Composition comprising a peptide and an inhibitor of viral neuraminidase

Abstract

Described is a composition comprising a peptide which consists of 7-17 adjacent amino acids and comprises the hexamer TX.sub.1EX.sub.2X.sub.3E, where X.sub.1, X.sub.2, and X.sub.3 can be any natural or non-natural amino acid, and the peptide is cyclized and does not exhibit TNF receptor binding activity, and an inhibitor of viral neuraminidase.

Claims

1. A composition, comprising: a peptide, selected from the group consisting of CGQRETPEGAEAKPWYC (SEQ ID NO: 1), QRETPEGAEAKPWY (SEQ ID NO: 5), PKDTPEGAEALKPWY (SEQ ID NO: 6) and CGPKDTPEGAELKPWYC (SEQ ID NO: 7), wherein the peptide has no Tumor necrosis factor (TNF) receptor binding activity and is cyclized, and an inhibitor of viral neuraminidase, wherein the inhibitor of viral neuraminidase is a sialic acid analog.

2. The composition of claim 1, wherein the peptide comprises the amino acid sequence CGQRETPEGAEAKPWYC (SEQ ID NO: 1) and is cyclized via the C-residues.

3. The composition of claim 2, wherein the peptide is cyclized via a disulfide bridge between said C-residues.

4. The composition of claim 1, further comprising a pharmaceutically acceptable carrier.

5. The composition of claim 4, wherein the pharmaceutically acceptable carrier is selected from the group consisting of water, sodium chloride, sodium phosphate, sodium acetate, sodium carbonate, citrate, glycine, glycylglycine, histidine, lysine, arginine, TRIS, sodium citrate, Ringer's solution, dextrose, mannite, trehalose, saccharose, sorbate, fructose, maltose, lactose or dextrane, Hank's solution, fixed oils, ethyl oleate, stabilizing agents, pharmaceutically acceptable proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids and amino acid copolymers.

6. The composition of claim 1, wherein the composition comprises, independently of each other, the peptide in an amount of 1 μg to 10 g, and the inhibitor of viral neuraminidase in an amount of 1 μg to 10 g.

7. The composition of claim 1, wherein the composition is in a liquid form and comprises, independently of each other, the peptide in an amount of 1 μg to 10 g, and the inhibitor of viral neuraminidase in an amount of 1 μg to 10 g, and is provided in a volume of 0.5 to 10 ml.

8. The composition of claim 1, wherein the composition is further defined as a nebulizable powder formulation or as a nebulizable liquid formulation.

9. The composition of claim 1, wherein the inhibitor of viral neuraminidase is selected from the group consisting of Laninamivir, its prodrug CS-8958, Zanamivir, Peramivir, Oseltamivir phosphate, Oseltamivir carboxylate, and Oseltamivir.

10. The composition of claim 9, wherein the inhibitor of viral neuraminidase is in a racemic, optically pure, salt-free, salt, enantiomeric or diastereomeric form.

11. A dry-powder inhaler comprising the composition of claim 1 and lactose particles, wherein the average particle size of said lactose particles is between 50 and 150 μm.

12. A set, comprising: a first container comprising a peptide, selected from the group consisting of CGQRETPEGAEAKPWYC (SEQ ID NO: 1), QRETPEGAEAKPWY (SEQ ID NO: 5), PKDTPEGAEALKPWY (SEQ ID NO: 6) and CGPKDTPEGAELKPWYC (SEQ ID NO: 7), wherein the peptide has no TNF receptor binding activity and is cyclized, and a second container comprising an inhibitor of viral neuraminidase, wherein the inhibitor of viral neuraminidase is a sialic acid analog.

13. The set of claim 12, wherein the peptide comprises the amino acid sequence CGQRETPEGAEAKPWYC (SEQ ID NO: 1) and is cyclized via the C-residues.

14. The set of claim 12, wherein the inhibitor of viral neuraminidase is selected from the group consisting of Laninamivir, its prodrug CS-8958, Zanamivir, Peramivir, Oseltamivir phosphate, Oseltamivir carboxylate, and Oseltamivir.

15. The set of claim 14, wherein the inhibitor of viral neuraminidase is in a racemic, optically pure, salt-free, salt, enantiomeric or diastereomeric form.

16. The set of claim 13, wherein the inhibitor of viral neuraminidase is selected from the group consisting of Laninamivir, its prodrug CS-8958, Zanamivir, Peramivir, Oseltamivir phosphate, Oseltamivir carboxylate, and Oseltamivir.

17. The set of claim 16, wherein the inhibitor of viral neuraminidase is in a racemic, optically pure, salt-free, salt, enantiomeric or diastereomeric form.

