Wanda Cromlish

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.

A Natural Disruption of the Secretory Group II Phospholipase A2 Gene in Inbred Mouse Strains

The synovial fluid or group II secretory phospholipase A2 (sPLA2) has been implicated as an important agent involved in a number of inflammatory processes. In an attempt to determine the role of sPLA2 in inflammation, we set out to generate sPLA2-deficient mice. During this investigation, we observed that in a number of inbred mouse strains, the sPLA2 gene was already disrupted by a frameshift mutation in exon 3. This mutation, a T insertion at position 166 from the ATG of the cDNA, terminates out of frame in exon 4, resulting in the disruption of the calcium binding domain in exon 3 and loss of both activity domains coded by exons 4 and 5. The mouse strains C57BL/6, 129/Sv, and B10.RIII were found to be homozygous for the defective sPLA2 gene, whereas outbred CD-1:SW mice had variable genotype at this locus. BALB/c, C3H/HE, DBA/1, DBA/2, NZB/B1N, and MRL lpr/lpr mice had a normal sPLA2 genotype. The sPLA2 mRNA was expressed at very high levels in the BALB/c mouse small intestine, whereas in the small intestine of the sPLA2 mutant mouse strains, sPLA2 mRNA was undetectable. In addition, PLA2 activity in acid extracts of the small intestine were approximately 40 times higher in BALB/c than in the mutant mice. Transcription of the mutant sPLA2 gene resulted in multiple transcripts due to exon skipping. None of the resulting mutant mRNAs encoded an active product. The identification of this mutation should not only help define the physiological role of sPLA2 but also has important implications in mouse inflammatory models developed by targeted mutagenesis.

The discovery of odanacatib (MK-0822), a selective inhibitor of cathepsin K.

Odanacatib is a potent, selective, and neutral cathepsin K inhibitor which was developed to address the metabolic liabilities of the Cat K inhibitor L-873724. Substituting P1 and modifying the P2 side chain led to a metabolically robust inhibitor with a long half-life in preclinical species. Odanacatib was more selective in whole cell assays than the published Cat K inhibitors balicatib and relacatib. Evaluation in dermal fibroblast culture showed minimal intracellular collagen accumulation relative to less selective Cat K inhibitors.

Mechanism of selective inhibition of human prostaglandin G/H synthase-1 and -2 in intact cells.

Selective inhibitors of prostaglandin synthase-2 (PGHS-2) possess potent anti-inflammatory, antipyretic, and analgesic properties but demonstrate reduced side-effects (e.g. gastrotoxicity) when compared with nonselective inhibitors of PGHS-1 and -2. We investigated the mechanism of the differential inhibition of human PGHS-1 (hPGHS-1) and -2 (hPGHS-2) in intact cells by nonsteroidal anti-inflammatory drugs (NSAIDs) and examined factors that contribute to the increased potency of PGHS inhibitors observed in intact cells versus cell-free systems. In intact Chinese hamster ovary (CHO) cell lines stably expressing the hPGHS isozymes, both PGHS isoforms exhibited the same affinity for arachidonic acid. Exogenous and endogenous arachidonic acid were used as substrates by both CHO [hPGHS-1] and CHO [hPGHS-2] cell lines. However, differences were observed in the ability of the hPGHS isoforms to utilize endogenous arachidonic acid released intracellularly following calcium ionophore stimulation or released by human cytosolic phospholipase A2 transiently expressed in the cells. Cell-based screening of PGHS inhibitors demonstrated that the selectivities and potencies of PGHS inhibitors determined using intact cells are affected by substrate concentration and differ from that determined in cell-free microsomal or purified enzyme preparations of PGHS isozymes. The mechanism of inhibition of PGHS isozymes by NSAIDs in intact cells involved difference in their time-dependent inhibition. Indomethacin displayed time-dependent inhibition of cellular hPGHS-1 and -2. In contrast, the selective PGHS-2 inhibitor NS-398 exhibited time-independent inhibition of hPGHS-1 but time-dependent inhibition of hPGHS-2 in intact cells. Reversible inhibition of cellular CHO [hPGHS-1] and CHO [hPGHS-2] was observed with the nonselective NSAIDs ibuprofen and indomethacin, whereas inhibition by the selective PGHS-2 inhibitor DuP-697 was reversible against hPGHS-1 but irreversible against hPGHS-2.

