Paul J. Marshall

Preclinical pharmacology of lumiracoxib: a novel selective inhibitor of cyclooxygenase-2

1 This manuscript presents the preclinical profile of lumiracoxib, a novel cyclooxygenase‐2 (COX‐2) selective inhibitor. 2 Lumiracoxib inhibited purified COX‐1 and COX‐2 with Ki values of 3 and 0.06 μM, respectively. In cellular assays, lumiracoxib had an IC50 of 0.14 μM in COX‐2‐expressing dermal fibroblasts, but caused no inhibition of COX‐1 at concentrations up to 30 μM (HEK 293 cells transfected with human COX‐1). 3 In a human whole blood assay, IC50 values for lumiracoxib were 0.13 μM for COX‐2 and 67 μM for COX‐1 (COX‐1/COX‐2 selectivity ratio 515). 4 Lumiracoxib was rapidly absorbed following oral administration in rats with peak plasma levels being reached between 0.5 and 1 h. 5 Ex vivo, lumiracoxib inhibited COX‐1‐derived thromboxane B2 (TxB2) generation with an ID50 of 33 mg kg−1, whereas COX‐2‐derived production of prostaglandin E2 (PGE2) in the lipopolysaccharide‐stimulated rat air pouch was inhibited with an ID50 value of 0.24 mg kg−1. 6 Efficacy of lumiracoxib in rat models of hyperalgesia, oedema, pyresis and arthritis was dose‐dependent and similar to diclofenac. However, consistent with its low COX‐1 inhibitory activity, lumiracoxib at a dose of 100 mg kg−1 orally caused no ulcers and was significantly less ulcerogenic than diclofenac (P<0.05). 7 Lumiracoxib is a highly selective COX‐2 inhibitor with anti‐inflammatory, analgesic and antipyretic activities comparable with diclofenac, the reference NSAID, but with much improved gastrointestinal safety.

Effect of selective phosphodiesterase type IV inhibitor, rolipram, on fluid and cellular phases of inflammatory response

The antiinflammatory activity of rolipram, a selective inhibitor of the cyclic AMP-specific phosphodiesterase (PDE IV), was studied. Rolipram did not inhibit 5-lipoxygenase activity but did inhibit human monocyte production of leukotriene B4 (LTB4, IC50 3.5 μM). Likewise, murine mast cell release of leukotriene C4 and histamine was inhibited. In vivo, rolipram inhibited arachidonic acid-induced inflammation in the mouse, while the lowKm-cyclic-GMP PDE inhibitor, zaprinast, did not inhibit. Rolipram had a modest effect on LTB4 production in the mouse, but markedly reduced LTB4-induced PMN infiltration. Beta-adrenergic receptor activation of adenylate cyclase was important for rolipram antiinflammatory activity since beta blockade abrogated arachidonic acid-induced inflammation. Thus, the antiinflammatory profile of rolipram is novel and may result from inhibition of PMN function and perhaps vasoactive amine release and leukotriene biosynthesis. These actions may be dependent upon endogenous beta-adrenergic activity and are likely mediated through inhibition of PDE IV.

Prostaglandin H synthase: distinct binding sites for cyclooxygenase and peroxidase substrates

Prostaglandin H synthase has two distinct catalytic activities: a cyclooxygenase activity that forms prostaglandin G2 from arachidonic acid; and a peroxidase activity that reduces prostaglandin G2 to prostaglandin H2. Lipid hydroperoxides, such as prostaglandin G2, also initiate the cyclooxygenase reaction, probably via peroxidase reaction cycle enzyme intermediates. The relation between the binding sites for lipid substrates of the two activities was investigated with an analysis of the effects of arachidonic and docosahexaenoic acids on the reaction kinetics of the peroxidase activity, and their effects on the ability of a lipid hydroperoxide to initiate the cyclooxygenase reaction. The cyclooxygenase activity of pure ovine synthase was found to have an apparent Km value for arachidonate of 5.3 microM and a Ki value (competetive inhibitor) for docosahexaenoate of 5.2 microM. When present at 20 microM neither fatty acid had a significant effect on the apparent Km value of the peroxidase for 15-hydroperoxyeicosatetraenoic acid: the values were 7.6 microM in the absence of docosahexaenoic acid and 5.9 microM in its presence, and (using aspirin-treated synthase) 13.7 microM in the absence of arachidonic acid and 15.7 microM in its presence. Over a range of 1 to 110 microM the level of arachidonate had no significant effect on the initiation of the cyclooxygenase reaction by 15-hydroperoxyeicosatetraenoic acid. The inability of either arachidonic acid or docosahexaenoic acid to interfere with the interaction between the peroxidase and lipid hydroperoxides indicates that the cyclooxygenase and peroxidase activities of prostaglandin H synthase have distinct binding sites for their lipid substrates.