18. A method of treating one or more symptoms of influenza comprising the steps of: obtaining a peptide, selected from the group consisting of CGQRETPEGAEAKPWYC (SEQ ID NO: 1), QRETPEGAEAKPWY (SEQ ID NO: 5), PKDTPEGAEALKPWY (SEQ ID NO: 6) and CGPKDTPEGAELKPWYC (SEQ ID NO: 7), wherein the peptide has no TNF receptor binding activity and is cyclized, and an inhibitor of viral neuraminidase; and treating one or more symptoms of influenza, wherein the inhibitor of viral neuraminidase treats one or more symptoms of influenza and the peptide treats pulmonary inflammation associated with influenza; wherein the inhibitor of viral neuraminidase is a sialic acid analog.

19. The method of claim 18, wherein the inhibitor of viral neuraminidase is selected from the group consisting of Laninamivir, its prodrug CS-8958, Zanamivir, Peramivir, Oseltamivir phosphate, Oseltamivir carboxylate, and Oseltamivir.

20. The method of claim 19, wherein the inhibitor of viral neuraminidase is in a racemic, optically pure, salt-free, salt, enantiomeric or diastereomeric form.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The invention will be explained in more detail by way of the following examples and drawings, to which it is not limited, of course. It is shown in:

(2) FIG. 1: the relative lung weight of C57BL/6 mice on days 3, 5, 7 and 9 following an infection with influenza A or (as a control) without infection;

(3) FIG. 2: the relative lung weight of C57BL/6 mice on days 5, 7 and 9 following an infection with influenza A and treatment with Oseltamivir, treatment with the composition according to the invention, and without treatment (with PBS, for a control);

(4) FIG. 3: the relative lung weight of C57BL/6 mice on days 7 and 9 following an infection with influenza A and treatment with Zanamivir, treatment with the composition according to the invention, and without treatment (with PBS, for a control).

DETAILED DESCRIPTION OF THE INVENTION

(5) While the making and using of various embodiments of the present invention are discussed in detail below, it should be appreciated that the present invention provides many applicable inventive concepts that can be embodied in a wide variety of specific contexts. The specific embodiments discussed herein are merely illustrative of specific ways to make and use the invention and do not delimit the scope of the invention.

(6) The present examples are provided to show in an approved experimental mouse model that the goal of the present invention has been achieved by administering to mice, which had been infected with influenza virus, a neuraminidase inhibitor or a combination of the neuraminidase inhibitor and the synthetic peptide AP301 (SEQ ID NO: 1).

Example 1

(7) Infection with influenza virus causes the development of a pulmonary inflammation. Laboratory mice (strain C57BL/6, 8 weeks old) were infected per-nasal with the influenza strain A (PR8/34) at a dose of 150 PFU. On days 3, 5, 7 and 9 following the infection, the lungs were taken out of 8 mice, respectively, and the relative lung weight was determined as a measure of the pulmonary inflammation.

(8) The examination showed that with increasing length of time following the mice's infection with the influenza virus the lung weight strongly increased compared to control lungs. The results are shown graphically in FIG. 1.

Example 2

(9) Treatment of the pulmonary inflammation by administering the neuraminidase inhibitor or administering a combination of neuraminidase inhibitor and peptide AP301. Laboratory mice (strain C57BL/6, 8 weeks old) were infected per-nasal with the influenza strain A (PR8/34) at a dose of 150 PFU. Subsequently, each test animal received an oral administration of 10 mg/kg Oseltamivir (neuraminidase inhibitor) and an intratracheal administration of 10 μg/test animal of peptide AP301. The treatment was repeated on test days 2 and 4. On days 5, 7 and 9 following the infection, the lungs were taken out of 30 mice, respectively, and the relative lung weight was determined as a measure of the pulmonary inflammation. The results are shown graphically in FIG. 2.

(10) The examination showed that the neuraminidase inhibitor exhibited just a moderate effect on reducing the pulmonary inflammation, as measured by the lung weight. If, however, peptide AP301 was administered to the mice infected by the influenza virus in addition to the neuraminidase inhibitor, the pulmonary inflammation was essentially more reduced.

Example 3

(11) Ex vivo assessment of proinflammatory properties of the AP301 peptide in human whole blood. An ex vivo safety pharmacological study concerning the AP301 peptide in human whole blood was performed to assess whether the AP301 peptide results in the release of the proinflammatory marker interleukin-6 (IL-6) from fresh human whole blood (i.e. whether APN 301 exhibits TNF-specific inflammatory activity (i.e. TNF receptor binding activity)). In this study, fresh human whole blood has been used, as it represents an approved predictive model system for the assessment of inflammatory response in vivo.

