METHODS AND PHARMACEUTICAL COMPOSITIONS FOR THE TREATMENT OF ACUTE EXACERBATIONS OF CHRONIC OBSTRUCTIVE PULMONARY DISEASE

Abstract

The present invention relates to methods and pharmaceutical compositions for the treatment of acute exacerbation of chronic obstructive pulmonary disease. In particular, the present invention relates to a method of treating acute exacerbation of chronic obstructive pulmonary disease in a subject in need thereof comprising administering the subject with a therapeutically effective amount of a flagellin polypeptide.

Claims

1. A method of treating acute exacerbation of chronic obstructive pulmonary disease in a subject in need thereof comprising administering the subject with a therapeutically effective amount of a flagellin polypeptide.

2. The method of claim 1 wherein the acute exacerbation of COPD is caused by a bacterial infection, by a viral infection or by air pollution.

3. The method of claim 2 wherein the bacterial infection is caused by Streptococcus pneumoniae, Haemophilus influenzae, or Moraxella catarrhalis.

4. The method of claim 1 wherein the subject experienced an acute exacerbation of COPD or is at risk of experiencing an acute exacerbation of COPD.

5. The method of claim 1 wherein the subject is a frequent exacerbator.

6. The method of claim 1 wherein the flagellin polypeptide has at least 70% of identity with SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3.

7. The method of claim 1 wherein the flagellin polypeptide comprises: a) a N-terminal peptide having at least 90% amino acid identity with the amino acid sequence starting from the amino acid residue located at position 1 of SEQ ID NO:3 and ending at an amino acid residue selected from the group consisting of any one of the amino acid residues located at positions 99 to 173 of SEQ ID NO:3; and b) a C-terminal peptide having at least 90% amino acid identity with the amino acid sequence starting at an amino acid residue selected from the group consisting of any one of the amino acid residues located at positions 401 to 406 of SEQ ID NO:3 and ending at the amino acid residue located at position 494 of SEQ ID NO:3, wherein: the said N-terminal peptide is directly linked to the said C-terminal peptide, or the said N-terminal peptide and the said C-terminal peptide are indirectly linked, one to the other, through a spacer chain.

8. The method of claim 7 wherein the said N-terminal peptide is selected from the group consisting of the amino acid sequences 1-99, 1-137, 1-160 and 1-173 of SEQ ID NO:3.

9. The method of claim 7 wherein said C-terminal peptide is selected from the group consisting of the amino acid sequences 401-494 and 406-494 of SEQ ID NO:3.

10. The method of claim 7 wherein said N-terminal and C-terminal peptides consist of the amino acid sequences 1-173 and 401-494 of SEQ ID NO:3, respectively.

11. The method of claim 7 wherein said N-terminal and C-terminal peptides consist of the amino acid sequences 1-160 and 406-494 of SEQ ID NO:3, respectively.

12. The method of claim 7 wherein said N-terminal and C-terminal peptides consist of the amino acid sequences 1-137 and 406-494 of SEQ ID NO:3, respectively.

13. The method of claim 7 wherein said N-terminal peptide and the said C-terminal peptide are indirectly linked, one to the other, through an intermediate spacer chain consisting of a NH2-GIy-AIa-AIa-GIy-COOH (SEQ ID NO:4) peptide sequence.

14. The method of claim 7 wherein the asparagine amino acid residue located at position 488 of SEQ ID NO:3 is replaced by a serine.

15. The method of claim 1 wherein the flagellin polypeptide is administered to the subject in combination with an antibiotic.

Description

FIGURES

[0037] FIG. 1—Alterations of IL-17 and IL-22 production in response to S. pneumoniae and not typeable heamophilus influenzae (NTHI) in COPD mice. Mice were chronically exposed to cigarette smoke over a period of 12 weeks and then intranasally challenged with 5×10.sup.4 CFU of Streptococcus pneumoniae (Sp) or not (Mock). IFNγ, IL-17 and IL-22 levels were evaluated in the BAL (A). Concentrations of 11-22 in the serum (B) and in supernatants from restimulated pulmonary cells (C) were measured 24h after Sp challenge. Mice chronically exposed to cigarette smoke over a period of 12 weeks were then intranasally challenged with 5×10.sup.7 CFU of NTHI or not (Mock). Results were expressed as mean±SEM (n>10 per group).

[0038] FIG. 2: Administration of flagellin decreased the bacterial load in S. pneumoniae infected COPD mice. Bacterial load was measured in the bronchoalveolar lavade (BAL) (A), the lung lysate (B) and the blood (C) of COPD (chronically exposed to cigarette smoke) and Air (controls) mice infected or not with 5×10.sup.4 cfu of streptococcus pneumoniae serotype 1 (SP1). A group of mice received 5 μg of flagellin (FLIC) by intraperitoneal route. Bacterial load was analyzed at day 1 and 3 after infection. Results were expressed as mean±SEM (n>6 per group).

