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Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

Objective

All γ-chain cytokines signal through JAK-3 and JAK-1 acting in tandem. We undertook this study to determine whether the JAK-3 selective inhibitor WYE-151650 would be sufficient to disrupt cytokine signaling and to ameliorate autoimmune disease pathology without inhibiting other pathways mediated by JAK-1, JAK-2, and Tyk-2.

Methods

JAK-3 kinase selective compounds were characterized by kinase assay and JAK-3–dependent (interleukin-2 [IL-2]) and –independent (IL-6, granulocyte–macrophage colony-stimulating factor [GM-CSF]) cell-based assays measuring proliferation or STAT phosphorylation. In vivo, off-target signaling was measured by IL-22– and erythropoietin (EPO)–mediated models, while on-target signaling was measured by IL-2–mediated signaling. Efficacy of JAK-3 inhibitors was determined using delayed-type hypersensitivity (DTH) and collagen-induced arthritis (CIA) models in mice.

Results

In vitro, WYE-151650 potently suppressed IL-2–induced STAT-5 phosphorylation and cell proliferation, while exhibiting 10–29-fold less activity against JAK-3–independent IL-6– or GM-CSF–induced STAT phosphorylation. Ex vivo, WYE-151650 suppressed IL-2–induced STAT phosphorylation, but not IL-6–induced STAT phosphorylation, as measured in whole blood. In vivo, WYE-151650 inhibited JAK-3–mediated IL-2–induced interferon-γ production and decreased the natural killer cell population in mice, while not affecting IL-22–induced serum amyloid A production or EPO-induced reticulocytosis. WYE-151650 was efficacious in mouse DTH and CIA models.

Conclusion

In vitro, ex vivo, and in vivo assays demonstrate that WYE-151650 is efficacious in mouse CIA despite JAK-3 selectivity. These data question the need to broadly inhibit JAK-1–, JAK-2–, or Tyk-2–dependent cytokine pathways for efficacy.

Interleukin-2 (IL-2), IL-4, IL-7, IL-9, IL-15, and IL-21 signal through their specific cognate receptor(s) and a common γ-chain receptor to promote lymphoid development and function, and they maintain homeostasis of the immune system by signaling via the well-characterized JAK/STAT pathway. JAK-3 uniquely associates with γ-chain, and in conjunction with JAK-1 plays an essential role in γ-chain–dependent signal transduction (1). Mutations in either γ-chain or JAK-3 have been identified in humans as a cause of severe combined immunodeficiency disease (SCID), resulting in dysfunctional T, B, and natural killer (NK) cells with no other defects (2, 3). JAK-3 is predominantly expressed in cells of hematopoietic lineage (4–6), and consequently these immunosuppressed patients can be successfully treated with stem cell transplants (7). JAK-3–deficient mice have a defect similar to that observed in humans. These observations suggest that selective JAK-3 inhibition should be sufficient for immunosupression without having effects outside the immune system.

In addition to the 6 cytokines that signal using JAK-3 and γ-chain, ∼30 other type I and type II cytokine receptors require the kinase activity of the other JAK family members. Tyk-2 deficiency results in a human primary immunodeficiency similar to the JAK-3 SCID phenotype; however, neither JAK-1–deficient nor JAK-2–deficient humans have been identified, and deficient mice are not viable (8, 9). Several of the cytokines that require JAK-1, JAK-2, and/or Tyk-2 activity, including IL-6, IL-12, and IL-23, play a pathogenic role in human rheumatoid arthritis (RA) (10, 11), suggesting that inhibiting the JAK family more broadly may be beneficial. However, the adverse effects of broader cytokine inhibition will need to be balanced against the potential efficacy.

Consistent with the human and mouse genetic data, an experimental JAK-3 inhibitor, CP-690,550, has demonstrated efficacy in clinical trials for multiple indications, including RA, psoriasis, and renal transplantation (12–14). Although CP-690,550 inhibits JAK-3, it also has an overlapping activity against JAK-1 and JAK-2 (15, 16). This fact raises the question of whether inhibition of JAK-3 alone is sufficient to disrupt cytokine signaling and to result in amelioration of autoimmune disease pathology. Here, we report the characterization of novel JAK-3 inhibitors, WYE-151650 and WYE-152038, that potently inhibit JAK-3 activity, compare favorably with CP-690,550 in ex vivo and in vivo selectivity assays, and are highly efficacious in a mouse model of collagen-induced arthritis (CIA).

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

Mice.

BALB/c mice were purchased from Taconic or Hilltop Lab Animals. DBA/1JBomTac and B6129SF2/J mice were obtained from Taconic and The Jackson Laboratory, respectively. Animals were kept at the animal facility of Wyeth Research or Ligand Pharmaceuticals in accordance with the Guide for the Care and Use of Laboratory Animals. All study protocols were approved by the Institutional Animal Care and Use Committee.

Compounds and reagents.

