Combination treatment for cystic fibrosis

Abstract

Cystic fibrosis (CF) is a genetic disease caused by mutations in the gene encoding the 5 CFTR Cl— channel and affects several organs, but the most severe consequences are observed in the lung. The inventors have now instigated the combination of Orkambi® and ANO1 TSB (SEQ ID NO:1) and show that said combination increases mucociliary clearance and chloride channel activity. Thus the combination represents an alternative treatment for CF subjects.

Claims

1. A method of treating cystic fibrosis in a subject in need thereof comprising administering to the subject a therapeutically effective combination of lumacaftor, ivacaftor and a nucleic acid molecule comprising the nucleic acid sequence TABLE-US-00004 (SEQ ID NO: 1) TTTTCTCCGTCTTTGGGACCT.

2. The method of claim 1 wherein the subject harbors at least one allelic mutation selected from class I, class II, class III, class IV or class V.

3. The method of claim 1 wherein the subject harbors at least one mutation selected from class I, class II, class III, class IV or class V in the first allele and at least one mutation selected from class I, class II, class III, class IV or class V in the second allele.

4. The method of claim 3, wherein the subject harbors at least a mutation of class II in the first allele and at least a mutation of class II in the second allele.

5. The method of claim 3 wherein the subject harbors at least a mutation of class I in the first allele and at least a mutation of class II in the second allele.

6. The method of claim 2 wherein the subject harbors at least one allelic mutation selected from F508del-CFTR, R117H CFTR, 2184delA CFTR, W1282X CFTR or G551D CFTR.

7. The method of claim 4 wherein the subject harbors at least a F508del mutation in the first allele and at least a F508del mutation in the second allele.

8. The method of claim 5 wherein the subject harbors at least a W1282X mutation in the first allele and at least a 2184delA mutation in the second allele.

9. The method of claim 1 wherein the nucleic acid molecule comprises LNA nucleotides, or morpholino nucleotides, or 2′-O-methyl modified nucleotides, or 2′-O-methoxyethyl modified nucleotides, or 2′-fluoro modified nucleotides.

10. The method of claim 1 wherein lumacaftor and ivacaftor are administered to the subject in the same pharmaceutical composition.

11. The method of claim 8 wherein lumacaftor and ivacaftor are administered in the form of tablets for oral administration.

12. The method of claim 1 wherein the nucleic acid molecule is delivered by any device adapted to introduce one or more therapeutic compositions into the upper and/or lower respiratory tract.

Description

FIGURES

(1) FIG. 1. Effect of TSB control, ANO1 TSB, Orkambi® or TSB ANO1+Orkambi® on mucus dynamics. Primary hAECB and fully differentiated human bronchial air-liquid-interface cultures, isolated from bronchial biopsies from CF (F508del/F508del) subjects were transfected with TSB control (control) or ANO1 TSB every day during 3 days and/or stimulated with VX-770 and VX-809 (Orkambi®) for 24 h. The movements of 100 beads were quantified for each condition and the average speed (μm/ms) was determined. Histograms represent the average values±SDs and were compared using ANOVA coupled with Dunnett's, Bonferroni's and Tukey's post-hoc test.

(2) FIG. 2. Effect of TSB control, ANO1 TSB, Orkambi® or TSB ANO1+Orkambi® on chloride channel activity. CFPAC cells were transfected with TSB control (control) or ANO1 TSB for 24 h and/or stimulated with VX-770 and VX-809 (Orkambi®) for 24 h and chloride channel activity assay was assessed. Quantification of global channel activity of CF pancreatic epithelial cells transfected with ANO1 TSB or a negative control and/or stimulated with Orkambi® (n=8, in triplicate). Histograms represent average values±SDs and the conditions were compared using ANOVA coupled with Dunnett's, Bonferroni's and Tukey's post-hoc test.

(3) FIG. 3: Effect of TSB control, ANO1 TSB, Orkambi or TSB ANO1+Orkambi on mucus dynamics. Primary hAECB and fully differentiated human bronchial air-liquid-interface cultures, isolated from bronchial biopsies from CF (2184Δa+W1282X) subjects were transfected with TSB control (control) or ANO1 TSB every day during 3 days and/or stimulated with VX-770 and VX-809 (Orkambi) for 24 h. The movements of 100 beads were quantified for each condition and the average speed (μm/ms) was determined. Histograms represent the average values±SDs and were compared using ANOVA coupled with Dunnett's, Bonferroni's and Tukey's post-hoc test

(4) FIG. 4: Effect of TSB control, ANO1 TSB, Orkambi or TSB ANO1+Orkambi on mucus dynamics. Primary hAECB and fully differentiated human bronchial air-liquid-interface cultures, isolated from bronchial biopsies from CF (1717-1G>A+F508Del) subjects were transfected with TSB control (control) or ANO1 TSB every day during 3 days and/or stimulated with VX-770 and VX-809 (Orkambi) for 24 h. The movements of 100 beads were quantified for each condition and the average speed (μm/ms) was determined. Histograms represent the average values±SDs and were compared using ANOVA coupled with Dunnett's, Bonferroni's and Tukey's post-hoc test.

