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  • The on target in vitro pharmacology of was


    The on target in vitro pharmacology of 18 was then further explored, first in respect of murine CRTh2 receptor activity (Table 5), where similar levels of activity were found in both a binding and functional assay compared with the human orthologue, consistent with our earlier observations in the sulfonamide series. Next, the shape change activity of 18 was probed in more detail (Table 6). Eosinophil shape change is fundamental in leukocyte migration from the microcirculation to the site of inflammation and is a PGD2 dependent process routinely used as a measure of functional CRTh2 activity. An attractive aspect of CRTh2 as a therapeutic target is the fact that a range of PGD2 metabolites and also non-PGD2 derived arachidonic ZSTK474 sale products such as 11-dehydro-thromboxane B2 can selectively activate the receptor. Thus it was important to confirm 18 as an antagonist of all these potential endogenous stimuli and this was readily established, with low nM potency found against all CRTh2 agonists tested, concordant with the effects against DK-PGD2. In addition, the specificity of the response was confirmed, with 18 found to be inactive against eotaxin stimulated shape change mediated through the CCR-3 receptor. Inhibition of human eosinophil shape change in whole blood has been reported by ourselves and others as a more physiologically relevant measure of CRTh2 antagonism, with potential application as an ex vivo clinical target engagement indicator. Accordingly, compound 18 elicited a potent inhibitory response to shape change elicited by both PGD2 and DK-PGD2 in whole blood (Table 6). The chronic inflammatory response characteristically observed in allergic asthma occurs by the selective accumulation of Th2 lymphocytes which further potentiate the inflammatory response by releasing Th2 cytokines such as IL-5 and IL-13. The ability to modulate inflammatory responses in this cell type, in addition to damping down eosinophilic activation responses, is an additional key element to the postulated CRTh2 antagonist therapeutic modality. As indicated in (Table 7), compound 18 exhibited nM potency for the blockade of DK-PGD2 induced IL-5 and IL-13 cytokine production from primary cultured human CD4+ Th2 lymphocytes. With potent activity established against primary human inflammatory cell types, we next characterized 18 in CRTh2-dependent rodent mechanistic and allergic disease models. Intra-tracheal installation of DK-PGD2 in IL-5 primed rats leads to a time and dose-dependent recruitment of leukocytes (particularly eosinophils) from circulation into the lung. It was also shown that this pulmonary eosinophilia was mediated by activation of the CRTh2 receptor and could be abrogated by blockade of that receptor using a CRTh2 antagonist. Compound 18 dosed orally in an adapted variant of this model (Fig. 2) showed statistically significant inhibition of eosinophil infiltration after bronchoalveolar lavage (BAL) at a minimum dose of 1mg/kg, with a trend to dose-dependency, thus confirming CRTh2 target engagement with a readout in a disease relevant tissue. In a follow up experiment, compound 18 was applied at lower doses of 0.3 and 0.1mg/kg, with plasma sampling concomitant with BAL determination. The exposure–effect relationship corrected for rat plasma protein binding (rat fu 0.34) is shown in Figure 3. The approximate ED50 for eosinophilia reduction was broadly similar to the in vitro human whole blood eosinophil SC potency, although we were unsuccessful in generating an in vitro shape change response in rat eosinophils to either PGD2 or DK-PGD2. It is unclear whether the rat CRTh2 receptor may be downregulated during cell isolation (we did not have access to a cloned rat CRTh2 assay system), but the lack of in vitro response is concordant with recently reported observations by Schmidt et al. during the characterization of AZD-1981. The fluorescein isothiocyanate (FITC)-induced model of allergic contact dermatitis in the mouse is associated with both expression of the CRTh2 receptor and the production of PGD2, as well as displaying Th2 lymphocyte dependency.23, 24 Compound 18 showed a dose dependent inhibition of ear edema induced by sensitisation and challenge with the FITC hapten, exhibiting a minimum effective dose of 10mg/kg (Fig. 4). Encouragingly, this level of efficacy achieved by blockade of a single mediator receptor was comparable to that observed with the glucocorticoid dexamethasone which modulates multiple inflammatory gene expression pathways.