The structures of JAK-3 inhibitors (WYE-151650, WYE-152038, and CP-690,550) are shown in Figure 1A, and their synthesis was described previously (16, 17). Compounds were dissolved in vehicle containing 0.5% methylcellulose and 0.25% Tween for in vivo studies, unless otherwise indicated. IL-2 was purchased from PeproTech, and granulocyte–macrophage colony-stimulating factor (GM-CSF) and IL-6 were obtained from R&D Systems. Alexa Fluor–conjugated anti–phospho–STAT-5 and anti–phospho–STAT-3 antibodies were purchased from BD Biosciences. Recombinant human JAK-1 and Tyk-2 were purchased from Carna Biosciences.

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Figure 1. Inhibition of JAK-3 by WYE-151650. A, Structures of WYE-151650, WYE-152038, and CP-690,550 are shown. B, Various concentrations of WYE-151650 were included in the JAK-3 kinase assay, and JAK-3 activity was measured. The mean ± SD 50% inhibition concentration was 0.9 ± 0.1 nM (3 repeated experiments). Peripheral blood mononuclear cell (PBMC) blasts were cultured in the presence of interleukin-2 (IL-2) (10 ng/ml) with WYE-151650 for 2 days. The number of viable cells was measured using CellTiter-Glo Luminescent Cell Viability Assay reagents. C, PBMC blasts were treated with WYE-151650 for 30 minutes, followed by stimulation with IL-2 (10 ng/ml) for an additional 15 minutes. Phospho–STAT-5 (arrow) was detected by immunoblotting. D, Human white blood cells were pretreated with WYE-151650 for 30 minutes prior to stimulation with IL-2. Cells were fixed, permeabilized, stained with fluorochrome-labeled anti–phospho–STAT-5 antibody, and analyzed using flow cytometry. E, Human or mouse blood was treated with WYE-151650 for 30 minutes and then incubated with IL-2 (10 ng/ml) for an additional 15 minutes. Cells were fixed, permeabilized, stained with fluorochrome-labeled anti–phospho–STAT-5 antibody, and analyzed using flow cytometry. FITC = fluorescein isothiocyanate.

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Kinase assays.

Recombinant human JAK-3 (amino acids [aa] 508–1124) and JAK-2 (aa 532–1132) were expressed using the Bac-to-Bac Baculovirus Expression Systems according to the instructions of the manufacturer (Invitrogen). Kinase activity was assessed by homogeneous time-resolved fluorescence assay, incubating biotinylated poly Glu-Ala-Tyr as the substrate, ATP (at experimentally determined Km for JAK kinases), compound, and kinase for 1-hour reactions quenched with EDTA. For kinase selectivity profiling assays, kinases (Invitrogen), fluorescence-labeled peptide substrates (Anaspec Fremont), compound, and ATP (at the experimentally determined Km) were incubated 1 or 2 hours depending on kinase activity and then quenched with EDTA, and phosphorylated substrate was quantified.

Cell proliferation assay.

Cryopreserved peripheral blood mononuclear cells (PBMCs; AllCells) were cultured in complete RPMI 1640 medium (supplemented with 10% fetal bovine serum, penicillin/streptomycin, 2 mML-glutamine, and 1 mM sodium pyruvate) plus 10 μg/ml lectin phytohemagglutinin (PHA) for 3 days, then in complete RPMI 1640 medium plus IL-2 and various concentrations of compound for 2 days. The number of viable cells was determined by using the CellTiter-Glo Luminescent Cell Viability Assay kit (Promega).

Immunoblotting.

Human white blood cells (WBCs) were generated from fresh human blood treated with hemolysis buffer to lyse and remove erythrocytes. Human WBCs or PHA-activated PBMCs were pretreated with compound for 30 minutes at 37°C, followed by stimulation with IL-2, GM-CSF, or IL-6 for 15 minutes. Total cell lysates from equivalent cell numbers were analyzed using immunoblotting (18).

Flow cytometry analysis.

Human or mouse blood aliquots or human WBCs were incubated with compound for 30 minutes at 37°C, and then treated with IL-2, GM-CSF, or IL-6 for an additional 15 minutes. To terminate the stimulation, cells were fixed with Lyse/Fix Buffer (BD Biosciences) for blood samples and 2% paraformaldehyde for WBC samples. The fixed cells were incubated with 90% methanol for 30 minutes on ice and then were incubated with fluorochrome-labeled anti–phospho-STAT antibodies overnight at 4°C. IL-2–induced STAT-5 phosphorylation, GM-CSF–induced STAT-5 phosphorylation, and IL-6–induced STAT-3 phosphorylation were analyzed and quantified using a FACSCanto flow cytometer (BD Biosciences).

IL-22–induced serum amyloid A (SAA) production in mice.

B6129SF2/J mice (n = 10) were dosed orally with vehicle (2% Tween 80, 0.5% methylcellulose), WYE-151650, or CP-690,550, or intraperitoneally with anti–IL-22 antibody (generated internally, clone Ab-01) (19). One hour later, mice were injected intraperitoneally with 10 μg of recombinant mouse IL-22 (20). Six hours later, mice were bled, and serum SAA levels were measured by enzyme-linked immunosorbent assay (ELISA; BioSource).

