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  • br Material and methods br Results br Discussion In


    Material and methods
    Discussion In vitro evidence indicates that LAS191859 is a potent CRTh2 antagonist as demonstrated in GTPγS binding studies with potency in the low nanomolar range and in addition it demonstrates a long receptor residence time. This potency is also maintained in cellular assays performed with human eosinophils and whole blood. In the functional assay measuring inhibition of PGD2-mediated GTPγS binding, LAS191589 depressed the agonist binding capacity producing a progressive reduction of maximal response at 2h of incubation that was observed even after 22h of incubation time therefore showing a clear functional insurmountability. This behavior can be related to several possible mechanisms of action including: slow dissociation of antagonist from the receptor, interaction of the antagonist with allosteric OSI-930 that induces changes in conformation in the receptor preventing proper agonist binding, irreversible covalent binding or even antagonist-mediated receptor internalization [35], [36]. We and others have described that CRTh2 antagonists can have slow dissociation kinetics from the receptor [37], [38], [39] and this was our first hypothesis to explain the observed unsurmountable behavior of our compound. We confirmed this hypothesis for LAS191589 in the dissociation studies for human and guinea pig CRTh2 receptors using the [35S]-GTPγS functional assay, demonstrating long receptor residence time for the human and guinea pig receptors, with half lives of 21h and 16h, respectively. Also, Schild plots discarded the allosteric effect as the unsurmontability profile was overcome with time. In the process of these studies it was intriguing to note that related compounds whose only structural difference was presence or absence of a single methyl group could make a huge difference in terms of receptor kinetics. Changes from around 2h up to 21h could be observed adding a methyl group improving dissociation half-life by an order of magnitude while maintaining same potency [40]. Considering reported CRTh2 antagonists, receptor kinetics makes LAS191859 unique, as reported dissociation half-lives are typically in the range of minutes compared with the 21h of LAS191859 [37], [38], [32]. Mechanistic in vivo studies of inhibition of the systemic eosinophilia in guinea pig showed that the compound is also very potent in vivo with an ID50 of 13.25μg/kg after 2h of compound administration. Administration of a single 0.3mg/kg oral dose of LAS191589 leads to a complete inhibition of DK-PGD2-induced systemic eosinophilia. Potencies reported for other CRTh2 antagonists in similar models showed lower in vivo potency: ID50 for AM211 was calculated to be 850μg/kg in a guinea pig model measuring blood leukocytosis [33] and for OC000459 the effect on systemic eosinophilia induced by DK-PGD2 in a rat model, ID50 was reported to be 40μg/kg [34]. Duration of action studies by dosing LAS191859 18h before the administration of the agonist showed a shift in potency but still an inhibition (approx. 60%) that was maintained at a time point where plasma levels were no longer relevant to block the receptor. These are relevant findings proving that a long receptor residence time in the CRTh2 receptor translates into a long in vivo duration of action without pharmacological active plasma levels. This finding indicates that a disconnection between the pharmacokinetic (PK) and the pharmacodynamic (PD) profiles has been observed for LAS191859 in guinea pig. Toxicological and safety studies performed to complete the profile of LAS191589 showed that so far, it is a safe compound. No issues have been detected in any of the studies performed in terms of cytotoxicity, cardiovascular safety, central nervous system safety and genotoxicity. No relevant adverse effects have been observed in 1, 4, and 14days toxicological studies in different preclinical species. Although it is premature to postulate that a slowly dissociating CRTh2 antagonist with the potential to produce long lasting receptor blockade will be an advantage for asthma treatment, other compounds with similar characteristics exist in the market. A number of drugs have slow dissociation rate, including among others, angiotensin receptor antagonists like candesartan (dissociation half-life: 11.5h), muscarinic M3 receptor antagonists like tiotropium and aclidinium (dissociation half-life: 62h and 29h, respectively) or histamine H1R antagonist such as desloratadine (dissociation half-life: >6h), that contribute to their therapeutic effect and duration of action [41], [42], [43], [44].