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INFLAMMATION, IMMUNOPHARMACOLOGY, AND ASTHMA

(S)-1-((S)-2-{[1-(4-Amino-3-chloro-phenyl)-methanoyl]-amino}-3,3-dimethyl-butanoyl)-pyrrolidine-2-carboxylic acid ((2R,3S)-2-ethoxy-5-oxo-tetrahydro-furan-3-yl)-amide (VX-765), an Orally Available Selective Interleukin (IL)-Converting Enzyme/Caspase-1 Inhibitor, Exhibits Potent Anti-Inflammatory Activities by Inhibiting the Release of IL-1 and IL-18

Departments of Chemistry (W.W., R.D.), Protein Sciences (M.N.), and Biology (P.F., G.K., U.A.G., K.K.), Drug Discovery Support Unit (C.D.), and Department of Modeling (P.C.), Strategic Development (J.C.R.R.), Vertex Pharmaceuticals, Inc., Cambridge, Massachusetts; and Protein Sciences (J.P.), Pharmacology (P.W.), Vertex Pharmaceuticals (Europe Ltd.), Oxfordshire, United Kingdom

Received July 21, 2006; accepted February 5, 2007.

	Abstract

Top Abstract Materials and Methods Results Discussion References

 
(S)-1-((S)-2-{[1-(4-Amino-3-chloro-phenyl)-methanoyl]-amino}-3,3-dimethyl-butanoyl)-pyrrolidine-2-carboxylic acid ((2R,3S)-2-ethoxy-5-oxo-tetrahydro-furan-3-yl)-amide (VX-765) is an orally absorbed prodrug of (S)-3-({1-[(S)-1-((S)-2-{[1-(4-amino-3-chlorophenyl)-methanoyl]-amino}-3,3-dimethyl-butanoyl)-pyrrolidin-2yl]-methanoyl}-amino)-4-oxo-butyric acid (VRT-043198), a potent and selective inhibitor of interleukin-converting enzyme/caspase-1 subfamily caspases. VRT-043198 exhibits 100- to 10,000-fold selectivity against other caspase-3 and -6 to -9. The therapeutic potential of VX-765 was assessed by determining the effects of VRT-043198 on cytokine release by monocytes in vitro and of orally administered VX-765 in several animal models in vivo. In cultures of peripheral blood mononuclear cells and whole blood from healthy subjects stimulated with bacterial products, VRT-043198 inhibited the release of interleukin (IL)-1 and IL-18, but it had little effect on the release of several other cytokines, including IL-1, tumor necrosis factor-, IL-6 and IL-8. In contrast, VRT-043198 had little or no demonstrable activity in cellular models of apoptosis, and it did not affect the proliferation of activated primary T cells or T-cell lines. VX-765 was efficiently converted to VRT-043198 when administered orally to mice, and it inhibited lipopolysaccharide-induced cytokine secretion. In addition, VX-765 reduced disease severity and the expression of inflammatory mediators in models of rheumatoid arthritis and skin inflammation. These data suggest that VX-765 is a novel cytokine inhibitor useful for treatment of inflammatory diseases.

ICE/caspase-1 is the first member identified in the caspase family of cysteine proteases that now has 14 known members, 11 of which are expressed in humans (for review, see Earnshaw et al., 1999). The caspases are typically divided into three subfamilies on the basis of sequence homology and function. The caspase-1 subfamily includes ICE/caspase-1 along with caspase-4 and caspase-5 in humans. This subfamily seems to be involved primarily in inflammatory response and the production of IL-1 and IL-18 (for review, see Martinon and Tschopp, 2004). Although ICE/caspase-1 directly cleaves and activates the cytokines, the other family members may participate in the proteolytic activation of ICE/caspase-1 in response to signaling from membrane receptors. The remaining caspases are divided into two subfamilies based on their key roles in the initiation and execution of programmed cell death, or apoptosis, in a variety of cell types. ICE/caspase-1 is constitutively expressed and highly inducible in macrophages, T cells, and neutrophils. ICE/caspase-1 expression is also induced under certain conditions in other cell types, such as keratinocytes (Zepter et al., 1997). The ICE/caspase-1 subfamily caspases do not seem to play a prominent direct role in apoptosis (Li et al., 1995; Smith et al., 1997), although they may play indirect roles through their influence on cytokine-mediated inflammatory responses that ultimately lead to apoptosis. In addition, Thalappilly et al. (2006) recently reported that activation of ICE/caspase-1 induces changes in the mitochondria leading to caspase-9 activation in apoptosis mediated by a phosphatase, suggesting a substantial role of ICE/caspase-1 in limited circumstances. ICE/caspase-1-deficient mice develop and reproduce normally, and they have normal T-cell development (Kuida et al., 1995; Li et al., 1995). Apoptotic pathways in mature T cells are unimpaired, and the only discernible defect in apoptosis is in Fas-mediated apoptosis in thymocytes (Kuida et al., 1995). Under normal rearing conditions up to 1 year of age, ICE/caspase-1-deficient mice exhibit no obvious increase in the incidence of infection or malignancy (Kuida et al., 1995; D. Boucher, K. Kuida, J. C. R. Randle, unpublished data).

VX-765 is an orally absorbed prodrug of VRT-043198 (Fig. 1), a potent and selective inhibitor of caspases belonging to the ICE/caspase-1 subfamily. VX-765 is converted rapidly to VRT-043198 under the action of plasma and liver esterases and also much more slowly in aqueous solution. This article describes studies performed to evaluate the therapeutic potential of VX-765 based on its inhibition of cytokine release by monocytes in vitro and clinical and biomarker responses in animal models of inflammatory, autoimmune joint, and skin disease in vivo.

