Timothy Joseph Paradis

CP-481,715, a Potent and Selective CCR1 Antagonist with Potential Therapeutic Implications for Inflammatory Diseases

The chemokines CCL3 and CCL5, as well as their shared receptor CCR1, are believed to play a role in the pathogenesis of several inflammatory diseases including rheumatoid arthritis, multiple sclerosis, and transplant rejection. In this study we describe the pharmacological properties of a novel small molecular weight CCR1 antagonist, CP-481,715 (quinoxaline-2-carboxylic acid [4(R)-carbamoyl-1(S)-(3-fluorobenzyl)-2(S),7-dihydroxy-7-methyloctyl]amide). Radiolabeled binding studies indicate that CP-481,715 binds to human CCR1 with a Kd of 9.2 nm and displaces 125I-labeled CCL3 from CCR1-transfected cells with an IC50 of 74 nm. CP-481,715 lacks intrinsic agonist activity but fully blocks the ability of CCL3 and CCL5 to stimulate receptor signaling (guanosine 5′-O-(thiotriphosphate) incorporation; IC50 = 210 nm), calcium mobilization (IC50 = 71 nm), monocyte chemotaxis (IC50 = 55 nm), and matrix metalloproteinase 9 release (IC50 = 54 nm). CP-481,715 retains activity in human whole blood, inhibiting CCL3-induced CD11b up-regulation and actin polymerization (IC50 = 165 and 57 nm, respectively) on monocytes. Furthermore, it behaves as a competitive and reversible antagonist. CP-481,715 is >100-fold selective for CCR1 as compared with a panel of G-protein-coupled receptors including related chemokine receptors. Evidence for its potential use in human disease is suggested by its ability to inhibit 90% of the monocyte chemotactic activity present in 11/15 rheumatoid arthritis synovial fluid samples. These data illustrate that CP-481,715 is a potent and selective antagonist for CCR1 with therapeutic potential for rheumatoid arthritis and other inflammatory diseases.

The anti-tumor activity of anti-CTLA-4 is mediated through its induction of IFNγ

Abstract The T-cell-specific receptor, CTLA-4, has been demonstrated to be a potent negative regulator of lymphocyte activation, the functional significance of which has been demonstrated in murine tumor models using blocking antibodies. However, the mechanism(s) involved in enhancing tumor regression has not been identified. In this study, we determined whether IFNγ was playing a role in this activity. In vitro, anti-CTLA-4 enhanced IFNγ production by lymph node cells obtained from tumor-bearing mice (351 pg/ml vs 77 pg/ml). Additionally, fibrosarcoma-challenged animals treated with anti-CTLA-4 had elevated levels of the IFN-inducible enzyme 2-5-OAS in draining lymph nodes (850 pM vs 260 pM for controls) and an increased amount of IFNγ in tumor lysates (at day 7, 620 pg/100 μg vs 160 pg/100 μg in controls). The importance of IFNγ was demonstrated by the ability of neutralizing antibodies to completely abrogate the anti-tumor effects of anti-CTLA-4. Moreover, fibrosarcoma cells were shown to be exquisitely sensitive to IFNγ-mediated class I upregulation and histological examination of tumors from anti-CTLA-4-treated mice revealed a trend toward increased tumor cell apoptosis and decreased angiogenesis. These studies have demonstrated that one mechanism for the anti-tumor effects of anti-CTLA-4 relates to its ability to augment IFNγ production, resulting in an increased expression of class I on the tumor, enhanced apoptosis, and a decrease in blood vessel growth.

THE CC CHEMOKINE MIP-1ALPHA INDUCES A SELECTIVE MONOCYTE INFILTRATION FOLLOWING INTRADERMAL INJECTION INTO NONHUMAN PRIMATES

The in vitro chemotactic activity of chemokines have been well documented. However, study of their in vivo effects where components of rolling, adherence and diapedesis are pre-requisites to leukocyte infiltration have not been examined in higher species. In this study, we examined the biological activity of the CC chemokine, MIP-1α, in rhesus monkeys. Following an intradermal injection, a significant cellular infiltrate and an increase in the number of inflamed vessels were observed. This response peaked at 24 h and was sustained for up to 48 hours after injection. Phenotypically, the specific infiltrate consisted exclusively of CD68+ monocytes with no increase in other cell types over the saline injected controls. These studies represent the first examination of the in vivo effects of MIP-1α in higher species and indicate that MIP-1α is a selective monocyte recruiting agent in vivo.