Angela Styhler

Development of a Liver-Targeted Stearoyl-CoA Desaturase (SCD) Inhibitor (MK-8245) to Establish a Therapeutic Window for the Treatment of Diabetes and Dyslipidemia

The potential use of SCD inhibitors for the chronic treatment of diabetes and dyslipidemia has been limited by preclinical adverse events associated with inhibition of SCD in skin and eye tissues. To establish a therapeutic window, we embarked on designing liver-targeted SCD inhibitors by utilizing molecular recognition by liver-specific organic anion transporting polypeptides (OATPs). In doing so, we set out to target the SCD inhibitor to the organ believed to be responsible for the therapeutic efficacy (liver) while minimizing its exposure in the tissues associated with mechanism-based SCD depletion of essential lubricating lipids (skin and eye). These efforts led to the discovery of MK-8245 (7), a potent, liver-targeted SCD inhibitor with preclinical antidiabetic and antidyslipidemic efficacy with a significantly improved therapeutic window.

The effects of phosphodiesterase type 4 inhibitors on tumour necrosis factor-α and leukotriene B4 in a novel human whole blood assay

The aim of this study was to assess the inhibitory activities of phosphodiesterase type 4 (PDE4) inhibitors on tumour necrosis factor‐α (TNF‐α) and leukotriene B4 (LTB4) production in a novel human whole blood assay. Lipopolysaccharide (LPS) stimulation of human whole blood caused a time dependent increase in TNF‐α and prostaglandin E2 (PGE2) plasma levels. Inhibition of LPS‐induced TNF‐α by the selective PDE4 inhibitor RP73401 was proportionally enhanced with endogenous PGE2 (maximal after 24 h). In contrast, blocking endogenous PGE2 production with indomethacin in blood stimulated with LPS for 24 h decreased the potency of RP73401 to that observed with a 4 h LPS incubation. Non‐selective and selective PDE4 inhibitors showed greater inhibition of LPS‐induced TNF‐α after 24 h compared to 4 h. Stereoselectivity was only achieved in the 24 h method. LPS‐stimulation of whole blood for either 30 min or 24 h followed by N‐formyl‐Met‐Leu‐Phe (fMLP) activation resulted in low plasma LTB4 levels. Combination of both treatments resulted in a greater than 7 fold increase in plasma LTB4 levels. Inhibition of the double LPS and fMLP‐activated LTB4 production was observed with non‐selective and PDE4‐selective inhibitors. Their LTB4 inhibitory potencies were similar to that observed in the 24 h LPS‐induced TNF‐α assay. Thus, stimulation of human whole blood with two LPS stimulations followed by fMLP gives rise to both TNF‐α and LTB4 and their inhibition by various compounds can be assessed in the same blood sample. Calcium ionophore (A23187) stimulation of whole blood resulted in plasma LTB4 levels similar to the double LPS and fMLP method. Inhibition of A23187‐induced LTB4 biosynthesis was also achieved by PDE4‐selective inhibitors as well as the direct 5‐lipoxygenase (5‐LO) inhibitor L‐739,010. These results confirm the anti‐inflammatory properties of PDE4 inhibitors. Thus, this novel human whole blood can be used to assess the biochemical efficacy of PDE4 inhibitors in human subjects.

In vitro metabolism considerations, including activity testing of metabolites, in the discovery and selection of the COX-2 inhibitor etoricoxib (MK-0663)

Characterization of the metabolites of the COX-2 inhibitor etoricoxib (MK-0663 and L-791,456) produced in vitro indicate formation of an N-oxide pyridine and hydroxymethyl pyridine that can further be glucuronidated or oxidized to an acid. Significant turnover is observed in human hepatocytes. Several CYPs are involved in the oxidative biotranformations and, from in vitro studies, etoricoxib is not a potent CYP3A4 inducer or inhibitor. Based on an in vitro whole blood assay, none of the metabolites of etoricoxib inhibits COX-1 or contributes significantly to the inhibition of COX-2.

The Discovery of Setileuton, a Potent and Selective 5-Lipoxygenase Inhibitor

The discovery of novel and selective inhibitors of human 5-lipoxygenase (5-LO) is described. These compounds are potent, orally bioavailable, and active at inhibiting leukotriene biosynthesis in vivo in a dog PK/PD model. A major focus of the optimization process was to reduce affinity for the human ether-a-go-go gene potassium channel while preserving inhibitory potency on 5-LO. These efforts led to the identification of inhibitor (S)-16 (MK-0633, setileuton), a compound selected for clinical development for the treatment of respiratory diseases.

Discovery of MK-0952, a selective PDE4 inhibitor for the treatment of long-term memory loss and mild cognitive impairment

The structure-activity relationship of a novel series of 8-biarylnaphthyridinones acting as type 4 phosphodiesterase (PDE4) inhibitors for the treatment of long-term memory loss and mild cognitive impairment is described herein. The manuscript describes a new paradigm for the development of PDE4 inhibitor targeting CNS indications. This effort led to the discovery of the clinical candidate MK-0952, an intrinsically potent inhibitor (IC(50)=0.6 nM) displaying limited whole blood activity (IC(50)=555 nM). Supporting in vivo results in two preclinical efficacy tests and one test assessing adverse effects are also reported. The comparative profiles of MK-0952 and two other Merck compounds are described to validate the proposed hypothesis.