Preclinical pharmacology of robenacoxib: a novel selective inhibitor of cyclooxygenase‐2

This manuscript reports the results of preclinical studies in the rat with robenacoxib, a novel selective cyclooxygenase (COX)-2 inhibitor. Robenacoxib selectively inhibited COX-2 in vitro as evidenced from COX-1:COX-2 IC50 ratios of 27:1 in purified enzyme preparations and >967:1 in isolated cell assays. Binding to COX-1 was rapid and readily reversible (dissociation t(1/2) << 1 min), whilst COX-2 binding was slowly reversible (t(1/2) = 25 min). In vivo, robenacoxib inhibited PGE2 production (an index of COX-2 inhibition) in lipopolysaccharide (LPS)-stimulated air pouches (ID50 0.3 mg/kg) and for at least 24 h in zymosan-induced inflammatory exudate (at 2 mg/kg). Robenacoxib was COX-1 sparing, as it inhibited serum TxB2 synthesis ex vivo (an index of COX-1 inhibition) only at very high doses (100 mg/kg but not at 2-30 mg/kg). Robenacoxib inhibited carrageenan-induced paw oedema (ID50 0.40-0.48 mg/kg), LPS-induced fever (ID50 1.1 mg/kg) and Randall-Selitto pain (10 mg/kg). Robenacoxib was highly bound to plasma protein (99.9% at 50 ng/mL in vitro). After intravenous dosing, clearance was 2.4 mL/min/kg and volume of distribution at steady-state was 306 mL/kg. Robenacoxib was preferentially distributed into inflammatory exudate; the AUC for exudate was 2.9 times higher than for blood and the MRT in exudate (15.9 h) was three times longer than in blood (5.3 h). Robenacoxib produced significantly less gastric ulceration and intestinal permeability as compared with the reference nonsteroidal anti-inflammatory drug (NSAID), diclofenac, and did not inhibit PGE2 or 6-keto PGF(1alpha) concentrations in the stomach and ileum at 30 mg/kg. Robenacoxib also had no relevant effects on kidney function at 30 mg/kg. In summary, results of preclinical studies in rats studies suggest that robenacoxib has an attractive pharmacological profile for potential use in the intended target species, cats and dogs.

Rapid Kinetics of Tyrosyl Radical Formation and Heme Redox State Changes in Prostaglandin H Synthase-1 and -2

Hydroperoxide-induced tyrosyl radicals are putative intermediates in cyclooxygenase catalysis by prostaglandin H synthase (PGHS)-1 and -2. Rapid-freeze EPR and stopped-flow were used to characterize tyrosyl radical kinetics in PGHS-1 and -2 reacted with ethyl hydrogen peroxide. In PGHS-1, a wide doublet tyrosyl radical (34–35 G) was formed by 4 ms, followed by transition to a wide singlet (33–34 G); changes in total radical intensity paralleled those of Intermediate II absorbance during both formation and decay phases. In PGHS-2, some wide doublet (30 G) was present at early time points, but transition to wide singlet (29 G) was complete by 50 ms. In contrast to PGHS-1, only the formation kinetics of the PGHS-2 tyrosyl radical matched the Intermediate II absorbance kinetics. Indomethacin-treated PGHS-1 and nimesulide-treated PGHS-2 rapidly formed narrow singlet EPR (25–26 G in PGHS-1; 21 G in PGHS-2), and the same line shapes persisted throughout the reactions. Radical intensity paralleled Intermediate II absorbance throughout the indomethacin-treated PGHS-1 reaction. For nimesulide-treated PGHS-2, radical formed in concert with Intermediate II, but later persisted while Intermediate II relaxed. These results substantiate the kinetic competence of a tyrosyl radical as the catalytic intermediate for both PGHS isoforms and also indicate that the heme redox state becomes uncoupled from the tyrosyl radical in PGHS-2.

