METHODS AND COMPOSITIONS FOR TREATING ASTHMA AND ALLERGIC DISEASES

Abstract

The present invention relates to allergy field. Several independent groups have recently investigated the implication of PCSK9 on inflammation and sepsis but none of them have determine its impact on allergies and/or asthma which is a global health burden. Inventors have obtained preliminary data on wild-type (PCSK9+/+) or PCSK9-deficient mice (PCSK9 −/−) and shown that, under basal condition and in the absence of a particular stimulus, PCSK9 deficiency significantly increases the percentage of regulatory T cells in the spleen, the mesenteric lymph nodes and Peyer's patches. Moreover, inventors have shown the effect of allergic challenge Non primary human bronchial epithelial cells on PCSK9 expression and secretion. Very interestingly, their first results obtained by Q-PCR showed that HDM and LPS increase PCSK9 mRNA levels. Accordingly, the present invention relates to inhibitors of PCSK9 for use in the treatment of asthma and/or allergic disease, such as food allergy.

Claims

1. A method for assessing or predicting the severity of asthma or the severity of an allergic disease in a subject, comprising i) determining the PCSK9 level in a biological sample obtained from the subject, ii) comparing the level determined at step i) with a predetermined reference value and iii) concluding that the subject has or is susceptible to have a severe asthma or severe allergic disease when the level of PCSK9 is higher than the predetermined reference value or concluding that the subject has not or is not susceptible to have a severe asthma or severe allergic disease when the level of PCSK9 is lower than the predetermined reference value.

2. The method according to claim 1 wherein the biological sample is a blood sample and/or a bronchoscopy and bronchoalveolar lavage (BAL) sample.

3. The method of claim 1 wherein the biological sample is a PBMC sample.

4. The method according to claim 1 wherein the PCSK9 level is measured by ELISA.

5. (canceled)

6. The method according to claim 12, wherein the allergic disease is a food allergy.

7. The according to claim 12, wherein the method comprises of predicting whether the subject is at risk of having severe asthma or a severe allergic disease by i) determining the PCSK9 level in a biological sample obtained from the subject, ii) comparing the level determined at step i) with a predetermined reference value and iii) concluding that the subject has or is susceptible to have severe asthma or severe allergic disease when the level of PCSK9 is higher than the predetermined reference value or concluding that the subject is not susceptible to have a severe asthma or severe allergic disease when the level of PCSK9 is lower than the predetermined reference value.

8. The method according to claim 12, wherein the inhibitor is an antibody.

9. The method according to claim 12, wherein the inhibitor is alirocumab, evolocumab or bococizumab.

10. The method according to claim 12, wherein the inhibitor is a small molecule.

11. The method according to claim 12, wherein the inhibitor is siRNA or antisense oligonucleotides.

12. A method of treating asthma and/or allergic disease in a subject in need thereof comprising of administering to said subject a therapeutically effective amount of an inhibitor of PCSK9.

13. A pharmaceutical composition comprising an inhibitor of PCSK9.

14. A method of screening a drug suitable for inhibiting PCSK9 comprising i) providing a test compound and ii) determining the ability of said test compound to inhibit the expression or activity of PCSK9.

Description

FIGURES

[0071] FIG. 1. Regulatory T cells are significantly more abundant in the spleen, mesenteric lymph nodes and Peyer's patches of PCSK9 −/− mice.

[0072] FIG. 2. HDM allergens increase the intracellular PCSK9 expression and possibly its secretion. Human primary BECs were cultured for 48 hours with HDM allergens or LPS (10 μg/ml). (A) PCSK9 mRNA levels (B) Intracellular PCSK9 protein and (C) “secreted” PCSK9 protein levels. Protein levels were assayed by ELISA. * P<0,05.

[0073] FIG. 3. PCSK9 deficiency attenuates the allergic responses. PCSK9+/+ (white circle) and PCSK9 −/− mice (black circle) were sensitized by intra-peritoneal injection of vehicle (white bars) or gliadins (black bars) and then challenged twice with water or gliadins by gavage. One hour after the second oral allergic challenge body weight (A), plasma PCSK9 concentrations (B) ear thickness (C) were measured and represented as dot plots and histograms representing mean ±SEM. One hour after the second oral allergic challenge spleen weight (D), splenic CD19+IgE+ cells percentage (E) and plasma gliadin specific IgE concentrations (F) were measured and represented as dot plots and bars representing mean ±SEM. Statistical significance was analyzed using non-parametric Mann-Whitney test with Graphprism® (Graphpad Software). * P<0,05; ** P<0,01; ***P<0,001; ****P<0,0001; NS:Non Significant.

EXAMPLE

[0074] Our laboratory has access to human primary bronchial epithelial cells (Human primary BECs) via the multicentre Cohort of Lung Transplantation (COLT-NCT00980967 study/CPP 2009-A00036-51). Briefly, these cells are dissociated from lung donor trachea or bronchi via overnight incubation at 4° C. with type XIV collagenase in HEPES-buffered RPMI. Humans BECs are then cultured in cnT17 media containing penicillin and streptomycin on human type IV collagen coated plates (19). Considering the PCSK9 expression in the gut and the lung and its potential role in immunity, we hypothesize that PCSK9 may play a role in both food allergy and progression towards respiratory pathological complications such as asthma. Preliminary data obtained in our laboratory on wild-type (PCSK9+/+) or PCSK9-deficient mice (PCSK9−/−) show that, under basal condition and in the absence of a particular stimulus, PCSK9 deficiency significantly increases the percentage of regulatory T cells in the spleen, the mesenteric lymph nodes and Peyer's patches (FIG. 1). Interestingly, the alteration of activation of these cells in allergy is considered as essential in the occurrence of chronic inflammation. Our driving hypothesis is that PCSK9 deficiency protects against food allergy & asthma.

[0075] Very interestingly, our first results obtained by Q-PCR showed that HDM and LPS increase PCSK9 mRNA levels. Similarly, HDM induced a strong rise of intracellular PCSK9 protein content. We also observed a non-significant trend for intracellular PCSK9 increase when cells were cultured with LPS (+53%). We also found an increase of PCSK9 concentration in the media after HDM or LPS treatment (FIG. 2). These first results are reassuring about the relevance of the use of this cell model and suggest that PCSK9 is expressed in bronchial epithelial cells and regulated during inflammatory process.

[0076] Then an allergy induction was proceeded. The allergy induction protocols were set up routinely at l'Institut du Thorax and were previously described in detail (17). We used PCSK9+/+ and PCSK9 −/− males and females mice fed since weaning with gluten free regular chow diet. In order to induce sensitization, 5 weeks old mice received 3 consecutive intraperitoneal injection with either vehicle or deamidated wheat gliadins (10 micrograms) on days 0, 10 and 20. Mice were then challenged twice by oral administration of 20 milligrams of deamidated gliadins. Results shown on FIGS. 3 & 4 were obtained 1 h after the second allergic challenge. We did observe that PCSK9.sup.+/+ mice under allergic condition (Alimentary Allergy, A A) have a reduced body weight compared to control PCSK9.sup.+/+ mice (FIG. 3A). We also observed a slight trend for body weight decrease in PCSK9.sup.−/− under allergic condition however it did not reach significancy (FIG. 3A). Interestingly, plasma PCSK9 levels are significantly increased after allergy induction in PCSK9.sup.+/+ mice (FIG. 3B). Gliadin challenge increased spleen weight (FIG. 3D) and IgE+ plasmocytes % (FIG. 3E) in both PCSK9.sup.+/+ and PCSK9 .sup.−/−mice. By contrast, the plasma gliadin specific IgE+ concentrations is significantly increased in allergy PCSK9.sup.+/+ mice but not in PCSK9.sup.−/− mice (FIG. 3F) suggesting that PCSK9 deficiency attenuates the allergic responses. To characterize the allergic symptoms, we measured ear thickness with a micrometer after the oral challenge. In control groups, the vehicle solution gavage did not affect ear thickness in both PCSK9.sup.+/+ and PCSK9 .sup.−/−mice. We show a significant increase in ear thickness after the oral challenge for the PCSK9.sup.+/+ mice sensitized and challenged with the deamidated form of gliadins (FIG. 3C). By contrast, no effect was observed in PCSK9.sup.−/−mice in the same condition (FIG. 3C).

REFERENCES

[0077] 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.

[0078] 1) Abifadel M. et al. Mutations in PCSK9 cause autosomal dominant hypercholesterolemia. Nat Genet 2003; 34:154-6.

[0079] 2) Seidah N G et al. PCSK9 : a key modulator of cardiovascular health. Circ Res 2014; 114:1022-36.

