Research ArticleAsthma

Lung-restricted inhibition of Janus kinase 1 is effective in rodent models of asthma

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Science Translational Medicine  21 Nov 2018:
Vol. 10, Issue 468, eaao2151
DOI: 10.1126/scitranslmed.aao2151
  • Fig. 1 Cell-based potency of iJak-381 and oJak-989.

    Mesoscale discovery (MSD) (A to D) or fluorescence-activated cell sorting (FACS) (E and F) analysis of pSTAT in response to cytokine stimulations. Stimulations, cell lines, and specific pSTAT readouts are indicated in the figure. TF1, trifactor-dependent 1; EPO, erythropoietin. (G and H) Quantitative reverse transcription polymerase chain reaction analysis of IL-13– or IL-4–induced CCL26 and POSTN induction in NHBE cells cultured in ALI cultures and preincubated with inhibitors. Error bars represent SD of duplicate or triplicate measurements. Numbers in graphs indicate mean IC50 values in nanomolar and 95% confidence intervals for iJak-381 (blue) and oJak-989 (red). DMSO, dimethyl sulfoxide; gMFI, geometric mean fluorescence intensity.

  • Fig. 2 PK profile of iJak-381 and oJak-989 with different delivery routes.

    Graphs show compound concentrations in the lung (solid circles) and plasma (open circles) upon oral delivery of iJak-381 (10 mg/kg) (A) or oJak-989 (100 mg/kg) (B), intranasal delivery of iJak-381 (0.3 mg/kg) (C) or oJak-989 (0.3 mg/kg) (D), and DPI administration of iJak-381 (10.7 mg/kg) (E). Black lines indicate IC50 values for the respective compound as determined in the IL-13–stimulated BEAS-2B cell-based assay and corrected for lung tissue binding (dashed) or plasma protein binding (solid). Mean and SD of three to six animals per group are shown.

  • Fig. 3 PD model to assess Jak1 inhibition in the lung.

    (A) Design of IL-13 PD model. Animals received oJak-989 via oral delivery, iJak-381 via intranasal drop delivery, or a range of iJak-381 doses via DPI. Thirty minutes later, 3 μg of IL-13 was administered intravenously (IV). Fifteen minutes later, lungs and plasma were collected for pStat6 and PK analysis. (B to D) pStat6 MSD analysis of total lung homogenate samples. *P < 0.05, **P < 0.01, ****P < 0.001. mpk, milligrams per kilogram. (E to G) Total drug concentrations in lung (solid bars) and plasma (open bars) of oJak-989 (red) and iJak-381 (blue). Lines indicate IC50 values for the respective compound as determined in the IL-13–stimulated BEAS-2B cell-based assay and corrected for lung tissue binding (solid) or plasma protein binding (dashed). Error bars represent SEM of eight animals per group. (H and I) Representative MALDI-MS images from lungs collected directly after either IN or DPI delivery. Pixel intensity scale is representative of the detected relative drug abundance and demonstrates that drug is concentrated in a single lobe after IN administration but evenly distributed upon DPI.

  • Fig. 4 Disease-relevant PD model.

    (A) Design of OVA pStat induction model. Animals were immunized with OVA/alum on day 0. Thirty-five days later, animals received either oJak-989 (100 mg/kg) via oral delivery or iJak-381 (10.7 mg/kg) via DPI. One hour after drug delivery, animals were challenged with inhaled OVA for 30 min. Two and a half hours after the end of the OVA challenge, lungs and plasma were collected. (B and C) pStat6 (B) and pStat3 (C) MSD analysis of total lung homogenate samples. *P < 0.05, **P < 0.05, ***P < 0.005, ****P < 0.001. (D and E) Total drug concentrations in lung (solid bars) and plasma (open bars) of oJak-989 (D) and iJak-381 (E). Lines indicate IC50 values for the respective compound as determined in the IL-13–stimulated BEAS-2B cell-based assay and corrected for lung tissue binding (solid) or plasma protein binding (dashed). Error bars represent SEM; n = 5 to 6 animals per group.

  • Fig. 5 Efficacy of iJak-381 and oJak-989 in a murine model of eosinophilic asthma.

    (A) Design of mouse OVA asthma model. Animals received either oJak-989 (100 mg/kg) or vehicle via oral delivery or iJak-381 (8.6 mg/kg; dry powder) or air only via inhalation 1 hour before each OVA challenge. An unimmunized, unchallenged group was also included (naïve). Twenty-four hours after the final challenge, BAL fluid (BALF), lungs, spleens, and plasma were collected (red arrow). QD, quaque die. (B and C) Analysis of total cell counts (B) and eosinophils (C) from BALF. (D to G) Fluidigm analysis of gene expression in the lung relative to the expression measured in naïve animals. (H and I) Total spleen cell count and the percentage of NK cells as determined by FACS analysis. (J and K) Decrease of NK cells in spleen (J) and blood (K) plotted against inhibition of BAL eosinophil response with linear regression of results with y-axis intercept constrained to the origin (combined results from four studies). (L and M) Resistance of the respiratory system (Rrs) measurements (L) and area under the curve (AUC) thereof (M) of OVA-challenged mice challenged with methacholine (70 mg/ml). Error bars represent SEM; n = 5 to 8 animals per group. ns, not significant. *P < 0.05, **P < 0.01, ***P < 0.005, ****P < 0.001.

  • Fig. 6 Efficacy of iJak-381 in an allergen-driven asthma model.