Clinical pharmacology of lumiracoxib : A selective cyclo-oxygenase-2 inhibitor

Lumiracoxib (Prexige®) is a selective cyclo-oxygenase (COX)-2 inhibitor developed for the treatment of osteoarthritis, rheumatoid arthritis and acute pain. Lumiracoxib possesses a carboxylic acid group that makes it weakly acidic (acid dissociation constant [pKa] 4.7), distinguishing it from other selective COX-2 inhibitors.Lumiracoxib has good oral bioavailability (74%). It is rapidly absorbed, reaching maximum plasma concentrations 2 hours after dosing, and is highly plasma protein bound. Lumiracoxib has a short elimination half-life from plasma (mean 4 hours) and demonstrates dose-proportional plasma pharmacokinetics with no accumulation during multiple dosing. In patients with rheumatoid arthritis, peak lumiracoxib synovial fluid concentrations occur 3–4 hours later than in plasma and exceed plasma concentrations from 5 hours after dosing to the end of the 24-hour dosing interval. These data suggest that lumiracoxib may be associated with reduced systemic exposure, while still reaching sites where COX-2 inhibition is required for pain relief.Lumiracoxib is metabolised extensively prior to excretion, with only a small amount excreted unchanged in urine or faeces. Lumiracoxib and its metabolites are excreted via renal and faecal routes in approximately equal amounts. The major metabolic pathways identified involve oxidation of the 5-methyl group of lumiracoxib and/or hydroxylation of its dihaloaromatic ring. Major metabolites of lumiracoxib in plasma are the 5-carboxy, 4′-hydroxy and 4′-hydroxy-5-carboxy derivatives, of which only the 4′-hydroxy derivative is active and COX-2 selective. In vitro, the major oxidative pathways are catalysed primarily by cytochrome P450 (CYP) 2C9 with very minor contribution from CYP1A2 and CYP2C19. However, in patients genotyped as poor CYP2C9 metabolisers, exposure to lumiracoxib (area under the plasma concentration-time curve) is not significantly increased compared with control subjects, indicating no requirement for adjustment of lumiracoxib dose in these subjects.Lumiracoxib is selective for COX-2 compared with COX-1 in the human whole blood assay with a ratio of 515: 1 in healthy subjects and in patients with osteoarthritis or rheumatoid arthritis. COX-2 selectivity was confirmed by a lack of inhibition of arachidonic acid and collagen-induced platelet aggregation. COX-2 selectivity of lumiracoxib is associated with a reduced incidence of gastroduodenal erosions compared with naproxen and a lack of effect on both small and large bowel permeability.Lumiracoxib does not exhibit any clinically meaningful interactions with a range of commonly used medications including aspirin (acetylsalicylic acid), fluconazole, an ethinylestradiol- and levonorgestrel-containing oral contraceptive, omeprazole, the antacid Maalox®, methotrexate and warfarin (although, as in common practice, routine monitoring of coagulation is recommended when lumiracoxib is co-administered with warfarin). As such, dose adjustments are not required when co-administering these agents with lumiracoxib. In addition, moderate hepatic impairment and mild to moderate renal impairment do not appear to influence lumiracoxib exposure.

Pharmacology of the pyrroloimidazole, SK&F 105809—I: Inhibition of inflammatory cytokine production and of 5-lipoxygenase- and cyclooxygenase-mediated metabolism of arachidonic acid

SK&F 105809 [2-(4- methylsulfinylphenyl)-3-(4-pyridyl)-6,7-dihydro-[5H]-pyrrolo[1,2- a] imidazole] was determined to be a prodrug for the sulfide metabolite SK&F 105561 [2-(4- methylthiophenyl)-3-(4-pyridyl)-6,7-dihydro-[5H]-pyrrolo[1,2-a] imidazole] which inhibited interleukin-1 (IL-1) production in vitro and both 5-lipoxygenase (5-LO) and prostaglandin H (PGH) synthase activities in vitro and ex vivo. SK&F 105561 inhibited partially purified 5-LO with a half-maximal concentration (IC50) of 3 microM. This inhibition was reversible, independent of preincubation time, and dependent on the concentration of the substrate arachidonic acid. SK&F 105561 also inhibited purified PGH synthase with the potency dependent on the level of peroxidase activity. The IC50 was 100 microM in the absence of peroxidase activity, whereas an IC50 of 3 microM was observed in the presence of peroxidase activity. Using human monocytes, SK&F 105561 inhibited A23187-stimulated prostaglandin E2 (PGE2) and leukotriene B4 (LTB4) production with IC50 values of 0.1 and 2 microM, respectively. In addition, IL-1 production by lipopolysaccharide-stimulated human monocytes was also inhibited (IC50 2 microM). Oral administration of SK&F 105809 to rats resulted in a dose-related generation of SK&F 105561 and in the inhibition of thromboxane B2 and LTB4 production ex vivo with a half-maximal dose (ED50) of 15 and 60 mg/kg, respectively. SK&F 105561 showed weak inhibitory activity on 12-lipoxygenase with an IC50 of greater than 200 microM. Neither SK&F 105561 nor SK&F 105809 inhibited the stimulated-turnover of arachidonic acid-containing phospholipids in human monocytes or the activity of cell-free phospholipases A2 and C. Moreover, neither SK&F 105561 nor SK&F 105809 antagonized the binding of LTB4 or leukotriene D4 to membrane receptors. From these results, SK&F 105561, the active principle of SK&F 105809, acts as an inhibitor of both inflammatory cytokine and eicosanoid production.

