(E)-2(R)-[1(S)-(Hydroxycarbamoyl)-4-phenyl-3-butenyl]-2 - isobutyl-2 -(methanesulfonyl)-4-methylvalerohydrazide (Ro 32-7315), a Selective and Orally Active Inhibitor of Tumor Necrosis Factor- Convertase

Tumor necrosis factor(TNF), a cytokine secreted by inflammatory cells, has been implicated in several inflammatory disease states. (E)-2(R)-[1(S)-(Hydroxycarbamoyl)-4-phenyl-3-butenyl]-2 isobutyl-2 -(methanesulfonyl)-4-methylvalerohydrazide (Ro 327315), is a potent, orally active inhibitor of the TNFconvertase (TACE), an enzyme responsible for proteolytic cleavage of the membrane bound precursor, pro-TNF. Ro 32-7315 inhibited a recombinant form of TACE (IC50 5.2 nM) with selectivity over related matrix metalloproteinases. In a cellular assay system, THP-1 cell line, and in human and rat whole blood, Ro 32-7315 significantly reduced lipopolysaccharide (LPS)-induced TNFrelease with IC50 values of 350 14 nM (n 5), 2.4 0.5 M (n 5), and 110 18 nM (n 5), respectively. Oral administration of Ro 32-7315 to Wistar rats caused a dose-dependent inhibition of LPS-induced release of systemic TNFwith an ED50 of 25 mg/kg. Treatment (days 0–14) of Allen and Hamburys hooded rats with Ro 32-7315 (2.5, 5, 10, and 20 mg/kg, i.p., twice daily) significantly reduced adjuvant-induced secondary paw swelling (42, 71, 83, and 93%, respectively) as compared with the vehicle group. In the Ro 32-7315-treated group, the reduced paw swelling was associated with improved lesion score and joint mobility. Furthermore, in a placebocontrolled, single-dose study, Ro 32-7315 given orally (450 mg) significantly suppressed ex vivo, LPS-induced TNFrelease in the whole-blood samples taken from healthy male and female volunteers (mean inhibition of 42% over a 4-h duration, n 6). These data collectively support the potential use of such a compound for the oral treatment of inflammatory disorders. Tumor necrosis factor-alpha (TNF), a cytokine produced primarily by activated monocytes and macrophages, is an important mediator of immuno-inflammatory responses and inducer of other pro-inflammatory cytokines (Vassali, 1992). TNFis produced as a 26-kDa membrane-bound pro-TNFthat undergoes a specific proteolytic cleavage between Ala-76 and Val-77 to release a 17-kDa soluble TNF(Kriegler et al., 1988). The enzyme that is responsible for this process is TNFconvertase (TACE), a metalloproteinase closely related to the matrix metalloproteinases (MMPs; Gearing et al., 1994; McGeehan et al., 1994; Mohler et al., 1994). MMPs are a large family of Zn endopeptidases that include 72and 92-kDa gelatinases, collagenases, stromelysins 1–3, matrilysin, macrophage metalloelastase, and membrane-bound MMP 1–4 (Birkedal-Hansen et al., 1993). They are expressed in immuno-inflammatory conditions such as rheumatoid arthritis (RA), sepsis, and inflammatory bowel disease and are collectively capable of degrading most connective tissue macromolecules under both physiological and pathological conditions (Buchan et al., 1988; Lewis et al., 1997). RA is a chronic, progressive inflammatory disease that causes substantial morbidity. Although the precise etiology of RA remains unknown, a great deal has been learned about the immunopathophysiology of the disease in recent years. Proinflammatory cytokines such as TNF, interleukin (IL)-1, and IL-6 have been suggested to play an important role in the pathogenesis of the RA. In addition to promoting inflammation, these cytokines are capable of directly mediating bone and cartilage destruction, the most prominent feature of RA (Buchan et al., 1988; Feldmann and Maini, ABBREVIATIONS: DMSO, dimethyl sulfoxide; ELISA, enzyme-linked immunosorbant assay; IL, interleukin; LPS, lipopolysaccharide; MMP, matrix metalloproteinase; Ro 32-7315, (E)-2(R)-[1(S)-(hydroxycarbamoyl)-4-phenyl-3-butenyl]-2 -isobutyl-2 -(methanesulfonyl)-4-methylvalerohydrazide; TACE, tumor necrosis factor alpha convertase; TNF, tumor necrosis factor-alpha; RA, rheumatoid arthritis; AHH/R, Allen and Hamburys hooded rats; Dpa, N-3-(2, 4-dinitrophenyl)-L-2, 3-diaminopropionyl. 0022-3565/02/3021-390–396$7.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 302, No. 1 Copyright © 2002 by The American Society for Pharmacology and Experimental Therapeutics 4209/990842 JPET 302:390–396, 2002 Printed in U.S.A. 390 at A PE T Jornals on O cber 2, 2017 jpet.asjournals.org D ow nladed from 1999). Several lines of evidence have identified TNFas a key mediator of chronic inflammatory diseases such as RA, inflammatory bowel disease, and sepsis (Espersen et al., 1991; Tracey et al., 1993). The randomized phase II and III clinical trials using a monoclonal antibody to TNF, infliximab, or the soluble TNFreceptor fusion protein, etanercept, significantly improved the disease activity in RA patients, thus, validating TNFas a therapeutic target for RA (Moreland et al., 1997; Maini et al., 1998; Lorenz et al., 2000). The inhibition of TACE offers an alternative point of therapeutic intervention and the possibility of a low molecular weight, orally active agent. Hydroxamic-acid-based MMP inhibitors have been reported to inhibit endotoxin-induced production of TNFby inhibiting the processing enzyme, TACE (Gearing et al., 1994; Mohler et al., 1994). However, there are very limited reports concerning their selectivity for TACE and oral efficacy in preclinical and clinical models. Ro 32-7315 (Fig. 1) is among the first of a new generation of potent, hydroxamicacid-containing TACE inhibitors with greater selectivity over the MMP family of enzymes. In addition to the potency and selectivity of this compound, a marked improvement in the in vivo biological profile compared with compounds such as BB 1101 (DiMartino et al., 1997; Barlaam et al., 1999) was observed. In this report, we describe the in vitro and in vivo biological profile of Ro 32-7315, a selective inhibitor of TACE. Furthermore, we considered the feasibility of using changes in LPS-induced TNFrelease for the ex vivo evaluation of TACE inhibitor pharmacodynamics. Using this approach, Ro 32-7315 was shown to cause a significant inhibition of TNFproduction ex vivo after oral dosing in healthy volunteers. The findings support the contention that this selective TACE inhibitor may have use as a potential oral treatment for inflammatory diseases. Materials and Methods