Erythropoietin (EPO)–induced reticulocyte generation in mice.

Female BALB/c mice (n = 9 or n = 10) were dosed orally with vehicle or compound once daily for 4 days (day 0 to day 3), starting 1 hour before subcutaneous injection of EPO on day 0. On day 4, mice were killed, and terminal blood samples were collected for analysis of reticulocytes and NK cells by flow cytometry. Reticulocytes were stained by thiazole orange. After the removal of erythrocytes, NK cells (allophycocyanin-CD49b+ [eBioscience] fluorescein–T cell receptor β–negative [BD Biosciences]) were quantified. CP-690,550 and WYE-151650 were dissolved in vehicle A (0.5% methylcellulose/0.25% Tween 80/10% ethanol) and vehicle B (0.5% methylcellulose/0.25% Tween 80), respectively.

IL-2–induced interferon-γ (IFNγ) production in mice.

BALB/c mice (n = 6) were dosed once via oral gavage with compounds. One hour after compound administration, the mice were injected intraperitoneally with a combination of IL-2 and biotinylated anti-mouse IFNγ capture antibody (BD Biosciences). Three hours later, mice were killed, and blood was collected. Serum IFNγ was quantified using an ELISA kit (BD Biosciences).

Sheep red blood cell (SRBC)–induced delayed-type hypersensitivity (DTH) model.

BALB/c mice (n = 10) were treated with vehicle or compounds orally for 7 days (day –1 to day 5). On day 0, mice were primed with a subcutaneous injection of SRBCs. On day 5, the right hind footpad was measured using a caliper. The right hind footpad was then challenged by injecting SRBCs. Foot pad swelling (ventrodorsal thickness) was measured 24 hours later. The difference in thickness prior to and after challenge of the right hind footpad was reported as the change in right hind footpad thickness.

Mouse CIA model.

Arthritis was induced in 10-week-old male DBA/1JBomTac mice by immunizations with a 1:1 emulsion of bovine type II collagen (Chondrex) and Freund's complete adjuvant (Sigma-Aldrich) on day 0 and a 1:1 emulsion of bovine type II collagen in Freund's incomplete adjuvant (Sigma-Aldrich) on day 21 as previously described (21). Treatment was initiated when >10% of mice demonstrated signs of disease. On the day treatment was initiated, the mice were randomly assigned to a treatment group (n = 14) and began receiving daily doses via oral gavage of test articles or vehicle once daily. Mice were scored for signs of arthritis daily for 22 days.

Hematoxylin and eosin–stained tissue sections from all 4 paws from each of the 14 mice/group were evaluated microscopically using methods modified from McKew et al (22), as follows: grade 0 = no abnormal findings; grade 1 = synoviocyte hypertrophy, slight synovial membrane fibrosis, slight-to-mild inflammatory cell infiltrates into the synovial membrane/articular capsule and/or joint space; grade 2 = grade 1 plus mild-to-moderate inflammatory cell infiltrates, pannus formation (if present) minimal with superficial cartilage erosion; grade 3 = grade 2 plus marked inflammatory cell infiltrates and fibrosis, mild-to-severe erosion of the cartilage extending into subchondral bone; and grade 4 = loss of joint integrity through erosion or destruction with bone remodeling, massive inflammatory cell infiltrates, fibrosis, and ankylosis.

Statistical analysis.

Student's t-test or one-way analysis of variance with Dunnett's multiple comparison test was performed to determine statistically significant differences between experimental groups. P values less than 0.05 were considered significant.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

In vitro activity.

WYE-151650 (Figure 1A) was tested in a panel of in vitro kinase and cell-based assays. WYE-151650 potently inhibited JAK-3 kinase activity in a dose-dependent manner with a 50% inhibition concentration (IC50) of 0.9 nM (Figure 1B). To demonstrate the cellular effects of WYE-151650 on JAK-3–mediated signaling, human PBMC blasts were stimulated with IL-2. IL-2 signals via a heterotrimeric receptor complex consisting of IL-2 receptor β (IL-2Rβ), IL-2Rα, and γ-chain. Following IL-2R engagement, the receptor-associated JAK-1 and JAK-3 kinases phosphorylate STAT-5 protein (23, 24). In human PBMC blasts, WYE-151650 inhibited both a functional readout of IL-2–induced proliferation with an IC50 of 50 nM (Figure 1B) and also inhibited IL-2–induced STAT-5 phosphorylation, a more proximal event (Figure 1C). Similar inhibition of IL-2–stimulated STAT-5 phosphorylation by WYE-151650 was detected using flow cytometric analysis in human WBCs (Figure 1D). Additionally, IL-21–induced STAT-5 phosphorylation, which is also mediated by JAK-1 and JAK-3 (25), was blocked by WYE-151650 (data not shown). The cellular activity of WYE-151650 was further demonstrated in the more physiologic setting of human and mouse blood, in which the IL-2–induced STAT-5 phosphorylation of lymphocytes was inhibited by WYE-151650 (Figure 1E). WYE-152038 (Figure 1A), a close structural analog of WYE-151650, showed in vitro potency similar to that of WYE-151650 and CP-690,550 (Figure 1A and Table 1).