(12) Summary of methodology. It was the goal of this study to determine the proinflammatory signalling capacity of the peptide AP301. Whole blood cultures were used and the secretion of interleukin-6 (IL-6), a very sensitive marker for proinflammatory stimulation, was quantified by ELISA.

(13) Test System

(14) Test system 25 ml of freshly taken heparinized blood from 5 healthy volunteers (HV) was used in the tests.

(15) Test item Identification: AP301 peptide (dose: 1 ng/ml to 10 μg/ml; single administration in solution) Description: White powder, purity 96%

(16) Whole blood cultures. Whole blood (WB) cultures are performed by pipetting 1 ml WB into wells of 24-well-plates. In each experiment, unstimulated and control-stimulated cultures were included.

(17) If possible, the substances and stimulants to be investigated were always added in an identical volume to each well of a given experiment, which is not greater than 10% of the total volume contained in a well. Unstimulated controls received PBS. Volume adjustments and dilutions for different treatments were also done with PBS.

(18) The content of each well was mixed and the plates incubated at 37° C. and 5% CO2 for 24 hours. After incubation the content of each well was transferred to a fresh 1.5 ml microtube and centrifuged at 8,000-9,000×g for 15 minutes. The supernatant of each sample was transferred individually to two 1.5 ml microtubes and kept at −20° C. until use.

(19) Detection of interleukin-6. Interleukin-6 was quantified by a specific ELISA (Human IL-6 ELISA Set, BD Biosciences, Cat. No. 555220) employing an anti-human IL-6 antibody as capture antibody, a biotinylated anti-human IL-6 detection antibody, avidin-horseradish peroxidase conjugate as enzyme reagent and recombinant IL-6 as standard. Absorbance measurement was performed at 450 nm using the Packard FusionReader.

(20) Data analysis. The results for each plate were stored and evaluated using the FusionDataAnalysis Software.

(21) Summary of study results. It was the goal of this study to determine the proinflammatory signalling capacity of the AP301peptide. Whole blood cultures were used and the secretion of IL-6, a very sensitive marker for proinflammatory stimulation, was quantified by ELISA.

(22) Whole blood samples of five healthy volunteers were either left unstimulated (negative control), stimulated with high and low doses of LPS (positive controls), or incubated with peptide with nine semi-logarithmic dilutions ranging from 10 μg/ml to 1 ng/ml. The results are shown in the following table:

(23) Table: Release of interleukin-6 from fresh human whole blood on addition of peptide AP301 and LPS

(24) TABLE-US-00002 AP301 peptide Positive control (LPS) Concentration concentration of IL-6 (pg/ml, n = 5) 0 (negative control) less than 0.5 less than 0.5 10 mg/ml less than 0.5 195.640 1 mg/ml less than 0.5 108.370 3 ng/ml less than 0.5  34.867 1 ng/ml less than 0.5 not determined

(25) The results clearly reveal that the AP301 peptide did not induce any detectable level of IL-6 secretion at any of the concentrations tested. The positive controls (LPS) resulted in a strong induction of IL-6 secretion.

(26) Discussion. The experiments have been performed to assess whether the AP301 peptide mediates the induction of a proinflammatory cascade. Readout parameter was the induced secretion of IL-6 in whole blood cultures from five healthy donors. The results clear showed that the AP301 peptide did not induce any detectable level of IL-6 in any donor's cultures. Therefore, it is demonstrated that the AP301 peptide did not induce a proinflammatory response in the chosen ex vivo model and, thus, does not exhibit any TNF receptor binding activity.

Example 4

(27) Treatment of pulmonary inflammation by administering neuraminidase inhibitor (Zanamivir) or administering a combination of neuraminidase inhibitor (Zanamivir) and peptide AP301. Laboratory mice (strain C57BL/6, 8 weeks old) were infected per-nasal with the influenza strain A (PR8/34) at a dose of 150 PFU. Subsequently, each test animal received a nasal administration of 1.5 mg/kg Zanamivir (neuraminidase inhibitor) and an intratracheal administration of 50 μg/test animal of peptide AP301. The treatment was repeated on test days 2 and 4.

(28) On days 7 and 9 following the infection, the lungs were taken out of 20 mice, respectively, and the relative lung weight was determined as a measure of the pulmonary inflammation. The results are shown graphically in FIG. 3.