Nicotinic acids: Liver-targeted SCD inhibitors with preclinical anti-diabetic efficacy

An in vitro screening protocol was used to transform a systemically-distributed SCD inhibitor into a liver-targeted compound. Incorporation of a key nicotinic acid moiety enables molecular recognition by OATP transporters, as demonstrated by uptake studies in transfected cell lines, and likely serves as a critical component of the observed liver-targeted tissue distribution profile. Preclinical anti-diabetic oGTT efficacy is demonstrated with nicotinic acid-based, liver-targeting SCD inhibitor 10, and studies with a close-structural analog devoid of SCD1 activity, suggest this efficacy is a result of on-target activity.

Peripheral Phosphodiesterase 4 Inhibition Produced by 4-[2-(3,4-Bis-difluoromethoxyphenyl)-2-[4-(1,1,1,3,3,3-hexafluoro-2-hydroxypropan-2-yl)-phenyl]-ethyl]-3-methylpyridine-1-oxide (L-826,141) Prevents Experimental Autoimmune Encephalomyelitis

Administration of phosphodiesterase 4 (PDE4) inhibitors suppresses the pathogenesis associated with experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). In the present study, we compared the effects of rolipram and 4-[2-(3,4-bis-difluoromethoxyphenyl)-2-[4-(1,1,1,3,3,3-hexafluoro-2-hydroxypropan-2-yl)-phenyl]-ethyl]-3-methylpyridine-1-oxide (L-826,141), a novel nonbrain penetrant PDE4 inhibitor, on the onset and severity of clinical signs in a chronic, nonrelapsing/remitting model of EAE. Both rolipram (10 mg/kg p.o.) and L-826,141 (3 mg/kg p.o.) reduced the severity of EAE relative to controls, whereas L-826,141 (3 mg/kg p.o.) also delayed disease onset. To assess whether L-826,141 prevented EAE progression after the first signs of clinical onset, rolipram (10 mg/kg p.o.) or L-826,141 (3 or 30 mg/kg p.o.) were administered 24 h after the first signs of EAE were observed. Only L-826,141 at a dose of 30 mg/kg p.o. significantly decreased the clinical severity of EAE compared with vehicle controls. Immunohistochemical detection of the neuronal activity marker Fos confirmed that L-826,141 did not reach concentrations in the central nervous system sufficient to activate central neurons. Lipopolysaccharide-induced tumor necrosis factor-α in whole blood and plasma concentrations of L-826,141 revealed that only the 30-mg/kg dose resulted in levels sufficient to produce a near complete inhibition of PDE4 activity in immune cells. Taken together, these results demonstrate that peripheral PDE4 inhibition, produced by L-826,141, prevents the progression of EAE after the first onset of clinical signs, and suggest that similar compounds may have clinical efficacy in the treatment of MS.

A Multiplexed Cell Assay in HepG2 Cells for the Identification of Delta-5, Delta-6, and Delta-9 Desaturase and Elongase Inhibitors

A multiplexed cell assay has been optimized to measure the activities of fatty acyl-CoA elongase, delta-5 desaturase (Δ5D), delta-6 desaturase (Δ6D), and delta-9 desaturase (Δ9D) together using 14C-labeled tracers in HepG2 cells, which express the human stearoyl-CoA desaturase-1 isoform (SCD1) exclusively. The Δ5 and Δ9 desaturase activities are indexed by the efficient conversion of [1-14C]-eicosatrienoic acid (C20:3, cis-8,11,14) to 14C-arachidonic acid (C20:4, cis-5,8,11,14) and the conversion of [1-14C]-stearic acid to 14C-oleic acid (C18:1, cis-9), respectively. CP-74006 potently blocks the Δ5D activity with an IC50 value of 20 nM and simplifies the metabolism of [1-14C]-α-linolenate (C18:3, cis-9,12,15) by accumulating 14C-eicosatetraenoic acid (C20:4, cis-8,11,14,17) as the major 14C-eicosatrienoic acid (C20:3, cis-11,14,17) and 14C-docosatetraenoic acid (C22:4, cis-10,13,16,19) as the minor metabolites through Δ6 desaturation and elongation. This simplified metabolite spectrum enables the delineation of the Δ6D activity by comparing the combined Δ6D/elongase activity index of the 14C-(C20:4/C18:3) ratio with the corresponding elongation index of the 14C-(C20:3/C18:3) ratio following compound treatment. SC-26196 and sterculic acid specifically inhibit the Δ6D and Δ9D activities with an IC50 value of 0.1 µM and 0.9 µM, respectively. This medium-throughput cell assay provides an efficient tool in the identification of specific desaturase and elongase inhibitors.

Optimization and structure-activity relationship of a series of 1-phenyl-1,8-naphthyridin-4-one-3-carboxamides: identification of MK-0873, a potent and effective PDE4 inhibitor.

A SAR study of a series of 1-phenyl-1,8-naphthyridin-4-one-3-carboxamides is described. Optimization of the series was based on in vitro potency against PDE4, inhibition of the LPS-induced production of TNF-alpha in human whole blood and minimizing affinity for the hERG potassium channel. From these studies, compounds 18 and 20 (MK-0873) were identified as optimized PDE4 inhibitors with good overall in vitro and in vivo profiles and selected as development candidates.

Substituted 2-pyridinemethanol derivatives as potent and selective phosphodiesterase-4 inhibitors

The synthesis and the phosphodiesterase-4 (PDE4) inhibitory activity of 2-pyridinemethanol derivatives is described. The evaluation of the structure-activity relationship (SAR) in this series of novel PDE4 inhibitors led to the identification of compound 9 which exhibits excellent in vitro activity, desirable pharmacokinetic parameters and good efficacy in animal models of bronchoconstriction.