., and Hancock, W. W. (2000) J. Clin. Invest. 105, 35– 44 20. Beinborn, M., Lee, Y. M., McBride, E. W., Quinn, S. M., and Kopin, A. S. (1993) Nature 362, 348 –350 21. Gether, U., Johansen, T. E., Snider, R. M., Lowe, J. A. d., Nakanishi, S., and Schwartz, T. W. (1993) Nature 362, 345–348 22. Kong, H., Raynor, K., Yano, H., Takeda, J., Bell, G. I., and Reisine, T. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 8042– 8046 23. Proudfoot, A. E. I., Power, C. A., Hoogewerf, A. J., Montjovent, M. O., Borlat, F., Offord, R. E., and Wells, T. N. C. (1996) J. Biol. Chem. 271, 2599 –2603 24. Simmons, G., Clapham, P. R., Picard, L., Offord, R. E., Rosenkilde, M. M., Schwartz, T. W., Buser, R., Wells, T. N. C., and Proudfoot, A. E. I. (1997) Science 276, 276 –279 25. Ng, H. P., May, K., Bauman, J. G., Ghannam, A., Islam, I., Liang, M., Horuk,


CXCR3 (38, 39) have been visualized in EAE or multiple sclerosis brains, the consequential versus causative ramifications for the development of multiple sclerosis pathology remain to be proven. Only by the use of specific chemokine receptor inhibitors to treat multiple sclerosis in human clinical trials will the significance of these molecules be elucidated. In summary, we have identified a potent, selective, small molecule functional antagonist for CCR1, BX 471. The inhibition of CCR1 by the compound appears to be reversible and surmountable. Studies in dogs demonstrated that the compound is orally available and relatively stable in plasma, and the compound appears to be effective in an EAE model of multiple sclerosis in the rat. We believe that BX 471 represents a CCR1 antagonist possessing high potential as a therapy among all the CCR1 inhibitors reported so far. Multiple sclerosis is a particularly devastating disease of the central nervous system. Other than immunosuppresive drugs like steroids, which have a myriad of side effects, or the use of interferons, no orally active drugs are available for its treatment. Safety studies with BX 471 in a variety of animal species have revealed no toxicity, hemodynamic, or central nervous system effects with the drug at doses way in excess of the blood plasma levels observed in this animal study (data not shown). Thus, a specific treatment with an orally available small molecule like BX 471 may represent a potential therapeutic alternative to the current drugs of choice in treating patients with multiple sclerosis.

19008


R., Hesselgesser, J., Snider, R. M., Perez, H. D., and Morrissey, M. M. (1999) J. Med. Chem. 42, 4680 – 4694 26. Kato, K., Yamamoto, M., Honda, S., and Fujisawa, T. (1997) in World (PCT) Patent WO-9724325 27. Naya, A., Owada, Y., Saeki, T., Ohkawi, K., and Iwasawa, K. (1998) in World (PCT) Patent W0 –9804554 28. Ebers, G. C. (1986) in Diseases of the Nervous System (Asbury, A. K., McKhann, G. M., and McDonald, W. I., eds), pp. 1268 –1281, Ardmore Medical Books, Philadelphia 29. Merrill, J. E. (1987) Immunol. Today 8, 146 –150 30. Benveniste, E. N. (1992) Am. J. Physiol. 32, C1–C16 31. Luster, A. D. (1998) N. Engl. J. Med. 338, 436 – 445 32. Cannella, B., Cross, A. H., and Raine, C. S. (1993) J. Neuroimmunol. 46, 43–55 33. Kent, S. J., Karlik, S. J., Cannon, C., Hines, D. K., Yednock, T. A., Fritz, L. C.,

and Horner, H. C. (1995) J. Neuroimmunol. 58, 1–10 34. Keszthelyi, E., Karlik, S., Hyduk, S., Rice, G. P., Gordon, G., Yednock, T., and Horner, H. (1996) Neurology 47, 1053–1059 35. Yednock, T. A., Cannon, C., Fritz, L. C., Sanchez-Madrid, F., Steinman, L., and Karin, N. (1992) Nature 356, 63– 66 36. Leger, O. J., Yednock, T. A., Tanner, L., Horner, H. C., Hines, D. K., Keen, S., Saldanha, J., Jones, S. T., Fritz, L. C., and Bendig, M. M. (1997) Hum. Antibodies 8, 3–16 37. Hesselgesser, J., and Horuk, R. (1999) J. Neurovirol. 5, 13–26 38. Balashov, K. E., Rottman, J. B., Weiner, H. L., and Hancock, W. W. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 6873– 6878 39. Sorensen, T. L., Tani, M., Jensen, J., Pierce, V., Lucchinetti, C., Folcik, V. A., Qin, S., Rottman, J., Sellebjerg, F., Strieter, R. M., Frederiksen, J. L., and Ransohoff, R. M. (1999) J. Clin. Invest. 103, 807– 815


Identification and Characterization of a Potent, Selective, and Orally Active Antagonist of the CC Chemokine Receptor-1 Meina Liang, Cornell Mallari, Mary Rosser, Howard P. Ng, Karen May, Sean Monahan, John G. Bauman, Imadul Islam, Ameen Ghannam, Brad Buckman, Ken Shaw, Guo-Ping Wei, Wei Xu, Zuchun Zhao, Elena Ho, Jun Shen, Huynh Oanh, Babu Subramanyam, Ron Vergona, Dennis Taub, Laura Dunning, Susan Harvey, R. Michael Snider, Joseph Hesselgesser, Michael M. Morrissey, H. Daniel Perez and Richard Horuk J. Biol. Chem. 2000, 275:19000-19008. doi: 10.1074/jbc.M001222200 originally published online March 29, 2000

Access the most updated version of this article at doi: 10.1074/jbc.M001222200

Click here to choose from all of JBC's e-mail alerts This article cites 36 references, 11 of which can be accessed free at http://www.jbc.org/content/275/25/19000.full.html#ref-list-1


Alerts: • When this article is cited • When a correction for this article is posted