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.

Selective inhibition of cyclooxygenase-1 and -2 using intact insect cell assays

We have utilized the baculovirus expression system to develop an in vitro intact cell assay for screening nonsteroidal anti-inflammatory drug (NSAID) inhibition of the two isozymes of human cyclooxygenase (prostaglandin endoperoxidase synthase, EC 1.14.99.1). Infected Spodoptera frugiperda (sf9) cells expressing either human cyclooxygenase-1 (hCOX-1) or human cyclooxygenase-2 (hCOX-2) were harvested 24 hr postinfection, a time point where all cells are viable and hCOX-1 or hCOX-2 are correctly processed. Cells were distributed to a 96-well plate, preincubated with various NSAIDs, and challenged with 10 microM arachidonic acid; then cyclooxygenase activity was assessed indirectly by prostaglandin E2-specific radioimmunoassay. The rank order of potency of NSAID-mediated inhibitions of hCOX-1 and hCOX-2 paralleled those that have been observed in other cell systems. This sf9 cell-based assay can be utilized for the identification of potent and selective inhibitors of hCOX-1 and/or hCOX-2. Compounds that preferentially inhibit hCOX-2 may provide novel NSAIDs that reduce inflammation while sparing the stomach and kidneys of toxic side-effects seen with current nonselective NSAIDs.

CHARACTERIZATION OF AUTOCRINE INDUCIBLE PROSTAGLANDIN H SYNTHASE-2 (PGHS-2) IN HUMAN OSTEOSARCOMA CELLS

Abstract. The human osteosarcoma 143.98.2 cell line was found to express high levels of prostaglandin synthase-2 (PGHS-2) without detectable levels of prostaglandin synthase-1 (PGHS-1) as measured by reverse transcriptase-polymerase chain reaction (RT-PCR) and immunoblot analysis. Maximal levels of PGHS-2 induction were attained when the cells were grown beyond confluence. The osteosarcoma cells also secrete IL-1, IL-1 and TNF in the culture medium. PGHS-2 expression was inducible by the exogenous addition of these cytokines as well as conditioned media from auto-induced cultures and inhibitable by treatment with dexamethasone. In contrast, undifferentiated U937 cells selectively express PGHS-1 as analyzed by RT-PCR and Western blotting. The effects of non-steroidal anti-inflammatory drugs (NSAIDs) on the cellular PGE2 production mediated by each isoform of human PGHS were determined using osteosarcoma and undifferentiated U937 cells. When cells were p tors to allow time-dependent inhibition prior to arachidonic acid stimulation, NS-398, CGP 28238, L-745,337, SC-58125 all behaved as potent (IC50 = 1 - 30 nM) and selective inhibitors of PGHS-2, in contrast to indomethacin, flurbiprofen or diclofenac which are potent inhibitors of both enzymes. DuP-697 and sulindac sulfide were also potent inhibitors of PGHS-2 but both compounds inhibited cellular PGHS-1 activity at higher doses (IC50 = 0.2 = 0.4 M). Time-dependent inhibition of PGE2 production in osteosarcoma cells was observed for indomethacin, diclofenac and etodolac. The synthesis of PGE2 by U937 cells was strongly dependent on exogenous arachidonic acid (100-fold stimulation) whereas confluent osteosarcoma cells also produced PGE2 without exogenous stimulus (7-fold stimulation by arachidonic acid). Osteosarcoma cells grown beyond confluence released m ore PGE2 from endogenous substrate than arachidonic acid stimulated undifferentiated U937 cells. These results indicate that osteosarcoma cells selectively express PGHS-2 with an autocrine regulation and effective utilization of endogenous arachidonic acid for PGE2 synthesis.