Investigating A Link Between Lipophilicity And Wash-Resistance

Some molecules continue to elicit a functional response even when free drug has been removed from the system. This ability to demonstrate long-lasting signalling, or wash-resistance, affects duration of drug action (1). Wash-resistance has been seen at the M1 receptor, notably with the M1 agonist xanomeline (2). M1 receptor agonists are potential therapies for Alzheimer’s Disease (3), where an extended duration of action could be beneficial. This study investigates the link between lipophilicity and wash-resistance.

Calculated partition coefficient (clogP) is anestimate of lipophilicity. Fifteen literature M1 receptor agonists were selected representinga range of clogP values (-6.2 to 5.8); acetylcholine, xanomeline, AC-42, 77-LH-28-1, AC-260584, a Merck M1 PAM (patentWO2010059773), cevimeline, a DaiNipponM1/M4agonist (4), n-desmethyl clozapine, AZD6088, milameline, McN-A-343, oxotremorine-M and iperoxo. An IPone assay (CisBio) was used to determine accumulation of IP1asmeasure of Gqactivityin response to M1 receptor activation. Briefly, CHO cells stably expressing the muscarinic M1 receptor were seeded at15,000 cells/well in 96 half-area plates. 16h after plating, media was removed and replaced with IPone buffer without LiCl (10mM HEPES, 1mM CaCl2, 0.5mM MgCl2, 4.2mM KCl, 146mM NaCl, 5.5mM D-glucose; pH7.4). Cells were treated withan EC80 of agonist(‘stimulated’) or with DMSO vehicle (‘basal’) and incubated (37°C) for 1h. LiCl prevents the breakdown of IP1 into myo-inositol, leading to accumulation of IP1. Therefore omitting LiCl prevents IP1accumulation and so no response is measured. Cells were subsequently washed (3 x 50µL) to remove compound, and 50µL of IPone buffer with 50mM LiCl added. As a control, cells that had not been pre-treated with challenge were treated with an EC80 of agonist (‘challenge’) after the wash step whilst, cells that were pre-treated with an EC80 of agonist, were only treated with vehicle after the wash step. After 30 min incubation (37°C) assay was terminated and read as per manufacturer’s instructions. An unpaired two-tailed t-test (Prism v6) was used to compare stimulated response to basal response. If p<0.05 the stimulated response was compared to challenge (unpaired two-tailed t-test).

Here, wash-resistance was defined as a significant increase in IP1 production in cells that had been pre-treated with an EC80of agonist (then washed) compared to basal. As LiCl was not presentin the pre-treatmentmedia, any response measured occurred after cells had been washed. No agonist with a clogP<3.0 exhibited wash-resistance (p>0.05). Xanomeline (clogP 4.4) was confirmed to be wash-resistant as, a significant increase in IP1 accumulation above basal (p<0.0001)was measured. Further statistical tests demonstrated that for xanomeline, the residual response was not significantly different to unwashed challenge (p>0.05). cLogP was not the only factor to cause wash resistance as AC-42, which had the highest cLogP tested (5.8), did not demonstrate wash-resistance (p>0.05). AC-260584 (clogP 4.9) appeared to show ‘partial’ wash resistance as the residual IP1 response was significantly higher than basal (p<0.001) but also significantly lower than the initial challenge (p<0.05).

A clogP ≥3.0 appears necessary, but not sufficient, for an agonist to exhibit wash-resistance at the M1 receptor. However, lipophilicity is not the only factor governing wash-resistance and further research is needed to determine what other parameters are important.

(1) De Lorme KC et al. (2006). Neurosci Lett. 410: 11-4.

(2) Christopoulos A et al. (1998). MolPharmacol. 53: 1120-30.

(3) Jiang S et al. (2014). Neurosci Bull. 30: 295-307.

(4) Takai K et al. (2014). Bioorg Med Chem Lett. 24: 3189-93.