Table 1. Cell-based activity and selectivity of JAK-3 inhibitors*
Assay/mediumWYE-151650WYE-152038CP-690,550
  • *

    Values are the mean ± SD 50% inhibition concentration in nM (number of repeated experiments). The JAK-3 inhibitors WYE-151650, WYE-152038, and CP-690,550 were evaluated in cytokine-induced cell proliferation and STAT phosphorylation assays using peripheral blood mononuclear cell (PBMC) blasts, white blood cells (WBCs), or human blood as described in Materials and Methods. IL-2 = interleukin-2; GM-CSF = granulocyte–macrophage colony-stimulating factor.

IL-2–induced cell proliferation/PBMCs50 ± 30 (4)77 ± 24 (4)56 ± 23 (6)
IL-2–induced STAT-5 phosphorylation/WBCs59 ± 18 (3)78 ± 8 (3)31 ± 5 (4)
GM-CSF–induced STAT-5 phosphorylation/WBCs1,700 ± 150 (3)2,000 ± 290 (3)1,100 ± 250 (4)
IL-6–induced STAT-3 phosphorylation/WBCs610 ± 10 (2)810 ± 10 (3)160 ± 40 (3)
IL-2–induced STAT-5 phosphorylation/blood580 ± 300 (4)730 ± 210 (3)57 ± 9 (5)
IL-6–induced STAT-3 phosphorylation/blood28,000 ± 3,000 (3)46,000 ± 8,000 (2)610 ± 150 (4)

Kinase selectivity.

Of the JAK family kinases, JAK-3 expression is restricted to cells primarily of hematopoietic origin (4–6). In contrast, JAK-1, JAK-2, and Tyk-2 are more ubiquitously expressed. WYE-151650 exhibited 36-, 14-, and 34-fold selectivity against JAK-1, JAK-2, and Tyk-2, respectively, in kinase assays using recombinant enzymes (data not shown). In addition, WYE-151650 was evaluated against a broad panel of 27 non-JAK family kinases and demonstrated >100-fold selectivity (data not shown).

In addition to enzymatic selectivity, functional selectivity was characterized in several cell-based assays. Human WBCs consist of various populations of immune cells that can be stimulated by different cytokines, whose signals are transduced by different combinations of JAK family members. For example, IL-2–induced STAT-5 phosphorylation is mediated by JAK-3 and JAK-1 (24), GM-CSF–induced STAT-5 phosphorylation is dependent on JAK-2 (26), and IL-6–induced STAT-3 phosphorylation is thought to involve JAK-1, JAK-2, and Tyk-2 (27). Figure 2A shows that WYE-151650 potently inhibited IL-2–induced STAT-5 phosphorylation in human WBCs, but was much less active in the GM-CSF–induced STAT-5 phosphorylation assay and in the IL-6–induced STAT-3 phosphorylation assay. In addition to immunoblotting analysis, intracellular phosphorylated STAT proteins were stained by fluorochrome-labeled antibodies, and the levels of STAT phosphorylation were quantified using flow cytometric analysis. WYE-151650 inhibited IL-2 signaling with an IC50 of 59 nM and showed 29-fold and 10-fold selectivity compared with GM-CSF– and IL-6–mediated signaling, respectively (Figure 2B and Table 1). Analogous IL-2 and IL-6 assays in whole blood showed a further enhancement in selectivity (Table 1). A similar profile of JAK family selectivity was observed for WYE-152038 (Table 1 and data not shown).

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Figure 2. In vivo and ex vivo selectivity. Human white blood cells were pretreated with WYE-151650 (WYE) for 30 minutes, followed by treatment with interleukin-2 (IL-2), granulocyte–macrophage colony-stimulating factor (GM-CSF), or IL-6 for an additional 15 minutes. A, Cells were lysed with sample buffer, and the lysates were analyzed using immunoblotting. B, Cells were fixed, and the plasma membranes were permeabilized. Phospho–STAT-5 or phospho–STAT-3 was stained using fluorochrome-conjugated antibody and quantified using flow cytometry. C and D, BALB/c mice were orally dosed with WYE-151650 (C) or CP-690,550 (CP) (D) at 10 mg/kg. The mice (n = 3) were killed at different time intervals after administration, and blood was harvested. The drug concentrations of WYE-151650 and CP-690,550 in the plasma were measured. The blood samples were challenged with IL-2 (20 ng/ml) or IL-6 (10 ng/ml) for 15 minutes. After the removal of erythrocytes, the intracellular phospho–STAT-3 and phospho–STAT-5 were analyzed and quantified using flow cytometry. Blood harvested from vehicle-treated mice was used as the control. Signals of IL-2–induced STAT-5 phosphorylation or IL-6–induced STAT-3 phosphorylation from unstimulated and stimulated samples were defined as 0% of the control (background) and 100% of the control, respectively. The drug concentrations in mouse plasma were determined by liquid chromatography–tandem mass spectrometry. Values in C and D are the mean ± SEM. IC50 = 50% inhibition concentration.