View larger version (14K): [in this window] [in a new window]   Fig. 1. Structure of VX-765 and its active metabolite VRT-043198.

	Materials and Methods

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VX-765 and VRT-043198. VX-765 and VRT-043198 were synthesized at Vertex Pharmaceuticals, Inc. (Cambridge, MA) as described in Wannamaker and Davies (2001) (Fig. 1).

Protease Enzyme Assays. Caspase-1, -3, -7, and -8 were produced at Vertex Pharmaceuticals, Inc. Caspase-6 and -9 were purchased from BD Biosciences PharMingen (San Diego, CA) and Chemicon International (Hampshire, UK), respectively. Caspase-4 was purchased from Eurogenetics (Tessenderlo, Belgium). Granzyme B was purchased from Alexis Biochemical (Carlsbad, CA). Cathepsin B from bovine spleen and trypsin from bovine pancreas were purchased from Sigma-Aldrich (St. Louis, MO). Enzyme inhibition was assayed by tracking of the rate of hydrolysis of an appropriate substrate labeled with either p-nitroaniline or aminomethyl coumarin (AMC) as follows: ICE/caspase-1, suc-YVAD-p-nitroanilide; caspase-4, Ac-WEHD-AMC; caspase-6, Ac-VEID-AMC; caspase-3, -7, -8, and -9, Ac-DEVD-AMC; and granzyme B, Ac-IEPD-AMC (Bachem Biosciences, King of Prussia, PA). Enzymes and substrates were incubated in a reaction buffer [10 mM Tris, pH 7.5, 0.1% (w/v) CHAPS, 1 mM dithiothreitol, and 5% (v/v) dimethyl sulfoxide] for 10 min at 37°C. Glycerol was added to the buffer at 8% (v/v) for caspase-3, -6, and -9 and granzyme B to improve stability of enzymes. The rate of substrate hydrolysis was monitored using a fluorometer. Assays for cathepsin B and trypsin were performed as described previously (Fox et al., 1992).

Peripheral Blood Mononuclear Cell and Whole Blood Assays. Buffy coat fractions from healthy volunteer donors were purchased from the Massachusetts General Hospital. PBMCs were prepared from the buffy coat fractions by a Ficoll gradient using Ficoll-Hypaque (Amersham Bioscience, Uppsala, Sweden) and washed twice in RPMI 1640 medium (JRH Biosciences, Lenexa, KS). Cells were then transferred to 96-well microtiter plates at 4.8 x 10⁵cells/well and stimulated with either 1 µg/ml of Escherichia coli LPS (O111:B4; Sigma-Aldrich) or 1:1000 dilution of Staphylococcus aureus-Cowan strain 1 (SAC) (2 mg/ml solution; Calbiochem, San Diego, CA). Plates were incubated overnight (16–20 h) at 37°C in 5% CO₂. For the whole blood assay, blood was drawn from healthy volunteers at Vertex Pharmaceuticals, Inc. using BD Biosciences (Franklin Lakes, NJ) Vacutainers. The blood was diluted with equal volume of RPMI 1640 medium, and 0.2 ml of the diluted blood was added to each of a 96-well costar plate. Cells were stimulated with 5 ng/ml E. coli LPS (O111:B4). At the end of an 18-h incubation period, the plates were shaken, centrifuged at 200g for 5 min, and the supernatant was removed for cytokine evaluation. IL-1, IL-18, and other cytokines in culture media were measured using specific ELISA kits (R&D Systems, Minneapolis, MN).

Phamacokinetic Analysis of VX-765 and VRT-043198 in Mice. Single doses of VX-765 (10, 21, 43, and 84 mg/kg) in vehicle (25% Cremophor EL in water; Sigma-Aldrich) were administered via oral gavage. Blood samples (approximately 0.25–0.3 ml) were collected before dose administration and 0.167, 0.25, 0.5, 1, 1.5, 2, 3, 4, 6, 8, and 24 h after dosing via the retroorbital sinus and processed for plasma. A high-performance liquid chromatography/mass spectrometry methodology was used to determine the concentration of VX-765 and VRT-043198 in plasma samples. Noncompartmental analysis was carried out using WinNonlin Pro, version 4.0.1 (Pharsight, Mountain View, CA).

Induction of IL-1 by Intravenous Injection of LPS. Naive male CD-1 mice (Charles River Laboratories, Inc., Wilmington, MA) 6 to 7 weeks of age (30–32 g body weight) were randomized and dosed by oral gavage with VX-765 1 h before i.v. injection of 2 mg/kg E. coli LPS (strain 0111:B4, lot 32K4092; Sigma-Aldrich). Vehicle (25% Cremophor EL in water) was dosed at 1 h before the LPS challenge as a control. Peripheral blood samples were harvested 2.5 h after the LPS challenge. The blood was allowed to clot overnight at 4°C, and then it centrifuged to obtain sera for analysis on levels of IL-1 by a specific ELISA system (R&D Systems). All studies were performed with six mice per group.

