Identification of N-acyl 4-(3-pyridonyl)phenylalanine derivatives and their orally active prodrug esters as dual acting α4ß1 and α4ß7 receptor antagonists.

From a series of N-acyl 4-(3-pyridonyl)phenylalanine derivatives of 4, the trifluoromethyl derivative 28 was identified as a potent, dual acting alpha4 integrin antagonist with activity in primate models of allergic asthma. Investigation of a series of prodrug esters led to the discovery of the morpholinopropyl derivative 48 that demonstrated good intestinal fluid stability, solubility and permeability. Compound 48 gave high blood levels of 28 when dosed orally in cynomolgus monkeys. Surprisingly, hydrolysis of 48 was rapid in liver microsomes from the pharmacological species, mouse, rat and monkey, but slow in dog and human; in vivo studies also indicated there was prolonged exposure to unchanged prodrug in dogs.

Identification of N-acyl 4-(5-pyrimidine-2,4-dionyl)phenylalanine derivatives and their orally active prodrug esters as dual-acting alpha4-beta1 and alpha4-beta7 receptor antagonists.

N-Acyl 4-(5-pyrimidine-2,4-dionyl)phenylalanine derivatives of type 4 were designed to replace the 2,6-dichlorobenzoylamine portion of compound 1 in order to identify novel compounds with improved potency against α4-integrins. Several derivatives were identified as very potent dual-acting α4-integrin, α4ß1 and α4ß7 antagonists. Investigation of a limited number of prodrug esters led to the discovery of the ethyl ester prodrug 42, which demonstrated good intestinal fluid stability and good permeability. Despite low solubility, 42 gave acceptable blood levels of 30 when dosed orally in non-human primates. Additionally, 42 had an overall excellent profile and was selected for clinical trials. Investigation of N-acyl 4-(5-pyrimidine-2,4-dionyl)phenylalanine derivatives led to the discovery of several very potent dual-acting α4-integrin antagonists. Ethyl ester prodrug 42 advanced to human clinical trials based on the excellent intestinal fluid stability, good permeability and superior efficacy in non-human primates.

The mRNA level of Charcot-Leyden crystal protein/galectin-10 is a marker for CRTH2 activation in human whole blood in vitro.

CRTH2 is one of the prostaglandin D2 receptors and plays a proinflammatory role in allergic diseases. Gene expression markers in whole blood induced by CRTH2 activation have not previously been reported. Using microarray analyses of 54 675 genes, we revealed modest gene expression changes in human whole blood stimulated in vitro by a selective CRTH2 agonist, DK-PGD2. Five genes were found to exhibit 1.5- to 2.6-fold changes in expression. The expression of Charcot-Leyden crystal protein/galectin-10 (CLC/Gal-10) in particular was consistently enhanced in human whole blood stimulated by DK-PGD2, as confirmed by quantitative real-time polymerase chain reaction analyses. DK-PGD(2)-induced increases in blood CLC/Gal-10 mRNA levels were largely attenuated by the CRTH2 antagonist CAY10471.Thus, the DK-PGD2-induced CLC/Gal-10 mRNA level can serve as a potential marker for monitoring pharmacodynamic effects of blood exposure to CRTH2 modulating agents.

Discovery of novel and potent leukotriene B4 receptor antagonists. Part 1.

The inhibition of LTB(4) binding to and activation of G-protein-coupled receptors BLT1 and BLT2 is the premise of a treatment for several inflammatory diseases. In a lead optimization effort starting with the leukotriene B(4) (LTB(4)) receptor antagonist (2), members of a series of 3,5-diarylphenyl ethers were found to be highly potent inhibitors of LTB(4) binding to BLT1 and BLT2 receptors, with varying levels of selectivity depending on the substitution. In addition, compounds 33 and 38 from this series have good in vitro ADME properties, good oral bioavailability, and efficacy after oral delivery in guinea pig LTB(4) and nonhuman primate allergen challenge models. Further profiling in a rat non-GLP toxicity experiment provided the rationale for differentiation and selection of one compound (33) for clinical development.

KU812 cells provide a novel in vitro model of the human IL-33/ST2L axis: functional responses and identification of signaling pathways.

Activation of interleukin-1 family receptor ST2L by its ligand interleukin-33 (IL-33) is an important component in inflammatory responses. Peripheral blood basophils, recognized as major effector cells in allergic inflammation that play a role in both innate and adaptive immunity, are activated by IL-33 through ST2L. However, studies are challenging due to the paucity of this cell population, representing less than 1% of peripheral blood leukocytes. We identified a basophil-like chronic myelogenous leukemia cell line, KU812, that constitutively expresses ST2L and demonstrates functional responses to IL-33 stimulation. IL-33 induced production of multiple inflammatory mediators in KU812 cells that were blocked by anti-ST2L and anti-IL-33 antibodies. The interaction of IL-33 and ST2L activated NF-kappaB, JNK, and p38 MAPK, but not ERK1/2 signaling pathways. Studies using pharmacological inhibitors to IKK-2 and MAP kinases revealed that one of the functional responses, IL-33-induced IL-13 production, was regulated through NF-kappaB, but not JNK or p38 MAPK signaling. The requirement of NF-kappaB was confirmed by IKK-2 knockdown using shRNA. KU812 represents the first human cell line-based in vitro model of the IL-33/ST2L axis and provides a valuable tool to aid in understanding the mechanism and significance of IL-33 and ST2L interaction and function.

Effects of LTB4 receptor antagonism on pulmonary inflammation in rodents and non-human primates.

Asthma, chronic obstructive pulmonary disease (COPD) and acute lung injury/acute respiratory distress syndrome (ALI/ARDS) are characterized by neutrophilic inflammation and elevated levels of leukotriene B4 (LTB4). However, the exact role of LTB4 pathways in mediating pulmonary neutrophilia and the potential therapeutic application of LTB4 receptor antagonists in these diseases remains controversial. Here we show that a novel dual BLT1 and BLT2 receptor antagonist, RO5101576, potently inhibited LTB4-evoked calcium mobilization in HL-60 cells and chemotaxis of human neutrophils. RO5101576 significantly attenuated LTB4-evoked pulmonary eosinophilia in guinea pigs. In non-human primates, RO5101576 inhibited allergen and ozone-evoked pulmonary neutrophilia, with comparable efficacy to budesonide (allergic responses). RO5101576 had no effects on LPS-evoked neutrophilia in guinea pigs and cigarette smoke-evoked neutrophilia in mice and rats. In toxicology studies RO5101576 was well-tolerated. Theses studies show differential effects of LTB4 receptor antagonism on neutrophil responses in vivo and suggest RO5101576 may represent a potential new treatment for pulmonary neutrophilia in asthma.