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Ex vivo activity and selectivity.

The pharmacokinetic study indicated that WYE-151650 was well absorbed, with an oral bioavailability of 36% in mice (data not shown). The correlation between compound concentration in the plasma and its inhibitory activity was studied up to 4 hours after oral administration in mice. Following oral dosing of either WYE-151650 or the clinical compound CP-690,550, whole blood samples were split into 3 sets. One set was used to measure compound concentration in the plasma. The other 2 sets were stimulated ex vivo by IL-2 or IL-6, and STAT-5 or STAT-3 phosphorylation, respectively, was measured.

Since the plasma concentration of WYE-151650 exceeded the IC50 measured in mouse whole blood for the in vitro IL-2–induced STAT-5 phosphorylation assay (790 nM or 350 ng/ml) (Figure 1E), the ex vivo IL-2–induced STAT-5 phosphorylation was significantly suppressed and remained inhibited during the course of the experiment (Figure 2C). In contrast, the concentration of WYE-151650 was always significantly lower than the IC50 obtained in the mouse whole blood in vitro IL-6–induced STAT-3 phosphorylation assay (28,000 nM or 12,000 ng/ml) (Figure 2C and Table 1), and no inhibition of ex vivo IL-6–induced STAT-3 phosphorylation was detected. In the same experiment, the rapid absorption and faster clearance of CP-690,550 led to more complete but transient inhibition of IL-2–mediated STAT-5 phosphorylation, but with the additional inhibition of IL-6–mediated STAT-3 phosphorylation during the time that plasma concentrations of CP-690,550 exceeded the whole blood IC50 for IL-6–mediated STAT-3 phosphorylation (Figure 2D and Table 1). These data showed that WYE-151650 reaches sufficiently high concentrations in a target tissue, blood, to result in significant inhibition of JAK-3 without an effect on JAK-1–, JAK-2–, or Tyk-2–mediated signaling. In contrast, the exposure of CP-690,550 results in additional inhibition of JAK-1, JAK-2, and Tyk-2.

In vivo potency and selectivity.

In vivo administration of IL-2 induces IFNγ production in mice (28). To demonstrate in vivo JAK-3 inhibition, mice were treated with WYE-151650 orally prior to challenge with IL-2. IL-2–induced IFNγ production was inhibited by WYE-151650 in a dose-dependent manner (Figure 3A). The median effective dose was estimated to be 1 mg/kg. WYE-152038 and CP-690,550 have similar potency in this IL-2 model (data not shown).

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Figure 3. Short-term in vivo selectivity. A, BALB/c mice (n = 6) were dosed with vehicle or WYE-151650 (WYE), and 1 hour later were injected with a mixture of interleukin-2 (IL-2) and anti-mouse interferon-γ (IFNγ) capture antibody. Three hours later, blood samples were collected, and IFNγ in the serum was measured. Values are the mean and SEM. ∗ = P < 0.01 versus vehicle. B, B6129SF2/J mice (n = 10) were dosed orally with vehicle, WYE-151650 (100 mg/kg), or CP-690,550 (CP; 100 mg/kg), or intraperitoneally with anti–IL-22 antibody (200 μg/mouse). One hour later, mice were injected intraperitoneally with 10 μg of recombinant mouse IL-22. Six hours later, the mice were bled, and serum amyloid A (SAA) concentrations in serum were measured. Bars show the mean ± SEM. ∗ = P < 0.01 versus vehicle + IL-22. C and D, BALB/c mice (n = 9 or n = 10) were dosed orally with vehicle, CP-690,550, or WYE-151650 once daily for 4 days, starting 1 hour before the subcutaneous injection of erythropoietin (EPO; 100 IU/mouse) on day 0. On day 4, blood samples were collected, and the populations of natural killer (NK) cells (C) and reticulocytes (D) were stained and analyzed and expressed as percentages within the erythrocyte and leukocyte populations, respectively. Values are the mean and SEM. ∗ = P < 0.05; ∗∗ = P < 0.01 versus vehicle + EPO, by one-way analysis of variance with Dunnett's multiple comparison test. Vehicle A = 0.5% methylcellulose/0.25% Tween 80/10% ethanol; vehicle B = 0.5% methylcellulose/0.25% Tween 80.