(5) FIG. 5: Effect of TSB control, ANO1 TSB, Orkambi or TSB ANO1+Orkambi on mucus dynamics. Primary hAECB and fully differentiated human bronchial air-liquid-interface cultures, isolated from bronchial biopsies from CF (F508/2183AA>G) subjects were transfected with TSB control (control) or ANO1 TSB every day during 3 days and/or stimulated with VX-770 and VX-809 (Orkambi) for 24 h. The movements of 100 beads were quantified for each condition and the average speed (μm/ms) was determined. Histograms represent the average values±SDs and were compared using ANOVA coupled with Dunnett's, Bonferroni's and Tukey's post-hoc test.

EXAMPLE

(6) Material & Methods

(7) Cell Culture

(8) Primary hAECB isolated from bronchial biopsies from CF (F508del/F508del) subjects were purchased from Epithelix SARL (Geneva, Switzerland). Fully differentiated ALI cultures (MucilAir,™), were cultured according to the provider's recommendations.

(9) CFPAC cell line were provided by Dr. Caroline Norez (STIM, Poitiers, France). This line was derived from a ductal adenocarcinoma (liver metastasis) from a subject with cystic fibrosis. Cells were cultured as recommended by ATCC (www.lgcstandards-atcc.org) in Iscove's Modified Dulbecco's Medium with fetal bovine serum at a final concentration of 10%. Cells were maintained at 37° C. in a humidified atmosphere of air with 5% CO.sub.2. All cells were tested for mycoplasma contamination (Lonza, Ambroise, France).

(10) Cell Transfections and Stimulation

(11) CFPAC cells were transfected with LNA-enhanced oligonucleotides targeting the miR-9 target site in the ANO1 3′UTR (ANO1 TSB) or with a miRCURY LNA microRNA inhibitor negative control (TSB control) (Exiqon, Denmark) using Interferin (Polyplus, Ozyme, France). CFPAC cells were stimulated for 24 h with VX-770 (100 nM) and VX-809 (1 μM) (Selleck Chemicals, Houston, USA) [1]. Twenty-four hours after the transfection and/or stimulation, the cells were processed for chloride activity.

(12) hAECB were transfected by adding medium containing LNA control or ANO1 TSB without any transfection reagent to ALI cells. After a 2-h incubation at 37° C., the medium was removed to restore the ALI condition. Freshly prepared LNA control or ANO1 TSB were added every day for three days, and mucus clearance assay was assessed 24 h post-treatment [2].

(13) ANO1, CFTR and Global Chloride Channel Activity Assay

(14) The chloride channel activity was assessed by t quenching of halide-sensitive YFP-H148Q/I152L protein (Thermo Fischer Scientific). The probe was transfected into the cells, and after 48 h of culture, conductance was stimulated for CFTR and ANO1 respectively with cAMP agonist cocktail and UTP (10 μM). For the global chloride channel activity, after 48 h of transfection with the probe, the conductance was stimulated with cAMP agonist cocktail and UTP (10 μM) together. I.sup.− solution (140 mM) was added, and the fluorescence was recorded using a plate reader as previously described [3]. The initial I.sup.− influx rate upon addition of each solution was computed from changes in YFP fluorescence data using non-linear regression. For quantitative analysis, the slope for fluorescence quenching, which correlates to the level of chloride conductance (I.sup.− uptake), was determined using linear regression. The rate of change (ΔF/min) was then calculated.

(15) Mucus Clearance Assay

(16) Thirty days after transfection and stimulation of hAECB cells with control TSB, ANO1 TSB, and/or Orkambi®, FluoSpheres Carboxylate-Modified Microspheres, 1.0 μm, yellow-green fluorescent (505/515) (Thermo Fisher Scientific) diluted in culture medium (1/50) were added to the apical face of the cultures. Movement of the beads was recorded under an Axiovert 200 microscope (Zeiss). Fluorescence images were collected every 3 ms and composed into a time-lapse image series using Axiovision software (version 4.6).

(17) Results

(18) We have instigated the combination of Orkambi® and ANO1 TSB (SEQ ID NO:1). The results are depicted in FIGS. 1, 2, 3, 4 and 5. Thus the combination increases mucociliary clearance (FIG. 1, FIG. 3) and chloride channel activity (FIG. 2). Surprisingly, we have found that the combination is not efficient with certain mutations (FIG. 4 and FIG. 5). Thus the combination of Orkambi® and ANO1 TSB (SEQ ID NO:1) represents an alternative treatment for CF subjects.

REFERENCES

(19) Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure. 1. Matthes E, Goepp J, Carlile G W, et al. Low free drug concentration prevents inhibition of F508del CFTR functional expression by the potentiator VX-770 (ivacaftor). Br J Pharmacol 2016; 173: 459-470. 2. Sonneville F, Ruffin M, Coraux C, et al. MicroRNA-9 downregulates the ANO1 chloride channel and contributes to cystic fibrosis lung pathology. Nature communications 2017; 8: 710. 3. Saint-Criq V, Ruffin M, Rebeyrol C, et al. Azithromycin fails to reduce inflammation in cystic fibrosis airway epithelial cells. Eur J Pharmacol 2012; 674: 1-6. 4. Harriet C., Kristin E. T., Olivier T., et al. Translating the genetics of cystic fibrosis to personalized medicine. Transl Res 2016; 168 40-49.