    (A) Design of the AAH model. Animals were challenged three times per week with human allergens as indicated. From days 9 to 14, animals received either oJak-989 (100 mg/kg) via oral delivery or iJak-381 (9.9 mg/kg) via DPI 1 hour before the allergen challenge. Control animals received allergens, but either received vehicle PO or were placed on towers containing only air. An unimmunized, unchallenged group was also included (naïve). Twenty-four hours after the final challenge, BALF, lungs, spleens, and plasma were collected (red arrow). HDM, house dust mite. (B to D) Analysis of total cell counts, eosinophils, and neutrophils from BALF. (E and F) Enzyme-linked immunosorbent assay (ELISA) analysis of Ccl11 and Cxcl1 in BALF harvested on day 15. (G and H) Total spleen cell and splenic NK cell percentage as determined by FACS analysis. *P < 0.05, **P < 0.01, ***P < 0.005, ****P < 0.001. Error bars represent SEM; n = 5 to 8 animals per group.

  • Fig. 7 Combination of iJak-381 with dexamethasone.

    (A) Experimental design is similar to the experiment shown in Fig. 6. From days 9 to 14, animals received either iJak-381 (9.2 mg/kg) via DPI or time-matched tower control treatment 1 hour before the allergen challenge and/or dexamethasone (Dex; 0.3 or 3.0 mg/kg) via intraperitoneal (IP) injection 30 min before allergen challenge. An unimmunized, unchallenged group was also included (naïve). Twenty-four hours after the final challenge, BALF, lungs, spleens, and plasma were collected (red arrow). (B) Analysis of cell counts, (C) eosinophils, and (D) neutrophils from BALF. (E) ELISA analysis of Cxcl1 in BALF harvested on day 15. (F) Spleen cell counts of experimental animals. *P < 0.05, **P < 0.01, ***P < 0.005, ****P < 0.001. Error bars represent SEM; n = 5 to 8 animals per group.

  • Fig. 8 Efficacy of iJak-381 in a guinea pig asthma model.

    (A) Design of guinea pig OVA asthma model. Animals were immunized twice with OVA (days 0 and 7). On day 28, animals received a single OVA challenge. One hour before the OVA challenge, animals received either air or iJak-381 at the indicated dose via DPI. Twenty-four hours after the final challenge, lungs, spleens, and plasma were collected (red arrow). (B) Representative images of hematoxylin and eosin–stained formalin-fixed lung sections. (C) Inflammation severity scores of lung sections. Histologic sections were subjectively scored on a scale of 0 to 5 for severity and distribution of inflammatory cell infiltrate: 0, normal; 1, minimal; 2, mild; 3, moderate; 4, marked; and 5, severe. **P < 0.01, ***P < 0.005. (D) Drug concentrations in lung tissue (solid bars) and plasma [not detectable (N.D.)] 24 hours after aerosol challenge. Lines indicate IC50 values for iJak-381 as determined in the IL-13–stimulated BEAS-2B cell-based assay and corrected for lung tissue binding (solid) or plasma protein binding (dashed). Mouse blood and lung tissue binding numbers were used for the calculations. Error bars represent SEM; n = 4 to 6 animals per group.

  • Table 1 Chemical and PK properties of iJak-381 and oJak-989.

    Ki inhibitory constants were determined at concentrations of ATP matched to the Michaelis constant (Km) of the respective kinase. Potencies and selectivities at 1 mM ATP were calculated as described in Materials and Methods. pKa, acid dissociation constant; MDCK, Madin-Darby canine kidney cells; Papp, apparent permeability; A:B, apical to basolateral.

    iJak-381oJak-989
    StructureEmbedded ImageEmbedded Image
    Molecular weight612.03286.33
    Kinetic solubility (μM)34.1196
    Calculated pKa6.44.5
    MDCK permeability
    (Papp A:B) (×10−6 cm/s)
    2.77.6
    Mouse hepatocyte clearance
    (% liver blood flow)
    8224
    Mouse lung protein binding (%)94.577.4
    Mouse plasma protein binding (%)98.345.2
    Ki (nM) as determined at ATP Km (JAK1/JAK2/JAK3/
    TYK2)
    0.26/0.62/20.8/3.152.93/75.9/114/30.8
    Calculated IC50 (nM) at 1 mM ATP (JAK1/JAK2/JAK3/
    TYK2)
    8.52/53.4/5998/24094.2/6563/32,873/2347
    Calculated JAK1 selectivity at 1 mM ATP (JAK2/
    JAK3/TYK2)
    6.3/704/2870/349/25

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/10/468/eaao2151/DC1

    Methods

    Fig. S1. Cell-based potency of inhibitors for IL-5–, TSLP-, and IgE-induced signals.

    Fig. S2. DPI system.

    Fig. S3. Co-dose PD model.

    Fig. S4. IL-13 and Gm-csf PD models.

    Fig. S5. Dose-response curve of iJak-381 in asthma model.

    Fig. S6. Complete blood count analysis of iJak-treated animals.

    Fig. S7. Drug concentrations in AAH model.

    Table S1. Biochemical selectivity of iJak-381 and oJak-989.

  • This PDF file includes:

    • Methods
    • Fig. S1. Cell-based potency of inhibitors for IL-5–, TSLP-, and IgE-induced signals.
    • Fig. S2. DPI system.
    • Fig. S3. Co-dose PD model.
    • Fig. S4. IL-13 and Gm-csf PD models.
    • Fig. S5. Dose-response curve of iJak-381 in asthma model.
    • Fig. S6. Complete blood count analysis of iJak-treated animals.
    • Fig. S7. Drug concentrations in AAH model.
    • Table S1. Biochemical selectivity of iJak-381 and oJak-989.

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