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To further examine the JAK family selectivity of WYE-151650 and CP-690,550, we developed 2 additional in vivo mouse models that elucidate the functional downstream effects of these compounds. In the first model, mice were dosed orally with WYE-151650 or CP-690,550 prior to IL-22 administration. IL-22 signals through IL-10R type II and IL-22R, which mediate STAT protein phosphorylation via Tyk-2 and JAK-1, respectively (29). IL-22 induces the release of acute-phase reactants such as haptoglobin, SAA, and α1-antichymotrypsin from the liver (30), and IL-22–dependent production of SAA can be detected in serum (31). An IL-22–neutralizing antibody completely blocked IL-22–mediated serum SAA production (Figure 3B). WYE-151650 had no effect on IL-22–dependent production of SAA, even at an exaggerated dose chosen to see off-target effects (mean ± SEM serum concentration 910 ± 459 ng/ml at 6 hours). However, CP-690,550 showed significant inhibition of IL-22–mediated SAA production, suggesting that this compound inhibited JAK-1– and/or Tyk-2–mediated signaling at high doses in vivo (mean ± SEM serum concentration 48.8 ± 18.4 ng/ml at 6 hours). Exposure concentrations for both compounds were at least 2-fold their respective whole blood IL-2 IC50 values at 6 hours. The difference observed in the ability of the compounds to inhibit the JAK-3–independent IL-22–mediated signaling could be due to differences in JAK-1 selectivity, differences in maximum concentration (Cmax), or a combination of both.

In the second functional model, EPO, a potent erythropoietic agent that signals exclusively through the JAK-2 pathway (9), was used to induce reticulocytosis in the peripheral blood of mice (32). The effect of the JAK-3 inhibitors, WYE-151650 and CP-690,550, on EPO-induced reticulocytosis was assessed as a measure of JAK-2 selectivity in vivo. At the same time in the same mice, NK cells were monitored to assess the effectiveness of JAK-3 inhibition. Survival and development of NK cells requires IL-15, which signals via a JAK-3–dependent pathway (33). Mice were given a single dose of EPO at the beginning of the study and treated with vehicle or test article once daily for 4 days prior to the measurement of reticulocytes and NK cells in the blood. As shown in Figure 3C, both WYE-151650 and CP-690,550 reduced NK cells in a dose-dependent manner at doses from 10 mg/kg to 100 mg/kg, with a comparable level of maximal inhibition (44%) for both compounds. At this dose range, CP-690,550 significantly suppressed EPO-induced reticulocytosis, while WYE-151650 showed no effect (Figure 3D). This JAK-2 inhibitory effect was seen even at the lower dose of 30 mg/kg for CP-690,550. The results clearly demonstrated that when WYE-151650 and CP-690,550 are dosed to give similar reduction of NK cells, CP-690,550 shows evidence of overlapping JAK-2 activity in this in vivo setting. Taken together, these data show that WYE-151650 exhibits in vivo functional JAK-3 selectivity over other JAK family members.

To investigate the efficacy of selective JAK-3 inhibition, WYE-151650 was used in a mouse DTH model. Mice were primed with a subcutaneous injection of SRBCs on day 0 and challenged with a right hind footpad injection of SRBCs on day 5. Compound was dosed once daily throughout the study. WYE-151650 significantly inhibited the DTH reaction in the right hind footpad in a dosage-dependent manner, and efficacy was observed with dosages as low as 3 mg/kg once daily (Figure 4). Dosing at the time of challenge was ineffective, suggesting that the JAK-3 inhibitor blocks the DTH response during the priming phase (data not shown). CP-690,550 was also efficacious with once-daily dosing at 30 mg/kg in this DTH model (Figure 4).

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Figure 4. Efficacy of WYE-151650 and CP-690,550 in a delayed-type hypersensitivity model. BALB/c mice (10 per group) were dosed orally with vehicle or with WYE-151650 or CP-690,550 at 30, 10, or 3 mg/kg once daily for 6 days. On day 0, mice were primed with a subcutaneous injection of sheep red blood cells (SRBCs; 2 × 107/100 μl phosphate buffered saline [PBS]). On day 5, the mice were challenged by injecting SRBCs (1 × 108/25 μl PBS) into the right hind footpad (RHFP). The change (delta) in thickness of the right hind footpad was determined. Values are the mean and SEM. ∗ = P < 0.05; ∗∗ = P < 0.005 versus vehicle, by Student's t-test.

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To characterize WYE-151650 in an autoimmune disease model, WYE-151650 was tested in the mouse CIA model, which has many immunologic and pathologic similarities to human RA. Efficacy was assessed in a semitherapeutic dosing regimen, which involved scoring and assignment to treatment groups when at least 10% of the mice showed signs of disease. All mice were scored daily in a blinded manner for signs of disease. On day 0, the vehicle-treated mice had a clinical severity score of 0.3 that gradually increased to 10.0 on day 22 (Figure 5A). Treatment with WYE-151650 significantly suppressed progression of the disease. The efficacy was dosage related with an efficacious dosage as low as 10 mg/kg once daily. Similar results were observed for CP-690,550 as previously reported in this model (34) (Figure 5B). Inflammation and damage to the paw were assessed microscopically. Treatment with WYE-151650 resulted in a reduction in the damage to the paw based on lower group mean histologic severity scores in the WYE-151650–treated groups compared with the vehicle-treated group (Figure 5C). The paws from vehicle-treated control mice had a group mean severity of 2.5, while the group mean histologic severity scores in the WYE-151650–treated mice were 1.4, 1.3, and 2.0 at dosages of 30, 10, and 3 mg/kg once daily, respectively.