Pharmacology of the pyrroloimidazole, SK&F 105809—II: Antiinflammatory activity and inhibition of mediator production in vivo

SK&F 105809 [2-(4-methylsulfinylphenyl)-3-(4-pyridyl)- 6,7-dihydro-[5H]-pyrrolo[1,2,a] imidazole] demonstrated unique antiinflammatory activities in murine models that are resistant to selective cyclooxygenase (CO) inhibitors. Both edema and inflammatory cell infiltration induced by the topical application of arachidonic acid to the mouse ear were decreased by SK&F 105809 (ED50 values of 44 mg/kg, p.o.). Polymorphonuclear leukocyte (PMN) infiltration following the intraperitoneal injection of either monosodium urate crystal or carrageenan was inhibited with ED50 values of 64 and 72 mg/kg, p.o., respectively. These inflammatory responses were unaffected by the selective cyclooxygenase inhibitor naproxen. SK&F 105809 also inhibited leukotriene B4 (LTB4) and prostaglandin E2 production in vivo in arachidonic acid-induced inflammatory exudates (ED50 values of 41 and 15 mg/kg, p.o., respectively). The inhibition of LTB4 production preceded the inhibition of PMN infiltration. The impact of inhibition of both 5-lipoxygenase (5-LO) and CO was seen with platelet-activating factor-induced vascular permeability which was inhibited markedly by SK&F 105809. However, the 5-LO inhibitor, phenidone, only strongly inhibited when coadministered with the selective CO inhibitor, indomethacin. In spite of a short half-life (14-18 min) for both SK&F 105809 and the active metabolite SK&F 105561 [2-(4- methylthiophenyl)-3-(4-pyridyl)-6,7-dihydro-[5H]-pyrrolo[1,2-a] imidazole], the pharmacological activity lasted at least 1.5 hr. The biochemical evidence of inhibition of interleukin-1 (IL-1) production and 5-LO and CO activity, in vitro, by the metabolite (SK&F 105561) seen in the companion paper (Marshall PJ, Griswold DE, Breton J. Webb EF, Hillegass LM, Sarau HM, Newton J Jr, Lee JC, Bender PE and Hanna N, Pharmacology of the pyrroloimidazole, SK&F 105809--I. Inhibition of inflammatory cytokine production and of 5-lipoxygenase- and cyclooxygenase-mediated metabolism of arachidonic acid. Biochem Pharmacol 42: 813-824, 1991) and inhibition of the fluid and cellular phases of the inflammatory response, in vivo, by SK&F 105809 suggest that this compound possesses a unique profile of activity.

Rapid kinetics of tyrosyl radical formation and heme redox state changes in prostaglandin H synthase-1 and -2

Hydroperoxide-induced tyrosyl radicals are putative intermediates in cyclooxygenase catalysis by prostaglandin H synthase (PGHS)-1 and -2. Rapid-freeze EPR and stopped-flow were used to characterize tyrosyl radical kinetics in PGHS-1 and -2 reacted with ethyl hydrogen peroxide. In PGHS-1, a wide doublet tyrosyl radical (34-35 G) was formed by 4 ms, followed by transition to a wide singlet (33-34 G); changes in total radical intensity paralleled those of Intermediate II absorbance during both formation and decay phases. In PGHS-2, some wide doublet (30 G) was present at early time points, but transition to wide singlet (29 G) was complete by 50 ms. In contrast to PGHS-1, only the formation kinetics of the PGHS-2 tyrosyl radical matched the Intermediate II absorbance kinetics. Indomethacin-treated PGHS-1 and nimesulide-treated PGHS-2 rapidly formed narrow singlet EPR (25-26 G in PGHS-1; 21 G in PGHS-2), and the same line shapes persisted throughout the reactions. Radical intensity paralleled Intermediate II absorbance throughout the indomethacin-treated PGHS-1 reaction. For nimesulide-treated PGHS-2, radical formed in concert with Intermediate II, but later persisted while Intermediate II relaxed. These results substantiate the kinetic competence of a tyrosyl radical as the catalytic intermediate for both PGHS isoforms and also indicate that the heme redox state becomes uncoupled from the tyrosyl radical in PGHS-2.