Summary /

Selective profiling of ML355 and selected analogs showed inactivity of ML355 against human reticulocyte 15-lipoxygenase-1 (15-LOX-1), human epithelial 15-lipoxygenase-2 (15 LOX-2) and arachidonate 5-lipoxygenase (5-LOX) but good inhibition against human platelet 12-lipoxygenase (12-LOX) with 0.29 μM potency.

Table 1. Selectivity profiling of ML355 and other top compounds. NT = not tested while NI = No inhibition.

Summary /

Previously reported inhibitors of 12-LOX (Table 2) such as baicalein (Svensson 2013) and nor-dihydroguairetic acid (NDGA) (Whitman 2002), “bromo-phenols” or “pyrazole derivatives” all possess several liabilities. These compounds are not only less potent, selective, but are also not easily amendable to further optimization. Our previously described 12-LOX inhibitor (ML127) demonstrates potent inhibition (<500 nM) and excellent selectivity but is not very tolerant of structural modifications (Kenyon 2011). Few regions of the molecule are amendable for optimization limiting available avenues for medicinal chemistry efforts. In addition, while the 8-HQ scaffold of ML127 has not yet emerged as a liability for the series, the known promiscuous metal chelation for related compounds of that type suggested one should proceed with caution. In comparison, the new ML355 probe series demonstrated potent (290 nM) activity towards 12-LOX and excellent selectivity against related enzymes 15-LOX-1, 5-LOX, 15-LOX-2, and COX ½ (Table 1). Additionally, this chemotype is structurally distinct from all previously reported inhibitors (including ML127) and it possesses a very drug-like scaffold. This series is readily amendable to structural modifications and displays clear and tractable SAR. Most importantly, ML355 exhibits a favorable in vitro ADME and in vivo PK profile with activity in disease relevant cell-based systems like diabetes through 12-HETE reduction in β-cells.

Table 2. Comparison of ML355 to previously identified 12-LOX inhibitors. ND = Not Determined.

Summary /

Mouse beta cells (BTC3) were treated with arachidonic acid and calcium ionophore (AA/IONO) alone or in the presence of ML355. Result showed inhibition of 12-HETE expression by the ML355 probe.

Figure 1. Dose dependent inhibition of stimulated 12-HETE by ML355. Graphed are the levels of 12-HETE expressed as a percentage of that detected in cells stimulated with AA/IONO alone. 12-HETE was measured by ELISA.

Summary /

Result showed inhibition of the of the 12-HETE expression on stimulated human islets upon treatment of ML355.

Figure 2. Histogram of 12-HETE expression (measured by ELISA) in human primary donor islets stimulated with Arachidonic acid and calcium ionophore (AA/IONO) alone or in the presence of 10 μM of ML355.

Summary /

ML355 demonstrated excellent microsomal stability with both rat (T1/2 >30 minutes) and mouse (T1/2 >300 minutes) and was found to be stable to mouse plasma over a 2 hour period (100% remaining). ML355 showed no degradation over various aqueous buffers (pH 2-9) and was stable to 5 mM glutathione suggesting excellent stability. One remaining liability is the aqueous solubility which is <5 μM, however improved solubility is observed in the assay buffer (qualitative analysis). ML355 showed moderate permeability in the Caco-2 assay (1.5 × 10 cm/s) and does not appear to be a substrate for Pgp given the efflux ratio of <2. In vivo PK studies where ML355 was administered as a solution via IV (3mpk) and PO (30mpk) demonstrated that ML355 is orally bioavailable (%F = 20) with good half-life (T1/2 = 2.9 hours). At 30 mpk dosing, ML355 achieves a Cmax of over 135 times the in vitro IC50 and remains over IC50 value for over 12 hours. The compound has low clearance (3.4 mL/min/kg) and good overall exposure (AUCinf ) of 38 μM. Although, the volume of distribution (V ) observed was low (0.55 L/kg), the rest of the PK profiling results suggested a reasonable distribution between tissue and blood. These profiling results provided informative data necessary to develop an appropriate dosing regimen for future in vivo studies.