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Figure 5. Inhibition of collagen-induced arthritis in mice by WYE-151650 and CP-690,550. DBA/1JBomTac mice were immunized with bovine type II collagen as described in Materials and Methods. A, When 10% of the mice exhibited disease, the mice were randomly assigned to different treatment groups (n = 14 per group) and dosed orally with vehicle or WYE-151650. Values are the mean ± SEM. B, In a separate experiment, mice were treated with vehicle or CP-690,550. Disease severity scores were assessed daily. Values are the mean ± SEM. The difference in disease severity scores between WYE-151650–treated mice and vehicle-treated mice was significant (P < 0.05, by Student's t-test) for 30 mg/kg and 10 mg/kg WYE-151650 on days 9–22, but not for 3 mg/kg WYE-151650. The difference between CP-690,550–treated mice and vehicle-treated mice was significant for 30 mg/kg CP-690,550 on days 18–22 and for 10 mg/kg CP-690,550 on days 19–22. C, Following killing with CO2 on day 23 after 22 days of treatment with vehicle or WYE-151650, all 4 paws of 14 mice from each of the groups were prepared, microscopically examined, and scored. Values are the mean and SEM. ∗ = P < 0.05; ∗∗ = P < 0.005 versus vehicle, by Student's t-test. D, Representative sections from a vehicle-treated mouse (a metatarsophalangeal joint from the right rear paw) and a 30 mg/kg WYE-151650–treated mouse (a proximal interphalangeal joint from the right front paw) are shown (hematoxylin and eosin stained; original magnification × 10).

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Figure 5D shows representative sections of the joints from mice treated with vehicle or WYE-151650 (30 mg/kg). In the vehicle-treated exemplar, >50% of the hyaline cartilage and subchondral bone of the apposed articular surfaces was resorbed, the articular cavity was obliterated, and apposed bone ends were spanned by deposits of fibrous connective tissue and newly formed woven bone. In contrast, there was only a mild inflammatory cell infiltrate within the periarticular soft tissue, joint capsule/synovial membrane, and articular cavity in the WYE-151650–treated exemplar. Taken together, the results indicated that microscopic assessment of disease severity in the paws correlated well with clinical severity scores, and that WYE-151650 was efficacious in the mouse CIA model of RA.

DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

Long-term treatment of RA with immunosuppressive drugs, such as calcineurin antagonists, mammalian target of rapamycin inhibitors, and other disease-modifying antirheumatic drugs (DMARDs), is limited by side effects, including nephrotoxicity and neurotoxicity (35), due, at least in part, to the ubiquitous tissue distribution of these molecular targets. In contrast, JAK-3 is restricted in expression to immune cells (4–6). Interestingly, the expression of JAK-3 and STATs in synovial tissue decreases in response to successful DMARD treatment (36). Therefore, selective JAK-3 inhibition has theoretical advantages over existing immunosuppressive drugs. Furthermore, the efficacy of CP-690,550 in the clinic for treating RA supports targeting JAK family kinases as a viable target for orally administered small molecule inhibitors (12). However, the exact JAK family kinase selectivity profile required for efficacy, while maintaining an optimal safety multiple, is unclear.

Despite excellent selectivity across the kinome in general, CP-690,550 was recently reported to have modest selectivity against JAK-1 (4-fold) and JAK-2 (3-fold) and considerable selectivity against Tyk-2 (357-fold) (16), which is consistent with our cell-free assays (5-fold versus JAK-1, 10-fold versus JAK-2, 20-fold versus Tyk-2). This is not unexpected, given that a crystallographic model of CP-690,550 at the active site of the JAK-3 kinase postulates that only 3 residues proximal to the inhibitor differ across the JAK family, and these differences are conservative mutations (16, 37). Nevertheless, based on precedent from JAK-3–deficient SCID patients and the potential limitations of inhibiting JAK-1, we have chosen to optimize for in vivo JAK-3 selectivity and have benchmarked the potential utility of WYE-151650 against that of CP-690,550 for measures of both in vivo efficacy and selectivity.

In translating selectivity against purified JAK-3 kinase to functional selectivity in vivo, multiple parameters must be considered, including the inherent enzymatic selectivity, the combination of JAK kinases downstream of each receptor complex, and the exposure at the target over a dosing period. WYE-151650 selectively inhibits JAK-3 with 13-fold, 36-fold, and 34-fold preference over that of JAK-2, JAK-1, and Tyk-2, respectively, by kinase assay. In comparison, CP-690,550 was similarly selective in our assays for JAK-2 and Tyk-2 but less selective versus JAK-1. Since JAK-1 pairs with JAK-3 in all γ-chain receptor–mediated signaling pathways (24), inhibitors of JAK-3 that also inhibit JAK-1 may exhibit increased cellular potency. However, JAK-1 inhibition will also interfere with signaling pathways mediated by other cytokines, such as the gp130 receptor cytokines (IL-6 family) and the cytokine type II receptors such as the IFNs and the IL-10 family (38).

