Roger Aki Fujimoto

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.

Multiple cyclophilins involved in different cellular pathways mediate HCV replication.

Three cyclophilin inhibitors (DEBIO-025, SCY635, and NIM811) are currently in clinical trials for hepatitis C therapy. The mechanism of action of these, however, is not completely understood. There are at least 16 cyclophilins expressed in human cells which are involved in a diverse set of cellular processes. Large-scale siRNA experiments, chemoproteomic assays with cyclophilin binding compounds, and mRNA profiling of HCV replicon containing cells were used to identify the cyclophilins that are instrumental to HCV replication. The previously reported cyclophilin A was confirmed and additional cyclophilin containing pathways were identified. Together, the experiments provide strong evidence that NIM811 reduces viral replication by inhibition of multiple cyclophilins and pathways with protein trafficking as the most strongly and persistently affected pathway.

Mechanism of resistance of hepatitis C virus replicons to structurally distinct cyclophilin inhibitors.

ABSTRACT The current standard of care for hepatitis C virus (HCV) infection, pegylated alpha interferon in combination with ribavirin, has a limited response rate and adverse side effects. Drugs targeting viral proteins are in clinical development, but they suffer from the development of high viral resistance. The inhibition of cellular proteins that are essential for viral amplification is thought to have a higher barrier to the emergence of resistance. Three cyclophilin inhibitors, the cyclosporine analogs DEBIO-025, SCY635, and NIM811, have shown promising results for the treatment of HCV infection in early clinical trials. In this study, we investigated the frequency and mechanism of resistance to cyclosporine (CsA), NIM811, and a structurally unrelated cyclophilin inhibitor, SFA-1, in replicon-containing Huh7 cells. Cross-resistance between all clones was observed. NIM811-resistant clones were selected only after obtaining initial resistance to either CsA or SFA-1. The time required to select resistance against cyclophilin inhibitors was significantly longer than that required for resistance selection against viral protein inhibitors, and the achievable resistance level was substantially lower. Resistance to cyclophilin inhibitors was mediated by amino acid substitutions in NS3, NS5A, and NS5B, with NS5A mutations conferring the majority of resistance. Mutation D320E in NS5A mediated most of the resistance conferred by NS5A. Taken together, the results indicate that there is a very low frequency and level of resistance to cyclophilin-binding drugs mediated by amino acid substitutions in three viral proteins. The interaction of cyclophilin with NS5A seems to be the most critical, since the NS5A mutations have the largest impact on resistance.

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.

Potent Nonimmunosuppressive Cyclophilin Inhibitors With Improved Pharmaceutical Properties and Decreased Transporter Inhibition

Nonimmunosuppressive cyclophilin inhibitors have demonstrated efficacy for the treatment of hepatitis C infection (HCV). However, alisporivir, cyclosporin A, and most other cyclosporins are potent inhibitors of OATP1B1, MRP2, MDR1, and other important drug transporters. Reduction of the side chain hydrophobicity of the P4 residue preserves cyclophilin binding and antiviral potency while decreasing transporter inhibition. Representative inhibitor 33 (NIM258) is a less potent transporter inhibitor relative to previously described cyclosporins, retains anti-HCV activity in cell culture, and has an acceptable pharmacokinetic profile in rats and dogs. An X-ray structure of 33 bound to rat cyclophilin D is reported.