[0080] 3) Cohen J C et al. Sequence variations in PCSK9, low LDL, and protection against coronary heart disease. N Engl J Med. 2006; 354:1264-72.

[0081] 4) Sabatine M. S. et al. Evolocumab and Clinical Outcomes in Patients with Cardiovascular Disease. N Engl J Med. 2017; 376:1713-1722.

[0082] 5) Cariou B et al. Role of PCSK9 beyond liver involvement. Curr Opin Lipidol. 2015 Jun; 26(3):155-6

[0083] 6) Seidah N. G. et al. The secretory proprotein convertase neural apoptosis-regulated convertase 1 (NARC-1): liver regeneration and neuronal differentiation. Proc Natl Acad Sci U S A. 2003; 100:928-33.

[0084] 7) Lagace T. A. et al. Secreted PCSK9 decreases the number of LDL receptors in hepatocytes and in livers of parabiotic mice. J Clin Invest. 2006; 116: 2995-3005.

[0085] 8) Feingold K. R. et al. Inflammation stimulates the expression of PCSK9. Biochem Biophys Res Commun. 2008; 374:341-4.

[0086] 9) Khademi F. et al. PCSK9 and infection: A potentially useful or dangerous association? J Cell Physiol. 2017 Jun; 1-8.

[0087] 10) Proto J. D.et al. Hypercholesterolemia induces T cell expansion in humanized immune mice. J Clin Invest. 2018 Apr. 30. pii: 97785. doi: 10.1172/JCI97785.

[0088] 11) Haldar P. et al. Cluster analysis and clinical asthma phenotypes. Am J Respir Crit Care Med 2008, 178:218-224.

[0089] 12) Dharmage S. C. et al. Atopic dermatitis and the atopic march revisited. Allergy 2014, 69:17-27.

[0090] 13) Lin Y. T. et al. Correlation of ovalbumin of egg white components with allergic diseases in children. J Microbiol Immunol Infect 2016; 49:112-8.

[0091] 14) Bieber T. et al. Atopic dermatitis: a candidate for disease-modifying strategy. Allergy 2012, 67:969-975.

[0092] 15) Bihouée T. et al. Food allergy enhances allergic asthma in mice. Respir Res. 2014; 15:142.

[0093] 16) Bouchaud G. et al. Consecutive Food and Respiratory Allergies Amplify Systemic and Gut but Not Lung Outcomes in Mice. J Agric Food Chem. 2015; 63: 6475-83.

[0094] 17) Bouchaud G. et al. Maternal exposure to GOS/inulin mixture prevents food allergies and promotes tolerance in offspring in mice. Allergy. 2016; 71:68-76.

[0095] 18) Castan L. et al. Food allergen-sensitized CCR9+ lymphocytes enhance airways allergic inflammation in mice. Allergy. 2018 Jan 9. doi: 10.111/all.13386.

[0096] 19) Pain M. et al. T Cells Promote Bronchial Epithelial Cell Secretion of Matrix Metalloproteinase-9 via a C-C Chemokine Receptor Type 2 Pathway: Implications for Chronic Lung Allograft Dysfunction. Am J Transplant. 2017; 17:1502-1514.

[0097] 20) Rusznak C. et al. Cigarette smoke potentiates house dust mite allergen-induced increase in the permeability of human bronchial epithelial cells in vitro. Am J Respir Cell Mol Biol. 1999 Jun; 20(6):1238-50.

[0098] 21) Zaid A. et al. Proprotein convertase subtilisin/kexin type 9 (PCSK9): hepatocyte-specific low-density lipoprotein receptor degradation and critical role in mouse liver regeneration. Hepatology. 2008 Aug; 48(2):646-54.

[0099] 22) Bonnefond A. et al. The loss-of-function PCSK9 p.R46L genetic variant does not alter glucose homeostasis. Diabetologia. 2015 Sep; 58(9):2051-5.

[0100] 23) Weider E. et al. Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) Single Domain Antibodies Are Potent Inhibitors of Low Density Lipoprotein Receptor Degradation. J Biol Chem. 2016 Aug 5; 291(32):16659-71.

[0101] 24) Sutherland S.D. et al. Cell of Origin of Small Cell Lung Cancer: Inactivation of Trp53 and Rb1 in Distinct Cell Types of Adult Mouse Lung. Cancer Cell, Jun. 14, 2011:19, 754-764.