WYE-151650 is more selective for IL-2–dependent signaling than for IL-6–dependent signaling, as shown in Table 1. IL-6–induced STAT-3 phosphorylation is thought to involve JAK-1, JAK-2, and Tyk-2 (27), in contrast to IL-2–induced STAT-5 phosphorylation, which is mediated by JAK-3 and JAK-1. The decreased potency of WYE-151650 for JAK-1 relative to JAK-3 by kinase assay results in 10-fold reduced potency for IL-6–mediated signaling relative to IL-2–mediated signaling in WBCs and in 48-fold reduced potency for IL-6–mediated signaling relative to IL-2–mediated signaling in whole blood (Table 1), suggesting that although IL-2–mediated signaling is potently inhibited, JAK-1 inhibition minimally contributes to this inhibition and JAK-1 inhibition is not required for potent inhibition of IL-2–mediated signaling.

The preferential inhibition of IL-2 compared with IL-6 for WYE-151650 is more pronounced in the ex vivo assessment, since the plasma exposure versus time profile also plays a role in selectivity. Whereas WYE-151650 reaches and maintains a plasma concentration in excess of the whole blood IL-2 IC50 for the duration of the experiment without approaching the IC50 in the off-target IL-6 assay, the rapid absorption and clearance of CP-690,550 results in a high multiple of the IL-2 IC50 that exceeds the IC50 in the IL-6 assay. In addition to our data showing that WYE-151650 does not inhibit IL-6–mediated signaling ex vivo, we have shown that WYE-151650 had no effect on IL-22–mediated signaling in vivo, supporting the interpretation that this compound does not inhibit the JAK-1 and/or Tyk-2 kinases in vivo. Taken together, our studies indicate that inhibition of JAK-1 and IL-6 is not necessary for efficacy in the mouse CIA model.

The anti–IL-6 receptor tocilizumab has recently been approved for RA in Europe (39), and anti–IL-6 receptor treatment shows efficacy in CIA (40, 41). The efficacy achieved in RA by blocking IL-6 receptor suggests that compounds targeting JAK-1 in addition to JAK-3 may have efficacy, at least in part, due to IL-6 inhibition. However, IL-6 inhibition may have additional effects, since increases in lipids were observed after treatment with tocilizumab (39). Elevated lipid levels are also observed for CP-690,550 (12), which could be due to inhibition of IL-6 signaling or to the degree of suppression of inflammation (39).

JAK-2 mediates signaling via the hematopoietic cytokines EPO, GM-CSF, and thrombopoietin (8, 9). Patients receiving the highest dosage of CP-690,550 (30 mg twice daily) had higher incidences of anemia, granulocytopenia, and thrombocytopenia than did the placebo group (12). It was suggested that these results could be consistent with reduced JAK-2 activity in vivo (12). In cellular assessments, CP-690,550 and WYE-151650 show 35-fold and 29-fold less activity, respectively, against JAK-2–dependent GM-CSF stimulation compared with JAK-3/JAK-1–dependent IL-2 stimulation (Figure 2B and Table 1). The in vivo JAK-2 selectivity of WYE-151650 was demonstrated in the EPO-induced reticulocytosis model in which treatment of mice with WYE-151650 reduced the percent of NK cells in the blood (Figure 3C) without effect on EPO-induced reticulocytosis (Figure 3D). In contrast, doses of CP-690,550 that showed similar inhibition of NK cells significantly reduced EPO-induced reticulocytosis, consistent with in vivo inhibition of JAK-2 likely driven by the high Cmax observed for this compound in mice (Figure 2D).

In summary, we have identified a novel and selective JAK-3 inhibitor, WYE-151650, that blocks in vitro JAK-3 kinase and functional activity in various cell types. When administered to mice orally, WYE-151650 reduces the clinical and microscopic manifestations of paw damage in a mouse CIA model, but has no effect on either IL-22– or EPO-mediated signaling. These results indicate that inhibition of JAK-3 alone is sufficient for efficacy in mouse CIA, and that selective JAK-3 inhibitors may become novel therapeutic agents for treatment of RA and other immune-related diseases.

AUTHOR CONTRIBUTIONS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Dr. Seidl had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

Study conception and design. Lin, Hegen, Quadros, Nickerson-Nutter, Appell, Cole, Tam, Ohlmeyer, Symanowicz, Wrocklage, Krykbaev, Xu, Mansour, Collins, Clark, Webb, Seidl.

Acquisition of data. Lin, Hegen, Nickerson-Nutter, Shao, Tam, Wang, Goodwin, Kimble, Quintero, Gao, Symanowicz, Wrocklage, Lussier, Schelling, Hewet, Xuan, Krykbaev, Togias, Harrison, Seidl.

Analysis and interpretation of data. Lin, Hegen, Quadros, Nickerson-Nutter, Tam, Goodwin, Symanowicz, Wrocklage, Lussier, Schelling, Xuan, Krykbaev, Xu, Collins, Clark, Seidl.

Acknowledgements

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

We would like to acknowledge Lynette Fouser for advice and reagents for the IL-22–mediated SAA model.

REFERENCES

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES