Lijing Xu

The discovery of rofecoxib, [MK 966, VIOXX®, 4-(4′-methylsulfonylphenyl)-3-phenyl-2(5H)-furanone], an orally active cyclooxygenase-2 inhibitor

The development of a COX-2 inhibitor rofecoxib (MK 966, Vioxx) is described. It is essentially equipotent to indomethacin both in vitro and in vivo but without the ulcerogenic side effect due to COX-1 inhibition.

Biochemical and pharmacological profile of a tetrasubstituted furanone as a highly selective COX-2 inhibitor

DFU (5,5‐dimethyl‐3‐(3‐fluorophenyl)‐4‐(4‐methylsulphonyl)phenyl‐2(5H)‐furanone) was identified as a novel orally active and highly selective cyclo‐oxygenase‐2 (COX‐2) inhibitor. In CHO cells stably transfected with human COX isozymes, DFU inhibited the arachidonic acid‐dependent production of prostaglandin E2 (PGE2) with at least a 1,000 fold selectivity for COX‐2 (IC50=41±14 nM) over COX‐1 (IC50>50 μM). Indomethacin was a potent inhibitor of both COX‐1 (IC50=18±3 nM) and COX‐2 (IC50=26±6 nM) under the same assay conditions. The large increase in selectivity of DFU over indomethacin was also observed in COX‐1 mediated production of thromboxane B2 (TXB2) by Ca2+ ionophore‐challenged human platelets (IC50>50 μM and 4.1±1.7 nM, respectively). DFU caused a time‐dependent inhibition of purified recombinant human COX‐2 with a Ki value of 140±68 μM for the initial reversible binding to enzyme and a k2 value of 0.11±0.06 s−1 for the first order rate constant for formation of a tightly bound enzyme‐inhibitor complex. Comparable values of 62±26 μM and 0.06±0.01 s−1, respectively, were obtained for indomethacin. The enzyme‐inhibitor complex was found to have a 1 : 1 stoichiometry and to dissociate only very slowly (t1/2=1–3 h) with recovery of intact inhibitor and active enzyme. The time‐dependent inhibition by DFU was decreased by co‐incubation with arachidonic acid under non‐turnover conditions, consistent with reversible competitive inhibition at the COX active site. Inhibition of purified recombinant human COX‐1 by DFU was very weak and observed only at low concentrations of substrate (IC50=63±5 μM at 0.1 μM arachidonic acid). In contrast to COX‐2, inhibition was time‐independent and rapidly reversible. These data are consistent with a reversible competitive inhibition of COX‐1. DFU inhibited lipopolysaccharide (LPS)‐induced PGE2 production (COX‐2) in a human whole blood assay with a potency (IC50=0.28±0.04 μM) similar to indomethacin (IC50=0.68±0.17 μM). In contrast, DFU was at least 500 times less potent (IC50>97 μM) than indomethacin at inhibiting coagulation‐induced TXB2 production (COX‐1) (IC50=0.19±0.02 μM). In a sensitive assay with U937 cell microsomes at a low arachidonic acid concentration (0.1 μM), DFU inhibited COX‐1 with an IC50 value of 13±2 μM as compared to 20±1 nM for indomethacin. CGP 28238, etodolac and SC‐58125 were about 10 times more potent inhibitors of COX‐1 than DFU. The order of potency of various inhibitors was diclofenac>indomethacin∼naproxen>nimesulide∼ meloxicam∼piroxicam>NS‐398∼SC‐57666>SC‐58125>CGP 28238∼etodolac>L‐745,337>DFU. DFU inhibited dose‐dependently both the carrageenan‐induced rat paw oedema (ED50 of 1.1 mg kg−1 vs 2.0 mg kg−1 for indomethacin) and hyperalgesia (ED50 of 0.95 mg kg−1 vs 1.5 mg kg−1 for indomethacin). The compound was also effective at reversing LPS‐induced pyrexia in rats (ED50=0.76 mg kg−1 vs 1.1 mg kg−1 for indomethacin). In a sensitive model in which 51Cr faecal excretion was used to assess the integrity of the gastrointestinal tract in rats, no significant effect was detected after oral administration of DFU (100 mg kg−1, b.i.d.) for 5 days, whereas chromium leakage was observed with lower doses of diclofenac (3 mg kg−1), meloxicam (3 mg kg−1) or etodolac (10–30 mg kg−1). A 5 day administration of DFU in squirrel monkeys (100 mg kg−1) did not affect chromium leakage in contrast to diclofenac (1 mg kg−1) or naproxen (5 mg kg−1). The results indicate that COX‐1 inhibitory effects can be detected for all selective COX‐2 inhibitors tested by use of a sensitive assay at low substrate concentration. The novel inhibitor DFU shows the lowest inhibitory potency against COX‐1, a consistent high selectivity of inhibition of COX‐2 over COX‐1 (>300 fold) with enzyme, whole cell and whole blood assays, with no detectable loss of integrity of the gastrointestinal tract at doses >200 fold higher than efficacious doses in models of inflammation, pyresis and hyperalgesia. These results provide further evidence that prostanoids derived from COX‐1 activity are not important in acute inflammatory responses and that a high therapeutic index of anti‐inflammatory effect to gastropathy can be achieved with a selective COX‐2 inhibitor.