Oxazolone-Induced Delayed-Type Hypersensitivity Responses in CD-1 Mice. Naive male CD-1 mice (Charles River Laboratories, Inc.) 6 to 7 weeks of age (30–32 g body weight; n = 9/group) were used in this study. The abdomen was shaved and 150 µl of a 5% (w/v) solution of oxazolone (Sigma-Aldrich) in a solvent composed of ethanol and acetone [4:1 (v/v)] was applied. Three days later, the mice were challenged with 10 µl of 1 to 3% oxazolone applied to each side of the right ear. The left ears were treated with the same volume of the solvent as control. Mice were then treated twice by oral gavage with either the vehicle (25% Cremophor EL in water) or VX-765 (10–100 mg/kg in a dosing volume of 10 ml/kg body weight) 24 and 36 h after the ear challenge. Prednisolone (5 mg/kg) was used as positive control and dosed orally. At 48 h after the ear challenge, 9-mm-diameter biopsy samples were collected from both right and left ears, and the samples were weighed. Edema in the oxazolone-challenged right ear was determined as the difference in weight between the right and control left ear biopsy samples. The biopsy samples were then homogenized individually in 1 ml of phosphate-buffered saline, pH 7, with a standardized cocktail of protease inhibitors (Roche Diagnostics, Indianapolis, IN). The homogenates were spun at 15,000 rpm for 15 min, and the resulting supernatants were analyzed by specific ELISA (R&D Systems) for IL-1, IL-18, IFN-, IL-4, monocyte chemotactic protein-1, monocyte inhibitory protein-1, monocyte inhibitory protein-2, myeloperoxidase, and nitric oxide.

Collagen-Induced Arthritis Model in Mouse. Naive male DBA/1 mice (The Jackson Laboratory, Bar Harbor, ME) at 8 to 10 weeks of age were immunized, intradermally at the base of the tail, 0.1-ml aliquots of a 1:1 (v/v) emulsion of Complete Freund's adjuvant (Sigma-Aldrich) and chick type II collagen (4 mg/ml 100 mM acetic acid; Elastin Products, Owensville, MO). The mice were immunized again with the same material 3 weeks later (Ku et al., 1996). Forepaw inflammation was monitored every other day and graded in a blinded manner semiquantitatively on a scale from 0 to 5: level 0, no evidence for inflammation; level 1, erythema around the wrist joint; level 2, erythema plus partial swelling of tissue around the wrist joint; level 3, erythema plus pronounced swelling of tissue around the wrist joint; level 4, erythema plus pronounced swelling of the wrist and palm; and level 5, erythema plus pronounced swelling of the wrist, palm, and fingers of each forepaw. In the prophylactic regimen, immediately following the second immunization mice were assigned to groups and treated by oral gavage twice daily (b.i.d.) with vehicle (25% Cremophor EL), VX-765 (10–100 mg/kg), or prednisolone (5 mg/kg). In the therapeutic regimen, the disease was allowed to progress until a portion of the mice (usually 60–70%) exhibited level 2 paw inflammation score in each front paw. Then, these mice were assigned to different treatment groups. Dosing materials were provided blinded to the investigator performing the drug treatment and disease scoring. The sum of the forepaw inflammation scores was recorded every second day. Area under the curve (AUC) of forepaw inflammation scores for each mouse was calculated using the trapezoidal rule. Statistical significance of treatment was analyzed based on AUC using the Wilcoxon rank sum test. At the end of studies, forepaws were collected for histological examination. The incidence of cartilage and bone lesions was noted, and a histological score was assigned to each joint as follows: level 1, infiltration in the synovium; level 2, level 1 plus erosion in the cartilage; level 3, level 2 plus erosion in the bone; and level 4, level 3 plus pannus formation.

	Results

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Selectivity of VRT-43198 against Caspases and Other Proteases. We evaluated in vitro the potency of VRT-043198 against ICE/caspase-1 and caspase-4 and its selectivity against representatives of the three subfamilies of caspases, and other proteases, including granzyme B and trypsin (serine proteases), and cathepsin B (cysteine protease). As shown in Table 1, VRT-043198 exhibited potent inhibition of ICE/caspase-1 (K_i = 0.8 nM) and caspase-4 (K_i < 0.6 nM) and at least 100-fold lower potency against other non-ICE subfamily caspases. VRT-043198 exhibited no significant inhibition of trypsin or cathepsin B and only weak inhibition of granzyme B (K_i = 9 µM).

Inhibition of Cytokine Release from Human PBMCs and Whole Blood. We evaluated the ability of VRT-043198 to inhibit the LPS-stimulated release of cytokines from human PBMCs and whole blood obtained from healthy volunteers. VRT-043198 inhibited IL-1 release from both PBMCs (n = 8) and whole blood (n = 4) with IC₅₀ values of 0.67 ± 0.55 and 1.9 ± 0.80 µM (mean ± S.D.), respectively. Additional experiments were conducted using PBMCs stimulated with SAC, which induces the release of large amounts of IL-1 as well as sufficient IL-18, IFN-, and TNF- to determine the inhibitory effects of VRT-043198. VRT-043198 dose-dependently inhibited production of IL-1, IL-18, and IFN-, but it did not affect TNF- release (Table 2).

VRT-043198 Lacks Potent Antiapoptotic Activity. VRT-043198 was evaluated in a hypoxia-induced apoptosis assay using the human neuroblastoma cell line NT2 where caspase-9 may play a role in the initiation of hypoxia- and ischemia-induced apoptosis. VRT-043198 did not alter ischemia-induced apoptosis at concentrations up to 100 µM (Supplemental Fig. 1), consistent with its low potency against caspase-9 (Table 1). In addition, the effects of VRT-043198 on Fas-induced apoptosis were evaluated in the Jurkat human T-cell line, where initiation of the apoptosis cascade is mediated by caspase-8 activity. VRT-043198 only affected cell death at concentrations of 200 µM (approximately 50% inhibition) (Supplemental Fig. 2). Comparison of these apoptosis studies with the cytokine inhibition studies suggests that approximately 100-fold lower concentrations of VRT-043198 are required for anti-inflammatory activity than for caspase-8-mediated antiapoptotic activity, consistent with the 100-fold difference in potency on isolated ICE/caspase-1 and caspase-8 enzymes (Table 1).

Oral Administration of VX-765 Resulted in High Plasma Concentrations of VRT-043198. To evaluate the pharmacokinetic properties of VX-765, plasma concentrations of VX-765 and VRT-043198 were monitored after oral dosing of VX-765 for 24 h in mice. Exposure of VRT-043198 was greater than that of VX-765 following oral administration of VX-765 at all dose levels tested (Table 3). The maximum plasma concentration (C_max) values of VRT-043198 observed in the mouse were higher than or close to in vitro IC₅₀ values for inhibition of IL-1 and IL-18 in human PBMCs and whole blood (Table 2; and see above).

VX-765 Inhibits LPS-Induced IL-1 Production in Vivo. Intravenous injection of E. coli LPS in mice provokes a spike of serum IL-1 levels within 2 to 2.5 h. To evaluate the inhibition of ICE/caspase-1-mediated IL-1 production in vivo, a single oral dose of VX-765 (25, 50, 100, or 200 mg/kg) was administered 1 h before i.v. LPS injection and peripheral blood samples were harvested at 2.5 h and assayed for IL-1 concentrations. VX-765 doses 50, 100, and 200 mg/kg significantly (Dunnett's ANOVA test; p < 0.05) reduced serum IL-1 levels by as much as 60%, whereas 25 mg/kg had a smaller effect (35% inhibition) that was not statistically significant (Fig. 2). It is noteworthy that the effect of VX-765 on the release of IL-1 induced by LPS reached a plateau at 100 mg/kg. Although the reason for this ceiling effect is not clear, we speculate that intravenous injection of LPS may cause substantial release of pro-IL-1, which was detected by the ELISA system used in this study. Alternatively, IL-1 immunoreactivity may be produced by a processing mechanism other than ICE/caspase-1. Finally, the release of IL-1 may be regulated by two phases, i.e., acute induction and processing of IL-1 followed by a subacute process of IL-1 production mediated through production of other cytokines and/or mediators such as TNF-, which is known to induce IL-1 production (Fong et al., 1989; Covert et al., 2005). It is quite possible that drug concentrations were not high enough to suppress the second process by the time it began.

View larger version (16K): [in this window] [in a new window]   Fig. 2. Dose response of VX-765 in LPS-induced IL-1 production in vivo. VX-765 (25, 50, 100, or 200 mg/kg) was administered by oral gavage 1 h before the intravenous LPS challenge. Blood samples were collected 2.5 h after the LPS challenge serum IL-1 was assayed by specific ELISA. Data are means ± S.D. Dunnett's ANOVA test was performed for statistical analysis (*, p < 0.05 compared with the LPS control group).

View larger version (18K): [in this window] [in a new window]   Fig. 3. Effects of VX-765 on ear swelling induced by oxazolone. Forty-eight hours after the challenge on the right ear, biopsy samples 9 mm in diameter were taken from both ears of each animal, and the samples were weighed. Data are expressed as the difference in weight between the right and left ear discs of animal (n = 9/group; means ± S.D.). Dunnett's ANOVA test was performed for statistical analysis (*, p < 0.05 compared with the vehicle-treated group). Similar results were obtained from at least two independent experiments.

View larger version (34K): [in this window] [in a new window]   Fig. 4. Effects of VX-765 on production of inflammatory mediators in biopsy samples from oxazolone-challenged mouse ears (n = 6/group). Cytokines (A–D), nitric oxide (E), myeloperoxidase (F), and chemokines (G–I) were quantified by specific ELISA kits. Data are means ± S.D. Dunnett's ANOVA test was performed for statistical analysis (*, p < 0.05 compared with control). Similar results were obtained from at least two independent experiments.

VX-765 Attenuates CIA Responses in Mice. Cytokines seem to play a pivotal role in the pathogenesis of rheumatoid arthritis (RA). IL-1 has been detected in arthritic joints of patients with rheumatoid arthritis, and administration of IL-1 is sufficient to induce arthritis in experimental animals (for review, see van den Berg, 2002). The efficacy of VX-765 administered orally twice daily was evaluated in CIA, a mouse model of rheumatoid arthritis, using both prophylactic and therapeutic treatment regimens. VX-765 was well tolerated even at 100 mg/kg twice daily for 28 days, and it did not show substantial changes in body weight (data not shown). In the prophylactic study, compounds and vehicle were dosed beginning after booster immunization. In the therapeutic study, treatment was started when erythema and partial swelling of the tissue around the wrist joint were apparent (level 2) in both forepaws. Figure 5, A and B, shows the mean forepaw inflammation scores, evaluated every other day. In both studies, VX-765 induced a dose-dependent, statistically significant reduction in the inflammation scores (Wilcoxon rank-sum test; p < 0.05), and 100 mg/kg VX-765 was as efficacious as 5 mg/kg prednisolone (Ku et al., 1996; Rioja et al., 2004). Histological analysis of forepaws at the end of both studies revealed good correlation between the degree of joint structural damage and the inflammation scores (Table 4; Fig. 6). In the therapeutic study, a majority of mice in the vehicle-treated group exhibited severe inflammation and extensive damage to bone and cartilage (Table 4; Fig. 6C). In contrast, mice treated with VX-765 showed pronounced protection from joint changes, with maximum benefit being similar to that observed with prednisolone (Table 4; Fig. 6D).

View larger version (34K): [in this window] [in a new window]   Fig. 5. Effects of VX-765 on forepaw inflammation in the mouse CIA model administered in either the prophylactic (A) or therapeutic (B) regimen. In the prophylactic study, mice (n = 4–6/group) were treated orally twice daily with vehicle, prednisolone (5 mg/kg) or VX-765 (25, 50, or 100 mg/kg) after the second immunization of the type II collagen for 28 days. In the therapeutic regimen, mice (9–10/group) were allowed to develop level 2 inflammation in both forepaws before assignment to treatment groups (n = 10/group), and then they were treated with either vehicle, prednisolone (5 mg/kg b.i.d.) or VX-765 (10, 25, 50, or 100 mg/kg b.i.d.) for 24 days. Paw inflammation was scored every other day. Scores are reported as the average of the sum of inflammation scores from both forepaws of each mouse in a given treatment group with standard deviation. Similar results were obtained from two independent experiments both in the prophylactic and therapeutic dosing regimens.

View larger version (154K): [in this window] [in a new window]   Fig. 6. Histological examination of representative forepaw wrist joints from the prophylactic and therapeutic studies. Joint issues were harvested at the end of studies and stained with hematoxylin and eosin. In the prophylactic study, a representative forepaw joint from a mouse treated with the vehicle (A) shows substantial cartilage erosion and synovial infiltration in the joint space, whereas a forepaw from an animal treated with 100 mg/kg VX-765 exhibits minimal signs of such changes (B). Bone erosion in addition to synovial infiltration and cartilage erosion is obvious in a representative joint from a mouse treated with the vehicle in the therapeutic study (C). These changes were not observed in a mouse treated therapeutically with 100 mg/kg VX-765 (D).

	Discussion

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Cytokines are implicated in a number of human immune and inflammatory diseases, and modulation of production of key cytokines has emerged as an important therapeutic approach. Increased production of IL-1 and IL-18 has been demonstrated in patients with inflammatory diseases such as psoriasis, RA, and Crohn's disease. A recombinant form of human IL-1Ra (anakinra) has been shown to be effective in clinical trials of RA, and it is approved for the treatment of RA (for review, see Braddock and Quinn, 2004). In addition, anakinra markedly suppressed inflammatory manifestations of autoinflammatory syndromes, including Muckle-Wells syndrome, neonatal onset multisystem inflammatory disease, familial cold autoinflammatory syndrome (FCAS) (for review, see Ting et al., 2006), and systemic onset juvenile idiopathic arthritis (Pascual et al., 2005) and adult Still's disease (Fitzgerald et al., 2005).

In this report, we characterized another approach to suppress excessive inflammation in diseases by inhibiting the ICE/caspase-1 subfamily of caspases essential for generation of biologically active IL-1 and IL-18. Previously, we developed an ICE/caspase-1 inhibitor, pralnacasan, and found it to be effective in several preclinical models and in RA patients (Ku et al., 2001; Rudolphi et al., 2003; Loher et al., 2004). VX-765 was developed from a structurally distinct class of caspase-1 inhibitors that share with pralnacasan the aspartyl-hemiacetal ester moiety that serves as a prodrug. VRT-043198, the active metabolite of VX-765, is a potent, specific inhibitor of the caspase-1 subfamily. VRT-043198 inhibits the release of IL-1 and IL-18 from human monocytes in vitro and their production in vivo in models of inflammation. Note that we recently studied PBMCs from FCAS patients, which produce high levels of IL-1 and IL-18 upon LPS stimulation. Consistent with the current report, VX-765 inhibited the release of IL-1 and IL-18 in FCAS patient PB-MCs and equally potent at inhibiting the release of IL-1 in both normal and FACS patient PBMCs (Stack et al., 2005). VRT-043198 exhibited little or no effect on cell apoptosis and proliferation, as expected based on observations in caspase-1-deficient mice. This activity profile endows VX-765 with beneficial activity in mouse models of dermatological and bone and joint inflammation and autoimmunity.

We used the mouse DTH model to evaluate the effect of VX-765 on skin inflammation and to provide support for its use in inflammatory skin disorders such as psoriasis and atopic dermatitis. DTH responses are typically measured as skin reactions to haptens and mediated by inflammatory cytokines (Askenase, 2001). In particular, IL-18 is constitutively expressed at high levels in macrophages, keratinocytes, and Langerhans cells (LCs) (Stoll et al., 1998; Naik et al., 1999). LCs are a key component of DTH responses in the skin against reactive molecules such as oxazolone and picryl chloride. IL-18 has been shown to play an essential role in migration of LCs into draining lymph nodes by an IL-1- and TNF--dependent mechanism (Cumberbatch et al., 2001). IL-18 has also been implicated in IL-12-driven IFN- production by T_H1 cells, thus contributing to DTH responses in the skin (Wang et al., 2002). As shown in Fig. 4, VX-765 inhibited the release of IL-1 and IL-18 in a dose-dependent manner, resulting in reduced production of IFN- and chemokines. Reduction of theses inflammatory mediators presumably contributed to diminished T_H1 responses and recruitment of neutrophils and macrophages, the latter reflected in decreased levels of myeloperoxidase and nitric oxide in the ear biopsy samples from mice treated with VX-765. Moreover, the highest dose of VX-765 suppressed production of IL-4, whereas prednisolone did not. In DTH responses, IL-4 is produced by natural killer T cells and promotes activation of a subset of B cells. Although IL-18 is an inducer of the T_H1 cytokine IFN-, IL-18 is known to induce the expression of the T_H2 cytokines IL-4 and IL-13 in T cells, natural killer cells, mast cells, and basophils (Hoshino et al., 2001). Thus, it is most likely that decreased IL-18 production by VX-765 resulted in reduction of IL-4 in the inflamed ear discs. Taken together, our results in the DTH model suggest that VX-765 may be useful in the treatment of inflammatory skin disorders such as psoriasis and atopic dermatitis.