[0039] FIG. 3: Administration of flagellin increased the IL-17 production at day 3 after infection by S. pneumoniae in COPD mice. Levels of IL17 were measured in the bronchoalveolar lavade (BAL) (A) and in supernatants of lung cells (B) from COPD (chronically exposed to cigarette smoke) and Air (controls) mice infected or not with 5×10.sup.4 cfu of streptococcus pneumoniae serotype 1 (SP1). A group of mice received 5 μg of flagellin (FLIC) by intraperitoneal route. Concentrations of IL-17 were analyzed by ELISA at day 1 and 3 after infection. Results were expressed as mean±SEM (n>6 per group).

[0040] FIG. 4: Administration of flagellin increased the IL-22 production at day 3 after infection by S. pneumoniae in COPD mice. Levels of IL-22 were measured in the bronchoalveolar lavade (BAL) (A) and in supernatants of lung cells (B) from COPD (chronically exposed to cigarette smoke) and Air (controls) mice infected or not with 5×104 cfu of streptococcus pneumoniae serotype 1 (SP1). A group of mice received 5 μg of flagellin (FLIC) by intraperitoneal route. Lung cells were cultivated for 48h in complete medium alone (unstimulated conditions) or were stimulated by fixed anti-CD3 antibody (Anti-CD3 conditions). Concentrations of IL-22 were analyzed by ELISA at day 1 and 3 after infection. Results were expressed as mean±SEM (n>6 per group).

[0041] FIG. 5: Administration of flagellin decreased the bacterial load at day 1 post infection by NTHI in COPD mice. Bacterial load was measured in the bronchoalveolar lavade (BAL) (A), the lung lysate (B) and the blood (C) of COPD (chronically exposed to cigarette smoke) and Air (controls) mice infected or not with 5×10.sup.7 cfu of not typable haemophilus influenza (NTHI). A group of mice received 5 μg of flagellin (FLIC) by intraperitoneal route. Bacterial load was analyzed at day 1 after infection. Results were expressed as mean±SEM (n>6 per group).

[0042] FIG. 6: Administration of flagellin increased the IL-22 production at day 1 after infection by NTHI in COPD mice. Levels of IL-22 were measured in the bronchoalveolar lavade (BAL) (A) and in supernatants of lung cells (B) from COPD (chronically exposed to cigarette smoke) and Air (controls) mice infected or not with 5×10.sup.7 cfu of not typable haemophilus influenza (NTHI). A group of mice received 5 μg of flagellin (FLIC) by intraperitoneal route. Lung cells were cultivated for 48h in complete medium alone (unstimulated conditions) or were stimulated by heat-killed NTHI (HK-NTHIconditions). Concentrations of IL-22 were analyzed by ELISA in lung cells collected at day 1 after infection. Results were expressed as mean±SEM (n>6 per group).

EXAMPLE

[0043] Introduction:

[0044] Exacerbation episodes due to bacterial infection are a common feature during a wide variety of lung inflammatory disorders such as COPD. In patients with COPD, acute exacerbation is mostly associated with bacterial infections mostly due to Haemophilus influenzae and Streptococcus pneumoniae. Pathologically, exacerbations of COPD are characterized by enhanced oedema, airway and systemic inflammation, resulting in more airflow limitation and gas exchange defects. A growing body of evidence indicates that host innate immune defenses are broadly suppressed during COPD. We have developed appropriate models to better define mechanisms responsible for bacterial susceptibility during the exacerbation of COPD underlying on mice chronically exposed to cigarette smoke (CS) during 12 weeks (Pichavant M et al., Mucosal Immunol, 2014). For this, C57/BL6 mice chronically exposed to CS were infected by the local administration of sub-lethal doses of H. influenzae and S. pneumoniae. As reported in the first paragraph, these models have allowed to identify a strong defect in Th17 cytokines (IL-17A and IL-22) production after bacterial infection in COPD mice. Since TLR agonists have been identified as potent immuno-stimulators and as promoter of lung anti-bacterial defenses particularly against S. pneumoniae (ref JCS), we have then tested the ability of flagellin (a TLR5 agonist) to limit the development of bacterial infection and their consequences on lung inflammation in COPD mice

[0045] Material & Methods

[0046] Cigarette Smoke Exposure

[0047] C57/BL6 mice were exposed to CS generated from 5 cigarettes per day, 5 days a week, and up to 12 weeks using a smoke machine (Emka, Scireq, Canada). Air mice were housed in similar conditions and were only exposed to filtered air.

[0048] Bacterial Infection

[0049] Two strains of bacteria were used to exacerbate COPD: Streptococcus pneumoniae (Sp) serotype 1, and non typable Haemophilus influenza (NTHI). Bacteria stocks were kept frozen at −80° C. Bacteria were defrost just before the infection, and the number of cfu was determined on chocolate plates. Infection was performed by intranasal route (50 μl/mouse). Mice infected with S. pneumoniae were sacrificed at day 1 and day 3. Mice infected with NTHI were sacrificed at day 1 and day 2.

[0050] Administration of Flagellin

[0051] Purified recombinant flagellin were prepared in endotoxin-free conditions. Five μg of flagellin was injected by intraperitoneal route just before the infection in all the treated mice and two days after the infection in mice infected by S. pneumoniae. The controls were injected with the same volume of PBS (100 μl) (Mock).