(29) The examination showed that the neuraminidase inhibitor (Zanamivir) exhibited just a moderate effect on reducing the pulmonary inflammation, as measured by the lung weight. If, however, peptide AP301 was administered to the mice infected by the influenza virus in addition to the neuraminidase inhibitor, the pulmonary inflammation was essentially more reduced.

(30) The peptides according to the invention exhibit a synergistic effect in the treatment of influenza infections in combination with a neuraminidase inhibitor.

(31) In WO 2010/099556 A1, the treatment of various pulmonary symptoms is documented, as mentioned, which is targeted to the hyperpermeability of epithelial cells and endothelial cells in such lung diseases. According to WO 2010/099556 A1 it was shown that the claimed peptides are excellently suited for preventing and treating these symptoms. Although, according to WO 2010/099556 A1, hyperpermeability of epithelial cells and endothelial cells can also be treated in cases of infections with influenza viruses (in the context of a pneumonia developing with this infection), the present invention is of course not suggested by this. The possibility of generally treating an influenza with a combination preparation, comprising this peptide, is an entirely new and inventive teaching as to the applicability of the peptide for treating a—facultatively—emerging symptom of influenza.

(32) The basic difference can also be derived from a view into the detailed experiments according to WO 2010/099556 A1: In the examples of WO 2010/099556 A1, it is experimentally demonstrated that the peptides in the lung tissue: i) influence the content of reactive oxygen, ii) the effect of the bacterial gram positive toxines “listeriolysine” and “pneumolysine” also influences hyperpermeability, a.o. by regulating the level of phosphorylated myosin light chain, infiltration of leucocytes, activated protein kinase C, iii) influence the body weight after an influenza infection, iv) influence the body temperature after an influenza infection, v) influence the survival rate of the test animals after an influenza infection.

(33) Throughout the whole WO 2010/099556 A1, however, there are no experimental hints as to an infection of test animals with the influenza virus leading to an alteration of the relative lung weight and as to halting and treating such a process by the administration of the peptide. Only now, by the present invention, it is shown that an infection of the lungs of test animals with the influenza virus leads to a substantial increase in the relative lung weight.

(34) The lung constitutes one of the most important organs of all. An increase in the relative lung weight is connected to a damage of the lung function which cannot be compensated for by any other organ. It is a property of a healthy lung to contain as many air-filled spaces (alveoles) as possible. At an increase of the relative lung weight, it must strongly be assumed that the share of air-filled alveoles will decline severely which will restrict the function of the lung. Therefore, the relative lung weight constitutes an essential factor for the treatment of influenza. The results shown in the experimental part of the present application thus demonstrate impressively the synergistic effect of the combination preparation according to the present invention, based on an extremely critical and relevant parameter.

(35) It has hitherto been known from neuraminidase inhibitors that these may suppress the proliferation of the influenza virus. Neuraminidase inhibitors do not cause a reduction of living influenza viruses. Hitherto, it could not be shown in the state of the art that influenza virus leads to an increase of the relative lung weight. Only with the present invention has it surprisingly been shown that the administration of a neuraminidase inhibitor after an influenza infection reduces the relative weight gain of the lung weight. Additionally, by the present invention could it be shown for the first time that a simultaneous treatment of test animals, which were infected with the influenza virus, with a neuraminidase inhibitor and a peptide according to WO 2010/099556 A1 in combination results in a significant and unforeseeable synergistic effect on the relative lung weight. While neuraminidase inhibitors inhibit the proliferation of influenza virus, without reducing the already living viruses, the synergistic combination of a neuraminidase inhibitor and a peptide according to the invention obviously leads to a significant improvement of the influenza treatment. None of the drugs (neuraminidase inhibitor and the peptide according to the invention) administered individually does by itself lead to the effect shown by the present invention.

(36) Therefore, the present invention could not be rendered obvious in any way by the results disclosed in WO 2010/099556 A1.

(37) TABLE-US-00003 Summary of sequences: SEQ ID NO: 1 CGQRETPEGAEAKPWYC SEQ ID NO: 2 KSPGGQRETPEGAEAKPWYE SEQ ID NO: 3 CGQREAPAGAAAKPWYC SEQ ID NO: 4 TPEGAE SEQ ID NO: 5 QRETPEGAEAKPWY SEQ ID NO: 6 PKDTPEGAELKPWY SEQ ID NO: 7 CGPKDTPEGAELKPWYC SEQ ID NO: 8 CGQKETPEGAEAKPWYC SEQ ID NO: 9 CGQRETPEGAEARPWYC SEQ ID NO: 10 CGQRETPEGAEAKPC SEQ ID NO: 11 CQRETPEGAEAKPWYC SEQ ID NO: 12 CGQRETPEGAEAKFWYC