The C3a Anaphylatoxin Receptor Is a Key Mediator of Insulin Resistance and Functions by Modulating Adipose Tissue Macrophage Infiltration and Activation

OBJECTIVE Significant new data suggest that metabolic disorders such as diabetes, obesity, and atherosclerosis all posses an important inflammatory component. Infiltrating macrophages contribute to both tissue-specific and systemic inflammation, which promotes insulin resistance. The complement cascade is involved in the inflammatory cascade initiated by the innate and adaptive immune response. A mouse genomic F2 cross biology was performed and identified several causal genes linked to type 2 diabetes, including the complement pathway. RESEARCH DESIGN AND METHODS We therefore sought to investigate the effect of a C3a receptor (C3aR) deletion on insulin resistance, obesity, and macrophage function utilizing both the normal-diet (ND) and a diet-induced obesity mouse model. RESULTS We demonstrate that high C3aR expression is found in white adipose tissue and increases upon high-fat diet (HFD) feeding. Both adipocytes and macrophages within the white adipose tissue express significant amounts of C3aR. C3aR−/− mice on HFD are transiently resistant to diet-induced obesity during an 8-week period. Metabolic profiling suggests that they are also protected from HFD-induced insulin resistance and liver steatosis. C3aR−/− mice had improved insulin sensitivity on both ND and HFD as seen by an insulin tolerance test and an oral glucose tolerance test. Adipose tissue analysis revealed a striking decrease in macrophage infiltration with a concomitant reduction in both tissue and plasma proinflammatory cytokine production. Furthermore, C3aR−/− macrophages polarized to the M1 phenotype showed a considerable decrease in proinflammatory mediators. CONCLUSIONS Overall, our results suggest that the C3aR in macrophages, and potentially adipocytes, plays an important role in adipose tissue homeostasis and insulin resistance.

FROM INDOMETHACIN TO A SELECTIVE COX-2 INHIBITOR Development of Indolalkanoic Acids as Potent and Selective Cyclooxygenase-2 Inhibitors

Abstract A series of potent and highly selective cyclooxygenase-2 inhibitors have been prepared by replacing the benzoyl group of indomethacin with a 4-bromobenzyl group, and by extending the acetic acid side chain. These compounds show anti-inflammatory activity in rats with no evidence of GI toxicity, even at high doses.

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.

Synthesis and biological evaluation of 5,6-diarylimidazo[2.1-b]thiazole as selective COX-2 inhibitors

Abstract A series of 5,6-diarylimidazo[2.1-b]thiazole compounds were prepared and their inhibitory potencies against COX-2 and Cox-1 enzymes were measured. This led to the identification of L-766,112 as a potent, orally active and selective inhibitor of the COX-2 enzyme.

A new series of selective COX-2 inhibitors: 5,6-diarylthiazolo[3,2-b][1,2,4]triazoles

A series of 5,6-diarylthiazolo[3,2-b][1,2,4]triazoles was prepared for evaluation of potency and selectivity against human COX-1 and COX-2 enzymes. This lead to the discovery of L-768,277, a potent and selective COX-2 inhibitor that also demonstrated good in vivo activity.

Synthesis and biological evaluation of 2,3-diarylthiophenes as selective cox-2 inhibitors. part II: Replacing the heterocycle

Abstract The thiophene ring of DuP 697 was replaced by a variety of heterocycles and the products were tested for their ability to inhibit human Cox-2 and Cox-1, the isozymes of cyclooxygenase.

SAR in the alkoxy lactone series: the discovery of DFP, a potent and orally active COX-2 inhibitor.

Extensive SAR has been established in the alkoxy lactone series and this has lead to the discovery of DFP (5,5-dimethyl-3-(2-propoxy)-4-methanesulfonylphenyl)-2(5H)-furanon e), a potent COX-2 inhibitor exhibiting in vivo efficacy in all models studied.

Reduction of animal usage by serial bleeding of mice for pharmacokinetic studies : application of robotic sample preparation and fast liquid chromatography-mass spectrometry

Typically, pharmacokinetic studies in mice require one animal per time point, thus resulting in differences due to dosing error, animal to animal variation and more importantly the euthanasia of a large number of animals. A method for the determination of pharmacokinetic data from serially bled mice to support early drug discovery is described. Sample analysis relies on liquid chromatography coupled with tandem mass spectrometry permitting robust and reproducible analysis requiring approximately 3 min per sample. Several parameters are discussed including the method of sample collection, preparation and analysis. The use of serially bled mice has lead to a remarkable reduction in animal usage and a corresponding reduction in compound required for such experiments. Using conventional methodology, a nine-point pharmacokinetic curve with four animals per time point would require 36 mice. With the method described below, only four mice in total are used and euthanasia is not required, permitting reuse after several weeks recovery and washout. Also, pharmacodynamic-pharmacokinetic correlation is possible and is demonstrated using a mouse model of diabetes.