Howard P. Ng

CCR1-specific non-peptide antagonist: efficacy in a rabbit allograft rejection model

The classic signs of acute cellular rejection during organ transplantation include the infiltration of mononuclear cells into the interstitium. This recruitment of leukocytes into the transplanted tissue is promoted by chemokines like RANTES. Since RANTES is a potent agonist for the CC chemokine receptor CCR1, we examined whether the CCR1 antagonist BX 471 was efficacious in a rabbit kidney transplant rejection model. BX 471 was able to compete with high affinity with the CCR1 ligands MIP-1alpha and RANTES for binding to HEK 293 cells expressing rabbit CCR1. BX 471 was a competitive antagonist of rabbit CCR1 in Ca(2+) flux studies. Two separate studies in which animals were subcutaneously implanted with slow release pellets of BX 471 demonstrated that animals implanted with BX 471 had increased survival compared with untreated controls or animals implanted with placebo. The mean survival time for the placebo group was 12.33+/-1.7 days. The animals in the BX 471 treated group had mean survival times of 16.9+/-2.1 and 16.0+/-1.7 days, respectively, for the two studies. Analysis of the combined data by Student t-test gave a P value of 0.03 that is significant at the 0.05 level. In addition, there was a marked reduction in the urea and creatinine levels in the BX 471 treated animals compared with the control and placebo groups in both studies. Finally, pathologic analysis of the kidneys in the rabbit renal transplantation model from animals in the different groups showed that BX 471 was similar to cyclosporin in its ability to prevent extensive infarction of transplanted kidneys. Based on the data from these studies, BX 471 shows clear efficacy at the single dose tested compared with animals treated with placebo.

Species selectivity of a small molecule antagonist for the CCR1 chemokine receptor.

The species specificity of a small molecule antagonist for the human CCR1 chemokine receptor, 2-2-diphenyl-5-(4-chlorophenyl)piperidin-1-yl)valeronitrile (CCR1 antagonist 1), has been examined using cloned CCR1 receptors from various species. The compound was able to bind to rabbit, marmoset, and human CCR1, and was able to block the functional activation of these receptors. However, it failed to significantly displace radiolabeled macrophage inflammatory protein-1alpha (MIP-1alpha) binding to mouse CCR1 at concentrations up to 10 microM. These data suggested that the antagonist binding site is well-conserved in rabbit, marmoset and human CCR1, but not in mouse CCR1. The functional selectivity and mechanism of action for CCR1 antagonist 1 were further characterized. CCR1 antagonist 1 blocked the increase in intracellular Ca(2+) stimulated by CCR1 agonists, but had no effect on N-formyl-Met-Leu-Phe (FMLP), monocyte chemotactic protein-1 (MCP-1) and stromal-derived factor 1alpha (SDF1alpha)-induced Ca(2+) mobilization, demonstrating functional selectivity for CCR1. Since CCR1 antagonist 1 is a functional antagonist of marmoset and rabbit CCR1 receptors, it should be possible to test its efficacy in animal models of disease.

Structure Function Differences in Nonpeptide CCR1 Antagonists for Human and Mouse CCR1

A useful strategy for identifying ligand binding domains of G protein-coupled receptors has been the exploitation of species differences in antagonist potencies. We have used this approach for the CCR1 chemokine receptor with a novel series of antagonists, the 4-hydroxypiperidines, which were discovered by high throughput screening of human CCR1 and subsequently optimized. The structure-activity relationships for a number of different 4-hydroxypiperidine antagonists for human and mouse CCR1 were examined by receptor binding and functional assays. These compounds exhibit major differences in their rank order of potency for the human and mouse chemokine receptor CCR1. For example, the initial lead template, BX 510, which was a highly potent functional antagonist for human CCR1 (Ki = 21 nM) was >400-fold less active on mouse CCR1 (Ki = 9150 nM). However, increasing the length of the linker between the piperidine and dibenzothiepine groups by one methylene group generated a compound, BX 511, which was equipotent for both human and mouse CCR1. These and other analogs of the lead template BX 510, which have major differences in potency for human and mouse CCR1, are described, and a model for their interaction with human CCR1 is presented.

Design, synthesis, and biological activity of novel factor Xa inhibitors: 4-aryloxy substituents of 2,6-diphenoxypyridines.

A novel series of triaryloxypyridines have been designed to inhibit factor Xa, a serine protease strategically located in the coagulation cascade. Inhibitor 5e has a K(I) against factor Xa of 0.12nM and is greater than 8000- and 2000-fold selective over two related serine proteases, thrombin and trypsin, respectively. The 4-position of the central pyridine has been identified as a site that tolerates various substitutions without deleterious effects on potency and selectivity. This suggests that the 4-position of the pyridine ring is an ideal site for chemical modifications to identify inhibitors with improved pharmacokinetic characteristics. This investigation has resulted in inhibitor 5d, which has an oral availability of 6% in dogs. The synthesis, in vitro activity, and in vivo profile of this class of inhibitors is outlined.

Automated Parallel Solid-Phase Synthesis of Non-Peptide CCR1 Receptor Antagonists

An automated, parallel, solid-phase synthesis and screening strategy using commercially available aryl acetic acids as starting materials has discovered novel, non-peptide CCR1 antagonists (K(i) < 100 nM).