The discovery of a selective and potent A2a agonist with extended lung retention.

Although the anti-inflammatory role of the A2a receptor is well established, controversy remains with regard to the therapeutic value for A2a agonists in treatment of inflammatory lung diseases, also as a result of unwanted A2a-mediated cardiovascular effects. In this paper, we describe the discovery and characterization of a new, potent and selective A2a agonist (compound 2) with prolonged lung retention and limited systemic exposure following local administration. To support the lead optimization chemistry program with compound selection and profiling, multiple in vitro and in vivo assays were used, characterizing compound properties, pharmacodynamics (PD), and drug concentrations. Particularly, pharmacokinetic-PD modeling was applied to quantify the effects on the cardiovascular system, and an investigative toxicology study in rats was performed to explore potential myocardial toxicities. Compound 2, in comparison to a reference A2a agonist, UK-432,097, demonstrated higher solubility, lower lipophilicity, lower plasma protein binding, high rat lung retention (28% remaining after 24 h), and was efficacious in a lung inflammatory rat model following intratracheal dosing. Despite these properties, compound 2 did not provide a sufficient therapeutic index, that is, separation of local anti-inflammatory efficacy in the lung from systemic side effects in the cardiovascular system. The plasma concentration that resulted in induction of hypotension (half maximal effective concentration; EC50 0.5 nmol/L) correlated to the in vitro A2a potency (rIC50 0.6 nmol/L). Histopathological lesions in the heart were observed at a dose level which is threefold above the efficacious dose level in the inflammatory rat lung model. In conclusion, compound 2 is a highly potent and selective A2a agonist with significant lung retention after intratracheal administration. Despite its local anti-inflammatory efficacy in rat lung, small margins to the cardiovascular effects suggested limited therapeutic value of this compound for treatment of inflammatory lung disease by the inhaled route.

Identification of novel substrates and structure-activity relationship of cellular uptake mediated by human organic cation transporters 1 and 2.

Recently the clinical importance of human organic cation transporters 1 (hOCT1/SLC22A1) and 2 (hOCT2/SLC22A2) in drug disposition, for example, clearance, toxicity, and drug-drug interactions, have been highlighted [Annu. Rev. Pharmacol. Toxicol. 2012, 52, 249-273; Nat. Rev. Drug Discovery 2010, 9 (3), 215-236]. Consequently, there is an extensive need for experimental assessment of structure-transport relationships as well as tools to predict drug uptake by these transporters in ADMET (absorption, distribution, metabolism, excretion, toxicity) investigations. In the present study, we developed a robust assay for screening unlabeled compound uptake by hOCT1 and hOCT2 using transfected HEK293 cells. For the first time, an extensive data set comprising uptake of 354 compounds is presented. As expected, there was a large overlap in substrate specificity between the two organic cation transporters. However, several compounds selectively taken up by either hOCT1 or hOCT2 were identified. In particular, a chemical series of phenylthiophenecarboxamide ureas was identified as selective hOCT1 substrates. Moreover, the drivers for transport differed: molecular volume was the most important determinant of hOCT1 substrates, whereas H-bonding parameters like polar surface area (PSA) dominated for hOCT2.

Explanation for main features of structure-genotoxicity relationships of aromatic amines by theoretical studies of their activation pathways in CYP1A2.

Aromatic and heteroaromatic amines (ArNH(2)) represent a class of potential mutagens that after being metabolically activated covalently modify DNA. Activation of ArNH(2) in many cases starts with N-hydroxylation by P450 enzymes, primarily CYP1A2. Poor understanding of structure-mutagenicity relationships of ArNH(2) limits their use in drug discovery programs. Key factors that facilitate activation of ArNH(2) are revealed by exploring their reaction intermediates in CYP1A2 using DFT calculations. On the basis of these calculations and extensive analysis of structure-mutagenicity data, we suggest that mutagenic metabolites are generated by ferric peroxo intermediate, (CYP1A2)Fe(III)-OO(-), in a three-step heterolytic mechanism. First, the distal oxygen of the oxidant abstracts proton from H-bonded ArNH(2). The subsequent proximal protonation of the resulting (CYP1A2)Fe(III)-OOH weakens both the O-O and the O-H bonds of the oxidant. Heterolytic cleavage of the O-O bond leads to N-hydroxylation of ArNH(-) via S(N)2 mechanism, whereas cleavage of the O-H bond results in release of hydroperoxy radical. Thus, our proposed reaction offers a mechanistic explanation for previous observations that metabolism of aromatic amines could cause oxidative stress. The primary drivers for mutagenic potency of ArNH(2) are (i) binding affinity of ArNH(2) in the productive binding mode within the CYP1A2 substrate cavity, (ii) resonance stabilization of the anionic forms of ArNH(2), and (iii) exothermicity of proton-assisted heterolytic cleavage of N-O bonds of hydroxylamines and their bioconjugates. This leads to a strategy for designing mutagenicity free ArNH(2): Structural alterations in ArNH(2), which disrupt geometric compatibility with CYP1A2, hinder proton abstraction, or strongly destabilize the nitrenium ion, in this order of priority, prevent genotoxicity.

Increasing selectivity of CC chemokine receptor 8 antagonists by engineering nondesolvation related interactions with the intended and off-target binding sites.

The metabolic stability and selectivity of a series of CCR8 antagonists against binding to the hERG ion channel and cytochrome Cyp2D6 are studied by principal component analysis. It is demonstrated that an efficient way of increasing metabolic stability and selectivity of this series is to decrease compound lipophilicity by engineering nondesolvation related attractive interactions with CCR8, as rationalized by three-dimensional receptor models. Although such polar interactions led to increased compound selectivity, such a strategy could also jeopardize the DMPK profile of compounds. However, once increased potency is found, the lipophilicity can be readjusted by engineering hydrophobic substituents that fit to CCR8 but do not fit to hERG. Several such lipophilic fragments are identified by two-dimensional fragment-based QSAR analysis. Electrophysiological measurements and site-directed mutagenesis studies indicated that the repulsive interactions of these fragments with hERG are caused by steric hindrances with residue F656.