RA is a chronic syndrome characterized by systematic inflammation of peripheral joints, potentially resulting in destruction of articular and periarticular structures. Although no animal model fully represents the pathophysiological changes in human RA patients, mouse CIA is widely used as a surrogate for analyzing pathogenic mechanisms of joint inflammation and evaluating therapeutic agents (Iwakura, 2002). Mouse CIA is induced by immunization with type II collagen to a susceptible strain of mouse, and it is mediated by both humoral and cellular immunity. Similarities between mouse CIA responses and human RA include linkage of disease to genes located in the histocompatibility locus, mononuclear cell infiltration, pannus development, fibrin deposition, erosion of cartilage, and bone destruction. In addition, inflammatory cytokines such as IL-1, TNF-, and IL-6 are also produced in CIA joints (Rioja et al., 2004). Although high levels of TNF- are produced in the synovial fluid of RA patients (Neidel et al., 1995), analyses of paw tissues from CIA studies in animals indicate that arthritic joints of CIA produce low levels of TNF- compared with IL-1 and IL-6 (Rioja et al., 2004). Consistent with these data, blockade of TNF- is partially effective at an early stage of CIA, whereas blocking of IL-1, either right after the onset or during established CIA, effectively suppresses progression of arthritis (Joosten et al., 1996). In mice deficient in TNF- or TNF receptor, the incidence of CIA is reduced. However, once initial signs of arthritis develop, the condition tends to progress to full-blown, destructive arthritis in the mice (Mori et al., 1996; Campbell et al., 2001). In contrast, IL-1/-deficient mice are markedly resistant to development of CIA, and mice deficient in either IL-1 or IL-1 exhibit reduced progression of CIA (Saijo et al., 2002). Thus, TNF- plays a role mainly in the early stage of CIA, whereas IL-1 plays a more pivotal role both in the onset and progression of the disease (van den Berg, 2002). Here, we show that caspase-1 inhibition with VX-765 delayed the onset of arthritis and suppressed progression of the disease when dosed prophylactically and that it reversed paw inflammation and prevented joint damage in established disease when dosed therapeutically. This suggests the potential of VX-765 for therapeutic benefit against established arthritis in RA patients. It is noteworthy that IL-1 is linked to osteoclast activation by inducing expression of receptor activator of the nuclear factor-B ligand, resulting in bone resorption and strong suppression of aggrecan synthesis, causing cartilage erosion (Goldring and Gravallese, 2000). This is highlighted by the fact that IL-1-deficient mice or mice treated with IL-1Ra do not develop joint erosion, whereas treatment with anti-TNF- antibodies or soluble TNF receptor protein in CIA models does not result in any measurable effect on cartilage or bone destruction (Wooley et al., 1993; Joosten et al., 1996; Saijo et al., 2002). Given that VX-765 treatment reduced histological changes in the prophylactic regimen and resulted in significant improvement of joint histology in the therapeutic regimen comparable with treatment with prednisolone (Table 4), VX-765 may act as a disease-modifying agent in the treatment of RA.

Several therapeutic agents targeting IL-1 and IL-18 have been tested in clinic for inflammation (for review, see Braddock and Quinn, 2004). The data described in this report confirm the therapeutic promise of a novel approach, selective ICE/caspase-1 inhibition. VX-765 is a potent and selective ICE/caspase-1 inhibitor that reduces the production of IL-1 and IL-18 both in vitro and in vivo in correlation with tissue-protective effects in animal models of inflammatory disease. VX-765 is currently in clinical trials in inflammatory and autoimmune indications, the results of which will be reported separately.

	Footnotes

 
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.106.111344.

ABBREVIATIONS: ICE, interleukin-converting enzyme; IL, interleukin; TNF, tumor necrosis factor; IFN, interferon; T_H, T-helper; IL-1Ra, interleukin-1 receptor antagonist; VRT-043198, (S)-3-({1-[(S)-1-((S)-2-{[1-(4-amino-3-chloro-phenyl)-methanoyl]-amino}-3,3-dimethyl-butanoyl)-pyrrolidin-2-yl]-methanoyl}-amino)-4-oxo-butyric acid; VX-765, (S)-1-((S)-2-{[1-(4-amino-3-chloro-phenyl)-methanoyl]-amino}-3,3-dimethyl-butanoyl)-pyrrolidine-2-carboxylic acid ((2R,3S)-2-ethoxy-5-oxo-tetrahydro-furan-3-yl)-amide; AUC, area under the curve; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]propanesulfonate; PBMC, peripheral blood mononuclear cell; LPS, lipopolysaccharide; SAC, S. aureus-Cowan strain 1; ELISA, enzyme-linked immunosorbent assay; DTH, delayed-type hypersensitivity; CIA, collagen-induced arthritis; ANOVA, analysis of variance; RA, rheumatoid arthritis; FCAS, familial cold autoinflammatory syndrome; LC, Langerhans cell.

The online version of this article (available at http://jpet.aspetjournals.org) contains supplemental material.

