Potent in vivo lung cancer Wnt signaling inhibition via cyclodextrin-LGK974 inclusion complexes

Abstract

Activation of the Wnt signaling pathway promotes lung cancer progression and contributes to poor patient prognosis. The porcupine inhibitor LGK974, a novel orally bioavailable cancer therapeutic in Phase I clinical trials, induces potent Wnt signaling inhibition and leads to suppressed growth and progression of multiple types of cancers. The clinical use of LGK974, however, is limited in part due to its low solubility and high toxicity in tissues that rely on Wnt signaling for normal homeostasis. Here, we report the use of host-guest chemistry to enhance the solubility and bioavailability of LGK974 in mice through complexation with cyclodextrins (CD). We assessed the effects of these complexes to inhibit Wnt signaling in lung adenocarcinomas that are typically driven by overactive Wnt signaling. 2D ¹H NMR confirmed host-guest complexation of CDs with LGK974. CD:LGK974 complexes significantly decreased the expression of Wnt target genes in lung cancer organoids and in lung cancer allografts in mice. Further, CD:LGK974 complexes increased the bioavailability upon oral administration in mice compared to free LGK974. In a mouse lung cancer allograft model, CD:LGK974 complexes induced potent Wnt signaling inhibition with reduced intestinal toxicity compared to treatment with free drug. Collectively, the development of these complexes enables safer and repeated oral or parenteral administration of Wnt signaling inhibitors, which hold promise for the treatment of multiple types of malignancies.

Fig. 1.

Schematic of CD:LGK974 complexation for oral delivery of Wnt inhibitors in vivo.

Fig. 2.

(a) Structure of α-cyclodextrin and modified sulfobutylether-α-cyclodextrin. (b) Structure of β-cyclodextrin and modified sulfobutylether-β-cyclodextrin. (c) Scheme of reversible host-guest complex formation. (d) Table of physicochemical properties of cyclodextrins.

Fig. 3.

(a) Partial 2D NOESY NMR spectrum of CD:LGK974. (b,c) Proposed geometric arrangement of LGK974 in the cavities of HPβCD and βSBECD based on the ROESY experiment.

Fig. 4.

Isothermal Titration Calorimetry experiments for (a) βSBECD (concentration: 60.0 mM) and (b) HPβCD (concentration: 60.0 mM) in LGK974 (concentration: 3.0 mM) (square) and nonlinear fitting by Wiseman Isotherm (line). (c) Thermodynamic parameters of CD-LGK974 interactions determined by isothermal titration calorimetry experiments (average stoichiometry N of the complex in solution, enthalpy ΔH°, entropy TΔS°, equilibrium constant K_eq). (d) Free LGK974, LGK974 suspension in CMC/Tween 80 and CD:LGK974 solubility in PBS.

Fig. 5.

(a) Schematic of the Caco-2 drug permeability assay. (b) Efflux ratio, (c) permeability coefficient (Papp), (d) experimental recovery, (e) transepithelial electrical resistance (TEER) and (f) lucifer yellow percentage of LGK974 and CD:LGK974 complexes after 2 h treatment.

Fig. 6.

Quantitative real-time PCR (qRT-PCR) analysis of (a) Axin2 and (b) Lgr5 transcripts in 3D cultures of primary KPT- LUAD cells following Wnt pathway inhibition with LGK974 or CD:LGK974 treatment (concentration: 100nM) every two days (three treatments total) over the course of one week. (c) Immunofluorescence staining for EdU (green), BrdU (red), and nuclei (blue) in 3D cultures of primary KPT- LUAD cells. (d) Calcein pixel intensity by organoid area of LGK974 and CD:LGK974 complexes. (e) Uptake and diffusion of fluorescent calcein in KPT- LUAD organoids after treatment with LGK974 and CD:LGK974. Organoids were incubated with 1μM calcein-AM and fluorescent images were taken 2h after incubation at 37 °C and 10% CO₂. Data are mean ± s.d.; one-way ANOVA with posthoc Bonferri (b); * P < .05; ** P < .01; *** P < .001 compared to control group. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 7.

(a) Blood concentration of LGK974 between 30 min and 180 min after oral administration of LGK974 and CD:LGK974 (dose: 10 mg/kg). (b) Suppression of 7TCF-driven bioluminescence by LGK974 or CD:LGK974 relative to tdTomato signal in mice harboring subcutaneous transplants of the KPT LUAD cell line. Treatment with 5mg/kg/d of LGK974 or CD:LGK974 over 7days, n =8 tumors, 4 mice per group. (c, d) tdTomato and 7TCF::Luciferase signals at baseline (0 h) and 48 h following treatment with LGK974 or CD:LGK974 complexes (dose: 5mg/kg). Quantitative real-time PCR analysis of (e) Axin2 and (f) Lgr5 transcripts in KPT-LUAD tumors 1 week following treatment with 5mg/kg/d LGK974 or CD:LGK974. Treatment was started at 1week post-tumor initiation. Data are mean ± s.d.; Two-way ANOVA (a, b); One-way ANOVA (e,f) * P < .05; ** P < .01; *** P < .001 compared to control (b, e, f) and LGK974 group (a, e, f).

Fig. 8.

Representative images of H&E staining of the intestine from mice treated with (a) PBS, (b) LGK974, (c) HPβCD:LGK974, or (d) βSBECD:LGK974 complexes at high dosages of 10mg/kg/d over 7days (Magnification: duodenum, 20×.Scale bars: 20×−100 μm. (e) Scoring for intestinal mucosal damage. *** P < .001 compared to the LGK974 group.