A novel, orally active CXCR1/2 receptor antagonist, Sch527123, inhibits neutrophil recruitment, mucus production, and goblet cell hyperplasia in animal models of pulmonary inflammation.

Sch527123 [2-hydroxy-N,N-dimethyl-3-[[2-[[1(R)-(5-methyl-2-furanyl)propyl]amino]-3,4-dioxo-1-cyclobuten-1-yl]amino]ben-zamide] is a potent, selective antagonist of the human CXCR1 and CXCR2 receptors (Gonsiorek et al., 2007). Here we describe its pharmacologic properties at rodent CXCR2 and at the CXCR1 and CXCR2 receptors in the cynomolgus monkey, as well as its in vivo activity in models demonstrating prominent pulmonary neutrophilia, goblet cell hyperplasia, and mucus production. Sch527123 bound with high affinity to the CXCR2 receptors of mouse (K(d) = 0.20 nM), rat (K(d) = 0.20 nM), and cynomolgus monkey (K(d) = 0.08 nM) and was a potent antagonist of CXCR2-mediated chemotaxis (IC(50) approximately 3-6 nM). In contrast, Sch527123 bound to cynomolgus CXCR1 with lesser affinity (K(d) = 41 nM) and weakly inhibited cynomolgus CXCR1-mediated chemotaxis (IC(50) approximately 1000 nM). Oral treatment with Sch527123 blocked pulmonary neutrophilia (ED(50) = 1.2 mg/kg) and goblet cell hyperplasia (32-38% inhibition at 1-3 mg/kg) in mice following the intranasal lipopolysaccharide (LPS) administration. In rats, Sch527123 suppressed the pulmonary neutrophilia (ED(50) = 1.8 mg/kg) and increase in bronchoalveolar lavage (BAL) mucin content (ED(50) =<0.1 mg/kg) induced by intratracheal (i.t.) LPS. Sch527123 also suppressed the pulmonary neutrophilia (ED(50) = 1.3 mg/kg), goblet cell hyperplasia (ED(50) = 0.7 mg/kg), and increase in BAL mucin content (ED(50) = <1 mg/kg) in rats after i.t. administration of vanadium pentoxide. In cynomolgus monkeys, Sch527123 reduced the pulmonary neutrophilia induced by repeat bronchoscopy and lavage (ED(50) = 0.3 mg/kg). Therefore, Sch527123 may offer benefit for the treatment of inflammatory lung disorders in which pulmonary neutrophilia and mucus hypersecretion are important components of the underlying disease pathology.

Pharmacological characterization of Sch527123, a potent allosteric CXCR1/CXCR2 antagonist.

In neutrophils, growth-related protein-alpha (CXCL1) and interleukin-8 (CXCL8), are potent chemoattractants (Cytokine 14:27-36, 2001; Biochemistry 42:2874-2886, 2003) and can stimulate myeloperoxidase release via activation of the G protein-coupled receptors CXCR1 and CXCR2. The role of CXCR1 and CXCR2 in the pathogenesis of inflammatory responses has encouraged the development of small molecule antagonists for these receptors. The data presented herein describe the pharmacology of 2-hydroxy-N,N-dimethyl-3-{2-[[(R)-1-(5-methyl-furan-2-yl)-propyl]amino]-3,4-dioxo-cyclobut-1-enylamino}-benzamide (Sch527123), a novel antagonist of both CXCR1 and CXCR2. Sch527123 inhibited chemokine binding to (and activation of) these receptors in an insurmountable manner and, as such, is categorized as an allosteric antagonist. Sch527123 inhibited neutrophil chemotaxis and myeloperoxidase release in response to CXCL1 and CXCL8 but had no effect on the response of these cells to C5a or formyl-methionyl-leucyl-phenylalanine. The pharmacological specificity of Sch527123 was confirmed by testing in a diversity profile against a panel of enzymes, channels, and receptors. To measure compound affinity, we characterized [(3)H]Sch527123 in both equilibrium and nonequilibrium binding analyses. Sch527123 binding to CXCR1 and CXCR2 was both saturable and reversible. Although Sch527123 bound to CXCR1 with good affinity (K(d) = 3.9 +/- 0.3 nM), the compound is CXCR2-selective (K(d) = 0.049 +/- 0.004 nM). Taken together, our data show that Sch527123 represents a novel, potent, and specific CXCR2 antagonist with potential therapeutic utility in a variety of inflammatory conditions.

Murine CXCR1 is a functional receptor for GCP-2/CXCL6 and interleukin-8/CXCL8.

Functional interleuin-8 (IL-8) receptors (IL-8RA and IL-8RB: CXCR1 and CXCR2, respectively) have been described in human, monkey, dog, rabbit, and guinea pig. Although three IL-8R homologues have been found in rat, only one of these, rat CXCR2, appears to be functional based on responsiveness to ligands. Similarly, CXC chemokines induce biological responses through the murine homolog of CXCR2, but the identification of functional rodent CXCR1 homologues has remained elusive. We have identified and characterized the mouse CXCR1 homologue (mCXCR1). Murine CXCR1 shares 68 and 88% amino acid identity with its human and rat counterparts, respectively. Similar to the tissue distribution pattern of rat CXCR1, we found murine CXCR1 mRNA expression predominantly in lung, stomach, bone marrow, and leukocyte-rich tissues. In contrast to previous reports, we determined that mCXCR1 is a functional receptor. We show predominant engagement of this receptor by mouse GCP-2/CXCL6, human GCP-2, and IL-8/CXCL8 by binding, stimulation of GTPgammaS exchange, and chemotaxis of mCXCR1-transfected cells. Furthermore, murine CXCR1 is not responsive to the human CXCR2 ligands ENA-78/CXCL5, NAP-2/CXCL7, GRO-alpha, -beta, -gamma/CXCL1-3, or rat CINC-1-3. In addition, we show concomitant elevation of mCXCR1 and its proposed major ligand, GCP-2, positively correlated with paw swelling in murine collagen-induced arthritis. This report represents the first description of a functional CXCR1-like receptor in rodents.

Characterization of peripheral human cannabinoid receptor (hCB2) expression and pharmacology using a novel radioligand, [35S]Sch225336.

Studies to characterize the endogenous expression and pharmacology of peripheral human cannabinoid receptor (hCB2) have been hampered by the dearth of authentic anti-hCB2 antibodies and the lack of radioligands with CB2 selectivity. We recently described a novel CB2 inverse agonist, N-[1(S)-[4-[[4-methoxy-2-[(4methoxyphenyl)sulfonyl] phenyl]sulfonyl] phenyl]ethyl]methane-sulfonamide (Sch225336), that binds hCB2 with high affinity and excellent selectivity versus hCB1. The precursor primary amine of Sch225336 was prepared and reacted directly with [(35)S]mesyl chloride (synthesized from commercially obtained [(35)S]methane sulfonic acid) to generate [(35)S]Sch225336. [(35)S]Sch225336 has high specific activity (>1,400 Ci/mmol) and affinity for hCB2 (65 pm). Using [(35)S]Sch225336, we assayed hemopoietic cells and cell lines to quantitate the expression and pharmacology of hCB2. Lastly, we used [(35)S]Sch225336 for detailed autoradiographic analysis of CB2 in lymphoid tissues. Based on these data, we conclude that [(35)S]Sch225336 represents a unique radioligand for the study of CB2 endogenously expressed in blood cells and tissues.

Pharmacological characterization of human S1P4 using a novel radioligand, [4,5-3H]-dihydrosphingosine-1-phosphate.

Sphingosine-1-phosphate (S1P) is a bioactive lipid that affects a variety of cellular processes through both its actions as a second messenger and via activation of a family of G protein-coupled receptors (S1P(1-5)). The study of S1P receptor pharmacology, particularly S1P(4), has been hindered by the lack of high-affinity radioligands with good specific activity. The studies presented herein characterize [(3)H]DH-S1P as a stable, high-affinity radioligand for S1P(4) pharmacology. Using a transfected Ba/F3 cell line selected for high hS1P(4) surface expression, we compared the consequences of different cellular backgrounds and commercial sources of sphingophospholipids on S1P(4) characterization. The development and subsequent use of the assay described has enabled us to extensively and definitively characterize the pharmacology of the human S1P(4) receptor.

Cloning and pharmacological characterization of CXCR1 and CXCR2 from Macaca fascicularis.

Two genes with high sequence homology to human CXCR1 (hCXCR1) and CXCR2 (hCXCR2) were cloned from blood of cynomolgus monkey (Macaca fascicularis). Comparison of the expression pattern of these receptors in different species demonstrated that, like in humans, cynomolgus CXCR1 (cCXCR1) and CXCR2 (cCXCR2) are highly expressed in blood. Membranes from transfected BaF3 cells expressing cCXCR1 bind interleukin (IL)-8 with an affinity similar to hCXCR1 (Kd values, 170 +/- 87 and 103 +/- 37 pM, respectively) and show low binding affinity to Gro-alpha. Cynomolgus CXCR2 also binds hIL-8 but with somewhat higher affinity than the hCXCR2 (46 +/- 28 and 220 +/- 14 pM, respectively). Surprisingly, cCXCR2 has a reduced binding affinity to hGro-alpha (3.7 +/- 2.2 nM), a specific ligand of hCXCR2 (540 +/- 140 pM). Furthermore, the CXCR2-specific antagonist SB225002 [N-(2-hydroxy-4-nitrophenyl)-N'-(2-bromophenyl)urea] is 10-fold more potent in inhibiting IL-8 binding to hCXCR2 than to cCXCR2, suggesting that some of the observed differences in the amino acid sequences of the human and monkey receptor affect ligand binding sites or the conformation of the receptor. Both cynomolgus receptors were functionally active in inducing guanosine 5'-O-(3-thio)triphosphate exchange on membranes in response to IL-8 and Gro-alpha and in mediating chemotactic activity of recombinant BA/F3 cells in response to IL-8 and Gro-alpha. These results identify the products of the novel cynomolgus genes as functional homologs of hCXCR1 and hCXCR2.

Impairment of leukocyte trafficking in a murine pleuritis model by IL-4 and IL-10.

We have characterized leukocyte migration to the pleural cavity in a methylated-BSA (mBSA)-induced model of murine delayed-type hypersensitivity and evaluated the ability of IL-4 and IL-10 to modulate this response. Neutrophils, macrophages, T cells, and dendritic cells migrated to the pleural cavity in a time-dependent fashion following direct intrapleural antigen challenge, with neutrophils comprising the majority of exudate leukocytes in the cavity within the first 24 h and the number of mononuclear cells increasing at later times. Real-time quantitative PCR analysis of infiltrating leukocytes revealed a marked elevation of steady-state mRNA levels of IL-1beta and TNFalpha and the chemokines KC, MIP-2, CXCL9, CXCL10, CXCL11, CCL2, CCL3, and CCL4 at 6 h postchallenge, which diminished over time. In contrast, gammaIFN mRNA levels were maximal at 24 h and CCL5 expression was sustained throughout 72 h. ELISA analysis of pleural exudate fluid revealed significant elevations of KC and CCL2 protein levels at 6 h postantigen challenge and a peak increase in gammaIFN protein at 24 h, confirming our mRNA observations. Administration of recombinant murine IL-4 or IL-10 prior to challenge significantly blocked cell trafficking to the pleural cavity as well as peak levels of exudate gammaIFN, with IL-4 being more potent in impairing these responses. IL-4 administration also increased the proportion of naive T cells in the pleural cavity, as judged by CD62L and CD45RB expression. These results indicate that this in vivo model demonstrates a pattern of events associated with Th1-mediated leukocyte trafficking and underscore the potential utility of this in vivo model for evaluating therapeutic inhibitors of leukocyte trafficking.

Pharmacological analysis of calcium responses mediated by the human A3 adenosine receptor in monocyte-derived dendritic cells and recombinant cells.

Extensive characterization of adenosine receptors expressed by human monocyte-derived dendritic cells (MDDCs) was performed with quantitative polymerase chain reaction, radioligand binding, and calcium signaling. Transcript for the A3 adenosine receptor was elevated more than 100-fold in immature MDDCs compared with monocyte precursors. A3 receptor transcript was substantially diminished, and A2A receptor transcript increased, by lipopolysaccharide maturation of MDDCs. Saturation binding of N(6)-(3-[(125)I]iodo-4-aminobenzyl)-adenosine-5'-N-methyluronamide ([(125)I]AB-MECA) to membranes from immature MDDCs yielded B(max) of 298 fmol/mg of protein and K(D) of 0.7 nM. Competition against [(125)I]AB-MECA binding confirmed the site to be the A3 receptor. Adenosine elicited pertussis toxin-sensitive calcium responses with EC(50) values ranging as low as 2 nM. The order of potency for related agonists was N(6)-(3-iodobenzyl)-adenosine-5'-N-methylcarboxamide (IB-MECA) >/= I-AB-MECA > 2Cl-IB-MECA >/= adenosine > 2-[p-(2-carboxyethyl)phenylethylamino]-5'-N-ethylcarboxyamidoadenosine (CGS21680). The order of efficacy was adenosine >/= CGS21680 > IB-MECA >/= I-AB-MECA > 2Cl-IB-MECA. Calcium responses to 2Cl-IB-MECA and CGS21680, and the lower range of adenosine concentrations, were completely blocked by 10 nM N-(2-methoxyphenyl)-N-[2-(3-pyridyl)quinazolin-4-yl]urea (VUF5574) but not by 7-(2-phenylethyl)-5-amino-2-(2-furyl)-pyrazolo-[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine (SCH58261) or 8-cyclopentyl-1,3-dipropylxanthine. Pretreatment with 100 nM 2Cl-IB-MECA eliminated responses to CGS21680 but not to monocyte inhibitory protein-1alpha. For comparison, dose-response functions were obtained from double-recombinant human embryonic kidney 293 cells expressing the human A3 receptor and a chimeric Galphaq-i3 protein, which was required to establish A3-mediated calcium signaling. The pharmacological profile of calcium signaling elicited by adenosine-related agonists in the double-recombinant cells was essentially identical to that obtained from immature MDDCs. Our results provide an extensive analysis of A3-mediated calcium signaling and unequivocally identify immature MDDCs as native expressers of the human A3 receptor.

Pharmacological characterization of the chemokine receptor, hCCR1 in a stable transfectant and differentiated HL-60 cells: antagonism of hCCR1 activation by MIP-1beta.

C-C chemokine receptor-1 (CCR1) has been implicated in mediating a variety of inflammatory conditions including multiple sclerosis and organ rejection. Although originally referred to as the MIP-1alpha/RANTES receptor, CCR1 is quite promiscuous and can be activated by numerous chemokines. We used radioligand binding and [35S]-GTPgammaS exchange assays in membranes from a cell line transfected to express CCR1 (Ba/F3-hCCR1) to characterize a panel of chemokines (HCC-1, MIP-1alpha, MIP-1beta, MIP-1delta, MPIF-1, MCP-2, MCP-3, and RANTES) as CCR1 ligands. In this recombinant model, these chemokines displaced 125I-MIP-1alpha with a wide range of potencies and, with the exception of MCP-2, acted as full agonists in stimulating [35S]-GTPgammaS exchange. We then assessed the utility of HL-60 cells cultured with known differentiating agents (PMA, DMSO, dibutyryl-cAMP or retinoic acid) for investigating CCR1 pharmacology. In [35S]-GTPgammaS exchange assays, membranes from cells cultured with retinoic acid (4-6 days) were the most responsive to activation by MIP-1alpha and MPIF-1. FACS analysis and comparative pharmacology confirmed that these activities were mediated by CCR1. Using [35S]-GTPgammaS exchange assays, intracellular calcium flux and/or whole cell chemotaxis assays in HL-60(Rx) cells, we validated that MIP-1alpha was the most potent CCR1 ligand (MIP-1alpha>MPIF-1>RANTES>or=MIP-1beta) although the ligands differed in their efficacy as agonists. MPIF-1 was the more efficacious (MPIF-1>RANTES=MIP-1alpha>>MIP-1beta). 125I-MIP-1beta binding in Ba/F3-hCCR1 and HL-60(Rx) membranes was competitively displaced by MIP-1alpha, MPIF-1 and MIP-1beta. The binding K(i) for these chemokines with 125I-MIP-1beta were essentially identical in the two membrane systems. Lastly, MIP-1beta antagonized [35S]-GTPgammaS exchange, Ca2+ flux and chemotaxis in HL-60(Rx) cells in response to robust agonists such as MIP-1alpha, RANTES and MPIF-1. Based on our results, we propose that MIP-1beta could function as an endogenous inhibitor of CCR1 function.