S. Charleson

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.

5-lipoxygenase-activating protein is an arachidonate binding protein.

5‐Lipoxygenase‐activating protein (FLAP) is an 18‐kDa integral membrane protein which is essential for cellular leukotriene (LT) synthesis, and is the target of LT biosynthesis inhibitors. However, the mechanism by which FLAP activates 5‐LO has not been determined. We have expressed high levels of human FLAP in Spodoptera frugiperda (Sf9) insect cells infected with recombinant baculovirus, and used this system to demonstrate that FLAP specifically binds [125I]L‐739,059, a novel photoaffinity analog of arachidonic acid. This binding is inhibited by both arachidonic acid and MK‐886, an LT biosynthesis inhibitor which specifically interacts with FLAP. These studies suggest that FLAP may activate 5‐LO by specifically binding arachidonic acid and transferring this substrate to the enzyme.

Urinary leukotriene E4 levels during early and late asthmatic responses

The sulphidopeptide leukotrienes C4 and D4 (LTC4, LTD4) are potent bronchoconstrictor mediators, released from human lung fragments after challenge with specific allergens in vitro. The purpose of this study was to measure urinary LTE4 (metabolite of LTC4 and LTD4) in subjects undergoing inhalation challenges with allergens or occupational sensitizing agents in the laboratory. Eighteen subjects with previously documented isolated early asthmatic responses (EARs), isolated late asthmatic responses (LARs), or dual (both early and late) asthmatic responses were studied. Urinary LTE4 levels increased in subjects who developed either isolated EARs (mean fall in FEV1, 27.98%) or early responses preceding LARs (mean fall in FEV1, 15.01%). The baseline levels of LTE4 were 150.26 (SEM, 49.5) pg/mg of creatinine in the isolated responders and 66.60 (SEM, 13.5) pg/mg of creatinine in the dual responders. These levels increased to 1816 (SEM, 606.1) pg/mg of creatinine (p = 0.041) and 174.80 (SEM, 40.1) pg/mg of creatinine (p = 0.025), respectively, after the EAR. The degree of maximal bronchoconstriction during the EAR correlated with the levels of LTE4 (r = 0.68; p = 0.001). No significant increase in urinary LTE4 levels occurred during the LAR. These results suggest that the LTE4 precursors, LTC4 and LTD4, are important bronchoconstrictor mediators causing EARs after allergen inhalation.

Multiple opiate receptors : Differential binding of μ, κ and δ agonists

Abstract Much data has accumulated suggesting that μ and δ opiate receptors represent distinct topographical units within the CNS. In addition, in vitro binding studies by the present authors also suggest that a unique κ receptor may be present in the brain. In this regard, [3H]ethylketocyclazocine binding to membrane preparations of rat brain was found to be potently displaced by κ partial μ and agonist/antagonist analgesics. However, μ, δ, σ and ϵ receptor agonists were much less active at this binding site, suggesting that the κ site possesses a different pharmacology from μ and δ sites. These data therefore support previous in vivo experiments which demonstrated a unique behavioral pharmacology and CNS distribution for κ-mediated analgesia.

Enkephalinase: selective peptide inhibitors.

A unique, CNS membrane bound enkephalinase is described with greatest activities being measured in the striatum of the mouse. This enzyme was resistant to inhibition by puromycin and bestatin which are potent aminopeptidase inhibitors and to the angiotensin converting enzyme inhibitors, captopril and the free acid of MK-421, which were also very weak inhibitors of aminopeptidase. However, the glycopeptide, phosphoramidon, and the hydroxamic acids, HO-NHCOCH(CH2CH(CH3)2)-CO-Ala-Gly-NH2 and HO-NHCOCH(CH2C6H5)-CO-Ala-Gly-NH2, were potent enkephalinase inhibitors with IC50s (nM) of 39, 3.1 and 8.4, respectively. These peptides remain to be tested invivo.

2-Pyridinyl-3-(4-methylsulfonyl)phenylpyridines: Selective and orally active cyclooxygenase-2 inhibitors

A series of novel 2-pyridinyl-3-(4-methylsulfonyl)phenylpyridines has been synthesized and evaluated with respect to their ability to inhibit the isozymes of cyclooxygenase, COX-1, and COX-2. Optimum COX-2 activity is observed by introduction of a substituent at C5 of the central pyridine. 5- Chloro-3-(4-methylsulfonyl)phenyl-2-(2-methyl-5-pyridinyl)pyridine 33 was identified as the optimum compound in this series.

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.

Production of leukotrienes in human skin and conjunctival mucosa after specific allergen challenge

The production of leukotrienes has been monitored in tear fluids from subjects following a conjunctival provocation test, and skin blister fluids following initiation of a Prausnitz‐Kustner reaction. In tear fluids elevated levels of leukotriene C4 (LTC4)‐immunoreactive material were measured following allergen challenge as compared to control tear fluid obtained by mechanical or reflex stimulation. Analysis by high performance liquid chromatography indicated the presence of LTC4, LTD4 and LTE4. In the skin, significantly elevated levels of LTC4‐immunoreactive material were measured following allergen challenge in the Prausnitz‐Kustner reaction. HPLC analysis indicated the presence of both LTC4 and LTD4. LTB4 immunoreactive material was detected both in the tear fluid and the skin tissue fluid. However, no significant increase occurred in either tissue after the allergic reactions. These results indicate that the SRS‐A leukotrienes are released in vivo in man following allergen challenge, and indicate these mediators may be important in human allergic diseases.

Leukotriene B4, polymorphonuclear leukocytes and inflammatory exudates in the rat.

Leukocyte numbers and Leukotriene B4- (LTB4-) and LTC4-immunoreactivity were measured in inflammatory exudates obtained from sponges impregnated with several irritants implanted subcutaneously in the rat. Sponges containing 1% uric acid, carrageenan or zymosan were implanted for 5h and compared to saline sponges. Increases in leukocyte numbers and LTB4-immunoreactivity were found in the presence of irritants, the highest concentrations being observed in the presence of zymosan. The presence of LTB4 was confirmed by liquid chromatographic (HPLC) analysis. A time course study was carried out with zymosan-impregnated sponges and the maximal rate of leukocyte infiltration was found to coincide with the maximal levels of LTB4-immunoreactivity. The LTC4-immunoreactivity was low and following analysis by HPLC was concluded to be unrelated to leukotrienes. The levels of LTB4-immunoreactivity, but not the numbers of leukocytes, were elevated compared to corresponding controls in sponges containing 0.01% ionophore A23187 (untreated rats) or in sponges containing zymosan (rats pretreated with indomethacin; 3 and 10 mg/kg p.o.). Impregnation of sponges with 3 X 10(-6)M LTB4 but not 3 X 10(-5) and 3 X 10(-7)M LTB4 induced a significant leukocyte migration. It was concluded that LTB4 can induce leukocyte migration into sponge exudates in the rat but that measurements of LTB4 in such exudates can not be correlated with the degree of leukocyte infiltration.