[0052] Cytokine Quantification

[0053] Mouse IL-2, IL-17, IL-22 and IFN-gamma concentrations were measured in BAL, lung lysates and supernatants of lung cell culture by ELISA (R&D systems and e-Biosciences).

[0054] Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) Analysis

[0055] Quantitative RT-PCR was performed to quantify mRNA of interest (Table 1). Results were expressed as mean±SEM of folds (2.sup.−ΔΔCt) of the gene expression using β-actin as a reference, and compared to controls (air) calculated for each experiment.

[0056] Results

[0057] IL-17 and IL-22 Response to Bacterial Infection is Altered in COPD Mice.

[0058] Infected COPD mice develop a strong lung infection with SP (associated with an increased inflammatory reaction) whereas naïve mice are able to clear the bacteria within 24 hours. This defect in bacterial clearance is associated with a lower production of both IL-17 and IL-22 in the BAL (FIG. 1A) and after restimulation of lung cells (FIG. 1C). IL-22 concentrations were also lower in the blood of infected COPD mice as compared with Air mice (FIG. 1B). The defect in IL-17 and IL-22 is related to a decreased percentage of NK, NKT cells as well as innate lymphoid cells (ILC) positive for these cytokines in the lung of infected COPD mice as compared to infected air-exposed mice. Moreover, the supplementation with recombinant IL-22 allows to accelerate the clearance of the bacteria and to limit the consequences of the infection by S. pneumoniae in COPD mice. In COPD mice, infection with NTHI is associated with a defect in the production of IL-22 as shown in the BAL fluid (FIG. 1D). This defect also involved the same cells as observed for SP (ILC, NK and NKT cells). Since the role of IL-22 is unknown during infection by NTHI, we observed that IL-22−/− mice reproduce a phenotype close to that of COPD mice (increased susceptibility, higher inflammatory response and lung remodeling). These data show that COPD mice are more susceptible to infection by H. influenzae and S. pneumoniae than control mice and a deficient production of IL-17 and/or IL-22 may favor the development of bacteria-induced COPD exacerbations.

[0059] Administration of Flagellin Limits the Consequences of Infection by S. pneumoniae in COPD Mice.

[0060] Administration of flagellin by intraperitoneal route markedly reduced the bacterial load in Air and COPD mice at day 1 and 3 after infection. The effect is more important in the BAL (FIG. 2A) and the blood (FIG. 2C) as compared with the lung (FIG. 2B).

[0061] Analysis of the cytokine burst in the lung from infected animals revealed that administration of flagellin did not markedly increased the production of inflammatory cytokines (IL-1β, IL-6 and TNF-alpha) in infected Air and COPD mice. In contrast, treatment with flagellin enhanced the concentrations of IL-17 at day 3 in BAL fluids and in supernatants of anti-CD3 stimulated lung cells from both Air and COPD mice (FIG. 3). The same profile was observed for the secretion of IL-22 (FIG. 4) although the effect seems stronger for this cytokine than for IL-17. The modulation at day 3 of cytokine production was not associated with an increased number of neutrophils in the lung of infected mice receiving flagellin.

[0062] These data show that treatment with flagellin limits the bacterial susceptibility in COPD mice. This effect was associated with an increased production of IL-17 and IL-22 in the lung of infected COPD mice, both cytokines being involved in lung defenses against bacteria. Moreover, treatment with flagellin in infected COPD mice was not associated with a burst of inflammatory cytokines and an amplified recruitment of neutrophils which might be deleterious for the lung function.

[0063] Administration of Flagellin Limits the Consequences of Infection by NTHI in COPD Mice.

[0064] Similar experiments were reproduced in Air and COPD mice infected with NTHI. As showed with S. pneumoniae, administration of flagellin reduced the bacterial load in the BAL, the lung compartment and the blood (FIG. 5). The decrease reached 55 and 65% in the BAL and lung lysate, respectively, whereas it was nearly total in the blood.

[0065] The effect of flagellin was measured on the cytokine production in NTHI-infected COPD mice. As previously reported with S. pneumoniae, treatment with flagellin did not increased the production of inflammatory cytokines (IL-1β, IL-6 and TNF-alpha) in the lung of infected Air and COPD mice. Moreover, the secretion of IL-17 was not modulated with this bacteria. In contrast, the synthesis of IL-22 in the BAL and the cultures of lung cells was markedly amplified in COPD mice at day 1 (FIG. 6). Similar findings were observed at day 2 post infection. The recruitment of neutrophils was not modulated by the treatment with flagellin.

[0066] Altogether, these data demonstrate that administration of flagellin is able to decrease the bacterial load in COPD mice infected with NTHI. Since we previously reported that IL-22 production is important for the control of this infection, we can suspect that the increase in IL-22 production might be implicated in the beneficial effect of flagellin in NTHI-infected COPD mice. This treatment did not obviously amplified the lung inflammation in NTHI-infected COPD mice.

REFERENCES

[0067] 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.