Address correspondence to: Dr. John C. R. Randle, Vertex Pharmaceuticals, Inc., 130 Waverly St., Cambridge, MA 02139. E-mail: john_randle{at}vrtx.com

	References

Top Abstract Materials and Methods Results Discussion References

 

Antonopoulos C, Cumberbatch M, Dearman RJ, Daniel RJ, Kimber I, and Groves RW (2001) Functional caspase-1 is required for Langerhans cell migration and optimal contact sensitization in mice. J Immunol 166: 3672–3677.[Abstract/Free Full Text]

Askenase PW (2001) Yes T cells, but three different T cells (alphabeta, gammadelta and NK T cells), and also B-1 cells mediate contact sensitivity. Clin Exp Immunol 125: 345–350.[CrossRef][Medline]

Braddock M and Quinn A (2004) Targeting IL-1 in inflammatory disease: new opportunities for therapeutic intervention. Nat Rev Drug Discov 3: 330–339.[CrossRef][Medline]

Campbell IK, O'Donnell K, Lawlor KE, and Wicks IP (2001) Severe inflammatory arthritis and lymphadenopathy in the absence of TNF. J Clin Investig 107: 1519–1527.[Medline]

Covert MW, Leung TH, Gaston JE, and Baltimore D (2005) Achieving stability of lipopolysaccharide-induced NF-B activation. Science (Wash DC) 309: 1854–1857.[Abstract/Free Full Text]

Cumberbatch M, Dearman RJ, Antonopoulos C, Groves RW, and Kimber I (2001) Interleukin (IL)-18 induces Langerhans cell migration by a tumour necrosis factor-- and IL-1-dependent mechanism. Immunology 102: 323–330.[CrossRef][Medline]

Dinarello CA (2002) The IL-1 family and inflammatory diseases. Clin Exp Rheumatol 20: S1–S13.[Medline]

Earnshaw WC, Martins LM, and Kaufmann SH (1999) Mammalian caspases: structure, activation, substrates, and functions during apoptosis. Annu Rev Biochem 68: 383–424.[CrossRef][Medline]

Fong Y, Tracey KJ, Moldawer LL, Hesse DG, Manogue KB, Kenney JS, Lee AT, Kuo GC, Allison AC, Lowry SF, et al. (1989) Antibodies to cachectin/tumor necrosis factor reduce interleukin 1 and interleukin 6 appearance during lethal bacteremia. J Exp Med 170: 1627–1633.[Abstract/Free Full Text]

Fitzgerald AA, LeClercq SA, Yan A, Homik JE, and Dinarello CA (2005) Rapid responses to anakinra in patients with refractory adult-onset Still's disease. Arthritis Rheum 52: 1794–1803.[CrossRef][Medline]

Fox T, de Miguel E, Mort JS, and Storer AC (1992) Potent slow-binding inhibition of cathepsin B by its propeptide. Biochemistry 31: 12571–12576.[CrossRef][Medline]

Goldring SR and Gravallese EM (2000) Pathogenesis of bone erosions in rheumatoid arthritis. Curr Opin Rheumatol 12: 195–199.[CrossRef][Medline]

Hoshino T, Kawase Y, Okamoto M, Yokota K, Yoshino K, Yamamura K, Miyazaki J, Young HA, and Oizumi K (2001) Cutting edge: IL-18-transgenic mice: in vivo evidence of a broad role for IL-18 in modulating immune function. J Immunol 166: 7014–7018.[Abstract/Free Full Text]

Iwakura Y (2002) Roles of IL-1 in the development of rheumatoid arthritis: consideration from mouse models. Cytokine Growth Factor Rev 13: 341–355.[CrossRef][Medline]

Joosten LA, Helsen MM, van de Loo FA, and van den Berg WB (1996) Anticytokine treatment of established type II collagen-induced arthritis in DBA/1 mice. A comparative study using anti-TNF, anti-IL-1/, and IL-1Ra. Arthritis Rheum 39: 797–809.[Medline]

Ku G, Faust T, Lauffer LL, Livingston DJ, and Harding MW (1996) Interleukin-1 converting enzyme inhibition blocks progression of type II collagen-induced arthritis in mice. Cytokine 8: 377–386.[CrossRef][Medline]

Ku G, Ford P, Raybuck SA, Harding MW, and Randle JCR (2001) Selective interleukin-1 converting enzyme (ICE/caspase-1) inhibition with pralnacasan (HMR 3480/VX-740) reduces inflammation and joint destruction in murine type II collagen-induced arthritis (CIA). Arthritis Rheum 44: S241.[CrossRef]

Kuida K, Lippke JA, Ku G, Harding MW, Livingston DJ, Su MS, and Flavell RA (1995) Altered cytokine export and apoptosis in mice deficient in interleukin-1 converting enzyme. Science (Wash DC) 267: 2000–2003.[Abstract/Free Full Text]

Li P, Allen H, Banerjee S, Franklin S, Herzog L, Johnston C, McDowell J, Paskind M, Rodman L, Salfeld J, et al. (1995) Mice deficient in IL-1-converting enzyme are defective in production of mature IL-1 and resistant to endotoxic shock. Cell 80: 401–411.[CrossRef][Medline]

Loher F, Bauer C, Landauer N, Schmall K, Siegmund B, Lehr HA, Dauer M, Schoenharting M, Endres S, and Eigler A (2004) The interleukin-1-converting enzyme inhibitor pralnacasan reduces dextran sulfate sodium-induced murine colitis and T helper 1 T-cell activation. J Pharmacol Exp Ther 308: 583–590.[Abstract/Free Full Text]

Martinon F and Tschopp J (2004) Inflammatory caspases: linking an intracellular innate immune system to autoinflammatory diseases. Cell 117: 561–574.[CrossRef][Medline]

Mori L, Iselin S, De Libero G, and Lesslauer W (1996) Attenuation of collagen-induced arthritis in 55-kDa TNF receptor type 1 (TNFR1)-IgG1-treated and TNFR1-deficient mice. J Immunol 157: 3178–3182.[Abstract]

Murray N, Zoerkler N, Brown T, and Bonhomme Y (1994) LCB 2183 inhibits the inflammation associated with oxazolone-induced contact sensitivity. Int J Immunopharmacol 16: 675–683.[CrossRef][Medline]

Naik SM, Cannon G, Burbach GJ, Singh SR, Swerlick RA, Wilcox JN, Ansel JC, and Caughman SW (1999) Human keratinocytes constitutively express interleukin-18 and secrete biologically active interleukin-18 after treatment with proinflammatory mediators and dinitrochlorobenzene. J Investig Dermatol 113: 766–772.[CrossRef][Medline]

Neidel J, Schulze M, and Lindschau J (1995) Association between degree of bone-erosion and synovial fluid-levels of tumor necrosis factor alpha in the knee-joints of patients with rheumatoid arthritis. Inflamm Res 44: 217–221.[CrossRef][Medline]

Pascual V, Allantaz F, Arce E, Punaro M, and Branchereau J (2005) Role of interleukin-1 (IL-1) in the pathogenesis of systemic onset juvenile idiopathic arthritis and clinical response to IL-1 blockade. J Exp Med 201: 1479–1486.[Abstract/Free Full Text]

Rioja I, Bush KA, Buckton JB, Dickson MC, and Life PF (2004) Joint cytokine quantification in two rodent arthritis models: kinetics of expression, correlation of mRNA and protein levels and response to prednisolone treatment. Clin Exp Immunol 137: 65–73.[CrossRef][Medline]

Rudolphi K, Gerwin N, Verzijl N, van der Kraan P, and van den Berg W (2003) Pralnacasan, an inhibitor of interleukin-1 converting enzyme, reduces joint damage in two murine models of osteoarthritis. Osteoarthr Cartil 11: 738–746.[CrossRef][Medline]

Saijo S, Asano M, Horai R, Yamamoto H, and Iwakura Y (2002) Suppression of autoimmune arthritis in interleukin-1-deficient mice in which T cell activation is impaired due to low levels of CD40 ligand and OX40 expression on T cells. Arthritis Rheum 46: 533–544.[CrossRef][Medline]

Smith DJ, McGuire MJ, Tocci MJ, and Thiele DL (1997) IL-1 convertase (ICE) does not play a requisite role in apoptosis induced in T lymphoblasts by Fas-dependent or Fas-independent CTL effector mechanisms. J Immunol 158: 163–170.[Abstract]

Stack JH, Beaumont K, Larsen PD, Straley KS, Henkel GW, Randle JCR, and Hoffman HM (2005) ICE/Caspase-1 inhibitor VX-765 blocks the hypersensitive response to an inflammatory stimulus in monocytes from FCAS patients. J Immunol 175: 2630–2634.[Abstract/Free Full Text]

Stoll S, Jonuleit H, Schmitt E, Muller G, Yamauchi H, Kurimoto M, Knop J, and Enk AH (1998) Production of functional IL-18 by different subtypes of murine and human dendritic cells (DC): DC-derived IL-18 enhances IL-12-dependent Th1 development. Eur J Immunol 28: 3231–3239.[CrossRef][Medline]

Thalappilly S, Sadasivam S, Radha V, and Swarup G (2006) Involvement of caspase 1 and its activator Ipaf upstream of mitochondrial events in apoptosis. FEBS J. 273: 2766–2778.[CrossRef][Medline]

Ting JP, Kastner DL, and Hoffman HM (2006) CATERPILLERs, pyrin and hereditary immunological disorders. Nat Rev Immunol 6: 183–195.[CrossRef][Medline]

van den Berg WB (2002) Is there a rationale for combined TNF and IL-1 blocking in arthritis? Clin Exp Rheumatol 20: S21–S25.[Medline]

Wang B, Feliciani C, Howell BG, Freed I, Cai Q, Watanabe H, and Sauder DN (2002) Contribution of Langerhans cell-derived IL-18 to contact hypersensitivity. J Immunol 168: 3303–3308.[Abstract/Free Full Text]

Wannamaker M and Davies R (2001) inventors; Vertex Pharma, assignee. Prodrug of an ICE inhibitor. World Patent WO0190063. 2001 Nov 29

Wooley PH, Dutcher J, Widmer MB, and Gillis S (1993) Influence of a recombinant human soluble tumor necrosis factor receptor FC fusion protein on type II collagen-induced arthritis in mice. J Immunol 151: 6602–6607.[Abstract]

Yoshimoto T, Takeda K, Tanaka T, Ohkusu K, Kashiwamura S, Okamura H, Akira S, and Nakanishi K (1998) IL-12 up-regulates IL-18 receptor expression on T cells, Th1 cells, and B cells: synergism with IL-18 for IFN- production. J Immunol 161: 3400–3407.[Abstract/Free Full Text]

Zepter K, Haffner A, Soohoo LF, De Luca D, Tang HP, Fisher P, Chavinson J, and Elmets CA (1997) Induction of biologically active IL-1-converting enzyme and mature IL-1 in human keratinocytes by inflammatory and immunologic stimuli. J Immunol 159: 6203–6208.[Abstract]

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